Handbook of Chitin and Chitosan: Volume 3: Chitin- and Chitosan-based Polymer Materials for Various Applications 012817966X, 9780128179666

Table of contents :
Contents
List of contributors
1. Polymer blends, IPNs, gels, composites, and nanocomposites from chitin and chitosan; manufacturing, and applications • Sabrina Sultana, Md. Shirajur Rahman, Md. Minhajul Islam, Md. Nurus Sakib and Md. Shahruzzaman
2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials • Inês Farinha and Filomena Freitas
3. Chitin and chitosan composites for wearable electronics and energy storage devices • Yasir Beeran Pottathara, Hanuma Reddy Tiyyagura, Zakiah Ahmad and Sabu Thomas
4. Investigation into the functional properties of cotton, wool, and denim textile materials finished with chitosan and the use of chitosan in textile-reinforced composites and medical textiles • Nilgün Becenen, Sevil Erdoğan and Elif Ecem Fındık
5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites for adsorption systems: environmental remediation and protein purification • Gabriel Ibrahin Tovar-Jimenez, Daniela Belén Hirsch, María Emilia Villanueva, Nicolás Urtasun, Federico Javier Wolman and Guillermo Javier Copello
6. Functional properties of chitin and chitosan-based polymer materials • Gisoo Maleki and Jafar M. Milani
7. Fundamentals of chitosan for biomedical applications • Mohammad Rahat Hossain, Abul K. Mallik and Mohammed Mizanur Rahman
8. Electrospun chitosan materials and their potential use as scaffolds for bone and cartilage tissue engineering • Adriana Hernández-Rangel, Gina Prado-Prone, Joseline J. Hidalgo-Moyle, Phaedra Silva-Bermudez and Keiko Shirai
9. Injectable polymeric gels based on chitosan and chitin for biomedical applications • Cong Xie, Wei Huang, Weiqing Sun and Xulin Jiang
10. Preparation and application of biomimetic and bioinspired membranes based on chitosan • Laxmi Gond, Preeti Pradhan and Anjali Bajpai
11. Chitin, chitosan, marine to market • G.M. Oyatogun, T.A. Esan, E.I. Akpan, S.O. Adeosun, A.P.I. Popoola, B.I. Imasogie, W.O. Soboyejo, A.A. Afonja, S.A. Ibitoye, V.D. Abere, A.O. Oyatogun, K.M. Oluwasegun, I.E. Akinwole and K.J. Akinluwade
12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications • Jafar M. Milani and Mohammad Hossein Naeli
13. Chitosan as biomaterial in drug delivery and tissue engineering • Poliana Pollizello Lopes, Eduardo Hiromitsu Tanabe and Daniel Assumpção Bertuol
14. Biomedical applications carboxymethyl chitosans • Shanta Biswas, Tanvir Ahmed, Md. Minhajul Islam, Md. Sazedul Islam and Mohammed Mizanur Rahman
15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing • Simone S. Silva, Joana M. Gomes, Luísa C. Rodrigues and Rui L. Reis
16. Chitin and chitosan composites for bone tissue regeneration • E.I. Akpan, O.P. Gbenebor, S.O. Adeosun and Odili Cletus
17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems • Rajitha Panonnummal, Nitheesh Antony and M. Sabitha
18. Future aspects of biomedical applications of chitin and chitosan in diseases associated with oxidative stress • Makoto Anraku, Shinsuke Ifuku, Daisuke Iohara, Fumitoshi Hirayama, Masaki Otagiri and Janusz M. Gebicki
19. Immunomodulatory activities of chitin and chitosan microparticles • Mostafa Haji Molla Hoseini, Sahar Sadeghi, Mahdieh Azizi and Ramin Pouriran
20. Chitosan/chitin-based composites for food packaging applications • Muhammad Zubair, Muhammad Arshad, Rehan Ali Pradhan and Aman Ullah
21. Modified release properties of glutathione-based chitosan films: Physical and functional characterization • Yhors Ciro, John Rojas, Cristian J. Yarce and Constaín H. Salamanca
22. Chitosan-based materials as templates for essential oils • Rut Fernández-Marín, Susana C.M. Fernandes, Colin McReynolds, Jalel Labidi and Ma Ángeles Andrés Sánchez
23. Chitosan and chitosan-based biomaterials for wound management • Md. Sazedul Islam, Md. Shirajur Rahman, Tanvir Ahmed, Shanta Biswas, Papia Haque and Mohammed Mizanur Rahman
24. Chitin and chitosan as promising immunostimulant for aquaculture • Dibyendu Kamilya and Md. Idrish Raja Khan
25. Chitosan-based materials for water and wastewater treatment • Muhammad Zubair, Muhammad Arshad and Aman Ullah
Index

Citation preview

HANDBOOK OF CHITIN AND CHITOSAN

This page intentionally left blank

HANDBOOK OF CHITIN AND CHITOSAN CHITIN- AND CHITOSAN-BASED POLYMER MATERIALS FOR VARIOUS APPLICATIONS

VOLUME 3 Edited by

SREERAG GOPI Center for Innovations and Technologies (CIT), ADSO Naturals Private Limited, Bangalore, India

SABU THOMAS Mahatma Gandhi University, Kottayam, India

ANITHA PIUS The Gandhigram Rural Institute (Deemed University), Dindigul, India

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-817966-6 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Susan Dennis Editorial Project Manager: Kelsey Connors Production Project Manager: Sujatha Thirugnana Sambandam Cover Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

Contents

List of contributors

xiii

1. Polymer blends, IPNs, gels, composites, and nanocomposites from chitin and chitosan; manufacturing, and applications

1

SABRINA SULTANA, MD. SHIRAJUR RAHMAN, MD. MINHAJUL ISLAM, MD. NURUS SAKIB AND MD. SHAHRUZZAMAN

1.1 Introduction 1.2 Chemical structure and preparation of chitin and chitosan 1.3 Polymer blends from chitin and chitosan 1.4 Interpenetrating polymer networks from chitin/chitosan 1.5 Chitin- and chitosan-based gels 1.6 Chitin- and chitosan-based composites 1.7 Chitin- and chitosan-based nanocomposites 1.8 Conclusions and future prospective References

2 4 6 9 14 22 27 32 33

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

43

INEˆS FARINHA AND FILOMENA FREITAS

2.1 Introduction 2.2 Chemically modified chitin 2.3 Chemically modified chitosan 2.4 Chemically modified chitinous polysaccharides 2.5 Conclusions References

44 46 53 62 63 64

3. Chitin and chitosan composites for wearable electronics and energy storage devices

71

YASIR BEERAN POTTATHARA, HANUMA REDDY TIYYAGURA, ZAKIAH AHMAD AND SABU THOMAS

3.1 Introduction 3.2 Physical and chemical properties of chitin and chitosan 3.3 Chitin and chitosan composites for advanced electronics 3.4 Energy storage devices 3.5 Conclusion Acknowledgments References

v

72 72 73 77 85 86 86

vi

CONTENTS

4. Investigation into the functional properties of cotton, wool, and denim textile materials finished with chitosan and the use of chitosan in textile-reinforced composites and medical textiles

89

˘ ¨ N BECENEN, SEVIL ERDOGAN NILGU AND ELIF ECEM FINDIK

4.1 Introduction 4.2 Chitin and chitosan 4.3 Application techniques and methods of chitosan to textile products 4.4 Properties of the chitosan-treated textile products 4.5 Use of chitosan in medical textiles 4.6 Use of chitosan in textile-reinforced composites 4.7 Conclusion Acknowledgment References

90 91 105 107 121 124 126 127 127

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites for adsorption systems: environmental remediation and protein purification

135

GABRIEL IBRAHIN TOVAR-JIMENEZ, DANIELA BELE´N HIRSCH, ´ S URTASUN, FEDERICO JAVIER WOLMAN AND MARI´A EMILIA VILLANUEVA, NICOLA GUILLERMO JAVIER COPELLO

5.1 5.2 5.3 5.4

Introduction Chitin adsorption properties Shaping chitin materials Optimizing adsorption performance by chemical and physical modifications 5.5 Concluding remarks and future considerations Acknowledgments References

6. Functional properties of chitin and chitosan-based polymer materials

136 138 148 154 166 167 168

177

GISOO MALEKI AND JAFAR M. MILANI

6.1 Introduction 6.2 Dietary activity 6.3 Antimicrobial activity 6.4 Emulsifying properties 6.5 Antioxidant activity 6.6 Flocculent and chelating 6.7 Future trends References

178 178 181 185 188 191 193 193

CONTENTS

7. Fundamentals of chitosan for biomedical applications

vii 199

MOHAMMAD RAHAT HOSSAIN, ABUL K. MALLIK AND MOHAMMED MIZANUR RAHMAN

7.1 Introduction 7.2 Processing of chitosan 7.3 Properties and limitations of chitosan 7.4 Modifications and modified form of chitosan in biomedical application 7.5 Biomedical application of chitosan and its derivatives 7.6 Conclusion References

8. Electrospun chitosan materials and their potential use as scaffolds for bone and cartilage tissue engineering

200 201 205 206 212 221 221

231

´ NDEZ-RANGEL, GINA PRADO-PRONE, JOSELINE J. HIDALGO-MOYLE, ADRIANA HERNA PHAEDRA SILVA-BERMUDEZ AND KEIKO SHIRAI

8.1 Introduction 8.2 Electrospinning 8.3 Chitosan 8.4 Regulatory issues of electrospinning scaffolds 8.5 Chitosan electrospun materials for bone tissue engineering 8.6 Chitosan electrospun materials for cartilage tissue engineering Acknowledgments References

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

232 241 249 255 256 266 269 269

281

CONG XIE, WEI HUANG, WEIQING SUN AND XULIN JIANG

9.1 Introduction 9.2 Injectable polymeric gels based on chitosan and chitin 9.3 Biomedical applications 9.4 Conclusions and future perspectives Acknowledgment References

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

282 283 294 298 299 299

307

LAXMI GOND, PREETI PRADHAN AND ANJALI BAJPAI

10.1 Introduction 10.2 Biomimetic and bioinspired membranes 10.3 Applications of biomimetic and bioinspired membranes 10.4 Chitosan References

308 308 310 316 331

viii

CONTENTS

11. Chitin, chitosan, marine to market

341

G.M. OYATOGUN, T.A. ESAN, E.I. AKPAN, S.O. ADEOSUN, A.P.I. POPOOLA, B.I. IMASOGIE, W.O. SOBOYEJO, A.A. AFONJA, S.A. IBITOYE, V.D. ABERE, A.O. OYATOGUN, K.M. OLUWASEGUN, I.E. AKINWOLE AND K.J. AKINLUWADE

11.1 Introduction 11.2 Origin and sources of chitin and chitosan 11.3 Synthesis of chitin and chitosan 11.4 Properties of chitin and chitosan 11.5 Potential applications of chitin and chitosan 11.6 Economic potential of chitin and chitosan 11.7 Conclusion References Further reading

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

342 343 345 354 359 367 370 370 381

383

JAFAR M. MILANI AND MOHAMMAD HOSSEIN NAELI

12.1 12.2 12.3 12.4 12.5 12.6 12.7

Introduction Application of chitin- and chitosan-based oleogels Preparation method and mechanism of chitin- and chitosan-based oleogels Chemical and physical modifications of chitin and chitosan Rheological properties of chitin- and chitosan-based oleogels Thermogravimetric properties of chitin- and chitosan-based oleogels Thermal behavior (differential scanning calorimetry thermogram) of chitin- and chitosan-based oleogels References

13. Chitosan as biomaterial in drug delivery and tissue engineering

384 386 387 389 397 400 402 404

407

POLIANA POLLIZELLO LOPES, EDUARDO HIROMITSU TANABE AND ˜ O BERTUOL DANIEL ASSUMPC ¸A

13.1 Introduction 13.2 General aspects of chitosan and functional features 13.3 Chitosan-based therapeutic systems 13.4 Chitosan as tissue supporting material 13.5 Conclusion References

14. Biomedical applications carboxymethyl chitosans

408 408 413 419 424 424

433

SHANTA BISWAS, TANVIR AHMED, MD. MINHAJUL ISLAM, MD. SAZEDUL ISLAM AND MOHAMMED MIZANUR RAHMAN

14.1 Introduction 14.2 Processing of chitosan 14.3 Properties and limitations of chitosan in the field of biomedical applications

434 435 436

CONTENTS

14.4 Preparative methods of various carboxymethyl chitosan 14.5 Properties of carboxymethyl chitosan 14.6 Application of carboxymethyl chitosan 14.7 Conclusion References

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

ix 437 441 445 463 463

471

SIMONE S. SILVA, JOANA M. GOMES, LUI´SA C. RODRIGUES AND RUI L. REIS

15.1 Introduction 15.2 Dissolution of chitin and chitosan using ionic liquids 15.3 Chitin 15.4 Chitosan 15.5 Processability of chitin and chitosan via ionic liquids 15.6 Chitin and chitosan-based architectures 15.7 Biomedical applications of chitin and chitosan in ionic liquids 15.8 Bone repair 15.9 Drug and gene delivery 15.10 Neuron repair 15.11 Final remarks and future trends Acknowledgments Abbreviators References

16. Chitin and chitosan composites for bone tissue regeneration

472 474 474 477 479 484 489 490 490 491 491 493 493 494

499

E.I. AKPAN, O.P. GBENEBOR, S.O. ADEOSUN AND ODILI CLETUS

16.1 Introduction 16.2 Bone tissue engineering 16.3 Insights to bone biology 16.4 Clinical requirements for bone tissue engineering scaffolds 16.5 Physical and mechanical requirements for bone tissue replacement 16.6 Chitin/chitosan-based materials for bone tissue engineering 16.7 Methods of fabrication of chitin and chitosan scaffolds 16.8 The dilemma of chitosan scaffold applicability 16.9 Conclusions References

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

500 501 504 506 509 513 528 537 540 541

555

RAJITHA PANONNUMMAL, NITHEESH ANTONY AND M. SABITHA

17.1 17.2 17.3 17.4

Introduction Chitosan-based nanosytems in drug therapy of neoplastic diseases Chitosan-based nanosystems in drug therapy of infectious diseases Blocking the supply of essential elements for microbes

556 561 566 567

x

CONTENTS

17.5 Chitosan-based nanosytems in drug therapy of inflammatory diseases 17.6 Tissue engineering applications of chitosan-based nanosytems 17.7 Conclusion References

18. Future aspects of biomedical applications of chitin and chitosan in diseases associated with oxidative stress

570 573 581 582

589

MAKOTO ANRAKU, SHINSUKE IFUKU, DAISUKE IOHARA, FUMITOSHI HIRAYAMA, MASAKI OTAGIRI AND JANUSZ M. GEBICKI

18.1 Introduction 18.2 Chitosan and its derivatives as antioxidants with protective effects for chronic renal failure 18.3 SDACNFs as a material for pharmaceutical formulation in diseases associated with oxidative stress 18.4 Therapeutic effects of PD/NFs-CDs gel in model mice 18.5 Conclusions Abbreviations References

19. Immunomodulatory activities of chitin and chitosan microparticles

590 591 597 601 604 605 605

609

MOSTAFA HAJI MOLLA HOSEINI, SAHAR SADEGHI, MAHDIEH AZIZI AND RAMIN POURIRAN

19.1 Chitinous microparticles and immune responses 19.2 Chitin and chitosan microparticles as adjuvant 19.3 Anticancer effect of chitin and chitosan microparticles 19.4 Allergy and chitinous microparticles 19.5 Inflammation and chitin, chitosan microparticles 19.6 Conclusion References

20. Chitosan/chitin-based composites for food packaging applications

610 614 619 623 627 629 631

641

MUHAMMAD ZUBAIR, MUHAMMAD ARSHAD, REHAN ALI PRADHAN AND AMAN ULLAH

20.1 Introduction 20.2 Various chitosan/chitin-based composites for food packaging applications 20.3 Chitin/chitosan and carbohydrates-derived composites 20.4 Chitin/chitosan composites with synthetic polymer 20.5 Inorganic materials-derived composites 20.6 Conclusion and future trends References

642 643 652 657 659 663 664

CONTENTS

21. Modified release properties of glutathione-based chitosan films: Physical and functional characterization

xi 671

YHORS CIRO, JOHN ROJAS, CRISTIAN J. YARCE AND CONSTAI´N H. SALAMANCA

21.1 Introduction 21.2 Experiment 21.3 Results and discussion 21.4 Conclusions Acknowledgments References

22. Chitosan-based materials as templates for essential oils

672 673 677 686 687 687

689

´ NDEZ-MARI´N, SUSANA C.M. FERNANDES, COLIN MCREYNOLDS, RUT FERNA ´ NGELES ANDRE´S SA ´ NCHEZ JALEL LABIDI AND MA A

22.1 Introduction 22.2 Chitosan-based essential oils coatings and films 22.3 Chitosan-based essential oils emulsions and (nano)gels 22.4 Chitosan (nano)capsules for essential oils encapsulation 22.5 Antioxidants activity in chitosan as templates for essential oils 22.6 Antibacterial activity in chitosan as templates for essential oils 22.7 Conclusions and future perspectives References

690 692 693 696 697 706 714 715

23. Chitosan and chitosan-based biomaterials for wound management

721

MD. SAZEDUL ISLAM, MD. SHIRAJUR RAHMAN, TANVIR AHMED, SHANTA BISWAS, PAPIA HAQUE AND MOHAMMED MIZANUR RAHMAN

23.1 Introduction 23.2 Wound healing and its different stages 23.3 Properties of chitosan advantageous for wound management 23.4 Different forms of chitosan-based biomaterials in wound management 23.5 Chitosan-based biocomposites for wound healing 23.6 Chitosan derivatives in wound management 23.7 Future aspects of chitosan-based material for wound management 23.8 Conclusion References

24. Chitin and chitosan as promising immunostimulant for aquaculture

722 723 727 731 739 749 752 753 753

761

DIBYENDU KAMILYA AND MD. IDRISH RAJA KHAN

24.1 24.2 24.3 24.4

Introduction Immunostimulatory effect of chitin on finfish Immunostimulatory effect of chitin on shellfish Immunostimulatory effect of chitosan on finfish

762 763 764 765

xii

CONTENTS

24.5 Immunostimulatory effect of chitosan on shellfish 24.6 Effect of chitin and chitosan on disease resistance 24.7 Mechanism of action of chitin and chitosan as an immunostimulant 24.8 Conclusions and future perspectives References

25. Chitosan-based materials for water and wastewater treatment

766 766 767 768 769

773

MUHAMMAD ZUBAIR, MUHAMMAD ARSHAD AND AMAN ULLAH

25.1 Introduction 25.2 Various forms of chitosan-based sorbents for water/wastewater remediation 25.3 Removal of different pollutants using chitosan-based material 25.4 Mechanism of adsorption 25.5 Summary and future perspective References

Index

774 775 783 792 800 801

811

List of Contributors V.D. Abere Department of Mineral Processing, National Metallurgical Development Centre, Jos, Nigeria S.O. Adeosun Department of Metallurgical and Materials Engineering, University of Lagos, Akoka, Nigeria A.A. Afonja Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria Zakiah Ahmad Faculty of Civil Engineering and Institute for Infrastructure Engineering and Sustainable Management, Universiti Teknologi Mara, Shah Alam, Malaysia Tanvir Ahmed Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh K.J. Akinluwade Department of Research and Development, Prototype Engineering Development Institute (National Agency for Science and Engineering Infrastructure, NASENI), Ilesa, Nigeria I.E. Akinwole Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria E.I. Akpan Institute for Kaiserslautern, Germany

Composite

Materials,

Technical

University,

Makoto Anraku Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan; DDS Research Institute, Sojo University, Kumamoto, Japan Nitheesh Antony Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Amrita Health Science Campus, Kochi, India Muhammad Arshad Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Mahdieh Azizi Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Anjali Bajpai Department of Chemistry, Government Science College, Jabalpur, India Nilgu¨n Becenen Textile Technology Programme, Edirne Vocational College of Technical Sciences, Trakya University, Edirne, Turkey Daniel Assumpc¸a˜o Bertuol Chemical Engineering Department, Federal University of Santa Maria—UFSM, Santa Maria, RS, Brazil Shanta Biswas Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

xiii

xiv

List of Contributors

Yhors Ciro Department of Pharmacy, School of Pharmaceutical and Food Sciences, University of Antioquia, Medellı´n, Colombia Odili Cletus Department of Metallurgical University of Lagos, Lagos, Nigeria

and

Materials

Engineering,

Guillermo Javier Copello Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquı´mica, Departamento de Quı´mica Analı´tica y Fisicoquı´mica, Junı´n 956, CABA, Argentina; CONICET Universidad de Buenos Aires (UBA), Instituto de Quı´mica y Metabolismo del Fa´rmaco (IQUIMEFA), Buenos Aires, Argentina ˘ Sevil Erdogan Laborant and Veterinary Health Programme, Ke¸san Vocational College, Trakya University, Edirne, Turkey T.A. Esan Department of Restorative Dentistry, Obafemi Awolowo University, Ile-Ife, Nigeria Ineˆs Farinha Portugal

73100 Lda., Edifı´cio Arcis, Rua Ivone Silva, 6, 4 piso, Lisboa,

Susana C.M. Fernandes CNRS/Univ Pau & Pays Adour/ E2S UPPA, Institut des Sciences Analytiques et de Physico-Chimie pour l’Environnement et les Materiaux, Anglet, France Rut Ferna´ndez-Marı´n Environmental and Chemical Engineering Departament, University of the Basque Country UPV/EHU, Donostia-San Sebastia´n, Spain Elif Ecem Fındık Department of Biotechnology and Genetic, Institute of Natural Sciences, Trakya University, Edirne, Turkey Filomena Freitas UCIBIO-REQUIMTE, Chemistry Department, Faculty of Sciences and Technology, Universidade NOVA de Lisboa, Campus da Caparica, Caparica, Portugal O.P. Gbenebor Department of Metallurgical and Materials Engineering, University of Lagos, Lagos, Nigeria Janusz M. Gebicki Department of Biological Sciences, Macquarie University, Sydney, Australia Joana M. Gomes 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimara˜es, Portugal; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimara˜es, Portugal Laxmi Gond India

Department of Chemistry, Government Science College, Jabalpur,

Papia Haque Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Adriana Herna´ndez-Rangel Biotechnology Department, Laboratory of Biopolymers and Pilot Plant of Bioprocessing of Agro-Industrial and Food By-Products, Autonomous Metropolitan University, Mexico City, Mexico Joseline J. Hidalgo-Moyle Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico

List of Contributors

xv

Fumitoshi Hirayama Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan; DDS Research Institute, Sojo University, Kumamoto, Japan Daniela Bele´n Hirsch Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquı´mica, Ca´tedra de Biotecnologı´a, Junı´n, Argentina; CONICET Universidad de Buenos Aires, Instituto de Nanobiotecnologı´a (NANOBIOTEC), Buenos Aires, Argentina Mostafa Haji Molla Hoseini Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Mohammad Rahat Hossain Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Wei Huang China

Hangzhou Singclean Medical Products Co., Ltd., Hangzhou, P.R.

S.A. Ibitoye Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria Shinsuke Ifuku Japan

Graduate School of Engineering, Tottori University, Tottori,

B.I. Imasogie Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria Daisuke Iohara Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan; DDS Research Institute, Sojo University, Kumamoto, Japan Md. Minhajul Islam Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Md. Sazedul Islam Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Xulin Jiang Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan, P.R. China Dibyendu Kamilya Department of Aquatic Health and Environment, College of Fisheries, Central Agricultural University, Lembucherra, Tripura, India Md. Idrish Raja Khan Department of Aquatic Health and Environment, College of Fisheries, Central Agricultural University, Lembucherra, Tripura, India Jalel Labidi Environmental and Chemical Engineering Departament, University of the Basque Country UPV/EHU, Donostia-San Sebastia´n, Spain Poliana Pollizello Lopes Chemical Engineering Department, University of Santa Maria—UFSM, Santa Maria, RS, Brazil

Federal

Gisoo Maleki Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad, Iran Abul K. Mallik Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

xvi

List of Contributors

Colin McReynolds CNRS/Univ Pau & Pays Adour/ E2S UPPA, Institut des Sciences Analytiques et de Physico-Chimie pour l’Environnement et les Materiaux, Anglet, France Jafar M. Milani Department of Food Science and Technology, Agricultural Sciences and Natural Resources University, Sari, Iran

Sari

Mohammad Hossein Naeli Department of Food Science and Technology, Sari Agricultural Sciences and Natural Resources University, Sari, Iran K.M. Oluwasegun Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria Masaki Otagiri Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan; DDS Research Institute, Sojo University, Kumamoto, Japan A.O. Oyatogun Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria G.M. Oyatogun Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria Rajitha Panonnummal Amrita School of Pharmacy, Amrita Vidyapeetham, Amrita Health Science Campus, Kochi, India

Vishwa

A.P.I. Popoola Deparment of Chemical, Metallurgical and Materials Engineering, Tshwane University of Technology, Pretoria, South Africa Yasir Beeran Pottathara Faculty of Mechanical Engineering, University of Maribor, Maribor, Slovenia Ramin Pouriran School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Preeti Pradhan Department of Chemistry, Government Science College, Jabalpur, India Rehan Ali Pradhan Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Gina Prado-Prone Postgraduate Studies and Research Division, Faculty of Dentistry, National Autonomous University of Mexico, Mexico City, Mexico Md. Shirajur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Mohammed Mizanur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Rui L. Reis 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimara˜es, Portugal; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimara˜es, Portugal; The Discoveries Centre for Regenerative and Precision Medicine, Guimara˜es, Portugal

List of Contributors

xvii

Luı´sa C. Rodrigues 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimara˜es, Portugal; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimara˜es, Portugal John Rojas Department of Pharmacy, School of Pharmaceutical and Food Sciences, University of Antioquia, Medellı´n, Colombia M. Sabitha Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Amrita Health Science Campus, Kochi, India Sahar Sadeghi Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Md. Nurus Sakib Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Constaı´n H. Salamanca Laboratorio de Disen˜o y Formulacio´n de Productos Quı´micos y Derivados, Departamento de Ciencias Farmace´uticas, Facultad de Ciencias Naturales, Universidad ICESI, Cali, Colombia ´ ngeles Andre´s Sa´nchez Environmental and Chemical Engineering Ma A Departament, University of the Basque Country UPV/EHU, Donostia-San Sebastia´n, Spain Md. Shahruzzaman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Keiko Shirai Biotechnology Department, Laboratory of Biopolymers and Pilot Plant of Bioprocessing of Agro-Industrial and Food By-Products, Autonomous Metropolitan University, Mexico City, Mexico Simone S. Silva 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimara˜es, Portugal; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimara˜es, Portugal Phaedra Silva-Bermudez Tissue Engineering, Cellular Therapy and Regenerative Medicine Unit, National Institute of Rehabilitation “Luis Guillermo Ibarra Ibarra”, Mexico City, Mexico W.O. Soboyejo Faculty of Engineering, Wisconsin Polytechnic Institute, Menomonie, WI, United States Sabrina Sultana Department of Arts and Sciences, Ahsanullah University of Science and Technology, Dhaka, Bangladesh Weiqing Sun Hangzhou Singclean Medical Products Co., Ltd., Hangzhou, P.R. China Eduardo Hiromitsu Tanabe Chemical Engineering Department, Federal University of Santa Maria—UFSM, Santa Maria, RS, Brazil

xviii

List of Contributors

Sabu Thomas International and Interuniversity Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, India Hanuma Reddy Tiyyagura Faculty of Mechanical Engineering, University of Maribor, Maribor, Slovenia Gabriel Ibrahin Tovar-Jimenez Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquı´mica, Departamento de Quı´mica Analı´tica y Fisicoquı´mica, Junı´n 956, CABA, Argentina; CONICET Universidad de Buenos Aires (UBA), Instituto de Quı´mica y Metabolismo del Fa´rmaco (IQUIMEFA), Buenos Aires, Argentina Aman Ullah Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Nicola´s Urtasun CONICET Universidad de Buenos Aires, Instituto de Nanobiotecnologı´a (NANOBIOTEC), Buenos Aires, Argentina; Universidad de Buenos Aires (UBA), Facultad de Ciencias Exactas y Naturales, Departamento de Fisiologı´a, Biologı´a Molecular y Celular, Junı´n, Argentina Marı´a Emilia Villanueva Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquı´mica, Departamento de Quı´mica Analı´tica y Fisicoquı´mica, Junı´n 956, CABA, Argentina; CONICET Universidad de Buenos Aires (UBA), Instituto de Quı´mica y Metabolismo del Fa´rmaco (IQUIMEFA), Buenos Aires, Argentina Federico Javier Wolman Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquı´mica, Ca´tedra de Biotecnologı´a, Junı´n, Argentina; CONICET Universidad de Buenos Aires, Instituto de Nanobiotecnologı´a (NANOBIOTEC), Buenos Aires, Argentina Cong Xie Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan, P.R. China; Nonpower Nuclear Technology Research and Development Center, Hubei University of Science and Technology, Xianning, P.R. China; Hangzhou Singclean Medical Products Co., Ltd., Hangzhou, P.R. China Cristian J. Yarce Laboratorio de Disen˜o y Formulacio´n de Productos Quı´micos y Derivados, Departamento de Ciencias Farmace´uticas, Facultad de Ciencias Naturales, Universidad ICESI, Cali, Colombia Muhammad Zubair Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

C H A P T E R

1 Polymer blends, IPNs, gels, composites, and nanocomposites from chitin and chitosan; manufacturing, and applications Sabrina Sultana1, Md. Shirajur Rahman2, Md. Minhajul Islam2, Md. Nurus Sakib2 and Md. Shahruzzaman2 1

Department of Arts and Sciences, Ahsanullah University of Science and Technology, Dhaka, Bangladesh, 2Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

O U T L I N E 1.1 Introduction

2

1.2 Chemical structure and preparation of chitin and chitosan 1.2.1 Chemical structure and preparation of chitin 1.2.2 Chemical structure and preparation of chitosan

4 4 5

1.3 Polymer blends from chitin and chitosan 1.3.1 Blends with synthetic polymers 1.3.2 Blends with natural polymers 1.3.3 Applications of chitin and chitosan blends

6 7 8 8

1.4 Interpenetrating polymer networks from chitin/chitosan 1.4.1 Chitosan-based full-interpenetrating polymer network and semiinterpenetrating polymer network systems

9

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00001-7

1

10

© 2020 Elsevier Inc. All rights reserved.

2

1. Polymer blends, IPNs, gels, composites, and nanocomposites

1.4.2 Interpenetrating polymer network hydrogels based on chitosan and synthetic ionic matrices 1.4.3 Interpenetrating polymer network hydrogels based on chitosan and synthetic nonionic matrices 1.4.4 Interpenetrating polymer network cryogels based on chitosan and synthetic matrices 1.4.5 Applications of chitin/chitosan-based Interpenetrating polymer networks

11 12 12 13

1.5 Chitin- and chitosan-based gels 1.5.1 Physically associated gels 1.5.2 Polyelectrolyte complexation 1.5.3 Covalently cross-linked gels 1.5.4 Applications of chitin- and chitosan-based gels

14 14 17 18 19

1.6 Chitin- and chitosan-based composites 1.6.1 Composites from synthetic fillers 1.6.2 Composites from natural fillers 1.6.3 Applications of chitin- and chitosan-based composites

22 22 23 25

1.7 Chitin- and chitosan-based nanocomposites 1.7.1 Nanocomposites from chitin and chitosan 1.7.2 Applications of chitin- and chitosan-based nanocomposites

27 28 29

1.8 Conclusions and future prospective

32

References

33

1.1 Introduction Natural polymers are the most widespread and effective materials, mainly due to their abundance, environmental concerns (renewability and biodegradability), and wide applicability. The demand of natural polymeric materials has been increasing over the last two decades because natural polymers have better biocompatibility and less toxic effects than most synthetic polymers. This has led to the utilization of their useful inherent properties for a wide range of applications in different fields [1,2]. Many studies have been carried out on the manufacturing of advanced polymeric materials, such as blends, gels, composites, and nanocomposites, by mixing natural polymers with other natural or synthetic polymers and fillers. Among the natural polymers, chitin and chitosan occupy a distinct position due to their abundance, versatility, ease of modification, and unique

Handbook of Chitin and Chitosan

1.1 Introduction

3

properties including biodegradability, biocompatibility, nontoxicity, antibacterial and antifungal properties, hydrophilicity [3
5], etc. In addition, the structural units of chitin and chitosan contain a reactive amino group that makes them much easier to be modified by chemical reactions than cellulose. These have made chitin and chitosan very useful and outstanding candidates for a broad range of applications in biomedical, pharmaceutical, chemical, agricultural, cosmetics, and environmental fields. There are many reviews focusing on the properties, modifications, and applications of chitin and chitosan [6,7]. A lot of derivatives of chitin with different functional properties have been synthesized by chemical modifications. Shalumon et al. [8] reported the synthesis of water-soluble carboxymethyl chitin and poly (vinyl alcohol) (PVA) blend by an electrospinning method which was used for tissue engineering applications. Morin and Dufresne [9] reported the preparation of nanocomposites from a colloidal suspension of chitin whiskers and poly(e-caprolactone) as the matrix. In another study, Madhumathi et al. developed novel chitin/nanosilver composite scaffolds that have excellent antibacterial activity against Staphylococcus aureus and Escherichia coli as well as good blood-clotting ability [10]. The results of their study suggested that α-chitin/nanosilver composite scaffolds could be used for wound-healing applications. In addition, SiO2chitin/carbon nanotubes (CNTs) bionanocomposites have also been reported by many researchers [11]. Recently, much attention has been paid to chitosan as a potential biopolymer due to its remarkable intrinsic properties [12,13]. Huang et al. reported the synthesis of various metal
chitosan nanocomposites including silver (Ag), gold (Au), platinum (Pt), and palladium (Pd) in aqueous solutions [14]. In another work, Berger and his coworkers prepared chitosan hydrogels by blending chitosan with other water-soluble nonionic polymers such as PVA [15]. Chitin and chitosan inherently have poor mechanical properties that results in difficulties for their use in bone repair and reconstruction. Therefore the mechanical properties of chitin and chitosan can be improved only when the addition of biomaterials like hydroxyapatite (HAp), bioactive glass ceramic (BGC), etc. is possible. Recently, our group reported the preparation of chitosan/nanohydroxyapatite bioceramic scaffold for spongy bone regeneration [16]. BGC are a group of osteoconductive silicate-based materials used for bone repair. Wheeler et al. reported BGC coating on the surface of titanium which was used for osteointegration [17]. Different types of preparation techniques for the manufacture of advanced polymeric materials with chitin and chitosan have been discussed by Paillet and Dufresne. These include casting and evaporating technique, freeze-drying and hot-pressing techniques, polymer grafting,

Handbook of Chitin and Chitosan

4

1. Polymer blends, IPNs, gels, composites, and nanocomposites

nonaqueous solvent dispersion technique, extrusion, impregnation, and electrospinning. This chapter intends to focus on the basic fundamentals of chitin and chitosan including their structure, preparation, and applications.

1.2 Chemical structure and preparation of chitin and chitosan Chitin is a naturally occurring polysaccharide found in the shells of living organisms such as crabs, lobsters, tortoise, shrimps, and insects [18] or it can be generated via fungal fermentation processes. And chitosan is a unique biobased polymer that is a derivative of chitin and forms the exoskeleton of arthropods. It is obtained by partial deacetylation of chitin using a chemical method or by enzymatic hydrolysis. In general, chitin with a degree of deacetylation of 70% or above is considered to be chitosan [19].

1.2.1 Chemical structure and preparation of chitin Chitin is a naturally occurring polymer consisting of 2-acetamido-2deoxy-D-glucose via a β(1-4) linkage (Fig. 1.1). Three forms of chitin are available, namely α-, β-, and γ-chitin, however, the structure of α-chitin has been investigated more extensively than that of either the β- or γ- form. Very few studies have been carried out on γ-chitin because γ-chitin may be a distorted version of either α- or β-chitin [20]. A suspension of chitin crystallite particles was first prepared by Marchessault et al. [21] in 1959. In this method, 2.5 N hydrochloric acid solution was used to treat purified chitin under reflux for 1 h. After the reflux, the excess acid was separated by a decantation process and then distilled water was added to obtain the suspension. It was observed from their method that the acid-hydrolyzed chitin spontaneously dispersed into rod-like particles that could be concentrated to a liquid

FIGURE 1.1 Chemical structure of chitin.

Handbook of Chitin and Chitosan

1.2 Chemical structure and preparation of chitin and chitosan

5

crystalline phase and self-assembled to a cholesteric liquid crystalline phase above a certain concentration [22]. Though chitins are present within numerous taxonomic groups, on the commercial scale they are usually extracted from marine crustaceans, mainly because a large amount of waste is available as a by-product of food processing.

1.2.2 Chemical structure and preparation of chitosan As the second most abundant natural biopolymer after cellulose, chitosan consists of β(1-4)-linked D-glucosamine with randomly located N-acetylglucosamine groups depending upon the degree of deacetylation of the polymer (Fig. 1.2). Deacetylation is normally conducted by repetitions of alkaline hydrolysis due to the resistance of such groups owing to the trans arrangement of the C2-C3 substituents in the sugar ring [23]. The conditions during deacetylation must be properly controlled so that the chitin may be deacetylated to chitosan resulting in a better yield. Generally, two different methods are known for the preparation of chitosan from chitin with varying degrees of acetylation. One of them is heterogeneous deacetylation of solid chitin and the other is homogeneous deacetylation of preswollen chitin under vacuum in an aqueous medium. In both cases, concentrated alkali solutions and long processing times are required for the deacetylation reaction which may vary depending on the heterogeneous or homogeneous conditions from 1 to nearly 80 h. To reduce the long processing times and the requirement for large amounts of alkali to deacetylate chitin, several alternative-processing methods have been developed. Examples of these include the use of successive alkali treatments using thiophenol in DMSO; thermomechanical processes using a cascade reactor operated under low alkali concentration; flash treatment under saturated steam; the use of microwave dielectric heating; and intermittent water washing. There is evidence that in certain bacteria and fungi, enzymatic deacetylation

FIGURE 1.2 Chemical structure of chitosan.

Handbook of Chitin and Chitosan

6

1. Polymer blends, IPNs, gels, composites, and nanocomposites

can take place [24]. Recently, a microwave technique for efficient deacetylation of chitin nanowhiskers to a chitosan nanoscaffold has also been reported [5]. Both chitin and chitosan can be prepared using common processes, which are given in Fig. 1.3.

1.3 Polymer blends from chitin and chitosan To improve the properties of chitin/chitosan and further diversify their applications, various strategies have been adopted, such as crosslinking, copolymerization, chemical modification, blending, and so on. In particular, modification of chitin/chitosan by means of blending is an attractive method that is commonly used for providing desirable polymeric materials with combined properties for particular applications [25]. Recently, blends of chitin and chitosan have attracted much attention due to their strong potential for replacing synthetic polymers in many applications, in addition to being renewable resources, nontoxic, inexpensive, and leaving behind biodegradable waste. Generally, two methods are available for blending chitosan: (1) solution blending [26] (dissolving in a solvent followed by evaporation); and (2) melt blending [27] (mixing under fusion conditions). Of the two methods, solution blending is the most applied method according to the literature due to its simplicity and suitability.

Crustaceans (shrimp, crab, lobster, krill, and squid)

Chitin Decalicification in dil. Aqueous HCLsolution (3–5% HCI w/v at room temperature) Deacetylation in hot conc. NaOH solution (40–50% w/v NaOH, at 90°C to 120°C for 4 to 5hrs.) Deproteination in dil. aqueous NaOH solution (3–5% w/v NaOH, 80°C to 900°C for a few hrs. or at room temperature overnight)

Chitosan Decolarization in 0.5% KMnO aqueous and oxalic acid aqueous or sunshine

FIGURE 1.3 Common process for the preparation of chitin and chitosan.

Handbook of Chitin and Chitosan

1.3 Polymer blends from chitin and chitosan

7

1.3.1 Blends with synthetic polymers To produce synthetic biodegradable polymers with good water absorbency and enhanced mechanical properties, blending of chitin and chitosan with synthetic polymers is the most convenient method nowadays. Among the synthetic polymers, PVA and polyethylene glycol (PEG) are the two frequently blended polymers [28] that possess better hydrophilicity, mechanical properties, and biocompatibility. Engelberg reported the preparation of biodegradable homogeneous films of chitosan blends by mixing PEG or PVA with a solution of chitosan acetate using a film casting method [29]. Moreover, Wiles et al. reported another film which was prepared by blending chitosan and PEG together. In another study Gong et al. evaluated the mechanical properties of blends of chitosan with polyurethane, prepared by a solvent casting method [30]. Chitosan hybrid materials were generated by mixing chitosan and viscose rayon using a mechanical blending method. Muzzarelli et al. reported chitin/chitosan blends by mixing chitin and chitosan with 1,6-diisocyanatohexane (polyurea (urethanes)) in DMA-LiCl solutions [31]. Also, Butler et al. [32] prepared chitosan-blended films containing glycerol (0.25% and 0.5%). In another study, the preparation and characterization of chitosan/ N-methylol nylon 6 blends in the form of membranes were reported for the separation of ethanol
water mixtures by the pervaporation method [33]. To enhance the separation performance, the blend membranes were mixed with H2SO4. However, the blending composition plays a vital role that affects the performance of the membranes. Srinivasa et al. studied the mechanical properties of chitosan films prepared by blending chitosan with polyols [34]. In another attempt, Nugraha et al. prepared chitosan films by blending chitosan with poly(lactic acid) (PLA). Chitosan blends with polyvinyl pyrrolidone (PVP) and PEG were reported by Zeng et al. [35]. They did FTIR, wide-angle X-ray diffraction (WAXD), and differential scanning calorimeter (DSC) to prove the compatibility of the chitosan/PVA blends. Sandoval et al. [36] studied the compatibility of two chitosan blends with PVA and poly(2-hydroxyethyl methacrylate) (P2HEM) through molecular dynamic simulations. Furthermore, Sarasam and Madihally [37] have reported the synthesis of chitosan/polycaproactone (PCA) blends. It was revealed from their study that the two polymers were successfully blended and used for tissue engineering applications. Chitosan was melt blended with polycaprolactone (PCL), poly(butylene succinate) (PBS), PLA, poly(butylene terephthalate adipate) (PBTA), and poly(butylene succinate adipate) (PBSA) [27]. The biodegradability of chitosan/nylon 11 was investigated by Kuo et al. [38]. From their study it was revealed that the addition of chitosan with nylon 11 greatly

Handbook of Chitin and Chitosan

8

1. Polymer blends, IPNs, gels, composites, and nanocomposites

affects the physical properties of nylon 11 that finally results in good biodegradability of blended films. In another work, Smitha et al. reported the preparation of chitosan/nylon 66 blends that showed better thermal stability.

1.3.2 Blends with natural polymers To bring new biomaterials with improved properties, the blending of chitin and chitosan with natural polymers has been projected as an attention-grabbing method to meet the requirements of specific applications. Various reports have been published on the blending of chitosan with natural polymers. Arvanitoyannis et al. [39] developed films by blending chitosan and gelatin using a casting and evaporation method. A series of chitosan/gelatin blend films has been reported by M. Cheng et al. [40]. The films showed a higher percentage of elongation-at-break together with a lower Young’s modulus. In another study, Ikejima et al. reported the development of completely biodegradable composites by blending chitin and chitosan with microbial poly(3-hydroxybutyric acid) (PHB) [41]. Kweon and his coworkers reported chitosan films by mixing Antheraea pernyi silk fibroin and acetic acid solution of chitosan [42]. They found that the thermal decomposition stability of chitosan was improved by blending with A. pernyi SF. Zhai et al. [43] reported antibacterial films prepared by blending chitosan with starch using a compression-molding process. Also, chitosan/starch blend films were prepared and characterized by Xu et al. [44]. Suyatma et al. [45] reported the preparation of biodegradable blend films from chitosan and PLA by a solution mixing and film casting method. Wu et al. [46] prepared antibacterial membranes from chitosan and cellulose blends using trifluoroacetic acid as a solvent. The chitosan/ cellulose blend membranes were used for wound-dressing applications. Mucha and Pawiak [47] reported the preparation of chitosan films by blending chitosan with hydroxylpropyl cellulose (HPC). The prepared chitosan/HPC films have better optical transparency and mechanical properties. The incorporation of corn-starch and dextran with chitosan using glutaraldehyde cross-linker was investigated by Wittaya-Areekul and Prahsarn [48]. The prepared film was used for wound-dressing applications.

1.3.3 Applications of chitin and chitosan blends According to the literature, chitin and chitosan blends are mainly applicable in pharmaceutical and biomedical fields. For example,

Handbook of Chitin and Chitosan

1.4 Interpenetrating polymer networks from chitin/chitosan

9

chitin/chitosan has been receiving great interest in drug delivery, colon or nasal delivery, and gene delivery applications. Because of their advantageous properties, such as nontoxicity, biocompatibility, biodegradability, and availability of terminal functional groups, chitin/chitosan blends have been widely used for controlled release of drugs in the area of drug delivery systems [49]. For various drug release applications, like oral and nasal drug delivery, various types of chitin/chitosan blends, such as microparticles, tablets, films, beads, and gels, have been proposed by many researchers [50,51]. For example, chitosan/collagen blends were used for controlled release for the architecture of membranes [52]. Chitin/chitosan blends have found growing interest in the tissue engineering field too. Chitin/chitosan-based scaffolds and gels have been reported for tissue engineering applications [53]. For example, chitosan/collagen blends have been used for the design of polymeric scaffolds for the in vitro culture of human cells, the tissue engineering of skin, and for implant fibers [54]. Kojima reported chitosan to be an antimicrobial agent because chitosan is a biopolymer that has been well-known to accelerate the healing of wounds in humans [55]. Furthermore, chitosan is widely used for the effective delivery of many pharmaceuticals. Due to its powerful chelating ability, chitosan-based powders, flakes, gel beads, composite membranes etc. were found to be used for heavy metal ion binders in environmental field [56]. Some chitosan-blended films have been developed for food packaging applications because of their antimicrobial activity, good water resistance, biodegradability, good thermal properties, biocompatibility, and nontoxicity [57].

1.4 Interpenetrating polymer networks from chitin/chitosan Interpenetrating polymer networks (IPN) have morphed into an innovative material due to their newfound versatilities and applications. IPNs can be regarded somewhat as “polymer blends” on the grounds that they generally contain two or more polymer components. But a true definition of an IPN is more complex [58]. According to IUPAC, “a polymer comprising two or more networks which are at least partially interlaced on a molecular scale but not covalently bonded to each other and cannot be separated unless chemical bonds are broken. A mixture of two or more preformed polymer networks is not an IPN” [59]. So, IPNs are combinations of polymers where at least one of them was synthesized or cross-linked in the immediate presence of another and they don’t have covalent bonds between them but still cannot be

Handbook of Chitin and Chitosan

10

1. Polymer blends, IPNs, gels, composites, and nanocomposites

separated without breaking chemical bonds. In simple words, an IPN can be considered to be an advanced multicomponent polymeric system with new properties. Based on the chemistry of their preparation, IPN hydrogels can be classified as: 1. Simultaneous IPN: this type of IPN develops when a mixture of the precursors of both polymers are prepared first and then two networks are fabricated simultaneously by self-governing, noninterfering routes such as chain and stepwise polymerization [58]. 2. Sequential IPN: this type of hydrogel is usually prepared by swelling a single-network hydrogel into a solution containing the mixture of monomer, initiator, and activator, with or without a cross-linker. If a cross-linker is used, a full IPN is obtained. But if no cross-linker is used, a polymer network embedded in another one results. This is called semi-IPN [58]. Chitosan is a natural, hydrophilic, biocompatible, and biodegradable polymer. Its abundancy in nature is second only to cellulose [60,61]. Chitosan is a deacetylated derivate of chitin. It is a linear cationic polysaccharide structurally made of β(1-4)-2-amino-2-deoxy-D-glucopyranose and β(1-4)-2-acetamido-2-deoxy-D-glucopyranose units. Chitin is converted into chitosan by partial deacetylation. This process of deacetylation of chitin results in a more water-soluble polysaccharide called chitosan. Chitosan has been used widely for various applications like drug delivery, tissue engineering, and cell scaffolds due to its biological properties like biodegradability, biocompatibility, and antibacterial activity [4]. Chitosan has also been applied as a bioadsorbent due to the presence of amino and hydroxyl functional groups in the structure [62].

1.4.1 Chitosan-based full-interpenetrating polymer network and semi-interpenetrating polymer network systems Chitosan-based full-IPN and semi-IPN systems have been synthesized either by selective cross-linking of chitosan in the presence of a preformed polyelectrolyte or by the synthesis of the cross-linked polyelectrolyte in the presence of chitosan. But full-IPNs have been mostly prepared by the postcross-linking of chitosan entrapped in a polyelectrolyte matrix. Cross-linking chitosan with a preformed polyelectrolyte like polyaniline was a pathway followed to prepare semi-IPN hydrogel based on chitosan. First a 2-wt.% chitosan solution was dissolved completely in acetic acid. Polyaniline was then dissolved in 0.5 wt.% 1-methyl-2pyrrolidone and acidified by HCl to a pH lower than 1. Chitosan and polyaniline solution was prepared by mechanical stirring for 24 h. After

Handbook of Chitin and Chitosan

1.4 Interpenetrating polymer networks from chitin/chitosan

11

that, the solution was poured into a petri dish and dried in a vacuum oven at 50 C for 48 h. The film was immersed in glutaraldehyde solution (methanol to deionized water 5 1:1) and cross-linking was carried out at a pH lower than 1. The hydrogel obtained was then washed with methanol and dried under vacuum for about 24 h [63]. Hydrogels of the cross-linked polyelectrolyte type in the presence of chitosan were prepared by free radical polymerization of acrylamide and acrylic acid (AA) in the presence of chitosan [64]. First a 1% chitosan solution was prepared in 1% acetic acid. Measured amounts of acrylamide and AA were added to the solution. Then 3.3 3 1022 M N, N-methylenebisacrylamide and 1.2 3 1023 M N,N,N0 ,N0 tetramethylenediamine in a 1:1 molar ratio with ammonium persulfate were added and it was stirred at room temperature for 10 min. The solution was inserted in a glass tube and degassed. After that, the glass tube was placed in a water batch at 50 C for 24 h.

1.4.2 Interpenetrating polymer network hydrogels based on chitosan and synthetic ionic matrices Synthetic ionic polymers have become one of the most popular matrices for fabricating IPN hydrogels based on chitosan. This is particularly prevalent with polymers containing either anionic centers like carboxylic groups like poly(AA) (PAA), poly(methacrylic acid) (PMAA), and poly(N-acryloylglycine), or cationic centers like quaternary ammonium and amine groups [65]. An IPN hydrogel based on chitosan and PAA was fabricated in an innovative way by free radical cross-linking copolymerization using crosslinker monomer N,N0 -methylenebisacryl amide (MBA) in the presence
absence of an electric field. First, a solution was prepared by dissolving AA, chitosan, and K2S2O8 in water at 55 C. Then MBA (0.13
0.19 wt.% in reaction system) was added in the solution. After that sodium hydroxide aqueous solution (20 wt.%) was added and stirred vigorously. Finally, the solution was moved rapidly into two plexiglas boxes (40 3 20 3 8 mm3). It was cured simultaneously in the presence
absence of an external parallel DC (direct current) electric field for 15.5 h starting from temperature 55 C to room temperature. The holdup time was 3.5 h at 55 C, then it was cooled down to room temperature and kept at this temperature for 12 h. In this system, plexiglas boxes were equipped with two copper foils as electrodes. At the start, DC electric fields were applied in the upright direction of the curing system for 1.5 h of the curing process with a high-voltage power regulator in the range of 0
30 kV. The parallel DC electric fields were 0.6, 1.0, 1.1, 1.2, and 1.5 kV/mm, respectively. In this way chitosangrafted PAA IPN hydrogel was prepared [66].

Handbook of Chitin and Chitosan

12

1. Polymer blends, IPNs, gels, composites, and nanocomposites

1.4.3 Interpenetrating polymer network hydrogels based on chitosan and synthetic nonionic matrices Nonionic monomers have been also used to prepare IPN hydrogels based on chitosan. Prominent monomers belonging to this category which have been used to prepare IPN are acrylamide, N-isopropylacrylamide, N, N-dimethylacrylamide, and 2-hydroxyehtyl methacrylate [67,68]. Poly(N-isopropylacrylamide) was used to prepare a hydrogel which exhibited a transition from the swollen to the collapsed state at a certain temperature. N-isopropylacrylamide and N,N0 -methylenebis(acrylamide) was added to 1.5 wt.% chitosan solution in 0.2 M 40 mL acetic acid. The solution was stirred for 30 min and degassed at 48 C for 1 h. After that, 400 μL N,N,N0 ,N0 -tetramethylethylenediamine and 400 μL ammonium persulfate were added to this solution. Molds consisting of two glass plates (10 3 10 cm) separated by a 0.9-mm Teflon frame were prepared. A measured amount of the solution was injected into these molds. Polymerization was carried out at room temperature for 48 h. After completion of the reaction, IPN hydrogels were removed from the molds and immersed in 250 mL of water or glutaraldehyde solution (0.1, 0.3, 0.5, or 0.7 vol.%) for 1 h. Then these IPN gels were washed with water for about 24 h. The disks were kept in water at room temperature after cutting with a cork borer [65,69].

1.4.4 Interpenetrating polymer network cryogels based on chitosan and synthetic matrices Cryogels are an innovative 3D macroporous polymeric gels. They are fabricated at temperatures well below the freezing point of the solvent used for their preparation. The pore size in cryogels varies between 1 and 100 μm. This is mainly influenced by the polymer itself, its concentration, and temperature. The main advantage of a porous cryogel network is that it is highly interconnected and provides rapid and nonrestricted mass transport of any solute [70]. Macroporous cryogels composed of a semi-interpenetrating network (semi-IPN) of polyacrylamide
chitosan were prepared by Kumar et al. First, 3.95 g acrylamide monomer and 1.058 g N,N0 -methylene-bisacrylamide were taken in 50 mL degassed water. Solutions of chitosan of 2, 2.25, and 2.4% were prepared by dissolving 0.6, 0.9, and 1.2 g chitosan respectively in 0.1 M acetic acid. These two solutions were then mixed and monomer (acrylamide 1 N,N0 -methylene-bis-acrylamide) concentration in the final solution was 5% and chitosan concentration remained at 0.6, 0.9, and 1.2%. Acrylamide/chitosan ratio in the final solution was 6:1, 4:1, and 3:1 (w/w) respectively. Then 100 μL N,N,N0 , N0 -tetramethylethylenediamine and 100 μL ammonium persulfate were

Handbook of Chitin and Chitosan

1.4 Interpenetrating polymer networks from chitin/chitosan

13

added to the solution and stirred. After that the solution was inserted into 2.5 mL syringes and immediately frozen at 212 C for 16 h. In this stage, polymerization was carried out and after completion of polymerization the cryogels were thawed at room temperature. Finally, the gels were washed to remove any unreacted monomers and vacuum dried [71].

1.4.5 Applications of chitin/chitosan-based Interpenetrating polymer networks Chitosan-based IPN systems have earned substantial interest as an attractive medium for drug delivery systems. These drug delivery systems can be prepared in the form of beads, microspheres, discs, or tabs. An IPN based on chitosan, polyacrylic acid, and polyacrylamide was prepared by free radical polymerization [64]. This IPN hydrogel was loaded with bovine serum albumin. Impressive bovine serum albumin loadings were possible in hydrolyzed hydrogels. This could be attributed to their high water content and porosity. The results showed that at lower pH (1.2) protein release from hydrogels was low (20% after 10 h). On the other hand, sustained release of protein could be observed at pH 6.8 and 7.4. The integrity of the protein remained unaffected at 6.8 and 7.4. Moreover, this hydrogel exhibited no noticeable cytotoxicity. A temperature and pH-sensitive IPN hydrogel based on poly(Nisopropylacrylamide) and chitosan was prepared by free radical polymerization and cross-linking. The poly(N-isopropylacrylamide)/chitosan IPN could be loaded with a large amount of diclofenac. It was possible to control the release of the drug and it was possible to sustain the drug release for more than 8 h in 0.9% NaCl solutions or pH 8 phosphate buffer. The IPNs which had lower degrees of chitosan cross-linking exhibited higher temperature-sensitive release patterns. Importantly, higher temperature did not considerably alter the release rate [72]. Chitosan was combined with a conducting polymer polyaniline to fabricate a semi-IPN network [63]. Interestingly, conducting polymers, such as polyaniline, have garnered interest for microelectronics applications. The potential of modifying chemical properties as well as the electronic properties of the conducting polymer have led to the development of new sensors. A chitosan/polyaniline semi-IPN has exhibited unique self-oscillating bending actuation at constant DC voltage. The IPN bends in opposite directions when immersed in either acidic or basic electrolytes. But the IPN system oscillates slowly when immersed in a neutral electrolyte. The bending behavior has been linked to ion movements and electrochemical processes involving the conducting polymer and the surrounding electrolyte. This could be of good use in

Handbook of Chitin and Chitosan

14

1. Polymer blends, IPNs, gels, composites, and nanocomposites

simple propulsion mechanisms in fishlike swimming robots or for simple pumps [63]. A similar IPN complex hydrogel elastomer with many fine polar groups has exhibited remarkable positive electroresponse. This system can be applied in sensors, actuators, switches, and drug delivery systems [66]. Cryogels based on chitosan can be potential matrices for cell growth in 3Dstructures and can be applied for various applications, such as tissue engineering, scaffolds for cell immobilizations in bioreactors, etc. A cryogel semi-IPN of polyacrylamide
chitosan was fabricated at subzero temperature [71]. This cryogel not only demonstrated adherence and proliferation of cells on the polymer surface but also a sustained growth of cells over the scaffolds for a period of 14 days. The 3D cryogel network with such impressive physical characteristics makes them very interesting materials for cell scaffolds in a perfusion bioreactor.

1.5 Chitin- and chitosan-based gels Chitin or its deacetylated derivative chitosan do not generally exist in gel form, rather they exist as fibrous forms, such as in protective layers or structures forming layers in nature. However, when synthesized, gels of chitin and chitosan have shown potential use in many fields of application.

1.5.1 Physically associated gels Chitin or chitosan gels can be prepared physically where the covalent bonding is absent. The gels obtained by such processes are generally reversible. Synthesis of these physical gels once was a challenging task but now the synthesized physical gels of chitin or chitosan are common. Parameters such as molecular weight, pH, bonds, composition, or ionic strength can be regulated to achieve different shapes and characteristics. The entanglement bonding is aided by the electrostatic and hydrophobic action as well as hydrogen bonds. The sol/gel transformation rates can be altered by regulating the pH and ionic interaction. Although the micro- or nanosized physical gels are easy to prepare, macrosized gelation is still a rigorous task [73]. Physical gels are more favorable than the chemically prepared gels mainly because they are comparatively low in toxicity. The free covalent cross-linkers of the chemically prepared gels often allow increased exposure to toxicity. On the other hand, physical gels often show weak mechanical properties. However, they can be tuned more easily and are

Handbook of Chitin and Chitosan

1.5 Chitin- and chitosan-based gels

15

FIGURE 1.4 Chitosan hydrogels formed by different bonds. — denotes additional polymers,— (bold) denotes Chitosan, K-K denotes covalent cross-linker; 1 denotes positive charge of chitosan. In ionic cross-linking, the positive charge of chitosan is ionically bonded with the negatively charged cross-linker. (A) Chitosan cross-linked with itself. (B) Hybrid polymer network. (C) Semi-interpenetrating network. (D) Ionic cross-linking of Chitosan. Source: Reprinted from J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. 57 (2004) 19
34 [74] with permission from Elsevier.

biodegradable [74]. This section covers examples of some of the physically associated gels synthesized and their characteristics (Fig. 1.4). Let us first discuss the physical gels of chitin or chitosan which are prepared without using other cross-linking agents. Generally, gelation is dependent on pKa values as well as D and A unit ratio. If the D unit and A unit are the same in number in a polymeric chain, chitosan will not precipitate. Hirano et al. [75] prepared a physical gel by reacetylation of a high degree deacetylated chitosan. The chitosan was acetylated by treating it with acetic anhydride. The anhydride worked as the acetyl group donor. The excess of the acetyl anhydride was important in making the gel. This experiment was further modified and optimized by Moore and Roberts [76]. They have tested it to see the temperature, composition, and concentration effect on gelation. The study shows that with the increase in temperature and concentration, the gelation occurs faster. They have also studied the reason behind the gelation process. They have found that the gelation is the result of cross-linking by the

Handbook of Chitin and Chitosan

16

1. Polymer blends, IPNs, gels, composites, and nanocomposites

hydrophobic reactions with micellar junctions. When the A unit sugar formation reaches 70%, switching the solution from polyelectrolyte to hydrophobic polysaccharide, the solubility decreases in the solution, thus forming the gel [77]. Physical gels of chitin have been explored and it has been found that with the proper solvent, chitin can form physical gels through nonsolvent addition which may result in bead-shaped, fiber-shaped, or bulk gels. Hasan et al. have synthesized bead-shape chitin physical gels grafted with poly(4-vinylpyridine). Chitin was dissolved in a solution of DMAc/5%LiCl solution. Then it was grafted and purified. Purification removed any unreacted or unwanted elements. The purification process was done using ethanol and water [78]. The use of cosolvents can also help physical gel formation. The use of methanol, for instance, can help the N-acetylation process by keeping the dielectric constant lower and preventing O-acetylation [79]. Another study showed the comparative analysis of ethanol and 1,2-propanediol as cosolvents. The gelation rate was faster when ethanol was used possibly because of the lower viscosity. However, after gelation, the gels showed no difference proving that cosolvent was added only to aid the process [80]. Chitin is generally insoluble in polar solvents. However, a study shows that it dissolves in solvents containing N,N-dimethyl acetamide
5% LiCl. Gelation of chitin can also be done thermally when dissolved in a solution of DMA and LiCl [81]. Li et al. have prepared a water-soluble hydroxypropyl chitin in NaOH solution. The synthesized gel had varied gelation temperatures that can be modulated by changing the propylene oxide to chitin feed ratio. The material was biocompatible as well as biodegradable and the researchers suggested that it could have a potential to be used for biomedical purposes [82]. Gel formation without using cross-linkers can also be done by the use of ammonia instead of alkaline solution. The gaseous alkaline ammonia creates an interphase uniformly and neutralized the amines of the chitosan and subsequently hydrophobic interactions and hydrogen bonds produced a gel. The A sugars are vital in this process providing the hydrophobic interactions [83]. Gels can also be made by changing temperature, also known as thermosensitive gelling. Chenite et al. have prepared a gel using a basic salt, glycerol phosphate, as a buffering and conditioning (hydrophobic interaction and hydrogen bonding) agent, and then heated the solution to form hydrogel. In the process, a strongly deacetylated chitosan was neutralized with beta-glycerol phosphate to keep chitosan soluble at pH 4. Then the temperature was increased and the solution changed to a hydrogel form. The whole gelation and its rate is pH and temperature dependent [84]. Some thermosensitive gels have seen problems in stability when stored, as well as inadequate mechanical strength in biomedical use. Thermosensitive gels

Handbook of Chitin and Chitosan

1.5 Chitin- and chitosan-based gels

17

made of chitosan and inorganic orthophosphate are found to be cytocompatible. Only the dibasic phosphate salts were able to form gels and the time and temperature for gel formation can be modulated by changing concentrations [85]. A large number of hydrogels are synthesized using cross-linkers and this is possibly the easiest way to form gels of chitin or chitosan. A common ionic cross-linker used in gelation is tripolyphosphates (TPP). Tripolyphosphates, a multivalent anion, generally is used in an acidic chitosan medium for making gels. Similar to TPP, pyrophosphate anions have also been used for gelation [86]. But a study shows that pyrophosphate gels are often not homogeneous and have weak mechanical properties [87]. Apart from these there are other ionic cross-linkers available such as phosphates and citrates. Phosphates and citrates help the gelation by means of electrostatic charge (Fig. 1.5).

1.5.2 Polyelectrolyte complexation Polyelectrolyte complexation is often associated with phase separation with counterion release. Two phases appear when chitosan is used to form

FIGURE 1.5 Preparation of chitosan aqueous hydrogel and hydroalcoholic gels polyelectrolyte complexation. Source: Reprinted from L. Rami, S. Malaise, S. Delmond, J.-C. Fricain, R. Siadous, S. Schlaubitz, et al., Physicochemical modulation of chitosan-based hydrogels induces different biological responses: interest for tissue engineering, J. Biomed. Mater. Res. Part A 102 (2014) 3666
3676 [88] with permission from John Wiley and Sons.

Handbook of Chitin and Chitosan

18

1. Polymer blends, IPNs, gels, composites, and nanocomposites

polyelectrolyte complex. The more viscous phase is known as coacervate which is enriched with polymer. In this process, water is often ejected when a complex is formed, and therefore it does not comply with the regular hydrogel notion where the gel usually absorbs a high amount of water [89]. Hyaluronic acid is often used to make chitosan-based polyelectrolyte complexes. One study shows the making of chitosan
hyaluronan nanogels where the hyaluronan is dropped into a chitosan solution. The nanogels synthesized from chitosan
hyaluronan gain entropy by releasing water and counterions from both the components [90,91]. Dextran sulfate is also used in making nanogels of such kind. Costalat et al. have synthesized a reversible chitosan
dextran sulfate nanogel where the ionic strength was modulated via dialysis [92]. In complex coacervation, precipitation may occur. To avoid it, oligomers are used in lieu of polymers as complexing agents. A study shows that mixtures of alginate oligomer could be used as a cross-linker for chitosan and chitosan oligomers could be used with alginate to start the gel formation [93]. Similar to the complex coacervation method in forming nanogels as discussed above where there are at least two oppositely charged polyelectrolytes present, there is also a simple coacervation method to form gel where only one macromolecule is present. Several types of chitosan/TPP gels can be found in the literature [14,94].

1.5.3 Covalently cross-linked gels Chitin- or chitosan-based hydrogels synthesized with covalent bondforming cross-linkers have the edge over other physical hydrogels with regard to several characteristics such as mechanical strength, degradation, stability, swelling ratio, and thermal sensitivity. Generally, three types of covalently cross-linked chitosan are found. The first of this kind is when chitosan is entangled with itself in covalent bonding and forms a hydrogel. The second type is the hybrid polymer network where chitosan is covalently cross-linked with a different type of polymeric chain structure unit. The third type is the IPN or IPN where a nonreactive polymer is entrapped in the cross-linked chitosan network or can be further cross-linked to produce two entangled cross-linked network. The former situation is known as semi-IPN and the latter is full IPN. In all these three stages, covalent bonds plays an important role in forming the gel network. Dialdehydes, such as glutaraldehyde, are very common cross-linkers added to form gels with covalent bonds. In glutaraldehyde
chitosan gel formation, the aldehyde is bonded to chitosan’s amino groups to form imine bonds that are covalently held together [95]. The dialdehydes give easy reaction with chitosan and do not generally need any additional

Handbook of Chitin and Chitosan

1.5 Chitin- and chitosan-based gels

19

components. However, dialdehydes may have drawbacks regarding toxicity because glutaraldehyde is often considered neurotoxic and glyoxal to be mutagenic. So any free dialdehydes could lead to a serious toxic acid when using these hydrogels. Examples of cross-linkers used other than the dialdehydes are oxalic acid, diethyl squarate, and genipin [15]. Genipin is a safe alternative to glutaraldehyde. It is found in nature and does not possess cytotoxicity. To form the HPN, polymers bearing reactive groups should be added to chitosan in the presence of a cross-linker. The cross-linker used generally is glutaraldehyde. Gelatine, collagen, etc. are used as the additional polymer with amine groups that are connected to chitosan via cross-linking agents [96]. For semi-IPN, the polymers are nonreacting and thus are entrapped in the chitosan gel. Examples of such a polymer is fibroin. For full IPN, cross-linking of the polymer and chitosan are necessary. In such process a second cross-linker is often used [97]. For polymeric gel formation with chitosan, polysaccharides such as maltodextrins and methylcellulose are often used. They are used prior to oxidizing with periodate to enable shiff bases with amino groups of chitosan [98] (Fig. 1.6). Although chitin is the most common natural amino polysaccharide, the hydrogels of chitin often show some weaknesses and their fabrication is difficult. The mechanical properties are often weak and chitin is often slightly dissolvable in the common solvents. Xu et al. have prepared a physically and chemically bonded chitin gel that shows great mechanical stability with good flexibility and toughness [99].

1.5.4 Applications of chitin- and chitosan-based gels Chitin- and chitosan-based gels have versatile uses due to their biocompatibility, lower toxicity, large-scale availability, and biodegradability. The most common uses of gels of chitin and chitosan are in drug delivery, wound healing, tissue engineering, and related biomedical applications such as scaffold preparation. Mayol et al. have synthesized a chitosan physical gel for woundhealing purposes. They heat-treated the chitosan powder in an autoclave and found that the thermal treatment changed the viscoelastic properties as well as the flow properties of the chitosan powder. The treated chitosan had better proliferation of fetal fibroblast as well as faster wound healing. The powder was sterilized in the autoclave at 121 C and at two atmospheric pressure. Then chitosan powder was added in different concentrations to acetic acid solution and the solution was passed through a cellulose membrane. The gel showed potential characteristics of a wound cavity filler [100].

Handbook of Chitin and Chitosan

FIGURE 1.6 Preparation of double cross-linked chitin hydrogel. Source: Reprinted from D. Xu, J. Huang, D. Zhao, B. Ding, L. Zhang, J. Cai, Highflexibility, high-toughness double-cross-linked chitin hydrogels by sequential chemical and physical cross-linkings, Adv. Mater. 28 (2016) 5844
5849 [99] with permission from John Wiley and Sons.

1.5 Chitin- and chitosan-based gels

21

Thermosensitive polymer hydrogels are especially of great interest in tissue and therapeutic engineering. A thermosensitive polymer was devised to study a drug release mechanism. PEG was grafted onto a chitosan backbone to produce a temperature-sensitive hydrogel and its drug release efficacy was tested by bovine serum albumin as a model protein. PEG was covalently bonded to a chitosan chain. The hydrogel thus created was an injectable liquid at low temperature but at body temperature the gel turns into a semisolid. If cross-linked with genipin, the drug release maintained a quasilinear model for up to 40 days [101]. A hybrid injectable hydrogel was produced without adding any chemical cross-linkers. The hydrogel was prepared using chitosan-grafted dexamethasone and cross-linked aldehyde hyaluronic acid by Schiff base reaction. The hydrogel showed better cell adhesion and provided better proliferation of stem cells than hydrogels prepared without dexamethasone. It has also been proved that the hydrogels have better water absorption and faster weight loss than the hydrogels without the dexamethasone. Furthermore, the addition of toxic cross-linking agents is absent in this process and hence it shows a good potential as an injectable cell carrier [102]. Chitosan is well-known for its antimicrobial characteristics. Making composites with other known elements of antimicrobial capability is a good strategy to find a more effective antimicrobial material. Silver particles are often used for their antimicrobial properties. Valencia et al. presented a study where chitosan gels were prepared with silver and gold nanoparticles. Compared with silver nanoparticle gels, the chitosan
gold nanoparticle gels have better thermal stability. The antimicrobial property shows that the gels resist the growth of S. aureus, S. mutans, and E. coli bacteria. They are biocompatible and do not show any negative or delayed reaction against wound healing, making them a viable option for treating bacterial infections [103]. Madhumathi et al. have studied a chitin/nanosilver composite’s effectiveness as scaffolds for wound dressing. The chitin/nanosilver composite scaffold was shown to prevent S. aureus and E. coli and showed blood-clotting behavior. Although the cytotoxicity of the scaffold in vitro was present, according to the researchers, they presumed that the composite might not be cytotoxic when used in vivo [104]. A similar type of study was carried out by Kumar et al. where they prepared a chitin hydrogel and nano-ZnO composite in bandages to check its effect against wound infection. The chitin hydrogel and zinc oxide nanoparticles are homogenously mixed and then freeze-dried. The material showed good adhesion, good swelling, and a bactericidal property. Unlike the previous case, this composite did not show any cytotoxicity when tested with cells derived from human skin [105]. Chitin and chitosan hydrogels are often applied for dye removal. A study shows chitin and chitosan hybrid hydrogel and SiO2 made

Handbook of Chitin and Chitosan

22

1. Polymer blends, IPNs, gels, composites, and nanocomposites

mesoporous materials that have been used to remove four dyes, that is, Remazol Black B, Neutral Red, Gentian Violet, and Erythrosine B. The study shows that the adsorption was pH dependent and mostly chemisorption. Only for Erythrosine B, the adsorption was physical adsorption for chitin/SiO2 matrix. The matrix containing chitin showed equal or better adsorption than the chitosan ones apart from in the case of highly charged dyes. This study was done to see the possibilities of replacing chitosan by means of chitin due to its low cost [106]. A chitin hydrogel has been prepared and tested to see the removal efficiency of malachite green (MG) dye. The chitosan (3 wt.%) was dissolved in 8 wt.% NaOH and 4 wt.% of urea. The chitosan was then cross-linked with epichlorohydrin. The resulting hydrogel showed around good adsorption following a pseudo-second-order kinetics model [107].

1.6 Chitin- and chitosan-based composites Composite materials are constructed from two or more materials and their overall characteristics are derived from the individual constituents. The search for new applications has called for development in the properties of chitin and chitosan through incorporation of chitin or chitosan polymer with reinforcement agents of other polymer to produce chitinand chitosan-based composite materials.

1.6.1 Composites from synthetic fillers Chitin and chitosan with synthetic fillers have primarily been developed for the production of highly resistive, biodegradable, and conductive products that allow material engineers to design promising materials with good thermal and mechanical properties. Composites made of chitosan incorporation with synthetic material are widely available. These investigations were made mostly to exploit the advantages of both chitosan and other components incorporated in it. A composite material was made from chitosan and β-cyclodextrin polymer by connecting them with maleoyl chain followed by cross-linking with glutaraldehyde for the separation of methyl orange (MO) from aqueous solution. This composite displayed superior selectivity to MO than methylene blue and rhodamine B. This behavior shows the electrostatic attraction of MO toward NH2 groups of chitosan, and host
guest interaction between MO and β-cyclodextrin. That is why this composite was considered as a potential adsorbent of MO owing to its high selectivity, efficiency, and biodegradability [108]. In another study, a composite of PVA and chitosan was synthesized for the adsorption of copper(II) and

Handbook of Chitin and Chitosan

1.6 Chitin- and chitosan-based composites

23

MG from an aqueous solution. It is worth mentioning that in a binary system of MG and Cu21 competitive adsorption was shown. Moreover, investigators also studied the desorption and reusability of this composite based on sequential adsorption
desorption cycles and found that the adsorbent could be regenerated several times [109]. The presence of synthetic materials in chitosan has high influence on the properties of composites. To obtain various desirable properties in composites various synthetic materials have been added by researchers. For instance, biosorbent composite material was developed from naturally available chitosan and synthetic polyvinyl chloride beads for the adsorption of copper(II) and nickel(II) ions from aqueous medium [110]. PVA has been introduced into chitosan to prepare a PVA
chitosan composite hydrogel for the separation of copper(II). This PVA
chitosan composite was further cross-linked by glutaraldehyde to enhance the adsorption ability of copper. Moreover, it was observed that the effectiveness of copper adsorbance increased with an increase in chitosan content with the polymeric network of the composite [111]. Similarly, PVA displayed a promising ability to remove cobalt ions from radioactive wastewater when PVA was blended with chitosan to form a magnetic composite. In the case of cobalt adsorption, the main role has been played by the functional groups
NH2 and
OH, as proved by FTIR and SEM-EDAX images of composites taken before and after the adsorption [112]. In a different study, a composite was prepared from cross-linked chitosan and PVA beads for the removal of cadmium(II) ion from aqueous solution. Even this composite adsorbent was discovered to be regenerable for subsequent use and this regeneration was achieved barely by EDTA solution. Neither the acidic (HCl or HNO3) nor the neutral solution was able to recover the cadmium ions by the method of desorption and this phenomenon clearly indicated that the adsorption occurred on the surface mostly via complexation [113].

1.6.2 Composites from natural fillers Natural filler such as mineral, polysaccharide-based, clay-based, and wood-based products have been suggested to replace the synthetic filler for better compatibility and functionality of chitin- and chitosan-based composite material. However, the harvesting and processing of natural filler is not as straightforward as in the case of synthetic filler production. In an investigation, poly(methacrylic acid)-grafted chitosan/bentonite composite (PMAA-g-CTS/B) was prepared by the researchers for the removal of thorium(IV) from aqueous solution as well as seawater via a batch adsorption technique [114]. The same group of researchers exploited the same composite for the removal of uranium(VI) from the

Handbook of Chitin and Chitosan

24

1. Polymer blends, IPNs, gels, composites, and nanocomposites

aqueous solution [115]. In other research, investigators have prepared a composite of chitosan and alginate to examine the removal of chromium(VI) from wastewater and asserted the composite to be an outstanding biosorbent for heavy metals [116]. Copper(II) and nickel(II) were significantly removed by a composite prepared from chitosan primarily coated on an inactive substance, the silicate ore perlite [117]. The adsorption capability of copper(II) was examined by another composite made of chitosan and sand. It has been announced by examiners that the increase of the amount of chitosan amplified the adsorption capability of copper ions [118]. Furthermore, another composite biosorbent prepared from chitosan-coated montmorillonite clay was found to be much more effective than other natural clays for the removal of tungsten from aqueous solution [119]. Cellulose is abundant and has a low density, renewability, high mechanical strength, large aspect ratio, and economic affordability [120]. The use of cellulose in composite formation is very popular. A composite was prepared using cross-linked chitosan and cellulose for the removal of copper ions from wastewater. The addition of cross-linked cellulose in the composite has given the composite additional mechanical strength and higher density where the cross-linking was attained by ethylene glycol diglycidyl ether (EGDE) [121]. Another composite was produced from chitosan and cellulose for the removal of Methylene Blue (a cationic dye) and Congo Red (an anionic dye) from aqueous Solution. The researchers deduced that these composite beads can eliminate Methylene Blue up to 58.2% and Congo Red up to 99.9% when treated individually. Electrostatic interactions, hydrogen bonds, and dipole
dipole forces between the functional groups of the polymeric beads and the dyes are expected to be the desired mechanism of adsorption [122]. Removal of various dyes using clay is very well-known, but a more invigorating result is found where a composite is prepared from a modified ball clay in combination with chitosan. This composite is capable of removing Methylene Blue from aqueous solution. Moreover, this composite adsorbent displayed 50% adsorption uptake capacity even after its fifth regeneration cycle, where the regeneration could be accomplished simply by an acidic solution of pH 4 [123]. In another study, researchers prepared a chitosan
bentonite hybrid composite for the removal of anionic and cationic dyes from aqueous solutions, which also facilitated separation of the adsorbents due to solid/liquid phase discrimination [124]. Azo dye-like Amido Black 10B was successfully removed by the same type of composite made of cross-linked chitosan and bentonite where the cross-linking reaction was executed between chitosan and glutaraldehyde [125]. Due to its antibacterial nature, biocompatibility, and being a suitable cell in growth, osteoconduction, and negligible foreign body

Handbook of Chitin and Chitosan

1.6 Chitin- and chitosan-based composites

25

reactions, chitosan has found its utilization in tissue engineering. A composite prepared from chitosan and HAp has found a predominant role in bone tissue engineering. HAp (Ca10(PO4)6(OH)2) is the major component of bones and one of the most constant forms, which has delivered the extra advantage of biocompatibility to the chitosan composite. The formulators have asserted the unparalleled mechanical properties of this composite and deduced the in vitro activity of this composite to elaborately explain the chemical interaction [126].

1.6.3 Applications of chitin- and chitosan-based composites The use of chitin has restrictions due to its low solubility in inorganic and organic solvent, whereas chitosan, the derivative of chitin, shows great potential in various fields of application due to its enhanced functionality and solubility in most organic solvents [127]. Moreover, chitosan is nontoxic in any concentration, as well as being biocompatible and biodegradable, which make it a precious material in the field of medicine [128]. Chitosan has been extensively studied as an adsorbent and for most of the studies chitosan has been used in the form of hydrogel beads, powder, or flakes. Chitosan has a natural inclination to form agglomerates or a gel in aqueous medium. Additionally, the presence of free amino and hydroxyl groups provides the chitosan the additional advantage of adsorbing heavy metals and various dyes on its surface. Chitin- and chitosan-based composites are currently used for medical implants, regenerated tissue, drug carriers, biosensors, as active adsorbents, and packaging materials. 1.6.3.1 Dye removal Bioremediation is a developing biological process that offers the possibility to destroy or render harmless various contaminants using natural biological activity. Removal of various categories of dye particles from environment is one of the criteria of bioremediation. The removal of cationic dyes like Methylene Blue was observed using a modified ball clay and chitosan composite which is regenerable by a simple acid solution of pH 4 [123]. Removal of anionic dye like Amaranth Red from colored effluent was also studied successfully on a chitosan
bentonite hybrid composite, but the composites displayed no such regeneration ability [124]. Both the cationic dye (Methylene Blue) and anionic dye (Congo Red) from aqueous solution can also be removed by a composite made of cellulose and chitosan. Moreover, those dyes can be removed from the solution individually or even simultaneously [122]. Among the other dyes, Methyl Orange (MO) was selectively removed from aqueous solution by a composite made

Handbook of Chitin and Chitosan

26

1. Polymer blends, IPNs, gels, composites, and nanocomposites

of chitosan and β-cyclodextrin due to the electrostatic attraction between the MO and the NH2 group of chitosan, and host
guest interaction between MO and β-cyclodextrin [108]. The proof of azo dye absorption on a composite of cross-linked chitosan and bentonite is also well founded where the best adsorption capacity were investigated at room temperature and natural pH [125]. Both MG and copper from the aqueous solution were readily removed with the help of a novel composite foam of PVA and chitosan, moreover, the PVA
chitosan composite demonstrated better adsorption than that shown by the PVA and chitosan individually [109]. 1.6.3.2 Heavy metals removal The removal of heavy metals using a chitosan-based composite is widespread. Heavy metals like chromium(VI) were removed by a chitosan
alginate composite very efficiently [116]. Thorium(IV) and uranium(VI) removal from wastewater and seawater was investigated and it was found that poly(methacrylic acid)-grafted chitosan/bentonite composite was an excellent biosorbent [114]. Significant removal of copper(II) and nickel(II) was observed by a composite prepared from chitosan and perlite, where the chitosan was coated on an inactive substance perlite [117]. In a different experiment, both the copper(II) and nickel(II) were observed to be removed by a composite made of chitosan and polyvinyl chloride [110]. PVA was also shown to be effective removing copper(II) ions when it was incorporated with chitosan to make a composite [111]. Cobalt ions have also been removed from radioactive wastewater by a magnetic composite prepared from PVA in combination with chitosan [112]. Cadmium ion removal from the aqueous solution by means of a composite made of cross-linked chitosan and PVA displayed a prolific result, where adsorption followed a path of complexation rather than physisorption [113]. 1.6.3.3 Wound healing Chitosan has great antimicrobial activity and its ability to mend skin injuries may be increased by more than a few fold by incorporating materials that might have some synergistic effect on destroying or inhibiting the growth of microorganisms and especially pathogenic microorganisms. The composite, nano-TiO2
chitosan with collagen artificial skin (NTCAS), is one of the earliest wound healers found in literature. In vivo tests of NTCAS exhibited better and faster recovery than other available wound-healing materials. It was stated that NTCAS was a prominent artificial skin substitute and was superior to any other market product in use [129].

Handbook of Chitin and Chitosan

1.7 Chitin- and chitosan-based nanocomposites

27

1.6.3.4 Tissue engineering Over last two decades, chitosan has been utilized as an artificial prosthesis to treat the loss or failure of an organ or bone tissue. Due to biodegradability and biocompatibility, a composite made of chitosan and HAp has become very popular. The researchers also paid attention to the incorporation of CNTs to enhance the mechanical strength of the composite [126]. 1.6.3.5 Drug delivery Chitosan has fungistatic, hemostatic, spermicidal, central nervous system depressant, anticholesteremic, antitumor, and immunoadjuvant properties. Moreover, it is biodegradable in normal body constituents, safe, nontoxic, and it binds to mammalian and microbial cells aggressively [130]. Chitosan was used to form tablets with sodium alginate for the extended release of trimetazidine hydrochloride (a highly watersoluble drug) by changing its formulation variables. As per the in vitro experiment carried out in simulated gastric fluid and simulated intestinal fluid it was concluded that the liberation of the drug can be modulated by regulating the quantity of composite as matrix [131]. Similar categories of experiments were pursued by a group of investigators where composites were prepared from chitosan and anionic polymers for the extended release of water-soluble drugs like sodium valproate and valproic acid. Xanthan gum, carrageenan, sodium carboxymethyl cellulose, and sodium alginate were the anionic polymers used extensively for this drug release experiment. It is noteworthy that the duration of drug release could be extended up to 24 h [132]. In another study, a composite gel of chitosan and cashew gum was prepared with an aim of adjustable discharge of pilocarpine hydrochloride [133].

1.7 Chitin- and chitosan-based nanocomposites In the area of nanotechnology, polymer matrix-based nanocomposites have generated a considerable amount of attention in the recent literature. Chitin and chitosan have been widely employed to fabricate nanocomposites due to their biodegradability, biocompatibility, low cost, availability, and so on. Though chitin is insoluble in most of the solvents, chitosan is readily soluble in dilute aqueous acidic solutions and is easily processed because of the presence of amino groups. A new class of chitin/chitosan nanocomposite materials has emerged recently, based on the incorporation of reinforcing fillers with dimensions in the nanometric scale. These natural nanomaterials are mostly minerals excavated from beneath the Earth’s surface. For instance, bentonite,

Handbook of Chitin and Chitosan

28

1. Polymer blends, IPNs, gels, composites, and nanocomposites

magnetite, montmorillonite, and halloysite have found significant uses in the formation of composites with chitin and chitosan.

1.7.1 Nanocomposites from chitin and chitosan Since it is positively charged chitosan generally becomes readily soluble in acidic to neutral solutions depending on the charge density and degree of deacetylation. Its inherited bioadhesive property allows it to attach to negatively charged surfaces such as the mucosal membrane. Additionally, it has antibacterial activity. With these properties of chitosan in mind several researchers have progressed several investigations on its biomedical applications. Among those examinations, preparation of a composite nanoTiO2
chitosan with collagen artificial skin (NTCAS) displayed encouraging characteristics of moderate water absorption, fine thickness, low density, moderate biodegradability, and favorable time-dependent biodegradability. All these characteristics of NTCAS have led the researchers to its potential application in wound healing [129]. The wound healing potential of the nanocomposite made of chitosan and bentonite has been extensively investigated in recent years. It is stated that the hydrophilic nature of bentonite enhanced the water absorption ability and mechanical strength. Additionally, the antibacterial activity of bentonite provided a synergistic effect on the healing ability of chitosan more efficiently [134]. Again, chitosan
montmorillonite nanocomposites were formulated by intercalating cationic biopolymer chitosan into Na1-montmorillonite through a cationic exchange process, and it showed great antimicrobial activity and enriched thermal stability [135]. Polyhedral oligomeric silsesquioxanes (POSS) has a three-dimensional rigid framework that closely resembles silica and it presents an exclusive prospect for the production of molecularly scattered nanocomposites [136]. A novel composite was fabricated by a solvent casting technique from chitosan and POSS for bone regeneration. Meanwhile, POSS incorporation in chitosan extensively enhanced the alkaline phosphatase (ALP) activity in liver function and bone development [128]. Again for bone engineering, biocomposite scaffolds were prepared by blending chitosan, alginate, and nanosilica following a freeze-drying method. The researchers stated that the presence of nanosilica in the scaffolds facilitated the regulated swelling ability and enhanced protein absorption. There was no observed cytotoxicity of these composites toward osteolineage cells [137]. The behavior of the adsorption of various dyes and heavy metals on composite surfaces was studied by various researchers. In these composites nanosized components were incorporated in chitosan. Applying a microemulsion method, a composite was prepared from nanosized γ-Fe2O3, kaolin, and chitosan and the adsorption behavior of Methyl

Handbook of Chitin and Chitosan

1.7 Chitin- and chitosan-based nanocomposites

29

Orange on the composite surface was observed [138]. A magnetic composite was prepared incorporating nanomagnetite and heulandites in cross-linked chitosan for the adsorption of copper(II) and arsenic(V) from aqueous solution. Here the mass ratio of chitosan, heulandites, and nanomagnetite was optimized for getting the best possible adsorption of both of these heavy metals [139]. In another study, the absorption of lead(II) and cadmium(II) as single and binary mixtures from water was tested by a functional adsorbent which was prepared by integrating chitosan with cross-linked polymethacrylic acid and halloysite nanotube. Higher adsorption of lead than cadmium was demonstrated when they were exposed to the composite separately. Likewise, their adsorption was reduced in a binary mixture [140]. Again in a different approach, chromium(VI) was removed from wastewater of the tanning industry by a nanobiocomposite prepared by the intercalation of chitosan in the interlayer space of bentonite [141]. In a composite preparation, the se of cellulose and its derivatives in combination with chitosan is advantageous for various purposes. A novel magnetic hydrogel nanocomposite was prepared by blending carboxylated cellulose nanofibrils (CCNFs) and PVA in chitosan. This composite was produced by an instantaneous gelation method for the adsorption of lead(II) from aqueous solution. Moreover, it was found that the composite hydrogel could be reused up to 4 times without a significant change in adsorption ability [142]. Other than nanosized minerals, the pure nanoform of metal incorporated in chitosan also displayed a prominent adsorption capacity for both heavy metals and dye particles. Such patterns were widely studied by several researchers. Nanoparticles of silver and gold were separately blended with chitosan and clay by a solution mixing method to prepare two separate hybrid composites, namely, (Ch)/AgNPs/clay and (Ch)/AuNPs/clay, for the removal of Cu(II) ions from aqueous solution [143].

1.7.2 Applications of chitin- and chitosan-based nanocomposites Because of their unique properties, chitin and chitosan have attracted scientific and industrial interest in several fields, such as biotechnology, pharmaceutics, biomedicine, packaging, wastewater treatment, cosmetics, and food science. 1.7.2.1 Dye removal A composite of nanosized γ-Fe2O3/kaolin/chitosan can be a valuable alternative for the removal of anionic dyes from industrial wastewater [138]. Completely biobased composites were prepared from cellulose

Handbook of Chitin and Chitosan

30

1. Polymer blends, IPNs, gels, composites, and nanocomposites

nanocrystals and chitosan by a freeze-drying technique followed by compacting. Many positive dyes like cellulose nanocrystals, Methyl Violet 2B and Victoria Blue 2B were removed by this composite, and the producer of this composite declared that the removal of dyes was expected to be driven by the electrostatic attraction between negatively charged CNCs and the positively charged dyes [144]. Magnetic bioadsorbents with high adsorption capacity and convenient recovery were prepared from chitosan/PVA hydrogel beads by an instantaneous gelation method. These composites removed commercial dyes like Congo Red significantly from the aqueous solution and can be considered to be a low-cost alternative to other adsorbents for the removal of dyes [145]. 1.7.2.2 Heavy metals removal Copper can be removed from the aqueous solution promisingly using hybrid nanocomposites of chitosan
silver nanoparticle
clay and chitosan
gold nanoparticle
clay [143]. Lead(II) and cadmium(II) can be removed as single and binary mixtures in water by a functional adsorbent made of PMA cross-linked chitosan and halloysite nanotube. For the removal of other different or similar ions from water this functional composite might be utilized comprehensively [140]. Again, lead can be removed from aqueous solution by a magnetic composite made of cellulose nanofibrils (CCNFs), PVA, and chitosan. In addition, this composite has regenerablity without considerable loss of its adsorption capacity [142]. Hexavalent chromium can be removed from a synthetic solution and wastewater from the tanning industry by a bentonite/chitosan nanobiocomposite [141]. 1.7.2.3 Wound healing The main purpose of wound dressing is to protect the wound from bacterial infection and subsequently promote healing. Chitosan-based substances can stimulate wound healing on their own and cause low or no scarring [146]. The dressing at the first phase of wound healing should support healing, ensure the solubilization of antimicrobial agent, and support the growth of fibroblasts. Chitosan-based composites meet all of those requirements. Moreover, an increased healing process was observed by the chitosan when utilized in the form of powder, granules, nano-/microparticles, or as a composite of other materials [147]. Chitosan composite hydrogel scaffolds infused with β-fibroblast growth factor microspheres tremendously accelerated the healing of chronic ulcers [148]. Hydrogel chitosan composites of alginate and chitosan fibers were prepared by a one-step wet spinning extrusion process for wound-care application. It was also observed that the incorporation of chitosan into fiber form improved the absorption of fibers in both saline and distilled water [149]. Wound healing, antibacterial activity, biocompatibility, and temperature responsiveness could be promoted through sequential

Handbook of Chitin and Chitosan

1.7 Chitin- and chitosan-based nanocomposites

31

grafting and plasma modification [150]. Hemocompatibility, an essential test for evaluating the interactions of medical devices having contact with blood, pointed toward a higher ability of chitosan
bentonite nanocomposite to be employed in contact with body fluid. Therefore chitosan
bentonite nanocomposite has been suggested as a potential candidate for wound-healing application [134]. 1.7.2.4 Biodegradable packaging materials The antimicrobial activity od chitosan is well-known. Many researchers have reported this activity when incorporating chitosan in other materials. For instance, a composite prepared by intercalating chitosan in montmorillonite had been tested on two representative food pathogenic bacteria—S. aureus (Gram 1 ) and E. coli (Gram 2 )—for determining antimicrobial activity. It was concluded that this composite material could be utilized as a biodegradable packaging materials with antimicrobial activity [135]. Nanocomposite films were prepared by casting chitosan and nanofibrillated cellulose for the purpose of medical and antibacterial packaging. Due to the addition of nanofibrillated cellulose in chitosan, the mechanical properties like flexibility and the thermal stability of the composite were enhanced significantly compared with native chitosan films [151]. Surface-deacetylated chitin nanofiberreinforced chitosan films were prepared. The composites were observed to develop the mechanical properties like tensile strength, Young’s modulus, and thermal expansion without compromising its flexibility transparency and antifungal activity, and thus they can be used as film in food and agricultural packaging applications [152]. 1.7.2.5 Bone regeneration Bone is composed of strong, rigid, organized, dense, and highly specialized connective tissue. For carrying out fundamental tasks, the bone mainly consists of various cells and several organic and inorganic matrices [153]. Even though chitosan and chitin have a lot of advantages such as biocompatibility and biodegradability, their uses are limited in indigenous form because of their instability and poor mechanical strength. Nevertheless, combining chitosan with other polymers to form composites can increase its mechanical properties and these composites can then be utilized as osteoconductive matrices. The suitable chitosan composite can promote the mineral deposition and cell growth [154]. For bone regeneration in the medical field a composite was prepared from chitosan and polyhedral oligomeric silsesquioxanes which could enhance the adsorption of plasma protein on the bone surface [128]. Alginate is an anionic polymer that is biocompatible, hydrophilic, and biodegradable under common physiological situation, and it is extensively used as an instant gel for bone tissue engineering [155]. As a

Handbook of Chitin and Chitosan

32

1. Polymer blends, IPNs, gels, composites, and nanocomposites

temporary skeleton to accommodate and stimulate new tissue growth, a composite scaffold is prepared by blending chitosan and alginate. The in vivo experiment has proven that the chitosan
alginate composite upheld swift vascularization and deposited connective tissue and calcified matrix within the entire scaffold structure [156]. Gelatin (G), a hydrolyzed form of collagen, has been broadly used for the fabrication of scaffolds in combination with other scaffold-forming materials. Nanocomposites were prepared from chitosan and gelatin to release simvastatine in bone implants. Simvastatin is reported to inhibit farnesyl pyrophosphate, mevalonate, and geranyl-pyrophosphate pathways and to be responsible for enhanced bone mineral density by diminishing osteoclast activity [157]. Chitosan-based scaffolds with various materials such as methylcellulose, tricalcium phosphate, PVA, nanoclays, and HAp have also been reported for bone tissue engineering. In almost every case, the nanocomposite scaffolds of chitosan were formulated with HAp, as carbonated HAp forms the major part of the inorganic matrix in the human bone [158
160]. 1.7.2.6 Drug delivery Chitosan has been used widely for the last few decades. Small drug molecules as well as large biomolecules, such as protein and nucleic acid are being delivered to animals for medical treatment [161]. Types of organ or tissue, the nature of the drug and the availability of the drug molecules decide which method is to be applied during drug loading. Drug encapsulation in chitosan and polymer-containing composites is one of the simplest methods for the delivery of drugs where crosslinking between various polymer chains takes place [162]. A composite was made from carboxymethyl chitosan by cross-linking with glutaraldehyde for a potential carrier for colon targeted drug delivery of ornidazole and it was found that the prepared hydrogel can be used as a potential carrier [163]. Another pH-responsive composite was produced from chitosan and carboxymethyl chitosan nanoparticles for the oral delivery of doxorubicin hydrochloride. In vitro and in vivo analysis of this composite suggested that intestinal adhesion and permeation of this drug can be controlled effectively and the composite polyelectrolyte complex is highly efficient and safe as an oral delivery system for doxorubicin hydrochloride [164].

1.8 Conclusions and future prospective Chitin and chitosan are unique biopolymers characterized by primary amines along the backbone. Such structure imparts to this amino polysaccharide not only highly structural possibilities but also particular

Handbook of Chitin and Chitosan

References

33

interactions with other natural and synthetic polymers. This chapter summarizes chitin- and chitosan-based materials, such as polymer blends, gels, IPNs, composites, nanocomposites, and their manufacturing processes and applications. Due to their biodegradability, biocompatibility, antibacterial and antifungal activity, and nontoxicity, chitin- and chitosan-based materials are used in a wide variety of applications including the biomedical, pharmaceutical, chemical, cosmetics, environmental, food, and agricultural fields. We expect that the extensive varieties of properties and processed materials will give rise to chitin- and chitosan-based materials as promising biomaterials for future development.

References [1] J. Akbuga, A biopolymer: chitosan, Int. J. Pharm. Adv. 1 (1) (1995) 3
17. [2] L. Ilium, Chitosan and its use as a pharmaceutical excipient, Pharm. Res. 15 (1998) 1326
1331. [3] Y. Dong, W. Qiu, Y. Ruan, Y. Wu, M. Wang, C. Xu, Influence of molecular weight on critical concentration of chitosan/formic acid liquid crystalline solution, Polym. J. 33 (2001) 387. [4] M.S. Islam, P. Haque, T.U. Rashid, M.N. Khan, A.K. Mallik, M.N.I. Khan, et al., Core
shell drug carrier from folate conjugated chitosan obtained from prawn shell for targeted doxorubicin delivery, J. Mater. Sci. 28 (2017) 55. [5] I. Aranaz, M. Mengı´bar, R. Harris, I. Pan˜os, B. Miralles, N. Acosta, et al., Functional characterization of chitin and chitosan, Curr. Chem. Biol. 3 (2009) 203
230. [6] C. Pillai, W. Paul, C.P. Sharma, Chitin and chitosan polymers: chemistry, solubility and fiber formation, Prog. Polym. Sci. 34 (2009) 641
678. [7] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603
632. [8] K. Shalumon, N. Binulal, N. Selvamurugan, S. Nair, D. Menon, T. Furuike, et al., Electrospinning of carboxymethyl chitin/poly (vinyl alcohol) nanofibrous scaffolds for tissue engineering applications, Carbohydr. Polym. 77 (2009) 863
869. [9] A. Morin, A. Dufresne, Nanocomposites of chitin whiskers from Riftia tubes and poly (caprolactone), Macromolecules 35 (2002) 2190
2199. [10] K. Madhumathi, P.T. Sudheesh Kumar, S. Abhilash, V. Sreeja, H. Tamura, K. Manzoor, et al., Development of novel chitin/nanosilver composite scaffolds for wound dressing applications, J. Mater. Sci. 21 (2010) 807
813. [11] J.K. Pandey, A.P. Kumar, M. Misra, A.K. Mohanty, L.T. Drzal, R. Palsingh, Recent advances in biodegradable nanocomposites, J. Nanosci. Nanotechnol. 5 (2005) 497
526. [12] S.-Y. Ong, J. Wu, S.M. Moochhala, M.-H. Tan, J. Lu, Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties, Biomaterials 29 (2008) 4323
4332. [13] J. Yang, F. Tian, Z. Wang, Q. Wang, Y.J. Zeng, S.Q. Chen, Effect of chitosan molecular weight and deacetylation degree on hemostasis, J. Biomed. Mater. Res. Part. B 84 (2008) 131
137. [14] Y. Huang, Y. Lapitsky, Salt-assisted mechanistic analysis of chitosan/tripolyphosphate micro- and nanogel formation, Biomacromolecules 13 (2012) 3868
3876. [15] J. Berger, M. Reist, J.M. Mayer, O. Felt, R. Gurny, Structure and interactions in chitosan hydrogels formed by complexation or aggregation for biomedical applications, Eur. J. Pharm. Biopharm. 57 (2004) 35
52.

Handbook of Chitin and Chitosan

34

1. Polymer blends, IPNs, gels, composites, and nanocomposites

[16] M.M. Rahman, M. Shahruzzaman, M.S. Islam, M.N. Khan, P. Haque, Preparation and properties of biodegradable polymer/nano-hydroxyapatite bioceramic scaffold for spongy bone regeneration, J. Polym. Eng 39 (2018) 134
142. [17] D. Wheeler, M. Montfort, S. Mcloughlin, Differential healing response of bone adjacent to porous implants coated with hydroxyapatite and 45S5 bioactive glass, J. Biomed. Mater. Res. 55 (2001) 603
612. [18] M.N.R. Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (2000) 1
27. [19] Q. Li, E. Dunn, E. Grandmaison, M.F. Goosen, Applications and properties of chitosan, J. Bioact. Compat. Polym. 7 (1992) 370
397. [20] G. Roberts, Chitin Chemistry, The Macmillan Press, Basingstoke, 1992. [21] J.-F. Revol, R. Marchessault, In vitro chiral nematic ordering of chitin crystallites, Int. J. Biol. Macromol. 15 (1993) 329
335. [22] J. Li, J. Revol, E. Naranjo, R. Marchessault, Effect of electrostatic interaction on phase separation behaviour of chitin crystallite suspensions, Int. J. Biol. Macromol. 18 (1996) 177
187. [23] S.-I. Park, Y. Zhao, Incorporation of a high concentration of mineral or vitamin into chitosan-based films, J. Agric. Food Chem. 52 (2004) 1933
1939. [24] T. Lertwattanaseri, N. Ichikawa, T. Mizoguchi, Y. Tanaka, S. Chirachanchai, Natural chelating polymers, Carbohydr. Res. 344 (2009) 331
335. [25] B. Smitha, S. Sridhar, A. Khan, Synthesis and characterization of proton conducting polymer membranes for fuel cells, J. Membr. Sci. 225 (2003) 63
76. [26] S. Yoshikawa, T. Takayama, N. Tsubokawa, Grafting reaction of living polymer cations with amino groups on chitosan powder, J. Appl. Polym. Sci. 68 (1998) 1883
1889. [27] V.M. Correlo, L.F. Boesel, M. Bhattacharya, J.F. Mano, N.M. Neves, R.L. Reis, Properties of melt processed chitosan and aliphatic polyester blends, Mater. Sci. Eng. A 403 (2005) 57
68. [28] M. Mucha, Rheological properties of chitosan blends with poly (ethylene oxide) and poly (vinyl alcohol) in solution, React. Funct. Polym. 38 (1998) 19
25. [29] I. Engelberg, J. Kohn, Physico-mechanical properties of degradable polymers used in medical applications: a comparative study, Biomaterials 12 (1991) 292
304. [30] P. Gong, L. Zhang, L. Zhuang, J. Lu, Synthesis and characterization of polyurethane
chitosan interpenetrating polymer networks, J. Appl. Polym. Sci. 68 (1998) 1321
1329. [31] R.A.A. Muzzarelli, P. Ilari, M. Tomasetti, Chitin-based poly(urea-urethane)s, J. Biomater. Sci. Polym. Ed. 6 (1995) 541
547. [32] B.L. Butler, P.J. Vergano, R.F. Testin, J.M. Bunn, J.L. Wiles, Mechanical and barrier properties of edible chitosan films as affected by composition and storage, J. Food Sci. 61 (1996) 953
956. [33] J.-J. Shieh, R.Y. Huang, Chitosan/N-methylol nylon 6 blend membranes for the pervaporation separation of ethanol
water mixtures, J. Membr. Sci. 148 (1998) 243
255. [34] P. Srinivasa, M. Ramesh, K. Kumar, R. Tharanathan, Properties and sorption studies of chitosan
polyvinyl alcohol blend films, Carbohydr. Polym. 53 (2003) 431
438. [35] M. Zeng, Z. Fang, C. Xu, Effect of compatibility on the structure of the microporous membrane prepared by selective dissolution of chitosan/synthetic polymer blend membrane, J. Membr. Sci. 230 (2004) 175
181. [36] C. Sandoval, C. Castro, L. Gargallo, D. Radic, J. Freire, Specific interactions in blends containing chitosan and functionalized polymers. Molecular dynamics simulations, Polymer 46 (2005) 10437
10442. [37] A. Sarasam, S.V. Madihally, Characterization of chitosan
polycaprolactone blends for tissue engineering applications, Biomaterials 26 (2005) 5500
5508.

Handbook of Chitin and Chitosan

References

35

[38] P.-C. Kuo, D. Sahu, H.H. Yu, Properties and biodegradability of chitosan/nylon 11 blending films, Polym. Degrad. Stab. 91 (2006) 3097
3102. [39] I.S. Arvanitoyannis, A. Nakayama, S.-I. Aiba, Chitosan and gelatin based edible films: state diagrams, mechanical and permeation properties, Carbohydr. Polym. 37 (1998) 371
382. [40] M. Cheng, J. Deng, F. Yang, Y. Gong, N. Zhao, X. Zhang, Study on physical properties and nerve cell affinity of composite films from chitosan and gelatin solutions, Biomaterials 24 (2003) 2871
2880. [41] T. Ikejima, K. Yagi, Y. Inoue, Thermal properties and crystallization behavior of poly (3-hydroxybutyric acid) in blends with chitin and chitosan, Macromol. Chem. Phys. 200 (1999) 413
421. [42] H. Kweon, I.C. Um, Y.H. Park, Structural and thermal characteristics of Antheraea pernyi silk fibroin/chitosan blend film, Polymer 42 (2001) 6651
6656. [43] M. Zhai, L. Zhao, F. Yoshii, T. Kume, Study on antibacterial starch/chitosan blend film formed under the action of irradiation, Carbohydr. Polym. 57 (2004) 83
88. [44] Y.X. Xu, K.M. Kim, M.A. Hanna, D. Nag, Chitosan
starch composite film: preparation and characterization, Ind. Crop. Prod. 21 (2005) 185
192. [45] N.E. Suyatma, A. Copinet, L. Tighzert, V. Coma, Mechanical and barrier properties of biodegradable films made from chitosan and poly (lactic acid) blends, J. Polym. Environ. 12 (2004) 1
6. [46] Y.-B. Wu, S.-H. Yu, F.-L. Mi, C.-W. Wu, S.-S. Shyu, C.-K. Peng, et al., Preparation and characterization on mechanical and antibacterial properties of chitsoan/cellulose blends, Carbohydr. Polym. 57 (2004) 435
440. [47] M. Mucha, A. Pawlak, Thermal analysis of chitosan and its blends, Thermochim. Acta 427 (2005) 69
76. [48] S. Wittaya-Areekul, C. Prahsarn, Development and in vitro evaluation of chitosan
polysaccharides composite wound dressings, Int. J. Pharm. 313 (2006) 123
128. [49] T. Chandy, C.P. Sharma, Chitosan-as a biomaterial, Biomater., Artif. Cell Artif. Organs 18 (1990) 1
24. [50] A. Berthold, K. Cremer, J. Kreuter, Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for antiinflammatory drugs, J. Control. Release 39 (1996) 17
25. ˘ Alternative approach to the preparation of chitosan beads, Int. [51] C. Aral, J. Akbuga, J. Pharm. 168 (1998) 9
15. [52] C.C. Leffler, B.W. Mu¨ller, Influence of the acid type on the physical and drug liberation properties of chitosan
gelatin sponges, Int. J. Pharm. 194 (2000) 229
237. [53] T.W. Chung, J. Yang, T. Akaike, K.Y. Cho, J.W. Nah, S.I. Kim, et al., Preparation of alginate/galactosylated chitosan scaffold for hepatocyte attachment, Biomaterials 23 (2002) 2827
2834. [54] H.R. Ramay, Z. Li, E. Shum, M. Zhang, Chitosan-alginate porous scaffolds reinforced by hydroxyapatite nano- and micro-particles: structural, mechanical, and biological properties, J. Biomed. Nanotechnol. 1 (2005) 151
160. [55] K. Kojima, Y. Okamoto, K. Miyatake, K. Yukisato, S. Minami, Collagen typing of granulation tissue induced by chitin and chitosan, Carbohydr. Polym. 37 (1998) 109
113. [56] B. Krajewska, Membrane-based processes performed with use of chitin/chitosan materials, Sep. Purif. Technol. 41 (2005) 305
312. [57] S. Tripathi, G.K. Mehrotra, P.K. Dutta, Physicochemical and bioactivity of crosslinked chitosan
PVA film for food packaging applications, Int. J. Biol. Macromol. 45 (2009) 372
376. [58] D. Myung, D. Waters, M. Wiseman, P.-E. Duhamel, J. Noolandi, C.N. Ta, et al., Progress in the development of interpenetrating polymer network hydrogels, Polym. Adv. Technol. 19 (2008) 647
657.

Handbook of Chitin and Chitosan

36

1. Polymer blends, IPNs, gels, composites, and nanocomposites

[59] A.D. Jenkins, P. Kratochvı´l, R.F.T. Stepto, U.W. Suter, Glossary of basic terms in polymer science (IUPAC recommendations 1996), Pure Appl. Chem 68 (1996) 2287
2311. [60] M.M. Islam, S. Biswas, M.M. Hasan, P. Haque, S.H. Rimu, M.M. Rahman, Studies of Cr (VI) adsorption on novel jute cellulose-kaolinite clay biocomposite, Desalin. Water Treat. 123 (2018) 265
276. [61] M.M. Islam, M.N. Khan, S. Biswas, T.R. Choudhury, P. Haque, T.U. Rashid, et al., Preparation and characterization of bijoypur clay-crystalline cellulose composite for application as an adsorbent, Adv. Mater. Sci. 2 (2017) 1
7. [62] S. Biswas, T.U. Rashid, A.K. Mallik, M.M. Islam, M.N. Khan, P. Haque, et al., Facile preparation of biocomposite from prawn shell derived chitosan and Kaolinite-Rich locally available clay, Int. J. Polym. Sci. 2017 (2017) 8. [63] S.J. Kim, S.R. Shin, G.M. Spinks, I.Y. Kim, S.I. Kim, Synthesis and characteristics of a semi-interpenetrating polymer network based on chitosan/polyaniline under different pH conditions, J. Appl. Polym. Sci. 96 (2005) 867
873. [64] M.B. Povea, W.A. Monal, J.V. Cauich-Rodrı´guez, A.M. Pat, N.B. Rivero, C.P. Covas, Interpenetrated chitosan-poly (acrylic acid-co-acrylamide) hydrogels. Synthesis, characterization and sustained protein release studies, Mater. Sci. Appl. 2 (2011) 509. [65] H. Wang, W. Li, Y. Lu, Z. Wang, Studies on chitosan and poly(acrylic acid) interpolymer complex. I. Preparation, structure, pH-sensitivity, and salt sensitivity of complex-forming poly(acrylic acid): chitosan semi-interpenetrating polymer network, J. Appl. Polym. Sci. 65 (1997) 1445
1450. [66] J. Chen, L.-X. Gao, X. Han, T. Chen, J. Luo, K. Liu, et al., Preparation and electroresponse of chitosan-g-poly (acrylic acid) hydrogel elastomers with interpenetrating network, Mater. Chem. Phys. 169 (2016) 105
112. [67] V.R. Babu, K.M. Hosamani, T.M. Aminabhavi, Preparation and in-vitro release of chlorothiazide novel pH-sensitive chitosan-N,N0 -dimethylacrylamide semiinterpenetrating network microspheres, Carbohydr. Polym. 71 (2008) 208
217. [68] P. Bonina, T. Petrova, N. Manolova, pH-sensitive hydrogels composed of chitosan and polyacrylamide
preparation and properties, J. Bioact. Compat. Polym. 19 (2004) 101
116. [69] I.M. El-Sherbiny, R.J. Lins, E.M. Abdel-Bary, D.R.K. Harding, Preparation, characterization, swelling and in vitro drug release behaviour of poly[N-acryloylglycinechitosan] interpolymeric pH and thermally-responsive hydrogels, Eur. Polym. J. 41 (2005) 2584
2591. [70] V.I. Lozinsky, F.M. Plieva, I.Y. Galaev, B. Mattiasson, The potential of polymeric cryogels in bioseparation, Bioseparation 10 (2001) 163
188. [71] E. Jain, A. Kumar, Designing supermacroporous cryogels based on polyacrylonitrile and a polyacrylamide
chitosan semi-interpenetrating network, J. Biomater. Sci., Polym. Ed. 20 (2009) 877
902. [72] C. Alvarez-Lorenzo, A. Concheiro, A.S. Dubovik, N.V. Grinberg, T.V. Burova, V.Y. Grinberg, Temperature-sensitive chitosan-poly(N-isopropylacrylamide) interpenetrated networks with enhanced loading capacity and controlled release properties, J. Control. Release 102 (2005) 629
641. [73] P. Sacco, F. Furlani, G. de Marzo, E. Marsich, S. Paoletti, I. Donati, Concepts for developing physical gels of chitosan and of chitosan derivatives, Gels 4 (2018) 67. [74] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. 57 (2004) 19
34. [75] S. Hirano, S. Kondo, Y. Ohe, Chitosan gel: a novel polysaccharide gel, Polymer 16 (1975) 622. [76] G.K. Moore, G.A.F. Roberts, Chitosan gels: 1. Study of reaction variables, Int. J. Biol. Macromol. 2 (1980) 73
77.

Handbook of Chitin and Chitosan

References

37

[77] G.K. Moore, G.A.F. Roberts, Chitosan gels: 2. Mechanism of gelation, Int. J. Biol. Macromol. 2 (1980) 78
80. [78] H. Oylum, E. Yilmaz, O. Yilmaz, Preparation of chitin-g-poly(4-vinylpyridine) beads, J. Macromol. Sci., Part A 50 (2013) 221
229. [79] S. Hirano, Y. Ohe, H. Ono, Selective N-acylation of chitosan, Carbohydr. Res. 47 (1976) 315
320. [80] L. Vachoud, N. Zydowicz, A. Domard, Formation and characterisation of a physical chitin gel, Carbohydr. Res. 302 (1997) 169
177. [81] E. Bianchi, E. Marsano, M. Baldini, G. Conio, A. Tealdi, Chitin
cellulose blends: phase properties in dimethylacetamide
LiCl, Polym. Adv. Technol. 6 (1995) 727
732. [82] C. Li, J. Hou, J. Gu, Q. Han, Y. Guan, Y. Zhang, Synthesis and thermal gelation of hydroxypropyl chitin, RSC Adv. 5 (2015) 39677
39685. [83] A. Montembault, C. Viton, A. Domard, Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent, Biomacromolecules 6 (2005) 653
662. [84] A. Chenite, M. Buschmann, D. Wang, C. Chaput, N. Kandani, Rheological characterisation of thermogelling chitosan/glycerol-phosphate solutions, Carbohydr. Polym. 46 (2001) 39
47. [85] H.T. Ta, H. Han, I. Larson, C.R. Dass, D.E. Dunstan, Chitosan-dibasic orthophosphate hydrogel: a potential drug delivery system, Int. J. Pharm. 371 (2009) 134
141. [86] P. Sacco, S. Paoletti, M. Cok, F. Asaro, M. Abrami, M. Grassi, et al., Insight into the ionotropic gelation of chitosan using tripolyphosphate and pyrophosphate as crosslinkers, Int. J. Biol. Macromol. 92 (2016) 476
483. [87] P. Sacco, M. Cok, F. Asaro, S. Paoletti, I. Donati, The role played by the molecular weight and acetylation degree in modulating the stiffness and elasticity of chitosan gels, Carbohydr. Polym. 196 (2018) 405
413. [88] L. Rami, S. Malaise, S. Delmond, J.-C. Fricain, R. Siadous, S. Schlaubitz, et al., Physicochemical modulation of chitosan-based hydrogels induces different biological responses: interest for tissue engineering, J. Biomed. Mater. Res. Part A 102 (2014) 3666
3676. [89] J. Nilsen-Nygaard, S. Strand, K. Va˚rum, K. Draget, C. Nordga˚rd, Chitosan: gels and interfacial properties, Polymers 7 (2015) 552. [90] P. Sacco, E. Decleva, F. Tentor, R. Menegazzi, M. Borgogna, S. Paoletti, et al., Butyrate-loaded chitosan/hyaluronan nanoparticles: a suitable tool for sustained inhibition of ROS release by activated neutrophils, Macromol. Biosci. 17 (2017) 1700214. [91] M. De La Fuente, B. Seijo, M.J. Alonso, Novel hyaluronan-based nanocarriers for transmucosal delivery of macromolecules, Macromol. Biosci. 8 (2008) 441
450. [92] M. Costalat, L. David, T. Delair, Reversible controlled assembly of chitosan and dextran sulfate: a new method for nanoparticle elaboration, Carbohydr. Polym. 102 (2014) 717
726. [93] T.T. Khong, O.A. Aarstad, G. Skja˚k-Bræk, K.I. Draget, K.M. Va˚rum, Gelling concept combining chitosan and alginate—proof of principle, Biomacromolecules 14 (2013) 2765
2771. [94] P. Calvo, C. Remun˜a´n-Lo´pez, J.L. Vila-Jato, M.J. Alonso, Novel hydrophilic chitosanpolyethylene oxide nanoparticles as protein carriers, J. Appl. Polym. Sci. 63 (1997) 125
132. [95] O.A.C. Monteiro, C. Airoldi, Some studies of crosslinking chitosan
glutaraldehyde interaction in a homogeneous system, Int. J. Biol. Macromol. 26 (1999) 119
128. [96] Y. Ji Yin, F. Zhao, X. Feng Song, K. De Yao, W.W. Lu, J. Chiyan Leong, Preparation and characterization of hydroxyapatite/chitosan
gelatin network composite, J. Appl. Polym. Sci. 77 (2000) 2929
2938.

Handbook of Chitin and Chitosan

38

1. Polymer blends, IPNs, gels, composites, and nanocomposites

[97] M. Wang, Y. Fang, D. Hu, Preparation and properties of chitosan-poly (N-isopropylacrylamide) full-IPN hydrogels, React. Funct. Polym. 48 (2001) 215
221. [98] M. Rinaudo, New way to crosslink chitosan in aqueous solution, Eur. Polym. J. 46 (2010) 1537
1544. [99] D. Xu, J. Huang, D. Zhao, B. Ding, L. Zhang, J. Cai, High-flexibility, high-toughness double-cross-linked chitin hydrogels by sequential chemical and physical crosslinkings, Adv. Mater. 28 (2016) 5844
5849. [100] L. Mayol, D. De Stefano, V. Campani, F. De Falco, E. Ferrari, C. Cencetti, et al., Design and characterization of a chitosan physical gel promoting wound healing in mice, J. Mater. Sci. 25 (2014) 1483
1493. [101] N. Bhattarai, H.R. Ramay, J. Gunn, F.A. Matsen, M. Zhang, PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release, J. Control. Release 103 (2005) 609
624. [102] J. Sun, C. Xiao, H. Tan, X. Hu, Covalently crosslinked hyaluronic acid-chitosan hydrogel containing dexamethasone as an injectable scaffold for soft tissue engineering, J. Appl. Polym. Sci. 129 (2013) 682
688. [103] C. Sa´mano-Valencia, G.A. Martı´nez-Castan˜o´n, F. Martı´nez-Gutie´rrez, F. Ruiz, J.F. Toro-Va´zquez, J.A. Morales-Rueda, et al., Characterization and biocompatibility of chitosan gels with silver and gold nanoparticles, J. Nanomater. 2014 (2014). 142. [104] K. Madhumathi, P.S. Kumar, S. Abhilash, V. Sreeja, H. Tamura, K. Manzoor, et al., Development of novel chitin/nanosilver composite scaffolds for wound dressing applications, J. Mater. Sci. 21 (2010) 807
813. [105] P.T. Kumar, V.K. Lakshmanan, R. Biswas, S.V. Nair, R. Jayakumar, Synthesis and biological evaluation of chitin hydrogel/nano ZnO composite bandage as antibacterial wound dressing, J. Biomed. Nanotechnol. 8 (2012) 891
900. [106] G.J. Copello, A.M. Mebert, M. Raineri, M.P. Pesenti, L.E. Diaz, Removal of dyes from water using chitosan hydrogel/SiO2 and chitin hydrogel/SiO2 hybrid materials obtained by the sol
gel method, J. Hazard. Mater. 186 (2011) 932
939. [107] H. Tang, W. Zhou, L. Zhang, Adsorption isotherms and kinetics studies of malachite green on chitin hydrogels, J. Hazard. Mater. 209
210 (2012) 218
225. [108] Y. Jiang, B. Liu, J. Xu, K. Pan, H. Hou, J. Hu, et al., Cross-linked chitosan/ β-cyclodextrin composite for selective removal of methyl orange: adsorption performance and mechanism, Carbohydr. Polym. 182 (2018) 106
114. [109] X. Li, Y. Li, S. Zhang, Z. Ye, Preparation and characterization of new foam adsorbents of poly(vinyl alcohol)/chitosan composites and their removal for dye and heavy metal from aqueous solution, Chem. Eng. J. 183 (2012) 88
97. [110] S.R. Popuri, Y. Vijaya, V.M. Boddu, K. Abburi, Adsorptive removal of copper and nickel ions from water using chitosan coated PVC beads, Bioresour. Technol. 100 (2009) 194
199. [111] T. Jamnongkan, K. Singcharoen, Towards novel adsorbents: the ratio of PVA/chitosan blended hydrogels on the copper (II) ion adsorption, Energy Procedia 89 (2016) 299
306. [112] Y. Zhu, J. Hu, J. Wang, Removal of Co2 1 from radioactive wastewater by polyvinyl alcohol (PVA)/chitosan magnetic composite, Prog. Nucl. Energy 71 (2014) 172
178. [113] M. Kumar, B.P. Tripathi, V.K. Shahi, Crosslinked chitosan/polyvinyl alcohol blend beads for removal and recovery of Cd(II) from wastewater, J. Hazard. Mater. 172 (2009) 1041
1048. [114] T.S. Anirudhan, S. Rijith, A.R. Tharun, Adsorptive removal of thorium(IV) from aqueous solutions using poly(methacrylic acid)-grafted chitosan/bentonite composite matrix: process design and equilibrium studies, Colloid. Surf. A: Physicochem. Eng. Asp. 368 (2010) 13
22.

Handbook of Chitin and Chitosan

References

39

[115] T.S. Anirudhan, S. Rijith, Synthesis and characterization of carboxyl terminated poly (methacrylic acid) grafted chitosan/bentonite composite and its application for the recovery of uranium(VI) from aqueous media, J. Environ. Radioactiv. 106 (2012) 8
19. [116] S. Gokila, T. Gomathi, P.N. Sudha, S. Anil, Removal of the heavy metal ion chromiuim(VI) using chitosan and alginate nanocomposites, Int. J. Biol. Macromol. 104 (2017) 1459
1468. [117] S. Kalyani, J.A. Priya, P.S. Rao, A. Krishnaiah, Removal of copper and nickel from aqueous solutions using chitosan coated on perlite as biosorbent, Sep. Sci. Technol. 40 (2005) 1483
1495. [118] M.-W. Wan, C.-C. Kan, C.-H. Lin, D. Buenda, C.-H. Wu, Adsorption of copper (II) by chitosan immobilized on sand, Chia-Nan Annu. Bull. 33 (2007) 96
106. [119] H. Gecol, P. Miakatsindila, E. Ergican, S.R. Hiibel, Biopolymer coated clay particles for the adsorption of tungsten from water, Desalination 197 (2006) 165
178. [120] D. Liu, X. Chen, Y. Yue, M. Chen, Q. Wu, Structure and rheology of nanocrystalline cellulose, Carbohydr. Polym. 84 (2011) 316
322. [121] N. Li, R. Bai, Copper adsorption on chitosan
cellulose hydrogel beads: behaviors and mechanisms, Sep. Purif. Technol. 42 (2005) 237
247. [122] A.L. Vega-Negron, L. Alamo-Nole, O. Perales-Perez, A.M. Gonzalez-Mederos, C. Jusino-Olivencia, F.R. Roman-Velazquez, Simultaneous adsorption of cationic and anionic dyes by chitosan/cellulose beads for wastewaters treatment, Int. J. Environ. Res. 12 (2018) 59
65. [123] M. Auta, B.H. Hameed, Chitosan
clay composite as highly effective and low-cost adsorbent for batch and fixed-bed adsorption of methylene blue, Chem. Eng. J. 237 (2014) 352
361. [124] G.L. Dotto, F.K. Rodrigues, E.H. Tanabe, R. Fro¨hlich, D.A. Bertuol, T.R. Martins, et al., Development of chitosan/bentonite hybrid composite to remove hazardous anionic and cationic dyes from colored effluents, J. Environ. Chem. Eng. 4 (2016) 3230
3239. [125] Q. Liu, B. Yang, L. Zhang, R. Huang, Adsorption of an anionic azo dye by crosslinked chitosan/bentonite composite, Int. J. Biol. Macromol. 72 (2015) 1129
1135. [126] J. Venkatesan, S.-K. Kim, Chitosan composites for bone tissue engineering—an overview, Mar. Drugs 8 (2010) 2252. [127] M. Ahmad, K. Manzoor, S. Ahmad, N. Akram, S. Ikram, 11 - Chitosan-based nanocomposites for cardiac, liver, and wound healing applications, in: A.M. Inamuddin, Asiri, A. Mohammad (Eds.), Applications of Nanocomposite Materials in Orthopedics, Woodhead Publishing, 2019. [128] S. Tamburaci, F. Tihminlioglu, Novel poss reinforced chitosan composite membranes for guided bone tissue regeneration, J. Mater. Sci. 29 (2017) 1. [129] C.-C. Peng, M.-H. Yang, W.-T. Chiu, C.-H. Chiu, C.-S. Yang, Y.-W. Chen, et al., Composite nano-titanium oxide
chitosan artificial skin exhibits strong woundhealing effect—an approach with anti-inflammatory and bactericidal kinetics, Macromol. Biosci. 8 (2008) 316
327. [130] R. Jayakumar, M. Prabaharan, S.V. Nair, S. Tokura, H. Tamura, N. Selvamurugan, Novel carboxymethyl derivatives of chitin and chitosan materials and their biomedical applications, Prog. Mater. Sci. 55 (2010) 675
709. [131] L. Li, J. Li, S. Si, L. Wang, C. Shi, Y. Sun, et al., Effect of formulation variables on in vitro release of a water-soluble drug from chitosan
sodium alginate matrix tablets, Asian J. Pharm. Sci. 10 (2015) 314
321. [132] Y. Shao, L. Li, X. Gu, L. Wang, S. Mao, Evaluation of chitosan
anionic polymers based tablets for extended-release of highly water-soluble drugs, Asian J. Pharm. Sci. 10 (2015) 24
30.

Handbook of Chitin and Chitosan

40

1. Polymer blends, IPNs, gels, composites, and nanocomposites

[133] J.S. Maciel, H.C.B. Paula, M.A.R. Miranda, J.M. Sasaki, R.C.M. De Paula, Reacetylated chitosan/cashew gum gel: preliminary study for potential utilization as drug release matrix, J. Appl. Polym. Sci. 99 (2006) 326
334. [134] N. Devi, J. Dutta, Preparation and characterization of chitosan-bentonite nanocomposite films for wound healing application, Int. J. Biol. Macromol. 104 (2017) 1897
1904. [135] Y.-S. Han, S.-H. Lee, K.H. Choi, I. Park, Preparation and characterization of chitosan
clay nanocomposites with antimicrobial activity, J. Phys. Chem. Solids 71 (2010) 464
467. [136] K. Pielichowski, J. Njuguna, B. Janowski, J. Pielichowski, Polyhedral oligomeric silsesquioxanes (POSS)-containing nanohybrid polymers, Supramolecular Polymers Polymeric Betains Oligomers, Springer Berlin Heidelberg, Berlin, Heidelberg, 2006. [137] J.A. Sowjanya, J. Singh, T. Mohita, S. Sarvanan, A. Moorthi, N. Srinivasan, et al., Biocomposite scaffolds containing chitosan/alginate/nano-silica for bone tissue engineering, Colloid. Surf. B 109 (2013) 294
300. [138] H.-Y. Zhu, R. Jiang, L. Xiao, Adsorption of an anionic azo dye by chitosan/kaolin/ γ-Fe2O3 composites, Appl. Clay Sci. 48 (2010) 522
526. [139] D.-W. Cho, B.-H. Jeon, C.-M. Chon, Y. Kim, F.W. Schwartz, E.-S. Lee, et al., A novel chitosan/clay/magnetite composite for adsorption of Cu(II) and As(V), Chem. Eng. J. 200-202 (2012) 654
662. [140] J. Maity, S.K. Ray, Chitosan based nano composite adsorbent—Synthesis, characterization and application for adsorption of binary mixtures of Pb(II) and Cd(II) from water, Carbohydr. Polym. 182 (2018) 159
171. [141] H. Moussout, H. Ahlafi, M. Aazza, C. El Akili, Performances of local chitosan and its nanocomposite 5%Bentonite/chitosan in the removal of chromium ions (Cr(VI)) from wastewater, Int. J. Biol. Macromol. 108 (2018) 1063
1073. [142] Y. Zhou, S. Fu, L. Zhang, H. Zhan, M.V. Levit, Use of carboxylated cellulose nanofibrils-filled magnetic chitosan hydrogel beads as adsorbents for Pb(II), Carbohydr. Polym. 101 (2014) 75
82. [143] E.M.S. Azzam, G. Eshaq, A.M. Rabie, A.A. Bakr, A.A. Abd-Elaal, A.E. El Metwally, et al., Preparation and characterization of chitosan-clay nanocomposites for the removal of Cu(II) from aqueous solution, Int. J. Biol. Macromol. 89 (2016) 507
517. [144] Z. Karim, A.P. Mathew, M. Grahn, J. Mouzon, K. Oksman, Nanoporous membranes with cellulose nanocrystals as functional entity in chitosan: removal of dyes from water, Carbohydr. Polym. 112 (2014) 668
676. [145] H.Y. Zhu, Y.Q. Fu, R. Jiang, J. Yao, L. Xiao, G.M. Zeng, Novel magnetic chitosan/ poly(vinyl alcohol) hydrogel beads: preparation, characterization and application for adsorption of dye from aqueous solution, Bioresour. Technol. 105 (2012) 24
30. [146] H. Ueno, T. Mori, T. Fujinaga, Topical formulations and wound healing applications of chitosan, Adv. Drug. Deliv. Rev. 52 (2001) 105
115. [147] Y. Shigemasa, S. Minami, Applications of chitin and chitosan for biomaterials, Biotechnol. Genet. Eng. Rev. 13 (1996) 383
420. [148] C.J. Park, S.G. Clark, C.A. Lichtensteiger, R.D. Jamison, A.J.W. Johnson, Accelerated wound closure of pressure ulcers in aged mice by chitosan scaffolds with and without bFGF, Acta Biomater. 5 (2009) 1926
1936. [149] I.R. Sweeney, M. Miraftab, G. Collyer, Absorbent alginate fibres modified with hydrolysed chitosan for wound care dressings—II. Pilot scale development, Carbohydr. Polym. 102 (2014) 920
927. [150] J.-P. Chen, C.-Y. Kuo, W.-L. Lee, Thermo-responsive wound dressings by grafting chitosan and poly(N-isopropylacrylamide) to plasma-induced graft polymerization modified non-woven fabrics, Appl. Surf. Sci. 262 (2012) 95
101.

Handbook of Chitin and Chitosan

References

41

[151] S.C.M. Fernandes, C.S.R. Freire, A.J.D. Silvestre, C. Pascoal Neto, A. Gandini, L.A. Berglund, et al., Transparent chitosan films reinforced with a high content of nanofibrillated cellulose, Carbohydr. Polym. 81 (2010) 394
401. [152] S. Ifuku, A. Ikuta, M. Egusa, H. Kaminaka, H. Izawa, M. Morimoto, et al., Preparation of high-strength transparent chitosan film reinforced with surfacedeacetylated chitin nanofibers, Carbohydr. Polym. 98 (2013) 1198
1202. [153] I. Cacciotti, Cationic and anionic substitutions in hydroxyapatite, in: I.V. Antoniac (Ed.), Handbook of Bioceramics and Biocomposites, Springer International Publishing, Cham, 2014. [154] H. Seeherman, R. Li, J. Wozney, A review of preclinical program development for evaluating injectable carriers for osteogenic factors, JBJS 85 (2003) 96
108. [155] T.A. Becker, D.R. Kipke, T. Brandon, Calcium alginate gel: a biocompatible and mechanically stable polymer for endovascular embolization, J. Biomed. Mater. Res. 54 (2001) 76
86. [156] Z. Li, H.R. Ramay, K.D. Hauch, D. Xiao, M. Zhang, Chitosan
alginate hybrid scaffolds for bone tissue engineering, Biomaterials 26 (2005) 3919
3928. [157] P. Gentile, V.K. Nandagiri, J. Daly, V. Chiono, C. Mattu, C. Tonda-Turo, et al., Localised controlled release of simvastatin from porous chitosan
gelatin scaffolds engrafted with simvastatin loaded PLGA-microparticles for bone tissue engineering application, Mater. Sci. Eng. C. 59 (2016) 249
257. [158] R. Niranjan, C. Koushik, S. Saravanan, A. Moorthi, M. Vairamani, N. Selvamurugan, A novel injectable temperature-sensitive zinc doped chitosan/β-glycerophosphate hydrogel for bone tissue engineering, Int. J. Biol. Macromol. 54 (2013) 24
29. [159] A. Olad, F. Farshi Azhar, The synergetic effect of bioactive ceramic and nanoclay on the properties of chitosan
gelatin/nanohydroxyapatite
montmorillonite scaffold for bone tissue engineering, Ceram. Int. 40 (2014) 10061
10072. [160] S. Saravanan, S. Nethala, S. Pattnaik, A. Tripathi, A. Moorthi, N. Selvamurugan, Preparation, characterization and antimicrobial activity of a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver for bone tissue engineering, Int. J. Biol. Macromol. 49 (2011) 188
193. [161] J.H. Park, G. Saravanakumar, K. Kim, I.C. Kwon, Targeted delivery of low molecular drugs using chitosan and its derivatives, Adv. Drug. Deliv. Rev. 62 (2010) 28
41. [162] N. Bhattarai, J. Gunn, M. Zhang, Chitosan-based hydrogels for controlled, localized drug delivery, Adv. Drug. Deliv. Rev. 62 (2010) 83
99. [163] S.S. Vaghani, M.M. Patel, C.S. Satish, Synthesis and characterization of pH-sensitive hydrogel composed of carboxymethyl chitosan for colon targeted delivery of ornidazole, Carbohydr. Res. 347 (2012) 76
82. [164] C. Feng, Z. Wang, C. Jiang, M. Kong, X. Zhou, Y. Li, et al., Chitosan/o-carboxymethyl chitosan nanoparticles for efficient and safe oral anticancer drug delivery: in vitro and in vivo evaluation, Int. J. Pharm. 457 (2013) 158
167.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

2 Chemically modified chitin, chitosan, and chitinous polymers as biomaterials Ineˆs Farinha1 and Filomena Freitas2 1

73100 Lda., Edifı´cio Arcis, Rua Ivone Silva, 6, 4 piso, Lisboa, Portugal, UCIBIO-REQUIMTE, Chemistry Department, Faculty of Sciences and Technology, Universidade NOVA de Lisboa, Campus da Caparica, Caparica, Portugal

2

O U T L I N E 2.1 Introduction

44

2.2 Chemically modified chitin 2.2.1 Carboxymethyl chitin and other chitin alkylated derivatives 2.2.2 Phosphorylated chitin 2.2.3 Sulfated chitin 2.2.4 Chitin graft copolymerization 2.2.5 Other chitin derivatives

46 46 48 49 49 50

2.3 Chemically modified chitosan 2.3.1 Carboxymethyl chitosan and other chitosan alkylated derivatives 2.3.2 Phosphorylated chitosan 2.3.3 Sulfated chitosan 2.3.4 Acyl-chitosan 2.3.5 Quaternary chitosan derivatives 2.3.6 Graft copolymerization of chitosan 2.3.7 Other chitosan derivatives

53 55 56 57 58 59 59 60

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00002-9

43

© 2020 Elsevier Inc. All rights reserved.

44

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

2.4 Chemically modified chitinous polysaccharides

62

2.5 Conclusions

63

References

64

2.1 Introduction Chitin is the second most abundant polysaccharide available in nature, after cellulose. Composed of N-acetyl-D-glucosamine and D-glucosamine units (Fig. 2.1), this polymer can be obtained from different sources, such as animals (crustaceous, insects, and mollusks), microorganisms (yeasts and filamentous fungi), or algae [2
4]. This crystalline polysaccharide, with three known polymorphisms (α-, β-, and γ- forms), hydrophobic and with a molecular weight of 1
2 3 105 Da [2,4
6], is attracting increasing attention, due to its low toxicity, biocompatibility, biodegradability, and biological activity [4,7,8]. However, due to its rigid structure, this biopolymer is insoluble in water and in most organic solvents. The most common solvents used for chitin solubilization are the solvent system dimethylacetamide (DMAc)/LiCl, solutions of LiOH or NaOH with or without urea, and more recently, ionic liquids [9
11]. Chitosan is the most common chitin derivative (Fig. 2.1). Obtained thought the deacetylation of the chitin macromolecule, by subjecting it to hot alkali (e.g., NaOH concentrated solutions) treatment, chitosan is characterized by a low acetylation degree, usually below 50% and molecular weight between 50 and 2000 kDa [4,8,9,12]. In contrast to chitin, chitosan is soluble in dilute solutions (usually 0.1
2% v/v) of acetic acid, hydrochloric acid, or citric acid [2,4,8
11]. However, chitosan shows limited solubility at pH above its pKa, 5.5
6.5 [12]. Fungal chitin and chitosan can also be found in the form of complexes, in which chitin or chitosan macromolecules are covalently linked to β-glucans [13,14]. They are extracted from yeasts and fungi cell walls by mechanical, chemical, and/or enzymatic methods [15]. Depending on the fungal source and the extraction methodology used, a chitin
glucan complex (CGC) or chitosan
glucan complex (ChGC) can be obtained with different chitin/chitosan:glucan ratios [16
19]. Chitin, chitosan, and their complexes have diverse properties that drive an increasing interest in their use for the development of hydrogels, nanofibers, micro/nanoparticles, scaffolds, or tablets [8,17,20
26]. For example, chitinous polysaccharides have fat and

Handbook of Chitin and Chitosan

2.1 Introduction

45

FIGURE 2.1 Deacetylation reaction of chitin to chitosan. Source: Based on F. Croisier, C. Je´roˆme, Chitosan-based biomaterials for tissue engineering, Eur. Polym. J. 49 (2013) 780
792.

cholesterol absorption capacities [27], can be used as coatings due to their wound-healing and antimicrobial activity [16,28
31], have the ability to remove metal ions from wastewaters [32
34], or may have electrolytic properties [35,36]. All of these characteristics make chitin and chitosan valuable biomaterials for several areas that include biomedicine [1,37
39], pharmaceutical products [17,40], food products [41], cosmetic formulations [42], agriculture [43,44], wastewater treatment [33,45], or energy [46]. However, the low solubility capacity of these polysaccharides is a drawback for their use in most of these application areas. Therefore the chemical modification of chitin and chitosan-based polysaccharides has been considered as an alternative. Besides enabling the improvement of chitinous polysaccharides solubility properties, they bring new advanced functional properties to these biopolymers [3,4,9,47]. In this section, a perspective of the most common chemical modifications that can be applied on chitin/chitosan and chitinous polysaccharides’ structures is given, as well as an insight into their derivatives’ functionalities and their use as new biomaterials.

Handbook of Chitin and Chitosan

46

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

2.2 Chemically modified chitin The development of chemically modified chitin biomaterials has attracted much attention nowadays, since it enables the use of novel compounds, with enhanced properties, especially related to the polymer’s improved solubility. The most common chitin derivatives reactions include carboxymethylation, phosphorylation, sulfation, acetylation, and graft copolymerization. In the chemical structure of chitin, the three regions more susceptible to interactions with functional groups of other compounds are the amino group in C2 and the hydroxyl groups in C3 and C6 (Fig. 2.2). The amino functional group can be modified through different reactions, such as acetylation, quaternization, alkylation, or graft reactions [9], to obtain chitin derivatives with distinctive properties and biological functions, including solubility in water. Modification of the two hydroxyl groups in C3 and C6 also contributes to the enhancement of the polymer’s solubility and allows participation in some chemical reactions such as O-acetylation, H-bonding with polar atoms, or grafting [9]. Some of these reactions involve the disruption of inter- and intramolecular hydrogen bonds, without cleaving glycosidic linkages, which is effective in making chitin soluble in water and in some other solvents [48]. Significant advances have been made regarding chitin’s chemical modification that have resulted in a wide range of structures. Moreover, chitosan, the most common chitin derivative, also has been the subject of several chemical modifications, as will be shown later on this chapter (see Section 2.3, Chemically Modified Chitosan). In Table 2.1, some examples of chitin derivatives are shown, along with their properties and applications.

2.2.1 Carboxymethyl chitin and other chitin alkylated derivatives One of the most attractive and used chitin derivatives is carboxymethyl chitin (CM-chitin). Chitin carboxymethylation involves a condensation

FIGURE 2.2 Chitin reaction sites for possible chemical modifications (marked in bold). Source: Based on C.K.S. Pillai, W. Paul, C.P. Sharma, Chitin and chitosan polymers: chemistry, solubility and fiber formation, Prog. Polym. Sci. 34 (2009) 641
678.

Handbook of Chitin and Chitosan

47

2.2 Chemically modified chitin

TABLE 2.1

Some chitin derivatives properties and applications.

Chitin derivative

Properties

Applications

Refs.

CM-chitin

Protects wound tissues from pathogens infections and enhance the growth of cells

Wound dressing

[39,48,49]

Hydrogel with pH-sensitive swelling behavior

Biomedicine devices

[50,51]

Controlled and sustained drug delivery at neutral conditions

Carrier for drug delivery applications

[52]

P-chitin

Metal chelating ability and Cu21 corrosion inhibition

Protective films onto metal surface, wastewater treatment

[53]

S-chitin

Anticoagulant, antisclerotic, antiviral, and lipoprotein lipase-releasing activities

Pharmaceutical applications

[54]

Chitin
acrylonitrile graft

Arsenic absorption capacity

Groundwater treatment

[55]

T-chitin

Antioxidant, anticholesterol, and bile acid binding capacity

Food supplement and pharma ingredient

[56]

reaction with monochloroacetic acid in isopropanol performed at 37 C, with a pretreatment with sodium hydroxide at 220 C (Fig. 2.3) [57]. The introduction of the carboxyl group into chitin’s structure leads to the formation of anionic derivatives, that can be useful in pharmaceutical, biomedical, or environmental applications. CM-chitin is soluble in water, biodegradable, biocompatible, and shows low toxicity [49,57]. Hydrogels of CM-chitin with good mechanical properties can be prepared by radiation cross-linking [50]. These

FIGURE 2.3 Reaction scheme of the CM-chitin synthesis. Source: Based on R. Jayakumar, M. Prabaharan, S.V. Nair, S. Tokura, H. Tamura, N. Selvamurugan, Novel carboxymethyl derivatives of chitin and chitosan materials and their biomedical applications, Prog. Mater. Sci. 55 (2010) 675
709.

Handbook of Chitin and Chitosan

48

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

hydrogels revealed a pH-sensitive swelling behavior, showing typical electrolyte properties under different ionic strengths, which are important characteristics for biomedical applications (Table 2.1) [50,51]. CM-chitin also presents antioxidant, antibacterial, and antitumoral activities [49,52,58]. This biopolymer can be used as a carrier for subcutaneous injections and for antitumoral drug delivery systems, and protects wound tissues against pathogens infections [48,49,52]. Moreover, CM-chitin also enhances cells’ growth and activates macrophages, important features for wound-healing dressings [39,48]. Beyond CM-chitin, it is also possible to produce other chitin derivates from alkylation reactions. For example, the production of hydroxyethyl chitin was reported by treatment of chitin with ethylene oxide, under alkaline conditions [3]. Water-soluble hydroxypropyl chitin and diethylaminoethyl chitin were also already obtained, with the latter presenting swelling properties even with nonpolar solvents (such as benzene) [3].

2.2.2 Phosphorylated chitin Phosphorylated chitin (P-chitin), another water-soluble chitin derivative, can be prepared by several methodologies. The most common phosphorylation reaction involves heat treatment of chitin with orthophosphoric acid and urea in dimethylformamide (DMF) (Fig. 2.4) [59]. Another way to obtain P-chitin is by bringing chitin into contact with phosphorous pentoxide in methane sulfonic acid (Fig. 2.4) or by chitin treatment with a mixture of orthophosphoric acid/triethyl phosphate/ phosphorous pentoxide [59]. Independently of the degree of substitution, P-chitin is soluble in water and exhibits biocompatible, antibacterial, and osteoinductive properties [59,60]. This chitin derivative also shows polyelectrolyte behavior and high metal absorption capacity [53,60]. The chelating capacity of P-chitin to copper ions (Cu21) was also reported, as well as

FIGURE 2.4 Reaction scheme of the P-chitin synthesis. Source: Based on R. Jayakumar, N. Selvamurugan, S.V. Nair, S. Tokura, H. Tamura, Preparative methods of phosphorylated chitin and chitosan-an overview, Int. J. Biol. Macromol. 43 (2008) 221
225.

Handbook of Chitin and Chitosan

2.2 Chemically modified chitin

49

the use of this biopolymer for corrosion inhibition of copper in aqueous chloride environments (Table 2.1) [53].

2.2.3 Sulfated chitin Sulfated chitin (S-chitin) is usually prepared by using sulfuric acid, tetrahydrofuran, and phosphorus pentose, at 220 C (Fig. 2.5) [54]. However, some attention must be made to polymer degradation due to the use of concentrated sulfuric acid, showed by its molecular weight reduction and low reaction yield [54]. S-chitin is an interesting material for pharmaceutical applications since this polymer is a structural analogue of heparin, a natural blood anticoagulant. Due to this structural similarity, S-chitin has anticoagulant, antisclerotic, and antiviral activities, and also reveals lipoprotein lipase-releasing activity. Moreover, this chitin derivative has antitumor activity and can be used in other application fields due to its metal chelating capacity [54,59].

2.2.4 Chitin graft copolymerization Graft copolymerization is a derivatization procedure commonly used to introduce polymeric side chains into chitin molecules, resulting in novel types of hybrid materials composed of natural polysaccharides and synthetic polymers [5]. This kind of chitin derivatization can be performed by using different methodologies, promoting the formation of graft chains by the generation of free radicals or free electrons. The reported studies include different grafting strategies: ion initiation with cerium (IV), the use of Fenton’s reagent, gamma radiation, and photoinduced methods [61]. In the ion initiation procedure, the use of cerium in its tetravalent state as an oxidizing agent creates free radicals through redox reactions, being usually used to initiate vinyl polymerizations [61]. The use of Fenton’s reagent involves the occurrence of redox reactions

FIGURE 2.5 Reaction scheme of the S-chitin synthesis. Source: Based on R. Jayakumar, N. Nwe, S. Tokura, H. Tamura, Sulfated chitin and chitosan as novel biomaterials, Int. J. Biol. Macromol. 40 (2007) 175
181.

Handbook of Chitin and Chitosan

50

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

between a mixture of potassium persulfate and ferrous ammonium sulfate with hydrogen peroxide, to produce hydroxyl radicals [61]. These radicals can also be obtained by gamma radiation, a high-energy procedure, where cation radicals are formed by electron abstraction, in an unselective way [61]. The graft radicals can also be formed by photoinduced methods (ultraviolet radiation) or by using other organic compounds, such as 2-azobisizobutironitril (AIBN), ammonium peroxy disulfate (APS), methyl methacrylate, methyl acrylate, or polypyrrole, as initiators [61,62]. Vinyl monomers are also commonly used for chitin grafting, such as acrylamide, acrylonitrile, and acrylic acid [5]. Chitin derivatives, such as tosyl-chitin, 6-mecarpto-chitin, or O-acetyl chitin [5], have also been reported. Cankaya [55] showed the production of chitin methacrylate from the chitin esterification with methacryloyl chloride (Fig. 2.6) and its free radical graft reaction with monomers, 1-vinylimidazole (VIM), methacrylamide (MAm), and 2-acrylamido-2methyl-1-propanesulfonic acid (AMPS). Chitin graft copolymerization has been revealed to be very useful to improve chitin’s solubility, especially in water, and also contributes to enhance its swelling properties and metals’ absorption capacity [5,63]. For example, Hanh et al. [63] reported an arsenic absorption capacity of 19.724 mg/g with chitin grafted with acrylonitrile for groundwater samples (Table 2.1).

2.2.5 Other chitin derivatives Besides the chitin derivatives discussed above, many other chitinderived compounds were also created and tested in several application areas. Some of these derivatives involve acylation, acetylation, tosylation, or silylation reactions. The acylation reaction of chitin is usually performed with acid chlorides, such as p-nitrobenzoic acid (NBAC) or myristic acid (MAC), in the presence of dimethylacetamide/LiCl and pyridine (Fig. 2.7) [64]. This reaction occurs between the chitin hydroxyl groups and the acyl-halides yielding to ester derivatives. Generally, these derivatives are more polar than chitin and their solubility is improved depending on the hydroxyl

FIGURE 2.6 Reaction scheme of the chitin methacrylate synthesis. Source: Based on N. Cankaya, Grafting studies of chitin, Sigma J. Eng. Nat. Sci. 37 (1) (2019) 111
117.

Handbook of Chitin and Chitosan

2.2 Chemically modified chitin

51

FIGURE 2.7 Reaction scheme of acyl-chitin synthesis, where R is
p-O2N-C6H4
or CH3(CH2)12
. Source: Based on V.A. Vasnev, A.I. Tarasov, G.D. Markova, S.V. Vinogradova, O.G. Garkusha, Synthesis and properties of acylated chitin and chitosan derivatives, Carbohydr. Polym. 64 (2006) 184
189.

groups substituted [48]. For example, butyryl-chitin does not swell in water but is soluble in acetone and in alcohols, while longer residues introduced into chitin’s structure resulted in an acetylated chitin with higher solubility in water [48]. Some acetylated chitin derivatives are used for the controlled release of herbicides [5]. Similarly, chitin acetylation also results in ester derivatives. Acetylated chitin is obtained by treating chitin with a mixture of acetic anhydride with perchloric acid, at room temperature, for at least 1 h (Fig. 2.8) [19]. In order to achieve a fully acetylated chitin, this reaction can be performed with other organic solvents, such as hydrogen chloride, or a mixture of trichloroacetic acid with 1,2-dichloroethane [5]. This chitin derivative can be used as a nanofiber and its modification using hydrophobic groups contributes to improve its hygroscopicity and adhesion properties with other hydrophobic matrices [19]. Beyond carboxymethylation, other methodologies are available to introduce carboxyl groups into chitin’s structure. TEMPO oxidation is one of them. This oxidation reaction is performed by contacting chitin with 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), sodium bromide, and sodium hypochlorite (Fig. 2.9) [65]. This new derivative, here designated as T-chitin, is soluble in water at several pH values, has a gel-forming capacity and a metal absorption ability [56,65,66]. For

FIGURE 2.8 Reaction scheme of acetylated chitin synthesis. Source: Based on S. Ifuku, Chitin and chitosan nanofibers: preparation and chemical modifications, Molecules 19 (2014) 18367
18380.

Handbook of Chitin and Chitosan

52

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

FIGURE 2.9 Reaction scheme of T-chitin synthesis. Source: Based on J. Huang, W.-W. Chen, S. Hu, J.-Y. Gong, H.-W. Lai, P. Liu, et al., Biochemical activities of 6-carboxy β-chitin derived from squid pens, Carbohydr. Polym. 91 (2013) 191
197.

example, Sun et al. [65] reported a T-chitin Cd(II) absorption capacity of 207.9 mg/g. T-chitin also has biological properties such as antioxidant and anticholesterol activities [56]. This chitin derivative can be used as a cholesterol-lowering adjuvant due to its high bile acid binding capacity (18
41 mg/g) in food supplements or as a pharma ingredient (Table 2.1) [56]. The tosyl (CH3C6H4SO2) group is a very reactive ester and chitin tosylation involves contacting chitin with tosyl chloride in chloroform (Fig. 2.10) [5]. Tosyl-chitin is usually used as a precursor for several other derivatives, namely radical graft reactions, to yield iodo-chitin or mercapto-chitin, for example [5,67]. Some of its properties are substitution degree dependent. For example, tosyl-chitin with a substitution degree lower than 0.3 resulted in a highly hydrophobic polymer that is partially soluble in water, while a tosyl-chitin with a substitution degree higher than 0.4 gave a hydrophobic polymer soluble in common organic solvents [5]. This kind of chitin derivatives can also be used as a support for enzyme immobilization processes [5]. The full substitution of chitin’s structure, by interfering with the strong intermolecular bonding, is another alternative to improve chitin’s solubility. Silylation is a possible reaction to achieve this kind of substitution on hydroxyl groups in C3 and C6 [5]. One of the most well-known silyl-chitin derivatives is trimethylsilylated chitin, produced by contacting chitin with a mixture of hexamethyldisilazane

FIGURE 2.10 Reaction scheme of tosyl-chitin synthesis. Source: Based on K. Kurita, Controlled functionalization of the polysaccharide chitin, Prog. Polym. Sci. 26 (2001) 1921
1971.

Handbook of Chitin and Chitosan

2.3 Chemically modified chitosan

53

and chlorotrimethylsilane in pyridine at 70 C (Fig. 2.11), resulting in a fully substituted polymer [5]. This silyl derivative is highly soluble and has high swelling capacity in organic solvents, with good reactivity. Chitin regeneration is an easy procedure that involves a reaction with acetic acid at room temperature. Trimethylsilylated chitin also shows film-forming ability when cast with acetone [5]. To attain a better control of the derivatization conditions and good regioselectivities under mild conditions, N-phthaloylation of chitin is one of the best derivatization options [5]. N-phthaloylation reaction involves the protection of the chitin amino group (in C2), by reaction of chitin with phthalic anhydride [5,19]. This procedure is commonly used to produce chitin nanofibers that have thermoresponsive properties and are dispersible in water and organic solvents. Due to their thermoresponsive properties, N-phthaloyl chitin films also exhibit UV-A, B, and C light adsorption capacity [19].

2.3 Chemically modified chitosan Chitosan, the most well-known chitin derivative, presents similar solubility limitations as chitin despite being soluble in diluted acid solutions. Beyond the polymer’s solubility, chitosan’s properties also depend on the deacetylation degree, exhibiting some limitations in reactivity and processability [54,59]. Similar to chitin, chitosan chemical derivatization is a valuable alternative to improve chitosan’s physicochemical properties. Most of the chitin derivatization procedures were already tested for chitosan, such as carboxymethylation, phosphorylation, sulfation, or graft copolymerization. Moreover, some chitin derivatizations include a deacetylation step to obtain chitosan in a first process step and, later, a second step of chitosan derivatization is performed. Table 2.2 presents some examples of chitosan derivatives, their properties, and their applications.

FIGURE 2.11 Reaction scheme of silyl-chitin (trimethylsilylated chitin) synthesis. Source: Based on K. Kurita, Controlled functionalization of the polysaccharide chitin, Prog. Polym. Sci. 26 (2001) 1921
1971.

Handbook of Chitin and Chitosan

54

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

TABLE 2.2 Some chitosan derivatives properties and applications. Chitosan derivative

Properties

Applications

Refs.

Cross-linked with gelatin, hydrogel that promotes cell attachment and fibroblasts growth

Wound healing

[68]

Conjugate with rhein, micelles form with low toxicity and antitumor activity

Oral delivery of waterinsoluble drugs

[69]

P-chitosan

Polyelectrolyte with controlled drug release capacity

Drug delivery systems (e.g., with ibuprofen) for oral administration

[70,71]

S-chitosan

Mercury and others metal ions recovery capacity

Treatment of contaminated effluents

[54]

Polymeric micelles or microcapsules with polyelectrolyte properties

Carrier for drug delivery systems

[54,70]

Trimethyl chitosan derivatives (quaternization)

Mucosal adsorption enhancer of peptides and small molecules

Gene delivery systems; microspheres for drug loading

[12,70]

PLLA-chitosan (graft copolymerization)

Water swelling capacity, mucoadhesive, and antibacterial properties

Tissue engineering scaffolds and drug carriers

[70]

Chitosan-graftpolyethylenimine

DNA binding activity and protection from nuclease attack

Gene carrier

[72]

PEG-chitosan derivatives

pH-sensitive swelling hydrogels/ nanoparticles with high hydrophobic drug loading capacity

Controlled drug release for cancer therapy

[73
75]

Acetylated chitosan

Water absorption capacity and in vitro degradation by lysosyme

Scaffold for biomedical applications

[76]

Batteries discharge characteristics, but with short lifetimes

Polymer batteries

[77]

Nontoxicity, mucoadhesive properties, and delivery capacity

Ocular or nasal drug delivery systems

[78,79]

Antitumor effect and drug bioavailability

Drug delivery for colon cancer treatment

[80]

Reduce bacterial infection risk and promote the reepithelialization of damage tissue

Membranes for wound dressing

[81]

CM-chitosan

Thiolated chitosan

Sulfonamide chitosan

Handbook of Chitin and Chitosan

2.3 Chemically modified chitosan

55

2.3.1 Carboxymethyl chitosan and other chitosan alkylated derivatives One of the most used chitosan derivatives is carboxymethyl chitosan (CM-chitosan). Chitosan’s carboxymethylation reaction can be performed in amino and/or hydroxyl groups to give N-, O-, or N,O-CM-chitosan [12,57]. For example, N-CM-chitosan can be prepared by using glyoxylic acid, in a reductive alkylation, regioselective for the amino group at C2, while O-CM-chitosan can be prepared using monochloroacetic acid, to obtain an amphiprotic ether derivative [5,12,57]. The simultaneous introduction of carboxymethyl groups into amino and hydroxyl sites of glucosamine units results in the formation of N,O-CM-chitosan. This reaction is performed by contacting chitosan with sodium hydroxide and monochloroacetic acid in isopropanol at 50 C (Fig. 2.12) [57,82]. Bukzem et al. [82] optimized this carboxymethylation process in an experimental design to achieve a substitution degree of 1.86% and 99.6% of polymer solubility in water. Chitosan carboxymethylated derivatives are water soluble, biocompatible, biodegradable, and present antibacterial, antimutagenic, and antioxidant activities [50,57,68,70]. O-CM-chitosan and N,O-CM-chitosan can be used as viscosity-inducing agents due to their rheology properties: Newtonian behavior in acidic conditions and pseudoplastic properties under neutral and alkaline conditions [83]. N,O-CM-chitosan can also be used as an amphoteric polyelectrolyte with anticancer activity and in the preparation of membranes for filtration processes [57]. Moreover, this chitosan derivative showed hemostatic activity by contributing to the decrease of the whole blood clotting time and lowering the plasma recalcification time [84]. Hydrogels of carboxymethylated chitosan can be prepared, with pH-sensitive swelling behavior and adhesive properties [50,70]. All these properties make these chitosan derivatives suitable for use in several application areas, such as pharmaceutical, wound healing, cosmetics, food preservation, tissue engineering, biomedicine, and metal absorption applications [12,57]. Wang et al. [69], for example, developed a CM-chitosan
rhein conjugate with micelles form, to be used as a

FIGURE 2.12 Reaction scheme of N,O-CM-chitosan synthesis. Source: Based on R. Jayakumar, M. Prabaharan, S.V. Nair, S. Tokura, H. Tamura, N. Selvamurugan, Novel carboxymethyl derivatives of chitin and chitosan materials and their biomedical applications, Prog. Mater. Sci. 55 (2010) 675
709.

Handbook of Chitin and Chitosan

56

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

potential drug carrier for oral delivery of water-insoluble antitumor drugs, such as paclitaxel (Table 2.2). CM-chitosan cross-linked with gelatin was used to produce hydrogels with swelling and water absorption capacity that promote cell attachment and a rapid growth of fibroblasts on this polymer, important features for a wound-healing material [68]. CM-chitosan can also be subjected to other derivatization reactions to further improve its physicochemical properties. As an example, the amphiphatic carboxymethyl hexanoyl chitosan was described by Lu et al. [85] to produce injectable in situ hydrogels blended with hyaluronic acid, for the controlled release of berberine, an antiarthritis compound. These gels were cytocompatible, pH-responsive, and prevented the progression of cartilage degradation, and have been revealed to be an interesting hydrogel for cartilage tissue engineering. Other examples of chitosan alkylated derivatives are N,N-dicarboxymethyl chitosan and N-carboxybutyl chitosan. N,N-dicarboxymethyl chitosan is produced by contacting chitosan with glyoxylic acid, sodium borohydride, and acetic acid, resulting in a chitosan derivative that forms transparent and resistant films, with capacity for chelating metal ions [57]. N-carboxybutyl chitosan is prepared with levulinic acid and sodium borohydride and is water soluble, with film-forming capacity and bacteriostatic activity, being an interesting material for wound healing and tissue repair promoters [5,57]. Beyond carboxymethylation, there are other alkylation reactions that can be used to obtain alkylated chitosan. N-alkylation of chitosan can be performed with mono- or disaccharides, such as glucose, fructose, rhamnose, lactose, or maltose [86,87]. Depending on the type of saccharide used, alkylated chitosan can be soluble in water and present antibacterial and antioxidant activities [86,87]. The use of Michael reaction is another way to obtain an alkylated chitosan, using acrylamides as N,N-dimethylacrylamide or N-isopropylacrylamide [88]. The resulting chitosan derivatives have antimicrobial activity, low crystallinity, and different water solubility levels [88].

2.3.2 Phosphorylated chitosan Similar to P-chitin, phosphorylated chitosan (P-chitosan) results from the reaction of chitosan with ortophosphoric acid or phosphorous pentoxide. P-chitosan can be prepared by heating chitosan with ortophosphoric acid and urea in DMF (Fig. 2.13), or by contacting chitosan with phosphorous pentoxide in methane sulfonic acid [59,71]. Grafting methods can also be applied in chitosan phosphorylation, by using mono(2-methacryloyl oxyethyl) acid phosphate, for example [59]. Depending on the chemical reaction used, chitosan can be mono- or disubstituted [70].

Handbook of Chitin and Chitosan

2.3 Chemically modified chitosan

57

FIGURE 2.13 Reaction scheme of P-chitosan synthesis. Source: Based on R. Jayakumar, N. Selvamurugan, S.V. Nair, S. Tokura, H. Tamura, Preparative methods of phosphorylated chitin and chitosan-an overview, Int. J. Biol. Macromol. 43 (2008) 221
225.

P-chitosan is water soluble in neutral conditions and has improved antimicrobial activity compared with chitosan [59,71]. This chitosan derivative can be used as a basis for new polyelectrolyte complexes developed for controlled drug release for oral administration, since the drug release in the acidic gastric fluid region of stomach is avoided [70,71]. For example, a drug delivery system with P-chitosan and ibuprofen as a model drug was reported by Mourya and Inamdar [70] (Table 2.2). Beyond P-chitosan, other chitosan phosphate derivatives are also found in literature. For example, chitosan-O-ethyl phosphonate is prepared using a mixture of potassium hydroxide with methanol and 2-chloro ethylphosphoric acid, under mild conditions [59]. N-methylene phosphonic chitosan is prepared using phosphoric acid and formaldehyde and results in a polymer with improved water solubility, without comprising its film-forming capacity and properties [59].

2.3.3 Sulfated chitosan Several methodologies can be used to obtain sulfated chitosan (S-chitosan), including the use of reagents such as sulfuric acid or chlorosulfonic acid, in homogeneous or heterogeneous conditions, in different reaction media that include DMF, tetrahydrofuran, or formic acid, or even under microwave radiation (Fig. 2.14) [70]. Depending on the

FIGURE 2.14

Reaction scheme of a N,O-S-chitosan synthesis. Source: Based on V.K. Mourya, N.N. Inamdar, Chitosan-modifications and applications: opportunities galore, React. Funct. Polym. 68 (2008) 1013
1051.

Handbook of Chitin and Chitosan

58

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

sulfation reaction conditions, S-chitin can be mono-, di-, or trisubstituted, being often N,O-disubstituted [54,70]. S-chitosan is an important type of chitosan derivative, with several reported biological activities. It has film-forming capacity with antithrombogenic properties, anticoagulant activity similar to heparin, as well as antiviral, antimicrobial, antioxidant, and enzyme inhibition activities [49,54,70]. Water-insoluble anticancer drugs, like Taxol, can be solubilized in S-chitosan micelles, demonstrating the ability of this polymer to be used as a drug carrier or polyelectrolyte in drug delivery systems (Table 2.2) [54,70]. Moreover, S-chitosan presents high metal absorption capacity, which is useful for metal ion recovery applications (Table 2.2) [54,70]. Similar to P-chitosan, chitosan sulfated derivatives can also be prepared from other chitosan derivatives. For example, a sulfated O-CM-chitosan is prepared from CM-chitosan [54]. Other sulfated chitosan derivatives include N-sulfofurfuryl chitosan or O-sulfated N-acetyl chitosan, among many others reported in the literature [54].

2.3.4 Acyl-chitosan Acylated chitosan can be obtained through different chemical reactions. The most common acylation procedure involves the reaction of chitosan with acyl chlorides and anhydrides [70]. Chitosan can be acylated with hexanoyl or decanoyl chlorides in pyridine/chloroform or in methane sulfonic acid, resulting in N,O-acyl chitosan (Fig. 2.15) [5,70]. Other procedures for chitosan acylation are the reaction with p-nitrobenzoic acid, with hydrochloric acid or myristic acid in an acetone
water system, for example [64]. The N-acylation of chitosan with anhydride acetic results in the regeneration of chitin [61]. Chitosan acylation results in the production of amorphous polymers, with chelation, flocculation, and biological functions [64,84]. O-acyl chitosan was designed for use as a biodegradable coating material, while N, O-acyl chitosan exhibits antifungal activity, depending on the acyl chain length [70,89]. The introduction of hydrophobic branches in the chitosan

FIGURE 2.15 Reaction scheme of O-acyl-chitosan synthesis. Source: Based on V.K. Mourya, N.N. Inamdar, Chitosan-modifications and applications: opportunities galore, React. Funct. Polym. 68 (2008) 1013
1051.

Handbook of Chitin and Chitosan

2.3 Chemically modified chitosan

59

macromolecule results in the formation of polymeric assemblies, including gels, liquid crystals, membranes, and fibers [70]. On the other hand, the introduction of aromatic rings results in a hydrophobic polymer, which is useful to coat silica gels or as a packing material for chromatography to separate racemic mixtures (e.g., amino acids) [5].

2.3.5 Quaternary chitosan derivatives Quaternary ammonium salts are another group of chitosan derivative. Different quaternization degrees of amino groups in chitosan can be achieved with methyl iodide in an alkaline solution with N-methylpyrrolidinone (NMP) as a first step reaction (Fig. 2.16) [70]. Then, the quaternization reaction proceeds with the use of other reagents, such as chloroacetyl chloride in dimethysulfoxide and the use of pyridine or amino-pyridine, in a third step reaction, as reported by Li et al. [90]. These quaternary chitosan derivatives are soluble in neutral and slightly alkaline solutions, showing antioxidant, antifungal, and low toxicity activities [12,90,91]. Moreover, they present improved absorption and mucoadhesive properties, compared to chitosan, making these polymers suitable for use in gene and drug delivery systems (Table 2.2) [12,48,70].

2.3.6 Graft copolymerization of chitosan Graft copolymerization is an attractive method to improve the physicochemical properties of chitosan. The properties of the resulting graft copolymers are broadly controlled by the characteristics of the side chains, including molecular structure, length, and number [70,71]. Chitosan grafting allows the formation of functional derivatives by covalent linkage of the graft into chitosan’s structure [71]. Similar to chitin, chitosan grating can be performed using different methodologies: free radical initiation, using radiation (gamma-radiation, UV light, or microwave), or by enzymatic methods [61,71]. The free radical method is one of the most common graft reactions used for chitosan derivatization. It involves grafting of vinyl

FIGURE 2.16 Reaction scheme of the first step of a chitosan quartenization (trimethyl chitosan synthesis) Source: Based on V.K. Mourya, N.N. Inamdar, Chitosan-modifications and applications: opportunities galore, React. Funct. Polym. 68 (2008) 1013
1051.

Handbook of Chitin and Chitosan

60

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

monomers into chitosan using free radical initiation [71]. This reaction is typically performed with 2,2’-azobisiobutyronitrile, cerium (IV), or by a redox system with Fenton’s reagent, which involves a redox reaction between ferrous ion and hydrogen peroxide, to produce hydroxyl radicals [5,61,71]. Several types of monomers are used as initiators, such as vinyl monomers (e.g., acrylonitrile, methyl methacrylate, methyl acrylate) or poly(ethylene glycol) and polyanilide [5]. For example, methyl acrylate and methyl methacrylate were grafted to chitosan with yields of 250%
300% and 400%
500%, respectively, on chitosan basis [61]. Graft copolymerization of chitosan after a primary derivatization was also reported, including CM-chitosan, N-carboxyethyl chitosan, or maleoyl chitosan [70]. Chitosan grafts may form hydrogels sensitive to pH, temperature, or both, with antibacterial and antioxidant properties [70,71]. These stimuli responsive hydrogels can be prepared by blending chitosan with synthetic thermoresponsive materials by several mechanisms of chemical cross-linking, using glutaraldehyde [71]. Graft chitosan with improved flocculation and swelling capacities, mucoadhesive properties, and water solubility was also reported [5,70,71]. In general, graft chitosan may be used in several application areas, such as drug delivery systems, tissue engineering, as an antibacterial agent for wound healing, or metal ions absorption [71]. For example, PLLA-chitosan hybrid scaffolds have been already used as tissue engineering scaffolds and drug carriers (Table 2.2) [71]. Jiang et al. [72] reported the use of chitosan-graft-polyethylenimine in gene delivery systems (Table 2.2). Polyaniline was successfully grafted onto chitosan for copper removal from aqueous solutions [92]. One of the most known chitosan graft copolymerizations is PEGylation. PEG
chitosan is produced by a two-step process, starting with PEG functionalization, by amino-group substitution, O-substitution, or graft copolymerization [12]. From chitosan PEGylation several derivatives can be obtained, such as PEG-aldehyde, PEG-carboxymethyl, PEGcarbonate, or PEG-iodide [12]. These copolymers present low toxicity, biocompatibility, biodegradability, and improved solubility in water and some organic solvents (e.g., dimethylsulfoxide, dimethylformamide, acetone) [12]. PEG
chitosan derivatives are being used as drug carrier nanoparticles, gene delivery systems, and in tissue engineering in cell adhesion applications (Table 2.2) [12,73
75].

2.3.7 Other chitosan derivatives Besides the chitosan derivatives described, many other have been already developed and used in several application areas. Acetylated, thiolated, or sulfonamide chitosan are included among other derivatives.

Handbook of Chitin and Chitosan

2.3 Chemically modified chitosan

61

Acetylation of chitosan is performed by contacting chitosan with acetyl chloride or anhydride acetic with methane sulfonic acid (Fig. 2.17) [93]. This reaction can be controlled by the amount of acid chlorides resulting in derivatives with various substitution degrees [5]. This chitosan derivative has film-forming capacity and is soluble in water, especially in the case of lower-molecular-weight structures [93,94]. The substitution degree also affects the absorption capacity of Cu(II), increasing in case of low substitution (higher hydrophilicity) [5]. Acetylated chitosan can be used in several areas, including biomedicine, packaging, the food industry, or even in batteries [76,77,94]. For example, Han et al. [76] developed a N-acetylated chitosan scaffold for biomedical applications (Table 2.2). Acetylated chitosan complexed in lithium nitrate was already used for polymer batteries development. However, its short lifetime and the small discharge currents obtained limit this polymer’s use in the batteries field (Table 2.2) [77]. The derivatization of the chitosan primary amino group with coupling reagents bearing a thiol function leads to the formation of thiolated chitosan derivatives (Fig.2.18) [71]. So far, four types of thiolated derivatives have been generated: chitosan
cysteine, chitosan
thioglycolic acid, chitosan
4-thiobutylamidine, and chitosan
thioethylamidine conjugates [71]. The protection of the thiol group enables the improvement of several polymer properties, such as mucoadhesive and gelling properties, permeation enhancement, biodegradability, and enzyme inhibition properties [71,95,96]. These properties make thiolated chitosan an important material for biomedical applications, such as drug delivery systems [95,96]. Thiolated chitosan nanoparticles were successfully used in ocular and nasal drug delivery systems [78,79]. Moreover, this chitosan derivative can be combined with curcumin or 5-fluorouracil (antitumor drugs) to develop antitumor nanoparticles for chemotherapeutic colon cancer treatments [80]. Sulfonamide and sulfonated chitosan were recently developed as new chitosan derivatives materials. Sulfonamide can be produced in a two-step reaction: (1) N-chloroacetyl sulfonamides production, and (2) sulfonamides chitosan derivatives [81]. Sulfonated chitosan is

FIGURE 2.17 Reaction scheme of O-acetyl-chitosan. Source: Based on H. Sashiwa, N. Kawasaki, A. Nakayama, E. Muraki, N. Yamamoto, S.-I. Aiba, Chemical modification of chitosan. 14:1 synthesis of water-soluble chitosan derivatives by simple acetylation, Biomacromolecules 3 (2002) 1126
1128.

Handbook of Chitin and Chitosan

62

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

FIGURE 2.18 Reaction scheme of a thiolated chitosan. Source: Based on N.M. Alves, J.F. Mano, Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications, Int. J. Biol. Macromol. 43 (2008) 401
414

produced by contacting chitosan with phenacyl bromide and sodium benzene sulfinate [97]. These chitosan derivatives are soluble in water, biocompatible and biodegradable, have antimicrobial and swelling properties, and metal ions absorption capacity [81,97
99]. For example, sulfone chitosan was used as a mercury(II) adsorbent, with an adsorption capacity of 122.47 mg/g [97]. Liu et al. [98] reported the use of sulfonated chitosan as an alternative for the control of bacterial pathogens in agriculture and food industry since it inhibits bacterial adhesion, biofilm formation, and metabolic activity of some bacterial species. Sulfonamide chitosan was already used as wound-dressing polymers, reducing bacterial infection risk and promoting the reepithelialization of tissue damage (Table 2.2) [81].

2.4 Chemically modified chitinous polysaccharides Besides chitin and chitosan, chitin/chitosan complexes of microbial origin were also subject to derivative reactions. These derivatizations were performed due to their properties’ similarities, especially regarding the solubilization capacity in water and organic solvents. Some of these chitinous polysaccharides derivatives include carboxymethylation, sulfoethylation, or conjugation of chitin
glucan complex (CGC)
aldehyde with quercetin. CGC extracted from Aspergillus niger cell-wall can be carboxymethylated by contacting the fungal biomass with sodium hydroxide 1 M in a hot alkaline treatment and the resulting CGC is treated with monochloroacetic acid in isopropanol [100]. After the carboxymethylation reaction, the CGC derivative shows antimutagenic activity, being an interesting material to prevent mycotic infections and can induce single and double breaks on supercoiled DNA [48,100,101]. Low-molecular-weight carboxymethylated CGC (57 kDa) also has metal absorption capacity, especially for copper ions (0.947 mmol/g), but also for zinc (0.397 mmol/g), nickel (0.0.253 mmol/g), and cobalt ions (0.155 mmol/g) [102]. Another example of an alkylated chitinous polysaccharide derivative is the carboxyethyl chitosan
glucan

Handbook of Chitin and Chitosan

2.5 Conclusions

63

complex (ChGC). This alkylated derivative is obtained by contacting ChGC with acrylic acid or with a mixture with sodium hydroxide, isopropanol, and 3-chloropropionic acid, resulting in a derivative with a substitution degree of 0.06
0.32 [102]. Carboxyethyl ChGC has metal absorption capacity, especially for copper ions (0.210 mmol/g) [102]. Another chitinous polysaccharide derivative is the sulfoethylated CGC. The CGC sulfoethylation can be performed in two ways: by a substitution reaction with haloalkylsulfonates, or by an addition reaction with vinylsulfonate or alkali sultones [103]. An example of a sulfoethylated CGC is O-(2-sulfoethyl) CGC, produced by using a substitution reaction with 2chloroethylsulfonate in isopropyl alcohol, which resulted in a polymer with metal absorption capacity in neutral solutions, especially for copper and chromium ions [102,103]. Sulfonated CGC is also known as a DNA damage inhibitor when induced by free radical oxidation initiated by iron (Fe21) [102]. CGC derivatives can be conjugated with bioactive compounds, as CGC
aldehyde
quercetin. Quercetin is a bioflavonoid with a potential application in a wide range of diseases (e.g., cardiovascular, antitumoral) [104]. The CGC
aldheyde
quercetin conjugate involves a two-step reaction. In the first step, the CGC
aldehyde is formed by a periodate oxidation with formic acid and sodium periodate. The second step, the conjugation reaction is performed by condensation with formic acid and quercetin in DMSO/ acetic acid [104]. Singh et al. [104] already demonstrates that this derivative is nontoxic and biocompatible for normal cell lines and cytotoxic in cancerous cell lines. Moreover, CGC
aldehyde
quercetin has a higher antioxidant activity when compared with CGC [104].

2.5 Conclusions Over the last years, a large number of chitin and chitosan derivatives were developed to improve these polysaccharides’ properties and, consequently, increase the number of potential applications for them. In this chapter, the most well-known chitin and chitosan derivates were reviewed with some of the recent findings and applications for most of them. However, new chitinous polymeric structures are under development and reports arise in scientific publications. Microbial chitinous polysaccharides were also the subject of these derivatization reactions, also being valuable biomaterials not only due to their improved properties, but also due to their biological and environment-friendly production processes (in contrast with chitin extraction from marine sources). Independently of the chitin and chitosan sources, these biomaterials are promising candidates to be used in the near future in several application areas, especially as carriers in drug delivery systems, in wound healing, or in tissue engineering. Handbook of Chitin and Chitosan

64

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

References [1] F. Croisier, C. Je´roˆme, Chitosan-based biomaterials for tissue engineering, Eur. Polym. J. 49 (2013) 780
792. ¨ zogul, J.M. Regenstein, Industrial applications of crustacean by[2] I. Hamed, F. O products (chitin, chitosan, and chitooligosaccharides): a review, Trends Food Sci. Technol. 48 (2016) 40
50. [3] K. Kurita, Chitin and chitosan: functional biopolymers from marine crustaceans, Mar. Biotechnol. 8 (2006) 203
226. [4] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603
632. [5] K. Kurita, Controlled functionalization of the polysaccharide chitin, Prog. Polym. Sci. 26 (2001) 1921
1971. [6] W.L. Teng, E. Khor, T.K. Tan, L.Y. Lim, S.C. Tana, Concurrent production of chitin from shrimp shells and fungi, Carbohydr. Res. 332 (2001) 305
316. [7] I. Giavasis, Bioactive fungal polysaccharides as potential functional ingredients in food and nutraceuticals, Curr. Opin. Biotechnol. 26 (2014) 162
173. [8] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources: structure, properties and applications, Mar. Drugs 13 (2015) 1133
1174. [9] C.K.S. Pillai, W. Paul, C.P. Sharma, Chitin and chitosan polymers: chemistry, solubility and fiber formation, Prog. Polym. Sci. 34 (2009) 641
678. [10] G. Ru, S. Wu, X. Yan, B. Liu, P. Gong, L. Wang, et al., Inverse solubility of chitin/chitosan in aqueous alkali solvents at low temperature, Carbohydr. Polym. 206 (2019) 487
492. [11] S.S. Silva, J.F. Mano, R.L. Reis, Ionic liquids in the processing and chemical modification of chitin and chitosan for biomedical applications, Green Chem. 19 (2017) 1208
1220. [12] L. Casettari, D. Vllasaliu, E. Castagnino, S. Stolnik, S. Howdle, L. Illum, PEGylated chitosan derivatives: synthesis, characterizations and pharmaceutical applications, Prog. Polym. Sci. 37 (2012) 659
685. [13] J. Arroyo, V. Farkaˇs, A.B. Sanz, E. Cabib, Strengthening the fungal cell wall through chitin-glucan cross-links: effects on morphogenesis and cell integrity, Cell. Microbiol. 18 (9) (2016) 1239
1250. ´ [14] X. Kang, A. Kirui, A. Muszynski, M.C.D. Widanage, A. Chen, P. Azadi, et al., Molecular architecture of fungal cell walls revealed by solid-state NMR, Nat. Commun. 9 (2018) 2747. [15] D. Liu, L. Ding, J. Sun, N. Boussetta, E. Vorobiev, Yeast cell disruption strategies for recovery of intracellular bio-active compounds—a review, Innovative Food Sci. Emerging Technol 36 (2016) 181
192. [16] A.M. Abdel-Mohsen, J. Jancar, D. Massoud, Z. Fohlerova, H. Elhadidy, Z. Spotz, et al., Novel chitin/chitosan-glucan wound dressing: isolation, characterization, antibacterial activity and wound healing properties, Int. J. Pharm. 510 (2016) 86
99. [17] G. Mun˜oz, C. Valencia, N. Valderruten, E. Ruiz-Dura´ntez, F. Zuluaga, Extraction of chitosan from Aspergillus niger mycelium and synthesis of hydrogels for controlled release of betahistine, React. Funct. Polym. 91
92 (2015) 1
10. [18] T. Wu, S. Zivanovic, F.A. Draughon, C.E. Sams, Chitin and chitosans
value-added products from mushroom waste, J. Agric. Food Chem. 52 (2004) 7905
7910. [19] S. Ifuku, Chitin and chitosan nanofibers: preparation and chemical modifications, Molecules 19 (2014) 18367
18380. [20] S. Ifuku, R. Nomura, M. Morimoto, H. Saimoto, Preparation of chitin nanofibers from mushrooms, Materials 4 (2011) 1417
1425. [21] R. Jayakumar, M. Prabaharan, S.V. Nair, H. Tamura, Novel chitin and chitosan nanofibers in biomedical applications, Biotechnol. Adv. 28 (2010) 142
150.

Handbook of Chitin and Chitosan

References

65

[22] B. Sun, S. Yu, D. Zhao, S. Guo, X. Wang, K. Zhao, Polysaccharides as vaccine adjuvants, Vaccine 36 (2018) 5226
5234. [23] P. Dechwongya, K. Sathirakul, B. Kristja´nsson, S. Chirachanchai, S. Honsawek, Preparation and characterization of demineralized bone matrix/chitosan composite scaffolds for bone tissue engineering, Chula. Med. J 63 (2) (2019) 119
126. [24] P. Quatresooz, C. Pie´rard-Franchimont, G. Szepetiuk, C. Devillers, G.E. Pie´rard, Fungal chitin-glucan scaffold for managing diabetic xerosis of the feet in menopausal women, Expert Opin. Pharmacother. 10 (14) (2009) 2221
2229. [25] A.A. Badwan, I. Rashid, M.M.H. Al Omari, F.H. Darras, Chitin and chitosan as direct compression excipients in pharmaceutical applications, Mar. Drugs 13 (2015) 1519
1547. [26] J. Rojas, Y. Ciro, L. Correa, Functionality of chitin as a direct compression excipient: an acetaminophen comparative study, Carbohydr. Polym. 103 (2014) 134
139. [27] H.E. Bays, J.L. Evans, K.C. Maki, M. Evans, V. Maquet, R. Cooper, et al., Chitinglucan fiber effects on oxidized low-density lipoprotein: a randomized controlled trial, Eur. J. Clin. Nutr. 67 (2013) 2
7. [28] R.M. Abdel-Rahman, R. Hrdina, A.M. Abdel-Mohsen, M.M.G. Fouda, A.Y. Soliman, F.K. Mohamed, et al., Chitin and chitosan from Brazilian Atlantic Coast: isolation, characterization and antibacterial activity, Int. J. Biol. Macromol. 80 (2015) 107
120. [29] A. Singh, P.K. Dutta, Extraction of chitin-glucan complex from Agaricus bisporus: characterization and antibacterial activity, J. Polym. Mater. 34 (1) (2017) 1
9. [30] R. Jayakumar, M. Prabaharan, P.T.S. Kumar, S.V. Nair, H. Tamura, Biomaterials based on chitin and chitosan in wound dressing applications, Biotechnol. Adv. 29 (2011) 322
337. [31] D.M. Martı´nez-Ibarra, D.I. Sa´nchez-Machado, J. Lo´pez-Cervantes, O.N. CampasBaypoli, A. Sanches-Silva, T.J. Madera-Santana, Hydrogel wound dressings based on chitosan and xyloglucan: development and characterization, J. Appl. Polym. Sci. 136 (2019) 47342. [32] I. Anastopoulos, A. Bhatnagar, D.N. Bikiaris, G.Z. Kyzas, Chitin adsorbents for toxic metals: a review, Int. J. Mol. Sci. 18 (2017) 114. [33] C. Gerente, V.K.C. Lee, P. Le Cloirec, G. McKay, Application of chitosan for the removal of metals from wastewaters by adsorption—mechanisms and models review, Crit. Rev. Environ. Sci. Technol. 37 (2007) 41
127. [34] W. Boulaiche, B. Hamdi, M. Trari, Removal of heavy metals by chitin: equilibrium, kinetic and thermodynamic studies, Appl. Water Sci. 9 (2019) 39. [35] Y. Wan, K.A.M. Creber, B. Peppley, V.T. Bui, Ionic conductivity of chitosan membranes, Polymer 44 (2003) 1057
1065. [36] V.L. Finkenstadt, Natural polysaccharides as electroactive polymers, Appl. Microbiol. Biotechnol. 67 (2005) 735
745. [37] D. Elieh-Ali-Komi, M.R. Hamblin, Chitin and chitosan: production and application of versatile biomedical nanomaterials, Int. J. Adv. Res. (Indore) 4 (3) (2016) 411
427. [38] R.A.A. Muzzarelli, Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone, Carbohydr. Polym. 76 (2009) 167
182. [39] R. Singh, K. Shitiz, A. Singh, Chitin and chitosan: biopolymers for wound management, Int. Wound J. 14 (6) (2017) 1276
1289. [40] S.A. Agnihotri, N.N. Mallikarjuna, T.M. Aminabhavi, Recent advances on chitosanbased micro- and nanoparticles in drug delivery, J. Controlled Release 100 (2004) 5
28. [41] D. Kulev, I. Negrutsa, Chitin-glucan complex—food additive with sorbent properties, J. Hyg. Eng. Des. 11 (2015) 53
56. [42] S. Gautier, E. Xhauflaire-Uhoda, P. Gonry, G.E. Pie´rard, Chitin
glucan, a natural cell scaffold for skin moisturization and rejuvenation, Int. J. Cosmet. Sci. 30 (2008) 459
469.

Handbook of Chitin and Chitosan

66

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

[43] C. Sun, D. Fu, L. Jin, M. Chen, X. Zheng, T. Yu, Chitin isolated from yeast cell wall induces the resistance of tomato fruit to Botrytis cinereal, Carbohydr. Polym. 199 (2018) 341
352. [44] A. Bornet, P.L. Teissedre, Chitosan, chitin-glucan and chitin effects on minerals (iron, lead, cadmium) and organic (ochratoxin A) contaminants in wines, Eur. Food. Res. Technol. 226 (2008) 681
689. [45] G. Crini, Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment, Prog. Polym. Sci. 30 (2005) 38
70. [46] X. Liu, C. Chen, Y. Wu, Investigation of N-doped carbon-coated lithium zinc titanate using chitin as a carbon source for lithium-ion batteries, Ionics 23 (2017) 889
896. [47] S.-K. Kim, J. Venkatesan, Chitin and chitosan derivatives, in: S.-K. Kim (Ed.), Chitin and Chitosan Derivatives—Advances in Drug Discovery and Developments, CRC Press, Boca Raton, FL, 2014, pp. 3
13. [48] J. Synowiecki, N.A. Al-Khateeb, Production, properties, and some new applications of chitin and its derivatives, Crit. Rev. Food Sci. Nutr. 43 (2) (2003) 145
171. [49] P.A. Felse, T. Panda, Studies on applications of chitin and its derivatives, Bioprocess Eng. 20 (1999) 505
512. [50] L. Zhao, H. Mitomo, N. Nagasawa, F. Yoshii, T. Kume, Radiation synthesis and characteristic of the hydrogels based on carboxymethylated chitin derivatives, Carbohydr. Polym. 51 (2003) 169
175. [51] H. Liu, Q. Yang, L. Zhang, R. Zhuo, X. Jiang, Synthesis of carboxymethyl chitin in aqueous solution and its thermo- and pH-sensitive behaviors, Carbohydr. Polym. 137 (2016) 600
607. [52] M.Z. Karagozlu, F. Karadeniz, S.-K. Kim, Anticancer effects of chitin and chitosan derivatives, in: S.-K. Kim (Ed.), Chitin and Chitosan Derivatives—Advances in Drug Discovery and Developments, CRC Press, Boca Raton, FL, 2014, pp. 183
189. [53] K.V. Kumar, B.V.A. Rao, N.Y. Hebalkar, Phosphorylated chitin as a chemically modified polymer for ecofriendly corrosion inhibition of copper in aqueous chloride environment, Res. Chem. Intermed. 43 (2017) 5811
5828. [54] R. Jayakumar, N. Nwe, S. Tokura, H. Tamura, Sulfated chitin and chitosan as novel biomaterials, Int. J. Biol. Macromol. 40 (2007) 175
181. [55] N. Cankaya, Grafting studies of chitin, Sigma J. Eng. Nat. Sci. 37 (1) (2019) 111
117. [56] J. Huang, W.-w Chen, S. Hu, J.-Y. Gong, H.-W. Lai, P. Liu, et al., Biochemical activities of 6-carboxy β-chitin derived from squid pens, Carbohydr. Polym. 91 (2013) 191
197. [57] R. Jayakumar, M. Prabaharan, S.V. Nair, S. Tokura, H. Tamura, N. Selvamurugan, Novel carboxymethyl derivatives of chitin and chitosan materials and their biomedical applications, Prog. Mater. Sci. 55 (2010) 675
709. [58] S. Khattak, F. Wahid, L.-P. Liu, S.-R. Jia, L.-Q. Chu, Y.-Y. Xie, et al., Applications of cellulose and chitin/chitosan derivatives and composites as antibacterial materials: current state and perspectives, Appl. Microbiol. Biotechnol. 103 (5) (2019) 1989
2006. [59] R. Jayakumar, N. Selvamurugan, S.V. Nair, S. Tokura, H. Tamura, Preparative methods of phosphorylated chitin and chitosan-an overview, Int. J. Biol. Macromol. 43 (2008) 221
225. [60] N. Nishi, A. Ebina, S.-I. Nishimura, A. Tsutsumi, O. Hasegawa, S. Tokura, Highly phosphorylated derivatives of chitin, partially deacetylated chitin and chitosan as new functional polymers: preparation and characterization, Int. J. Biol, Macromol. 8 (1986) 311
317. [61] D.W. Jenkins, S.M. Hudson, Review of vinyl graft copolymerization featuring recent advances toward controlled radical-based reactions and illustrated with chitin/chitosan trunk polymers, Chem. Rev. 101 (2001) 3245
3273.

Handbook of Chitin and Chitosan

References

67

[62] A.T. Ramaprasad, D. Latha, V. Rao, Synthesis and characterization of polypyrrole grafted chitin, J. Phys. Chem. Solids 104 (2017) 169
174. [63] T.T. Hanh, H.T. Huy, N.Q. Hien, Pre-irradiation grafting of acrylonitrile onto chitin for adsorption of arsenic in water, Radiat. Phys. Chem. 106 (2015) 235
241. [64] V.A. Vasnev, A.I. Tarasov, G.D. Markova, S.V. Vinogradova, O.G. Garkusha, Synthesis and properties of acylated chitin and chitosan derivatives, Carbohydr. Polym. 64 (2006) 184
189. [65] X. Sun, J. Zhu, Q. Gu, Y. You, Surface-modified chitin by TEMPO-mediated oxidation and adsorption of Cd(II), colloids and surfaces a: physicochem, Eng. Aspects 555 (2018) 103
110. [66] R.A.A. Muzzarelli, C. Muzzarelli, A. Cosani, M. Terbojevich, 6-Oxychitins, novel hyaluronan-like regiospecifically carboxylated chitins, Carbohydr. Polym. 39 (1999) 361
367. [67] Y. Chen, Y. Ye, L. Wang, Y. Guo, H. Tan, Synthesis of chitosan C6-substituted cyclodextrin derivatives with tosyl-chitin as the intermediate precursor, J. Appl. Polym. Sci. 125 (S2) (2012) E378
E383. [68] C. Yang, L. Xu, Y. Zhou, X. Zhang, X. Huang, M. Wang, et al., A green fabrication approach of gelatin/CM-chitosan hybrid hydrogel for wound healing, Carbohydr. Polym. 82 (2010) 1297
1305. [69] X. Wang, Y. Guo, L. Qiu, X. Wang, T. Li, L. Han, et al., Preparation and evaluation of carboxymethyl chitosan-rhein polymeric micelles with synergistic antitumor effect for oral delivery of paclitaxel, Carbohydr. Polym. 206 (2019) 121
131. [70] V.K. Mourya, N.N. Inamdar, Chitosan-modifications and applications: opportunities galore, React. Funct. Polym. 68 (2008) 1013
1051. [71] N.M. Alves, J.F. Mano, Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications, Int. J. Biol. Macromol. 43 (2008) 401
414. [72] H.-L. Jiang, Y.-K. Kim, R. Arote, J.-W. Nah, M.-H. Cho, Y.-J. Choi, et al., Chitosangraft-polyethylenimine as a gene carrier, J. Controlled Release 117 (2007) 273
280. [73] H. Wang, J. Di, Y. Sun, J. Fu, Z. Wei, H. Matsui, et al., Biocompatible PEG-chitosan @ carbon dots hybrid nanogels for two-photon fluorescence imaging, near-infrared light/pH dual-responsive drug carrier, and synergistic therapy, Adv. Funct. Mater. 25 (2015) 5537
5547. [74] J. Lin, Y. Li, Y. Li, H. Wu, F. Yu, S. Zhou, et al., Drug/dye-loaded, multifunctional PEG-chitosan-iron oxide nanocomposites for methotraxate synergistically selftargeted cancer therapy and dual model imaging, ACS Appl. Mater. Interfaces 7 (22) (2015) 11908
11920. [75] J. Jiao, X. Li, S. Zhang, J. Liu, D. Di, Y. Zhang, et al., Redox and pH dual-responsive PEG and chitosan-conjugated hollow mesoporous silica for controlled drug release, Mater. Sci. Eng. C 67 (2016) 26
33. [76] T. Han, N. Nwe, T. Furuike, S. Tokura, H. Tamura, Methods of N-acetylated chitosan scaffolds and its in vitro biodegradation by lysozyme, J. Biomed. Sci. Eng. 5 (2012) 15
23. [77] N.S. Mohamed, R.H.Y. Subban, A.K. Arof, Polymer batteries fabricated from lithium complexed acetylated chitosan, J. Power Sources 56 (1995) 153
156. [78] X. Zhu, M. Su, S. Tang, L. Wang, X. Liang, F. Meng, et al., Synthesis of thiolated chitosan and preparation nanoparticles with sodium alginate for ocular drug delivery, Mol. Vision 18 (2012) 1973
1982. [79] G. Shahnaz, A. Vetter, J. Barthelmes, D. Rahmat, F. Laffleur, J. Iqbal, et al., Thiolated chitosan nanoparticles for the nasal administration of leuprolide: bioavailability and pharmacokinetic characterization, Int. J. Pharm. 428 (2012) 164
170. [80] A. Anitha, N. Deepa, K.P. Chennazhi, V.-K. Lakshmanan, R. Jayakumar, Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated

Handbook of Chitin and Chitosan

68

[81]

[82]

[83]

[84] [85]

[86]

[87] [88] [89]

[90]

[91]

[92]

[93]

[94] [95]

[96] [97]

[98]

2. Chemically modified chitin, chitosan, and chitinous polymers as biomaterials

chitosan nanoparticles towards colon cancer treatment, Biochim. Biophys. Acta 1840 (2014) 2730
2743. O.M. Dragostin, S.K. Samal, M. Dash, F. Lupascu, A. Paˆnzariu, C. Tuchilus, et al., New antimicrobial chitosan derivatives for wound dressing applications, Carbohydr. Polym. 141 (2016) 28
40. A.L. Bukzem, R. Signini, D.M. dos Santos, L.M. Lia˜o, D.P.R. Ascheri, Optimization of carboxymethyl chitosan synthesis using response surface methodology and desirability function, Int. J. Biol. Macromol. 85 (2016) 615
624. S. Tungtong, S. Okonogi, S. Chowwanapoonpohn, W. Phutdhawong, S. Yotsawimonwat, Solubility, viscosity and rheological properties of water-soluble chitosan derivatives, Maejo Int. J. Sci. Technol. 6 (2) (2012) 315
322. J. Zhang, W. Xia, P. Liu, Q. Cheng, T. Tahirou, W. Gu, et al., Chitosan modification and pharmaceutical/biomedical applications, Mar. Drugs 8 (2010) 1962
1987. K.-Y. Lu, Y.-C. Lin, H.-T. Lu, Y.-C. Ho, S.-C. Weng, M.-L. Tsai, et al., A novel injectable in situ forming gel based on carboxymethyl hexanoyl chitosan/hyaluronic acid polymer blending for sustained release of berberine, Carbohydr. Polym. 206 (2019) 664
673. G.-q Ying, W.-y Xiong, H. Wang, Y. Sun, H.-z Liu, Preparation, water solubility and antioxidant activity of branched-chain chitosan derivatives, Carbohydr. Polym. 83 (2011) 1787
1796. T.-C. Yang, C.-C. Chou, C.-F. Li, Antibacterial activity of N-alkylated disaccharide chitosan derivatives, Int. J. Food Microbiol. 97 (2005) 237
245. G. Ma, D. Yang, H. Tan, Q. Wu, J. Nie, Preparation and characterization of N-alkylated chitosan derivatives, J. Appl. Polym. Sci. 109 (2008) 1093
1098. M.E.I. Badawy, E.I. Rabea, T.M. Rogge, C.V. Stevens, G. Smagghe, W. Steurbaut, et al., Synthesis and fungicidal activity of new N,O-acyl chitosan derivatives, Biomacromolecules 5 (2004) 589
595. Q. Li, C. Zhang, W. Tan, G. Gu, Z. Guo, Novel amino-pyridine functionalized chitosan quaternary ammonium derivatives: design, synthesis, and antioxidant activity, Molecules 22 (2017) 156. L. Wei, Q. Li, Y. Chen, J. Zhang, Y. Mi, F. Dong, et al., Enhanced antioxidant and antifungal activity of chitosan derivatives bearing 6-O-imidazole-based quaternary ammonium salts, Carbohydr. Polym. 206 (2019) 493
503. E. Igberase, P. Osifo, A. Ofomaja, The adsorption of copper (II) ions by polyaniline graft chitosan beads from aqueous solution: equilibrium, kinetic and desorption studies, J. Environ. Chem. Eng. 2 (1) (2014) 362
369. H. Sashiwa, N. Kawasaki, A. Nakayama, E. Muraki, N. Yamamoto, S.-I. Aiba, Chemical modification of chitosan. 14:1 synthesis of water-soluble chitosan derivatives by simple acetylation, Biomacromolecules 3 (2002) 1126
1128. F. Feng, Y. Liu, B. Zhao, K. Hu, Characterization of half N-acetylated chitosan powders and films, Procedia Eng. 27 (2012) 718
732. A. Anitha, N. Deepa, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Development of mucoadhesive thiolated chitosan nanoparticles for biomedical applications, Carbohydr. Polym. 83 (2011) 66
73. A. Bernkop-Schnu¨rch, S. Du¨nnhaupt, Chitosan-based drug delivery systems, Eur. J. Pharm. Biopharm. 81 (3) (2012) 463
469. Y.O. Al-Ghamdi, K.A. Alamry, M.A. Hussein, H.M. Marwani, A.M. Asiri, Sulfone-modified chitosan as selective adsorbent for the extraction of toxic Hg(II) metal ions, Adsorpt. Sci. Technol. 37 (1
2) (2019) 139
159. Y. Liu, Y. Jiang, J. Zhu, J. Huang, H. Zhang, Inhibition of bacterial adhesion and biofilm formation of sulfonated chitosan against Pseudomonas aeruginosa, Carbohydr. Polym. 206 (2019) 412
419.

Handbook of Chitin and Chitosan

References

69

[99] Z. Sun, C. Shi, X. Wang, Q. Fang, J. Huang, Synthesis, characterization, and antimicrobial activities of sulfonated chitosan, Carbohydr. Polym 155 (2017) 321
328. [100] E. Machova´, K. Kvapilova´, G. Kogan, J. Sǎndula, Effect of ultrasonic treatment on the molecular weight of carboxymethylated chitin-glucan complex from Aspergillus niger, Ultrason. Sonochem. 5 (1999) 169
172. [101] G. Kogan, P. Rauko, E. Machova´, Fungal chitin-glucan derivatives exert protective or damaging activity on plasmid DNA, Carbohydr. Res. 338 (2003) 931
935. [102] Y.A. Skorik, A.V. Pestov, Y.G. Yatluk, Evaluation of various chitin-glucan derivatives from Aspergillus niger as transition metal adsorbents, Bioresour. Technol. 101 (2010) 1769
1775. [103] L.A. Nud’ga, V.A. Petrova, A.D. Ben’kovich, G.A. Petropavlovskii, Comparative study of reactivity of cellulose, chitosan, and chitin-glucan complex in sulfoethylation, Russ. J. Appl. Chem. 74 (1) (2001) 145
148. [104] A. Singh, P.K. Dutta, H. Kumar, A.K. Kureel, A.K. Rai, Synthesis of chitin-glucanaldehyde-quercetin conjugate and evaluation of anticancer and antioxidant activities, Carbohydr. Polym. 193 (2018) 99
107.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

3 Chitin and chitosan composites for wearable electronics and energy storage devices Yasir Beeran Pottathara1, Hanuma Reddy Tiyyagura1, Zakiah Ahmad2 and Sabu Thomas3 1

Faculty of Mechanical Engineering, University of Maribor, Maribor, Slovenia, 2Faculty of Civil Engineering and Institute for Infrastructure Engineering and Sustainable Management, Universiti Teknologi Mara, Shah Alam, Malaysia, 3International and Interuniversity Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, India

O U T L I N E 3.1 Introduction

72

3.2 Physical and chemical properties of chitin and chitosan

72

3.3 Chitin and chitosan composites for advanced electronics 3.3.1 Stretchable and wearable devices

73 74

3.4 Energy storage devices 3.4.1 Supercapacitors 3.4.2 Batteries 3.4.3 Solar cells

77 77 82 84

3.5 Conclusion

85

Acknowledgments

86

References

86

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00003-0

71

© 2020 Elsevier Inc. All rights reserved.

72

3. Chitin and chitosan composites for wearable electronics and energy storage devices

3.1 Introduction Chitin is a natural biopolymer, which is biocompatible and biodegradable and useful in many applications like biomedical, electronics, photography, and textiles [1,2]. The main advantages of chitin and its derivates are the low cost and easily availability. The chitin-based composites like chitin beads, chitin nanofibers, and chitin hydrogels are used in drug delivery and tissue engineering applications. In recent times, chitin and its derivates have gained much importance in the field of electronic applications as sensors and energy storage applications [3]. Chitin nanofiber papers are being used as flexible green electronics in solar cells and organic light-emitting diodes [4]. Aksoy et al. prepared low-cost chitin derivatives with high conductivity, for the construction of Schottky diodes, which are useful for photoelectrical applications [5]. Schauer et al. reported a variation of the piezoelectric effect of the chitin electrospun nanofibers [6]. Chitosan is a term used to denote members of a family of natural cationic biopolymers derived from the deacetylation of chitin. Chitosan has received considerable interest in biological, industrial, textile, food, and pharmaceutical applications due to its nontoxic nature and biodegradability [7]. Chitosan-based based nanocomposites, like chitosan
carbon nanotubes (CNTs) [8], have been used as chitosan-based electrolytes in biosensors and as electrochromic devices [9]. Chitosan capped with gold nanoparticles has been used in heavy metal ion sensor applications [10], to fabricate the biophotonic field-effect transistors [11,12] and nanoelectronic devices [13]. In this chapter, the main aim is to present an overview specifically of the current advancements of chitin and its derivatives (chitosan) for different electronic applications, such as solar cells, fuel cells, and energy storage devices.

3.2 Physical and chemical properties of chitin and chitosan Chitin and its derivative chitosan are natural biopolymers which are extracted from crabs, prawns, and shrimps. Chitin consists of β-(1,4)linked 2-acetamido-2 deoxy-β-D-glucose1 which is insoluble in water and hydrophobic in nature [2]. Chitosan, consisting of (1,4)-linked 2-amino-deoxy-β-D-glucan, is obtained by partial N-deactylation of chitin (Fig. 3.1). The degree of acetylation of Chitosan is characterized by the molar fraction of N-acetylated units. Chitosan, in its crystalline form, is usually insoluble in aqueous solution above a pH of 7 [15]. Chitosan is most favored among all

Handbook of Chitin and Chitosan

3.3 Chitin and chitosan composites for advanced electronics

73

FIGURE 3.1 Preparation and chemical structures of chitin and chitosan. Source: Reproduced with permission from W. Suginta, P. Khunkaewla, A. Schulte, Chem. Rev. 113 (2013) 5458
5479 [14]. Copyright (2013). American Chemical Society.

other natural polymers because of its availability and economic issues (low cost compared to other biopolymers) [16]. Chitosan exhibits hydrophilicity which promotes cell adhesion and proliferation [17] and it can easily be processed into various forms like fibers, sponges, beads, and more complex forms (it can easily be microstructured to threedimensional (3D) complex forms) for orthopedic applications.

3.3 Chitin and chitosan composites for advanced electronics Numerous biological materials are widely explored in the field of materials science, chemistry, and physics to fabricate and expand functional electronic devices. The exciting features of chitin and chitosan offer inspiration to researchers for the development of advanced materials. The alteration of these natural building blocks into numerous formats may possibly extend the successful creation of emerging green electronics devices. In advanced electronics, there is a high demand for functional devices with high conductivity, lightweight nature, environment-friendliness, and flexibility [18
23]. Chitin/chitosan conductive composites offer a wide range of exceptional properties including high electrical conductivity,

Handbook of Chitin and Chitosan

74

3. Chitin and chitosan composites for wearable electronics and energy storage devices

mechanical robustness, and biocompatible and biodegradable characteristics [24
27]. By incorporating conductive materials, the limited applicability of chitin/chitosan in advanced electronic devices due to the lack of electrical conductivity has been overcome. Chen et al. reported a conductive nanocomposite gel film of chitin nanofibers and multiwalled CNTs with a conductivity of 9.3 S/cm for foldable electronic applications [24]. Air-dried composite aerogels of reduced graphene oxide (GO) and chitosan were fabricated by Li and his team [25]. These aerogels exhibit high electrical conductivity, environmental stability, and exceptional elasticity. Strong, tough, and conductive artificial nacre based on GO and chitosan was demonstrated by Cheng et al. with four and 10 times higher tensile strength and toughness than that of natural nacre [26]. In the following sections, we concentrate on the recent advancements of conductive chitin/ chitosan composites in wearable electronics and energy storage devices.

3.3.1 Stretchable and wearable devices In the field of stretchable and wearable electronics, there is a high demand for scalable sensors with high sensitivity to external forces in addition to high stretchability and good electrical conductivity [28]. Chitinous protein complexes were introduced to many composite systems due to their mechanical robustness and structural integrity. Hong et al. demonstrated chitin nanofibers and silk fibroin (β-sheet) for emerging wearable devices and advanced electronics [29]. The chitin nanofiber
silk fibroin hybrids have been proven as a film-type wireless heater, contact-lens-type glucose sensor, and scratch-resistant transparent display cover window. Recently, Huang et al. reported conducting flexible aerogel composites containing polyaniline (PANI), chitosan, and bacterial cellulose as piezoresistive sensors, as shown in Fig. 3.2, with high sensitivity of 1.41 k/Pa and low-pressure detection of 32 Pa [30]. The entrenching of polydimethylsiloxane (PDMS) into this aerogel further enabled the piezoresistive sensors as wearable devices. Highly sensitive strain sensors based on chitosan
carbon black aerogels with high electrical conductivity were fabricated by Liu et al. for identifying various human activities such as breathing, and joint bending [31]. Zhang et al. extracted starch and chitosan (SC)-based substrates from potato and crab shells, respectively, and demonstrated a flexible transparent electrode by interconnecting conductive nanocomposites [32]. A continuous 3D interconnected conductive network of transparent electrodes was designed, as shown in Fig. 3.3, with single-walled CNTs (SCNT), pristine graphene (PG), and poly(3,4-ethylenedioxythiophene) (PEDOT) chains. These fabricated flexible transparent electrodes exhibited a sheet resistance of 46 Ω/sq with a transmittance of 83.5% at a wavelength of 550 nm. This

Handbook of Chitin and Chitosan

3.3 Chitin and chitosan composites for advanced electronics

75

FIGURE 3.2 Photographs of piezoresistive sensor aerogels (left) chitosan/bacterial cellulose and (right) chitosan/bacterial cellulose with polyaniline. Source: Reprinted with permission from J. Huang, D. Li, M. Zhao, H. Ke, A. Mensah, P. Lv, et al., Chem. Eng. J. (2019) Copyright (2019). Elsevier.

FIGURE 3.3 Schematic design of the fabrication process of the flexible 3D interconnected SCNT 2 PG 2 PEDOT-based transparent electrodes. Source: Reprinted with permission from J. Miao, H. Liu, Y. Li, X. Zhang, ACS Appl. Mater. Interfaces (2018) Copyright (2018). American Chemical Society.

biodegradable transparent electrode is a promising material for nextgeneration wearable green optoelectronics, transient electronics, and edible electronics. Strain sensors based on spirally structured natural rubber (NR) latex with carbon black (CB) and chitin nanocrystals (CNC) were reported by Liu et al. [33]. The high strength and aspect ratio with low density of chitin nanocrystals offer a high reinforcement impact for the sensor fabrication. The strain sensitivity of NR/CNC-CB sensors at large strain are shown in Fig. 3.4. The fabricated strain sensor offers excellent stability and elasticity under a maximum strain of 200% and the conductivity

Handbook of Chitin and Chitosan

76

3. Chitin and chitosan composites for wearable electronics and energy storage devices

FIGURE 3.4 Changes in relative resistance under a 50% strain stretching
releasing cycle (A). Relative resistance variation under gradually increasing step strain from 1% to 9% strain (B). Stretch
release cycles for 25% and 50% (C). Dynamic stretch
release cycle response of the sensor for various strains 25%
200% (D). Photographs showing a lighted LED light changing with the strain of NR/CNC-CB strain sensor composites under a 50% strain stretch
release cycle (E). Source: Reprinted with permission from Y. Liu, F. Wu, X. Zhao, M. Liu, ACS Sustain. Chem. Eng. (2018) Copyright (2018). American Chemical Society.

can be changed rapidly with the strain; it could be designed as a wearable device. Chitosan-based CNTs composite fibers were introduced by Park and his team through cross-linking of polyurethane (PU) with 4,40-methylenebis (phenyl isocyanate) (MDI) [34]. The newly developed

Handbook of Chitin and Chitosan

3.4 Energy storage devices

77

composite fibers exhibited outstanding electrical conductivity of 7.3 S/ cm and good mechanical strength 88.2 MPa.

3.4 Energy storage devices Advanced electronic devices based on chitin and chitosan would be able to produce biodegradable, portable, lightweight, and flexible energy conversion and storage devices including supercapacitors (SCs), batteries, and solar cells [35]. Energy storage devices have become an important research topic in terms of their environment-friendliness, lightweight nature, sustainability, performance, manufacturing costs, and mechanical properties. Natural biomaterials such as cellulose, chitin, and chitosan have drawn increasing interest in the field of energy storage as novel and effective strategies to address these challenges. In this section, we focus on the latest advancements of chitin and chitosan composites in the field of SCs, Lithium-ion batteries (LiBs), and, solar cells.

3.4.1 Supercapacitors Supercapacitors are promising components of energy storage devices, and have attracted extensive attention due to their rapid charge and discharge rates, high power density, and long life [36]. Supercapacitors require high power and energy density, biocompatibility, and renewability, in addition to their safety aspects. The generation of new biobased supercapacitors from chitin and chitosan materials by exploring their peculiar properties were widely reported recently [37
51]. As mentioned earlier, chitin and chitosan materials have the advantageous properties of being lightweight and easy to handle but they are not valuable due to their low conductivity and rapid dissolution into the electrolyte. Chitosan, a deacylation product from chitin, is the most abundant biopolymer with an amino group in the repeating unit on Earth [52]. Numerous studies have proved that chitin and chitosan can be used as sources for the preparation of nitrogen-doped carbon materials for supercapacitor applications. The presence of amide and amino groups in chitin and chitosan can incorporate nitrogen functionalities into carbon frameworks [53]. Zhang and coworkers fabricated hierarchically porous carbon microspheres (HCMs) by pyrolyzing chitin microspheres made up from a chitin/chitosan blend solution, in which chitosan was used as a forming agent of nanopores/nanochannels to construct the microspheres [37]. The obtained HCM exhibited a specific surface area

Handbook of Chitin and Chitosan

78

3. Chitin and chitosan composites for wearable electronics and energy storage devices

of 1450 m2/g. Polyaniline (PANI) nanoclusters were deposited on the surface of HCM to establish it as an electrode material in supercapacitors. The supercapacitor based on HCM 2 PANI exhibited high rate capability and retained over 64% of the capacitance as the scan rate increased from 2 to 500 mV/s. The whole formation process of CCMPANI is schematically depicted in Fig. 3.5. As shown in Fig. 3.5A, chitin/chitosan microspheres (CCMs) consisting of nanofibers were obtained by an emulsion method from chitin/chitosan solution, leading to the formation of micro- and mesopores in the chitin nanofibers. Further carbonization of porous microspheres at 800 C under argon atmosphere provided chitin carbon microspheres (HCMs). Successively, PANI nanoclusters were deposited on the nanofibers of HCMs to obtain HCM 2 PANI. Recently, the interest in chitin and chitosan for supercapacitors has moved toward the formulation of nitrogen-doped porous carbons with hierarchical porosity. The occurrence of nitrogen in the carbon network modifies the polarity of its surface, along with its electronic properties. Nitrogen-doped porous carbons from chitosan were reported Zhong

FIGURE 3.5 Schematic images of the formation process (A), two-electrode system (B), and porous structure of the nanofibers (C) for hierarchically porous carbon microspheres
polyaniline (HCM 2 PANI). Source: Reprinted with permission from L. Gao, L. Xiong, D. Xu, J. Cai, L. Huang, J. Zhou, et al., ACS Appl. Mater. Interfaces (2018) Copyright (2018). American Chemical Society.

Handbook of Chitin and Chitosan

3.4 Energy storage devices

79

et al. with superior supercapacitance performances through hydrothermal carbonization [38]. The physicochemical and supercapacitance performances of the obtained porous carbon structure were achieved by the proper control of hydrothermal carbonization by the means of increasing turbostratic structure, specific surface area, doped nitrogen content, active nitrogen species, and balancing micro- and mesoporosity. The nitrogen-doped carbon offers an ultrahigh specific capacitance of 406 6 36 F/g in a three-electrode system and an ultrahigh energy density of 23.6 6 3.1 Wh/kg. Xie et al. fabricated nitrogen-doped hierarchically porous carbon materials via carbonization and activation [49]. The obtained porous carbon materials exhibited a specific surface area of 3300 m2/g and a pore volume of up to 2.24 cm3/g. The assembled supercapacitor based on the obtained porous carbon brings a high energy density of 12.7 W h/kg and a power density of 25 W/kg with a high cycling stability of 94% capacitance retention after 20,000 cycles. Nitrogenrich hierarchically porous carbon was effectively produced through hydrothermal carbonization and self-activation of a chitosan
transition metal ion (Zn21) complex [39]. The nitrogen-rich porous carbon displays a high specific capacitance of 228.7 F/g at 1 A/g and an excellent energy density of 25.7 Wh/kg at the power density of 500 W/kg. A high surface area of 3312 m2/g of mesoporous activated carbon was synthesized by Lian and coworkers from chitosan biomass and showed excellent capacitive behavior in a range of acidic, neutral, and alkaline liquid electrolytes [47]. The chitosan precursor offers a naturally porous template to which carbonization and KOH chemical activation can be applied to create a nanoporous carbon structure as shown in Fig. 3.6. Fig. 3.6A and B show a “honeycomb” arrangement of large macropores indicative of the natural porosity of the chitosan precursor, whereas Fig. 3.6C and D illustrate the effect of KOH activation leading to a dense network of smaller porosity. Nitrogen-doped hierarchical porous carbon materials were also reported by Mi and coworkers using a chitosan
polyethylene glycol (PEG) blend via a facile carbonization
activation process for high-performance supercapacitors [43]. In this method, chitosan was employed as a nitrogencontaining carbon precursor, while PEG was a porogen. The as-fabricated porous carbon materials exhibited a high specific capacitance of 356 F/g in 1 M H2SO4 and 271 F/g in 2 M KOH at a current density of 1 A/g. The assembled symmetric supercapacitors offer an exceptional cycling stability with 97% and 94% retention for KOH and H2SO4 respectively after 10,000 cycles at 1 A/g. Some reports established the nitrogen and boron codoping of activated carbon through carbonization, KOH activation, and then the hydrothermal doping reaction process with chitosan as the renewable carbon source, and these were demonstrated for high-performance supercapacitor devices [44]. Zhang et al. designed a nitrogen-enriched carbon nanofiber aerogels from chitin nanofiber aerogels as the precursor [40]. The

Handbook of Chitin and Chitosan

80

3. Chitin and chitosan composites for wearable electronics and energy storage devices

FIGURE 3.6 SEM images of the porous chitosan activated carbon: (A) and (B) before KOH activation; (C) and (D) after KOH activation. Source: Reprinted with permission from M. Genovese, H. Wu, A. Virya, J. Li, P. Shen, K. Lian, Electrochim. Acta (2018). Copyright (2018). Elsevier.

uniformly structured ultrafine nanofiber network in addition to the nitrogen-rich composition offers the as-prepared aerogels a large specific capacitance of 221 F/g at the current density of 1.0 A/g and good capacitance retention of 92% over 8000 cycles in a 6.0 mol/L KOH electrolyte, as shown in Fig. 3.7. Microporous oxygen and nitrogen-doped activated carbon were developed by using chitin from the gladius of squid fish for the fabrication of aqueous and flexible supercapacitor electrodes with 100% specific capacitance retention after 25,000 subsequent charge/discharge cycles in 1 M H2SO4 electrolyte [46]. Zhang and coworkers reported 3D porous nitrogendoped graphene aerogels by using GO and chitosan via a self-assembly process by a rapid method [41]. A nanocomposite of zinc-doped iron oxide with chitosan and GO was fabricated by Vedhi and his group by a simple solution mixing
evaporation method, which exhibited a respectable capacitance nature for supercapacitor applications [42]. In another study, Badhulika et al. demonstrated cuprous oxide nanocubesdecorated reduced GO immersed in a chitosan matrix for electrode material supercapacitor applications [45]. Cobalt molybdate (CoMoO4) was modified

Handbook of Chitin and Chitosan

3.4 Energy storage devices

81

FIGURE 3.7 Cyclic voltammograms of nitrogen-enriched carbon nanofiber aerogels (NCNAs) at a scan rate of 20 mV/s (A). Cyclic voltammograms at different scan rates (B) and charge
discharge curves at different current densities (C) of NCNA-900. Specific capacitances of NCNAs at different current densities (D). Cycling performance of NCNA900 (insert charge
discharge curves before and after cycles) (E). Electrochemical impedance spectra of NCNAs under the influence of a voltage of 5 mV (insert: magnified 0
2 Ω region) (F). Source: Reprinted with permission from B. Ding, S. Huang, K. Pang, Y. Duan, J. Zhang, ACS Sustain. Chem. Eng. (2018) Copyright (2018). American Chemical Society.

by chitosan cross-linked with glutaraldehyde employed as a cathode material in an aqueous hybrid capacitor reported by Minakshiu et al. for a high-performance and low-cost energy storage device [50]. Stepniak and coworkers fabricated a chitosan/sponge chitin-based membrane via the casting method for electrochemical applications by using the demineralized

Handbook of Chitin and Chitosan

82

3. Chitin and chitosan composites for wearable electronics and energy storage devices

skeleton of the marine demosponge Ianthella basta as a source for a chitinous network [48]. Overall, the successful manufacturing of chitin and chitosanbased supercapacitors will greatly extend the lifetime of future energy storage devices, providing good economic and operational safety performance.

3.4.2 Batteries There is a high demand for low-cost and environment-friendly Li/ Na-ion batteries with high energy density to maintain sustainability for the extensive use of renewable energy in the future. Along with their use in supercapacitors, chitin and chitosan are of growing interest for electrodes, separators, and electrolytes for Li/Na-ion batteries [54
61]. In the following sections, we summarize the recent advances of Li/Naion batteries based on chitin and chitosan materials. The polymer binder plays a crucial part in the electrochemical performance of the electrodes of Li/Na batteries. Chitosan has been reported as a potential binder for the construction of cathode and anode electrodes for Li/Na-ion batteries [56
61]. As compared to polyvinylidene fluoride (PVDF), a conventional binder of Li-ion batteries, chitosan presents enhanced structural and morphological properties in addition to a high ionic conductivity [56]. The discharge capacity of the cells containing chitosan and PVDF binders at different C-rates are shown in Fig. 3.8. The electrode with chitosan binder attains a higher discharge capacity of 159.4 mAh/g with an excellent capacity retention ratio of 98.38% compared to the electrode with the PVDF binder, which had a discharge capacity of 127.9 mAh/g and a capacity retention ratio of 85.13%. Alginate
carboxymethyl chitosan composite was investigated by Yang and coworkers as a water-soluble binder for Si anodes in Liion batteries, and it exhibits an excellent cycling stability of 750 mAh/g after 100 cycles [57]. The obtained composite offers a porous scaffold network through the electrostatic interaction between carboxylate of alginate and protonated amines of chitosan which preserves a unified electrode structure in cycling process. Tang et al. introduced chitosan oligosaccharides as an electrode binder for lithium-ion batteries with improved electrochemical performances in terms of the first coulombic efficiency, cycling behavior, rate capability and long-life cycle than conventional PVDF binder [58]. Cross-linked chitosan was reported as a binder in Si/graphite electrodes for Li-ion batteries and offers higher initial coulombic efficiency and stable cycling performance [59]. This could be the effect of the polymeric network formed from cross-linking, and this network can effectively accommodate large volume changes of silicon particles and keep the other electrode components connected during cycling leading to excellent cycling stability. Lee and his group

Handbook of Chitin and Chitosan

3.4 Energy storage devices

83

FIGURE 3.8 The rate performance of electrodes for Li-ion batteries prepared using different binders at room temperature. Source: Reprinted with permission from K. Prasanna, T. Subburaj, Y.N. Jo, W.J. Lee, C.W. Lee, ACS Appl. Mater. Interfaces (2015) Copyright (2018). American Chemical Society.

reported that, through the cross-linking of amino groups of chitosan and the dialdehyde of glutaraldehyde, chitosan forms a 3D network to limit the movement of Si particles for the Si anode of Li-ion batteries [60]. Goodenough et al. also reported a polymer network binder based on the cross-linking of chitosan and glutaraldehyde for an Sb anode in Na-ion batteries [61]. The obtained polymer binder network accommodates a large volume change of the Sb anode upon sodiation/desodiation, heading to an outstanding cycling stability and high coulombic efficiency. Cross-linking of chitosan with glutaraldehyde were further reported for the fabrication of electrolytic manganese dioxide (EMD) nanoflakes as an electrode material for alkaline batteries [59]. In the presence of glutaraldehyde, chitosan influences the nucleation and growth of the EMDs during electrodeposition and exhibits a better discharge capacity upon cycling. The electrolyte composite for lithium battery should be compatible with components of the cell and should generate enough ionic conductivity over a temperature range of 220 C
50 C. Gel polymer electrolytes, a combination of liquid electrolytes and solid polymer electrolytes, offer the advantages of both electrolytes. A 3D cross-linked chitosan
poly(ethylene glycol) diglycidyl ether-based gel polymer electrolyte network was designed by Wen and coworkers with an improved mechanical strength of 5.5 MPa [55]. Chitosan’s special chemical structure and functional groups provides it with the ability to disaggregate lithium salts, adsorb the organic

Handbook of Chitin and Chitosan

84

3. Chitin and chitosan composites for wearable electronics and energy storage devices

solvents, and promote the migration of lithium ions [62]. The designed gel electrolyte exhibits a good lithium ionic conductivity of 2.74 3 1024 S/cm with an excellent lithium-ion transfer number of 0.869 at 25 C. Moreover, chitin-derived biochars obtained through pyrolysis were reported as cathode materials in lithium-sulfur batteries [63]. In addition to electrode materials and electrolytes, separators play an important role of Li/Na-ion batteries in terms of their safety and performance. Separators act as electronically isolating layers between the electrodes of Li/Na-ion batteries to avoid an internal short. Yu and coworkers fabricated a sustainable separator for Li/Na-ion batteries through the self-assembly of prawn shell-derived chitin nanofibers [54]. The chitin nanofiber-based separator exhibits improved performance in the LiFePO4/Li cell and Na3V2(PO4)3/Na cell than the commercial polypropylene separator. The chitin/chitosan-based separators offer eco-efficient and environmentfriendly separators with high mechanical strength, excellent thermal stability, and good electrolyte wettability for Li/Na-ion batteries.

3.4.3 Solar cells There is a huge interest for a wide range of functional materials such as photovoltaic devices from low-cost and nontoxic materials through the conversion of waste biomass. Photovoltaic devices, such as dyesensitized solar cells (DSSC), organic solar cells, perovskite solar cells, and quantum-dot (QD) solar cells, aim to reduce the production costs and/or obtain power conversion efficiencies greater than the Shockley
Queisser limit [64]. In this section, we summarize the recent progress of chitin and chitosan-based solar cells. The amine groups on chitosan make it a promising candidate in organic solar cells as cathode interlayers. Furthermore, the pHdependent solubility of chitosan permits it to form stable films on device surfaces. Wang and his group employed chitosan and its derivatives as cathode interlayer materials in inverted organic solar cells by employing an electrostatic Layer-by-Layer (eLbL) self-assembly technique [65]. This technique was demonstrated to be a suitable approach to obtain continuous films with full surface coverage, uniformity, and controlled thickness in the nanoscale. The performance of the organic solar cell—the J
V graph—is shown in Fig. 3.9. The chitosan eLbL films as cathode interlayer in inverted organic solar cells offer a power conversion efficiency of 9.34%. This is approximately a 200% improvement over cells with no cathode interlayer. To overcome the drawbacks of liquid electrolytes, such as solvent leakage, corrosion, and high volatility in DSSCs, quasi-solid-state electrolytes were reported. Yahya et al. demonstrated the utilization of

Handbook of Chitin and Chitosan

85

3.5 Conclusion

Current density (mA/cm2)

20 No interlayer Spin coat eLbL film eLbL film*

10

0

–10

–20 –0.2

0

0.2

0.4

0.6

0.8

Voltage (V)

FIGURE 3.9 Current density vs voltage curves of inverted organic solar cells with spin-coated chitosan and electrostatic Layer-by-Layer self-assembled chitosan derivative films with different thickness as cathode interlayers. Bare ITO without an interlayer is also included.

quasi-solid-state electrolytes with chitosan, poly(vinylidene fluoridehexafluoropropylene), 1-methyl-3-propylimidazolium iodide ionic liquid, and iodide/tri-iodide redox salts in DSSCs and exhibiting the highest power conversion efficiencies of 1.23% with ionic conductivity of 5.367 3 1024 S/cm [66]. The performance of carbon nanodots derived from chitin and chitosan as sensitizers for TiO2- based nanostructured solar cells was demonstrated by Briscoe et al. [67]. The obtained chitinderived carbon nanodots through hydrothermal carbonization exhibits a solar power conversion efficiency of 0.22%. Titirici and coworkers produced carbon quantum dots from chitin, chitosan, and glucose and used them to sensitize ZnO nanorods to visible light for solid-state nanostructured solar cells [68]. A layer combining chitosan and chitinderived carbon quantum dots fabricated devices with the highest efficiency of 0.077%. The light-harvesting efficiency of the devices could be improved further by coating thicker layers but without increasing the series resistance, or by increasing the visible light absorption.

3.5 Conclusion Chitin and chitosan are natural biopolymers, extracted from crustaceans for industrial applications. These biopolymers have properties

Handbook of Chitin and Chitosan

86

3. Chitin and chitosan composites for wearable electronics and energy storage devices

like nontoxicity, biocompatibility, renewability, and biodegradability, which help in the application of biosensors, solar cells, and batteries. In summary, the potential applications of chitin and its composites for stretchable and wearable electronics and energy storage applications (supercapacitors, solar cells, and batteries) have been highlighted in this chapter. Firstly, the properties of high stretchability and good electrical conductivity of chitin and its composites are useful in the application of flexible electronics, sensors, and energy storage devices. Secondly, chitosan N-doped porous carbon materials are used as high-performance supercapacitors. Thirdly, chitosan binders are useful in batteries, and amino groups in the chitosan help in the production of solar cells.

Acknowledgments Zakiah Ahmad would like to acknowledge the financial support by the TNB Research Sdn Bhd. Malaysia (grant no: 100-IRMI P. 37/7/ 20160419007) and Universiti Teknologi Mara, Selangor Malaysia.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]

A. Chortos, Z. Bao, Mater. Today (2014). M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603
632. N. Amdursky, E.D. Głowacki, P. Meredith, Adv. Mater. 31 (2019) 1
28. J. Jin, D. Lee, H.-G. Im, Y.C. Han, E.G. Jeong, M. Rolandi, et al., Adv. Mater. 28 (2016). 5141
5141. ˙ Uzun, G. Topal, Y.S. Ocak, O ¨ . Aksoy, I. ¨. C O ¸ elik, D. Batibay, Polym. Bull. (2018). R.M. Street, T. Huseynova, X. Xu, P. Chandrasekaran, L. Han, W.Y. Shih, et al., Carbohydr. Polym. (2018). H. Reddy Tiyyagura, R. Rudolf, S. Gorgieva, R. Fuchs-Godec, V.R. Boyapati, K.M. Mantravadi, et al., Prog. Org. Coatings (2016). Y.C. Tsai, S.Y. Chen, H.W. Liaw, Sensors Actuators B Chem. (2007). E. Eren, Solid State Ionics (2019). A. Sugunan, C. Thanachayanont, J. Dutta, J.G. Hilborn, Sci. Technol. Adv. Mater. (2005). C. Zhong, Y. Deng, A.F. Roudsari, A. Kapetanovic, M.P. Anantram, M. Rolandi, Nat. Commun. (2011). H. Liu, Q. Wan, Nanoscale (2012). N.R. Hosseini, J.S. Lee, Adv. Funct. Mater. (2015). W. Suginta, P. Khunkaewla, A. Schulte, Chem. Rev. 113 (2013) 5458
5479. V. Sencadas, D.M. Correia, A. Areias, G. Botelho, A.M. Fonseca, I.C. Neves, et al., Carbohydr. Polym 87 (2012) 1295
1301. F. Pishbin, A. Simchi, M.P. Ryan, A.R. Boccaccini, Surf. Coatings Technol 205 (2011) 5260
5268. R.A.A. Muzzarelli, Carbohydr. Polym. 76 (2009) 167
182. D.A. Gopakumar, A.R. Pai, Y.B. Pottathara, D. Pasquini, L. Carlos De Morais, M. Luke, et al., ACS Appl. Mater. Interfaces (2018). Y.B. Pottathara, V. Bobnar, M. Finˇsgar, Y. Grohens, S. Thomas, V. Kokol, Polymer (Guildf) (2018).

Handbook of Chitin and Chitosan

References

87

[20] M. Hasan, D.A. Gopakumar, V. Arumughan, Y.B. Pottathara, K.S. Sisanth, D. Pasquini, et al., Polymers (Basel) 11 (2019). [21] P.T. Yasir Beeran, V. Bobnar, S. Gorgieva, Y. Grohens, M. Finˇsgar, S. Thomas, et al., RSC Adv. 6 (2016) 49138
49149. [22] O. Koshy, Y.B. Pottathara, S. Thomas, B. Petovar, M. Finsgar, Curr. Anal. Chem. (2017). [23] Y.B. Pottathara, S. Thomas, N. Kalarikkal, T. Griesser, Y. Grohens, V. Bobnar, et al., New J. Chem. (2019). [24] C. Chen, C. Yang, S. Li, D. Li, Carbohydr. Polym. (2015). [25] Y. Zhang, L. Zhang, G. Zhang, H. Li, ACS Appl. Mater. Interfaces (2018). [26] S. Wan, J. Peng, Y. Li, H. Hu, L. Jiang, Q. Cheng, ACS Nano (2015). [27] B.C. Ozkan, T. Soganci, H. Turhan, M. Ak, Electrochim. Acta (2019). [28] S. Gong, W. Schwalb, Y. Wang, Y. Chen, Y. Tang, J. Si, et al., Nat. Commun. (2014). [29] M.S. Hong, G.M. Choi, J. Kim, J. Jang, B. Choi, J.K. Kim, et al., Adv. Funct. Mater. (2018). [30] J. Huang, D. Li, M. Zhao, H. Ke, A. Mensah, P. Lv, et al., Chem. Eng. J (2019). [31] Y. Liu, H. Zheng, M. Liu, Mater. Des. (2018). [32] J. Miao, H. Liu, Y. Li, X. Zhang, ACS Appl. Mater. Interfaces (2018). [33] Y. Liu, F. Wu, X. Zhao, M. Liu, ACS Sustain. Chem. Eng. (2018). [34] H.U. Lee, C. Park, J.Y. Park, RSC Adv. (2016). [35] X. Wang, K. Jiang, G. Shen, Mater. Today (2015). [36] M. Salanne, B. Rotenberg, K. Naoi, K. Kaneko, P.L. Taberna, C.P. Grey, et al., Nat. Energy (2016). [37] L. Gao, L. Xiong, D. Xu, J. Cai, L. Huang, J. Zhou, et al., ACS Appl. Mater. Interfaces (2018). [38] X. Tong, Z. Chen, H. Zhuo, Y. Hu, S. Jing, J. Liu, et al., Carbohydr. Polym. (2019). [39] J. Zhou, H. Wang, W. Yang, S. Wu, W. Han, Carbohydr. Polym. (2018). [40] B. Ding, S. Huang, K. Pang, Y. Duan, J. Zhang, ACS Sustain. Chem. Eng. (2018). [41] Y. Zhang, J.Y. Zhu, H.B. Ren, Y.T. Bi, L. Zhang, Chinese Chem. Lett. (2017). [42] R.B. Suneetha, P. Selvi, C. Vedhi, Vacuum (2019). [43] Y. Ba, W. Pan, S. Pi, Y. Zhao, L. Mi, RSC Adv. (2018). [44] Z. Lin, X. Xiang, S. Peng, X. Jiang, L. Hou, J. Electroanal. Chem. (2018). [45] A. Gopalakrishnan, N. Vishnu, S. Badhulika, J. Electroanal. Chem. (2019). [46] C.J. Raj, M. Rajesh, R. Manikandan, K.H. Yu, J.R. Anusha, J.H. Ahn, et al., J. Power Sources (2018). [47] M. Genovese, H. Wu, A. Virya, J. Li, P. Shen, K. Lian, Electrochim. Acta (2018). [48] I. Stepniak, M. Galinski, K. Nowacki, M. Wysokowski, P. Jakubowska, V.V. Bazhenov, et al., RSC Adv. (2016). [49] J. Cheng, Q. Xu, X. Wang, Z. Li, F. Wu, J. Shao, et al., Sustain. Energy Fuels (2019). [50] R. Ramkumar, M. Minakshi, Dalt. Trans (2015). [51] S. Anandhavelu, V. Dhanasekaran, V. Sethuraman, H.J. Park, J. Nanosci. Nanotechnol. (2017). [52] A. liwak, N. Dı´ez, E. Miniach, G. Gryglewicz, J. Appl. Electrochem. (2016). [53] J. Deng, M. Li, Y. Wang, Green Chem. (2016). [54] T.W. Zhang, B. Shen, H. Bin Yao, T. Ma, L.L. Lu, F. Zhou, et al., Nano Lett. (2017). [55] D. Xu, J. Jin, C. Chen, Z. Wen, ACS Appl. Mater. Interfaces (2018). [56] K. Prasanna, T. Subburaj, Y.N. Jo, W.J. Lee, C.W. Lee, ACS Appl. Mater. Interfaces (2015). [57] Z.H. Wu, J.Y. Yang, B. Yu, B.M. Shi, C.R. Zhao, Z.L. Yu, Rare Met. (2016). [58] H. Tang, Q. Weng, Z. Tang, Electrochim. Acta (2015). [59] X. Zhao, C.-H. Yim, N. Du, Y. Abu-Lebdeh, J. Electrochem. Soc. (2018). [60] C. Chen, S.H. Lee, M. Cho, J. Kim, Y. Lee, A.C.S. Appl, Mater. Interfaces (2016).

Handbook of Chitin and Chitosan

88

3. Chitin and chitosan composites for wearable electronics and energy storage devices

[61] H. Gao, W. Zhou, J.H. Jang, J.B. Goodenough, Adv. Energy Mater. (2016). [62] D. Xu, B. Wang, Q. Wang, S. Gu, W. Li, J. Jin, et al., ACS Appl. Mater. Interfaces (2018). [63] G. Magnacca, F. Guerretta, A. Vizintin, P. Benzi, M.C. Valsania, R. Nistico`, Appl. Surf. Sci. (2018). [64] W. Shockley, H.J. Queisser, J. Appl. Phys. (1961). [65] K. Zhang, R. Xu, W. Ge, M. Qi, G. Zhang, Q.H. Xu, et al., Nano Energy (2017). [66] W.Z.N. Yahya, W.T. Meng, M. Khatani, A.E. Samsudin, N.M. Mohamed, E-Polymers (2017). [67] A. Marinovic, L.S. Kiat, S. Dunn, M.M. Titirici, J. Briscoe, ChemSusChem (2017). [68] J. Briscoe, A. Marinovic, M. Sevilla, S. Dunn, M. Titirici, Angew. Chemie. Int. Ed (2015).

Handbook of Chitin and Chitosan

C H A P T E R

4 Investigation into the functional properties of cotton, wool, and denim textile materials finished with chitosan and the use of chitosan in textile-reinforced composites and medical textiles Nilgu¨n Becenen1, Sevil Erdog˘an2 and Elif Ecem Fındık3 1

Textile Technology Programme, Edirne Vocational College of Technical Sciences, Trakya University, Edirne, Turkey, 2Laborant and Veterinary Health Programme, Ke¸san Vocational College, Trakya University, Edirne, Turkey, 3Department of Biotechnology and Genetic, Institute of Natural Sciences, Trakya University, Edirne, Turkey

O U T L I N E 4.1 Introduction

90

4.2 Chitin and chitosan 4.2.1 General information 4.2.2 Synthesis and characterization of chitosan 4.2.3 Factors affecting the functional properties of chitosan 4.2.4 Antimicrobial activity of chitosan

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00004-2

89

91 91 93 96 101

© 2020 Elsevier Inc. All rights reserved.

90

4. Investigation into the functional properties of cotton, wool, and denim textile materials

4.3 Application techniques and methods of chitosan to textile products 105 4.3.1 Coating of shrimp chitosan on textile surfaces 105 4.4 Properties of the chitosan-treated textile products 4.4.1 Surface properties 4.4.2 Antibacterial activity

107 107 113

4.5 Use of chitosan in medical textiles

121

4.6 Use of chitosan in textile-reinforced composites

124

4.7 Conclusion

126

Acknowledgment

127

References

127

4.1 Introduction Textile production is a highly complex process that requires many processing steps and the introduction of suitable properties for use, as well as the use of advanced technology and the use of suitable substances. Biotechnology products, environmentalist approaches, functional designs, evaluation of recycled production products, and the development of textiles for different sectors led to the formation of new concepts, such as “Technical Textile,” “Ecological Textile,” “Smart Textile,” and “Industrial Textile,” and new textile products. By the 2030s, textile materials are predicted to play an important role in many production areas, leading to a better life on Earth [1]. In the last century, textile production has considerably increased with the rise in global population and innovations in chemistry. The textile industry uses around 8000 chemicals in different production stages, which can pollute natural bodies of water due to unsorted discharge and poor management. With a worldwide population of 7 billion people and an average of 7 kg of clothing per person, the use of chemicals reaches 5 billion kg per year. Most of these chemicals can turn into harmful metabolites. In order to solve this problem of the textile industry, the use of techniques such as biowaste sources, biodegradable chemicals, or digital coloring, where there is no water use or waste discharge, may be a solution [2]. Textile production has to continue to develop in the direction of environment-friendly textile products and technologies by taking into account the reduction of waste and ecological impact; specifically, recycling technologies are gaining importance in this respect. Textile manufacturers

Handbook of Chitin and Chitosan

4.2 Chitin and chitosan

91

have channeled the industry to the use of biotechnology, which is the cleaner option that provides economic benefits as well as environmental benefits [3]. Thus on the one hand, it can minimize the use of raw materials and energy in production activities and textile finishing enterprises, while on the other hand, it can both save resource usage and prevent environmental pollution at the source by using sustainable biomaterials. Organisms such as crab, shrimp, and mushrooms are consumed as food in large quantities throughout the world. However, their wastes are left unutilized. With recycling, the shells of these crustacean organisms are recovered by chemical and biological methods and are transformed into new products such as chitin and chitosan. Chitosan, a deacetylated derivative of chitin, is more widely used in food packaging and other food technologies, wastewater treatment, agriculture, biomedical applications, pharmacology, cosmetics, personal care products, and other biotechnological applications because of its higher solubility compared to chitin [4,5]. Chitosan is a natural, biodegradable, biocompatible, and commercially available polymer that is capable of superior film formation with low toxicity and has antibacterial and antifungal properties [6]. The superior properties of the chitosan, along with its diverse and widespread usage, make it very attractive for the textile industry. The intended use of chitosan is as follows: durable antimicrobial finishing agent, surface modification agent, binder and cross-linker, sizing agent and desizing agent, antifelting finish, providing resistance to shrinkage, improving tensile properties, improving dyeability properties, salt-free dyeing, UV radiation protection, antistatic agent, increasing the durability of textile prints, removal of dye and heavy metals from wastewater, wound dressing, drug-release chitosan-coated fibers, and synthetic intelligent textiles [7
9]. The study reviewed in this chapter aimed to give antibacterial properties to woolen, woven, and denim fabrics, which are widely used in clothing production, using coatings containing chitosan. For this purpose, chitosan produced from the waste shells of shrimp was used to coat the fabrics. Antibacterial activity tests on the chitosan-coated fabric samples were carried out in accordance with international standards before and after washing with household soap, and the results were compared with each other. The use of chitosan in medical textiles and textile-reinforced composites is discussed with a literature review.

4.2 Chitin and chitosan 4.2.1 General information 4.2.1.1 Definition of chitin and chitosan structure Chitin is a linear polysaccharide containing nitrogen. In nature, it is found in three different crystalline forms as α-, β-, and γ-chitin [10,11].

Handbook of Chitin and Chitosan

92

4. Investigation into the functional properties of cotton, wool, and denim textile materials

The alpha form, with its antiparallel chain structure, is the most common one. It is commonly found in many organisms, such as insects, mushrooms, and crustaceans [12,13]. β-Chitin has a parallel chain structure, and it has been found in marine organisms, such as squid and tube worms [14,15]. γ-Chitin contains both parallel and antiparallel chains together, and it is derived from fungi and yeast [16]. Chitosan is a derivative polymer obtained by deacetylation of chitin. These two polysaccharides consist of varying amounts of glucosamine (2-amino-2-deoxy-β-D-glucopyranose) and N-acetylglucosamine (2-acetamido-2-deoxy-β-D-glucopyranose) residues [16]. Since chitosan contains a greater amount of glucosamine (GlcN) units than chitin, it has a higher solubility in acidic solutions [17]. The fact that chitosan is easily soluble in weak acids and can be easily modified by reactive groups and transformed into new derivative materials has led to the common use of this biomaterial in various textile, medical, industrial, and ecological applications [17
22]. Chitosan exhibits linear polyelectrolytes with a high charge density, due to its cationic structure, and chelates toxic metal ions; therefore it serves as an excellent flocculant in water treatment [23]. Because of its high molecular weight and reactive amino and hydroxyl groups, chitosan is highly viscose and is capable of forming a film, making it an appropriate coating material. Besides these properties, it also has antimicrobial, antioxidant, antitumor, and anticancer properties and is used in various biomedical applications for therapeutic purposes [22]. It has been reported that the antimicrobial efficacy and the wound-healing and film-forming abilities of chitosan can be utilized in the development of ocular bandage lenses [24]. In addition, chitosan has exhibited great potential in controlled drug release, tissue engineering, and contact lens production [24]. Chitosan is also added to skincare products, hair lotions, and some cosmetic products due to its moisture holding capacity, protective film-forming ability, and nutritional properties. By binding anionic substances such as bile acid or free fatty acids, it can function as a cancer inhibitor and can lower cholesterol. It can also be used in enzyme and DNA immobilization and in the design of enzyme, antibody, and antigen-based biosensors [4,16,18
21]. In recent years, the biomaterial market has become a powerful market attracting both researchers and consumers, depending on the increase in biomaterial applications and the development of alternative treatment methods with natural materials. The global market size of chitosan was $3.19 billion in 2015, and significant growth is anticipated during the prescribed period in the chitosan market due to the increased applications in the water treatment, cosmetic, and medical industries [25]. Pathak [26] reported that the global chitin and chitosan market, which was $2.0 billion in 2016, is expected to reach $4.2 billion in 2021, with a compound annual growth rate of 15.4% from 2016 to 2021.

Handbook of Chitin and Chitosan

93

4.2 Chitin and chitosan

4.2.1.2 Chitin and chitosan sources Chitin is a protective and supportive structure commonly found in both terrestrial and aquatic organisms. It is found in the outer skeleton, the tubular trachea system, and the peritrophic matrix lining the digestive system of insects, as well as in the exoskeleton of crustaceans, the skeletons of sponges, the inner skeletons of mollusks such as squid and cuttlefish, the jaws and beaks of some cephalopods, the cell walls of mushrooms, and is also in nematodes, algae, and yeasts [10,11,14,27
30]; chitosan is only naturally produced by some Mucoraceae fungi [31]. The most commonly used chitin sources are crustacean organisms such as crabs, shrimp, and lobsters. These organisms are evaluated for the production of chitin due to their high chitin content and the necessity for reduction of their waste, which is a major source of pollution. However, recent studies have revealed that the source of chitin affects its physicochemical and biological properties and its deacetylated derivative, chitosan. Thus the attention of researchers was directed toward different living species (Table 4.1). Moreover, many species have become prominent as alternative and sustainable chitin sources, since they can be cultivated and produced in the desired amounts.

4.2.2 Synthesis and characterization of chitosan Chemical and biological synthesis methods can be used in the production of chitin and chitosan. Chemical methods include demineralization, deproteinization, and decolorization stages. The concentrations TABLE 4.1

Dry weight chitin contents of some groups of organisms.

Chitin sources

Dry weight chitin contents (%)

References

Squid species

35.8
40.5

[32,33]

Cuttlefish

11.7

[33]

Crab species

13
27.4

[33,34]

Shrimp species

16.75
37.2

[14,33
35]

Lobster

21.26

[14]

Other insect species

2.59
38

[36
38]

Scorpion

12.4

[39]

Millipede

17
18

[40]

Bryozoan

13.3

[41]

Mushroom species

2.4
36.72

[41
43]

Handbook of Chitin and Chitosan

94

4. Investigation into the functional properties of cotton, wool, and denim textile materials

and types of acids and bases applied in these steps and the treatment times with acids and bases widely vary. Because the chemical method is an easy and fast method of isolation, commercial chitosan is largely produced by the chemical synthesis method [44]. Various chitosan derivatives are synthesized by biological and enzymatic methods [45]. Isolation of chitin by deproteinization and demineralization with the help of proteases or organic acid bacteria and deacylation of chitin by chitin deacetylase enzyme to perform chitosan production are examples of green synthesis methods using fermentation technology [5,44,46]. Research suggests that biological methods are promising for the production of chitosan, because they allow the recovery of bioactive compounds and are cost-efficient. In addition, biological synthesis methods are preferred to produce high-value commercial by-products and to overcome the negative effects of the chemical synthesis method, such as energy consumption, undesired protein components, and damage to the environment due to the acids and bases used [5,44]. 4.2.2.1 Isolation of chitin from shrimp and production of chitosan In this study, the isolation of chitin from the shrimp was carried out in three stages using a chemical synthesis method (Fig. 4.1). The first one is the demineralization stage that is done to remove minerals from the shell of the organism. At this stage, 20 g of the crushed shrimp shell was treated with 300 mL of 2 M hydrochloric acid solution for 4 h at 75 C, stirring at 1000 rpm. It was then filtered through filter paper and washed with distilled water until the pH was neutral. In the second step, the filtered sample was treated with 200 mL of 2 M NaOH for 18 h at 60 C
70 C to remove the proteins in the structure. After the deproteinization step, the filtrate was rinsed again with deionized water until reaching a pH of around 7. The final stage in the isolation of the chitin is the decolorization step. In this step, the isolated chitin sample was decolorized with 140 mL of a mixture containing 20 mL of chloroform, 40 mL of methanol, and 80 mL of distilled water. The sample was incubated for 30 min to remove the remaining pigment and other residues in the structure of the chitin. The chitin was then washed again with water and dried at 50 C in an oven. Demineralization 2M HCI 60°C, 4 h, 800 rpm

Deacetylation

Deproteinization 2M NaOH 70°C, 18 h, 800 rpm Shrimp

70% NaOH 150° C, 3 h, 800 rpm Chitin

Chitosan

FIGURE 4.1 Diagram showing the production of chitin and chitosan by chemical method.

Handbook of Chitin and Chitosan

4.2 Chitin and chitosan

95

Twelve grams of dry chitin was used to produce chitosan (Fig. 4.1). The chitin sample was refluxed with 180 mL of 70% NaOH solution for 3.5 h at 150 C and 800 rpm. At the end of this time, the sample was washed with deionized water until the pH was neutral. The chitosan obtained was then dried on a petri dish at 60 C for 24 h. 4.2.2.2 Characterization of shrimp chitin and chitosan 4.2.2.2.1 Fourier-transform infrared spectroscopy

The chemical structure of shrimp chitosan was examined by Fouriertransform infrared (FTIR) spectroscopy in the range of 4000
625 cm21 using a Perkin Elmer ATR FTIR device. The results revealed that the chitin from shrimp was in alpha form. In FTIR spectroscopy, α-chitin displays three peaks around 1650, 1620, and 1550 cm21, and β-chitin shows a single peak around 1650 cm21 [11,47]. These absorption peaks in the α-chitin are ascribed to CQO secondary amide stretch (Amide I), CQO secondary amide stretch (Amide I), and N
H bend and C
N stretch (Amide II), respectively [10,14]. In this study, the FTIR spectrum of the chitin obtained from shrimp reflects the characteristics of the α-chitin by revealing three peaks at 1653, 1620, and 1552 cm21. The I. carbonyl [ν (CQO)] band and II. amide [ν(NH2)] band that are characteristic peaks for chitosan are known to be around 1650 cm21 and 1590 cm21, respectively [48]. In this study, chitosan produced by deacetylation of shrimp chitin revealed these two bands, which are characteristic for chitosan at 1651 and 1589 cm21. 4.2.2.2.2 Scanning electron microscope results

Surface images of chitin and chitosan from shrimp were taken at different magnifications using a EVO LS 10 scanning electron microscope (SEM) (Fig. 4.2A and B). The surface morphology of shrimp chitin exhibits a structure consisting of uniformly distributed regular pores of the same size. The shrimp chitosan presents a structure containing nanofibers as well as pores. This morphological structure is also described in blue crab chitosan [49]. (A)

(B)

FIGURE 4.2

SEM images showing the surface morphology of (A) chitin (2500X), and (B) chitosan (40000X) from shrimp.

Handbook of Chitin and Chitosan

96

4. Investigation into the functional properties of cotton, wool, and denim textile materials

3.500

25.00 100.0

3.000 90.0 2.500

80.0

2.000

70.0

1.500

60.0

108.5°C 92.8%

20.00

0.500 0.000 –0.500

50.0

10.00

339.2°C 47.5%

40.0 30.0

DTA (uV/mg)

1.000

TG (%)

DTG (mg/min)

15.00

5.00

20.0 10.0

0.00

–1.000

625.6°C –0.2%

0.0 –1.500 100.0

200.0

300.0

400.0 500.0 Temp (°C)

600.0

700.0

FIGURE 4.3 Thermogravimetric degradation curve of shrimp chitosan. 4.2.2.2.3 Thermo gravimetric analysis

The thermal decomposition curve of shrimp chitosan was recorded by heating between 30 C and 700 C in an inert atmosphere with an EXSTAR S11 7300 device. The results of TGA analysis showed that shrimp chitosan lost mass in three stages (Fig. 4.3). Due to the evaporation of water in the sample, a mass loss of 7.2% was observed in the first stage between 0 C and 108 C. In the second stage, a mass loss of 52.5% was observed between 108 C and 330 C. This mass loss is attributed to the breakdown of the antiparallel chains in chitin [36]. The third mass loss occurred between 339 C and 625 C, and it was reported that this was due to the degradation of residual carbon [50,51]. The minimum degradation temperature of the shrimp chitosan (DTGmin) was recorded as 339 C. This value was similar to those of chitosan samples from adult and nymphal grasshoppers (302 C and 308 C) [52] and blue crabs (306 C) [49].

4.2.3 Factors affecting the functional properties of chitosan 4.2.3.1 Physical and chemical properties of chitosan The main factors determining the functional properties of chitosan are its cationic structure and reactive groups. The reactive hydroxyl and amino groups of chitosan enable it to be produced in a wide variety of derivatives by modifications. Thus chitosan becomes functional and makes it a sought-after biomaterial in various fields of application [22].

Handbook of Chitin and Chitosan

97

4.2 Chitin and chitosan

The physical and chemical properties of chitosan may vary according to the source and synthesis methods [11,16,53,54]. Its fibrillar and porous structure, molecular weight, solubility, degree of deacetylation, cationic structure, and viscosity also affect the biological properties of chitosan, such as antibacterial and antitumoral activities [18,19,55]. The degree of deacetylation is defined as the average number of D-glucosamine units in chitosan and plays a key role in various applications of chitosan. Goosen [56] reported that the isolation method used also changes the degree of deacetylation of chitosan from 50% to 95%. The degree of deacetylations of chitosan from different organisms is also different [16] (Table 4.2), and it affects the solubility, viscosity, crystallinity, porosity, and tensile strength of chitosan [63
65]. Chitosan fibers, which have a high affinity to water due to the hydroxyl and the amine groups in their structure, can be used in the production of textiles to be used in underwear and bedding materials or in the production of materials that can be used in face masks in the cosmetic industry [66]. The high degree of deacetylation makes chitosan an effective antimicrobial and antioxidant agent [67,68]. TABLE 4.2 The degree of acetylation and deacetylation of chitin and chitosan produced from different sources. Chitin and chitosan sources

Degree of acetylation (DA) %

Degree of deacetylation (DD) %

References

Shrimp shell

88.5
94.3

81.23

[34,35,40]

Crab shells

78.6
97

83.3
93.3

[34,57,58]

Loligo chenisis (squid)

82

,20

[32]

Grasshopper species

108.5
180.71

64 and 22

[52,59]

Potato beetle (adult and larvae)

108 and 232

82 and 76

[13]

Zophobas morio larvae

82.39
101.39




[60]

Tenebrio molitor (mealworm)




90

[38]

Scorpion

99.3

70.5

[39]

Pill millipedes

93.1 and 93.4

Periwinkle and snail shells

[40] 77 and 60

[61]

Plumatella repens (Bryozoa)

100.5




[41]

Fungal chitosan




78.1
90.2

[62]

Mushroom chitin and chitosan

70

80.29

[50,51]

Handbook of Chitin and Chitosan

98

4. Investigation into the functional properties of cotton, wool, and denim textile materials

TABLE 4.3 Molecular weights of chitosan derived from different organisms. Chitosan sources

Molecular weights (Mw) of chitosans (kDa)

References

Crab shells

483
526 and 1360

[57,70]

Shrimp shell

512.06

[35]

Metapenaeus stebbingi (shrimp)

2.20

[71]

Loligo chenisis (squid)

8454

[32]

Adult and nymph grasshoppers

7.2 and 5.6

[52]

Honey bee

200
250

[72]

Potato beetle (adult and larvae)

2.722 and 2.676

[13]

Scorpion

3.22

[39]

Fungal chitosans

437, 403 and 383

[62]

Molecular weight is another important physicochemical property. Li et al. [69] reported that the molecular weight of natural chitin is around 1000 kDa and that of chitosan varies from 2 to 1200 kDa (Table 4.3). The molecular weight of chitin and chitosan differ according to the species, production method, degree of deacetylation, and degree of polymerization [9,20]. Kumirska et al. [16] reported that molecular weight affects solubility, crystallinity, viscosity, and antimicrobial, antioxidant, and antitumor activities of chitin and chitosan. Mourya and Inamdar [73] stated that high molecular weight and high viscosity may affect the application of chitosan in some cases, and the chitosan is depolymerized to oligosaccharides to overcome this problem. It has been reported that low-molecular-weight chitosan has better antimicrobial activity and higher antioxidant activity against bacteria, yeast, and fungi than other chitosan [74
76]. Chitin has a more crystalline structure than chitosan due to the acetamide groups in its structure [9]. Aranaz et al. [21] emphasized that the crystallinity and purity of chitin is determined by the source (Table 4.4). The authors also point out that purity is an important parameter to be controlled in food and medical applications. The absorption ability of chitin and chitosan is inversely proportional to the crystallinity values [78]. Previous studies reported that chitin and chitosan, which have a low crystalline index, are effectively used in water treatment [21]. Since the solubility of chitin and chitosan directly affects the biological properties, it is an important factor that limits the use of chitin and chitosan in various applications [16]. Because the glucosamine (GlcN) units in the chitosan are more than those of chitin, chitosan dissolves

Handbook of Chitin and Chitosan

99

4.2 Chitin and chitosan

TABLE 4.4

Crystalline index (CrI) values of chitin from different organisms.

Chitin source

Crystalline index value of chitin (%)

References

Shrimp

64.1
89.68

[34,40]

Callinectes sapidus (blue crab)

85

[49]

Loligo chenisis (squid)

62.1

[32]

Palomena prasina and Holotricha parallela (insect)

84.9 and 89.05

[41,77]

Potato beetle (adult and larvae)

76 and 72

[13]

Male and female grasshopper species

75
80

[59]

Scorpion

75.8

[39]

Pill millipedes

85.04 and 85.6

[40]

Plumatella repens (Bryozoa)

60.1

[41]

Mushroom species

58.5
64

[41,43,50]

better than chitin in dilute acid solutions [17] such as formic acid, acetic acid, tartaric acid, and citric acid. Chitin and chitosan with different surface morphologies are used in very different application areas, such as food, medicine, pharmacology, textile, and wastewater treatment, depending on the convenience of their use [21]. Fibrous chitosan is preferred more in the textile industry, whereas porous chitosan is preferred in tissue engineering and drug delivery [21,29,79]. Thermal stability is an important physical feature in terms of the use of chitin and chitosan in thermal therapies and the resistance of chitin and chitosan to various heat treatments [80]. It is necessary to know the thermal decomposition temperature of chitosan used in application areas such as biosensor technology and fireproof garment production. Table 4.5 presents the maximum thermal decomposition temperatures of chitin and chitosan from different organisms. 4.2.3.2 Factors related to synthesis The synthesis method affects and diversifies the product quality of the obtained chitosan. It has been emphasized that chitosan isolated by the biological method is convenient for numerous applications because it is highly viscous and bioactive [5,44,46]. The quality of chitin and chitosan obtained by the biological method is better and homogeneous. Furthermore, chitin produced by this method is more pure, healthy, and harmless to the environment. In textiles, viscosity is important for forming patterns in printing pastes and is regulated with chemicals. Especially in fabrics that come in

Handbook of Chitin and Chitosan

100

4. Investigation into the functional properties of cotton, wool, and denim textile materials

TABLE 4.5 Maximum degradation temperatures of chitin and chitosan from different sources. Chitin and chitosan sources

DTGmax value of chitin ( C)

DTGmax value of chitosan ( C)

References

Krill, crab, and shrimp

380, 380, and 365




[12]

Callinectes sapidus (blue crab)

390

306

[49]

Silkworm crysalites (insect)




300

[36]

Palomena prasina (insect)

386




[41]

Potato beetle (adult and larvae)

379 and 307

289 and 292

[13]

Male and female grasshopper species

350
387




[59]

Scorpion

384

302

[39]

Snail shell and periwinkle chitin

398.90 and 404.41




[61]

Plumatella repens (Bryozoa)

355




[41]

Mushroom species

275
354

229
273.2

[43,51,81]

contact with the body and those used in medical textiles, high-viscosity chitosan produced by biological methods can be used in place of these chemicals. Thus with the decrease in the number and amount of chemicals used in textile printing, more natural and low-cost products can be produced. In chitins isolated by chemical methods, physicochemical properties, which are important in determining the usage areas of chitin and chitosan, may vary according to the synthesis method [20]. Previous studies reported that the physicochemical properties of chitosan solutions, such as molecular weight and degree of acetylation, can be controlled by manipulating the solution conditions (temperature, pH, ionic strength, concentration, solvent) [16]. Gutirrez [82] reported that the prolonged duration of N-deacetylation caused an increase in the number of amino groups in the C-2 position of chitosan, which positively affected the antioxidant activity of chitosan. Shepperd et al. [54] suggested that the flocculant capacity of squid pen chitosan can be controlled by reducing the degree of acetylation. It is very important to provide standard material properties in industrial textiles. For this reason, the use of chemically produced chitosan is appropriate in the production of industrial textiles and composites, as the material properties can be standardized by controlling the synthesis conditions.

Handbook of Chitin and Chitosan

4.2 Chitin and chitosan

101

4.2.4 Antimicrobial activity of chitosan Properties such as biocompatibility, biodegradability, nontoxicity, and antimicrobial activity make chitosan a preferred material for biomedical textile applications. In recent years, studies have concentrated on determining the antimicrobial efficacy of chitosan obtained from different sources and coated on various textile surfaces. Goy et al. [55] reported that chitosan has a bactericidal or bacteriostatic effect and suggested that chitosan can exhibit its antimicrobial efficacy with three different mechanisms. In the first mechanism, chitosan molecules carrying positively charged amine groups have an electrostatic interaction with negatively charged cell membranes of microorganisms [55]. This electrostatic interaction can occur in two different ways. The first is disruption of intracellular osmotic pressure by changing the permeability of the membrane [83], and the second is causing intracellular electrolytes to leak out by hydrolyzing the peptidoglycans in the cell wall of the microorganisms [84,85]. In this case it is suggested that the increase in cationic amine groups will increase the antimicrobial activity of chitosan [86]. The second mechanism is the binding of chitosan to the microorganism DNA by crossing the bacterial cell wall and inhibiting mRNA and protein synthesis [55,84]. The third mechanism is the chelation of metal ions, which is a more effective mechanism, especially at high pH values [55]. It has been reported that chitosan binds to the nutrients required for microorganisms via its amine groups that have active metal binding ability hence, preventing microbial growth [87,88]. In this chapter, antimicrobial activity of shrimp chitosan on various bacteria and fungi species was investigated using the microbroth dilution method. 4.2.4.1 Antimicrobial assay of shrimp chitosan The antimicrobial activity of chitosan obtained from shrimp against Gram-negative bacteria Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19115, and Salmonella typhimurium ATCC 14028, Gram-positive bacterium Bacillus cereus ATCC 11778, and yeast Candida albicans ATCC 10231 was investigated by the CLSI (Clinical Laboratory Standards Institute) microbroth dilution method [89]. Microorganisms were incubated in tryptic soy broth (TSB) for 24 h at 37 C and adjusted to 0.5 on the McFarland scale. Ampicillin and gentamicin were used in bacterial cultures as antibiotic controls, and amphotericin B was used in yeast cultures. For sterilization, antibiotics and solute stock solutions were filtered through a 0.45 sterile filter. A mixture of distilled water and 1% acetic acid was used as the solvent. Meanwhile, antibiotic concentrations were set at 1000 μg/mL, and the antimicrobial agent concentrations ranged from 31.25 to 1000 μg/mL.

Handbook of Chitin and Chitosan

102

4. Investigation into the functional properties of cotton, wool, and denim textile materials

Bacteria and yeast cultures were inoculated into each well. All microplates were incubated at 37 C for 24 and 48 h. Absorbances were measured at 600 nm, and the vitality rates of microorganisms were determined. 4.2.4.2 Antimicrobial analysis results of shrimp chitosan The viability rates of microorganisms incubated with shrimp chitosan solutions were measured after 24 h and 48 h of contact times, and the antimicrobial activities of chitosan at different concentrations on these microorganisms were determined (Tables 4.6 and 4.7 and Figs. 4.4 and 4.5). After 24 h of incubation with chitosan, the percent viabilities of E. coli and S. typhimurium were lowest at a concentration of 1000 μg/mL, while they were the highest at a concentration of 125 μg/mL. A chitosan concentration TABLE 4.6 Percent viability of chitosan-treated microorganisms after 24 h of incubation. Microorganism species

Viability (%) Concentration

E. coli

S. typhimurium

S. aureus

L. monocytogenes

B. cereus

C. albicans

1000 μg/mL

12,870

25,558

105,303

75,649

23,943

124,701

500 μg/mL

43,944

65,034

111,020

114,864

53,443

70,243

250 μg/mL

39,415

47,993

72,349

78,906

42,827

69,775

125 μg/mL

77,260

78,969

50,509

55,807

70,020

66,021

62.5 μg/mL

67,327

68,541

120,428

47,902

80,779

84,181

31.25 μg/mL

61,851

64,806

119,497

49,032

77,910

117,631

Antibiotic

19,059

22,490

36,871

12,298

14,131

42,310

Control

100

100

100

100

100

100

TABLE 4.7 Percent viability of the microorganisms treated with chitosan after 48 h of incubation. Microorganism species

Viability (%) Concentration

E. coli

S. thphimurium

S. aureus

L. monocytogenes

B. cereus

C. albicans

1000 μg/mL

18,269

64,254

97,669

173,733

69,306

100,730

500 μg/mL

52,049

96,559

82,824

196,290

73,531

47,943

250 μg/mL

51,158

75,611

54,819

122,619

69,520

57,044

125 μg/mL

95,630

128,224

36,261

85,148

129,222

50,615

62.5 μg/mL

92,660

119,519

114,994

74,467

144,111

93,950

31.25 μg/mL

85,015

111,445

108,173

80,679

135,773

115,635

Antibiotic

20,636

34,434

44,478

24,586

22,612

43,998

Control

100

100

100

100

100

100

Handbook of Chitin and Chitosan

103

4.2 Chitin and chitosan

% viability on 24th hour 140 120 100 80 60 40 20 A nt ib io tic Co nt ro l

μg /m L .2

31

62

S. aureus

5

μg /m L

μg /m L 5

S. typhimurium

.5

μg /m L 0

25

E. coli

12

μg /m L 0

50

10

00

μg /m L

0

B. cereus

L. monocytogenes

C. albicans

FIGURE 4.4 Percent viability of bacteria after 24 hours of incubation in various chitosan solutions.

% viability on 48th hour 250 200 150 100 50

S. typhimurium

S. aureus

A nt ib io tic Co nt ro l

31

.2

5

μg /m L

μg /m L

62

.5

μg /m L

12

5

μg /m L 0

E. coli

25

μg /m L 0

50

10

00

μg /m L

0

L. monocytogenes

B. cereus

C. albicans

FIGURE 4.5 Percent viability of bacteria after 48 hours of incubation in various chitosan solutions.

of 1000 μg/mL appears to be more effective on E. coli than the antibiotics. In contrast to E. coli and S. typhimurium, the most effective chitosan concentration on S. aureus was found to be 125 μg/mL, whereas the concentration at which the antibacterial activity was minimal was 62.5 μg/mL. For L. monocytogenes, the situation was different. The most effective antibacterial concentration for this bacterium was 62.5 μg/mL, while the least effective concentration was 500 μg/mL. The chitosan concentration with the highest antibacterial effect on B. cereus was 1000 μg/mL, as in E. coli and S. typhimurium, while the lowest antibacterial activity was observed

Handbook of Chitin and Chitosan

104

4. Investigation into the functional properties of cotton, wool, and denim textile materials

at a concentration of 62.5 μg/mL, as in S. aureus and L. monocytogenes. A chitosan concentration of 125 μg/mL had the highest antifungal activity on yeast C. albicans. Unlike for the bacterial species, the lowest antifungal activity was observed at the highest chitosan concentration. After 48 h of incubation, chitosan solutions at the same concentration had similar antimicrobial activity on the same bacteria and fungus species as in 24 h of contact time. However, the vitality of the bacteria was higher than those with 24 h of contact time. The most effective concentration on the yeast C. albicans was 500 μg/mL. In contrast to 24-h contact time, the lowest antimicrobial activity was observed at the lower concentrations (31.25, 62.5, and 125 μg/mL). In addition, at the end of the 48-h incubation period, the 1000 μg/mL chitosan solution had higher antimicrobial activity against E. coli than the antibiotics, whereas the concentration of 125 μg/mL had higher activity against S. aureus. Kaya et al. [49] determined the antimicrobial activity of blue crab chitosan against six human pathogens and three fish pathogens by disk diffusion and microdilution methods. The authors reported that the lowest concentration of crab chitosan showed higher antibacterial activity (zone diameter; 20.21 mm) against Bacillus subtilis RSKK 244 than another strain of the same bacterium. The authors also stated that the antimicrobial activity of chitosan differs according to the species, and even the strains, of the microorganism tested, as well as the degree of deacetylation of chitosan. Kaya et al. [39] tested the antimicrobial activity of low-molecular-weight scorpion chitosan (LMWSC) against nine pathogenic microorganisms (seven bacteria and two yeasts) and compared the results with those of mediummolecular-weight commercial chitosan (MMWCC). The results of the study showed that the inhibitory effect of the LMWSC on pathogen microorganisms L. monocytogenes and C. albicans was higher than the MMWCC, and the scorpion chitosan showed species-specific antimicrobial activity. However, the paper revealed that MMWCC had a higher inhibitory effect on the bacterium B. subtilis compared to LMWSC. The disk diffusion method has shown that both types of chitosan were more effective on bacteria B. subtilis and Salmonella enteritidis than the commonly used commercial antibiotic gentamicin (CN, 10 mg/disk). As a result, shrimp chitosan showed antimicrobial activity on all microorganisms; however, antimicrobial activity was found to differ according to the concentration of chitosan, duration of contact, and bacterial and yeast species. The antibacterial effect of shrimp chitosan was specific to bacterial species. These results were similar to those presented by Kaya et al. [39,49]. No significant correlation was observed between the concentrations of chitosan solutions and the antimicrobial activity of chitosan. In contrast, the effective concentration of chitosan was different according to different bacteria and yeast species. Different concentrations of shrimp chitosan were more effective than antibiotics against some bacteria. Therefore in order to ensure an effective

Handbook of Chitin and Chitosan

4.3 Application techniques and methods of chitosan to textile products

105

antimicrobial activity, it is necessary to determine the effective chitosan concentration and chitosan type for each microorganism. In addition, when the antibacterial activity of textile products is determined, chitosan concentration and contact time should be indicated.

4.3 Application techniques and methods of chitosan to textile products The processes used to improve the use, feel, appearance, and functional properties of textile materials are called finishing processes. Finishing processes can be performed in two ways: as mechanical and physical finishing processes and as chemical finishing processes. Chemical finishing is the application of chemical substances to the fabric with the purpose of providing the desired functional properties. Water repellency, dirt repellency, flame retardancy, and antimicrobial finishing processes are some of the chemical finishing products [90]. The application techniques and methods of chitosan used in chemical finishing processes in the production of textile products varies according to the type of product to be used and the intended use. As it is a viscous solution, the most suitable method for the use of chitin and chitosan in chemical finishing is to coat with a stripper. Coating method: In functional textile production, coatings are processes applied with biomaterials or chemicals that can be applied to the surface of the fabric and that can be produced in the form of a viscous liquid. The drying process, which allows the coating to form a thin film on the fabric surface, is followed by the fixing process that allows the film to be bonded to the fabric. With this method, it is possible to produce an uninterrupted, full-length product [91]. Coating with a stripper: In this method, the viscous coating solution is directly and uniformly applied to the fabric with the help of a fixed stripper. Depending on the type of fabric and solution used, the strippers may be pointed or rounded.

4.3.1 Coating of shrimp chitosan on textile surfaces 4.3.1.1 Materials and methods Textile samples: Four types of fabrics were used: 1. 2. 3. 4.

Patterned fabric woven from dyed 100% wool yarn White woven fabric made of pretreated 100% cotton yarn Raw woven fabric made of 100% cotton yarn Dyed denim fabric made of 99% cotton and 1% elastane yarn (the warp yarn was dyed with blue indigo and the weft thread was not dyed).

Handbook of Chitin and Chitosan

106

4. Investigation into the functional properties of cotton, wool, and denim textile materials

4.3.1.1.1 Acrylic cross-binder resin

The acrylic cross-binder resin is a chemical substance that strengthens the bond between the finishing agent used and the fiber. In this study, an alkylphenol ethoxylate (APEO)-free and formaldehyde-free, elastic waterbased emulsion, which is self-cross-linking at low temperatures and developed for coating and textile printing applications, was used to strengthen the bond between chitosan and the fabric. This product is characterized by high chemical and mechanical stability and good coating properties. The acrylic cross-linker resin (Bind ELS 35) used in this study is an acrylic copolymer emulsion. This product is not classified as dangerous, according to directives 67/548/EEC and 1999/45/EC. Furthermore, according to 67/548/EEC, it does not contain any substances harmful to human health or the environment. 4.3.1.1.2 Preparation of shrimp chitosan for application

The solution containing 5% chitosan and 2% CH3COOH was stirred on a magnetic stirrer at 224 rpm at 25 C for 24 h. Chitosan obtained from shrimp was used to coat fabrics in two different forms to obtain durable antimicrobial properties. 4.3.1.1.3 Application of shrimp chitosan on textile surfaces without crosslinker

Chitosan solutions at 5% concentration dissolved in 2% CH3COOH were applied to the cotton, wool, raw, dyed, and printed fabric samples using a sliding coating method, then dried in the oven at 80 C and fixed for 2 min at 150 C. The coating process was carried out on a flat surface by means of a stripper (Fig. 4.6). 4.3.1.1.4 Application of shrimp chitosan on textile surfaces with acrylic crosslinker (bind ELS 35)

We used an acrylic binder that can be cross-linked at low temperatures to strengthen the bonding of shrimp-derived chitosan with fabric samples and to increase the resistance of antimicrobial activity to multiple household soap washes. Solutions containing 5% chitosan dissolved in 2% CH3COOH and 5% acrylic binder were applied to the cotton, wool, raw, dyed, and printed fabric samples using a sliding coating (A)

(B)

FIGURE 4.6 Images of chitosan application on the multifiber fabric. (A) Before (B) After.

Handbook of Chitin and Chitosan

4.4 Properties of the chitosan-treated textile products

107

FIGURE 4.7 Beginning of the application of chitosan containing acrylic binder on the 100% wool fabric.

method (Fig. 4.7). Then, the coated fabrics were dried in an oven at 80 C and fixed at 150 C for 2 min.

4.4 Properties of the chitosan-treated textile products It is difficult for the chitosan to be fixed to and remain on textile surfaces for a long time. This limits the use of chitosan, which is a natural and healthy material, in textiles. Therefore various cross-linking agents were used to eliminate this limitation. For this reason, glutaraldehyde, potassium permanganate, citric acid, butane tetracarboxylic acid, and sodium hypophosphite have been used as various cross-linking agents [9]. Acrylic binder, which has a high cross-linking ability and is widely used in the textile industry, was used for the first time in this study as a cross-linking agent for chitosan, and its bonding properties were tested. In order to reveal the effect of cross-linkers more clearly, 5% acrylic binder was mixed with 5% chitosan in the same ratio and coated on cotton, wool, and denim textile products using a stripper.

4.4.1 Surface properties Surface analysis of chitosan-coated dyed denim, undyed woven raw fabric, patterned wool fabric, and pretreated white cotton fabric samples were made in the central laboratory of Trakya University (TUTAGEM). The bond formation of shrimp chitosan on fabric samples was visualized using a Perkin Elmer ATR FTIR device and ZEISS EVO LS 10 SEM microscope. The presence of chitosan and acrylic binder on the coated fabrics was determined with a BRUKER X FLASH 6110 XRD device. 4.4.1.1 FTIR spectroscopy analysis results The FTIR spectra of the shrimp chitosan and acrylic binder used in fabric coating are shown in Fig. 4.8.

Handbook of Chitin and Chitosan

4000

3500

3000

2500

2000

Wave number (cm

1731 1638 1449 1379 1253 1160 1115 1025

2129

(B)

3351

2960

1075 1027 986 892

(A)

661

1651 1589 1422 1374 1149

Transmittance (%)

2868

4. Investigation into the functional properties of cotton, wool, and denim textile materials

3354 3286

108

1500

1000 500

–1)

FIGURE 4.8 FT-IR spectra of (A) shrimp chitosan, and (B) acrylic binder.

The ATR-FTIR spectra of cotton and wool surfaces coated with chitosan only and chitosan 1 cross-linker are given in Fig. 4.9. The presence of the characteristic peaks of both chitosan (Fig. 4.8A) and acrylic binder (Fig. 4.8B) were observed in the fabric coating. This shows that the chitosan and chitosan 1 cross-linker is coated on 100% cotton and 100% wool fabric surfaces. These results support the SEM images. The characteristic chitosan peak around 1650 cm21 remained the same in almost all samples, whereas the peak around 1590 cm21 decreased to around 1550 cm21. The most severe peak around 1731 cm21 of the acrylic binder was observed in all other coated fabrics excluding only chitosan-coated fabric samples, while the peak around 1638 cm21 was lost in the chitosan 1 acrylic cross-linker-coated samples. This change in peaks indicates that chitosan binds with acrylic binder and cellulose fibers. 4.4.1.2 Scanning electron microscope/EDX analysis results 4.4.1.2.1 Raw calico fabric

This is the untreated fabric from the weaving loom. Since the fabric was woven from 100% cotton yarns, it has natural and chemical impurities on it, such as sizing, oil, etc. SEM images of raw calico fabric samples showed that the coating with chitosan and acrylic binder was not smooth and it completely remained on the fabric surface (Fig. 4.10). The fibers were not visible (mag. 500 3 ). This was due to the hydrophobicity of the raw fabric. 4.4.1.2.2 White calico fabric

Processes initially applied for preparing the product for dyeing-finishing in textile finishing are called prefinishing processes. Thanks to the pretreatment process, the success of the textile finishing is increased with applications

Handbook of Chitin and Chitosan

109

700

851

1153 1112 1026 700

851

1154 1063 1022

700

851

1154 1023 1154 1023

1378 1251 1020

1641 1541 1377 1318 1152

2870

3249

(E)

1378 1251

1657 1554 1657 1554

(D)

1729

2923

3281

1729

2923

3281

Transmittance (%)

(C)

1378 1250

1652 1553

(B)

1730

2928

3263

1728

2917

3378

(A)

851 759 701

1657 1543 1447 1378 1241

4.4 Properties of the chitosan-treated textile products

4000

3500

3000

2500

2000

Wave number

(cm–1)

1500

1000 500

FIGURE 4.9 FTIR spectra of fabric samples coated with chitosan and acrylic binder. (A) Acrylic binder 1 chitosan-coated white fabric, (B) Acrylic binder 1 chitosan-coated raw fabric, (C) Acrylic binder 1 chitosan-coated dyed denim fabric, (D) Acrylic binder 1 chitosan-coated woolen fabric, and (E) Chitosan-coated dyed denim fabric.

such as precleaning to remove the dirt and oil from the fabric and kieringbleaching to make the fabric hydrophilic and white. Since our sample is pretreated and cleaned, SEM images also confirmed that the coating including acrylic binder and chitosan on the fabric surface was homogeneous and smooth (Fig. 4.11). Fibers were visible despite the coating (mag. 500 3 ). 4.4.1.2.3 Dyed denim fabric

Coloring of the textile surfaces and textile dyeing processes are carried out by treating the product with the dyestuff solution and various

Handbook of Chitin and Chitosan

110

4. Investigation into the functional properties of cotton, wool, and denim textile materials

20 μm

EHT = 10.00 kV WD = 7.0 mm

Detector = SE1 Mag = 500 X

Date : 26 dec 2018 Time : 12:45:34

FIGURE 4.10 SEM image of raw calico fabric coated with chitosan and acrylic binder.

20 μm

EHT = 10.00 kV WD = 10.0 mm

Detector = SE1 Mag = 500 X

Date : 26 dec 2018 Time : 13:21:37

FIGURE 4.11 SEM image of undyed calico fabric coated with chitosan and acrylic binder.

auxiliary chemicals (wetting agent, salt, alkali, and acid). In dyeing, water-dissolved or dispersed dyestuff is absorbed by the textile product. Since denim fabrics are made of indigo-dyed yarn, they have a surface consisting of dye and auxiliary chemicals. As can be seen in Fig. 4.12A, the chitosan was able to penetrate into the fabric, and the fibers can be seen on the surface (mag. 100 3 ). The carboxyl groups of chitosan were bound with the hydroxyl groups in cellulose, and chitosan could diffuse into the fabric fibers. The coating with chitosan and the acrylic binder

Handbook of Chitin and Chitosan

111

4.4 Properties of the chitosan-treated textile products

A

EHT = 10.00 kV WD = 8.0 mm

B

Detector = SE1 Mag = 100 X

EHT = 10.00 kV WD = 9.0 mm

Detector = SE1 Mag = 100 X

FIGURE 4.12 SEM images of (A) the only chitosan-coated denim fabric, and (B) denim fabric coated with chitosan and acrylic binder.

remained on the surface of the fabric (Fig. 4.12B). The fibers were not visible and the surface was completely covered (mag. 100 3 ). As a result of the bonding of the acrylic binder with chitosan, the molecular weight increased and the coating could not diffuse into the fiber. This is also supported by the FTIR analysis shown in Fig. 4.9. 4.4.1.2.4 Woolen fabric

This is a plaid, tight, and cross-woven fabric, with one surface that is pile. Although the fabric surface was pilous, chitosan was able to penetrate into the fibers (Fig. 4.13A, mag. 500 3 ). Fibers were visible. Chitosan and crosslinker together formed a thicker layer on the fabric surface (Fig. 4.13B, mag. 500 3 ). However, unlike denim and raw calico fabrics, the coating with chitosan and acrylic binder completely remained on the fabric surface, but still, it was not too dense to see the fibers. It was observed that the coating was compatible with the wool fiber and penetrated into the wool fiber. When considering the SEM images of fabric samples, it was concluded that chitosan can be used to coat the fabric surface with the riding-coating technique. In the coatings with chitosan only, chitosan was able to penetrate into the fiber, regardless of the fiber type and the textile production processes that the fabric was subjected to before coating. The fibers were visible despite the coating layer. In chitosan coatings made with acrylic binders, the coating remained on the surface of denim and raw calico fabric samples. The surface was completely covered with chitosan and cross-linker, and the fibers were not visible. However, chitosan and acrylic binder were able to penetrate into the fiber from the fabric surface in pretreated, uncolored fabric samples and patterned woolen fabric samples. Fibers were visible on the surface. The results show that the type of fabric, the chemicals used in the

Handbook of Chitin and Chitosan

112

4. Investigation into the functional properties of cotton, wool, and denim textile materials

production of fabrics, or the impurities on the fabric affect the penetration of chitosan into the fabric. The elemental analyses of coatings on cotton, wool, and denim fabrics were done with an EDX device integrated into the SEM device and the amount of carbon, oxygen, and other elements found in the coatings were determined as percentage by weigh (Figs. 4.14
4.16). EDX analysis was used in surface characterization of coatings to demonstrate that chitosan 1 cross-linker can bond together on the fabric surface and to identify the components of the cross-linker. According to the EDX analysis results, chitosan 1 cross-linker-coated fabric samples were found to have a higher carbon content than chitosan-coated fabric samples. Oxygen contents of the chitosan-coated fabric samples were higher than those of chitosan 1 cross-linker-coated fabric samples. In chitosan coatings containing acrylic cross-linkers, the highest sulfur content was recorded on dyed denim fabric, while no sulfur was found in chitosan-coated fabric samples. A

20 μm

B

EHT = 10.00 kV WD = 7.5 mm

Detector = SE1 Mag = 500 X

Date : 26 dec 2018 Time : 13:48:33

20 μm

EHT = 10.00 kV WD = 7.5 mm

Detector = SE1 Mag = 500 X

Date : 26 dec 2018 Time : 14:42:14

FIGURE 4.13 SEM images of (A) chitosan-coated wool fabric, and (B) woolen fabric coated with chitosan and acrylic binder.

FIGURE 4.14 EDX spectrum and distribution of elements of (A) white calico fabric coated with chitosan 1 crosslinker, and (B) raw calico fabric coated with chitosan 1 crosslinker.

Handbook of Chitin and Chitosan

4.4 Properties of the chitosan-treated textile products

113

FIGURE 4.15

EDX spectrum and distribution of elements of (A) chitosan-coated woolen fabric, and (B) chitosan 1 acrylic binder-coated woolen fabric.

FIGURE 4.16 EDX spectrum and distribution of elements of dyed denim fabric coated with (A) chitosan, and (B) chitosan 1 acrylic binder.

4.4.2 Antibacterial activity Microorganisms are found in the air we breathe, in our bodies, in the soil, and on all surfaces we touch. Bacteria can cause health problems, such as infection, disease, and odor, as well as the degradation and staining of textile products. Natural fibers like cotton are more susceptible to microorganismbased problems than synthetic fibers due to their porous, hydrophilic nature. On the other hand, the human body provides heat, moisture, and nutrients to bacteria on clothes that are in direct contact with it, so it offers an excellent environment and favorable conditions for bacterial growth. Antimicrobial substances can be applied to the textile fibers in various ways. New antimicrobial application methods and strategies are being developed to be more effective and durable. However, if antimicrobial agents with adverse dermal effects are used, such a system cannot be used in applications requiring skin contact. Research on developing environment-friendly antimicrobial agents based on natural products for use in textile applications has received worldwide attention.

Handbook of Chitin and Chitosan

114

4. Investigation into the functional properties of cotton, wool, and denim textile materials

Chitosan has antimicrobial activity against unwanted bacteria and fungi due to its polycationic structure. It can be used as an antimicrobial agent in a wide range of sectors where hygiene is important, such as food, health, and textile. In addition, several chitosan derivatives were also synthesized and used as antimicrobial agents in textiles [92]. These include chitooligosaccharide, N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride, N-p-(N-methylpyridine) methylated chitosan chloride, and N-4-(3-(trimethylammonio) propoxy) benzylated chitosan chloride. Most of these derivatives contain a quaternary ammonium group that provides antimicrobial activity. Another derivative is O-acrylamidomethyl-N((2-hydroxyl-3-trimethylammonium) propyl) chitosan chloride, in which the acrylamidomethyl group fiber is reactive and forms a covalent bond with cellulose in cotton, and excellent durability is achieved. The use of chitosan and its derivatives as antimicrobial agents in textile materials has attracted great interest. Chitosan can be chemically bound on cotton fabric using cross-linking agents, such as glutaric dialdehyde and polycarboxylic acids. When the cotton fabric is padded with a mixture of chitosan and citric acid and then cured at a high temperature, chitosan citrate forms a nonformaldehydebased, durable layer with antimicrobial properties for the fabric [93
95]. As a multifunctional textile finishing agent, chitosan can provide functions such as antimicrobial activity, improving dyeing properties, and antistatic activity in a single application. However, the disadvantage in these applications is that chitosan at a high concentration forms a film, reducing the air permeability on the surface of the fabric and hardening the fabric after application. New techniques developed for chitosan application should allow the production of textile products that can benefit from the excellent antimicrobial properties of chitosan without losing the textile properties desired by the users [96]. 4.4.2.1 Antibacterial analysis of textile products coated with chitosan and chitosan 1 acrylic binding resin (bind ELS 35) The antibacterial activity of the fabric samples finished with chitosan and chitosan 1 acrylic binder against S. aureus ATCC 6538 was investigated by AATCC 147 and AATCC 100 testing methods [97,98]. S. aureus is a Gram-positive coccus bacterium with a diameter of 0.5
1.5 μm. It is an inert, nonspore-forming, and facultative anaerobic bacterium. S. aureus is a human pathogenic bacterium that has become increasingly important in recent years due to the increase in antibiotic resistance. It has been reported that multidrug-resistant strains of this bacterium occur due to excessive antibiotic use [99]. S. aureus is a pathogen that causes a wide range of clinical infections; approximately 30% of the human population has been colonized by S. aureus [100].

Handbook of Chitin and Chitosan

4.4 Properties of the chitosan-treated textile products

115

This bacterium is found naturally in the skin and in the nasopharynx, however, it may cause local infections in the skin, nose, urethra, vagina, and gastrointestinal tract, most of which are small and nonlifethreatening [101]. 4.4.2.1.1 AATCC 147 (antibacterial activity assessment of textile materials)

This is a qualitative method applied to determine the bacteriostatic effect on textile materials. It is used to roughly detect the activity in the growth of the inoculated organism. In addition, the presence of antimicrobial agent that does not make chemical bonding with textile samples and the durability of antibacterial agent on the fabric surface after washing can also be determined by this method [102]. Before the analysis, chitosan and chitosan 1 cross-linker-coated fabric samples were sterilized stored at 120 C, 1.5 atm for 20 min. The appropriate bacterial concentration selected for the assay was diluted to 2 3 105. 25 3 50 mm textile samples coated with chitosan and chitosan 1 acrylic binder whose antibacterial activity will be tested were placed on the agar surface plotted with the inoculum of S. aureus ATCC 6538. Then, they were incubated at 37 C for 24 h. After incubation, it was checked whether or not an inhibition zone (mm) was formed under and around the fabrics. While this method is suitable for fine woven fabrics, it does not always give accurate results because it does not allow for a close contact with the agar in curled or bulky fabrics. Quantitative methods are more suitable for these type of fabrics. Therefore the antimicrobial activity analyses of the fabric samples in this study were repeated with the AATCC 100 method and the results were compared with each other. 4.4.2.1.2 AATCC 100 (assessment of antibacterial finishes on textile materials)

The AATCC 100 is a quantitative method used to detect bactericidal effects. It is used to evaluate the degree of antibacterial activity of coatings on textile materials. The fabric samples were circularly cut to have a diameter of 48 mm. Then, they were sterilized for 15 min at 121 C and 1.5 atm prior to the experiment. An untreated control sample was also analyzed. The prepared fabric samples were inoculated with 1 mL of a solution containing 2 3 105 cfu/mL bacteria in screw-capped tubes. Samples were studied at 0 and 24 h of contact time. At the end of the contact period, the fabric samples were removed from the tubes, placed in Mu¨ller
Hinton liquid medium, and mixed by vortexing. Thus the bacteria colonized to the surface of the fabric were allowed to pass into the liquid medium. In order to count the bacteria, the medium was diluted to suitable dilutions and cultivated on agar

Handbook of Chitin and Chitosan

116

4. Investigation into the functional properties of cotton, wool, and denim textile materials

medium. Petri dishes inoculated with S. aureus were incubated at 37 C for 24 h. The number of bacteria colonies was multiplied by the dilution factor, and the amount of reproduction was determined. The number of bacteria in the samples was determined and the percent bacterial reduction (R%) values were calculated according to the formula:   ðB 2 AÞ R% 5 3 100 B where A is the number of bacteria recovered from the inoculated treated test specimen swatches incubated over the desired contact period (24 h), and B is the number of bacteria recovered from the test specimen swatches immediately after inoculation (0 h). 4.4.2.2 Testing the resistance of antibacterial activity to washing with soap solution The resistance of the antibacterial activity of shrimp chitosan coated on textile surface to washing with soap solution was investigated. For this purpose, chitosan and chitosan 1 acrylic binder resin (Bind ELS 35) were applied to the pretreated white fabric samples. In addition, considering that the properties of the cross-linker can affect the washing resistance of antibacterial activity, we also chitosan-coated pretreated white fabric samples together with a different cross-linker, Special Coating & Lamination Binder (KEMILINE NW 160). Fabric samples coated with chitosan and cross-linkers were then washed once with soap solution using the domestic washing method described below. Fabric samples coated with solutions containing 5% chitosan or 5% chitosan 1 5% acrylic binders were washed at 40 C using 4 g/L granular soap (suitable for automatic washing machines). After washing, the fabric samples were rinsed for 10 min and dried at 60 C. Washed and dried fabric samples were tested for antibacterial activity in textile samples according to the AATCC 147: 2016 standard. 4.4.2.3 Antibacterial activity results of chitosan-coated textile products In this section, chitosan obtained from shrimp was used to coat dyed denim fabric, undyed woven raw fabric, yarn dyed wool fabric, and pretreated white cotton fabric samples, both with acrylic binder and without acrylic binder. The antibacterial properties of chitosan-coated fabric samples were examined in accredited test laboratories and in accordance with international standards. The results of the antimicrobial analyses of fabric samples coated with chitosan and acrylic binder according to the AATCC 100 and AATCC 147 methods are presented in Table 4.8 and Fig. 4.17.

Handbook of Chitin and Chitosan

117

4.4 Properties of the chitosan-treated textile products

TABLE 4.8 Antimicrobial analysis results of the chitosan and chitosan 1 crosslinker-coated different types of fabrics (Fabrics 1: patterned fabric woven from 100% wool dyed yarn and coated with chitosan 1 cross-linker, 2: pretreated white woven fabric made of 100% cotton yarn and coated with chitosan 1 cross-linker, 3: raw woven fabric produced from 100% cotton yarn, coated with chitosan 1 cross-linker, 4: dyed denim fabric produced from 99% cotton and 1% elastane yarn coated with chitosan 1 cross-linker, 5: patterned fabric woven from 100% wool dyed yarn coated with chitosan, 6: dyed denim fabric produced from 99% cotton and 1% elastane yarn and coated with chitosan). Methods

AATCC 147

Fabric type

Bacterial growth under the fabric and inhibition zone formation

Number of bacteria at 0 h contact time (cfu/ mL)

Number of bacteria after 24 h contact time (cfu/mL)

%R

No

1.43 3 10

4 3 10

97.20

No

1.43 3 10

,100

No

1.43 3 10

8.4 3 10

41.26

No

1.43 3 10

,100

99.99

No

1.43 3 10

,100

No

1.43 3 10

3 3 10

Fabric 1 Fabric 2 Fabric 3 Fabric 4 Fabric 5 Fabric 6

AATCC 100

5 5 5 5 5 5

3

99.99 4

99.99 8

0

According to the AATCC 147 test method, after the 24-h contact of chitosan and the chitosan 1 acrylic binder coated all fabric samples with S. aureus, no bacterial growth was observed under the fabrics and no inhibition zone was formed around fabrics (Table 4.8 and Fig. 4.17). Palamutc¸u et al. [103] suggested that there is no organism growth under the fabric sample if the antimicrobial agent was chemically bonded with the fabric, but that microorganisms are expected to grow around the fabric. Our result indicates that the chitosan was bound to all the fabric samples both alone and together with acrylic binder. Chitosan is compatible with natural fibers, such as cotton and wool, and can bind with them. The carboxyl group (
COOH) of the chitosan and the hydroxyl group (
OH) of the cellulose form a fiber bond and chitosan is tightly coated on the fabric. This prevents chitosan from diffusing into the environment. The R values obtained according to the AATCC 100 method used for quantitative determination of the antibacterial activity of the fabric samples are rated as excellent if R $ 99.99, good if 99 , R , 99.99, and acceptable if 0 , R , 99 [103]. At the end of the 24 h of contact time with S. aureus, the antibacterial activity of the 100% woolen fabric sample was acceptable (97.20%) and the number of cfu was less than 100. The antibacterial activity of the

Handbook of Chitin and Chitosan

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

(J)

(K)

(L)

FIGURE 4.17 Antimicrobial analysis results of the chitosan and chitosan 1 crosslinker coated different types of fabrics according to AATCC147 (pictures on the left) and AATCC 100 (pictures on the right) methods. (a and b: patterned wool fabric coated with chitosan 1 crosslinker, c and d: pretreated white cotton fabric coated with chitosan 1 crosslinker, e and f: raw cotton fabric coated with chitosan 1 crosslinker, g and h; dyed denim fabric coated with chitosan 1 crosslinker, i and j; patterned wool fabric coated with chitosan, k and l: dyed denim fabric coated with chitosan).

4.4 Properties of the chitosan-treated textile products

119

pretreated 100% cotton fabric sample coated with chitosan 1 cross-linker was very high (99.99%), and the number of cfu dropped below 100 after 24 h contact time with S. aureus. The dyed denim fabric coated with chitosan 1 cross-linker had a very high antibacterial activity (99.99%) and after 24 h of contact with S. aureus, the number of cfu fell below 100. However, the antibacterial activity of raw woven fabric produced from 100% cotton yarn, coated with chitosan 1 cross-linker against S. aureus was found to be minimal (41.26%) compared to other fabric samples. It is thought that the antibacterial activity of the raw fabric was low due to both the structure of the fabric and the different chemicals coming from the yarn and weaving process. As is observed in the raw fabric sample, the chemicals on the fabric affect the antibacterial activity of chitosan. The antibacterial activity of chitosan coated on textile surfaces varied according to the fabric type. The chitosan-coated patterned fabric woven from 100% wool dyed yarn showed very good antibacterial activity (99.99%) against S. aureus. At the end of the 24 h of contact time, the number of cfu fell below 100. The dyed denim fabric produced from 99% cotton and 1% elastane yarn and coated with chitosan showed no antibacterial activity (0%), and after 24 h of contact time with S. aureus, the number of cfu increased from 1.43 3 105 to 3 3 108 (Table 4.8). The uncoated fabric samples showed no antibacterial activity, and bacterial growth was observed under the fabric sample. Control samples showed high antibacterial activity (99.99%) and there was no bacterial growth under the sample, and an inhibition zone occurred around the sample. Overall, various fabric samples with different coatings were found to show significant antimicrobial activity against the human pathogen bacterium S. aureus. The antibacterial activity of chitosan was reduced due to the structure of the raw fabric and dyed denim fabric made of cotton and the impurities on the fabric surfaces. Thus the results revealed that the impurities and chemicals on the fabric’s surface and fabric type affect the antibacterial activity of chitosan. Scacchetti et al. [104] tested the antimicrobial activity of cotton fabric coated with chitosan
zeolite (CS-SZ) composite against two bacteria (E. coli and S. aureus) and two fungi (C. albicans and Trichophyton rubrum) using the AATCC 100: 2012 test method and evaluated the results according to percent reduction rates. Although, the CS-SZ composite showed antimicrobial activity against both types of microorganisms, it was more effective on bacteria than fungi. These results were similar to the antibacterial activity of cotton, wool, and denim fabrics coated with chitosan and chitosan 1 cross-linker against S. aureus in this study. In a study by Gaffer et al. [105], 1% (w/v) chitosan powder and sulfadimidine dye mixture (0.5 g) were suspended in acetic acid (2% v/v), then the mixture was vigorously stirred. The resulting solution was

Handbook of Chitin and Chitosan

120

4. Investigation into the functional properties of cotton, wool, and denim textile materials

used to impregnate fabric samples, and the fabric was dried at 80 C, then fixed at 180 C for 4 min. After these processes, the fabric sample was tested for antibacterial activity against E. coli and S. aureus bacteria. The treated cotton fabrics showed a greater efficacy against S. aureus than against E. coli. S¸ ahan and Demir [106] applied synthesized nanochitosan particles to woolen fabrics and compared the samples to normal chitosan in terms of their properties. In addition, silver-loaded nanochitosan particles were applied by different methods and examined for their antibacterial activities. The antibacterial activity of both chitosan and nanochitosan were great against Gram-positive (S. aureus) and Gram-negative bacteria (E. coli, Klebsiella pneumoniae). The antibacterial efficacy of nanochitosan was greater than normal chitosan due to the larger surface area and the presence of a greater number of amino groups that were able to interact with bacteria and fungi. 4.4.2.4 The evaluation of antibacterial activity after washing The antimicrobial activity of pretreated white fabric samples coated with chitosan only, chitosan 1 acrylic binder, or special coating and lamination binder 1 chitosan were analyzed after washing once with soap. AATCC 147 test results showed that pretreated white fabric samples that were coated with chitosan only and chitosan 1 acrylic binder (Bind ELS 35) had lost their antibacterial activity against S. aureus after washing. The antimicrobial effect against S. aureus of the pretreated white fabric sample coated with a special coating and lamination binder 1 chitosan continued after one wash. Therefore the antibacterial efficacy of fabrics coated with chitosan only was not resistant to washing with a soap solution at 40 C. For an antibacterial effect that is resistant to washing, chitosan must be bonded to the fabric with the help of binders; however, this wash resistance varies according to the properties of the binder used. In a similar study, a polyurethane binder was used to improve the washing resistance of the antibacterial effect of chitosan. However, the authors stated that the binder masked the amino groups of chitosan, and thus reduced the antimicrobial activity of the fabrics treated with chitosan 1 polyurethane binder [107]. Montazer et al. [108] applied chitosan together with N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC), citric acid (CA), butane tetracarboxylic acid (BTCA), and glutaraldehyde (GA) mixtures to cotton fabrics, and they tested the antimicrobial activity against multiple washes. A covalent bond is formed between the crosslinkers and the chitosan and cellulose. After 15 washes, the best antimicrobial activity was achieved with GA.

Handbook of Chitin and Chitosan

4.5 Use of chitosan in medical textiles

121

The shrimp chitosan provided antibacterial activity to all fabrics that it coated, both alone and with cross-linker; however, the wash resistance of shrimp chitosan on textile surfaces was very low. Although chitosan was applied both alone and together with an acrylic cross-linker, it was separated from the fabric surface after one wash with soap solution, and the antimicrobial activity was lost. Literature studies have shown that the wash resistance of chitosan-coated fabrics using different binders is different. In this case, the chemicals found in the cross-linker are thought to be effective against washing by affecting the binding properties of chitosan.

4.5 Use of chitosan in medical textiles Medicine is one of the areas that has increasingly benefited from the innovations offered by the textile industry, both in fiber and in product. Medical textiles, including medical clothing, surgical coatings, bedclothes, urine retaining pads, ladies’ sanitary pads, fabrics/cleaning cloths, and surgical socks, are produced from monofilament or multifilament yarns, woven, knitted, nonwoven, and composite textile materials. These medical textile products should treated with as few chemicals as possible, should be nonallergenic, and should be suitable for health. Therefore it is important to use natural and biocompatible materials to provide functionality to medical products. Chitosan is a biomaterial that has a variety of unique biological properties, including being biocompatible, biodegradable, nontoxic, antimicrobial, an antioxidant, antitumoric, antiinflammatory, a painkiller, cholesterol lowering, antiinflammatory, mucosal adhesive, showing macrophage activity, and able to increase adsorption [16,21]. Therefore its use in the medical sector is becoming more and more important. Chitosan is used in medical textiles to give antimicrobial properties to textile products, for drug release in nanogel and microcapsule forms in bandages, as wound dressings, plasters, and as a wound-healing agent. Pielka et al. [109] coated a nonwoven commercial polypropylene material with dibutyrylchitin and chitin films and produced a textile dressing to accelerate wound healing. The dry bandage coating consisted of approximately 40% dibutyrylchitin and 30% chitin. Then, the bandages were sterilized with ethylene oxide. After in vitro and in vivo tests, the authors observed that the bandages coated with both dibutyrylchitin and chitin accelerated the wound-healing process and did not cause any cytotoxic effect or primary irritation. They suggested that the nonwoven commercial polypropylene material could be considered a cover material that promotes wound healing.

Handbook of Chitin and Chitosan

122

4. Investigation into the functional properties of cotton, wool, and denim textile materials

Berger et al. [110] investigated the availability of covalent and ionically cross-linked chitosan hydrogels for medical or pharmacological applications. While covalently bonded hydrogels can be used in implants or bandages, ionic bonded hydrogels are particularly useful in drug delivery systems. Hu et al. [111] also suggested that smart hydrogels can be used for moisturizing, wound healing, and antiaging in the development of therapeutic functional textiles that are in contact with the skin. The development of complementary and therapeutic materials in the field of medical textiles are being continuously investigated. Wang et al. [112] remarked that various innovative coated fabrics containing anion producing agents and borage oil for the treatment of atopic dermatitis (AD) have been developed, and they presented various information on the use of chitosan in medical textile products. Hui et al. [113,114] developed a functional textile therapy by successfully loading herbal medicines into the chitosan and sodium alginate composite microcapsules by an emulsion cross-linking method. They used the pad-dry-cure method to coat the microcapsules onto cotton fabric. The authors observed that textiles coated with drug-containing microcapsules delivered a perfectly controlled drug release for 7 days. However, they found that functional textiles that were produced failed to treat AD, since there was significant drug loss during microcapsulation. Varan [115] coated high elasticity fabric samples that were used for rehabilitation of burn scar management with chitosan and then tested the physical and mechanical properties, wear performance, and antibacterial activities of the fabric. The author stated that the cross-linking of chitosan to the fabric fibers caused a decrease in the elasticity of the fabric by reducing the movement of the fabric fibers; however, the fabric was been found to maintain its elasticity to assist in the rehabilitation of burn scar management. There was a slight increase in moisture recovery of fabric samples. Antimicrobial activity tests also showed that all samples had very good antimicrobial activity. Souza et al. [116] evaluated the thermal properties, moisture management, and surface friction of surgical cotton gauze coated by chitosan in different ratios by weight. The results of the study revealed that the surgical gauze coated with chitosan caused low capillarity and created good moisture and wound leakage absorption capacity. The medical gauze coated with 0.125% chitosan by weight provided the best conditions for the maintenance of the wound microenvironment and made an effective coating. In addition, the 0.125% chitosan improved the functional properties of the gauze, such as thermal conductivity, air permeability, moisture management, surface friction, and pain relief, and thus accelerated wound healing. Lam et al. [117] developed a comfortable and skin-protective garment for epidermolysis bullosa patients. For this purpose, they examined the

Handbook of Chitin and Chitosan

4.5 Use of chitosan in medical textiles

123

fabric comfort properties of a chitosan/cotton blend knitted jersey fabric, such as bending, compression, thermal conductivity, and surface friction. They suggested that the fabric containing more than 50% chitosan in its composition formed a colder surface when it came into contact with the skin of epidermolysis bullosa patients. Thus it provided comfort to the patients by reducing both the bandage-induced overheating and the pain caused by painful blisters. The authors concluded that the cotton blend knitted jersey fabric containing chitosan was functional in the development of a smooth, soft, and comfortable skin-protective textile material that reduced friction between the fabric and skin and that provided thermal conductivity. Rasel et al. [118] developed a viscose fabric with an antibacterial surface using chitosan-encapsulated iodine to prevent bacterial contamination in the undergarments of apparel workers. They found that the functionalized fabric provided a good antibacterial and antioxidative surface, as well as providing perspiration control. Since the chitosan kept the iodine copolymer within the capsule, the coating did not cause any side effects, and the antibacterial coating was active for a long time during the contact of the fabric with the skin. Zemljiˇc et al. [119] coated the pure and oxidized cellulose viscous fabrics with iodine embedded in chitosan nanoparticles to give them antimicrobial and antioxidant properties. Modification via the dispersion of chitosan
iodine nanoparticles gave the viscous fabrics antimicrobial and antioxidant properties and good coating stability, and the preliminary oxidation process made it a more functionally effective fabric. The functional groups introduced into the fabric served as binding sites for both chitosan and iodine functionalization, creating a stronger interaction between the fabric and the coating. The authors observed that the combination of iodine and chitosan was synergistic and that chitosan nanoparticles acted as a transport system for iodine. The authors suggested that the viscose fabric coated with chitosan nanoparticles could be used in the production of medical textiles, such as gauzes, plasters, napkins, etc. They also stated that when this fabric is used in the production of garments such as underwear, it would provide better sweat control. Xu et al. [120] prepared durable antimicrobial cotton fabrics via onepot modification using a solution of colloidal silver nanoparticles (AgNPs) stabilized with carboxymethyl chitosan and using a carboxymethyl chitosan as a binder. The authors stated that the adhesion between the AgNPs and the cotton fiber surface strengthened due to the coordination bonds between the AgNPs and amine groups of carboxymethyl chitosan and due to the ester bonds between the carboxyl groups of carboxymethyl chitosan and the hydroxyl groups of cellulose; thus Ag NPSs were firmly fixed to the cotton fiber surface. The authors

Handbook of Chitin and Chitosan

124

4. Investigation into the functional properties of cotton, wool, and denim textile materials

stated that this application provided significant antibacterial activity to the modified cotton fabric against S. aureus and E. coli, resulting in a bacterial reduction rate of more than 94%, even after 50 washes. Literature studies showed that medical textile applications of chitosan focused on issues that include the treatment of skin diseases, preparation of moisturizing and wound-healing hydrogels, preparation of drug-carrying microcapsules, improvement of mechanical and physical properties of medical textiles, pain reduction, the treatment of burn wounds, and the rehabilitation of burn scar management. It has been reported that chitosan significantly improves the thermal properties, air permeability, moisture management, and surface friction of wound dressings. For this reason, the number of studies on the use of chitosan in medical textile production is increasing in order to develop soft, comfortable, perspiration controlling, skin-protective clothing with antimicrobial properties. Since chitosan derivatives are more suitable for modification, they should be tested to provide functionality to medical textiles. In addition, most of the studies on medical textiles have not been tested at the clinical level. Therefore it is clear that there is a need for further clinical practice testing.

4.6 Use of chitosan in textile-reinforced composites Materials formed by the combination of two or more solids that do not intermingle are called composites. The purpose of creating a composite material is to combine the beneficial features of two or more materials, or to reveal a new feature. For this reason, nano-, micro-, or macrolevel heterogeneous mixtures are used to form nano-, micro-, or macrocomposite biomaterials, respectively. It is necessary to combine various textile reinforcements with a natural and biocompatible material such as chitosan in order to improve properties such as therapeutic, antibacterial, abrasion resistance, flame retardant, dye preservative, thermal resistance, and UV protection. Biobased nanocomposite films can be successfully developed using cellulose haircrystals as a booster phase and chitosan as matrix. Nanocomposite films combined with cellulose hair-crystals show excellent thermal stability and water vapor resistance [121]. Wang et al. [122] synthesized chitosan/clay bionanocomposites using montmorillonite clay and chitosan in different ratios. The Congo Red adsorption capacity of bionanocomposites was measured separately at different pHs and temperatures in aqueous solutions. They found that the adsorption capacity of chitosan/clay bionanocomposites was higher than

Handbook of Chitin and Chitosan

4.6 Use of chitosan in textile-reinforced composites

125

that of chitosan and montmorillonite and suggested that it would be an effective adsorbent in the removal of Congo Red in dirty waste solutions. Wang et al. [123] applied a ZnO/carboxymethyl chitosan (ZnO/CMCS) composite on a cotton fabric using the pad-dry-cure method using cold oxygen plasma in order to create a durable UV resistance and antibacterial activity. Plasma modification has been reported to significantly increase the thermal properties and UV resistance of cotton fabric and the wash resistance of antibacterial activity in direct proportion to the increase in ZnO/CMCS composite concentration on the fabric. Tayel et al. [124] prepared a natural composite material from collagen, chitosan, and oak galls extract. Then, they coated this material on cotton fabric and examined the antimicrobial activity of cotton fabric and the durability of the coating material. The composite had more antimicrobial activity against the tested pathogens compared to the individual materials and retained most of the antimicrobial effectiveness even after two washes. The authors emphasized that this natural composite could be used in the production of skin preservatives and hygienic textiles. Fan et al. [125] made a multifunctional coating, consisting of polyacrylate emulsion, fragrant ethanol solution, SiO2 dispersion, and chitosan, and coated it on cotton fabrics and leather surfaces using a layer-by-layer (LBL) spraying technique. In this multifunctional coating, polyacrylate emulsion was used in base coating, SiO2 particles in the middle coating as a reinforcement, and chitosan in the top layer as an antibacterial agent. The results revealed that cotton fabrics and leathers coated with multifunctional coating have long-term antibacterial activity and odor-releasing properties, as well as a good abrasion and wash resistance. In addition, although the coatings are known to reduce the hygienic properties of the coated fabrics, in this study the hygiene characteristics improved. Rehan et al. [126] synthesized chitosan-based Cs/AgNPs and Cs/ AgNPs/clay nanocomposites via UV radiation. They then coated these nanocomposites on the surface of a cotton fabric as a finishing process. Nanoparticles were successfully deposited on the cotton fabric surface. The nanocomposite significantly improved the strength, uniform morphology, thermal stability, water retention capacity, antimicrobial activity, flammability, UV protection, and odor release of the cotton fabric. Scacchetti et al. [104] finished the cotton surface with silver zeolite (SZ), SZ combined with chitosan film, or chitosan
zeolite (CS-SZ) synthesized by a gelation process with sodium tripolyphosphate. They investigated the thermal and antimicrobial properties of cotton functionalized with CS-SZ composite material. The results showed that CS-SZ composites exhibited antimicrobial activity against E. coli, S. aureus, C. albicans, and T. rubrum. The authors also stated that the use of the phase-exchange materials stored in the microcapsule as a textile finishing agent will improve the thermoregulation properties of the textile materials.

Handbook of Chitin and Chitosan

126

4. Investigation into the functional properties of cotton, wool, and denim textile materials

Raza and Anwar [127] produced polylactic acid-mediated chitosan nanocomposites in order to obtain a polyester fabric with improved functional properties. The authors considered this nanocomposite to be an antibacterial finishing agent for hydrophobic textile fabrics, such as polyester. The various combinations of polylactic acid-mediated chitosan nanocomposite was used to coat the woven polyester fabric via a cross-linker. They found that all combinations of nanocomposites exhibited antibacterial activity against E. coli and S. aureus strains, and the activity improved with the increase in the amount of nanocomposites. The authors suggest that functionalized polyester fabric can be used for wrinkle removal, water filtration, and material packing, as well as in medical and antibacterial textiles. El Hage et al. [128] produced chitosan-based flame-retardant films containing various amounts of aluminum trihydroxide (ATH) fillers and tested their functional properties. The authors observed a significant improvement in the thermal behavior of chitosan films containing 33% (by weight) or more aluminum trihydroxide filler, while observing that their mechanical properties were weakened. Chitosan only and chitosan/aluminum trihydroxide filler (67/33% w/w) were used as binder in biocomposites. The chitosan/aluminum trihydroxide filler system improved the flame retardancy of the biocomposite by creating a barrier effect. The authors suggested the potential of the chitosan/ ATH (67/33% w/w) system for the design of nonflammable insulation materials and proposed that the obtained biocomposites be used as flame retardants for biobased building insulation materials. As can be seen from the literature studies, chitosan has been used successfully in various composite materials for different purposes and has improved the various functional properties of the composite. In addition, since chitosan is of biological origin and is a natural material, its use, especially in textile products that are in contact with skin, is advantageous compared to other chemical and synthetic composites. Studies are underway to develop chitosan-reinforced composites with better properties.

4.7 Conclusion Surface analyses confirmed that shrimp chitosan was successfully applied to the surfaces of various fabrics. The bond between chitosan and fabric was affected by the type of fabric, the chemicals used in fabric production, and the impurities on the fabric. Therefore we recommend that the fabric should be subjected to a thorough pretreatment prior to coating with chitosan. Further, shrimp chitosan is an effective antimicrobial agent, both alone and in textile applications, and is more effective than

Handbook of Chitin and Chitosan

References

127

antibiotics against some bacterial species. The effective concentrations of chitosan differed by bacterial species. In order to ensure successful antimicrobial activity, the effective chitosan type and concentration used against each bacterium and fungus should be determined. In addition, the literature review revealed that the antimicrobial activity of chitosan is affected by the chitosan source, degree of deacetylation, molecular weight, chitosan concentration, and antimicrobial testing method. It was observed that the resistance to washing with soap solution of the chitosan used to coat textile surfaces was very low, and thus the antibacterial activity was lost after washing. The cross-linking agents that are highly wash-resistant and compatible with chitosan should be used, or pretreatments such as oxygen plasma, oxidation, or UV radiation can be applied to overcome this problem. Chitosan can also be used as an antibacterial agent suitable for unwashable and disposable medical textile products, such as masks, gauze, bandages, adhesive plaster, wound dressings, and food packaging. Current research focuses on the use of chitosan textile composites in medical textile production. Ongoing studies aim to determine the therapeutic properties, such as antibacterial activity, thermal resistance, UV protection, elasticity, and drug delivery, as well as to increase the moisture retention, air permeability, and softness of textiles and give them the properties of low surface friction, pain relief, and wound healing.

Acknowledgment ¨ BAP 2018/198 by Trakya This study was funded in the scope of the research project TU University Scientific Research Projects Unit.

References [1] X. Tao (Ed.), Smart Fibres, Fabrics and Clothing, first ed., Woodhead Publishing, Cambridge, 2001, p. 336. [2] U. Nimkar, Sustainable chemistry: a solution to the textile industry in a developing world, Curr. Opin. Green. Sustain. Chem. 9 (2018) 13
17. [3] ENV/JM/MONO, Analysis and assessment of current protocols to develop harmonised test methods and relevant performance standards for the efficacy testing of treated articles/treated materials. ,http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote 5 env/jm/mono(2007)4&doclanguage 5 en., 2007 (accessed 10.05.19). [4] J. Venkatesan, S.K. Kim, Marine biomaterials, part A, 53, in: S.K. Kim (Ed.), Springer Handbook of Marine Biotechnology, Springer-Verlag Berlin Heidelberg, Mumbai, 2015, pp. 1
10. [5] T. Philibert, B.H. Lee, N. Fabien, Current status and new perspectives on chitin and chitosan as functional biopolymers, Appl. Biochem. Biotechnol. 181 (4) (2017) 1314
1337. ˙ ¨ zelliklerinin Incelenmesi, [6] S. Bener, Kitosan/grafen Oksit Kompozit Filmlerinin Fiziksel O ˙ ¨ niversitesi, Fen Bilimleri Enstitu¨su¨, 2016. Yu¨ksek Lisans Tezi, Istanbul Teknik U [7] D. Enescu, Use of chitosan in surface modification of textile materials, Rom. Biotechnol. Lett. 13 (6) (2008) 4037
4048.

Handbook of Chitin and Chitosan

128

4. Investigation into the functional properties of cotton, wool, and denim textile materials

[8] F. Ferrero, M. Periolatto, Chitosan coating on textile fibers for functional properties, in: V.K. Thakur, M.K. Thakur, M.R. Kessler (Eds.), Handbook of Composites from Renewable Materials, vol. 4, Scrivener Publishing LLC, Beverly, 2017, pp. 165
198. [9] J. Roy, F. Salau¨n, S. Giraud, A. Ferri, J. Guan, Chitosan-Based Sustainable Textile Technology: Process, Mechanism, Innovation, and Safety in Biological Activities and Application of Marine Polysaccharides, IntechOpen Limited, London, 2017 (Chapter 12). [10] M.K. Jang, B.G. Kong, Y.I. Jeong, C.H. Lee, J.W. Nah, Physicochemical characterization of α-chitin, β-chitin, and γ-chitin separated from natural resources, J. Polym. Sci. Part. A: Polym. Chem. 42 (2004) 3423
3432. [11] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (7) (2006) 603
632. [12] Y. Wang, Y. Chang, L. Yu, C. Zhang, X. Xu, Y. Xue, et al., Crystalline structure and thermal property characterization of chitin from Antarctic krill (Euphausia superba), Carbohydr. Polym. 92 (2013) 90
97. ¨ zu¨saglam, ˘ [13] M. Kaya, T. Baran, S. Erdogan, A. Mentes, M. A¸san O Y.S. Cakmak, Physicochemical comparison of chitin and chitosan obtained from larvae and adult Colorado potato beetle (Leptinotarsa decemlineata), Mater. Sci. Eng. 45 (2014) 72
81. [14] F.A. Al Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf, Carbohydr. Polym. 77 (2009) 410
419. [15] R. Jindan, R. Zalal, S. Hudson, W.M. King, β-chitin/chitosan obtained from Loligo and Humboldt squids effectiveness as wound dressing material, J. Donghua Univ. 30 (5) (2013) 406
409. [16] J. Kumirska, M.X. Weinhold, J. Thoming, P. Stepnowski, Biomedical activity of chitin/chitosan based materials influence of physicochemical properties apart from molecular weight and degree of N-acetylation, Polymers 3 (2011) 1875
1901. [17] E. Khor, L. Lim, Implantable applications of chitin and chitosan, Biomaterials 24 (2003) 2339
2349. [18] R.A.A. Muzzarelli, Clinical and biochemical evaluation of chitosan for hypercholesterolemia and overweight control, EXS 87 (1999) 293
304. [19] P.K. Dutta, J. Dutta, V.S. Tripathi, Chitin and chitosan: chemistry, properties and applications, J. Sci. Ind. Res. 63 (2004) 20
31. [20] S.O. Fernandez-Kim, Physicochemical and Functional Properties of Crawfish Chitosan as Affected by Different Processing Protocols (A Master thesis), Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College, Seoul National University, 2004. [21] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, et al., Functional characterization of chitin and chitosan, Curr. Chem. Biol. 3 (2009) 203
230. [22] A. Anitha, S. Sowmya, P.T. Sudheesh Kumar, S. Deepthi, K.P. Chennazhi, H. Ehrlich, et al., Chitin and chitosan in selected biomedical applications, Prog. Polym. Sci. 39 (9) (2014) 1644
1667. [23] R. Jayakumar, D. Menon, K. Manzoor, S.V. Nair, H. Tamura, Biomedical applications of chitin and chitosan nanomaterials-a short review, Carbohydr. Polym. 82 (2010) 227
232. [24] T.L.B. Ha, T.M. Quan, D.N. Vu, D.M. Si, in: J.A. Andrades (Ed.), Naturally Derived Biomaterials: Preparation and Application, Regenerative Medicine and Tissue Engineering, IntechOpen Limited, London, 2013 (Chapter 11). [25] Chitosan market size analysis industry research report, 2018
2025. ,https://www. grandviewresearch.com/industry-analysis/global-chitosan-market., 2017 (accessed 10.05.19).

Handbook of Chitin and Chitosan

References

129

[26] A. Pathak, Chitin and chitosan derivatives: technologies, applications and global markets. ,https://www.bccresearch.com/market-research/plastics/chitin-chitosan-derivatives-markets-report.html., 2017 (accessed 10.05.19). [27] R.A.A. Muzzarelli, C. Jeuniaux, G.W. Gooday, Chitin in Natu¨re and Technology, Plenum Publishing Corporation, New York, 1986. [28] H. Merzendorfer, L. Zimoch, Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases, J. Exp. Biol. 206 (2003) 4393
4412. [29] J. Synowiecki, N.A. Al-Khateeb, Production, properties, and some new applications of chitin and its derivatives, Crit. Rev. Food Sci. Nutr. 43 (2) (2003) 145
171. [30] H. Ehrlich, M. Maldonado, K.D. Spindler, C. Eckert, T. Hanke, R. Born, et al., First evidence of chitin as a component of the skeletal fibers of marine sponges: Part I Verongidae (demospongia: Porifera), J. Exp. Zool. Part B 308 (4) (2007) 347
356. [31] G.A.F. Roberts, Chitin Chemistry, second ed., MacMillan, London, 1998. [32] H.N. Cuong, N.C. Minh, N.V. Hoa, T.S. Trung, Preparation and characterization of high purity β-chitin from squid pens (Loligo chenisis), Int. J. Biol. Macromol. 93 (2016) 442
447. [33] F. Arrouze, M. Essahli, M. Rhazi, J. Desbrieres, A. Tolaimate, Chitin and chitosan: study of the possibilities of their production by valorization of the waste of crustaceans and cephalopods rejected in Essaouira, J. Mater. Environ. Sci. 8 (2017) 2251
2258. [34] S. Hajji, I. Younes, O. Ghorbel-Bellaaj, R. Hajji, M. Rinaudo, M. Nasri, et al., Structural differences between chitin and chitosan extracted from three different marine sources, Int. J. Biol. Macromol. 65 (2014) 298
306. [35] H. Zhang, S. Yun, L. Song, Y. Zhang, Y. Zhao, The preparation and characterization of chitin and chitosan under large-scale submerged fermentation level using shrimp by-products as substrate, Int. J. Biol. Macromol. 96 (2017) 334
339. [36] A.T. Paulino, J.I. Simionato, J.C. Garcia, J. Nozaki, Characterization of chitosan and chitin produced from silkworm chrysalides, Carbohydr. Polym. 64 (1) (2006) 98
103. [37] E. Kovaleva, A. Pestov, D. Stepanova, L. Molochnikov, Characterization of chitin and its complexes extracted from natural raw sources, AIP Conf. Proc. 1772 (2016) 050007. Available from: https://doi.org/10.1063/1.4964577. [38] Y.S. Song, Y.M.W. Kim, C. Moon, D.J. Seo, Y.S. Han, Y.H. Jo, et al., Extraction of chitin and chitosan from larval exuvium and whole body of edible mealworm, Tenebrio molitor, Entomol. Res. 48 (2018) 227
233. [39] M. Kaya, M. Asan-Ozusaglam, S. Erdogan, Comparison of antimicrobial activities of newly obtained low molecular weight scorpion chitosan and medium molecular weight commercial chitosan, J. Biosci. Bioeng. 121 (6) (2016) 678
684. [40] C.N. Ambarish, K.R. Sridhar, Isolation and characterization of chitin from exoskeleton of Pill-Millipedes, Trends Biomater. Artif. Organs 29 (2) (2015) 155
159. [41] M. Kaya, V. Baublys, I. Satkauskien, B. Akyuz, E. Bulut, V. Tubelyt, First chitin extraction from Plumatella repens (Bryozoa) with comparison to chitins of insect and fungal origin, Int. J. Biol. Macromol. 79 (2015) 126
132. [42] M.T. Yen, J.L. Mau, Preparation of fungal chitin and chitosan from shiitake stipes, Fungal Sci. 21 (1, 2) (2006) 1
11. [43] S. Erdogan, M. Kaya, I. Akata, Chitin extraction and chitosan production from cell wall of two mushroom species (Lactarius vellereus and Phyllophora ribis), AIP Conf. Proc. 1809 (2017) 020012. Available from: https://doi.org/10.1063/1.4975427. [44] S. Kaur, G.S. Dhillon, Recent trends in biological extraction of chitin from marine shell wastes: a review, Crit. Rev. Biotechnol. 35 (2015) 44
61. [45] A. Aljawish, I. Chevalot, J. Jasniewski, J. Scher, L. Muniglia, Enzymatic synthesis of chitosan derivatives and their potential applications, J. Mol. Catal. B Enzyme 112 (2015) 25
39.

Handbook of Chitin and Chitosan

130

4. Investigation into the functional properties of cotton, wool, and denim textile materials

[46] K. Brar, G. Dhillon, R. Soccol, Biotransformation of Waste Biomass into High Value Biochemicals, 504, Springer, 2013, pp. 1
29. [47] R.L. Lavall, O.B.G. Assis, S.P. Campana-Filho, Beta-chitin from the pens of Loligo sp.: extraction and characterization, Bioresour. Technol. 98 (2007) 2465
2472. [48] S. Chatterjee, M. Adhya, A.K. Guha, B.P. Chatterjee, Chitosan from Mucor rouxii: production and physico-chemical characterization, Process. Biochem. 40 (2005) 395
400. [49] M. Kaya, F. Dudakli, M. Asan-Ozusaglam, Y.S. Cakmak, T. Baran, A. Mentes, et al., Porous and nanofiber a-chitosan obtained from blue crab (Callinectes sapidus) tested for antimicrobial and antioxidant activities, LWT-Food Sci. Technol. 65 (2016) 1109
1117. [50] A. Hassainia, H. Satha, S. Boufi, Chitin from Agaricus bisporus: extraction and characterization, Int. J. Biol. Macromol. 117 (2018) 1334
1342. [51] N. Mesa Ospina, S.P. Ospina Alvarez, D.M. Escobar Sierra, D.F. Rojas Vahos, P.A. Zapata Ocampo, C.P. Ossa Orozco, Isolation of chitosan from Ganoderma lucidum mushroom for biomedical applications, J. Mater. Sci. Mater. Med. 26 (3) (2015) 135. Available from: https://doi.org/10.1007/s10856-015-5461-z. [52] S. Erdogan, M. Kaya, High similarity in physicochemical properties of chitin and chitosan from nymphs and adults of a grasshopper, Int. J. Biol. Macromol. 89 (2016) 118
126. [53] M. Kaya, K.O. Tozak, T. Baran, G. Sezen, I. Sargın, Natural porous and nano fiber chitin structure from Gammarus Argaeus (Gammaridae Crustacea), EXCLI J. 12 (2013) 503
510. [54] R. Shepherd, S. Reader, A. Falshaw, Chitosan functional properties, Glycoconj. J. 14 (4) (1997) 535
542. [55] R.C. Goy, D. de Britto, O.B.G. Assis, Review of the antimicrobial activity of chitosan, Polı´meros 19 (3) (2009) 241
247. [56] M.F.A. Goosen, Applications of Chitin and Chitosan, Technomic Publishing, Lancaster, PA, 1997, pp. 3
29. [57] M.S. Benhabiles, R. Salah, H. Lounici, N. Drouiche, M.F.A. Goosen, N. Mameri, Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste, Food Hydrocoll. 29 (2012) 48
56. [58] B.K. Park, M.M. Kim, Applications of chitin and its derivatives in biological medicine, Int. J. Mol. Sci. 11 (2010) 5152
5164. [59] A. Wassila, L. Adour, A. Amrane, Chitin extraction from crustacean shells using biological methods, Food Technol. Biotechnol. 51 (2013) 12
25. [60] Y. Qin, Y. Deng, Y. Hao, N. Zhang, X. Shang, Marine bioactive fibers: alginate and chitosan fibers-a critical review, J. Text. Eng. Fash. Technol. 1 (6) (2017) 228
231. [61] G.J. Tsai, W.H. Su, H.C. Chen, C.L. Pan, Antimicrobial activity of shrimp chitin and chitosan from different treatments and applications of fish preservation, Fish. Sci. 68 (2002) 170
177. [62] P.J. Park, J.Y. Je, H.G. Byun, S.H. Moon, S.K. Kim, Antimicrobial activity of heterochitosans and their oligosaccharides with different molecular weights, J. Microbiol. Biotechnol. 14 (2) (2004) 317
323. [63] M.T. Yen, J.H. Yang, J.L. Mau, Physicochemical characterization of chitin and chitosan from crab shells, Carbohydr. Polym. 75 (2009) 15
21. [64] M. Hamdi, S. Hajji, S. Affes, W. Taktak, H. Maalej, M. Nasri, et al., Nasri, development of a controlled bioconversion process for the recovery of chitosan from blue crab (Portunus segnis) exoskeleton, Food Hydrocoll. 77 (2018) 534
548. ˙ I. Sargin, G. Arslan, A. Mol, et al., [65] M. Kaya, E. Leleˇsius, R. Nagrockaite, Differentiations of chitin content and surface morphologies of chitins extracted from male and female grasshopper species, PLoS One 10 (1) (2015) e0115531. Available from: https://doi.org/10.1371/journal.pone.0115531.

Handbook of Chitin and Chitosan

References

131

[66] C.Y. Soon, Y.B. Tee, C.H. Tan, A.T. Rosnita, A. Khalina, Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in varying sodium hydroxide concentration, Int. J. Biol. Macromol. 108 (2018) 135
142. [67] O.P. Gbenebor, E.I. Akpan, S.O. Adeosun, Thermal, structural and acetylation behavior of snail and periwinkle shells chitin, Prog. Biomater. 6 (2017) 97
111. [68] M.T. Yen, J.L. Mau, Physico-chemical characterization of fungal chitosan from shiitake stipes, LWT-Food Sci. Technol. 40 (2007) 472
479. [69] H.Y. Zhou, X.G. Chen, M. Kong, C.S. Liu, D.S. Cha, J.F. Kennedy, Effect of molecular weight and degree of chitosan deacetylation on the preparation and characteristics of chitosan thermosensitive hydrogel as a delivery system, Carbohydr. Polym. 73 (2008) 265
273. [70] A. Kucukgulmez, M. Celik, Y. Yanar, D. Sen, H. Polat, A. Kadak, Physicochemical characterization of chitosan extracted from Metapenaeus stebbingi shells, Food Chem. 126 (2011) 1144
1148. [71] S.V. Nemtsev, O.Y. Zueva, M.R. Khismatullin, A.I. Albulov, V.P. Varlamov, Isolation of chitin and chitosan from honeybees, Appl. Biochem. Microbiol. 40 (2004) 39
43. [72] Q. Li, E.T. Dunn, E.W. Grandmaison, M.F.A. Goosen, Applications and properties of chitosan, J. Bioact. Compat. Polym. 7 (1992) 370
397. [73] V.K. Mourya, N.N. Inamdar, Chitosan-modifications and applications: opportunities galore, React. Funct. Polym. 68 (2008) 1013
1051. [74] V.E. Tikhonov, E.A. Stepnova, V.G. Babak, I.A. Yamskov, J. Palma-Guerrero, H.B. Jansson, et al., Bactericidal and antifungal activities of a low molecular weight chitosan and its N-/2(3)-(dodec-2-enyl)succinoyl/-derivatives, Carbohydr. Polym. 64 (1) (2006) 66
72. [75] L. Sun, Y. Du, L. Fan, X. Chen, J. Yang, Preparation, characterization and antimicrobial activity of quaternized carboxymethyl chitosan and application as pulp-cap, Polymers 47 (2006) 1796
1804. [76] V. Jarmila, E. Vavrı´kova, Chitosan derivatives with antimicrobial, antitumour and antioxidant activities-a review, Curr. Pharm. Des. 17 (32) (2011) 3596
3607. [77] M. Ioelovich, Crystallinity and hydrophility of chitin and chitosan, Res. Rev. J. Chem. 3 (3) (2014) 7
14. [78] S. Liu, J. Sun, L. Yu, C. Zhang, J. Bi, F. Zhu, et al., Extraction and characterization of chitin from the beetle Holotrichia parallela Motschulsky, Molecules 17 (2012) 4604
4611. [79] M.N.V.R. Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 40 (2000) 1
27. [80] M. Wysokowski, I. Petrenko, A.L. Stelling, D. Stawski, T. Jesionowski, H. Ehrlich, Poriferan chitin as a versatile template for extreme biomimetics, Polymers 7 (2) (2015) 235
265. [81] M. Kaya, I. Akata, T. Baran, A. Mente¸s, Physicochemical properties of chitin and chitosan produced from medicinal fungus (Fomitopsis pinicola), Food Biophys. 10 (2014) 162
168. [82] T.J. Gutierrez, Chitosan applications for the food industry, in: A. Shakeel, I. Saiqa (Eds.), Chitosan, Scrivener Publishing LLC, Beverly, 2017, pp. 183
232. [83] F. Shahidi, J.K.V. Arachchi, Y.J. Jeon, Food applications of chitin and chitosans, Trends Food Sci. Technol. 10 (2) (1999) 37
51. [84] N.R. Sudarshan, D.G. Hoover, D. Knorr, Antibacterial action of chitosan, Food Biotechnol. 6 (3) (1992) 257
272. [85] F. Devlieghere, A. Vermeulen, J. Debevere, Chitosan: antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables, Food Microbiol. 21 (6) (2004) 703
714. ¨ .V. Ru´narsson, T. Nevalainen, T. [86] M. Ma´sson, J. Holappa, M. Hja´lmarsdo´ttir, O Ja¨rvinen, Antimicrobial activity of piperazine derivatives of chitosan, Carbohydr. Polym. 74 (2008) 566
571.

Handbook of Chitin and Chitosan

132

4. Investigation into the functional properties of cotton, wool, and denim textile materials

[87] R.G. Cuero, G. Osuji, A. Washington, N-Carboxymethyl chitosan inhibition of aflatoxin production: role of zinc, Biotechnol. Lett. 13 (1991) 441
444. [88] S. Roller, N. Covill, The antifungal properties of chitosan in laboratory media and apple juice, Int. J. Food Microbiol. 47 (1999) 67
77. [89] CLSI-M07-A9, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard, ninth ed. ,file:///C:/Users/ KULLANICI/Downloads/03-CLSI-M07-A9-2012%20(1).pdf., 2012 (accessed 10.05.19). [90] W.D. Schindler, P.J. Hauser, Chemical Finishing of Textiles, first ed., Woodhead Publishing, Cambridge, 2004. [91] A.K. Sen, Coated Textiles: Principles and Applications, second ed., CRC Press, Boca Raton, FL, 2007. [92] B. Simoncic, B. Tomsic, Structures of novel antimicrobial agents for textiles-a review, Text. Res. J. 80 (16) (2010) 1721
1737. [93] Y.S. Chung, K.K. Lee, J.W. Kim, Durable press and antimicrobial finishing of cotton fabrics with a citric acid and chitosan treatment, Text. Res. J. 68 (10) (1998) 772
775. [94] H.S. Seong, J.P. Kim, S.W. Ko, Preparing chito-oligosaccharides as antimicrobial agents for cotton, Text. Res. J. 69 (7) (1999) 483
488. [95] Z. Zhang, L.J. Chen, Y. Haung, D. Chen, Antibacterial properties of cotton fabrics treated with chitosan, Text. Res. J. 73 (12) (2003) 1103
1106. [96] Y. Qin, Functional modifications of chitosan fibers, Chem. Fibers Int. 3 (2006) 159
161. [97] AATCC TM147-2016, Antibacterial activity assessment of textile materials: parallel streak method, ,https://members.aatcc.org/store/tm147/554/., 2016 (accessed 10.05.19). [98] AATCC TM100-2012, Antibacterial finishes on textile materials: assessment of antibacterial activity finishes on textile materials, ,https://members.aatcc.org/store/ tm100/513/., 2012 (accessed 10.05.19). [99] F.D. Lowy, Is Staphylococcus aureus an intracellular pathogen, Trends Microbiol. 8 (2000) 341
344. [100] H.F. Wertheim, D.C. Melles, M.C. Vos, W. van Leeuwen, A. van Belkum, H.A. Verbrugh, et al., The role of nasal carriage in Staphylococcus aureus infections, Lancet Infect. Dis. 5 (12) (2005) 751
762. [101] J.A. Shulman, A.J. Nahmias, Staphylococcal infections: clinical aspects, in: J.O. Cohen (Ed.), The Staphylococci, Wiley, New York, 1972, pp. 457
482. [102] M. Akaydin, M. Kalkanci, A research on antibacterial properties of woven fabrics using for hospital clothing, SDU J. Sci. 9 (1) (2014) 20
34. [103] S. Palamutc¸u, R. Keskin, N. Devrent, M. S¸ engu¨l, B. Hasc¸elik, et al., Fonksiyonel tekstiller II: antimikrobiyal tekstiller, Tekst. Teknolo. Elektron. Derg. 3 (3) (2009) 95
108. [104] F.A.P. Scacchetti, E. Pinto, G.M.B. Soares, Thermal and antimicrobial evaluation of cotton functionalized with a chitosan
zeolite composite and microcapsules of phase-change materials, J. Appl. Polym. Sci. 135 (2018) 46135. Available from: https://doi.org/10.1002/app.46135. [105] H.E. Gaffer, M. Gouda, E. Abdel-Latif, Antibacterial activity of cotton fabrics treated with sulfadimidine azo dye/chitosan colloid, J. Ind. Text. 0 (00) (2012) 1
8. Available from: https://doi.org/10.1177/1528083712441959. [106] G. S¸ ahan, A. Demir, Tekstil terbiyesinde nano boyutta kitosanin ye¸sil uygulamasi, Tekst. Konfeksiyon 26 (4) (2016) 414
420. [107] H.S. Seong, H.S. Whang, S.W. Ko, Synthesis of a quaternary ammonium derivative of chito-oligosaccharides as antimicrobial agent for cellulosic fibers, J. Appl. Polym. Sci. 76 (2000) 2009
2015.

Handbook of Chitin and Chitosan

References

133

[108] M. Montazer, G. Afjeh, Simultaneous X-linking and antimicrobial finishing of cotton fabric, J. Appl. Polym. Sci. 103 (2007) 178
185. [109] S. Pielka, D. Paluch, J. Staniszewska-Kus, B. Zywicka, L. Solski, L. Szosland, et al., Wound healing acceleration by a textile dressing containing dibutyrylchitin and chitin, Fibres Text. East Europe 11 (2:41) (2003) 79
84. [110] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. 57 (1) (2004) 19
34. [111] J. Hu, H. Meng, G. Li, S.I. Ibekwe, A review of stimuli-responsive polymers for smart textile applications, Smart Mater. Struct. 21 (2012) 053001. Available from: https://doi.org/10.1088/0964-1726/21/5/053001. [112] W. Wang, P.C.L. Hui, C.W. Kan, Review functionalized textile based therapy for the treatment of atopic dermatitis, Coatings 7 (2017) 82. Available from: https://doi. org/10.3390/coatings7060082. [113] P.C.L. Hui, W.Y. Wang, C.W. Kan, F.S.F. Ng, C.E. Zhou, E. Wat, et al., Preparation and characterization of chitosan/sodium alginate (CSA) microcapsule containing cortex moutan, Colloids Surf. A 434 (2013) 95
101. [114] P.C. Hui, W.Y. Wang, C.W. Kan, F.S. Ng, E. Wat, V.X. Zhang, et al., Microencapsulation of traditional Chinese Herbs-Penta Herbs extracts and potential application in healthcare textiles, Colloids Surf. B 111 (2013) 156
161. [115] N.Y. Varan, The effects of chitosan antimicrobial treatments on the physical and mechanical properties and wear performances of highly elastic fabrics used for burn scar management, J. Text. Inst. 109 (1) (2018) 39
45. [116] J. Souza, J. Matos, M. Fernandes, A. Zille, R. Fangueiro, Coated chitosan onto gauze to efficient conditions for maintenance of the wound microenvironment, Procedia Eng. 200 (2017) 135
140. [117] N.Y.K. Lam, M. Zhang, C. Yang, C.P. Ho, L. Li, A pilot intervention with chitosan/ cotton knitted jersey fabric to provide comfort for epidermolysis bullosa patients, Text. Res. J. 88 (6) (2018) 704
716. [118] M. Rasel, S.H. Bhuiyan, M.S. Sadi, I.R. Eshita, R. Mia, M.T. Ahmed, et al., An approach for development of medical textiles: safety issue of garments (female) worker, American, J. Mater. Sci. 8 (2) (2018) 27
31. ˇ [119] L.F. Zemljiˇc, Z. Perˇsin, O. Sauperl, A. Rudolf, M. Kosti´c, Medical textiles based on viscose rayon fabrics coated with chitosan-encapsulated iodine: antibacterial and antioxidant properties, Text. Res. J. 88 (22) (2018) 2519
2531. [120] Q.B. Xu, W.S. Zheng, P.P. Duan, J.N. Chen, Y.Y. Zhang, F.Y. Fu, et al., One-pot fabrication of durable antibacterial cotton fabric coated with silver nanoparticles via carboxymethyl chitosan as a binder and stabilizer, Carbohydr. Polym. 204 (2019) 42
49. [121] Q. Li, J. Zhou, L. Zhang, Structure and properties of the nanocomposite films of chitosan reinforced with cellulose whiskers, J. Polym. Sci. Part B: Polym. Phys. 47 (2009) 1069
1077. [122] L. Wang, A. Wang, Adsorption characteristics of Congo Red onto the chitosan/ montmorillonite nanocomposite, J. Hazard. Mater. 147 (2007) 979
985. [123] C. Wang, J. Lv, Y. Ren, Q. Zhou, J. Chen, T. Zhi, et al., Cotton fabric with plasma pretreatment and ZnO/Carboxymethyl chitosan composite finishing for durable UV resistance and antibacterial property panel, Carbohydr. Polym. 138 (2016) 106
113. [124] A.A. Tayel, R.A. Ghanem, S.H. Moussa, M. Fahmi, H.M. Tarjam, N. Ismail, Skin protectant textiles loaded with fish collagen, chitosan and oak galls extract composite, Int. J. Biol. Macromol. 117 (2018) 25
29. [125] Q. Fan, J. Maa, Q. Xua, W. Ana, R. Qiua, Multifunctional coatings crafted via layerby-layer spraying method, Prog. Org. Coat. 125 (2018) 215
221.

Handbook of Chitin and Chitosan

134

4. Investigation into the functional properties of cotton, wool, and denim textile materials

[126] M. Rehan, M.E. El-Naggara, H.M. Mashaly, R. Wilken, Nanocomposites based on chitosan/silver/clay for durable multi-functional properties of cotton fabrics, Carbohydr. Polym. 182 (2018) 29
41. [127] Z.A. Raza, F. Anwar, Fabrication of poly(lactic acid) incorporated chitosan nanocomposites for enhanced functional polyester fabric, Polı´meros 28 (2) (2018) 120
124. [128] R. El Hage, Y. Khalaf, C. Lacoste, M. Nakhl, P. Lacroix, A. Bergeret, A flame retarded chitosan binder for insulating miscanthus/recycled textile fibers reinforced biocomposites, J. Appl. Polym. Sci. 136 (2018) 47306. Available from: https://doi. org/10.1002/APP.47306.

C H A P T E R

5 Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites for adsorption systems: environmental remediation and protein purification Gabriel Ibrahin Tovar-Jimenez1,2, Daniela Bele´n Hirsch3,4, Marı´a Emilia Villanueva1,2, Nicola´s Urtasun4,5, Federico Javier Wolman3,4 and Guillermo Javier Copello1,2 1

Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquı´mica, Departamento de Quı´mica Analı´tica y Fisicoquı´mica, Junı´n 956, CABA, Argentina, 2CONICET
Universidad de Buenos Aires (UBA), Instituto de Quı´mica y Metabolismo del Fa´rmaco (IQUIMEFA), Buenos Aires, Argentina, 3Universidad de Buenos Aires (UBA), Facultad de Farmacia y Bioquı´mica, Ca´tedra de Biotecnologı´a, Junı´n, Argentina, 4CONICET
Universidad de Buenos Aires, Instituto de Nanobiotecnologı´a (NANOBIOTEC), Buenos Aires, Argentina, 5Universidad de Buenos Aires (UBA), Facultad de Ciencias Exactas y Naturales, Departamento de Fisiologı´a, Biologı´a Molecular y Celular, Junı´n, Argentina

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00005-4

135

© 2020 Elsevier Inc. All rights reserved.

136

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

O U T L I N E 5.1 Introduction

136

5.2 Chitin adsorption properties 5.2.1 Chitin chemistry 5.2.2 Chitin physics

138 139 144

5.3 Shaping chitin materials 5.3.1 Chitin supports for adsorptive processes 5.3.2 Batch systems 5.3.3 Continuous-flow systems

148 150 151 152

5.4 Optimizing adsorption performance by chemical and physical modifications 5.4.1 Discussion of some relevant developments 5.4.2 Pristine chitin 5.4.3 Cross-linking 5.4.4 Composites, nanocomposites, and fillers 5.4.5 Derivatization

154 154 154 156 158 163

5.5 Concluding remarks and future considerations

166

Acknowledgments

167

References

168

5.1 Introduction The global need to replace the synthetic polymers derived from petroleum by eco-friendly and sustainable materials surpasses the green wave by finding better results with high yields at low costs of biopolymers. Thus the future markets for biopolymers are significantly increased due to its sustainability. Application of chitinous products as adsorbent polymeric matrices is an expanding field for science and industry [1]. Chitin was first identified in 1884 and can be depicted as the second most abundant natural biopolymer, after cellulose [2]. Chitin exists in marine media and especially in the exoskeleton of crustaceans, cartilage of mollusks, cuticles of insects, and cell walls of microorganisms. Chitosan is a type of natural polyaminosaccharide, obtained by deacetylation of chitin, that is extensively used because of its easy physical and chemical manipulation. Chitin

Handbook of Chitin and Chitosan

137

5.1 Introduction

TABLE 5.1 Timeline from the Scopus database for chitin and chitosan for adsorption systems. Years

Chitin

Chitosan

1970
79

2

1

1980
89

19

21

1990
99

47

174

2000
09

133

634

2010
18

200

2232

and its derivatives are promising materials due to their macromolecular structure, nontoxicity, biocompatibility, biodegradability, and low-cost. These characteristics allow their use for potential applications in many fields, such as biotechnology, medicine, cosmetics, and the food industry. Interestingly, some of these fields involve adsorption processes [3,4]. A seminal work of Muzzarelli in 1970 described the synthesis and adsorption evaluation of chitin and chitosan for the removal of radioactive metals from seawater. Then, it was not until 1980 that new studies of the interactions of metal ions, proteins, and dyes with chitin were reported with higher frequency [5
7]. This trend is presented in Table 5.1. The growth of the number of publications has been exponential, from the adsorption of pollutants in wastewater (organic and inorganic) to chromatographic purification processes of proteins. Following the results of a search by timeline in the Scopus database (using the terms “chitosan”/“chitin” in combination with “sorption” or “isotherm”) in 2018 for each paper involving chitin, 12 involving chitosan were published. The trend of recent publications suggests a greater interest in chitosan. However, there is a bet on increasing the use of chitin due to its high chemical and mechanical stability among other reasons presented in this chapter. The evolution of the areas of interest for chitin is focused on dyes and heavy metals, however other topics are still being studied, according to the criteria mentioned from Scopus (Fig. 5.1). On the other hand, many other published papers are based on chitin and their adsorption properties in the design of new materials. For example, an invention from 2017 involves the use of chitin for cigarette filters, and claims that the biopolymer diminishes the harmful components of the combustion gas and avoids throat discomfort and oral ulcer symptoms [8]. We can also find patents for the cosmetic and therapeutic use of chitin, or for conventional water purification uses or soil treatment, including protein purification [9
12]. Therefore chitin and its modifications have important industrial and commercial uses that can be exploited.

Handbook of Chitin and Chitosan

138

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

FIGURE 5.1 Evolution of papers’ fields for chitin 1998
2018 in adsorption systems. Source: Scopus.

This chapter summarizes and brings a critical evaluation of the of the advantages and disadvantages of the properties and applications of chitin for sorption processes. The chapter also discusses potential physical and chemical modifications and future considerations.

5.2 Chitin adsorption properties Adsorption is a phenomenon that occurs between two chemical species. One, typically with lower molecular weight and mobilities of the gaseous or liquid phase; the other with higher molecular weight and many times with a solid-like behavior. Although this is not always true, this simplification can help to describe most of the adsorption systems. In these systems, mainly, a heterogeneous phase adsorption phenomenon takes place, in which the smaller and mobile sorbate reach the larger and quasistatic adsorbent. In this generalization, the adsorbent assumes the description of a surface. Adsorption processes may occur by diverse mechanisms. Chemisorption and physisorption mainly explain the interaction of an ion or a small molecule with a single adsorption site. Whereas physisorption involves

Handbook of Chitin and Chitosan

5.2 Chitin adsorption properties

139

low-energy interactions in which the original species remain intact, such as the ones that occur through van der Waals interactions, chemisorption occurs with higher interaction energies, such as in electrostatic interaction, and with the possibility of a transformation in the original species involved, such as in covalent bonding. Up to this point, this is a basic description of the adsorption process and it is worth taking into account that many systems are complex and the adsorbate may be a large molecule, such as in the case of a protein, and the adsorbent cannot be solely considered as an infinitely flat surface. When the adsorbates are large molecules, multiple interaction points can be present and the description of the interaction mechanism will probably involve a combination of several possible interactions. For example, when a small peptide is the adsorbate and the adsorbent presents several and heterogeneous adsorption sites, such as for the case of alginate, not only the electrostatic interaction between the protonated amino terminal of the peptide and the carboxylate groups of the alginate should be considered. Interaction by hydrogen bonding of alginate hydroxyl groups with the moieties capable of hydrogen bonding formation from the peptide should be considered to occur together with the electrostatic attraction. In addition, when multiple interactions are present simultaneously, the spatial distance among the interaction sites at the adsorbate and at the adsorbent imply that the adsorbate needs to acquire a specific positioning in order to let the multiple interactions occur. This would happen upon a simple molecule rotation or through more complex conformational rearrangements. From another perspective, when crystallinity hinders the diffusion of adsorbates, or when adsorbates are large molecules, or the structure of the adsorbent presents small pores, other phenomena became relevant, such as intraparticle diffusion, which accounts for a differential mobility of the sorbate within the interior of the adsorbent, or surface diffusion, which accounts for the mobility of the sorbate from site to site on the adsorbent surface [13]. Although adsorption phenomena involve complex processes that could not be easily separated in several isolated phenomena, in the present subsection we will present the properties of the chitin-based materials in two focused aspects: one regarding how chitin’s chemistry affects adsorption processes, and other dealing with the role of chitinbased materials’ physics in the adsorption processes.

5.2.1 Chitin chemistry The chemical structure of chitin consists of several (1
4)-linked 2-acetamido-2-deoxy-β-D-glucopyranose units. Its structure is comparable

Handbook of Chitin and Chitosan

140

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

to cellulose, with the difference that at the C2 position, chitin has an acetamide group (
NHCOCH3) instead of a hydroxyl. Although some of the glucopyranose residues are in the deacetylated form as 2-amino-2-deoxyβ-D-glucopyranose, it is considered a homopolymer. The degree of acetylation (DA) is typically 0.90, showing the presence of some amino groups (5%
15%) [14]. When chitin DA, becomes low enough that amine protonation leads to a soluble polysaccharide, it is considered as a different chemical entity, known as chitosan [15]. In nature, chitin exists in three different forms. The average molecular weights of α-chitin, β-chitin, and γ-chitin, calculated using the relative viscosity method, are approximately 701, 612, and 524 kDa, respectively [16]. Chitin is highly crystalline in comparison to other natural polymers, and is hydrophobic and insoluble in water and most organic solvents. It is soluble in hexafluoroisopropanol, hexafluoroacetone, and chloroalcohols in conjugation with aqueous solutions of mineral acids and dimethylacetamide containing 5% lithium chloride [17]. Besides, the degree of N-acetylation has a noticeable influence on chitin solubility and solution properties [18]. The poor solubility of chitin is the main limiting factor in its utilization for the development of applicable products [19]. Despite this limitation, many articles describing chitin-based materials for the adsorption of different molecules can be found in the literature. These studies provide information to understand the chemistry of the adsorption of different molecules onto this polysaccharide. Several studies investigated the interaction between chitin and metal ions by infrared spectroscopy (FT-IR), showing that the adsorption involved the acetylamino and hydroxyl groups [20
22]. Although the N-acetylD-glucosamine groups of the chitin could act as specific chemical binding sites for metal ions through the formation of ion complexes, they are not expected to be as efficient as the free amine sites present in chitosan. That is, the free amine groups in chitosan are much better ligands for the binding of metal ions than the N-acetylated amine groups [23]. In spite of this, many articles showing metal adsorption onto chitin can be found in the literature. Many metal ions, such as Zn21, Cd21, Cu21, Fe31, Cr31, and Pb21 have been adsorbed onto chitin [24]. Some of the basic parameters which strongly influence the whole process are (1) the pH value of the solution; (2) the contact time; (3) the initial ion concentration; (4) the temperature; (5) the agitation speed; (6) the volume of the adsorbate solution; (7) the ionic strength of the solution; and (8) the adsorbent dosage, etc. Most studies report that the Langmuir isotherm model and pseudo-second-order model were found to fit better to the adsorption data. Moreover, Weber and Morris and Elovich kinetic models are essential for projecting possible applications in real

Handbook of Chitin and Chitosan

5.2 Chitin adsorption properties

141

wastewaters under real environmental conditions [25]. In this regard, Karthikeyan et al. tested the removal of Fe31 by chitin, arriving at interesting conclusions. When chloride ion concentration increased, the removal percentage of Fe31 decreased due to the greater formation of soluble chloro-complexes and as a consequence there was less free Fe31 [26]. These chloro-complexes have less affinity than free metal ions toward chitin. A similar effect occurs in the presence of nitrate. Chitin has also been utilized as an adsorbent for a variety of organic substrates that usually contaminate water such as phenol, emerging contaminants, and dyes [27
33]. The interaction between chitin and these molecules varies according to its nature. Some studies proposed mechanisms involving nonionic interactions and the formation of hydrogen bonds [34,35]. Hydrophobic interaction and physical adsorption was also proposed as the main mechanisms [29]. As can be expected from the above stated, chitin chemical nature favors weak interaction with the adsorbates. This makes difficult the direct detection and characterization of chitin
adsorbate interactions. One of the most used techniques for interaction characterization is FT-IR spectroscopy. Although it is a widespread technique for this purpose, many times researchers tend to assign interactions when band shifts of less than 5 cm21 are detected from the chitin to the chitin
adsorbate spectrum. This interpretation can be tricky since variations in the spectrum baseline may lead to small band shifts. Also, many times the overprocessing of the spectra by routine methods, such as baseline correction or smoothing, may also lead to artificial band shifts. In this regard, Raman spectroscopy is less widespread but more accurate. This is probably due to the instrument’s lower availability and the fact that most interactions involve hydrophilic groups that present low signals in the Raman spectrum. Nevertheless, when the interaction can be detected by Raman spectroscopy, shifts of 1 cm21 in dispersive instruments and 0.5 cm21 in FT instruments are indicative of interaction-induced band shifts or structural alterations, also, probably induced by the adsorbate interaction. In this regard Saini et al. [36] proposed that the adsorption of p-nitrophenol onto chitin would occur by weak interactions because no band shifts or the appearance of new or disappearance of preexisting bands could be detected by FT-IR; meanwhile, in the FT-Raman spectra (Fig. 5.2), a shift in the amide I band could be detected from 1657.5 to 1653.7 cm21 when p-nitrophenol is adsorbed onto chitin [36]. This is a conformation-sensitive band with particular symmetry and peak maximum for α-chitin and β-chitin spectra. Thus it was proposed that the shift observed accounts for a restricted conformation driven by a hydrogen bonding between the aOH of the nitrophenol and the CQO or aNaH of the acetoamide group of chitin.

Handbook of Chitin and Chitosan

142

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

FIGURE 5.2 FT-Raman spectra of chitin and chitin-p-nitrophenol-loaded powders in

the range of 2000
1200 cm21. Source: Adapted from S.S. Saini, G.J. Copello, A.L.J. Rao, HPLCUV platform for trace analysis of three isomeric mononitrophenols in water with chitin based solid phase extraction, Anal. Methods 9 (2017) 4143
4150 by permission of The Royal Society of Chemistry.

Like many of other structural natural polymers, chitin has a close relation with proteins in animals, for example, in the exoskeleton of crustaceans. This implies that in each organism there are one or more proteins that tightly interact with this polysaccharide. This efficient pairing is possible through years of biological evolution. Taking advantage of this, many researchers have studied which proteins are capable of interacting and being adsorbed to chitin. This interaction has been studied, involving different applications, such as drug (therapeutic proteins and peptides) delivery systems, protein purification, and more recently the purification of a recombinant protein (human g-type lysozyme) [37
40]. Moreover, some studies report protein immobilization for bioprocess development [41]. Chitin
protein interaction occurs in nature with glycoside hydrolases (GHs) family 18. GHs catalyze the hydrolysis of glycosidic bonds and are key enzymes in carbohydrate metabolism. Efficient degradation of recalcitrant polysaccharides, such as chitin and cellulose, is accomplished due to synergistic enzyme cocktails consisting of accessory enzymes and mixtures of GHs with different modes of action and active site topologies. The substrate binding sites of chitinases and cellulases often have surface exposed aromatic amino acids and a tunnel or cleft topology [42,43].

Handbook of Chitin and Chitosan

5.2 Chitin adsorption properties

143

Chitinases are GHs that catalyze the conversion of chitin, a β-1,4 linked polymer of N-acetyl glucosamine (GlcNAc), into chitobiose units. The catalytic domains of GH family 18 chitinases have a characteristic (β/α)8 TIM barrel fold. Family 18 chitinases conduct hydrolysis through a unique substrate-assisted mechanism in which the N-acetyl group of the sugar bound in subsite 21 acts as the nucleophile [44]. Family GH18 chitinases degrade chitin with retention of the stereochemistry at the anomeric carbon [45,46]. In family 18 chitinases, only one catalytic carboxylate was identified as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. However, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center [47]. Lysozyme (muramidases) is a hydrolytic enzyme with many industrial applications that hydrolyzes β-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine, and also shows affinity for chitin. The surface characteristics of chitin play a significant role in lysozyme adsorption. Particularly, more porous surfaces enhance the interaction of biological molecules increasing the adsorptive capacity, most probably because of easier diffusion, whereas nonporous chitin adsorbs less lysozyme [48]. Furthermore, chitin
protein interactions can be dissociated and the protein eluted under nondenaturing conditions. Chitin is specifically affected by carboxyl and/or methyl groups of acetic acid suggesting lysozyme
acetic acid competition, making this an excellent eluent for the adsorbed protein [49]. The free divalent metal ions Mn(II) and Fe(II) caused significant reductions in the hydrolytic activity of GH18 chitinase. This inhibitory effect probably involves the interaction of these divalent cations with the negatively charged carboxylate residues in the chitinase active center [50]. This assumption is based on previous observations that divalent metal ions are able to form stable complexes with carboxylic groups at the active sites of enzymes, as investigated in hen egg-white lysozyme [51]. Therefore it can be assumed that some free metal ions in solution can hinder protein adsorption in chitin-based chromatographic systems. This interaction of the enzymes with divalent ions may be useful if the ions are incorporated into the polymeric chitin matrix. Divalent ions, such as Cu(II), Ni(II), and Fe(II), can enhance the performance of chitinous polymeric matrices used in separation and purification of biological molecules since they exhibit affinity for pendant groups (e.g., imidazole, indole, and thiol) present on certain amino acids. For example, lysozyme adsorption onto Cu(II)-free chitin-fixed pellets was reported to be much lower than onto Cu(II)-immobilized chitin-fixed pellets [48]. This observation demonstrated the contribution of Cu(II) to the interactions of lysozyme molecules with chitin surface.

Handbook of Chitin and Chitosan

144

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

Finally, molecular dynamics (MD) simulation has been used for studying the intermolecule interactions at the atomic level. The behavior and properties of proteins in the process of the interaction with polymers have been characterized using this technique [52
54]. Yahyaei et al. applied an MD simulation for the elucidation of the complexation between follicle stimulating hormone (FSH) and chitin polymers at the molecular level, finding that the secondary structure of FSH was not affected by the polymers [39]. This tool can provide important information for understanding interactions with new molecules or modified matrices.

5.2.2 Chitin physics As stated above, chitin physical properties have a relevant role in the overall adsorption process. For example, this polysaccharide and the adsorbate response to temperature will influence its performance in a particular situation. Since rising or lowering the temperature is associated with an energetic cost, industrial effluent adsorption, and wastewater treatment are usually performed at the location season temperature. Nevertheless, for adsorption processes associated with high addedvalue products, the gain in efficiency may surpass the cost of controlling the temperature of the process. Chitin can be subjected to high temperatures if necessary. The basic range of the thermal degradation of chitin is 300 C
460 C, the thermal resistance depends on the origin of this biopolymer and can be arranged as follows: krill chitin . shrimp chitin . crab chitin . squid chitin. The monoclinic β-chitin obtained from squid is thermally much less stable than the orthorhombic α-chitins originating from krill, crab, and shrimp. The size and perfection of crystallites are the most important factors influencing the thermal stability [55]. The similarity of thermogravimetric analysis (TGA) profiles indicate that there is no influence of the average molecular mass, the degree of crystallinity, and the DA on the thermal resistance. Meanwhile, by differential scanning calorimetry analysis (DSC) the specific thermal behavior associated with the crystalline structure was observed. The temperature for an exothermic peak above 200 C is higher in α-chitin than in β-chitin. Because γ-chitin has an antiparallel and parallel structure, that is, a combination of α and β conformations, this exothermic peak appears between those of α-chitin and β-chitin [16,55]. The following crystalline polymorphs of chitin have so far been found using X-ray diffraction measurements: the most abundant “α-chitin,” the less abundant “β-chitin,” and barely abundant “γ-chitin” forms [56
58]. The differences among chitin polymorphs are due to the

Handbook of Chitin and Chitosan

5.2 Chitin adsorption properties

145

arrangement of the chains in the crystalline regions: α-chitin has a structure of antiparallel chains, β-chitin has intrasheet hydrogen-bonding by parallel chains, and γ-chitin, being a combination of α- and β-chitin, has both parallel and antiparallel structures. Because of these differences, each chitin polymorph has different properties specific to it. For example, β-chitin is more soluble and more reactive in solvents and has a greater affinity toward them; it is also more susceptible to swelling than α-chitin. β-Chitin is also more amenable to N-deacetylation than α-chitin [59]. Chitin from different species, or extraction processes, may have different morphologies, such as the size of pores, the thickness of particles, and other parameters that influence the sorption kinetics. Chitin of butterfly, cockroach, or spider has particular morphologies and porosities that allow chitin to be used in extreme biomimetics synthesis [60
62]. Extreme biomimetics, a recent field of materials chemistry, is inspired by materials found in extremophile organisms, instead of aiming to implement their actual biosynthesis. Most species associated with hydrothermal vent habitats are invertebrates, many of which have a chitin-based exoskeleton that is often biomineralized. The structural rigidity—in chitin arising from a highly regular polymer and a considerable sum of intermolecular interactions leading to high crystallinity—yields enhanced stability, capable of withstanding high pressure and elevated temperatures [63]. This property has been seized for the extreme biomimetic synthesis of hybrid materials, by performing inorganic hydrothermal condensation of several metal oxides [64]. Examples include the preparation of chitin/SiO2, chitin/ZnO, chitin/ZrO2, chitin/GeO2, chitin/Fe2O3, and chitin/(Ti, Zr)O2 [65
70]. The majority of the latter examples were generated at very mild hydrothermal conditions, specifically at 90 C or 120 C. Other subsectors of extreme biomimetics, such as bioelectrometallurgy, propose chitin of poriferan origin for the electrochemical deposition of nanocrystallites of Cu and Cu2O; due to their high resistance to chemical treatments and exceptional thermostability, metallized chitinous scaffolds could be used in various processes such as the cleaning of wastewater from a wide range of organic compounds, including dyes such as methylene blue (MB) and methyl orange (MO) [71]. These structures have qualified as promising candidates for sophisticated applications, by means of the functionalization of these biomimetic structures of chitin with inorganic compounds. Recently they have used in the design of quartz crystal microbalance biosensors and metal
organic framework (MOFs) crystallites on chitin for the airfiltration of toxic industrial gases [72,73]. Studies show that sorbent biopolymers improve their capacity when they have lost their crystalline structure, therefore the accessibility to amorphous regions increases the capacity of the sorbent to absorb metal

Handbook of Chitin and Chitosan

146

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

ions [74]. Moreover, amorphous regions may sometimes implicate higher polymer mobility and, therefore higher affinities toward the adsorbate due to the precise arrangement of polymeric chains for the interaction. This is a key point in the use of chitin, because of its high crystallinity. This is why many sorption studies are carried out on chitin derivatives for greater ease, such as chitosan and its oligosaccharides. Novel methods such as instant catapult steam explosion (ICSE) reduce the crystallinity of chitin without affecting molecular weight, thermostability, and acetylation [75]. Chitin gel formation by CaBr2 xH2O/CH3OH led to porous chitins with continuous channel substructure by regeneration from gels. This lower crystallinity resulted in more accessible sites for the adsorbate, resulting in an increase in swelling from 50% to 150% and the adsorption capacity went from 30 to 560 mg/g for MB removal [76,77]. Similar structures can also be obtained with chitin hydrogel from a saturated solution of CaCl2 2H2O/CH3OH, known as the Tamura method [78]. A nanocomposite chitin
graphene oxide (GO) obtained via the wet-spinning process was analyzed by SAXS. A profile typical of collapsed structure and mass fractal was observed for the dry material. Upon hydration, chitin adopts an expanded rod-like conformation, reflecting an opening of the channels because of swelling [30]. In batch adsorption processes, the kinetic equilibrium is relevant, not regarding capacity but by means of the time that the whole process will take. Obviously, shorter times are always desired. Nevertheless, if capacity is the main feature to seek in a batch system, the kinetic equilibrium will not be taken into account. On the other hand, for continuous adsorption systems, the kinetic equilibrium is always a relevant feature. Slow kinetic equilibrium in continuous-flow systems will let the adsorbate pass through the column without being adsorbed and the capacity will drop. As a consequence, lower influent flows will be used in order to compensate for this effect, therefore leading to longer processing times. In this regard, McKay, Blair, and Gardner studied the performance of chitin particles fixed-bed columns in the adsorption of dyes [79]. The breakthrough profiles for the columns filled with particles of smaller diameter showed a sharper breakthrough compared to the columns with larger particles, which indicated a faster adsorption rate. The authors expected this effect due to the higher external surface area available for adsorption onto small particles and also because the average intraparticle diffusion paths are shorter. In two different approaches, Ma et al. [80] and Gonzalez et al. [30] prepared chitin-based matrices containing graphene oxide (GO) nanosheets as the main adsorbent for continuous-flow systems [30,80]. In both works, the strategy to obtain high-performance systems was to expand chitin’s structure into porous matrices. Ma et al. used a





Handbook of Chitin and Chitosan

5.2 Chitin adsorption properties

147

high-pressure homogenizer to obtain chitin nanofibrils (CNF) and also chitosan nanofibrils. Both were mixed with GO and then freeze-dried. During the material synthesis, the authors observed that the chitosan nanofibril
GO composite presented flocculated sections and led to loose blocks instead of a monolithic material, such as the one obtained when using the CNF
GO composites. The authors claimed that this effect would be due to the more charged surface of chitosan compared with chitin. Therefore they decided to continue their flow assays solely with the CNF
GO composite. In this regard, Gonza´lez et al. preferred to study the chitin
GO nanocomposites in the swelled particulate format. The authors observed that chitin
GO particles needed to be sieved down to 500 μm for optimum performance. Smaller particle sizes lead to high backpressure or even occlusion of the column system. In addition, with the increase in the flow, they observed an increase in adsorption capacity (determined by the Thomas model) but a decrease in the breakthrough time. It was proposed that this effect can be justified by the adsorption mechanism of ciprofloxacin on this material, where at low flow rates the adsorbate would interact mainly with the exposed surface and larger pores, and inner pores are slowly filled. In contrast, higher flows represent a higher availability of ciprofloxacin for the external sites that rapidly saturate and the remaining adsorbate would be forced to interact with the surface of the inner pores at lower kinetic rates, probably driven by an intraparticle diffusion mechanism. The structure of the matrix has a significant role in the performance of an adsorbent. The three-dimensional (3D) structure defines the surface area and porosity. The former is often related to the adsorption capacity. For a given mass of polymeric adsorbent, the expansion of the network would mean that fewer chains are interacting with other polymer chains and are prone to interacting with solutes of the surrounding media. Thus greater surface area implies more available sorption sites. The latter, the porosity, is related to the kinetics of the process, by means of intraparticle diffusion, involving intrapore and channel diffusion. Larger pores would be associated with larger diffusivity than smaller pores. Also, the presence of pores with similar sizes to the analyte would add tortuosity to its path toward the adsorption site. Moreover, when the target molecule is a protein with a diameter of several nanometers, macroporous ( . 50 nm) or gigaporous matrices would be desired in order to let the adsorbate reach every adsorption site available without the need for extremely long equilibrium times. The research done on chitin materials shows that this polysaccharide is not the exception and obtaining hydrogels and aerogels, or fibrillar structures, can endow the matrix with great surface area and porosity. As a starting point, chitin flakes can be considered as nonporous with a low surface area, reported to be around 1.236 m2/g [81]. Other researchers

Handbook of Chitin and Chitosan

148

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

have prepared microspheres in order to increase the surface area of the material. This is the case of the study presented by Xu et al. in which microspheres presented a specific surface area of 115.7 m2/g [82]. Silva et al. prepared porous gels by dissolving chitin in ionic liquids [83]. They reported surface area values between 100 and 150 m2/g and porosities from 0.2 to 0.3 cm3/g for the different treatments. Higher surface areas were obtained by Ding et al. in chitin aerogels obtained by dissolution in NaOH/urea and freeze
thawing cycles and posterior coagulation in ethanol before freeze-drying [84]. These aerogels presented specific surface areas between 320 and 370 m2/g and porosities from 1.2 to 2 cm3/g. All the before mentioned surface area values have been reported to be calculated using nitrogen adsorption isotherms and the Brunauer
Emmett
Teller (BET) model. Pore dimensions have been seldom reported for this kind of materials since this method allows the accurate determination of pore parameters when the pore diameters are in the order of nanometers. For larger pores, such as in gigaporous matrices, other methods need to be used, such as mercury intrusion porosimetry.

5.3 Shaping chitin materials An efficient adsorption process requires not only optimal affinity and capacity toward the sorbate. As mentioned above, when the kinetics of the process, mechanical stability of the matrix or recovery and recycling of the material enters into consideration, the format of the adsorbent is extremely relevant and can play a determinant role in the success of the adsorption process and the system’s life cycle. In nature, chitin exists in its own form, which many times differs from a final product with desirable properties. Chitin is present as part of the exoskeleton of arthropods and as part of the fungal cell wall. It is present in the shells of crustaceans such as crabs and shrimps, the exoskeletons of krill, the cuticles of insects, and it is also a specific component in many other living organisms [55]. In arthropods several CNF with different lengths are wrapped together with specific proteins to form a chitin
protein structure that is embedded in a mineral
protein-based matrix forming several planar layers that are stacked. Each layer rotates from the previous stacked layer at a constant angle determining a helicoidal structure forming the exoskeleton [85]. In fungi chitin is often associated with glucans and proteins that form a basket-like scaffold around the cell [86]. Once chitin is extracted from its natural source, several structures can be achieved using either a chitin solution as the starting material or directly from suspensions of the semicrystalline polymer composed of nanofibrils, nanowhiskers, or nanoparticles [85,87,88]. Although the

Handbook of Chitin and Chitosan

5.3 Shaping chitin materials

149

FIGURE 5.3 Structures that can be achieved using chitin once is extracted from their natural source.

dissolution of chitin is one of the main difficulties when working with this material, there are several alternatives to address this issue, such as employing ionic liquids [89,90], solutions of N,N-dimethylacetamide/ LiCl [91], methanol/CaCl2 [92], deep eutectic solvents [93], highly polar fluorinated solvents [94], or inorganic salts aqueous solutions and NaOH/urea aqueous solutions [85]. By using different techniques and protocols, chitin solutions could be formatted into a 3D network structure with different forms, like films, membranes, fibers, microspheres, hydrogels, aerogels, etc. (Fig. 5.3). These regeneration methods involve the sol
gel transition of chitin and can be achieved by different strategies, such as solvent exchange, dialysis, pH adjustment, crystal transformation, and physical, ionic, or chemical crosslinking [95,96]. For example, chitin films and membranes are usually prepared by casting a chitin solution in a glass container and then immersing them into a nonmiscible chitin solvent to allow film/membrane coagulation [97]. The same principle could be used to produce chitin fibers using a typical wet-spinning procedure [98,99]. Microspheres can be generated by adjusting the pH to 7.0 of a stirring chitin solution prepared using NaOH/Urea/H2O in a relationship of 11:4:85. Modifying the grade of stirring and the composition of the chitin solution by adding an emulsifier and an organic phase, different sizes of microspheres can be obtained [82,100]. Hydrogels—3D materials with abundant water—can be formed

Handbook of Chitin and Chitosan

150

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

by cross-linking polymer chains through physical, ionic, or covalent interactions and the final structure often depends on the recipient where the sol
gel transition occurs [101]. Another possibility for the use of chitin is to obtain nanofibrils, nanowhiskers, and/or nanoparticles and then use them to generate a 3D network structure. The methods to obtain these nanobuilding blocks can be classified as “top-down” and “bottom-up.” A top-down method implies the direct isolation of the nanostructure (extraction) from biological source material using mechanical and/or chemical treatments [87]. On the other hand, a bottom-up approach implies the assembly of several chitin molecules into this nanobuilding blocks (nanofibrils, nanowhiskers, and/or nanoparticles) [102]. The assembling can be triggered by changes in pH, solvent, temperature, and external forces (electric and magnetic fields), among others (Fig. 5.3). Depending on the natural source and the extraction protocol, α-chitin, β-chitin, and/or ɣ-chitin nanofibrils with different degrees of acetylation, with a diameter in a range of 1.5
25 nm and a length between 3 and 200 nm can be obtained [87]. Using suspensions of these nanobuilding blocks it is possible to generate nanostructured hydrogels, aerogels, films, membranes, fibers, and nano/microspheres. Another consideration to take into account is the possibility to combine chitin with other materials during the dissolution or suspension of chitin, prior to the formation of the 3D structures (Fig. 5.3). Chitin can interact with inorganic salts, metals, natural and synthetic polymers, organic compounds, carbon materials, etc. In each case, the combination of chitin with these elements determines a new kind of material with unique mechanical, chemical, and physical properties, different from each element separately [85]. The use of chitin in its different forms and in combination with other materials opens unlimited possibilities of new materials for a wide diversity of applications. According to the application or process in which it will be applied, and taking also into consideration the cost and the added value of the final product, among other important factors, the most favorable structure and combination of materials can be selected. Even though there are many applications for chitin, in agro, food, or pharma fields, this work focuses on the adsorptive applications, specifically involving chromatography for bioproduct purification, enzyme immobilization, and treatment of industrial pollutants, among other adsorptive processes [102].

5.3.1 Chitin supports for adsorptive processes From the adsorption perspective, the requirements of an adsorbent material are ideally the following: high adsorption capacity, high

Handbook of Chitin and Chitosan

5.3 Shaping chitin materials

151

chemical and biological resistance, high productivity (defined as the amount of solute adsorbed per unit of time and amount of material), low cost, and regulatory suitability for the proposed application. Depending on the specific application, it could be necessary to maximize other attributes, such as a high elution of the previously adsorbed solute, conditioning for chromatographic applications and purification of bioproducts, as well as the reusability of the matrix in multiple successive cycles [103]. As discussed previously, chitin can chemically interact with several metal ions, organic substances that pollute natural waters, such as phenol, drugs, dyes, proteins, and peptides. Several studies reported the interaction between chitin and these compounds, showing that the adsorption involves hydrophobic interactions, hydrogen bondings, and/or van der Waals interactions with the acetamide group and/or the hydroxyl groups of the chitin. Moreover, some proteins can be adsorbed due to their affinity to chitin, where multiple kinds of interactions are usually combined [104
106]. Typically, a support for an adsorptive process could be used in a batch or in a continuous-flow system. In both of them, the target molecule is concentrated in the adsorbent, since the volume of the adsorbent is by far smaller than the volume of the solution processed—an important fact in the cases of contaminant removal, which is maximized by an elevated adsorptive capacity of the materials. Different chitin structures are studied for this kind of applications, both as the main component of the adsorptive support and as a blend with other polymers, among other approaches. However, some considerations have to be taken into account for the design, to scale up, and to achieve a successful implementation of these processes.

5.3.2 Batch systems In a batch system, the adsorbent (in this case a chitin-based structure) is in contact with the target molecule in a stirred tank (Fig. 5.4). The adsorption process takes place for a certain amount of time and the system may—or may not—reach the equilibrium condition. The target molecule diffuses from the solution to the hydrated film around the adsorbent (interadsorbent and film diffusion), diffuses inside the pore (intraparticle diffusion, if it has a porous structure), and then chemically interacts with the adsorbent according to its binding kinetics determined for each type of interaction [107]. Therefore batch mode adsorption is a time-consuming process since each step of the process implies recovering the adsorbent, emptying and filling the tank with the different solutions. Usually, the support used in this kind of process presents

Handbook of Chitin and Chitosan

152

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

FIGURE 5.4 Adsorption modes (A) stirred tank, (B) expanded bed adsorption, (C) fixed packed bed, (D) fluidized bed, (E) adsorptive membrane, and (F) monolith.

an internal porous structure in order to maximize the adsorptive surface area and the loading capacity of the material. A batch system is selected over a continuous-flow system usually when the solution to be processed has high viscosity and/or has large particles in the solution which may occlude a packed column. In other cases, like when high volumes of starting material need to be processed as a source of a bioproduct, the batch condition may be preferred to reduce the required time for the adsorption. By increasing the relation of matrix amount per sample volume, the time required to complete the adsorption of the same amount of adsorbate is reduced [108]. Diffusion limits the velocity of the process. In the case of small molecules like dyes and metal ions, diffusion is faster than in the case of big molecules like proteins. The use of small chitin particles as a support increases the contact area and minimizes the required time for the diffusion of molecules in the system, but the separation of the support from the solution is difficult and expensive since smaller particles have a lower sedimentary coefficient. On this matter, there are several chitin structures used for batch systems: nanofibrils, nanowhiskers, nanoparticles, hydrogels, and microspheres. Chitin is studied for this kind of system both as the main component of the adsorbent and as a blend with other polymers.

5.3.3 Continuous-flow systems Different formats for continuous-flow adsorptive systems can be found in literature and can be divided into two groups; one group is represented by continuous-flow systems where the mass transfer is predominantly governed by diffusion, this is the case of a fluidized bed, expanded bed adsorption, and fixed-bed modes (Fig. 5.4A
D). The other group is represented by continuous-flow systems where the mass transfer is predominantly governed by convection; this is the case for adsorptive membrane and monoliths supports (Fig. 5.4E and F).

Handbook of Chitin and Chitosan

5.3 Shaping chitin materials

153

In the case of fluidized bed, expanded bed adsorption, and fixed-bed modes, as was previously mentioned for the batch systems, the target molecule should diffuse from the solution to the hydrated film around the adsorbent (interadsorbent and film diffusion), diffuse inside the pore (intraparticle diffusion), and then bind to the adsorbent according to its binding kinetics [107]. All these steps would have to happen in the time of residence of the target molecule in the system. Likewise in batch systems, the use of smaller adsorbent particles minimizes the diffusion times and accelerates the process. Nevertheless, in the case of the fixed-bed mode, with smaller particle sizes, the backpressure rises pressing against the physical integrity of the support. Usually, the support has a compressible porous structure that may collapse. For a fluidized bed, it is not possible to use smaller particles since a high sedimentary coefficient is needed for the maintenance of the fluidized bed without the loss of the support matrix. Another parameter to take into account is the processability of the solutions that pass through the fixed-bed support, especially in the case of wastewater treatment with the objective of adsorbing a specific contaminant or bulk contaminants. For example, for a fixed-bed column with 200-μm-diameter particles, the solutions should be filtered using a pore size of about 4% of the particle diameter to prevent plugging the packed bed. In this case, filtration with an 8-μm mesh is enough to avoid the plugging of the column [109]. This implies the previous clarification and even filtration of the starting material to prevent the plugging of the fixed-bed by larger particles than the voids between the particles of the adsorbent, an additional step that increases the process cost. When using a fluidized bed or an expanded bed adsorption mode this situation is avoided since the suspension of the bed prevents the clogging of the column and the rise of backpressure [110]. Adsorptive membrane supports involve the utilization of nano/ microfiltration membranes by the immobilization of different ligands on their internal pore structure, resulting in a support that can interact with a specific target molecule or group of molecules. A monolith is a highly interconnected porous support made of one piece of material, usually with the form of a column and with interconnected channels. In these support systems, mass transfer is predominantly governed by convection rather than by diffusion. The use of these systems eliminates pore diffusion as the main limitation since mass transport by convection occurs through the pores. The target molecule has to diffuse from the hydrated film at the pore walls and then bind to the specific ligand with its specific binding kinetics. As film diffusion is usually several orders of magnitude faster than pore diffusion, mass transport limitations are drastically reduced in adsorptive membrane and monolith systems [111]. In these convective systems, the flow velocity could be

Handbook of Chitin and Chitosan

154

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

increased considerably without a decrease in the adsorption capacity. These adsorptive membranes could be implemented using tangential flow filtration mode, increasing the productivity, and allowing the processing of larger volumes in a shorter time even with particulate material present in the feedstock sample. However, fouling problems and an improvement in the adsorptive capacity should be resolved before its industrial implementation [107]. On this matter, there are several chitin structures used for continuous-flow systems: hydrogels, microspheres, and membranes. Chitin is studied for these kinds of systems both as the main component of the adsorbent and as a blend with other polymers. These cases will be reviewed in the following section.

5.4 Optimizing adsorption performance by chemical and physical modifications 5.4.1 Discussion of some relevant developments The adsorption or separative performance for a specific adsorbate is not merely defined by the adsorption capacity onto the adsorbent. The adsorption or separative process is influenced by many variables of dissimilar nature. For example, for batch systems, an extremely long adsorptive equilibrium period would be undesired for the industrial recovery of valuable analytes. Also, chemical modification of adsorptive matrices by means of fine chemicals that increase selectivity or capacity would imply an increase in the cost of cheap matrices, probably leading to the inapplicability of the systems for the treatment of hundreds of litters of industrial effluents. Taking this into account, the present section will critically review the reported strategies directed to the optimization of adsorption processes based in chitin matrices.

5.4.2 Pristine chitin The high availability and renewability together with the low cost of its manufacturing highlight the advantages of developing pure chitin matrices without extra processing. Any further treatments may raise the cost of the final material and decrease the sustainability of the process. Up to this day, few chitin-based products are commercialized. Pinto et al. have evaluated the performance of commercial chitin-based adsorbents (Chitorem SC-20 and Chitorem SC-80 from JRW Bioremediation LLC) [112]. This material consists of low processed crab shell products rather than pure chitin. They were studied as an alternative for mining residues water remediation by the adsorption of heavy metals. The

Handbook of Chitin and Chitosan

5.4 Optimizing adsorption performance by chemical and physical modifications

155

surface area was determined for both matrices by the BET method, resulting in 10.28 m2/g for the SC-20 and 4.19 m2/g for the SC-80. At a load of 2 g/L, SC-20 removed almost all the iron (120 mg/L), zinc (79 mg/L), and lead (1.1 mg/L) and partially removed cadmium (96% of 1.3 mg/L), manganese (64% of 52 mg/L), cobalt (54% of 0.78 mg/L), and copper (42% of 72 mg/L). In the case of SC-80, adsorption capacities were analyzed from single metal solutions: lead (up to 1.24 mg/g), cadmium (up to 1.81 mg/g), and cobalt (up to 0.93 mg/g). Also, when evaluating the use of nonprocessed chitin-based materials— mainly food industry waste—to highly chemically modified materials— such as selective adsorbent derivatized chitin—one of the most costeffective strategies would be the treatment of chitin flakes for their direct use as adsorbents. In this regard, Dotto et al. modified chitin flakes surface to improve the adsorption characteristics toward cobalt using ultrasoundassisted (UA) and supercritical CO2 technologies (SCO2). After treatment, chitin particle size decreased which led to the increase of the material surface area. Interestingly, the porosity, pore volume, and average pore radius increased considerably, which may be related to the loss in crystallinity of the treated samples. These modifications improved cobalt adsorption from aqueous solutions. Although the equilibrium times do not vary appreciably, the adsorption capacities rise 68% when treated with ultrasound and 26% when treated with supercritical CO2 [27,28]. The ultrasound-assisted treatment of chitin’s surface proved that it is effective not only for small adsorbates such as Co21 but also for bigger molecules such as MB. Once again, treated chitin presented more adequate characteristics for adsorption purposes than raw chitin, such as higher surface area, higher porosity, lower crystallinity, and a more rough surface [27,28]. From a different point of view, pristine chitin can be treated to obtain nanowhiskers. As one of the examples of this strategy, Dhananasekaran et al. embraced the synthesis of α-chitin nanowhiskers for the adsorption of MB, bromophenol blue, and coomassie brilliant blue [113]. Chitin powder was soaked in 3 M HCl for 90 min at 90 C and then centrifuged in order to collect the pellets; this procedure was repeated three times. The pellets were suspended in distilled water, dialyzed until pH 6.0, and homogenized using a tissue homogenizer. Mechanical disruption and ultrasonication were performed to obtain the nanoparticle size. The authors presented a maximum capacity of 6.9 mg/g for MB, 22.7 mg/g for bromophenol blue, and 8.5 mg/g for coomassie brilliant blue according to the Langmuir isotherm model, and a capacity in equilibrium of 9.4, 24.4, and 13.1 mg/g, respectively, according to the pseudo-second-order kinetics. These values were high in comparison with other reported by-products with adsorptive capacities from the industrial and agricultural wastes used to bound different dyes. The authors assigned this difference to the change in surface area, particle

Handbook of Chitin and Chitosan

156

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

size, and physical properties of the nanomaterial due to the hydrolysis step that the polymer was submitted to in order to obtain the nanoparticle dimension. The fact that the matrix is smaller allows diffusion times to decrease making the process quicker. However, the recovery of the material after the adsorptive or washing steps is more difficult due to a low sedimentation coefficient. Several groups studied these kinds of supports for enzyme immobilization for different biotechnological purposes. Xu et al. took advantage of the affinity between triple-functional heparinase I and chitin to immobilize this enzyme in chitin microspheres (Fig. 5.3) [105]. The resin could be used up to eight times to produce low-molecular-weight heparin with a yield higher than 90%. Jiang et al. immobilized lysozyme on chitin nanowhiskers, which resulted in a superior enzymatic activity than free lysozyme [114]. Another alternative application to enzyme immobilization is using this kind of material as a loading material for drug delivery systems. In this matter, Wang et al. developed chitin microspheres to load anthocyanins for colon-specific delivery [115]. The adsorption capacity according to the Langmuir isotherm was 2718 mg/g which was the greatest value reported up to this publication. Recently, Chakravarty et al. used the ionic liquid 1-ethyl-3methylimidazolium acetate as solvent media for the preparation of chitin membranes. The synthesis was performed by a first step consisting of the dissolution of chitin in an ionic liquid, followed by its coagulation by adding deionized water. The resulting materials were thoroughly characterized. The drying methods and the concentration of chitin used defined many of the membrane properties, such as mechanical strength, porosity, and swelling. These proposed methods provide a starting point for the design and fabrication of a family of chitin membranes with potentially broad applicability including adsorption of proteins, dyes, or metals [116].

5.4.3 Cross-linking A proper chemical and mechanical stability of a matrix not only may allow its applicability in heterogeneous phase adsorption systems without the leaking of matrix residues to the liquid media but also may allow the reusability of this matrix. Cross-linking is one of the classic strategies to endow a material with these properties. Particularly, pristine chitin properties make cross-linking seldom used for these polysaccharide-based materials. Cross-linking has been reported for specific cases where chitin modification achieves soluble derivatives, such as chitosan or highly phosphorylated chitin. The latter was reported by Nishi et al. [117,118].

Handbook of Chitin and Chitosan

5.4 Optimizing adsorption performance by chemical and physical modifications

157

These researchers have obtained phosphorylated and soluble chitin. Although the introduction of phosphoryl groups granted a potential improvement for the adsorption of cations, as will be discussed below, the solubility of these derivatives imply a drawback for their use in heterogeneous phase adsorption, making their separation from the adsorption media difficult and making impossible their use in packed-bed columns. In order to overcome this drawback, the authors have crosslinked the soluble derivatives by reacting the unsubstituted hydroxyl groups with an acyl dichloride, the adipoyl chloride. During the reaction, the cross-linked product became insoluble and precipitated. Thus regaining the insolubility of the original chitin. One of the most common cross-linkers used for the chemical stabilization of polysaccharide matrices is the bidentate ligand epychlorohydrin. This electrophilic reagent can cross-link chitin hydroxyl groups in alkaline conditions. Tang et al. also developed an adsorbent based on chitin to remove malachite green from wastewaters using batch systems [119]. The adsorbent was a chitin hydrogel produced in a similar way to the matrices developed by Wang et al. previously discussed. Chitin was mixed with a solution of 8 wt.% NaOH/4 wt.% urea/88 wt.% water and was kept at 220 C for 8 h. The solution was thawed and stirred at room temperature, and the freezing/thawing cycle was repeated three times. The obtained solution had 3% chitin and 1 g of this solution was cross-linked with 0.05 mL epichlorohydrin. Afterward, the solution was kept at room temperature for 1 h to obtain the hydrogel. The main strength of the adsorbent was endowed by the presence of chitin, due to its low chain mobility. Even though the adsorbent was not shaped like 3D matrices, as in the previous examples, the authors made a thorough study of the mass transport for the batch adsorption of the dye. First, the adsorption isotherms were analyzed both by Langmuir and Freundlich models at different temperatures. The authors showed that the amount of malachite green adsorbed increased with increasing temperature and that the isotherms had a better fit for the Langmuir model (indicating monolayer adsorption), with a maximum capacity of 0.092 mmol/g and a dissociation constant of 2.15 L/mmol at 303K. In this work adsorption kinetics were analyzed using the pseudo-first-order, the pseudo-second-order, and the intraparticle diffusion model. The results showed that the adsorption system obeyed the pseudosecond-order kinetics model and the intraparticle diffusion model suggested that pore diffusion was not the step controlling the overall rate of mass transfer at the beginning of the adsorption; the external mass transfer was the rate-limiting process in the beginning and then the intraparticle diffusion. The malachite green could be adsorbed by the chitin adsorbent within 4 h.

Handbook of Chitin and Chitosan

158

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

5.4.4 Composites, nanocomposites, and fillers 5.4.4.1 Interpenetrated polymers and chitin grafting The combination of polymers with other polymers or particles is an interesting strategy for improving chitin-based materials. Many times, if the combination is carefully optimized, grafting or the obtaining of interpenetrated polymers can endow the final material with the advantageous properties of both components. In this regard, Duan et al. developed a film with chitin and lignin, with a high adsorption capacity for Fe(III) and Cu(II) from aqueous media [120]. The film samples were prepared by dissolving lignin and chitin in an ionic liquid consisting of 1-butyl-3-methylimidazolium and γ-valerolactone without any chemical modification and alteration of the two biopolymers. The chitin was essential for obtaining the film. In fact, it was not possible to obtain a film from lignin, and the flexibility of the composite films was attributed to its presence. The investigation of lignin/chitin film as a biosorbent was performed for Fe(III) and Cu(II) aqueous solutions at room temperature. The lignin/chitin film biosorbents showed advantages such as high adsorption capacity for metal ions, especially at low concentration, stability in aqueous solution, easy to use, facile desorption, and reusability, which are all requirements for their application in water purification processes. In a different approach, Zhou et al. and Liang et al. have blended two of the most insoluble natural polymers to form beads: chitin and cellulose [121,122]. This property allowed Zhou et al. to prepared a fixed-bed column for the removal of heavy metal ions that can be easily regenerated using with 0.1 mol/L HCl aqueous solution after the adsorption cycle. The authors report that after four adsorption/desorption cycles the column capacity has only varied by 5% of its initial process parameters. Another possibility is to form blend membranes suitable for different applications. Liang et al. reported a series of biodegradable cellulose/ chitin blend membranes prepared from blend solution of cellulose and chitin in 9.5 wt.% NaOH/4.5 wt.% thiourea aqueous solutions and then coagulating them with 5.0 wt.% (NH4)2SO4 [121]. Their results revealed that all the membranes synthesized had a porous-like structure. The introduction of chitin exhibited great influence on the morphology and crystalline structure of the blend membranes, resulting in materials with different permeability. One of the main target proteins for chitin-based adsorbents is lysozyme. This enzyme is an ubiquitous protein and it is present in secretions, body fluids, and animal and human tissues [123]. Lysozyme catalyzes the lysis of bacterial cell wall sugars, specifically the β bonds

Handbook of Chitin and Chitosan

5.4 Optimizing adsorption performance by chemical and physical modifications

159

between muramic acid and N-acetylglucosamine, and due to this fact chitin-based chromatographic materials were proposed for its purification [124]. In this regard, an affinity chromatographic matrix based on a composite hydrogel retaining noncovalently bound chitin in-between layers of a silicon oxide network was made by Wolman et al. for the purification of lysozyme directly from undiluted egg white [125]. In order to synthesize this matrix, chitin was diluted in methanol saturated with CaCl2. This solution was mixed with 20 parts of water and vigorously stirred to get a hydrogel after the mixture was centrifuged. The hydrogel was then blended with an equal volume of tetraethoxysilane (TEOS) solution, allowing the polymerization and the subsequent sectioning in 2 mm 3 2 mm beads. The effect of chitin in the silica polymerization process was seen in the porous structure of particles. The nitrogen sorption isotherms showed a mesoporous structure instead of the microporous one, commonly seen for many TEOS-based matrices. This matrix had a surface area of 142 m2/g and a total pore volume of 0.295 cm3/g. The adsorption isotherm was analyzed with the Langmuir model, achieving a maximum capacity of 117.1 mg of lysozyme per gram of matrix, and a dissociation constant of 0.73 mg/mL. The matrix adsorption kinetic was analyzed both for pure lysozyme solutions and for egg white, reaching a plateau after 4 and 10 h, respectively. The authors explained that the differences in the results from these two starting materials were due to the high viscosity of the egg white which affected the diffusion rate. Different eluents were tested and 0.1 M acetic acid was selected as the best one after presenting a global yield of 64%. In this study the authors took into account the reusability of the matrix by measuring the adsorption capacity after three cycles of hen egg white processing and they suggested that the resulting matrix had the advantage of being easily recovered—even in a high viscosity solution as egg white—by sedimentation because of its size, mechanical resistance, and density. The approach of enzyme immobilization to chitin-based materials was also followed by Kumari et al. by immobilizing cutinase of Aspergillus sp. RL2Ct to an acrylamide grafted copolymer of chitosan and chitin [126]. In this case, the immobilized enzyme had a good binding efficiency (78.8%) and was more stable than the free enzyme; the matrix was recycled 64 times without considerable loss of activity. Other enzymes were also immobilized in chitin-based materials such as protease and lipase on a chitin
starch matrix [127]. 5.4.4.2 Fillers In a completely different approach to cross-linkers, the obtaining of composites by the use of fillers is of recent interest, especially when

Handbook of Chitin and Chitosan

160

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

nanoobjects are employed. As mentioned above, an efficient way to reinforce a material is by cross-linking. The increase in the mechanical resistance of these materials is associated with the degree of crosslinking. Nevertheless, many cross-linkers interact directly with adsorption sites of the adsorbent, thus leading to the loss of reactive sites, changes in the adsorption selectivity or even adding toxicity to the material [128]. Aiming to overcome these disadvantages, the reinforcement can be pursued using fillers. In a similar manner, as the fibers do at the macroscopic level, even at low loads, the insertion of nanofillers between the polymer chains may achieve great reinforcement. In the last two decades, the use and types of nanofillers (such as nanoclays, talc, silica, and carbon allotropes) has been rising and increasing the offer of new nanocomposites [129]. Therefore the most widespread aim of fillers is to endow the polymers with desirable mechanical properties. Nevertheless, in some cases, when the fillers consist of the functional part of the material, the polymer acts as the containing matrix or support that facilitates the use of the former in heterogeneous phase applications, such as adsorption or catalysis. Different nanocomposites have been developed combining chitin with a great variety of nanoobjects. Ma et al. and Gonza´lez et al. created chitin/GO composites [30,80,130]. These materials have been achieved by two different synthetic strategies. Ma et al. prepared composite foams with 3D porous network structures by embedding CNF in a GO suspension. Then, they filled a fritted empty column and freeze-dried the suspension. They obtained a self-contained adsorption system and used it for the removal of MB, MO, Pb(II), and p-chloroaniline dye from aqueous solution through batch adsorption and column adsorption. The introduction of CNF significantly improved the mechanical strength, which was mainly related to the high crystalline and porous network formed by interconnected microribbons of CNF in GO sheets. Also, the interfacial adhesion arose from electrostatic interaction and the hydrogen bond between GO and CNF played a relevant role in the material stability. The results of column adsorption showed that the removal efficiency of MB was still kept at about 90% after three cycles. In a different approach, Gonza´lez et al. developed a chitin hydrogel reinforced with GO [34,131]. The chitin:GO ratio ranged from proportions where chitin was the main component to ones where GO exceeded the chitin amount. The rheological behavior of the material was shown to become more solid-like with increasing GO content. A batch adsorption study of this material was tested using two kinds of widely used dyes: Remazol Black (RB) as an acid dye model and Neutral Red (NR) as a basic dye model. It was demonstrated that the adsorption capacities of chitin and the composites against both pollutants are pH and Chi:GO proportion dependent. Despite the lower adsorption capacity for the

Handbook of Chitin and Chitosan

5.4 Optimizing adsorption performance by chemical and physical modifications

161

acidic dye of the composite in comparison to the Chi gel, the GO addition would be necessary to endow the material with optimal mechanical properties. Hence, the amount of GO in the material would imply a compromise between mechanical resistance and adsorption behavior. The optimal desorption pH values for RB and NR were 9.0 and 10.0, respectively; this would mean an advantage if reuse of the adsorbents is pursued. This material was also tested for the continuous-flow adsorption of ciprofloxacin. A low flow resistance was observed, due to high and wide size porosity. This allowed working at different flow rates. The tested medium conditions and the real water samples experiments showed that the type of interaction, both electrostatic attraction or π
π stacking, and hence breakthrough times, are strongly dependent on the media pH and the cation composition. Different nanoobjects have been used for chitin reinforcement and to create new adsorption sites. For example, Ramos et al. have developed chitin hydrogels combined with TiO2 nanoparticles for arsenic adsorption [132]. The reutilization of the hybrid gel was assessed up to five adsorption cycles. This was possible due to the chemical stability of chitin in the alkaline media needed for the regeneration of TiO2 NPs. In this work, chitin presented negligible adsorption capacity toward As(V). Thus its main role in this material is the one of a supportive matrix that allows the use of the NPs in a heterogeneous phase adsorption system. Otherwise, the use of NPs for batch systems would be difficult when it comes to recovering all the As-loaded nanoparticles from large water volumes. With a similar aim, Wisser et al. have developed a biological chitin
metal
organic framework composite for air filtration [73]. MOFs are novel and promising materials for filtration applications. However, MOF particles need to be incorporated into stable and manageable support matrices for this type of application. They have used a marine sponge skeleton which is based on chitin fibers as a matrix for MOF growing and application. This system showed that chitin does not interfere with the ammonia adsorption capacity of the MOF, resulting in a capacity proportional to the amount of MOF loaded in the composite. From a different point of view, Safarik and Safarikova developed a magnetic chitin adsorbent by the acetylation of magnetic chitosan particles for the purification of lysozyme from egg white [133]. The matrix was synthesized by mixing a chitosan acid solution with a magnetite powder suspension and then generating the particles by the addition of NaOH. After that, the conversion of chitosan to chitin was performed by treatment with acetic anhydride. The adsorption capacity reported was 2.5 mg of lysozyme per 1 mL of adsorbent and the authors reported that it was necessary to dilute the hen egg white before its processing.

Handbook of Chitin and Chitosan

162

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

Chitin microspheres and magnetic chitin microspheres were developed by Wang et al. using the sol
gel transition method, that involves the dispersion of chitin in a mixture of NaOH, urea, and distilled water in the ratio 11:4:85 followed by freezing the suspension for 4 h at 30 C and vigorous stirring at room temperature [100]. The freezing/thawing cycles were repeated twice and 30 mL of the chitin solution was dropped in 100 mL paraffin oil with 1 g Span-80 with slow stirring at 0 C for 3 h. The microspheres were formed with the adjustment of the suspension to pH 7.0 and the Span-80 was extracted with alcohol at 60 C for 24 h three times. The magnetic characteristic was added by the incorporation of Fe3O4 to the chitin solution. The particles presented a homogeneous surface with a porous structure with an average diameter of 1 μm. These microparticles were applied for immobilizing α-amylase and as an adsorbent for the removal of MB from aqueous solutions. The magnetic chitin microspheres were previously coated with polydopamine for the α-amylase immobilization using glutaraldehyde as cross-linker. The amount of covalently bound enzyme was 49.6 mg per gram of microspheres. Unlike free enzyme, it is easy to quickly remove the enzyme immobilized in magnetic particles from the reaction medium solution using an external magnet. To investigate the reusability, the immobilized α-amylase was used for 10 consecutive cycles, retaining 95% of its initial activity. Also, the magnetic chitin microspheres created were modified by nucleophilic substitution with Reactive Black 5 (an anionic dye) under alkaline conditions in order to adsorb cationic dyes. These dyes are widely used in the chemical industry and are harmful to human health. The matrix adsorbed 100% of MB from a solution of 10 mg/L within minutes and presented an adsorption capacity of 46 mg/g when the initial concentration of the solution was 80 mg/L [100]. The sol
gel transition method was also used by Xu et al. for the fabrication of nanocomposite chitin/clay microspheres developed for the removal of organic dyes [82]. In this case, chitin was dissolved in NaOH/urea solution at low temperature, followed by the addition of clay nanosheets through vigorous agitation. The solution was dropped into an isooctane and Span 85 suspension on an ice bath. The porous properties were analyzed by nitrogen adsorption isotherms and Brunauer
Emmett
Teller (BET), the surface area was 115.7 for chitin microspheres and 55.2 m2/g for chitin microspheres with 70% clay. Although the surface area decreased with the addition of clay, the matrix still presented accessible channels for the diffusion of the target molecule and much stronger adsorption. Removal of organic dyes was studied, and adsorption isotherms were analyzed by the Langmuir model (best fit), indicating that the adsorption of MB onto the matrix was via a monolayer. The maximum adsorption capacity obtained was 152.2 mg/g, allowing complete and rapid removal of the dye in aqueous

Handbook of Chitin and Chitosan

5.4 Optimizing adsorption performance by chemical and physical modifications

163

solutions at low concentrations (10 mg/g). The matrix showed high stability during five cycles of adsorption/desorption, using 1 M NaOH solution for the desorption step.

5.4.5 Derivatization Substantial interest has been paid for the chemical modifications of chitin in order to enhance its sorption capacity. However, as it has been previously exposed, chitin solubility is poor in most solvents. This fact has hindered the development of derivatization techniques of the polysaccharide. The most widely used techniques are via the deacetylation in order to obtain chitosan, whose application as a sorbent have been studied thoroughly. However, in this chapter, other modifications would be reviewed since, given the popularity of chitosan, it deserves a dedicated study. As previously mentioned, derivatization often needs liquid phase reactions and chitin solubility diminishes derivatization reaction yields. Therefore many attempts to functionalize this polysaccharide involve its partial deacetylation toward more soluble compounds [134]. As an example, the study by Hanh et al. can be mentioned, where partially deacetylated chitin, with a deacetylation degree near 40%, was graftcopolymerized with acrylonitrile (AN) by a γ-ray preirradiation method. The cyano groups grafted onto chitin were converted into amidoxime by hydroxylamine to enhance the adsorption of metal ions. The content of arsenic in groundwater was absolutely adsorbed by the treated chitin packed in the column [135]. Specifically, onto chitin, Yang et al. developed chitin nanofibers grafted with cysteine to create adsorption sites for arsenic oxoanion (AsO22) removal [136]. The arsenic adsorption performance of thiol-modified chitin nanofibers was evaluated under different pH conditions and at different metal ion concentrations, where the maximum adsorption capacity was found to be 149 mg/g at pH 5 7.0 using the Langmuir model. The authors claim that this adsorption capacity was higher than any chitin/ chitosan-based hydrogel or bead absorbent systems reported before. Also by adding thiol residues to chitin, Shao et al. modified chitin in tetrahydrofuran with l-cysteine in the presence of sulfuric acid as a catalyst, in order to adsorb Cu21, Cd21, Pb21, Zn21, and Ni21 [137]. The removal capacity was better for cys
chitin than for chitin, suggesting the success of the modification process and reinforcing the advantages of adding thiols to chitin. Heterocyclic amines can interact with metal cations to form stable coordination complexes. Karthik et al. has used this capability in order to obtain a chemically modified chitin with polypyrrole and used it as an adsorbent for the removal of Pb(II) and Cd(II) ions from

Handbook of Chitin and Chitosan

164

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

aqueous solution [138]. The batch equilibrium method was adopted to study the adsorption process. The adsorption of selected metal ions onto modified chitin was significantly affected by the solution pH because the polypyrrole moiety presented the main site of interaction. Thus chitin serves as a backbone for the adsorbent residues. The maximum removal was obtained at pH 6 for both Pb(II) and Cd(II) ions. The authors proposed that the mechanism for the uptake of Pb(II) and Cd (II) ions by the treated material could be ion exchange and electrostatic attraction followed by complexation. Seeking anion adsorption, Karthik et al. used polypyrrolefunctionalized chitin to adsorb chromate [139]. The results showed that the absorption was pH dependent, at an acid pH value the adsorption was higher than at a basic pH value. This might have been due to the antagonistic effect between
OH2 and chromate ions at alkaline pH values and the repulsion among chromate ions and the negative surface of the adsorbent. The presence of ions such as SO422, HCO32, and NO32 negatively affected the chromium removal suggesting a competitive relation (antagonism). With the aim of introducing negatively charged moieties that may enhance cation ions adsorption onto chitin, xanthation and phosphorylation were assessed by Kim et al. [140]. In this development, phosphorylation was performed directly onto chitin flakes in order to modify their surface whereas xanthation required a cross-linking step using epychlorohydrin in order to “harden” chitin walls as stated by the authors. Although the authors report a better adsorption performance for the xanthated samples, due to the addition of an epychlorohydrin residue it is difficult to evaluate if the xanthation occurred onto chitin residues or the epychlorohydrin ones. Taking this into account, it is clear that xanthation improves Pb(II) adsorption, showing a 23% increase in capacity, meanwhile the phosphorylation only increases the adsorption capacity of the material by 5%. Nishi et al., have also studied chitin phosphorylation and achieved highly phosphorylated chitin by using P2O5 in methanesulfonic acid [117]. As mentioned above, this phosphorylation leads to soluble polymers which needed to be cross-linked for cation heterogeneous phase adsorption [118]. In this case, the phosphorylation was achieved directly onto chitin residues. They studied highly phosphorilated chitin metal binding ability to Mg21, Ca21, Sr21, Ba21, Mn21, Ni21, Cu21, Zn21, and Cd21. Generally, alkaline-earth metals and Mn21 were adsorbed strongly to phosphorylated chitin rather than pristine chitin and deacetylated chitins. On the contrary, transition elements, aside from Mn21, strongly adsorbed to deacetylated chitins rather than to the phosphorylated. In an adsorptive membrane, the flow velocity could be increased considerably without a decrease in the adsorption capacity since the mass

Handbook of Chitin and Chitosan

5.4 Optimizing adsorption performance by chemical and physical modifications

165

transport phenomenon is governed by convection. An interesting approach performed by Zeng and Ruckenstein in the synthesis of chitin membranes was the development of chitosan-based membranes by the casting technique followed by the acetylation of the free amino groups of the glucosamine residues to convert them to N-acetyl glucosamine [141]. The synthesis was initiated by the dissolution of chitosan powder (1%) in acid acetic solution added with different sizes and amounts of silica particles. The suspension was then vigorously mixed and poured onto a rimmed glass plate and the liquid evaporated. The dried membrane was then submerged into a NaOH solution at 80 C in order to dissolve the silica particles and generate the pores of the system. These chitosan membranes were cross-linked with epichlorohydrin and converted into chitin membranes by treatment with acetic anhydride (5%) in methanol. The porosity of the membranes was controlled by changing the ratio of the silica to chitosan and the measured parameter used was the permeation flow rate of the membranes. This system was later published by the group as a chromatography convective matrix for the recovery and purification of lysozyme from artificial protein mixtures and from a 10 times diluted egg white [142]. To this purpose, they constructed a membrane cartridge by stacking four chitin membranes with a membrane effective area of 13.8 cm2 and a specific surface area of 1.6 m2/g. Equilibrium adsorption studies for lysozyme were performed according to the Langmuir model, presenting a maximum adsorption capacity of 40 mg/ mL and a dissociation constant of 0.25 mg/mL. Breakthrough curves were carried out for pure lysozyme and pure ovoalbumin solutions, showing a low adsorption capacity for ovoalbumin—the major contaminant protein present in egg white—and an increased adsorption capacity for lysozyme. This fact reveals that the chitin-based membrane is more specific toward lysozyme. Under the studied conditions 41.4 mg of lysozyme was eluted while 0.18 mg was eluted in the case of ovoalbumin when processing the diluted egg white. The same macroporous chitin affinity membranes were tested for the purification of albumin, wheat germ agglutinin (WGA), and concanavalin A (Con A) [106,143,144]. In the case of WGA purification, a chitin membrane cartridge was constructed by stacking 10 flat membranes, with an average pore size of 16 μm, a total thickness of 5 μm, an effective membrane area of 13.8 cm2, and a hydraulic permeability of 0.41 cm/min.psi. According to the Langmuir model, the adsorption isotherm resulted in a maximum adsorption capacity of 176.6 mg WGA/g chitin membrane and the dissociation constant was 0.56 mg WGA/mL. Different eluents were considered but the best results were obtained using 1 M CH3COOH aqueous solution, recovering 93% of the WGA. For the elution different flow rates of 2, 5, and 8 mL/min were tested and the same effect was observed as in the lysozyme case, regarding its

Handbook of Chitin and Chitosan

166

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

impact on the amount of protein recovered; the WGA concentrations in the eluent were 1.10, 0.67, and 0.65 mg/mL, respectively. This purification process was also affected, to a lesser extent, by the initial amount of target protein, and global yields between 80% and 90% were achieved. It is important to highlight that in this study the flow rate for the adsorptive step was not analyzed, so there is no evidence of convective mass transport over diffusion, which is one of the main advantages of using membrane systems. The adsorption behavior of Con A on macroporous chitosan or chitin membranes was studied under static and dynamic conditions [143]. Concanavalin A dynamic capacities for both membranes were 15 mg Con A/g dry membrane. However, since the recovery of Con A was 52% and 30% for chitosan and chitin membranes, respectively, the authors concluded that Con A can be coupled to the surfaces of macroporous chitosan and chitin membranes as a ligand without chemical activation for use in the purification of other proteins [143]. In the case of albumin, chitin and chitosan membranes were further modified chemically by the immobilization of Cibacron Blue F3GA, Procion Red HE-3B, or Procion Blue MX-R as potential dye ligands. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for human serum albumin (HSA) than bovine serum albumin (BSA), and than the other dye-modified membranes. About 8.4 mg of HSA were absorbed by 1 mL of Cibacron Blue F3GAchitosan membrane from a 0.05 M Tris
HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had lower dye content and a lower protein adsorption capacity than the chitosan membranes. Using human plasma, high purity HSA could be efficiently obtained by using the Cibacron Blue F3GA chitosan membranes. The separation of HSA from human plasma was performed both by recirculating several times or using a single-pass mode operation, obtaining 18.6 and 12.5 mg HSA from 1 mL human plasma, respectively. The purity of the HSA eluted with 0.5 N NaSCN in buffer from Cibacron Blue F3GA
chitosan membranes was high according to the electrophoresis studies. These membranes exhibited reproducibility during four successive operations cycles (equilibration/adsorption/desorption/regeneration) indicating that these membranes are stable and reusable [144].

5.5 Concluding remarks and future considerations As was previously mentioned, the application of a support for adsorptive processes should satisfy several demands. The adsorbent should be physically and chemically stable, allow high flow rate operations, and be

Handbook of Chitin and Chitosan

5.5 Concluding remarks and future considerations

167

stable to pressure (especially for packed-bed column, membrane, and monolith systems). It also should be able to withstand cleanup and/or sanitization conditions, be cheap and readily available, be sufficiently stable to be reused repeatedly, be porous enough to allow the entry of small and large molecules, have high adsorption capacity, be nontoxic, and not leach any chemical groups over time [145]. Even though the use of chitin as an adsorptive material could be commercially interesting, there are several aspects that need to be further studied. One of these aspects is the recovery and reusability of the support after the adsorptive process. Many supports have been developed for the treatments of industrial pollutants where the depletion of the contaminant is achieved but the separation or elution of the target molecule from the adsorbent is not studied. Although in some cases the elimination of the contaminant is easier when attached to the matrix by incineration, this is a key factor when thinking in a commercial support since the materials are usually not cheap and the costs are partially absorbed by the guarantee of several cycles of use without affecting the yield of the process. Not only is the elution of the adsorbed molecule necessary to accomplish this, but also matrix recovery, which could be a challenge when the selected structure is in nano/microscale in a batch system. A possible solution to this fact is working with magnetic particles which are easily recovered with a magnet after the adsorptive or washing step, but the cost of this kind of technology is usually high making it not scalable in most cases. Another challenge, especially for those processes in which the productivity must be increased and the time involved must be reduced, is the development of convective adsorptive matrices able to bind the selected molecule with high speed. The need for high adsorptive capacity materials which also fulfill the convective conditions is an active topic and many novel approaches are taking place using blend materials. Another interesting aspect with potential applications in protein production by biotechnological means is the construction of fusion proteins in which the DNA encoding for the protein of interest is fusioned to the chitin binding domain of the chitinase enzyme from yeast [104]. Those recombinant and chimeric proteins could be immobilized or purified with many of the supports described in this chapter. Though chitin as a starting material is generally considered cheap because it is obtained from a natural source as crustaceans, all these previously mentioned facts could affect the costs of using these materials at the industrial scale.

Acknowledgments G.T.J. and D.B.H. are grateful for the doctoral fellowship granted by Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET). This work was supported with grants

Handbook of Chitin and Chitosan

168

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

from Universidad de Buenos Aires (UBACYT 20020170100125BA, UBACYT 20020170100023BA) and Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (PICT 2015-0714, PICT 2016-1997).

References ¨ zogul, J. Regenstein, Industrial applications of crustacean by-products [1] I. Hamed, F. O (chitin, chitosan, and chitooligosaccharides): a review, Trends Food Sci. Technol. 48 (2016) 40
50. [2] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. structure, properties and applications, Mar. Drugs 13 (2015) 1133
1174. [3] E. Salehi, P. Daraei, A. Arabi Shamsabadi, A review on chitosan-based adsorptive membranes, Carbohydr. Polym. 152 (2016) 419
432. [4] V. Zargar, M. Asghari, A. Dashti, A review on chitin and chitosan polymers: structure, chemistry, solubility, derivatives, and applications, ChemBioEng. Rev. 2 (2015) 204
226. [5] V.G. Bendikene, Jh.H. Pesliakas, V.S. Vesa, Chromatography of serine proteases on chitin and its derivatives, Prikl. Biokhim. Mikrobiol. 17 (1981) 441
447. [6] G. McKay, H.S. Blair, J.R. Gardner, Adsorption of dyes on chitin. I. Equilibrium studies, J. Appl. Polym. Sci. 27 (1982) 3043
3057. [7] R.A.A. Muzzarelli, Uptake of nitrosyl 106-ruthenium on chitin and chitosan from waste solutions and polluted sea-water, Water Res. 4 (1970) 451
455. [8] B. Jiafeng, F. Shou’Ai, W. Kang, L. Hong, L. Shaohua, T. Zhiming, Be Applied to Nanometer Chitin Function Central Line and for Cigarette Filter Rod of Filter Rod, CN206576286U, 2017. [9] R. Cuero, D. McKay, Compositions and Methods for Removal of Toxic Metals and Radionuclides, US7309437B2, 2007. [10] Y.-K. Li, Y.-J. Wu, Vectors, Methods, Systems and Kits for Protein Purification, US8236529B2, 2012. [11] C. Muzzarelli, P. Morganti, Preparation of Chitin and Derivatives Thereof for Cosmetic and Therapeutic Use, US8383157B2, 2013. [12] J. Sorenson, S. Kent, Emplacement of Treatment Agents Using Soil Fracturing for Remediation of Subsurface Environmental Contamination, US7179381B2, 2007. [13] Z. Xu, J. Cai, B. Pan, Mathematically modeling fixed-bed adsorption in aqueous systems, J. Zhejiang Univ. Sci. A 14 (2013) 155
176. [14] E. Franca, R. Lins, L. Freitas, T. Straatsma, Characterization of chitin and chitosan molecular structure in aqueous solution, J. Chem. Theory Comput. 4 (2008) 2141
2149. [15] S. Hajji, I. Younes, O. Ghorbel-Bellaaj, R. Hajji, M. Rinaudo, M. Nasri, et al., Structural differences between chitin and chitosan extracted from three different marine sources, Int. J. Biol. Macromol. 65 (2014) 298
306. [16] M.-K. Jang, B.-G. Kong, Y.-I. Jeong, C.H. Lee, J.-W. Nah, Physicochemical characterization of α-chitin, β-chitin, and γ-chitin separated from natural resources, J. Polym. Sci. Part Polym. Chem. 42 (2004) 3423
3432. [17] M.N. Ravi Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (2000) 1
27. [18] P. Sorlier, A. Denuzie`re, C. Viton, A. Domard, Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan, Biomacromolecules 2 (2001) 765
772. [19] P.K. Dutta, J. Dutta, V. Tripathi, Chitin and chitosan: chemistry, properties and applications. J Sci Ind Res 63 (2004) 20
31.

Handbook of Chitin and Chitosan

References

169

[20] J.-K. Kim, D.H. Kim, S.H. Joo, B. Choi, A. Cha, K.M. Kim, et al., Hierarchical chitin fibers with aligned nanofibrillar architectures: a nonwoven-mat separator for lithium metal batteries, ACS Nano 11 (2017) 6114
6121. [21] X. Pei, Y. Deng, B. Duan, T. Chan, J. Lee, A. Lei, et al., Ultra-small Pd clusters supported by chitin nanowires as highly efficient catalysts, Nano Res. 11 (2018) 3145
3153. [22] C. Xiong, Adsorption of cadmium (II) by chitin, J. Chem. Soc. Pak. 32 (2010). [23] R. Maruca, B.J. Suder, J.P. Wightman, Interaction of heavy metals with chitin and chitosan. III. Chromium, J. Appl. Polym. Sci. 27 (1982) 4827
4837. [24] I. Anastopoulos, A. Bhatnagar, D. Bikiaris, G. Kyzas, Chitin adsorbents for toxic metals: a review, Int. J. Mol. Sci. 18 (2017) 114. [25] C. Londono-Zuluaga, H. Jameel, R.W. Gonzalez, L. Lucia, Crustacean shell-based biosorption water remediation platforms: status and perspectives, J. Environ. Manage. 231 (2019) 757
762. [26] G. Karthikeyan, N.M. Andal, K. Anbalagan, Adsorption studies of iron(III) on chitin, J. Chem. Sci. 117 (2005) 663
672. [27] G. Dotto, J.M. Cunha, C.O. Calgaro, E.H. Tanabe, D.A. Bertuol, Surface modification of chitin using ultrasound-assisted and supercritical CO2 technologies for cobalt adsorption, J. Hazard. Mater. 295 (2015) 29
36. [28] G. Dotto, J. Santos, I. Rodrigues, R. Rosa, F. Pavan, E. Lima, Adsorption of methylene blue by ultrasonic surface modified chitin, J. Colloid Interface Sci. 446 (2015) 133
140. [29] A.Y. Dursun, C ¸ .S. Kalayci, Equilibrium, kinetic and thermodynamic studies on the adsorption of phenol onto chitin, J. Hazard. Mater. 123 (2005) 151
157. [30] J.A. Gonza´lez, J.G. Bafico, M.E. Villanueva, S.A. Giorgieri, G.J. Copello, Continuous flow adsorption of ciprofloxacin by using a nanostructured chitin/graphene oxide hybrid material, Carbohydr. Polym. 188 (2018) 213
220. [31] T. Kamal, Y. Anwar, S.B. Khan, M.T.S. Chani, A.M. Asiri, Dye adsorption and bactericidal properties of TiO2/chitosan coating layer, Carbohydr. Polym. 148 (2016) 153
160. [32] O. Tepe, Adsorption of remazol brillant green 6B (RBG 6B) on chitin: process optimization using response surface methodology, Global Nest J 20 (2) (2017) pp 257
268. [33] G. Todde, S.K. Jha, G. Subramanian, M.K. Shukla, Adsorption of TNT, DNAN, NTO, FOX7, and NQ onto cellulose, chitin, and cellulose triacetate: insights from density functional theory calculations, Surf. Sci. 668 (2018) 54
60. [34] J.A. Gonzalez, M.E. Villanueva, M.L. Peralta Ramos, C.J. Perez, L.L. Piehl, G.J. Copello, Chitin based hybrid composites reinforced with graphene derivatives: a nanoscale study, RSC Adv. 5 (2015) 63813
63820. [35] N.P. Raval, P.U. Shah, D.G. Ladha, P.M. Wadhwani, N.K. Shah, Comparative study of chitin and chitosan beads for the adsorption of hazardous anionic azo dye congo red from wastewater, Desalination Water Treat. 57 (2016) 9247
9262. [36] S.S. Saini, G.J. Copello, A.L.J. Rao, HPLC-UV platform for trace analysis of three isomeric mononitrophenols in water with chitin based solid phase extraction, Anal. Methods 9 (2017) 4143
4150. [37] P. Huang, Q. Sun, W. Shi, W. Du, X. Li, N. Zhang, Efficient production of human goose-type lysozyme 2 in the methylotrophic yeast Pichia pastoris, J. Biotechnol. 275 (2018) 44
52. [38] M. Kaya, I. Sargin, V. Aylanc, M.N. Tomruk, S. Gevrek, I. Karatoprak, et al., Comparison of bovine serum albumin adsorption capacities of α-chitin isolated from an insect and β-chitin from cuttlebone, J. Ind. Eng. Chem. 38 (2016) 146
156. [39] M. Yahyaei, F. Mehrnejad, H. Naderi-manesh, A.H. Rezayan, Protein adsorption onto polysaccharides: comparison of chitosan and chitin polymers, Carbohydr. Polym. 191 (2018) 191
197. [40] L. Zhang, J. Pan, S. Dong, Z. Li, The application of polysaccharide-based nanogels in peptides/proteins and anticancer drugs delivery, J. Drug Target. 25 (2017) 673
684.

Handbook of Chitin and Chitosan

170

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

[41] M.-L. Pham, T. Leister, H.A. Nguyen, B.-C. Do, A.-T. Pham, D. Haltrich, et al., Immobilization of β-galactosidases from lactobacillus on chitin using a chitin-binding domain, J. Agric. Food Chem. 65 (2017) 2965
2976. [42] G. Davies, B. Henrissat, Structures and mechanisms of glycosyl hydrolases, Structure 3 (1995) 853
859. [43] A. Hamre, E. Frøberg, V. Eijsink, Sørlie, Thermodynamics of tunnel formation upon substrate binding in a processive glycoside hydrolase, Arch. Biochem. Biophys. 620 (2017) 35
42. [44] B. Synstad, S. Gaseidnes, D.M.F. van Aalten, G. Vriend, J.E. Nielsen, V.G.H. Eijsink, Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase, Eur. J. Biochem. 271 (2004) 253
262. [45] A. Perrakis, I. Tews, Z. Dauter, A.B. Oppenheim, I. Chet, K.S. Wilson, et al., Crystal ˚ resolution, Structure 2 (1994) 1169
1180. structure of a bacterial chitinase at 2.3 A [46] A.C. Terwisscha van Scheltinga, S. Armand, K.H. Kalk, A. Isogai, B. Henrissat, B.W. Dijkstra, Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and x-ray structure of a complex with allosamidin evidence for substrate assisted catalysis, Biochemistry 34 (1995) 15619
15623. [47] F. Fukamizo, Chitinolytic enzymes: catalysis, substrate binding, and their application, Curr. Protein Pept. Sci. 1 (2000) 105
124. [48] E. Bulut, I. Sargin, O. Arslan, M. Odabasi, B. Akyuz, M. Kaya, In situ chitin isolation from body parts of a centipede and lysozyme adsorption studies, Mater. Sci. Eng. C 70 (2017) 552
563. [49] E. Tabata, A. Kashimura, S. Wakita, M. Sakaguchi, Y. Sugahara, Y. Imamura, et al., Acidic chitinase-chitin complex is dissociated in a competitive manner by acetic acid: purification of natural enzyme for supplementation purposes, Int. J. Mol. Sci. 19 (2018) 362. [50] M. Lobo, F. Silva, P. Landim, P. da Cruz, T. de Brito, S. de Medeiros, et al., Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli, BMC Biotechnol. 13 (2013) 46. [51] S.J. Perkins, L.N. Johnson, P.A. Machin, D.C. Phillips, Crystal structures of hen eggwhite lysozyme complexes with gadolinium(III) and gadolinium(III)-N-acetyl-D-glucosamine, Biochem. J. 181 (1979) 21
36. [52] D.W. Borhani, D.E. Shaw, The future of molecular dynamics simulations in drug discovery, J. Comput. Aided Mol. Des. 26 (2012) 15
26. [53] M. De Vivo, M. Masetti, G. Bottegoni, A. Cavalli, Role of molecular dynamics and related methods in drug discovery, J. Med. Chem. 59 (2016) 4035
4061. [54] J. Thewalt, D.P. Tieleman, Biophysical experiments and simulation in nanoparticlebased drug delivery systems, J. Drug Target. 24 (2016) 768
773. ´ ´ [55] D. Stawski, S. Rabiej, L. Herczynska, Z. Draczynski, Thermogravimetric analysis of chitins of different origin, J. Therm. Anal. Calorim. 93 (2008) 489
494. [56] Y.-W. Cho, J. Jang, C.R. Park, S.-W. Ko, Preparation and solubility in acid and water of partially deacetylated chitins, Biomacromolecules 1 (2000) 609
614. [57] M. Kaya, M. Mujtaba, H. Ehrlich, A.M. Salaberria, T. Baran, C.T. Amemiya, et al., On chemistry of γ-chitin, Carbohydr. Polym. 176 (2017) 177
186. [58] R. Minke, J. Blackwell, The structure of α-chitin, J. Mol. Biol. 120 (1978) 167
181. ´ [59] J. Kumirska, M. Czerwicka, Z. Kaczynski, A. Bychowska, K. Brzozowski, J. Tho¨ming, et al., Application of spectroscopic methods for structural analysis of chitin and chitosan, Mar. Drugs 8 (2010) 1567
1636. [60] M. Kaya, T. Baran, Description of a new surface morphology for chitin extracted from wings of cockroach (Periplaneta americana), Int. J. Biol. Macromol. 75 (2015) 7
12. [61] M. Kaya, B. Bitim, M. Mujtaba, T. Koyuncu, Surface morphology of chitin highly related with the isolated body part of butterfly (Argynnis pandora), Int. J. Biol. Macromol. 81 (2015) 443
449.

Handbook of Chitin and Chitosan

References

171

˘ [62] M. Kaya, O. Seyyar, T. Baran, S. Erdogan, M. Kar, A physicochemical characterization of fully acetylated chitin structure isolated from two spider species: with new surface morphology, Int. J. Biol. Macromol. 65 (2014) 553
558. [63] M. Unterlass, Geomimetics and extreme biomimetics inspired by hydrothermal systems—what can we learn from nature for materials synthesis? Biomimetics 2 (2017) 8. [64] M. Wysokowski, I. Petrenko, A. Stelling, D. Stawski, T. Jesionowski, H. Ehrlich, Poriferan chitin as a versatile template for extreme biomimetics, Polymers 7 (2015) 235
265. [65] M. Wysokowski, M. Motylenko, V.V. Bazhenov, D. Stawski, I. Petrenko, A. Ehrlich, et al., Poriferan chitin as a template for hydrothermal zirconia deposition, Front. Mater. Sci. 7 (2013) 248
260. [66] M. Wysokowski, M. Motylenko, J. Beyer, A. Makarova, H. Sto¨cker, J. Walter, et al., Extreme biomimetic approach for developing novel chitin-GeO2 nanocomposites with photoluminescent properties, Nano Res. 8 (2015) 2288
2301. [67] M. Wysokowski, M. Motylenko, D. Rafaja, I. Koltsov, H. Sto¨cker, T.J. Szalaty, et al., Extreme biomimetic approach for synthesis of nanocrystalline chitin-(Ti, Zr)O2 multiphase composites, Mater. Chem. Phys. 188 (2017) 115
124. [68] M. Wysokowski, M. Motylenko, H. Stocker, V.V. Bazhenov, E. Langer, A. Dobrowolska, et al., An extreme biomimetic approach: hydrothermal synthesis of [small beta]chitin/ZnO nanostructured composites, J. Mater. Chem. B 1 (2013) 6469
6476. [69] M. Wysokowski, M. Motylenko, J. Walter, G. Lota, J. Wojciechowski, H. Sto¨cker, et al., Synthesis of nanostructured chitin
hematite composites under extreme biomimetic conditions, RSC Adv 4 (2014) 61743
61752. [70] M. Wysokowski, A. Piasecki, V.V. Bazhenov, D. Paukszta, R. Born, P. Schupp, et al., Poriferan chitin as the scaffold for nanosilica deposition under hydrothermal synthesis conditions, J. Chitin Chitosan Sci. 1 (2013) 26
33. [71] I. Petrenko, V.V. Bazhenov, R. Galli, M. Wysokowski, J. Fromont, P.J. Schupp, et al., Chitin of poriferan origin and the bioelectrometallurgy of copper/copper oxide, Int. J. Biol. Macromol. 104 (2017) 1626
1632. [72] S. Vogt, M. Kelkenberg, T. No¨ll, B. Steinhoff, H. Scho¨nherr, H. Merzendorfer, et al., Rapid determination of binding parameters of chitin binding domains using chitincoated quartz crystal microbalance sensor chips, Analyst 143 (2018) 5255
5263. [73] D. Wisser, F.M. Wisser, S. Raschke, N. Klein, M. Leistner, J. Grothe, et al., Biological chitin-MOF composites with hierarchical pore systems for air-filtration applications, Angew. Chem. Int. Ed. 54 (2015) 12588
12591. [74] E. Guibal, L. Dambies, C. Milot, Roussy, Influence of polymer structural parameters and experimental conditions on metal anion sorption by chitosan, Polym. Int. 48 (1999) 671
680. [75] Z. Tian, S. Wang, X. Hu, Z. Zhang, L. Liang, Crystalline reduction, surface area enlargement and pore generation of chitin by instant catapult steam explosion, Carbohydr. Polym. 200 (2018) 255
261. [76] Y.-L. Cao, Z.-H. Pan, Q.-X. Shi, J.-Y. Yu, Modification of chitin with high adsorption capacity for methylene blue removal, Int. J. Biol. Macromol. 114 (2018) 392
399. [77] R. Tajiri, A. Mihata, K. Yamamoto, J. Kadokawa, Facile preparation of chitin gels with calcium bromide dihydrate/methanol media and their efficient conversion into porous chitins, RSC Adv. 4 (2014) 5542. [78] H. Tamura, H. Nagahama, S. Tokura, Preparation of chitin hydrogel under mild conditions, Cellulose 13 (2006) 357
364. [79] G. McKay, H.S. Blair, J.R. Gardner, The adsorption of dyes onto chitin in fixed bed columns and batch adsorbers, J. Appl. Polym. Sci. 29 (1984) 1499
1514. [80] Z. Ma, D. Liu, Y. Zhu, Z. Li, Z. Li, H. Tian, et al., Graphene oxide/chitin nanofibril composite foams as column adsorbents for aqueous pollutants, Carbohydr. Polym. 144 (2016) 230
237.

Handbook of Chitin and Chitosan

172

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

[81] B. Benguella, Cadmium removal from aqueous solutions by chitin: kinetic and equilibrium studies, Water Res 36 (2002) 2463
2474. [82] R. Xu, J. Mao, N. Peng, X. Luo, C. Chang, Chitin/clay microspheres with hierarchical architecture for highly efficient removal of organic dyes, Carbohydr. Polym. 188 (2018) 143
150. [83] S.S. Silva, A.R.C. Duarte, A.P. Carvalho, J.F. Mano, R.L. Reis, Green processing of porous chitin structures for biomedical applications combining ionic liquids and supercritical fluid technology, Acta Biomater. 7 (2011) 1166
1172. [84] B. Ding, J. Cai, J. Huang, L. Zhang, Y. Chen, X. Shi, et al., Facile preparation of robust and biocompatible chitin aerogels, J. Mater. Chem. 22 (2012) 5801. [85] B. Duan, Y. Huang, A. Lu, L. Zhang, Recent advances in chitin based materials constructed via physical methods, Prog. Polym. Sci. 82 (2018) 1
33. https://doi.org/ 10.1016/j.progpolymsci.2018.04.001. [86] N.A. Gow, J.-P. Latge, C.A. Munro, The fungal cell wall: structure, biosynthesis, and function. Microbiol. Spectr. 5 (2017) 1
25. https://doi.org/10.1128/microbiolspec. FUNK-0035-2016. [87] S. Ling, W. Chen, Y. Fan, K. Zheng, K. Jin, H. Yu, et al., Biopolymer nanofibrils: structure, modeling, preparation, and applications, Prog. Polym. Sci. 85 (2018) 1
56. https://doi.org/10.1016/j.progpolymsci.2018.06.004. [88] J. Synowiecki, N.A. Al-Khateeb, Production, properties, and some new applications of chitin and its derivatives, (2003). [89] W.-T. Wang, J. Zhu, X.-L. Wang, Y. Huang, Y.-Z. Wang, Dissolution behavior of chitin in ionic liquids, J. Macromol. Sci. Part B 49 (2010) 528
541. [90] H. Xie, S. Zhang, S. Li, Chitin and chitosan dissolved in ionic liquids as reversible sorbents of CO2, Green Chem. 8 (2006) 630
633. [91] P.R. Austin, Solvents for and Purification of Chitin, US3892731A, University of Delaware, 1975. [92] H. Tamura, M. Sawada, H. Nagahama, T. Higuchi, S. Tokura, Influence of amide content on the crystal structure of chitin, Holzforschung 60 (2006) 480
484. [93] M. Sharma, C. Mukesh, D. Mondal, K. Prasad, Dissolution of α-chitin in deep eutectic solvents, RSC Adv. 3 (2013) 18149
18155. [94] H. Tamura, Destruction of rigid crystalline structure to prepare chitin solution, Adv. Chitin Sci. 7 (2004) 84
87. [95] R. Jayakumar, D. Menon, K. Manzoor, S. Nair, H. Tamura, Biomedical applications of chitin and chitosan based nanomaterials—a short review, Carbohydr. Polym. 82 (2010) 227
232. [96] R.A. Muzzarelli, New techniques for optimization of surface area and porosity in nanochitins and nanochitosans, in: Chitosan for Biomaterials II, Springer, 2011, pp. 167
186. [97] B. Duan, C. Chang, B. Ding, J. Cai, M. Xu, S. Feng, et al., High strength films with gas-barrier fabricated from chitin solution dissolved at low temperature, J. Mater. Chem. A 1 (2013) 1867
1874. [98] P. Das, T. Heuser, A. Wolf, B. Zhu, D. Demco, S. Ifuku, et al., Tough and catalytically active hybrid biofibers wet-spun from nanochitin hydrogels, Biomacromolecules 13 (2012) 4205
4212. [99] Y. Huang, Z. Zhong, B. Duan, L. Zhang, Z. Yang, Y. Wang, et al., Novel fibers fabricated directly from chitin solution and their application as wound dressing, J. Mater. Chem. B 2 (2014) 3427
3432. [100] Y. Wang, Y. Li, S. Liu, B. Li, Fabrication of chitin microspheres and their multipurpose application as catalyst support and adsorbent, Carbohydr. Polym. 120 (2015) 53
59.

Handbook of Chitin and Chitosan

References

173

[101] D. Xu, J. Huang, D. Zhao, B. Ding, L. Zhang, J. Cai, High-flexibility, high-toughness double-cross-linked chitin hydrogels by sequential chemical and physical crosslinkings, Adv. Mater. 28 (2016) 5844
5849. [102] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603
632. [103] M.F. Baieli, N. Urtasun, D.B. Hirsch, L.F. Bracco, M.V. Miranda, O. Cascone, et al., The use of chitosan mini-spheres for high value protein purification from raw materials, in: J. Phillips (Ed.), Chitin: Properties, Applications and Research, Nova Science Publishers Inc., New York, 2017. [104] M.J.M. Kuranda, Immobilization and Purification Offusion Proteins Using Chitin Binding Ability, US5258502A, 1993. [105] S. Xu, X. Zhang, B. Duan, J. Chen, An engineered platform based on chitin-affinity immobilization for producing low molecular weight heparin, Carbohydr. Polym. 177 (2017) 297
305. [106] X. Zeng, E. Ruckenstein, Macroporous chitin affinity membranes for wheat germ agglutinin purification from wheat germ, J. Membr. Sci. 156 (1999) 97
107. [107] N. Urtasun, M.F. Baieli, D.B. Hirsch, M.V. Miranda, O. Cascone, F.J. Wolman, Challenges in dairy whey protein purification, Handbook on Protein Purification: Industry Challenges and Technological Developments, Nova Science Publishers Inc., 2018. [108] C.J. Fee, A. Chand, Design considerations for the batch capture of proteins from raw whole milk by ion exchange chromatography, Chem. Eng. Technol. 28 (2005) 1360
1366. [109] M. Etzel, Manufacture and use of dairy protein fractions, J. Nutr 134 (2004) 996S
1002S. [110] Q.-Y. Du, D.-Q. Lin, Q.-L. Zhang, S.-J. Yao, An integrated expanded bed adsorption process for lactoferrin and immunoglobulin G purification from crude sweet whey, J. Chromatogr. B 947
948 (2014) 201
207. [111] J. Thoemmes, M.-R. Kula, Membrane chromatography—an integrative concept in the downstream processing of proteins, Biotechnol. Prog. 11 (1995) 357
367. [112] P.X. Pinto, S.R. Al-Abed, D.J. Reisman, Biosorption of heavy metals from mining influenced water onto chitin products, Chem. Eng. J. 166 (2011) 1002
1009. [113] S. Dhananasekaran, R. Palanivel, S. Pappu, Adsorption of methylene blue, bromophenol blue, and coomassie brilliant blue by alpha-chitin nanoparticles, J. Adv. Res. 7 (2016) 113
124. [114] S. Jiang, Y. Qin, J. Yang, M. Li, L. Xiong, Q. Sun, Enhanced antibacterial activity of lysozyme immobilized on chitin nanowhiskers, Food Chem. 221 (2017) 1507
1513. [115] Y. Wang, J. Li, B. Li, Chitin microspheres: a fascinating material with high loading capacity of anthocyanins for colon specific delivery, Food Hydrocoll 63 (2017) 293
300. [116] S. Chakrabarti, B. Dutta, Photocatalytic degradation of model textile dyes in wastewater using ZnO as semiconductor catalyst, J. Hazard. Mater. 112 (2004) 269
278. [117] N. Nishi, A. Ebina, S. Nishimura, A. Tsutsumi, O. Hasegawa, S. Tokura, Highly phosphorylated derivatives of chitin, partially deacetylated chitin and chitosan as new functional polymers: preparation and characterization, Int. J. Biol. Macromol. 8 (1986) 311
317. [118] N. Nishi, Y. Maekita, S. Nishimura, O. Hasegawa, S. Tokura, Highly phosphorylated derivatives of chitin, partially deacetylated chitin and chitosan as new functional polymers: metal binding property of the insolubilized materials, Int. J. Biol. Macromol. 9 (1987) 109
114.

Handbook of Chitin and Chitosan

174

5. Chitin blends, interpenetrating polymer networks, gels, composites, and nanocomposites

[119] H. Tang, W. Zhou, L. Zhang, Adsorption isotherms and kinetics studies of malachite green on chitin hydrogels, J. Hazard. Mater. 209
210 (2012) 218
225. [120] Y. Duan, A. Freyburger, W. Kunz, C. Zollfrank, Lignin/chitin films and their adsorption characteristics for heavy metal ions, ACS Sustain. Chem. Eng. 6 (2018) 6965
6973. [121] S. Liang, L. Zhang, J. Xu, Morphology and permeability of cellulose/chitin blend membranes, J. Membr. Sci. 287 (2007) 19
28. [122] D. Zhou, L. Zhang, J. Zhou, S. Guo, Development of a fixed-bed column with cellulose/chitin beads to remove heavy-metal ions, J. Appl. Polym. Sci. 94 (2004) 684
691. [123] G. Lesnieroswski, J. Kijowski, Lysozyme, in: R. Huopalahti, R. Lo´pez-Fandin˜o, M. Anton, R. Schade (Eds.), Bioactive Egg Compounds, Springer-Verlag, Berlin Heidelberg, 2007, pp. 33
42. [124] F. Wolman, M.F. Baieli, N. Urtasun, A. Navarro del Can˜ izo, M.V. Miranda, O. Cascone, Strategies to recover and purify lysozyme from egg white, in: Lysozymes: Sources, Functions and Role in Disease, Nova Science Publishers Inc., Hauppauge, NY, 2013, pp. 241
250. [125] F. Wolman, G. Copello, A. Mebert, A. Targovnik, M. Miranda, A. Navarro del Can˜izo, et al., Egg white lysozyme purification with a chitin
silica-based affinity chromatographic matrix, Eur. Food Res. Technol. 231 (2010) 181
188. [126] V. Kumari, S. Kumar, I. Kaur, T.C. Bhalla, Graft copolymerization of acrylamide on chitosan-co-chitin and its application for immobilization of Aspergillus sp. RL2Ct cutinase. Bioorg. Chem. 70 (2017) 34
43. https://doi.org/10.1016/j. bioorg.2016.11.006 ¨ zacar, Z. O ¨ zacar, 2018. Characterization and immo[127] W.A. Mehdi, A.A. Mehde, M. O bilization of protease and lipase on chitin-starch material as a novel matrix. Int. J. Biol. Macromol. 117, 947
958. https://doi.org/10.1016/j.ijbiomac.2018.04.195 [128] G. Crini, Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment, Prog. Polym. Sci. 30 (2005) 38
70. [129] D.M. Marquis, C. Chivas-Joly, E´. Guillaume, Properties of Nanofillers in Polymer, INTECH Open Access Publisher, 2011. [130] J.A. Gonza´lez, M.E. Villanueva, L.L. Piehl, G.J. Copello, Development of a chitin/ graphene oxide hybrid composite for the removal of pollutant dyes: adsorption and desorption study, Chem. Eng. J. 280 (2015) 41
48. [131] J.A. Gonzalez, M.F. Mazzobre, M.E. Villanueva, L.E. Diaz, G.J. Copello, Chitin hybrid materials reinforced with graphene oxide nanosheets: chemical and mechanical characterisation, RSC Adv. 4 (2014) 16480
16488. [132] M.L. Peralta Ramos, J.A. Gonza´lez, S.G. Albornoz, C.J. Pe´rez, M.E. Villanueva, S.A. Giorgieri, et al., Chitin hydrogel reinforced with TiO2 nanoparticles as an arsenic sorbent, Chem. Eng. J. 285 (2016) 581
587. ˇ r´ık, M. Safaraˇ ˇ [133] I. Safaˇ r´ıkova´, Batch isolation of hen egg white lysozyme with magnetic chitin, J. Biochem. Biophys. Methods 27 (1993) 327
330. [134] K. Kurita, Controlled functionalization of the polysaccharide chitin, Prog. Polym. Sci. 26 (2001) 1921
1971. [135] T.T. Hanh, H.T. Huy, N.Q. Hien, Pre-irradiation grafting of acrylonitrile onto chitin for adsorption of arsenic in water, Radiat. Phys. Chem. 106 (2015) 235
241. [136] R. Yang, Y. Su, K.B. Aubrecht, X. Wang, H. Ma, R.B. Grubbs, et al., Thiolfunctionalized chitin nanofibers for As (III) adsorption, Polymer 60 (2015) 9
17. [137] J. Shao, Y. Yang, C. Shi, Preparation and adsorption properties for metal ions of chitin modified by L-cysteine, J. Appl. Polym. Sci. 88 (2003) 2575
2579. [138] R. Karthik, S. Meenakshi, Chemical modification of chitin with polypyrrole for the uptake of Pb(II) and Cd(II) ions, Int. J. Biol. Macromol. 78 (2015) 157
164.

Handbook of Chitin and Chitosan

References

175

[139] R. Karthik, S. Meenakshi, Synthesis, characterization and Cr(VI) uptake studies of polypyrrole functionalized chitin, Synth. Met. 198 (2014) 181
187. [140] S.-H. Kim, H. Song, G.M. Nisola, J. Ahn, M.M. Galera, C.H. Lee, et al., Adsorption of lead(II) ions using surface-modified chitins, J. Ind. Eng. Chem. 12 (2006) 469
475. [141] X. Zeng, E. Ruckenstein, Control of pore sizes in macroporous chitosan and chitin membranes, Ind. Eng. Chem. Res. 35 (1996) 4169
4175. [142] E. Ruckenstein, X. Zeng, Macroporous chitin affinity membranes for lysozyme separation, Biotechnol. Bioeng. 56 (1997) 610
617. ˘ [143] M. Gu¨mu¨s¸ derelioglu, P. Agi, Adsorption of concanavalin A on the wellcharacterized macroporous chitosan and chitin membranes, React. Funct. Polym. 61 (2004) 211
220. [144] E. Ruckenstein, X. Zeng, Albumin separation with cibacron blue carrying macroporous chitosan and chitin affinity membranes, J. Membr. Sci. 142 (1998) 13
26. [145] D.B. Hirsch, N. Urtasun, M.F. Baieli, M.V. Miranda, O. Cascone, F.J. Wolman, Dye affinity chromatography: a low-cost tool for protein purification, in: J.C. Taylor (Ed.), Chemistry Research, Nova Science Publishers Inc, New York, 2018, pp. 147
166.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

6 Functional properties of chitin and chitosan-based polymer materials Gisoo Maleki1 and Jafar M. Milani2 1

Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad, Iran, 2Department of Food Science and Technology, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

O U T L I N E 6.1 Introduction

178

6.2 Dietary activity 6.2.1 Hypocholesterolemic effect 6.2.2 Prebiotics ingredients 6.2.3 Calcium absorption acceleration effect in vivo

178 178 180 180

6.3 Antimicrobial activity

181

6.4 Emulsifying properties

185

6.5 Antioxidant activity

188

6.6 Flocculent and chelating

191

6.7 Future trends

193

References

193

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00006-6

177

© 2020 Elsevier Inc. All rights reserved.

178

6. Functional properties of chitin and chitosan-based polymer materials

6.1 Introduction Chitin and its main derivative chitosan have been receiving increased consideration among the novel families of biological macromolecules due to their versatile biological activities which can be employed in food, agriculture, medicine, pharmaceutical, personal care product, and environmental sectors [1]. Potential and usual applications of chitin, chitosan, and their derivatives are estimated to be more than 200 [2]. Chitin is obtained from crustaceans’ exoskeletons, in shellfish processing industries, after demineralization and deproteinization treatments. The by-products of these industries are about 75% of the total weight of crustaceans such as shrimp, crabs, prawns, lobster, and krill [3]. Therefore acceptable waste management options are needed to turn these by-products, which make a large environmental concern, into applicable chitin and its derivatives in different industries. However, one of the limitations in the use of this biopolymer on a large scale is its water insolubility. Therefore water-soluble derivatives have been produced of which chitosan is the most important [4]. Chitin and its derivatives are reproducible, biocompatible, biodegradable, and nontoxic compounds that have many biological properties, such as dietary [5], antimicrobial [6], antioxidant [7], emulsifying [8], flocculent and chelating [9], and prebiotic [10] properties. Functional properties of chitin and chitosan vary from product to product due to the season, the quality of shell, species present, climate, and processing method. This has led to biotechnology research regarding the production of chitin and chitosan from various sources, such as exoskeleton of insects and cell wall of fungi [11,12]. In this chapter, some information about significant functional properties of chitin and chitosan is discussed to draw attention to these crustacean shell wastes’ by-products, which are starting to be used commercially, and provide a better understanding of the relationship between the physicochemical properties of these two polymers and their functional properties, as well as their biological activities.

6.2 Dietary activity 6.2.1 Hypocholesterolemic effect Chitosan is regarded as a useful dietary ingredient since it is influential in reducing plasma cholesterol level, which retards and cures cardiovascular diseases. Chitosan is being compared to and suggested as a substitute for cholestyramine which has proved to be a very effective factor in treating patients with increased serum cholesterol. However, the mechanisms of action may be different [13].

Handbook of Chitin and Chitosan

6.2 Dietary activity

179

It has been reported that chitosan has significant hypocholesterolemic activity in various experimental animals while high-cost hydrolyzed chitosan oligomers did not show a cholesterol-lowering activity [14]. However, chitosan is too highly viscous to be used in physiological and functional foods. Therefore the relationship between the cholesterollowering effect and the average molecular weight of chitosan needs to be established. This compound reportedly has the potential to reduce blood cholesterol in rats. Sugano et al. [14] studied rat groups fed on a cholesterolenriched diet in order to study the hypocholesterolemic activity of chitosan hydrolysates with different molecular weights and viscosity. It was reported that the cholesterol-lowering activity of the partial hydrolysates with molecular weights of 8000 and 20,000 Da was equal to or greater than that of the parent chitosan with a molecular weight of approximately 300,000 Da, whereas chitin does not display such an effect. The hypocholesterolemic activity of chitosan might be due to decreased cholesterol absorption and interference with bile acid absorption, the same mechanism as dietary fibers. Information regarding the digestion and absorption of chitin and chitosan in the GI tract is limited. In an in vivo study in the canine GI tract, it was shown that chitin did not undergo any changes in weight and shape, whereas chitosan showed a 10% decrease in weight and formed a film [5]. Compared with the control diet, those fed chitosan-containing diets generally lowered body weights and feed intakes, resulted in poorer feed conversion, reduced plasma lipid concentrations, reduced duodenal digestibilities of nutrients, ileal digestibilities of crude fat, organic matter, and nonstarch polysaccharide residues, and reduced cecal short-chain fatty acid concentrations. In comparison with the chitin-containing diet, chitosancontaining diets tended to reduce body weights and feed intakes, resulting in poorer feed conversion, reduced plasma lipid concentrations, reduced postprandial triacylglycerol response, increased duodenal nutrient digestibilities, increased duodenal digesta dry matter content, and reduced ileal digestibility of crude fat [15]. The safety of chitosan or chitin has not been well-documented. Both chitin and chitosan have been reported to bind essential elements from solution, and chitosan is used to remove suspended organic materials in waste treatment. These observations again suggest a reciprocal relationship between cholesterol and essential element status may occur with the ingestion of chitosan. Chitin and chitosan at very high levels of intake may cause physical insult to the intestinal tract of rats. Also, apparent absorption of Fe is impaired when chitin and chitosan are fed at dietary levels of 10% and 20%. However, such high levels of intake are atypical. At dietary levels below 5%, neither compound appears to compromise element absorption [13].

Handbook of Chitin and Chitosan

180

6. Functional properties of chitin and chitosan-based polymer materials

6.2.2 Prebiotics ingredients Several materials which are not metabolized and from sources other than plants have been recognized as consumed in the tract, for example, mucopolysaccharides from animal tissue, microbial cell walls, exoskeletons of arthropods, and synthetic bulking agents. Two unconventional, but potential sources of DF are chitin and chitosan. Chitin, along with cellulose and collagen, is the principal skeletal or support matrices in all living systems [13]. Chitin and chitosan have been used as a source of dietary fiber [16]. They are considered to be functional foods because they are not digested by intestinal enzymes so they play a role as prebiotics. Prebiotics are nondigestible food ingredients which beneficially affect the host by selectively stimulating the growth of beneficial bacteria and/or by suppressing the growth of harmful bacteria in the colon which have the potential to improve the host health [17]. Usually, prebiotics are oligosaccharides. Therefore chitooligosaccharides (COS) also stimulate beneficial gut species (Bifidobacterium and Lactobacillus sp.) offering prebiotic activity. Despite this property, in a mixed culture system no increase in the Bifidus counts was observed [18]. However, in pure cultures COS stimulated Bifidobacterium bifidum and Lactobacillus sp.; they resulted in greater cell numbers and showed prebiotic effects in low concentrations [19]. In spite of showing bactericidal effects on certain lactic acid bacterial species in the previous study [20], the bactericidal effects of COS against the bifidobacteria and lactic acid bacteria have not been reported in this study. In vitro studies have shown that COS can bind four to five times their weight of micellar lipids, leading to some slimming claims and control of obesity by blocking fat absorption. COS can also act as thickeners and stabilizing agents [21]. COS have a bifidogenic effect at concentrations between 0.1% and 0.5%, whereas they have a growth stimulatory effect on L. casei and L. brevis at a concentration of 0.1%. Since these COS are fully deacetylated, they are not digestible by the intestinal enzymes, thus increasing the applicability of COS as prebiotics. Pan et al. [22] studied the COS prebiotic activity in the mouse model system. The COS used in this experiment were 90% deacetylated and the DP ranged from 3 to 6. According to this study, the concentrations of more favorable bifidobacteria and lactobacilli increased while decreasing the concentrations of unfavorable enterococcus and Enterobacteriaceae in the cecum of mice treated with COS for 14 days. According to these results, the COS can potentially be utilized as prebiotics in the food industry.

6.2.3 Calcium absorption acceleration effect in vivo It has been reported that dietary chitosan affected calcium metabolism in animals. Jeon et al. [23] studied the in vivo calcium absorption

Handbook of Chitin and Chitosan

6.3 Antimicrobial activity

181

acceleration effect by chitosan oligomers and revealed that chitosan oligomers from trimers to heptamers lowered fecal calcium excretion and enhanced the breaking force of femur in rats. Compared with cellulose-fed rats, rats fed a 1%
5% chitosan diet showed more wholebody retention of radioactive calcium (47Ca) [24]. Feeding rats with chitosan for 2 weeks led to a decrease in mineral absorption and bone mineral content. Although chitosan retards calcium absorption, chitosan oligomers increase the absorption of calcium and other minerals in vivo [25].

6.3 Antimicrobial activity The antimicrobial activity of any substance is always directed toward its applicability. Recently, research has focused on the development of materials with film-forming potential and antimicrobial characteristics to help enhance food safety and shelf life. Antimicrobial packaging is one of the most promising active packaging systems that has been found to be highly effective in inhibiting spoilage and pathogenic microorganisms as food contaminants [26]. The antimicrobial property of chitosan and its derivatives, with conflicting results, has received considerable attention in recent years due to imminent problems associated with synthetic chemical agents. It is proven that microbial changes are responsible for food loss and hence various chemical and physical processes have been suggested to extend the shelf life of foods. The availability of suitable antimicrobials, new polymer materials, regulatory concerns, and appropriate testing methods limit the application of an antimicrobial packaging system for food [27]. Currently, polymeric bioactive films including antimicrobial factors have been found to be very influential and practical in such applications. The antimicrobial activity of chitin, chitosan, and their derivatives against different groups of microorganisms, such as bacteria, yeast, and fungi, has received considerable attention in recent years [28,29]. The addition of chitosan to food inhibits microorganisms’ growth and avoids poor appearance, off-flavors, and economic losses. Adding chitosan to cheese led to the improvement of its mycological quality; fungal growth was inhibited and hence shelf life was extended [30]. Chitosan shows a wide range of antimicrobial activity against both Gram1 and Gram2 bacteria and fungi [31]. In a study on the mode of antimicrobial action of chitosan (250 ppm at pH 5.3) by monitoring the uptake of the hydrophobic probe 1-N-phenyl naphthylamine, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium showed significant uptake which was reduced (in E. coli, Salmonella) or abolished (P. aeruginosa) by MgCl2. Chitosan

Handbook of Chitin and Chitosan

182

6. Functional properties of chitin and chitosan-based polymer materials

also sensitized P. aeruginosa and Salmonella to the lytic effect of sodium dodecyl sulfate. Electrophoretic and chemical analyses of the cell-free supernatants revealed no release of LPS or other membrane lipids. Electron microscopic observations showed that chitosan caused extensive cell surface alterations and covered the outer membrane with vesicular structures, resulting in the loss of the barrier functions [32]. This property of chitosan is useful in food preservation. To enhance the antibacterial potency of chitosan, thiourea chitosan was prepared by reacting chitosan with ammonium thiocyanate followed by complexing with Ag1 [33]. It has been reported that quaternary ammonium salt of chitosan exhibits good antibacterial activities, for example, diethylmethylchitosan chloride showed higher antibacterial activity than chitosan. Muraki et al. [34] reported that partially N-lauroyl (PNL)-COS (DP 7
8), PNL(GlcN)7, and PNL-(GlcN)8, with a degree of N-lauroylation of about 50% had a fairly strong antibacterial activity against the growth of E. coli, compared to all (GlcN)n and PNL-(GlcN)n with a chain length of less than seven residues. Chitosan also presents antifungal activity, as observed in the inhibition of the growth of Fusarium solani, a typical plant pathogenic fungus. In the field of agriculture, chitin and chitosan are expected to be fungus control agents. The addition of chitin and chitosan to the soil and the spraying of chitosan solutions on vegetables were claimed in patents to be effective for cultivation, and several forms of powders and solutions are produced commercially. Because of the virucidal and fungicidal activities of chitosan, it sometimes prevents plant diseases and improves crops. Seed coating with chitosan enhanced the germination ratio and growth rate, leading to enhanced crops [35]. Novel N,O-acyl chitosan derivatives were more active against the gray mold fungus Botrytis cinerea and the rice leaf blast fungus Pyricularia oryzae. Hydroxypropyl chitosan grafted with maleic acid sodium killed over 99% of Staphylococcus aureus and E. coli within 30 min of contact at a concentration of 100 ng/mL. Hydroxypropyl chitosan was a potent inhibitor of Azatobacter mali, Fusarium oxysporum, and Pyricularia piricola. The degree of substitution (DS) of hydroxypropyl group also influenced the antifungal activity. Regarding these antifungal mechanisms, it was reported that these chitosan derivatives directly interfered with fungal growth and activated several defense processes, such as accumulation of chitinases, synthesis of proteinase inhibitors, and induction of callous synthesis. It was also noted that the antibacterial activity of chitosan derivatives increased with an increasing chain length of the alkyl substituent, and this was attributed to the increased hydrophobicity [5].

Handbook of Chitin and Chitosan

6.3 Antimicrobial activity

183

Chitosan induced a plant-defense enzyme, chitinase, in plant tissues that degrades fungal cell walls, induces the accumulation of the antifungal phytoalexin pisatin in pea pods, and elicits the antimicrobial phytoalexin and pisatin in pea pods (Pisum sativum) [36]. Allan et al. [37] reported that chitosan has a higher antifungal activity than chitin because positively charged amino groups on chitosan retard the growth of fungi or microbacteria by polyelectrolyte complexes with negatively charged carboxyl anion groups present in their cell walls. Furthermore, chitosan oligomers have been shown to inhibit the growth of several phytopathogens. Hirani et al. [38] studied the relationship between the degree of polymerization of chitosan and the degree of pathogen inhibition. They showed that chitosan oligomers (DP 2
8), as well as partially hydrolyzed chitosan with low molecular weight, possessed stronger growth inhibition than high-molecularweight chitosan against several phytopathogens, including Fusarium oxymoron, Phomopsis fukushi, and Alternaria alternata. Kendra et al. [39] explained that some chitosan oligomers with the biological activity present in pea/Fusarium interactions appear to inhibit fungal growth. Uchida et al. [40] found that oligomers with a higher molecular weight, which were slightly hydrolyzed with chitosanase, were more active in both antifungal and antibacterial activities than native chitosan and lower-molecular-weight oligomers. With respect to antimicrobial activity of partially hydrolyzed chitosan oligomers, Jeon et al. [23] reported that the highest-molecular-weight oligomers (MW 5000
10,000 Da), among the three fractions of oligomers produced and separated using the ultrafiltration membrane enzymatic reactor system, showed the strongest bactericidal and fungicidal activities against most pathogens tested. Ueno et al. [41] also reported that chitosan oligomers with a MW of less than 2000 Da did not easily suppress microbial growth, while an oligomer with MW 9300 Da almost completely suppressed microbial growth at a very low concentration. The antimicrobial properties of chitosan in a liquid medium will be poorly represented in complex food systems where the interaction of chitosan with other components may modulate its activity [42]. Being soluble in aqueous acetic acid and locating at the interface, chitosan is greatly applicable as an antimicrobial agent in food emulsions [43]. Despite large concentrations of oil in emulsions which prevent growth, these emulsions may contain spoilage and pathogenic microorganisms in the nonlipid phase. Mayonnaise has been chosen as a model system where three target microorganisms have been inoculated. The most effective MW of chitosan has been shown to vary with the microorganism tested [42]. Studies of the effect of solubility of chitosan revealed that the waterinsoluble chitin exhibited the antimicrobial effect, whereas watersoluble chitosan itself had no significant antimicrobial effect against

Handbook of Chitin and Chitosan

184

6. Functional properties of chitin and chitosan-based polymer materials

both bacteria and yeast [44]. However, Chung et al. [45] have reported the metal ion chelating capacity and antibacterial activity of a chitosan
glucosamine derivative prepared by the Maillard reaction. This derivative appeared to be more effective than other chitosan or chitosan derivatives as a natural antimicrobial agent. The Maillard reaction has been used to develop biofunctional biopolymers as food preservatives with a wide range of antimicrobial effects. Chitosans of different degrees of polymerization were mixed with lysozyme and gluten peptides and conjugated through this reaction. The results demonstrate that high MW chitosan conjugates were very effective in improving the bactericidal activity of proteins or peptides compared to low MW chitosan conjugates. It has been shown that the Maillard reaction can be successfully used to generate products from β-lactoglobulin and chitosan, which exhibit better bactericidal properties compared with β-lactoglobulin alone [46]. Several mechanisms have been suggested regarding the antimicrobial activity of chitosan. Cationic groups of chitosan interact with anionic groups on the cell surface leading to the formation of an impermeable layer around the cell preventing the transport of essential solutes [47]. Electron microscopy analysis revealed that the site of action is the outer membrane of Gram2 bacteria. The permeabilizing effect has been observed at the slightly acidic condition in which chitosan is protonated, but this permeabilizing effect of chitosan is reversible [32]. The second mechanism involves the inhibition of the RNA and protein synthesis by permeation into the cell nucleus. Liu et al. [48]have observed labeled chitosan oligomers with MW from 8 to 5 kDa inside the E. coli cell and they showed good antibacterial activities. In this case, the MW is the crucial property. Other mechanisms have also been suggested. Chitosan may interfere with microbial growth by acting as a chelating agent rendering metal, trace elements, or essential nutrients unavailable for the organism to grow at the normal rate. Chitosan is also able to interact with flocculating proteins, but this action is highly pH-dependent. Several authors have proposed that the antimicrobial action of chitosan against filamentous fungi could be explained by a more direct disturbance of membrane function. However, it is not clear whether the antimicrobial activity of chitosan is caused by growth inhibition or cell death [8]. Antibacterial activities were found to increase in the order of N,O-CM
chitosan, chitosan, and O-CM
chitosan. The first product, where amino and hydroxyl groups have been substituted by carboxymethyl groups, contains fewer amino fragments. In the case of O-CM
chitosan, its number of amino groups is not changed. Moreover, its carboxyl group may have reacted with the amino groups intra- or intermolecularly and charged these groups. The authors concluded that

Handbook of Chitin and Chitosan

6.4 Emulsifying properties

185

the antibacterial activities of chitosan and carboxymethylated derivatives depend on the effective number of
NH31 groups [48]. Several studies prove that the higher the positive charge of chitosan, the stronger are is the binding to bacterial cell walls [49]. The relationship between MW, number of charges and antimicrobial activity has been pointed out [50]. It has been shown that O-CM chitosan derived from degraded chitosan was more effective than plain chitosan. This was attributed to the interaction of the COOH group with the NH2 group intra- or intermolecularly to impart a charge, the number of
NH3 groups becoming larger. Regarding native chitosan, an excessive concentration of amino groups on O-CM
chitosan develops a structure that involves cross-linking through strong intramolecular hydrogen bonding leading to a reduced number of amino groups available to attach to bacterial surfaces. In contrast, some authors have not found a clear relationship between the degree of deacetylation and antimicrobial activity. These authors suggest that the antimicrobial activity of chitosan is dependent on both the chitosan and the microorganism used. Park et al. [51] studied the antimicrobial activity of heterochitosans and hetero-COs with different degrees of deacetylation and MWs against three Gram2 bacteria and five Gram1 bacteria and found that the 75% deacetylated chitosan showed more effective antimicrobial activity compared with that of 90% and 50% deacetylated chitosan. The antimicrobial properties of chitosan have also been employed as active edible packaging [52]. Biofilms have been produced from chitosan that prolong the shelf life of food products. Antimicrobial coating of vegetables, fruits, grains, and fish retard microbial invasion as chitosan acts as a protective barrier to enhance the sensory and nutritional quality of the food [8,53]. Besides being a protective barrier, edible biopolymer films can be considered as carriers of bioactive compounds to improve food quality. Polymeric bioactive films could be combined with different antimicrobial agents, such as organic acids, bacteriocins (nisin and lacticin), essential oils (thymol, p-cymene, and cinnamaldehyde), proteins (conalbumin), antibiotics, fungicides, and chelating agents, to reduce food spoilage by pathogenic microorganisms and increase shelf life [54,55]. Chitosan-based edible films are biodegradable and can be consumed along with the product in the package. Moreover, they form transparent films with good mechanical properties to protect the appearance and the quality of the product inside [56].

6.4 Emulsifying properties Chitosan produces w/o/w emulsions without adding any surfactant, because this biopolymer is composed of a mixture of molecules with

Handbook of Chitin and Chitosan

186

6. Functional properties of chitin and chitosan-based polymer materials

different DD: some less deacetylated molecules may stabilize the water droplets inside the oil drops, while the hydrophilic ones stabilize the oil drops in the multiple emulsion formed [57]. The influence of the DD of chitosan on the emulsification of sunflower oil has been studied in HCl solutions. The resulting distribution was unimodal at low DD (75%) and high DD (95%) for all used concentrations. At intermediate DD, distribution was unimodal only when the most concentrated solution was used. When chitosan concentration increased, emulsion viscosity as a function of time was more stable [58]. Laplante et al. [59] studied the emulsion-stabilizing properties of various chitosans in the presence of whey protein isolate (WPI) and reported that a low deacetylated chitosan more efficiently stabilized the emulsion. There is a positive correlation between the increase of stability against syneresis and the increase of viscosity of chitosan. The most unfavorable effect from a low MW preparation on stability is mainly explained by a loss of interfacial coadsorption efficiency. When studying other variables, such as pH, ionic strength, and the WPI/chitosan ratio, a low deacetylated chitosan displayed better behavior, showing the predominance of electrostatic interactions in the interfacial stabilization [8]. Emulsions stabilized by surfactant
chitosan membranes have been shown to have better stability to pH, ionic strength, thermal processing, and freezing than emulsions stabilized by surfactant alone, which was attributed to the increase in electrostatic and steric repulsion between the droplets [60]. The influence of the molecular characteristics of chitosan on the properties of oil-in-water emulsions has been studied. The ζ-potential and mean diameter of the particles in the secondary emulsions were not strongly influenced by chitosan MW; however, with the lowest DD (40%) the fraction of droplets that were aggregated was considerably lower [61]. A study was carried out on the effect of chitosan on the emulsifying capacity of egg yolk with four chitosans from crab shell from different companies (Table 6.1) [62]. Although chitin and chitosan alone do not produce emulsions [63], the emulsifying capacity of egg yolk increased with the addition of chitosan compared with the control. Increase in emulsifying capacity (IEC) was more notable with 0.5% chitosan (IEC 5 7%) than with 0.1 or 0.3% chitosan (IEC 5 2%). However, no notable differences (P..05) in emulsifying capacity with chitosan products were observed at each concentration. It is concluded that any chitosan, regardless of physicochemical characteristics, could be utilized to increase the emulsifying capacity of egg yolk in the preparation of foods such as mayonnaise. An increase in the emulsifying capacity of egg yolk with the addition of chitosan also was observed by Lee [64], who reported that the emulsifying capacity of egg yolk increased B10%
13% with the addition of 0.1%
0.2%

Handbook of Chitin and Chitosan

187

6.4 Emulsifying properties

TABLE 6.1 Effects of various chitosan products and their concentrations on emulsifying capacity of egg yolk [62]. Emulsifying capacity (mL of soybean oil added/g of egg yolk)a at chitosan concentration of Chitosan product

0% (control)

0.1%

0.3%

0.5%

Keumho Chemical Co. (Seoul, Korea)

80.5 6 1.3

82.4 6 1.0

82.4 6 2.1

86.2 6 1.5

Sigma Chemical Co. (St. Louis)

80.5 6 1.3

82.0 6 1.8

82.2 6 1.7

86.4 6 0.4

Pronova Biopolymer (Raymond)

80.5 6 1.3

82.2 6 1.1

82.0 6 2.1

86.6 6 0.8

DuPont (Wilmington)

80.5 6 1.3

82.8 6 2.9

83.5 6 1.7

84.6 6 1.7

80.5

82.3

82.5

85.9

Av

Mean 6 standard deviation of duplicate determinations.

a

chitosan based on egg yolk weight. This study has clearly demonstrated that both physicochemical characteristics and functional properties, except for emulsifying capacity, of commercially available chitins and chitosans differ with products. Thus relationships between the functional properties and characteristics of chitin/chitosan products must be constantly monitored for proper quality control in order that chitin or chitosan is used as a functional ingredient. It is observed that alkylated chitosans are very important as amphiphilic polymers based on polysaccharides. The first derivative having these characteristics was a C-10-alkyl glycoside branched chitosan with a high DS (DS 5 1.5), which gelled when heated over 50 C [65]. Using the reductive amination, a series of amphiphilic derivatives were produced with different chain lengths (Cn from 3 to 14) and controlled DS (usually lower than 10% to maintain water solubility in acidic conditions). This technique was also used to introduce n-lauryl chains [66]. Alkylated chitosans with good solubility in acidic conditions (pH ,6) have a number of very interesting properties. First, they exhibit surface activity and they were compared with corresponding lowmolecular-weight surfactants [67]. For the same amount of alkyl chains with the same length, they have a relatively low effect on lowering the surface tension but they enhance the stability of the interfacial film [68]. It was illustrated that there is a complete difference between the simple surfactant and modified chitosan behavior. Secondly, they cause a considerable increase in the viscosity of aqueous solution due to hydrophobic interchain interactions. Particularly for C-12 chain length and a DS B5%, a physical gel is produced. This gel formation depends on pH and salt concentration. These gels are obtained from a balance between

Handbook of Chitin and Chitosan

188

6. Functional properties of chitin and chitosan-based polymer materials

electrostatic repulsions between the positively charged chitosan chains and hydrophobic attraction due to alkyl chains, mainly in relation with their length. The hydrophobic domains formed in the systems are important to adsorb hydrophobic molecules such as pyrene (a fluorescent probe is used to show these domains in dilute solution). The addition of cyclodextrins, which are known to complex the alkyl chains of surfactants, can destroy these associations. It is notable that alkyl chitosans are compatible with neutral and cationic surfactants. It was shown that cationic surfactant adsorbed on the alkyl chain grafted on chitosan, promotes its solubilization [69].

6.5 Antioxidant activity Reactive oxygen species such as H2O2, hydroxyl radicals, and superoxides lead to oxidative stress, which is correlated with various pathologies: cancer, cardiovascular disease, premature aging, rheumatoid arthritis, and inflammation [10]. Chitin and chitosan have shown antioxidant effects so they could be added as ingredients to produce functional food which could prevent age-related and diet-related diseases [70]. Moreover, the oxidation of food containing unsaturated lipids causes off-flavors and rancidity. Usually, synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), are used in this case. However, such compounds are possibly hazardous, and thus natural antioxidants have become preferred owing to their lack of toxicity [71]. Chitosan has shown a significant scavenging capacity against different radical species, the results being comparable to those obtained with commercial antioxidants. Samples prepared from crab shell chitin with DD of 90%, 75%, and 50% were evaluated on the basis of their abilities to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, superoxide radical, and alkyl radical. The results revealed that chitosan with higher DD exhibited the highest scavenging activity. On the other hand, chitosans of different size, as well as their sulfate derivatives, were assayed against superoxide and hydroxyl radicals. A negative correlation was found between chitosan MW and antioxidant activity (Table 6.2). Scavenging effect of chitosan sulfated derivatives was stronger on peroxide radicals but the chitosan of lowest MW showed considerably more ferrous ion-chelating potency than others. Chitosan can be potentially considered as a natural antioxidant to stabilize lipid-containing foods and enhance shelf life because it chelates metal ions in food systems. It may retard lipid oxidation by chelating ferrous ions present in the system, thus eliminating their prooxidant activity or their conversion to ferric ion [8].

Handbook of Chitin and Chitosan

6.5 Antioxidant activity

TABLE 6.2

189

Flocculation mechanisms of chitosan-based flocculants [9].

Mechanism

Description

Simple charge neutralization

Efficient reduction of the thickness of the electric double layer and full charge neutralization.

Charge patching

Unevenly distributed surface charges are incompletely neutralized.

Bridging

Adsorption and connection of the primary flocs on soluble linear large-MW flocculants.

Sweeping

Enmeshing and entrapping of small colloidal pollutants by large flocs or polymeric precipitates.

Illustration

This activity has been also studied in COS. Chitobiose and chitotriose have proved to be more potent than three reference compounds (aminoguanidine, pyridoxamine, and Trolox) in scavenging hydroxyl radicals while glucosamine and the corresponding N-acetylchitooligosaccharides did not show any capacity. Electron spin resonance spectrometry has been used to follow the scavenging activity of chitooligosaccharide mixtures fractionated by ultrafiltration. This activity was shown to be dependent on the MW, the fraction 1
3 kDa having the highest radical scavenging effect. When the DD was considered, a correlation between scavenging activity over all tested free radicals with the increment of deacetylation values of COS was found. Therefore it has been pointed out that the free amino groups in the hetero-COS play an important role in free radical scavenging activity, probably by forming stable macromolecule radicals [72]. This capacity

Handbook of Chitin and Chitosan

190

6. Functional properties of chitin and chitosan-based polymer materials

of oligosaccharides has been further assayed in vivo. Yang et al. [73] assayed two different MW COS (1.1 and 0.5 kDa) against H2O2 released from polymorphonuclear leukocytes stimulated by phorbol12-myristate-13-acetate in rats. They found that the radical scavenging capacity was higher for the first COS. Apple juice has been used as an aqueous model system to study the antioxidative activity of chitosans with different MWs. Low-MW chitosan exhibited stronger scavenging activity than medium- or high-MW chitosan and ascorbic acid, which was used as a positive scavenger. However, the authors conclude that in vivo antioxidant activity and the various antioxidant mechanisms must be further investigated [74]. Lipid oxidation of unsaturated fatty acids widely occurs in seafood. This oxidation is catalyzed by the high concentrations of prooxidants such as hemoglobin and metal ions in the fish muscle. The iron bound to fish tissue proteins such as myoglobin, hemoglobin, ferritin, and transferrin may be released during storage and cooking, thus activating oxygen and initiating lipid oxidation. The antioxidant effect of chitosan varies with its molecular weight, concentration, and viscosity [75]. It is attributed to differences in the availability of net cationic amino groups in the molecule, which impart intermolecular electrostatic repulsive forces leading to an increase in the hydrodynamic volume of the extended chain conformation [76]. Chitosans obtained from crab shell wastes were tested on herring flesh (Clupea harengus). Chitosan with different viscosity was used (360, 57, and 14 cP) to treat the fish samples. The corresponding viscosity average molecular weights were 1.8 3 106, 9.6 3 105, and 6.6 3 105 Da. All three biopolymers showed antioxidant effects such as lowering peroxide values, TBARS, and total volatile aldehydes. However, the highest activity was observed with the low-viscosity chitosan (14 cP). These results were compared with those obtained with commercial antioxidants [BHA, BHT, and tertiary butylhydroquinone (TBHQ)] and it seemed that the 14 cP sample was more effective than the higher viscosity chitosan because of the similarity of its action with the conventional antioxidants [10]. Kim and Thomas [77] also observed similar results with chitosan with different molecular weights (30, 90, and 120 kDa) in Atlantic salmon (Salmo salar) based on the measurement of 2-thiobarbituric acidreactive substances (TBARS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH)scavenging activity. The 30 kDa chitosan showed the highest scavenging activity compared to 90 and 120 kDa chitosan. The increase in the concentration of 30 kDa chitosan resulted in an increase of total amino groups responsible for scavenging more radicals. Fatty (herring) and lean (cod) fishes have been used as model systems to assay the antioxidant activity of three chitosans prepared with different deacetylation times for the same sample. The lowest viscosity

Handbook of Chitin and Chitosan

6.6 Flocculent and chelating

191

chitosan presented the highest antioxidant effect. This was attributed to the lower chelation by high-viscosity (high MW) chitosan, as the intramolecular electric repulsive forces would increase the hydrodynamic volume by extended chain conformation. However, the DD was pointed to as another factor that may be involved in the chelation ability of chitosans [8]. Chitin has been employed as an active carrier for cosmetic products. Morganti et al. [78] embedded chitin-nanofibrils (CN) with antioxidant ingredients (melatonin, lutein, and ectoin). They reported that CN was able to enhance the penetration of the active ingredients through the skin layer. Having synergic radical scavenging activity, the combination of chitin and antioxidants could be used to protect the skin against solar radiation responsible for photoaging and wrinkles. Tests with volunteers showed a tolerance for the CN, the absence of erythema, and improved skin barrier functions [18].

6.6 Flocculent and chelating The rapid development of modern industries throughout the world has been accompanied by the production of various wastewaters containing different types of dissolved and undissolved contaminants and therefore of increasingly severe water pollution [79]. Among all techniques addressed to this problem, coagulation/flocculation is one of the most commonly used to achieve solid
liquid separation, based on its cost-effectiveness and ease of operation [80,81]. Chitin and chitosan adsorb dyes, aromatic hydrocarbons, and proteins. They also adsorb metal cations such as copper, mercury, cadmium, iron, manganese, nickel, zinc, lead, and silver, but chitosan exhibits higher affinity due to the free amino groups. Chitosan collects even highly toxic organomercury compounds and is a candidate for adsorbents to remove toxic heavy metals from industrial wastewater [35], acting as a coagulant, flocculant, coagulant aid, and as a component in composite flocculants to efficiently treat wastewaters containing different types of dissolved and undissolved inorganic, organic, and biological contaminants, including suspended solids, heavy metals, humic acid, dyes, algae, and bacteria [82,83]. The flocculation mechanisms of chitosan could be classified as simple charge neutralization, charge patching, bridging, and sweeping. It is notable that flocculation processes usually are conducted using two or more of these mechanisms [84]. The flocculation efficiency of chitosan-based flocculants in wastewater treatment is highly dependent on their structures. As mentioned above, the DD and the MW are two very important structural factors

Handbook of Chitin and Chitosan

192

6. Functional properties of chitin and chitosan-based polymer materials

for chitosan. When chitosan is used as a flocculant, the DD is related to the charge density (short-range structure) and highly influences the charge neutralization efficiency, which together with the MW accounts for the long-range structure, that is, the morphology and conformation of the polymer chain in aqueous medium. The latter features are intrinsically related to bridging and sweeping flocculation mechanisms. The DS of the functional groups and grafting ratio of chitosan-based flocculants also determine their final flocculation efficiency. In addition, all of these structural factors interact with the various environmental factors (dose, pH, ionic strength, and temperature) to influence the performance of the flocculants [9]. Several studies have been done regarding this issue. Nair et al. [85] used chitosan for the removal of mercury from solutions. Peniche-Covas et al. [86] reported the adsorption kinetics of mercuric ions using chitosan. The results indicate that the efficiency of adsorption of Hg by chitosan depends upon the period of treatment, the particle size, initial concentration of Hg21, and quantity of chitosan. Jha et al. [87] studied the adsorption of Cd21 on chitosan powder over the concentration range of 1
10 ppm using various particle sizes by adopting a similar procedure as for the removal of mercury. Chitin and other water-soluble derivatives are useful flocculents for anionic waste streams. Weltrowski et al. [88] used chitosan N-benzyl sulfonate derivatives as sorbents for the removal of ions in an acidic medium. The selective adsorption capacity for metal ions of amidoximated chitosan bead-g-PAN copolymer has been studied by [89]. These researches reveal that chitosan naturally has selectivity for heavy metal ions and is efficient for the treatment of wastewater. Chitosan was used for the removal of Cu21, Hg21, Ni21, and Zn21 within the temperature range 25 C
60 C, at near neutral pH [73]. Manica et al. [90] used chitosan flakes of 0.4
4 mm for the removal of Cr(III) from wastewater. The decrease in the size of the flakes resulted in the increased adsorption capacity and they concluded that metal ions were preferably adsorbed on the outer surface of chitosan in the removal of Cr(III) from the wastewater. Sakiyama et al. [91] proposed a mechanism for complex formation with copper at pH .5, in agreement with X-ray data on chitosan
copper stretched films. This chelation depends on the physical properties of chitosan (powder, gel, fiber, film). Higher degrees of deacetylation of chitin resulted in better chelation. Thus chelation is related to the 2 NH2 content and distribution. It is also related to the DP of oligochitosans; the complex starts to form as DP .6. The two proposed forms are [Cu (2NH2)]21, 2OH2, H2O and [Cu (2NH2)2]21, 2OH2. The first complex is formed at pH between 5 and 5.8, while the second forms above pH 5.8.

Handbook of Chitin and Chitosan

References

193

The nature of the cation is very important in the mechanism of interaction. The affinity of chitosan for cations absorbed on film shows selectivity following the order: Cu12 . . Hg12 . Zn12 . Cd12 . Ni12 . Co12 BCa12 ; Eur13 . Nd13 . Cr13 BPr13 In another study, chitosan powder was dispersed in silver nitrate solution or used to fill a column to adsorb mercuric ions from a chloride solution [92]. It was shown that the conditions for using chitosan (50 mesh particles of chitosan or chemically cross-linked beads of chitosan) also play a large role in the adsorption and on the kinetics of retention [65].

6.7 Future trends Obtaining chitin and their derivatives from marine processing wastes (crustacean wastes) continuously keep growing. Chitosan and its derivatives have many notable advantages that warrant further studies leading to their broad-ranging application. They are known as effective, safe, and cost-effective flocculants with applications in a wide variety of settings, including the treatment of industrial effluents as well as potable water. However, maximizing the applications of chitosan-based flocculants first requires in-depth and molecular-level investigations of their mechanisms of action with respect to their structural characteristics.

References [1] A. Hemantaranjan, A future perspective in crop protection: chitosan and its oligosaccharides, APAR 1 (2014) 1
8. [2] M.N.V.R. Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (2000) 1
27. [3] S.M. Kuddus, R.I.Z. Ahmad, Isolation of novel chitinolytic bacteria and production optimization of extracellular chitinase, J. Genet. Eng. Biotechnol. 11 (2013) 39
46. [4] J. Du, E. Tan, H.J. Kim, A. Zhang, R. Bhattacharya, K.J. Yarema, Comparative evaluation of chitosan, cellulose acetate, and polyethersulfone nanofiber scaffolds for neural differentiation, Carbohydr. Polym. 99 (2014) 483
490. [5] K.V. Harish Prashanth, R.N. Tharanathan, Chitin/chitosan: modifications and their unlimited application potential-an overview, Trend Food Sci. Technol. 18 (2007) 117
131. [6] R.C. Goy, D. de Britto, O.B.G. Assis, A review of the antimicrobial activity of chitosan, Polimeros 19 (2009) 241
247. [7] M.T. Yen, J.H. Yang, J.L. Mau, Antioxidant properties of chitosan from crab shells, Carbohydr. Polym. 74 (2008) 840
844.

Handbook of Chitin and Chitosan

194

6. Functional properties of chitin and chitosan-based polymer materials

[8] I. Aranaz, M. Mengı´bar, R. Harris, I. Pan˜os, B. Miralles, N. Acosta, et al., Functional characterization of chitin and chitosan, Curr. Chem. Biol. 2009 (3) (2009) 203
230. [9] R. Yang, H. Li, M. Huang, H. Yang, A. Li, A review on chitosan-based flocculants and their applications in water treatment, Water Res. 95 (2016) 59
89. ¨ zogul, J.M. Regenstein, Industrial applications of crustacean by[10] I. Hamed, F. O products (chitin, chitosan, and chitooligosaccharides): a review, Trend Food Sci. Technol. 48 (2016) 40
50. [11] G.S. Dhillon, S. Kaur, S.K. Brar, M. Verma, Green synthesis approach: extraction of chitosan from fungus mycelia, Crit. Rev. Biotechnol. 33 (2013) 379
403. [12] M. Kaya, T. Baran, S. Erdogan, A. Mentes, M.A. Ozusaglam, Y.S. Cakmak, Physicochemical comparison of chitin and chitosan obtained from larvae and adult Colorado potato beetle (Leptinotarsa decemlineata), Mater. Sci. Eng. 45 (2014) 72
81. [13] D.T. Gordon, C.B. Williford, Chitin and chitosan: influence on element absorption in rats. in: A. Furda (Ed.), Unconventional Sources of Dietary Fiber. American Chemical Society, Washington, DC, 1983, pp. 155
184. [14] M. Sugano, S. Watanabe, A. Kishi, M. Izume, A. Ohtakara, Hypocholesterolemic action of chitosans with different viscosity in rats, Lipids 23 (1988) 187
191. [15] A. Razdan, D. Pettersson, Effect of chitin and chitosan on nutrient digestibility and plasma lipid concentrations in broiler chickens, Br. J. Nutr. 72 (1994) 211
288. [16] W. Xia, P. Liu, J. Zhang, J. Chen, Biological activities of chitosan and chitooligosaccharides, Food Hydrocolloid. 25 (2010) 170
179. [17] G.R. Gibson, M.B. Roberfroid, Dietary modulation of the human colonic microbiota introducing the concept of prebiotics, J. Nutr. 125 (1995) 1401
1412. [18] P. Morganti, G. Morganti, A. Morganti, Transforming nanostructured chitin from crustacean waste into beneficial health products: a must for our society, Nanotechnol. Sci. Appl. 4 (2011) 123
129. [19] H.W. Lee, Y.S. Park, J.S. Jung, W.S. Shin, Chitosan oligosaccharides, dp 2-8, have prebiotic effects on the Bifidobacterium bifidium and Lactobacillus species, Anaerobe 8 (2002) 319
324. [20] Y.J. Jeon, P.J. Park, S.K. Kim, Antimicrobial effects of chitooligosaccharides produced by bioreactor, Carbohydr. Polym. 44 (2001) 71
76. [21] I.S. Arvanitoyannis, The use of chitin and chitosan for food packaging applications, in: E. Chiellini (Ed.), Environmentally Compatible Food Packaging, Woodhead Publishing, Cambridge, 2008, pp. 137
157. [22] X. Pan, F. Chen, T. Wu, et al., Prebiotic oligosaccharides change the concentration of short change fatty acids and the microbial population of mouse bowel, J. Zhejiang Univ. Sci. B 10 (2009) 258
263. [23] Y.J. Jeon, P.J. Park, H.G. Byun, S.K. Kim, Production of chitosan oligosaccharides using chitin-immobilized enzyme, Chitin Chitosan 2 (3) (1998) 60. [24] M. Wada, Y. Nishimura, Y. Watanabe, T. Takita, S. Innami, Accelerating effect of chitosan intake on urinary calcium excretion by rats, Biosci. Biotech. Biochem. 61 (1997) 1206
1208. [25] K. Deuchi, O. Kanauchi, M. Shizukuish, E. Kobayashi, Continuous and massive intake of chitosan affects mineral and fat-soluble vitamin status in rats fed on a highfat diet, Biosci. Biotech. Biochem. 59 (1995) 1211
1216. [26] E. Salleh, I. Muhamad, N. Khairuddin, Preparation, characterization and antimicrobial analysis of antimicrobial starch-based film incorporated with chitosan and lauric acid, Asian Chitin J. 3 (2007) 55
68. [27] T. Jin, H. Zhang, Biodegradable polylactic acid polymer with nisin for use in antimicrobial food packaging, J. Food Sci. 73 (3) (2008) 127
134. [28] M. Barikani, E. Oliaei, H. Seddiqi, H. Honarkar, Preparation and application of chitin and its derivatives: a review, Iran Polym. J. 23 (2014) 307
326.

Handbook of Chitin and Chitosan

References

195

[29] C. Sethulekshmi, Chitin and its benefits, Int. J. Adv. Res. Biol. Sci. 1 (2014) 171
175. [30] E.M. El-Diasty, Z. Nesreen, A.M. Hoda, Using of chitosan as antifungal agent in Kariesh Cheese, NY Sci. J. 5 (2012) 5
10. [31] A.B. Vishu Kumar, M.C. Varadaraj, L.R. Gowda, R.N. Tharanathan, Characterization of chitooligosaccharides prepared by chitosanolysis with the aid of papain and pronase, and their bactericidal action, Biochem. J. 391 (2005) 167
175. [32] I.M. Helander, E.L. Nurmiaho-Lassila, R. Ahvenainen, J. Rhoades, S. Roller, Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria, Int. J. Food Microbiol. 71 (2001) 235
244. [33] S. Chen, G. Wu, H. Zeng, Preparation of high antimicrobial activity thiourea chitosaneAg 1 complex, Carbohydr. Polym. 60 (2005) 33
38. [34] E. Muraki, S. Aiba, Antibacterial activity of partially N-Lauroyl chitooligosaccharides. Proceedings from the 2nd Asia Pacific Chitin Synposium, Bangkok, pp 283
285, 1996. [35] K. Kurita, Chitin and chitosan: functional biopolymers from Marine crustaceans, Mar. Biotechnol. 8 (2006) 203
226. [36] Y. Jeon, F. Shahidi, S. Kim, Preparation of chitin and chitosan oligomers and their applications in physiological functional foods, Food Rev. Int. 16 (2) (2000) 159
176. [37] G.R. Allan, L.A. Hadwiger, The fungicidal effect of chitosan on fungi of varying cell wall composition, Exp. Mycol. 3 (1979) 285
287. [38] S. Hirano, N. Nagao, Effects of chitosan, pectic acid, lysozyme, and chitinase on the growth of several phytopathogens, Agric. Biol. Chem. 53 (1989) 3065
3066. [39] D.F. Kendra, L.A. Hadwiger, Characterization of the smallest chitosan oligomer that is maximally antifungal toFusarium solani and elicits pisatin formation in Pisum sativum, Exp. Mycol. 8 (1984) 276
281. [40] Y. Uchida, M. Izume, A. Ohtakara, Preparation of chitosan oligomers with purified chitosanase and its application, in: G. Skjak-Braek, T. Anthonsen, P. Sandford (Eds.), Chitin and Chitosan: Sources, Chemistry, Biochemistry, Physical Properties and Applications, Elsevier, London, 1989, pp. 373
382. [41] K. Ueno, T. Yamaguchi, N. Sakairi, N. Nishi, S. Tokura, Antimicrobial activity by fractionated chitosan oligomers, in: A. Domard, G.A.F. Roberts, K.M. Varum (Eds.), Advances in Chitin Science, vol. II, Jacques Andre Publisher, Paris, 1997, pp. 156
161. [42] H. Oh, Y. Kim, E. Chang, J. Kim, Antimicrobial characteristics of chitosans against food spoilage microorganisms in liquid media and mayonnaise, Biosci. Biotechnol. Biochem. 65 (11) (2001) 2378
2383. [43] S. Zivanovic, C.C. Basurto, S. Chi, P.M. Davidson, J. Weiss, Molecular weight of chitosan influences antimicrobial activity in oil-in-water emulsions, Food Protect. 67 (5) (2004) 952
959. [44] C. Qin, H. Li, Q. Xiao, Y. Liu, J. Zhu, Y. Du, Water-solubility of chitosan and its antimicrobial activity, Carbohydr. Polym. 63 (3) (2006) 367
374. [45] Y.C. Chung, Y.H. Li, C.C. Chen, Pollutant removal from aquaculture wastewater using the biopolymer chitosan at different molecular weights, Environ. Sci. Health A 40 (2005) 1775
1790. [46] B. Miralles, A. Martı´nez-Rodrı´guez, A. Santiago, J. van de Lagemaat, A. Heras, The occurrence of a Maillard-type protein-polysaccharide reaction between [beta]-lactoglobulin and chitosan, Food Chem. 100 (3) (2007) 1071
1075. [47] F. Khoushab, M. Yamabhai, Chitin research revisited, Mar. Drugs 8 (2010) 1988
2012.

Handbook of Chitin and Chitosan

196

6. Functional properties of chitin and chitosan-based polymer materials

[48] X. Liu, L. Yun, Z. Dong, L. Zhi, D. Kang, Antibacterial action of chitosan and carboxymethylated chitosan, J. Appl. Polym. Sci. 79 (7) (2001) 1324
1335. [49] D.V. Gerasimenko, I.D. Avdienko, G.E. Bannikova, O.Y. Zueva, V.P. Varlamov, Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms, Appl. Biochem. Microbiol. 40 (3) (2004) 253
257. [50] K. Kim, R. Thomas, C. Lee, H. Park, Antimicrobial activity of native chitosan, degraded chitosan, and O-carboxymethylated chitosan, J. Food Protect. 66 (2003) 1495
1498. [51] P.J. Park, J.Y. Je, H.G. Byun, S.H. Moon, S.K. Kim, Antimicrobial activity of heterochitosans and their oligosaccharides with different molecular weights, J. Microbiol. Biotechnol. 14 (2) (2004) 317
323. [52] R.A.A. Muzzarelli, C. Muzzarelli, Chitosan chemistry: relevance to the biomedical sciences, Adv. Polym. Sci. 186 (2005) 151
209. [53] S. Sinha, S. Chand, P. Tripathi, Microbial degradation of chitin waste for production of chitosanase and food related bioactive compounds, Appl. Biochem. Microbiol. 50 (2014) 125
133. [54] J. Dutta, S. Tripathi, P.K. Dutta, Progress in antimicrobial activities of chitin, chitosan and its oligosaccharides: a systematic study needs for food applications, Food Sci. Technol. Int. 18 (1) (2012) 3
34. [55] G. Maleki, N. Sedaghat, E.J. Woltering, M. Farhoodi, M. Mohebbi, Chitosanlimonene coating in combination with modified atmosphere packaging preserve postharvest quality of cucumber during storage, J. Food Meas. Charac. 12 (2018) 1610
1621. [56] H.J. Park, Y.J. Byun, Y.T. Kim, W.S. Whiteside, H.J. Bae, Processes and applications for edible coating and film materials from agropolymers, in: J.H. Han (Ed.), Innovations in Food Packaging, Academic Press, London, 2013, pp. 257
275. [57] P. Schulz, M. Rodrı´guez, L. Del Blanco, M. Pistonesi, E. Agullo´, Emulsification properties of chitosan, Coll. Polym. Sci. 276 (12) (1998) 1159
1165. [58] M.S. Rodriguez, L.A. Albertengo, E. Agullo, Emulsification capacity of chitosan, Carbohydr. Polym. 48 (3) (2002) 271
276. [59] S. Laplante, S.L. Turgeon, P. Paquin, Emulsion stabilizing properties of various chitosans in the presence of whey protein isolate, Carbohydr. Polym. 59 (4) (2005) 425
434. [60] T. Aoki, E. Decker, D. McClements, Influence of environmental stresses on the stability of O/W emulsions containing droplets stabilized by multilayered membranes produced by a layer-by-layer electrostatic deposition technique, Food Hydrocolloid. 19 (2) (2005) 209
220. [61] S. Mun, E.A. Decker, D.J. McClements, Effect of molecular weight and degree of deacetylation of chitosan on the formation of oil-inwater emulsions stabilized by surfactant-chitosan membranes, J. Colloid Interface Sci. 296 (2) (2006) 581
590. [62] Y.I. Cho, H.K. No, S.P. Meyers, Physicochemical characteristics and functional properties of various commercial chitin and chitosan products, J. Agric. Food Chem. 46 (9) (1998) 3839
3843. [63] D. Knorr, Functional properties of chitin and chitosan, J. Food Sci. 47 (1982) 593
595. [64] S.H. Lee, Effect of chitosan on emulsifying capacity of egg yolk, J. Korean Soc. Food Nutr. 25 (1996) 118
122. [65] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603
632. [66] R.A.A. Muzzarelli, N. Frega, M. Miliani, C. Muzzarelli, M. Cartolari, Interactions of chitin, chitosan, N-lauryl chitosan and N-dimethylaminopropyl chitosan with olive oil, Carbohydr. Polym. 43 (2000) 263
268.

Handbook of Chitin and Chitosan

References

197

[67] V.G. Babak, M. Rinaudo, Physico-chemical properties of chitin-surfactant complexes, in: R.A.A. Muzzarelli, C. Muzzarelli (Eds.), Chitosan in Pharmacy and Chemistry, ATEC, Grottammare, Italy, 2002, pp. 277
284. [68] J. Desbrie`res, M. Rinaudo, Interactions between chitin derivatives and surfactants, in: M.A. El-Nokaly, H.A. Soini (Eds.), Polysaccharide Applications: Cosmetics and Pharmaceuticals. ACS Symposium Series 737, pp. 199
213, 1999. [69] M. Rinaudo, R. Auzely, C. Vallin, I. Mullagaliev, Specific interactions in modified chitosan systems, Biomacromolecules 6 (2005) 2396
2407. [70] G. Kerch, The potential of chitosan and its derivatives in prevention and treatment of age-related diseases, Mar. Drugs 13 (2015) 2158
2182. [71] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. Structure, properties and applications, Mar. Drugs 13 (2015) 1133
1174. [72] J.Y. Je, P.J. Park, S.K. Kim, Free radical scavenging properties of heterochitooligosaccharides using an ESR spectroscopy, Food Chem. Toxicol. 42 (3) (2004) 381
387. [73] Y. Yang, R. Shu, J. Shao, G. Xu, X. Gu, Radical scavenging activity of chitooligosaccharide with different molecular weights, Eur. Food Res. Technol. 222 (1) (2006) 36
40. [74] P.J. Chien, F. Sheu, W.T. Huang, M.S. Su, Effect of molecular weight of chitosans on their antioxidative activities in apple juice, Food Chem. 102 (4) (2007) 1192
1198. [75] J.Y.V.A. Kamil, Y.J. Jeon, F. Shahidi, Antioxidative activity of chitosans of different viscosity in cooked comminuted flesh of herring (Clupea harengus), Food Chem. 79 (2002) 69
77. [76] H.Y. Lin, C.C. Chou, Antioxidative activities of water soluble disaccharides of chitosan derivatives, Food Res. Int. 37 (2004) 883
889. [77] K. Kim, R. Thomas, Antioxidative activity of chitosans with varying molecular weights, Food Chem. 101 (2007) 308
313. [78] P. Morganti, G. Fabrizi, P. Palombo, M. Palombo, E. Ruocco, A. Cardillo, et al., Chitin-nanofibrils: a new active cosmetic carrier, J. Appl. Cosmetol. 26 (2008) 113
128. [79] M.A. Shannon, P.W. Bohn, M. Elimelech, J.G. Georgiadis, B.J. Marinas, A.M. Mayes, Science and technology for water purification in the coming decades, Nature 452 (7185) (2008) 301
310. [80] V.H. Dao, N.R. Cameron, K. Saito, Synthesis, properties and performance of organic polymers employed in flocculation applications, Polym. Chem. 7 (1) (2016) 11
25. [81] J.Q. Jiang, The role of coagulation in water treatment, Curr. Opin. Chem. Eng. 8 (2015) 36
44. [82] G.Y. Chen, L. Zhao, Y. Qi, Y. Cui, Chitosan and its derivatives applied in harvesting microalgae for biodiesel production: an outlook, J. Nanomater. 2014 (4) (2014) 1
9. [83] C. Wan, M.A. Alam, X.Q. Zhao, X.Y. Zhang, S.L. Guo, S.H. Ho, et al., Current progress and future prospect of microalgal biomass harvest using various flocculation technologies, Bioresour. Technol. 184 (2015) 251
257. [84] F. Renault, B. Sancey, P.M. Badot, G. Crini, Chitosan for coagulation/flocculation processes-an eco-friendly approach, Eur. Polym. J. 45 (2009) 1337
1348. [85] K.G.R. Nair, P. Madhavan, Chitosan for removal of mercury from water, Fish. Tech. 21 (1984) 109
112. [86] C. Peniche-covas, L.W. Alwarez, W. Arguelles-Monal, The adsorption of mercuric ions by chitosan, J. Appl. Polym. Sci. 46 (1987) 1147
1150. [87] N. Jha, I. Leela, A.V.S. Prabhakar Rao, Removal of cadmium using chitosan, J. Environ. Eng. 114 (1988) 962
974.

Handbook of Chitin and Chitosan

198

6. Functional properties of chitin and chitosan-based polymer materials

[88] M. Weltrowski, B. Martel, M. Norcellet, Chitosan N-benzyl sulfonate derivatives as sorbents for removal of metal ions in an acidic medium, J. Appl. Polym. Sci. 59 (1996) 647
654. [89] D.W. Kang, H.K. Choi, D.K. Kweon, Selective adsorption capacity for metal ions of amidoximated chitosan bead-g-PAN copolymer, Polymer (Korea) 20 (1996) 989
995. [90] R. Maruca, B.J. Suder, J.P. Wightmen, Interaction of heavy metals with chitin and chitosan III chromium, J. Appl. Polym. Sci. 27 (1982) 4827
4837. [91] T. Sakiyama, C.H. Chu, T. Fuji, T. Yano, Preparation of polyelectrolyte complex gel from chitosan and k-carrageenan and its pH sensitivity swelling, J. Appl. Polym. Sci. 50 (1993) 2021
2025. [92] A. Apocella, B. Cappello, M.A. Del Nobile, M.I. Larotonda, G. Mensitieri, L. Nicolao, Poly (ethylene oxide) PEO and different molecular weight PEO blends as monolithic devices for drug release, Biomaterials 14 (1993) 83
90.

Handbook of Chitin and Chitosan

C H A P T E R

7 Fundamentals of chitosan for biomedical applications Mohammad Rahat Hossain, Abul K. Mallik and Mohammed Mizanur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

O U T L I N E 7.1 Introduction

200

7.2 Processing of chitosan

201

7.3 Properties and limitations of chitosan

205

7.4 Modifications and modified form of chitosan in biomedical application 7.4.1 Modification methods 7.4.2 Modified forms

206 207 209

7.5 Biomedical application of chitosan and its derivatives 7.5.1 Tissue engineering 7.5.2 Drug delivery 7.5.3 Wound healing 7.5.4 Regenerative medicine 7.5.5 Gene therapy 7.5.6 Biosensing 7.5.7 Other applications

212 212 217 218 219 219 220 221

7.6 Conclusion

221

References

221

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00007-8

199

© 2020 Elsevier Inc. All rights reserved.

200

7. Fundamentals of chitosan for biomedical applications

7.1 Introduction Chitosan is the partial or fully deacetylated form of chitin, a linear semicrystalline polymer and the most naturally abundant polysaccharide after cellulose [1,2]. It was first reported by Rouget in 1859 [3]. It is a copolymer composed of N-acetyl-D-glucosamine and D-glucosamine units found in different degree of acetylated moieties, generally composed of less than 20% β-(1,4)-2-acetamido-D-glucopyranose and more than 80% β-(1,4)-2-amino-Dglucopyranose [4]. The molecular structure of chitosan comprises one amino group and two hydroxyl groups in a repeating glycoside residue and happens to be one of the major cationic polymers found in nature [5] (Fig. 7.1). When the fraction of glucosamine units rises above 50% then it is known as chitosan and the number of glucosamine units is then known as the degree of deacetylation (DD) [6]. The solubility of the parent chitin is much less, in fact it is nearly insoluble, to that of chitosan in most of the common solvents. When the pH of the solvent gets above 7.0, chitosan tends to be insoluble in its crystalline nature [7]. But chitosan shows good solubility properties below pH 6.0 as the amino groups gets quaternized and turn into positively charged

O1´ C1´ O5 N2´

C2´ C5´

O6´ C6´

C3´

C4´ ψ´ O1

O3´

φ C1 O5

C2

N2

C5 C3

C4

O6 χ

C6

O3

FIGURE 7.1 Chemical structure of chitosan. Individual atoms are numbered. Dashed lines denote O3
O5 hydrogen bonds. Two dihedral angles (ϕ, ψ) defining the main chain conformation and one dihedral angle χ defining the O6 orientation are indicated. Source: Reprinted from P. Agrawal, G.J. Strijkers, K. Nicolay, Chitosan-based systems for molecular imaging. Adv. Drug Deliv. Rev. 62 (2010) 42
58. https://doi.org/10.1016/j.addr.2009.09.007 with the permission from Elsevier.

Handbook of Chitin and Chitosan

7.2 Processing of chitosan

201

polyelectrolytes. Yi et al. showed the variable nature of chitosan with respect to the pH of the solvents [8]. This transition in nature in terms of solubility occurs at pKa values of pH 6
6.5 and the pKa value is related to the DD of chitosan. One study shows that molecular weight can also affect the properties of chitosan [9]. However, due to its low solubility in most common solvents, chitosan offers limited application. To diversify its application, chitosan can undergo some modifications on the backbone of its molecular structure. The molecular structure facilitates these modification methods due to the presence of the functional groups [10]. After such modifications through chemical modification methods, physical interactions (inorganic composites and polyelectrolyte complexes), and other methods such as the use of avidin
biotin interaction, it presents itself as a potential candidate for different applications especially in the biomedical field. The intrinsic properties of chitosan such as biocompatibility, biodegradability [11,12], antimicrobial activity [13], anticancer activity [14], wound-healing capability [15], controlled drug delivery [16], gene therapy [17], biosensing capability [18], blood anticoagulant activity, and many more, has drawn much attention toward itself for use as scaffold material in tissue engineering [19], membranes [20], nanoparticles [21], beads, nanofibers [22], hydrogel [23], and other forms. Besides, all of the hope and its reputation in biomedical fields, it is also successfully being explored in other industrial fields like pulp and paper, cosmetics, and wastewater treatment. Chitosan has been generally formed from chitin from two marine crustaceans’ sources: shrimps and crabs [24]. The shells of these two sources are removed before food processing and packaging. Chitin is then obtained from the shells, followed by some chemical or biological processes to obtain chitosan. Chitin can also be found in the internal skeleton of squid and cuttlefish and the exoskeleton of insects. But these sources are not considered as major sources for chitosan [25]. However, this chapter will focus only on the fundamentals of chitosan and also some of its derivatives in biomedical applications by reviewing associated research works.

7.2 Processing of chitosan Chitosan is mainly extracted from crab and shrimp shells. In industrial processing, this polymer is treated with acid, followed by an alkaline treatment to remove calcium carbonates and proteins, respectively. Sometimes, decolorization and purification steps are added to the process to eliminate pigments and other impurities [26]. The process which involves the removal of calcium carbonates is known as decalcification or the demineralization process, and the process

Handbook of Chitin and Chitosan

202

7. Fundamentals of chitosan for biomedical applications

which removes proteins is known as the deproteinization process. The decolorization process mainly eliminates pigments like astaxanthin and β-carotene by using various solvents, such as sodium hypochlorite, acetone, and hydrogen peroxide [27]. Deacetylation is usually performed to obtain chitosan from chitin (Fig. 7.2). The demineralization process is treated with dilute hydrochloric acid (HCl) solution. This reacts with exoskeleton components, mainly calcium carbonate and chloride, to give off CO2 , the quantity of which basically indicates the amount of inorganic compound present. Then the obtained material is further filtered and washed and then dried in an oven overnight. Then the dried material undergoes alkaline treatment using a dilute NaOH solution to operate a deproteinization step. After some time, the material is then washed off to remove excess amounts of used NaOH and dried to obtain a product named chitin. The resulting chitin then undergoes a deacetylation step to obtain chitosan by removing acetyl groups. This reaction proceeds at an elevated temperature in the presence of concentrated sodium hydroxide (NaOH) solution. Then the purification step is performed to bring it to neutrality and obtain the desired chitosan without any impurities. The extraction process can be classified into two categories, which are chemical method and biological method. The chemical method involves the following advantages: 1. Short processing time 2. High DD% in final product 3. Obtain a product of completely free from organic salts It shows some disadvantageous effects too. As it involves the use of hazardous strong acid and bases, it is not an environment-friendly method. The other perspective is that the solubilized mineral and protein cannot also be used as a nutrient either. That is why more and more attention has been drawn to biological methods in recent years. This biological method involves the use of lactic acid
producing bacteria and proteases from bacteria for the demineralization and deproteinization steps, respectively. Chitin deacetylase is used for deacetylation in an enzymatic method [28]. Commercially, chitosan has a DDA range of 70%
95% and a molecular weight of 104
106 g/mol [29]. However, there are plenty of studies being undertaken on the extraction process of chitosan of different origins by these methods. An extraction process of chitosan from Catharsius molossus residue was described [30]. For the demineralization process 1.3 M of HCl solution at 80 C and for the deproteinization process 4.0 M of NaOH solution at 90 C were used for about 30 min and 6 h, respectively. Then the obtained chitin was soaked in 18 M of NaOH solution at room temperature for 24 h and dried at 90 C for 7 h.

Handbook of Chitin and Chitosan

CH2

CH2

Demineralization alkali treatment

NH HO O

O OH

NH O HO

OH

O

OH

O

Demineralization acid treatment

Chitosan

Chitin

CH2

O

HO O

O O

NH OH

O

Deacetylation alkali treatment

NH2 HO O

O

O HO

NH2

O O

O

HO O

NH2 OH

OH

CH2

FIGURE 7.2 Extraction process of chitin and chitosan. Source: Reprinted from H. El Knidri, R. Belaabed, A. Addaou, A. Laajeb, A. Lahsini, Extraction, chemical modification and characterization of chitin and chitosan, Int. J. Biol. Macromol. 120 (2018) 1181
1189. https://doi.org/10.1016/j.ijbiomac.2018.08.139 with the permission from Elsevier.

204

7. Fundamentals of chitosan for biomedical applications

Kumari et al. described the extraction process of chitosan from fish scales [27]. In this process, 1.0 M of HCl solution was used for the demineralization step for about 15
180 min and 0.5 N NaOH was used for the deproteinization step for 18 h at ambient temperature. Then the purified chitin was dried at 110 C in an oven. The deacetylation of chitin is done by steeping in strong NaOH at room temperature before heating to reduce the duration of deacetylation step which previously took about 20 h. The whole process is given in Fig. 7.3. Srinivasan et al. described an extraction method of chitosan by the biological method from shrimp Litopenaeus vannamei wastes [31]. In this case, lactic acid was used for demineralization for 36 h at 21 C and an enzymatic hydrolysis with viscozyme and alcalase was carried out for the removal of protein. The deacetylation step was treatment with 40% of NaOH solution for 4 h at 110 C. Rao et al. described a fermentation process of shrimp waste by Lactobacillus plantarum 541 with and without pH control [32]. There were four acids tested (glacial acetic, citric, hydrochloric, and lactic acids) and among them acetic and citric acid showed better results. About 75% deproteinization and 86% demineralization of biowaste was achieved at pH 6.0 in the presence of glucose. But in case of no pH control about 68.1% deproteinization and 64.1% and demineralization was achieved.

FIGURE 7.3 Overall processes for preparation of chitosan from fish scales. Source: Reprinted from S. Kumari, P. Rath, A. Sri Hari Kumar, T.N. Tiwari, Extraction and characterization of chitin and chitosan from fishery waste by chemical method. Environ. Technol. Innov. 3 (2015) 77
85. https://doi.org/10.1016/j.eti.2015.01.002 with the permission from Elsevier.

Handbook of Chitin and Chitosan

7.3 Properties and limitations of chitosan

205

A good comparative analysis was carried out by Khanafari and Marandi who described the extraction method of chitosan by both chemical and microbial methods, and the obtained results showed a better result for the microbial method than the chemical method of extraction [33].

7.3 Properties and limitations of chitosan Chitosan is a biocompatible [34] and natural polymer [35], that is safe, nontoxic [36], and biodegradable [4]. Chitosan has many physicochemical as well as biological properties. The physicochemical properties include solubility, reactivity, adsorption, and crystallinity, whereas the biological properties include biodegradability, antimicrobial activity, cytocompatibilty, lack of toxicity, fungicidal effects, hemostatic action, and woundhealing stimulation [37,38]. It also possesses a chelating effect to bind metal ions, proteins, tumor cells, fat, cholesterol, etc. Furthermore, the luxury of showing anticholestemic activity, antioxidant activity, macrophage activation, antiinflammatory activity, angiogenesis stimulation, macrophage activation, mucoadhesion, antitumor, granulation, and scar formulation ability makes it a potential candidate for numerous applications in agricultural, environmental, and biomedical fields [39]. The structure of chitosan makes it more susceptible to chemical activity as it has reactive amino groups, a linear polyamine structure, and available reactive hydroxyl groups. Biodegradation is a required property since it is very pivotal in drug delivery and tissue regeneration applications. As the DD decreases, the rate of biodegradation increases [40] and the degree of crystallinity decreases. So, the DD should be controlled to attain the required properties of chitosan. However, there is also an effect of the distribution of acetyl and acetamide groups and the length of chains. Kofuji et al. showed the homogeneous distribution of acetyl groups reduces the rate of enzymatic degradation [41]. In another study, Shigemasa et al. showed the effect of randomly distributed acetamide groups on chitin and chitosan obtained from squid pen and shrimp shell [42]. The aligned molecular structure of crab tendon makes it more mechanically strong. This crab tendon basically comprises chitin and the deacetylated form was found by treating it with 50% NaOH solution at 100 C followed by ethanol treatment. After the process, the obtained chitosan retained its tensile strength and was further enhanced by treating with heat at 120 C [43]. On the other hand, the DD of chitosan also affects the viscosity of the chitosan and unbranched highmolecular-weight chitosan works as an efficient viscosity enhancing agent in an acidic environment [16]. Moreover, crystallinity shows its maximum appearance at 0% DD which is chitin and 100% DD which is chitosan [44]. The viscosity also is attributed to different biomedical

Handbook of Chitin and Chitosan

206

7. Fundamentals of chitosan for biomedical applications

applications such as wound healing and osteogenesis enhancement. In the presence of available amino groups, the interaction between cell and chitosan increases with the increase of DD of chitosan which eventually contributes to cell adhesion and proliferation [16]. Antimicrobial activity of chitosan was first found with fungi by Allan and Hadwiger [45]. Since then there are glut of reports that have been abridged in a number of research papers. Nishimura et al. showed in a study that chitosan with 70% DD can perform successfully against Escherichia coli and Sendai virus [46]. However, the activity of chitosan happens to be affected by the degree of disaccharides substitution and the types of disaccharides present in the molecule [47]. The strong electronic charge contributes to the anticancer activity of chitosan [48]. Although the molecular mechanism of chitosan is still unclear in terms of antitumor activity, numerous studies have been going on in vivo and in vitro [4]. The interaction between positively charged chitosan and cell membrane proceeds with the rearrangement of the tight junction associated proteins, and appears to be instrumental for considering chitosan as a potential permeation enhancer [49]. Howling et al. described the wound-healing properties of chitosan by reporting the capability to stimulate fibroblast production with the help of the fibroblast growth factor [50]. It has also been used as a construction material for tissue scaffolds due to its biodegradability and porous structure as well as its low immunogenic activity [51]. However, there are some major disadvantages of chitosan which force the scientist to modify its structure for the better performance of chitosan in many diversified field of applications. The main limitation is the lesser solubility of chitosan in water. That brings out a limited field to operate with chitosan. That is why extensive investigations are being carried out to enhance the solubility, as well as other properties like targeting specific tissues, controlling drug delivery, improving antibacterial activity, anticancer activity, and antimicrobial efficacy, in addition to attaining the desired characteristics by experimenting with modifications on the backbone of chitosan’s structure. The obtained results from those experiments are also awakening hopes to explore further in biomedical and other fields. Some modification methods will be also discussed in a later part of this chapter.

7.4 Modifications and modified form of chitosan in biomedical application This section of the chapter will highlight on different modification methods which have been applied on the backbone of the chitosan structure to impart the desired property and to provide the different forms which will be eventually used for major applications in the biomedical field.

Handbook of Chitin and Chitosan

7.4 Modifications and modified form of chitosan in biomedical application

207

7.4.1 Modification methods As we stated earlier, chitosan has some unique properties which make it a potential candidate for many application but due to its lack of solubility in most commonly used solvents, it is most likely to offer much lower efficiency in those fields of application. Most of the commercial applications, however, remain covered by the unmodified forms of chitosan. Much modified chitosan has already been in operation and there are many that are reported or proposed to explore. These modified chitosans have been formed using some common modification methods or techniques such as self-assembly, sol
gel method, ultrasonication, microcontact printing. Nanocarriers of chitosan have been prepared by different methods, such as ionic gelation, mini emulsion, and spray drying methods, but the most advantageous one is molecular self-assembly as it provides the organized molecular structure of the desired functional group without any additional modification steps. Self-assembly methods are divided into two categories: 1. Monocomponent systems, in which only the molecules from chitosan are involved in self-assembly. 2. Multicomponent systems, in which other molecules along with chitosan can participate [52] Self-assembly of molecules has been affected in a good way by hydrogen bonds, ionic bonds [53], hydrophobic interaction [53], and van der Waals interaction of molecules. The combined effect of these forces becomes the driving force for self-assembly [52]. In a study, a bionanocomposite film was prepared from chitosan and montmorillonite hybrid blocks by the self-assembly method [54]. Sol
gel is another popular method for the modification of chitosan. Novel hybrid macroporous scaffolds were prepared by foaming a mixture of polymer solution and bioactive glass with a sol
gel precursor solution. Blending PVA solution with chitosan and undergoing reaction with bioactive glass reagents was also done to investigate bioartificial polymeric hybrids through sol
gel methods. The obtained hybrid showed better mechanical, morphological, and cell viability properties nonetheless [55]. In another study, chopped silk fibers and electrospun silk fibers were introduced into chitosan/glycerophosphate by gelation techniques to make scaffolds for hyaline cartilage regeneration [56]. Chitosan and silica nanocomposites were also prepared by the sol
gel method to adsorb vanadium, molybdenum, and chromium oxonions from aqueous solution [57]. As a potential wound dressing, a chitosan membrane was prepared by phase inversion method of immersion
precipitation. In this

Handbook of Chitin and Chitosan

208

7. Fundamentals of chitosan for biomedical applications

experiment, a solution of acetic acid (0.5 wt.%) and NaOH (2 wt. %)-Na2 CO3 (0.05 wt.%) was used as solvent and nonsolvent, respectively. The preparation was performed by using a casting process. The thickness of the skin surface and the porosity of the sponge-like sublayer were controlled by perevaporation at 50 C for 10
15 min. This obtained asymmetric membrane performed with excellent antimicrobial activity along with the permeation of oxygen and fluid drainage ability. This membrane is also beneficial to control evaporative water loss. This modification method of chitosan is responsible for a porous cellular structure of the membrane. The preparation and structure of the membrane is shown in Fig. 7.4 [58] Miguel et al. describes some preparation techniques of asymmetric membrane including dry/wet phase inversion method, scCO2-assisted phase inversion, electrospinning, and bioprinting. The authors mentioned the wet phase inversion and scCO2-assisted phase inversion method as a simple technique and green technique, respectively. Bioprinting techniques provide the customized and exact required tissue engineered constructs, whereas the membranes obtained from electrospinning method are dry and solvent-free and more importantly several polymers can be used as well [59].

(A)

Alkaline medium

Coagulation tank

Chitosan solution

(B)

Skin top-layer (dense)

Sponge-like sublayer (macroporous)

FIGURE 7.4 Preparation and structure of asymmetric membrane (A) preparation of asymmetric membrane (B) structure of asymmetric membrane. Source: Reprinted from F.L. Mi, S.S. Shyu, Y.B. Wu, S.T. Lee, J.Y. Shyong, R.N. Huang, Fabrication and characterization of a sponge-like asymmetric chitosan membrane as a wound dressing. Biomaterials, 2001. https://doi. org/10.1016/S0142-9612(00)00167-8 with the permission from Elsevier.

Handbook of Chitin and Chitosan

7.4 Modifications and modified form of chitosan in biomedical application

209

Microcontact printing is a technique for the fabrication of nanostructured macromolecules, such as dendrimers, peptides, and conducting polymers [60]. This method of modification, however, can run alongside other modification methods simultaneously and the nanofibers of many ranges can be obtained. Recently, mechanical treatment method of modification has also drawn attention for the fabrication of nanofibers [61].

7.4.2 Modified forms Chitosan is the only cationic polysaccharide which is obtained from biomass [62]. But due to its limitations regarding reactivity and processability, some modification of chitosan has been done over the years. However, there are many forms of chitosan which are processed for different fields of application. In biomedical fields, chitosan is generally processed into membranes, scaffolds, nanoparticles, hydrogels, nanofibers, etc. In this section, these different forms of chitosan and modified chitosan will be discussed. 7.4.2.1 Scaffolds The biodegradable material by which cells, genes, and proteins are delivered in terms of tissue engineering is known as a scaffold [63]. The functions that a scaffold provide are listed below: 1. Structural stability and integrity 2. Restructured design during proliferation of donor cells and growth of the host tissue 3. Controlled diffusion of the gas and nutrients between parenchymal cells 4. Controlled behavior of the cell within by maintaining mechanical forces occurred in tissue 5. Sufficient regrouping and nurturing of engineered tissue [64] In a study, Madihally and Matthew reported that the sponge form facilitates the scaffold structure the most [7]. There are many ways to form scaffolds. However, there are three prolific points which need to be taken into consideration in terms of scaffold design [65]. First is the surface of a scaffold. Sometimes it is necessary to modify the scaffold surface to enhance the cell interaction. There has been much research work in the past focusing on the modification method of the surface. And still there are many works going on. Recently in a study, the surface of chitosan films was modified with a fibronectin fragment
DNA aptamer complex [66]. The result showed much better osteoblastic cell activity and facilitated a better understand of protein biological activity on cell proliferation. Secondly, it is essential to control the release of growth factors from scaffolds. In a recent work, it is reported that the conventional incorporation of growth factors into scaffolds actually limited activity such as

Handbook of Chitin and Chitosan

210

7. Fundamentals of chitosan for biomedical applications

vasculogenesis, proliferation, and differentiation, etc. on a long span of time. There was a proposed modification that involved the scaffold being conjugated with an anti-His-tag antibody which enables the scaffolds to be reloaded after some time once again in vivo and in vitro, and also provides the controlled release of required growth factors. The report also justified its statement by releasing angiogenic factors in a static cell culture or under flow in a microfluidics device and on endothelial cells [67]. The third point described the fact of controlled and sound mass transportation through scaffolds. Diffusivity and permeability defines mass transportation in terms of scaffolds. Oxygen diffusion to cells is very pivotal because partial oxygen pressure affects cell differentiation [68]. The mentioned properties of chitosan are just admirable in this regard to form a scaffold and serve the purposes. 7.4.2.2 Nanomaterials Nanomaterials have drawn much more attention as they have great potential to be used as biomedical materials. Nanoparticles, nanogel, nanofibers, nanoceramics, nanocrystals, and nanocomposites are the forms of nanomaterials. These forms are often used in tissue engineering, gene therapy, drug delivery, cancer diagnosis, antimicrobial application, bioimaging applications, etc. Tissue engineering works with damaged tissue by remodeling it either through regeneration, transplantation, or treatment with living cells either in vivo or in vitro. That is why the materials to be used should facilitate cell proliferation, cell differentiation, cell migration, and cell adhesion. Chitosan and its modified forms meet that demand quite brilliantly and perform better in the field of tissue engineering [69]. Nanomaterials are frequently used in drug delivery systems. A drug delivery system would deliver the drug to the targeted site or cell in a controlled facile way. And this control is done by combining drugs with a substrate with biological, mechanical, and certain chemical properties [70]. The unique properties of chitosan make it a proper candidate for this purpose as well. Different forms of nanomaterial are prepared by blending chitosan with a substrate to provide the desired activity or enhance the existing one [71]. One study reported that CTS-g-lactic acid/sodium montmorillonite nanocomposite hybrids showed improved mechanical modulus, strength, and the ibuprofen drug release rate [72]. In the case of gene therapy, the insufficient cellular uptake with DNA instability causes the main drawback in this field. But when chitosan is applied to solve such problems, it is evident that chitosan not only protects DNA but also transports it to targeted cells [73]. One report suggested that chitosan shows lower transfection efficiency than other viral vectors, which justifies its use in this field [74].

Handbook of Chitin and Chitosan

7.4 Modifications and modified form of chitosan in biomedical application

211

7.4.2.3 Hydrogels Hydrogels are soft materials which are able to swell in water or biological fluids and generally composed of three-dimensional networks of hydrophilic polymers [75]. It can be of two types: 1. Physical gels: the chains of polymers are held together by cross-links, H-bond, etc. 2. Chemical gels: the chains are held together by covalent bonds. They are also called “smart materials” as they are sensitive to external milieu such as pH, light, temperature, etc. and the controlled release of drug and growth factors can be done accordingly. The swelling property allows diffusion and permeation as well [76]. There have been plenty of studies focusing on the chitosan-based hydrogels as smart biomaterials because of the unique properties of chitosan [23,77]. Drug delivery, tissue engineering, etc. are the field of application of hydrogels. Recently, the application of chitosan-based injectable ionic hydrogel is reported to be efficient in inducing a high temperature for microwave tumor ablation [78]. 7.4.2.4 Membranes Membranes, in particular asymmetric membranes, have become a very promising approach in terms of wound healing. The structural similarities between membranes and the dermal and epidermal layers provide an extra edge to membranes over other approaches. Moreover, the external layer of a membrane protects the wound from the external adverse environment including bacterial infection, microorganism attacks, etc. The internal porous layer provides the required cell proliferation, cell adhesion, and cell migration as well. There are many techniques employed to prepare membranes, such as electrospinning, phase inversion, and bioprinting. The biocompatibility, antibacterial activity, hemostaticity, and healing properties of chitosan makes it more preeminent in the field of wound healing, skin tissue regeneration, etc. [59]. A very recent study reported a novel technique to prepare a biologically inspired asymmetric topological chitosan (ATCS) membrane which is supported by a nanoporous anodic aluminum oxide (AAO) template to facilitate the biocompatibility of membrane. The study also compared this membrane with a symmetric chitosan membrane and the analogy showed that the asymmetric one had better tensile strength but the symmetric one possessed a greater extent of degradation. However, in vitro studies the asymmetric one showed superiority in terms of cytocompatibility. The asymmetric chitosan membrane showed better mechanical performances as well [79].

Handbook of Chitin and Chitosan

212

7. Fundamentals of chitosan for biomedical applications

7.5 Biomedical application of chitosan and its derivatives This segment of the chapter will discuss the diverse applications of chitosan and its derivatives which have been of use in various biomedical fields, such as tissue engineering, drug delivery, and wound healing.

7.5.1 Tissue engineering 7.5.1.1 Cartilage repair In recent years, when tissue engineering is operated for the repair of articular cartilage, it proceeds with the detachment of either articular chondrocytes or their precursor cells [80]. There has been so much work going on regarding this approach as the choice of biomaterial is critical to the justification of each one. The ideal one should be the one which can mimic the environment of the cartilage matrix. However, chitosan has been studied as a contributor in this regard in several research works over the years and shows some excellent efficiency as well. Suh and Matthew looked at the suitable mechanical properties which arise from the pore size and pore orientation of chitosan scaffolds [80]. Zhao et al. described its suitable analogous structures to GAGs that happen to stimulate the bioprocesses accordingly and are also involved in the synthesis of chondroitin sulfate, hyaluronic acid, and type II collagen. However, they acknowledged some of the drawbacks as well [81]. Moreira Teixeira et al. prepared a chitosan (CHT)/poly(3-caprolactone) (PCL) blend three-dimensional (3D) fiber-mesh scaffold as a possible support structure for articular cartilage. The scaffolds were prepared from microfibers of different polymeric solutions mainly of three types: 100:0 (100CHT), 75:25 (75CHT), and 50:50 (50CHT) wt.% CHT/PCL. This PCLmodified chitosan allows a higher fiber surface roughness to be achieved and also controls the swelling ratio, thus enhancing the mechanical properties. He studied a comparative analysis between those solutions and the SEM analysis implies the cell attachment, proliferation, and metabolic activity of each scaffold formulation which was carried by wet-spinning method [82]. In another study, Liu et al. showed chitosan in its hydrogel form as a scaffold for chondrocytes to repair articular cartilage imperfections. They prepared the hydrogels by adding chitosan with β-sodium glycerophosphate (GP) and hydroxy-ethyl cellulose (HEC) which showed satisfactory results for both in vivo and in vitro techniques. They concluded that this hydrogel could support matrix accumulation and could become a reliable technique for repairing articular cartilage defects [83]. Neethu et al. also studied another chitosan-based hydrogel which was prepared by combining chitosan with hyaluronic acid dialdehyde. They approached this experiment in two ways: one solely with gel and another

Handbook of Chitin and Chitosan

7.5 Biomedical application of chitosan and its derivatives

213

with gel encapsulated in chondrocytes. However, both the approaches showed similar results [84]. Articular cartilage has a limited repair capability and that’s why attention has been drawn to reconstruction of the cartilage recently [56]. Mirahmadi and Tafazzoli-shadpour modified hydrogels with two different types of silk fibers which reveal better mechanical properties and also suggests it as a possible solution to this problem [56]. 7.5.1.2 Bone tissue engineering Recent tissue engineering research is based on the seeding of cells onto the porous biodegradable polymer matrixes and that is why it is so important to focus on the availability of good biomaterials to serve as a temporary matrix [85]. When the biodegradable substitute gets incorporated into the targeted area to fix, it gives support as well as an environment to regenerate bone tissue [86]. The justification of using chitosan in bone tissue engineering is done by its competence to hold up mineral-rich matrix deposition by osteoblast and also its osteoconduction, biocompatibility, and biodegradability [87]. The reactive primary amines and primary and secondary hydroxyl groups present on chitosan are responsible for the addition of side groups, peptides, or amino acids, which ease the fictionalization for chitosan [88]. The innate cationic nature induces the electrostatic interaction with other anionic molecules [85]. As pH gets lower, chitosan tends to form gel [16], which is why it is used as an adjuvant with bone cement to increase its injectability without affecting the setting time much [89]. However, calcium phosphate
chitosan composite were fabricated and investigated as a scaffold for bone tissue engineering by Zhang et al. [90] and the mechanical property of the ceramic phase was seen to be enhanced via matrix reinforcement without affecting the osteoblast phenotype. Wen et al. constructed a novel iron foam-based calcium phosphate
chitosan as a coating for a biodegradable scaffold material which allows increasing the bioactive property through electrophoretic deposition and afterwards converting it into a phosphate buffer solution (PBS) [91]. Xu and Simon developed calcium phosphate cement scaffolds by incorporating chitosan and mannitol. The obtained scaffolds showed an in situ self-hardening ability, better porosity, better mechanical property, and fast setting ability [19]. Maji et al. prepared a gelatin
chitosan
nanobioglass 3D porous scaffold for the purpose of bone tissue engineering. He prepared 58 S bioactive glass by the sol
gel method and the gelatin chitosan bioglass was prepared by using this synthesized 58 S bioactive glass of varying amount. This whole process was done by the freeze-drying method and the gelatin was collected from Sigma Aldrich (USA) to carry out this experiment. The obtained scaffold was characterized by particle size analysis using dynamic light scattering (DLS), X-ray diffraction (XRD) analysis, Fourier-transform infrared spectroscopy (FTIR)

Handbook of Chitin and Chitosan

214

7. Fundamentals of chitosan for biomedical applications

analysis, scanning electron microscopic (SEM) analysis, compression test, swelling index, porosity test, and of course in vitro biodegradability. In this case, the obtained scaffold showed about 80% porosity, higher compressive strength, proliferation, adhesion, and a quick capability to convert into artificial bone grafts [92,93]. Novel biodegradable chitosan
gelatin scaffolds were prepared for alveolar bone tissue treatment. Pulieri et al. found a micro-molding method was suitable for tissue reconstruction when they were carrying out the blending of chitosan and gelatin of different compositions [94]. A hybrid polymer network using β-tricalcium phosphate (β-TCP) with chitosan and gelatin was developed by Yin et al. through a freeze-drying method which was found to be a scaffold of improved compressive modulus and yield strength with good biocompatibility [95]. Kavya et al. modified pure chitosan and chitosan
gelatin scaffolds by incorporating nano-SiO2 which showed much improved characterization properties such as mechanical integrity, cell adhesion, proliferation, and differentiation than chitosan and chitosan
gelatin scaffolds [96]. Hydroxyapatite is considered to be the main mineral constituent of natural bones [69]. The nanohydroxyapatite forms natural nanocomposite when it is incorporated in the collagen triple helix [97]. Zhao et al. developed a 3D biomimetic hydroxyapatite/chitosan
gelatin composite scaffold by phase separation method and they focused on the 3D spacing for seeding osteoblastic cells in vitro which resulted in improved proliferation, differentiation, and an approachable way to produce osteoinductive material to repair bone defects [98]. However, in case of in vivo biodegradation of chitosan
hydroxyapetite composite material, it has been found to show osteoconductive properties as well and can easily be replaced by newly formed bone tissue [99]. Carboxymethyl chitosan also shows good prospects as a scaffold material as it degrades when new tissue forms with a reduced effect of inflammation and toxic degradation [100]. Carboxymethyl also forms injectable gels with gelatin in the presence of nanohydroxyapatite susceptible to tyrosinase/p-cresol for in situ treatment of irregular bone defects [69]. A rheological study with FTIR analysis showed the degree of cross-linking and strength as a function of concentration of carboxymethyl and its application on mice also suggested a great deal of dependency in terms of stability on concentration and cross-linking property. Liuyun et al. prepared a novel composite scaffold made of from nanohydroxyapatite, chitosan, and carboxymethyl cellulose which is named as n-HA/CS/CMC by the freeze-dying method. They studied the properties by infrared absorption spectra (IR), transmission electron microscope, SEM, universal material testing machine, PBS soaking experiment, MG63 cells, and mesenchymal stem cells (MSCs) culture experiment in vitro, and reported the scaffold to be biocompatible and nontoxic. In the case of in vivo treatment, they found the same biocompatibility for tissue and proposed it to be a potential scaffold for bone tissue

Handbook of Chitin and Chitosan

7.5 Biomedical application of chitosan and its derivatives

215

engineering [101]. Yang et al. developed hydrogels made of gelatin and carboxymethyl chitosan which were fabricated by radiation-induced cross-linking and a study showed its controllable biodegradability [77]. Vitro studied the progress of hydrogel in regard to its gelatination mechanism and suitability. He showed its compatibility with BMSC and he concluded CH
GP
HEC hydrogel to be a suitable injectable delivery vehicle for in vitro bone tissue engineering [23]. 7.5.1.3 Skin tissue engineering Skin tissue engineering is obviously a branch of tissue engineering but recently it has captured more interest as the number of treatments using chitosan has increased. Because of its good biodegradability, it is efficient enough to form a porous scaffold which can be a viable method to work as a medium for tissue engineering [7]. There has been also some modification work done in the recent past. Dhandayuthapani et al. introduced gelatin into chitosan by an electrospinning method to form nanofibers which are eventually used as a scaffold. In this work, some characterization testing was done by SEM and FTIR to see the morphology as well as the comparability with natural human skin; the outcome was quite good [22]. This method was further confirmed [102]. The researchers formed the chitosan
gelatin scaffold by a freezing and lyophilizing method and claimed it to be an ideal skin substitute by showing its in vitro fibroblast proliferation and cocultivation with keratinocytes. Peng et al. worked with a composite nano-TiO2 -chitosan with collagen artificial skin and showed it to be a promising artificial skin substitute indicating the unique bactericidal effect of nano-TiO2 and immuneenhancing effect of chitosan [103]. Qian et al. also worked with a sponge of chitosan and sodium alginate and found successful application on skin tissue engineering [104]. Boucard et al. [105] worked to form a hydrogel of chitosan which depicts the possibility of using it in vivo as it promotes skin generation quite successfully. 7.5.1.4 Liver tissue engineering The requirement for new therapies for acute and chronic liver has immensely increased due to the insufficient donor organs for orthotropic liver transplantation [106]. In the fields of tissue engineering for fulminant hepatic failure (FHF), a bioartificial (BAL) liver is a convenient method. The patient plasma is circulated extracorporeally through a bioreactor, in which are found metabolically active liver cells, in order to develop a BAL device. The proper choice of cell sources is essential and the primary hepatocytes of cells are more suitable for BAL devices. As such many research works have been developing BAL devices which efficiently allow the hepatocyte to carry out many activities [86]. The BAL devices require a suitable ECM for hepatocytes culture. However, the selection of chitosan

Handbook of Chitin and Chitosan

216

7. Fundamentals of chitosan for biomedical applications

as a scaffold material for hepatocytes culture is due to the structural similarity to the GAGs—a component of the liver ECM [107]. Wang et al. prepared a collagen/chitosan matrix (CCM) which was made by a cross-linking method using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in N-hydroxysuccinimide (NHS) and a 2-morpholinoethane sulfonic acid (MES) buffer system. The CCM matrix showed a moderate mechanical strength, good hepatocyte compatibility, and excellent blood compatibility [108]. However, an implantable bioartificial liver (IBL) has severe complexities, since thrombus formation can lead to occlusion and decrease membrane efficiency [86]. A superior blood compatible chitosan/ collagen/heparin matrix in IBL applications was subsequently developed by Wang et al. An ammonia-treated CCM was also prepared and it showed good cytocompatibilty and enhanced biostability [109]. Dvir-Ginzberg et al. worked with a porous alginate scaffold in which he showed a better cell seeding capability of hepatocytes, eventually resulting in increased cell density. The hydrophilic nature and pore structure also contributes to the use of alginate matrices as scaffolds [110]. Multivalent galactose residues can also be used as another strategy in liver tissue engineering due to its ability to bind the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes [86]. Li et al. prepared a fructose modified surface for the culture of hepatocytes by a lyophilization method. It shows an enhancing effect on liver-specific metabolic activities and also improves cell density to a satisfactory level [111]. 7.5.1.5 Nerve tissue engineering As mature neurons do not undergo cell division, they possess little capacity for cell replication. That is why they complicate successful rehabilitation in the case of nerve injury. So it is very difficult to treat an impaired nervous system and as a consequence it affects other parts of the body [112]. By directing the regenerative nerve fibers into the proper endoneurial tubes the repair of nerve lesions has been going on. Strategies for nerve repair have been classified into two categories: (1) bridging—includes grafting and tubulization techniques; (2) end-to-end suturing of the nerve stumps. The former techniques were more effective because of less tension across the repair site [113]. Various kinds of artificial structures have been suggested for repair nerve injuries but there isn’t enough internal space to cohere the nerve fibers and Schwann cell (SCs). To solve the issue it has been found that the artificial tubes should contain a biodegradable matrix to shorten the nerve gap and facilitate the SCs and neuritis migration by providing a framework [86]. Many studies have showed that chitosan is a candidate as a material for nerve repair because of its inherent properties such as biocompatibility, biodegradability, antitumor, and antibacterial activity [114,115].

Handbook of Chitin and Chitosan

7.5 Biomedical application of chitosan and its derivatives

217

Saderi et al. prepared gold nanoparticle-doped electrospun PCL/chitosan nanofibrous scaffolds, since nanofibrous scaffolds could be a promising candidate for this cause. The gold nanoparticles possess unique physicochemical properties which increases the conductivity of scaffold to increase electrical signal transfer between neural cells. In this experiment, AuNPs were doped into a PCL/chitosan matrix, and improved cell attachment and higher proliferation was obtained to suggest it to be a candidate for peripheral nerve regeneration [116]. Salehi et al. used alginate/chitosan (alg/chit) hydrogel for the transplantation of olfactory ectomesenchymal stem cells (OE-MSCs) for peripheral nerve regeneration [117]. Recently, Singh et al. prepared clinically relevant aNGCs comprising of an antioxidant polyurethane (PUAO) conduit which is filled with aligned chitosan
gelatin (CG) cryogel filler for peripheral nerve regeneration and it was found it was compatible for large gaps providing different cues in a single device [118].

7.5.2 Drug delivery Chitosan has become a very good carrier of numerous active agents in the field of drug delivery in recent years [119]. In the forms of hydrogel system, drug conjugate, biodegradable release system, and PEC for many components, chitosan is being used in various biomedical applications such as gene therapy and bioimaging. Also, the chitosan-based system is much more fruitful in the delivery of proteins/peptides, growth factors, antibiotics, and antiinflammatory drugs [16]. Ahn et al. worked with a conventional inflammation-reducing drug, triamcinolone acetonide (TAA) [C24 H31 FO6 ], in a chitosan
polyacrylic acid (PAA) membrane that resulted in an efficient transmucosal drug delivery system. The system was found to be pH responsive [20]. Incorporation of chitosan microspheres into indomethacin offers a sustainable release pattern for indomethacin [120]. The preparation of this chitosan-based system can be done only by using aqueous solvents. Another study shows that the incorporation of chitosan microspheres in betamethasone disodium phosphate (C22 H28 FNa2 O8 P) offers good drug stability (,1% hydrolysis product), high entrapment efficiency (95%), and positive surface charge (37.5 mV) [121]. Here, the larger the betamethasone amount, the greater the yield and size particle, and, on the other hand, the larger the microspheres, the lower the zeta potential and density of particles. In a very recent work, Barbosa et al. prepared fucodian/chitosan nanoparticles for the delivery of methotrexate which is found to have a good skin inflammatory potential [122]. In case of anticancer drugs, Azab et al. prepared a chitosan-based hydrogel with 131I-norcholesterol (131I-NC) and investigated its efficiency in a breast cancer xenograft mouse model. The result was found to be very

Handbook of Chitin and Chitosan

218

7. Fundamentals of chitosan for biomedical applications

significant, preventing about 69% of tumor recurrence and spreading. Also, there was systemic distribution of the radioisotope after implantation of hydrogel [14]. One study reported a salicylic acid grafted chitosan oligosaccharide nanoparticle delivered paclitaxel quite efficiently also [21]. Recently, chitosan-coated magnetic nanoparticles, such as chitosan
citric acid-Fe3 O4 -CUR quartets, have been successfully investigated in terms of an appropriate drug delivery method and the findings are really impressive as they show better and controlled release of CUR. It also contributes to the improvement of therapeutic agents for chemotherapy agents and proves to be useful as an MRI contrast agent for early cancer diagnosis [123]. In another case, Shariatinia and Mazloom-Jalali justified the chitosan nanocomposite system containing graphene as an ifosfamide anticancer drug delivery system by using molecular dynamic simulations [71]. Hadjianfar et al. proposed polycaprolactone/chitosan blend nanofibers to be an appreciable way to deliver 5-flurouracil or generally an anticancer drug delivery system [124]. Yadav et al. further explored by upgrading the anticancer activity of chitosan
curcumin nanoparticles with computational methodologies like molecular docking, BBD-RSM, and MD simulation for the polymer section [125]. An examination was performed to treat the inner ear by using a chitosan drug delivery system which was loaded with a very small amount of a drug named neomycin. The delivery system was operated in the inner ear across the “round window membrane” (RWM) and the loaded system was injected into cavity of the middle ear of albino guinea pigs (n 5 35). The obtained result was satisfactory enough and showed the successful release of neomycin with a concentration-dependent ototoxic effect on the region of cochlear hair cells. That is why it was concluded as a possible antibiotic of treating inner ears [126].

7.5.3 Wound healing To be an ideal dressing it should heal as well as protect the wound from bacterial infection. Various chitosan-based materials have been used in such a cause, as they heal on their own without much scarring. They provide enhanced vascularization and chitooligomers seem to be implicated in better collagen fibril incorporation into extracellular matrix. The chitosan hydrogels can also deliver a therapeutic payload to the location which is an added advantage with respect to other material dressings. Kratz et al. investigated the effect of heparin/chitosan complexes on the stimulation of reepithelialization of wound on human skin in an in vitro model. The obtained result was significant but the effectiveness depends only on the concentration of heparin in heparin
chitosan gel [127]. Ishihara et al. also worked with photocross-linkable chitosan hydrogel in the presence of UV-irradiation and observed an accelerated healing of full

Handbook of Chitin and Chitosan

7.5 Biomedical application of chitosan and its derivatives

219

thick wound [128]. It is further developed by introducing fibroblast growth factor-2 by Obara et al. to have a more accelerated healing process [15]. Azad et al. prepared chitosan membrane with a 75% DD and thickness of 10 μm and the progress in wound healing was justified by the comparison with clinical and histological examination. The result showed a better reepithelialiszation and regeneration of the granular layer. The membrane was used in nonmesh or mesh form [129].

7.5.4 Regenerative medicine Development of modern science and technology has made a huge progression in the field of regenerative medicine [130]. The regenerative medicine is composed of cell, nutrient, and scaffolds [131]. The basic requirement of scaffolds is the following: 1. Materials should be biocompatible and easy to be molded into different shapes. 2. Based on the tissue structure and metabolic demands, cell interaction with the material should be characterized. 3. Performance of matrices should be evaluated by means of quantitative molecular and histological assays. Cell therapy, tissue engineering, and gene therapy suits chitosan for being used as a biomaterial for scaffold for its innate properties [130]. In another case, Lahiji et al. proposed a novel formulation of nanoparticles of chitosan and chondroitin sulfate for controlled release of nutrient-like platelet lysates. It is found that the interaction between polysaccharides and proteins, which are entrapped, shows exact similarities with chondroitin and proteins in native ECM [132]. Silva et al. worked with a membrane composed of chitosan and Aloe vera and the blended membrane showed satisfactory properties like degradation, roughness, and mechanical strength. It was also found to have excellent antibacterial proficiency and it showed good cell compatibility with primary human dermal fibroblasts in vitro [133].

7.5.5 Gene therapy Low transfection efficiency of naked acid injection in vitro and in vivo is a major issue and for that reason various nucleic acid deliveries have been investigated. However, the chitosan-based delivery system is one which has stood out in terms of nonviral gene therapy [13]. The orthodox polygalactosamine shows some reluctance compared to chitosan
DNA complexes. Though it is very easy to synthesize, it possesses a lower transfection efficiency [134]. On the other hand, the stability of this complex depends on several factors, for example, the chain

Handbook of Chitin and Chitosan

220

7. Fundamentals of chitosan for biomedical applications

length and quantity of chitosan. Larger amounts of chitosan provide greater stability and the larger chain length provides greater control of the ability of the polyplex-delivering therapeutic gene vectors to cells [17]. Masotti et al. prepared spherical nanoparticles of chitosan
DNA by incorporating 30% DNA to increase release time [135]. In one study, Dowaidar et al. worked with a cell-penetrating peptide which is conjugated with chitosan-modified iron oxide nanoparticles to enhance gene delivery of oligonucleotide [136]. In another study, Baghdan et al. prepared lipid-coated chitosan
DNA nanoparticles by ionic gelation technique. The chitosan, ultrapure and low molecular weight, was cross-linked by polyanionic tripolyphosphate to better entrap the plasmid DNA into the nanoparticles. The novel developed lipochitoplexes provided better pDNA protection and lower cytotoxicity. The transfection efficiency was increased as well [137]. Wu et al. prepared a multifunctional gene delivery nanovector made of a chitosan backbone and polyethylenimine (PEI) arms. Branched PEI was 2000 Da, and was used to sustain a balance between biocompatibility and transfection efficiency. The arm was attached to arginine
glycine
aspartate (RGD)/twin-arginine translocation (TAT) conjugated via polyethylene glycol (PEG). These RGD/TAT peptides provided increased target ability and cellular uptake. The characterization was done by FTIR and 1HNMR. The biocompatibility, condensation capability of DNA, and excellent gene transfection efficiency made it a potential candidate as a multifunctional copolymer in tumor therapy [138]. In another case, PEI/chitosan-TBA copolymers were used in hypoxia inducible bidirectional shRNA expression for colorectal cancer gene therapy [139]. Liu et al. worked with chitosan graft poly (L-lysine) dendrons to deliver MMP-9shRNA plasmid to HNE-1 cells for gene therapy. It showed higher bioavailability, biocompatibility, and excellent gene transfection as well. These characteristics made it a potential and viable candidate for application in gene therapy to tumors [140]. A very recent work has been done by Jaiswal et al. on methyl methacrylate-modified chitosan (CSMMA). The study was done in mammalian cancer cell lines (A549, HeLa, HepG2). The obtained CSMMA has been found to be a good gene delivery agent because of its excellent transfection efficiency [141].

7.5.6 Biosensing Chitosan has become an exemplary polymer recently because of its good biodegradability and biocompatibility properties, and it is gaining more and more attention in the field of biosensing. For example, glucose biosensing was performed with graphene/AuNPs/chitosan nanocomposite films by Shan et al. [142]. There have been so many studies going on regarding

Handbook of Chitin and Chitosan

References

221

the application of modified chitosan in biosensing. Chitosan-wrapped carbon nanotubes have found potential use in the biosensing of immobilized glucose oxide without any reagent and electrochemistry [18]. The study was earlier influenced by introducing p-benzoquinone [143]. In another study, a bioactive electrode, made of chitosan in which TiO2 is dispersed, showed improved surface porosity and current voltage properties. The study also suggested that there had been an increased resistance for charge transfer due to immobilization of HRP on CS/ TiO2 . Recently, Abdelhamid et al. [144] have worked with chitosanmodified quantum dots in the presence of hydrogen peroxide for selective biosensing of Staphylococcus aureus and found rapid biosensing for its innate enzymatic characteristics.

7.5.7 Other applications Chitosan has gained much more attention in recent years as a functional biopolymer in many biomedical fields, such as bioimaging, antibacterial agents, and targeted drug delivery. The inclusion of imaging agents such as Fe3 O4 for MRI into self-assembled nanoparticle enhances hepatocyte targeted imaging [145].

7.6 Conclusion Due to the inherent properties of chitosan, it can be used in biomedical applications, wastewater treatment, pulp and paper industry, and so on. However, some limitations of chitosan are also very evident. That is why modification of the structure of chitosan has gained much more attention in the recent past. In this chapter, some modification methods and some frequently used forms of chitosan have been discussed. Application in different biomedical fields along with the properties justifies the fundamental potential of chitosan being used as one of the most promising biomaterials in the field. There is the hope and belief that in the future it will become a very useful biomaterial and the abundant sources and unique properties promise that as well.

References [1] W.W. Thein-Han, R.D.K. Misra, Biomimetic chitosan
nanohydroxyapatite composite scaffolds for bone tissue engineering, Acta Biomater. 5 (2008) 1182
1197. Available from: https://doi.org/10.1016/j.actbio.2008.11.025. [2] L. Hu, X. Meng, R. Xing, S. Liu, X. Chen, Y. Qin, et al., Design, synthesis and antimicrobial activity of 6-N-substituted chitosan derivatives, Bioorg. Med. Chem. Lett 26 (2016) 4548
4551. Available from: https://doi.org/10.1016/j.bmcl.2015.08.047.

Handbook of Chitin and Chitosan

222

7. Fundamentals of chitosan for biomedical applications

[3] V. Dodane, V.D. Vilivalam, Vale´rie-1998-pharmaceutical appli.pdf 1. ,https://doi. org/10.1016/S1461-5347(98)00059-5., 1998. [4] P. Zou, X. Yang, J. Wang, Y. Li, H. Yu, Y. Zhang, et al., Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides, Food Chem. 190 (2016) 1174
1181. Available from: https://doi.org/10.1016/j.foodchem.2015.06.076. [5] P. Agrawal, G.J. Strijkers, K. Nicolay, Chitosan-based systems for molecular imaging, Adv. Drug Deliv. Rev. 62 (2010) 42
58. Available from: https://doi.org/10.1016/j. addr.2009.09.007. [6] M. Dornish, D. Kaplan, O. Skaugrud, Standards and guidelines for biopolymers in tissueengineered medical products: ASTM alginate and chitosan standard guides. American Society for Testing and Materials, Ann. N. Y. Acad. Sci., 944, 2001, pp. 388
397. [7] S.V. Madihally, H.W.T. Matthew, Porous chitosan scaffolds for tissue engineering, Biomaterials 20 (1999) 1133
1142. Available from: https://doi.org/10.1016/S01429612(99)00011-3. [8] H. Yi, L.Q. Wu, W.E. Bentley, R. Ghodssi, G.W. Rubloff, J.N. Culver, et al., Biofabrication with chitosan, Biomacromolecules 6 (2005) 2881
2894. Available from: https://doi.org/ 10.1021/bm050410l. [9] H. Zhang, S.H. Neau, In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation, Biomaterials 22 (2001) 1653
1658. Available from: https://doi.org/10.1016/S0142-9612(00)00326-4. [10] V.K. Mourya, N.N. Inamdar, Chitosan-modifications and applications: opportunities galore, React. Funct. Polym. 68 (2008). Available from: https://doi.org/10.1016/j. reactfunctpolym.2008.03.002. [11] E.R.S. Kenawy, M.M. Azaam, K.M. Saad-Allah, Synthesis and antimicrobial activity of α-aminophosphonates containing chitosan moiety, Arab. J. Chem 8 (2015) 427
432. Available from: https://doi.org/10.1016/j.arabjc.2013.12.029. [12] M.N.V.R. Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A.J. Domb, Chitosan chemistry and pharmaceutical perspectives, Chem. Rev. 104 (2004) 6017
6084. Available from: https://doi.org/10.1021/cr030441b. [13] S.A. Agnihotri, N.N. Mallikarjuna, T.M. Aminabhavi, Recent advances on chitosanbased micro- and nanoparticles in drug delivery, J. Control. Release 100 (2004) 5
28. Available from: https://doi.org/10.1016/j.jconrel.2004.08.010. [14] A.K. Azab, J. Kleinstern, V. Doviner, B. Orkin, M. Srebnik, A. Nissan, et al., Prevention of tumor recurrence and distant metastasis formation in a breast cancer mouse model by biodegradable implant of 131I-norcholesterol, J. Control. Release 123 (2007) 116
122. Available from: https://doi.org/10.1016/j.jconrel.2007.07.014. [15] K. Obara, M. Ishihara, T. Ishizuka, M. Fujita, Y. Ozeki, T. Maehara, et al., Photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2 stimulates wound healing in healing-impaired db/db mice, Biomaterials 24 (2003) 3437
3444. Available from: https://doi.org/10.1016/S0142-9612(03)00220-5. [16] M. Dash, F. Chiellini, R.M. Ottenbrite, E. Chiellini, Chitosan—a versatile semisynthetic polymer in biomedical applications, Prog. Polym. Sci. 36 (2011) 981
1014. Available from: https://doi.org/10.1016/j.progpolymsci.2011.02.001. [17] S. Mansouri, P. Lavigne, K. Corsi, M. Benderdour, E. Beaumont, J.C. Fernandes, Eur. J. Pharm. Biopharm. 57 (2004) 1
8. Available from: https://doi.org/10.1016/S09396411(03)00155-3. [18] Y. Zhou, H. Yang, H.Y. Chen, Direct electrochemistry and reagentless biosensing of glucose oxidase immobilized on chitosan wrapped single-walled carbon nanotubes, Talanta 76 (2008) 419
423. Available from: https://doi.org/10.1016/j.talanta.2008.03.028. [19] H.H.K. Xu, C.G. Simon, Fast setting calcium phosphate-chitosan scaffold: mechanical properties and biocompatibility, Biomaterials 26 (2005) 1337
1348. Available from: https://doi.org/10.1016/j.biomaterials.2004.04.043.

Handbook of Chitin and Chitosan

References

223

[20] J.S. Ahn, H.K. Choi, C.S. Cho, A novel mucoadhesive polymer prepared by template polymerization of acrylic acid in the presence of chitosan, Biomaterials 22 (2001) 923
928. Available from: https://doi.org/10.1016/S0142-9612(00)00256-8. [21] X.H. Wei, Y.P. Niu, Y.Y. Xu, Y.Z. Du, F.Q. Hu, Hong Yuan, Salicylic acid-grafted chitosan oligosaccharide nanoparticle for paclitaxel delivery, J. Bioact. Compat. Polym. 25 (2010) 319
335. Available from: https://doi.org/10.1177/0883911510363548. [22] B. Dhandayuthapani, U.M. Krishnan, S. Sethuraman, Fabrication and characterization of chitosan-gelatin blend nanofibers for skin tissue engineering, J. Biomed. Mater. Res. Part B Appl. Biomater (2010). Available from: https://doi.org/10.1002/jbm.b.31651. [23] C.I. Vitro, Hydrogels for the culturing 24, 625
638. ,https://doi.org/10.1177/ 0885328208100536., n.d. [24] H. El Knidri, R. Belaabed, A. Addaou, A. Laajeb, A. Lahsini, Extraction, chemical modification and characterization of chitin and chitosan, Int. J. Biol. Macromol. 120 (2018) 1181
1189. Available from: https://doi.org/10.1016/j.ijbiomac.2018.08.139. [25] I. Aranaz, R. Harris, A. Heras, Chitosan amphiphilic derivatives. chemistry and applications, Curr. Org. Chem. 14 (2010) 308
330. Available from: https://doi.org/ 10.2174/138527210790231919. [26] Y.S. Puvvada, S. Vankayalapati, S. Sukhavasi, Extraction of chitin from chitosan from exoskeleton of shrimp for application in the pharmaceutical industry, Int. Curr. Pharm. J. 1 (2012) 258
263. Available from: https://doi.org/10.3329/icpj.v1i9.11616. [27] S. Kumari, P. Rath, A. Sri Hari Kumar, T.N. Tiwari, Extraction and characterization of chitin and chitosan from fishery waste by chemical method, Environ. Technol. Innov. 3 (2015) 77
85. Available from: https://doi.org/10.1016/j.eti.2015.01.002. ¨ zogul, J.M. Regenstein, Industrial applications of crustacean by[28] I. Hamed, F. O products (chitin, chitosan, and chitooligosaccharides): a review, Trends Food Sci. Technol. 48 (2016) 40
50. Available from: https://doi.org/10.1016/j.tifs.2015.11.007. [29] C.M. Moura, J.M. de Moura, N.M. de Soares, L.A. Pinto, A. de, Evaluation of molar weight and deacetylation degree of chitosan during chitin deacetylation reaction: used to produce biofilm, Chem. Eng. Process. Process Intensif. 50 (2011) 351
355. Available from: https://doi.org/10.1016/j.cep.2011.03.003. [30] J. Ma, C. Xin, C. Tan, Preparation, physicochemical and pharmaceutical characterization of chitosan from Catharsius molossus residue. Int, J. Biol. Macromol. 80 (2015) 547
556. Available from: https://doi.org/10.1016/j.ijbiomac.2015.07.027. [31] H. Srinivasan, V. Kanayairam, R. Ravichandran, Chitin and chitosan preparation from shrimp shells Penaeus monodon and its human ovarian cancer cell line, PA-1, Int. J. Biol. Macromol. 107 (2018) 662
667. Available from: https://doi.org/10.1016/j. ijbiomac.2017.09.035. [32] M.S. Rao, J. Mun˜oz, W.F. Stevens, Critical factors in chitin production by fermentation of shrimp biowaste, Appl. Microbiol. Biotechnol. 54 (2000) 808
813. Available from: https://doi.org/10.1007/s002530000449. [33] A. Khanafari, R. Marandi, Recovery of chitin and chitosan from shrimp waste by, Iran J. Environ. Healt. 5 (2008) 19
24. [34] R.A.A. Muzzarelli, C. Muzzarelli, Chitosan chemistry: relevance to the biomedical sciences, Adv. Polym. Sci 186 (2005) 151
209. Available from: https://doi.org/10.1007/ b136820. [35] G.D. Mogo¸sanu, A.M. Grumezescu, Natural and synthetic polymers for wounds and burns dressing, Int. J. Pharm 463 (2014) 127
136. Available from: https://doi.org/ 10.1016/j.ijpharm.2013.12.015. [36] A. Hector, S.H. Chotirmall, G.M. Lavelle, B. Mirkovi´c, D. Horan, L. Eichler, et al., Chitinase activation in patients with fungus-associated cystic fibrosis lung disease, J. Allergy Clin. Immunol 138 (2016). Available from: https://doi.org/10.1016/j. jaci.2016.01.031. 1183-1189.e4.

Handbook of Chitin and Chitosan

224

7. Fundamentals of chitosan for biomedical applications

[37] N.H. Foda, H.M. El-Laithy, M.I. Tadros, Implantable biodegradable sponges: effect of interpolymer complex formation of chitosan with gelatin on the release behavior of tramadol hydrochloride, Drug Dev. Ind. Pharm. 33 (2007) 7
17. Available from: https://doi.org/10.1080/03639040600975188. [38] E. Khor, L.Y. Lim, Implantable applications of chitin and chitosan, Biomaterials 24 (2003) 2339
2349. Available from: https://doi.org/10.1016/S0142-9612(03)00026-7. [39] J. Kumirska, M.X. Weinhold, J. Tho¨ming, P. Stepnowski, Biomedical activity of chitin/chitosan based materials- influence of physicochemical properties apart from molecular weight and degree of N-acetylation, Polymers (Basel) 3 (2011) 1875
1901. Available from: https://doi.org/10.3390/polym3041875. [40] S. Hirano, H. Tsuchida, N. Nagao, N-acetylation in chitosan and the rate of its enzymic hydrolysis, Biomaterials 10 (1989) 574
576. Available from: https://doi.org/ 10.1016/0142-9612(89)90066-5. [41] K. Kofuji, C.J. Qian, M. Nishimura, I. Sugiyama, Y. Murata, S. Kawashima, Relationship between physicochemical characteristics and functional properties of chitosan, Eur. Polym. J 41 (2005) 2784
2791. Available from: https://doi.org/ 10.1016/j.eurpolymj.2005.04.041. [42] Y. Shigemasa, K. Saito, H. Sashiwa, H. Saimoto, Enzymatic degradation of chitin and partially deacetylated chitins, Int. J. Biol. Macromol 16 (1994) 43
49. [43] I. Yamaguchi, S. Itoh, M. Suzuki, M. Sakane, A. Osaka, J. Tanaka, The chitosan prepared from crab tendon I: the characterization and the mechanical properties, Biomaterials 24 (2003) 2031
2036. Available from: https://doi.org/10.1016/S0142-9612(02)00633-6. [44] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, et al., Functional characterization of chitin and chitosan, Curr. Chem. Biol. 3 (2009) 203
230. [45] C.R. Allan, L.A. Hadwiger, The fungicidal effect of chitosan on fungi of varying cell wall composition, Exp. Mycol. 3 (1979) 285
287. Available from: https://doi.org/ 10.1016/S0147-5975(79)80054-7. [46] I.C. Nishimura, S. Nishimura, N. Nishi, I. Saiki, S. Tokura, I. Azuma, Immunological activity of chitin, Vaccine 2 (1984) 93
99. [47] T.C. Yang, C.C. Chou, C.F. Li, Antibacterial activity of N-alkylated disaccharide chitosan derivatives, Int. J. Food Microbiol 97 (2005) 237
245. Available from: https:// doi.org/10.1016/S0168-1605(03)00083-7. [48] K.O. Suzuki, T. Mikami, Y. Okawa, A. Tokoro, M. Suzuki. Note antitumor effect of hexaN-acetylchitohexaose and chitohexaose, Carbohydrate research 151 (1986), 403
408. [49] J. Smith, E. Wood, M. Dornish, Effect of chitosan on epithelial cell tight junctions, Pharm. Res. 21 (2004) 43
49. [50] G.I. Howling, P.W. Dettmar, P.A. Goddard, F.C. Hampson, M. Dornish, E.J. Wood, The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro, Biomaterials 22 (2001) 2959
2966. Available from: https://doi. org/10.1016/S0142-9612(01)00042-4. [51] M.H. Ho, D.M. Wang, H.J. Hsieh, H.C. Liu, T.Y. Hsien, J.Y. Lai, et al., Preparation and characterization of RGD-immobilized chitosan scaffolds, Biomaterials 26 (2005) 3197
3206. Available from: https://doi.org/10.1016/j.biomaterials.2004.08.032. [52] Y. Yang, S. Wang, Y. Wang, X. Wang, Q. Wang, M. Chen, Advances in selfassembled chitosan nanomaterials for drug delivery, Biotechnol. Adv. 32 (2014) 1301
1316. Available from: https://doi.org/10.1016/j.biotechadv.2014.07.007. [53] P.J. Rossky, Exploring nanoscale hydrophobic hydration, Faraday Discuss. 146 (2010) 13
18. Available from: https://doi.org/10.1039/c005270c. [54] H. Bin Yao, Z.H. Tan, H.Y. Fang, S.H. Yu, Artificial nacre-like bionanocomposite films from the self-assembly of chitosan-montmorillonite hybrid building blocks, Angew. Chem. Int. Ed 49 (2010) 10127
10131. Available from: https://doi.org/ 10.1002/anie.201004748.

Handbook of Chitin and Chitosan

References

225

[55] H.S. Mansur, H.S. Costa, Nanostructured poly(vinyl alcohol)/bioactive glass and poly(vinyl alcohol)/chitosan/bioactive glass hybrid scaffolds for biomedical applications, Chem. Eng. J. 137 (2008) 72
83. Available from: https://doi.org/10.1016/j. cej.2007.09.036. [56] F. Mirahmadi, M. Tafazzoli-shadpour, Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fi bers for cartilage tissue engineering, Mater. Sci. Eng. C 33 (2013) 4786
4794. Available from: https://doi.org/10.1016/j.msec.2013.07.043. [57] T.M. Budnyak, I.V. Pylypchuk, V.A. Tertykh, E.S. Yanovska, D. Kolodynska, Synthesis and adsorption properties of chitosan-silica nanocomposite prepared by sol-gel method, Nanoscale Res. Lett. 10 (2015) 1
10. Available from: https://doi. org/10.1186/s11671-014-0722-1. [58] F.L. Mi, S.S. Shyu, Y.B. Wu, S.T. Lee, J.Y. Shyong, R.N. Huang, Fabrication and characterization of a sponge-like asymmetric chitosan membrane as a wound dressing, Biomaterials (2001). Available from: https://doi.org/10.1016/S0142-9612(00)00167-8. [59] S.P. Miguel, A.F. Moreira, I.J. Correia, Chitosan based-asymmetric membranes for wound healing: a review, Int. J. Biol. Macromol. 127 (2019) 460
475. Available from: https://doi.org/10.1016/j.ijbiomac.2019.01.072. [60] A.P. Quist, Æ.E. Pavlovic, Oscarsson, Recent advances in microcontact printing, Anal. Bioanal. Chem. 381 (2005) 591
600. Available from: https://doi.org/10.1007/ s00216-004-2847-z. [61] Ifuku shinsuke, Nanoscale. ,https://doi.org/10.1039/c2nr30383c., 2012. [62] E. Vunain, A.K. Mishra, B.B. Mamba, Fundamentals of chitosan for biomedical applications, Chitosan Based Biomater 1 (2016) 3
30. Available from: https://doi.org/ 10.1016/B978-0-08-100230-8.00001-7. [63] L.G. Cima, J.P. Vacanti, C. Vacanti, D. Ingber, D. Mooney, R. Langer, Tissue engineering by cell transplantation using degradable polymer substrates, J. Biomech. Eng 113 (2008) 143. Available from: https://doi.org/10.1115/1.2891228. [64] Vacanti, Langer, Upton, Marler, Transplantation of cells in matrices for tissue regeneration, Adv. Drug Deliv. Rev. 33 (1998) 165
182. [65] S.J. Hollister, Scaffold design and manufacturing: from concept to clinic, Adv. Mater. 21 (2009) 3330
3342. Available from: https://doi.org/10.1002/adma.200802977. [66] M. Saccani, L. Parisi, C. Bergonzi, A. Bianchera, C. Galli, G.M. Macaluso, et al., Surface modification of chitosan films with a fibronectin fragment-DNA aptamer complex to enhance osteoblastic cell activity: a mass spectrometry approach probing evidence on protein behavior, Rapid Commun. Mass Spectrom. 33 (2019) 336
342. Available from: https://doi.org/10.1002/rcm.8335. [67] L.S. Carasso, I. Benhar, T. Dvir, Universal biofactor-releasing scaffold enabling in vivo reloading, Nano Lett. 19 (2019) 1838
1843. Available from: https://doi.org/ 10.1021/acs.nanolett.8b04927. [68] C. Domm, M. Schu¨nke, K. Christesen, B. Kurz, Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension, Osteoarthr. Cartil 10 (2002) 13
22. Available from: https://doi.org/10.1053/joca.2001.0477. [69] D. Mishra, B. Bhunia, I. Banerjee, P. Datta, S. Dhara, T.K. Maiti, Enzymatically crosslinked carboxymethyl-chitosan/gelatin/nano- hydroxyapatite injectable gels for in situ bone tissue engineering application, Mater. Sci. Eng. C 31 (2011) 1295
1304. Available from: https://doi.org/10.1016/j.msec.2011.04.007. [70] A. Bernkop-Schnu¨rch, S. Du¨nnhaupt, Chitosan-based drug delivery systems, Eur. J. Pharm. Biopharm. 81 (2012) 463
469. Available from: https://doi.org/10.1016/j. ejpb.2012.04.007. [71] Z. Shariatinia, A. Mazloom-Jalali, Chitosan nanocomposite drug delivery systems designed for the ifosfamide anticancer drug using molecular dynamics simulations, J. Mol. Liq. 273 (2019) 346
367. Available from: https://doi.org/10.1016/j.molliq.2018.10.047.

Handbook of Chitin and Chitosan

226

7. Fundamentals of chitosan for biomedical applications

[72] D. Depan, A.P. Kumar, R.P. Singh, Cell proliferation and controlled drug release studies of nanohybrids based on chitosan-g-lactic acid and montmorillonite, Acta Biomater. 5 (2009) 93
100. Available from: https://doi.org/10.1016/j. actbio.2008.08.007. [73] K. Annu, Manzoor, S. Ahmad, A. Soundarajan, S. Ikram, S. Ahmed, Chapter 30—chitosan based nanomaterials for biomedical applications, Handbook of Nanomaterials for Industrial Applications, Elsevier Inc, 2018. Available from: https://doi.org/10.1016/ B978-0-12-813351-4.00031-6. [74] B. Layek, J. Singh, Caproic acid grafted chitosan cationic nanocomplexes for enhanced gene delivery: effect of degree of substitution, Int. J. Pharm. 447 (2013) 182
191. Available from: https://doi.org/10.1016/j.ijpharm.2013.02.052. [75] M.K. Lima-Teno´rio, E.A. Go´mez Pineda, N.M. Ahmad, H. Fessi, A. Elaissari, Magnetic nanoparticles: in vivo cancer diagnosis and therapy, Int. J. Pharm 493 (2015) 313
327. Available from: https://doi.org/10.1016/j.ijpharm.2015.07.059. [76] M.C.G. Pella´, M.K. Lima-Teno´rio, E.T. Teno´rio-Neto, M.R. Guilherme, E.C. Muniz, A.F. Rubira, Chitosan-based hydrogels: from preparation to biomedical applications, Carbohydr. Polym. 196 (2018) 233
245. Available from: https://doi.org/10.1016/j. carbpol.2018.05.033. [77] C. Yang, L. Xu, Y. Zhou, X. Zhang, X. Huang, M. Wang, et al., A green fabrication approach of gelatin/CM-chitosan hybrid hydrogel for wound healing, Carbohydr. Polym. 82 (2010) 1297
1305. Available from: https://doi.org/10.1016/j.carbpol.2010.07.013. [78] J. Wang, D. Wang, H. Yan, L. Tao, Y. Wei, Y. Li, et al., An injectable ionic hydrogel inducing high temperature hyperthermia for microwave tumor ablation, J. Mater. Chem. B 5 (2017) 4110
4120. Available from: https://doi.org/10.1039/c7tb00556c. [79] Y. Jiang, Y. Deng, Y. Tu, B. Ay, X. Sun, Y. Li, et al., Chitosan-based asymmetric topological membranes with cell-like features for healthcare applications, J. Mater. Chem. B 7 (2019) 2634
2642. Available from: https://doi.org/10.1039/c8tb03296c. [80] J.F. Suh, H.W.T. Matthew, Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering : a review, Biomaterials 21 (2000). [81] W. Zhao, X. Jin, Y. Cong, Y. Liu, J. Fu, Degradable natural polymer hydrogels for articular cartilage tissue engineering, J. Chem. Technol. Biotechnol. 88 (2013) 327
339. Available from: https://doi.org/10.1002/jctb.3970. [82] L.S. Moreira Teixeira, C.A. Van Blitterswijk, R.L. Reis, N.M. Alves, L. Moroni, M. Karperien, et al., Chitosan/poly(ε-caprolactone) blend scaffolds for cartilage repair, Biomaterials 32 (2010) 1068
1079. Available from: https://doi.org/10.1016/j. biomaterials.2010.09.073. [83] T. Liu, Q. Lin, C. Duan, T. Hao, N. Wen, J. Cao, et al., The support of matrix accumulation and the promotion of sheep articular cartilage defects repair in vivo by chitosan hydrogels, Osteoarthr. Cartil 18 (2010) 257
265. Available from: https://doi.org/ 10.1016/j.joca.2009.08.007. [84] M. Neethu, M. Pv, A. Sabareeswaran, N. Prabha, Chitosan-hyaluronic acid hydrogel for cartilage repair, Int. J. Biol. Macromol. 26 (2017). Available from: https://doi.org/ 10.1016/j.ijbiomac.2017.03.142. [85] N.M. Alves, J.F. Mano, Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications, Int. J. Biol. Macromol. 43 (2008) 401
414. Available from: https://doi.org/10.1016/j.ijbiomac.2008.09.007. [86] I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo, I.Y. Park, B.C. Kim, et al., Chitosan and its derivatives for tissue engineering applications, Biotechnol. Adv. 26 (2008) 1
21. Available from: https://doi.org/10.1016/j.biotechadv.2007.07.009. [87] A. Di Martino, M. Sittinger, M.V. Risbud, Chitosan: a versatile biopolymer for orthopaedic tissue-engineering, Biomaterials 26 (2005) 5983
5990. Available from: https://doi.org/10.1016/j.biomaterials.2005.03.016.

Handbook of Chitin and Chitosan

References

227

[88] S.K.L. Levengood, M. Zhang, Chitosan-based scaffolds for bone tissue engineering, J. Mater. Chem. B 2 (2014) 3161
3184. Available from: https://doi.org/10.1039/ c4tb00027g. [89] L. Leroux, Z. Hatim, M. Fre`che, J.L. Lacout, Effects of various adjuvants (lactic acid, glycerol, and chitosan) on the injectability of a calcium phosphate cement, Bone 25 (1999). Available from: https://doi.org/10.1016/S8756-3282(99)00130-1. [90] Y. Zhang, M. Ni, M. Zhang, B. Ratner, Calcium phosphate—chitosan composite scaffolds for bone tissue engineering, Tissue Eng. 9 (2003) 337
345. Available from: https://doi.org/10.1089/107632703764664800. [91] Z. Wen, L. Zhang, C. Chen, Y. Liu, C. Wu, C. Dai, A construction of novel ironfoam-based calcium phosphate/chitosan coating biodegradable scaffold material, Mater. Sci. Eng. C 33 (2013). Available from: https://doi.org/10.1016/j. msec.2012.10.009. [92] K. Maji, S. Dasgupta, K. Pramanik, A. Bissoyi, Preparation and evaluation of gelatin-chitosan-nanobioglass 3D porous scaffold for bone tissue engineering, Int. J. Biomater (2016) 2016. Available from: https://doi.org/10.1155/2016/9825659. [93] M. Peter, N.S. Binulal, S.V. Nair, N. Selvamurugan, H. Tamura, R. Jayakumar, Novel biodegradable chitosan-gelatin/nano-bioactive glass ceramic composite scaffolds for alveolar bone tissue engineering, Chem. Eng. J. 158 (2010) 353
361. Available from: https://doi.org/10.1016/j.cej.2010.02.003. [94] E. Pulieri, V. Chiono, G. Ciardelli, G. Vozzi, A. Ahluwalia, C. Domenici, et al., Chitosan/gelatin blends for biomedical applications, J. Biomed. Mater. Res. Part A 86 (2008) 311
322. Available from: https://doi.org/10.1002/jbm.a.31492. [95] Y. Yin, F. Ye, J. Cui, F. Zhang, X. Li, K. Yao, Preparation and characterization of macroporous chitosan-gelatin/β -tricalcium phosphate composite scaffolds for bone tissue engineering, J. Biomed. Mater. Res. Part A 67 (2003) 844
855. Available from: https://doi.org/10.1002/jbm.a.10153. [96] K.C. Kavya, R. Jayakumar, S. Nair, K.P. Chennazhi, Fabrication and characterization of chitosan/gelatin/nSiO2composite scaffold for bone tissue engineering, Int. J. Biol. Macromol. 59 (2013) 255
263. Available from: https://doi.org/10.1016/j. ijbiomac.2013.04.023. [97] M.M. Stevens, J.H. George, ,MM Stevens.PDF., 1135
1138, 2005. [98] F. Zhao, Y. Yin, W.W. Lu, J.C. Leong, W. Zhang, J. Zhang, et al., Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosangelatin network composite scaffolds, Biomaterials 23 (2002) 3227
3234. Available from: https://doi.org/10.1016/S0142-9612(02)00077-7. [99] S.N. Danilchenko, O.V. Kalinkevich, M.V. Pogorelov, A.N. Kalinkevich, A.M. Sklyar, T.G. Kalinichenko, et al., Characterization and in vivo evaluation of chitosanhydroxyapatite bone scaffolds made by one step coprecipitation method, J. Biomed. Mater. Res. Part A (2011). Available from: https://doi.org/10.1002/jbm.a.33017. [100] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, Biomedical applications of carboxymethyl chitosans, Carbohydr. Polym. (2013). Available from: https://doi.org/ 10.1016/j.carbpol.2012.07.076. [101] J. Liuyun, L. Yubao, X. Chengdong, Preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering, J. Biomed. Sci. 16 (2009) 1
10. Available from: https://doi. org/10.1186/1423-0127-16-65. [102] J. Mao, L. Zhao, K. De Yao, Q. Shang, G. Yang, Y. Cao, Study of novel chitosangelatin artificial skin in vitro, J. Biomed. Mater. Res. Part A 64 (2003) 301
308. Available from: https://doi.org/10.1002/jbm.a.10223. [103] C.C. Peng, M.H. Yang, W.T. Chiu, C.H. Chiu, C.S. Yang, Y.W. Chen, et al., Composite nano-titanium oxide-chitosan artificial skin exhibits strong wound-healing effect—an

Handbook of Chitin and Chitosan

228

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113] [114]

[115]

[116]

[117]

[118]

7. Fundamentals of chitosan for biomedical applications

approach with anti-inflammatory and bactericidal kinetics, Macromol. Biosci. 8 (2008) 316
327. Available from: https://doi.org/10.1002/mabi.200700188. Z. Qian, M. Dai, X. Zheng, X. Xu, X. Kong, X. Li, et al., Chitosan-alginate sponge: preparation and application in curcumin delivery for dermal wound healing in rat, J. Biomed. Biotechnol (2009). Available from: https://doi.org/10.1155/2009/595126. N. Boucard, C. Viton, D. Agay, E. Mari, T. Roger, Y. Chancerelle, et al., The use of physical hydrogels of chitosan for skin regeneration following third-degree burns, Biomaterials 28 (2007) 3478
3488. Available from: https://doi.org/10.1016/j. biomaterials.2007.04.021. In-Yong Kim, Seog-Jin Seo, Hyun-Seuk Moon, Hu-Lin Jiang, You-Kyoung Kim, Factors affecting liver functions in liver tissue engineering.pdfC.-S.C. Tissue Eng. Regen. Med 3 (2006). 37
33. J. Li, J. Pan, L. Zhang, X. Guo, Y. Yu, Culture of primary rat hepatocytes within porous chitosan scaffolds, J. Biomed. Mater. Res. Part A 67 (2003) 938
943. Available from: https://doi.org/10.1002/jbm.a.10076. X.H. Wang, D.P. Li, W.J. Wang, Q.L. Feng, F.Z. Cui, Y.X. Xu, et al., Crosslinked collagen/chitosan matrix for artificial livers, Biomaterials 24 (2003) 3213
3220. Available from: https://doi.org/10.1016/S0142-9612(03)00170-4. X. Wang, Y. Yan, Z. Xiong, F. Lin, R. Wu, R. Zhang, et al., Preparation and evaluation of ammonia-treated collagen/chitosan matrices for liver tissue engineering, J. Biomed. Mater. Res. Part B Appl. Biomater 75 (2005) 91
98. Available from: https://doi.org/10.1002/jbm.b.30264. M. Dvir-Ginzberg, I. Gamlieli-Bonshtein, R. Agbaria, S. Cohen, Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability, morphology, and function, Tissue Eng. 9 (2003) 757
766. Available from: https://doi.org/10.1089/107632703768247430. J. Li, J. Pan, L. Zhang, Y. Yu, Culture of hepatocytes on fructose-modified chitosan scaffolds, Biomaterials 24 (2003) 2317
2322. Available from: https://doi.org/ 10.1016/S0142-9612(03)00048-6. C.A. Heath, G.E. Rutkowski, The development of bioartificial nerve grafts for peripheral-nerve regeneration, Trends Biotechnol. 16 (1998) 163
168. Available from: https://doi.org/10.1016/S0167-7799(97)01165-7. G. Ciardelli, V. Chiono, Materials for peripheral nerve regeneration, Macromol. Biosci. 6 (2006) 13
26. Available from: https://doi.org/10.1002/mabi.200500151. Z. Yinghui, L. Jianchun, Z. Nanming, G. Haipeng, Z. Xiufang, G. Yandao, Studies on nerve cell affinity of chitosan-derived materials, J. Biomed. Mater. Res. 52 (2002) 285
295. Available from: https://doi.org/10.1002/1097-4636(200011)52:2 , 285::aid-jbm7 . 3.3.co;2-7. Y. Yuan, P. Zhang, Y. Yang, X. Wang, X. Gu, The interaction of Schwann cells with chitosan membranes and fibers in vitro, Biomaterials 25 (2004) 4273
4278. Available from: https://doi.org/10.1016/j.biomaterials.2003.11.029. N. Saderi, M. Rajabi, B. Akbari, M. Firouzi, Z. Hassannejad, Fabrication and characterization of gold nanoparticle-doped electrospun PCL/chitosan nanofibrous scaffolds for nerve tissue engineering, J. Mater. Sci. Mater. Med 29 (2018). Available from: https://doi.org/10.1007/s10856-018-6144-3. M. Salehi, Z. Bagher, S.K. Kamrava, A. Ehterami, R. Alizadeh, M. Farhadi, et al., Alginate/chitosan hydrogel containing olfactory ectomesenchymal stem cells for sciatic nerve tissue engineering, J. Cell. Physiol (2019) 1
12. Available from: https:// doi.org/10.1002/jcp.28183. A. Singh, P.A. Shiekh, M. Das, J. Seppa¨la¨, A. Kumar, Aligned chitosan-gelatin cryogel-filled polyurethane nerve guidance channel for neural tissue engineering: fabrication, characterization, and in vitro evaluation, Biomacromolecules 20 (2019) 662
673. Available from: https://doi.org/10.1021/acs.biomac.8b01308.

Handbook of Chitin and Chitosan

References

229

[119] M. de la Fuente, M. Ravin˜a, P. Paolicelli, A. Sanchez, B. Seijo, M.J. Alonso, Chitosanbased nanostructures: a delivery platform for ocular therapeutics, Adv. Drug Deliv. Rev. 62 (2010) 100
117. Available from: https://doi.org/10.1016/j.addr.2009.11.026. [120] S. Shiraishi, T. Imai, M. Otagiri, Controlled release of indomethacin by chitosanpolyelectrolyte complex: optimization and in vivo/in vitro evaluation, J. Control. Release 25 (1993) 217
225. Available from: https://doi.org/10.1016/0168-3659(93) 90080-O. [121] Y.C. Huang, M.K. Yeh, S.N. Cheng, C.H. Chiang, The characteristics of betamethasoneloaded chitosan microparticles by spray-drying method, J. Microencapsul. 20 (2003) 459
472. Available from: https://doi.org/10.1080/0265204021000058456. [122] A.I. Barbosa, S.A. Costa Lima, S. Reis, Development of methotrexate loaded fucoidan/chitosan nanoparticles with anti-inflammatory potential and enhanced skin permeation, Int. J. Biol. Macromol. 124 (2019) 1115
1122. Available from: https:// doi.org/10.1016/j.ijbiomac.2018.12.014. [123] H. Danafar, F. Asadi, A. Sharafi, H. Manjili, Preparation and evaluation of ph sensitive novel anticancer drug carrier based on magnetic chitosan quartets, Drug Res. (2019). Available from: https://doi.org/10.1055/a-0855-8464. [124] M. Hadjianfar, D. Semnani, J. Varshosaz, Polycaprolactone/chitosan blend nanofibers loaded by 5-fluorouracil: an approach to anticancer drug delivery system, Polym. Adv. Technol. 29 (2018) 2972
2981. Available from: https://doi.org/ 10.1002/pat.4417. [125] P. Yadav, A. Bandyopadhyay, A. Chakraborty, K. Sarkar, Enhancement of anticancer activity and drug delivery of chitosan-curcumin nanoparticle via molecular docking and simulation analysis, Carbohydr. Polym. 182 (2018) 188
198. Available from: https://doi.org/10.1016/j.carbpol.2017.10.102. [126] A. Saber, S.P. Strand, M. Ulfendahl, Use of the biodegradable polymer chitosan as a vehicle for applying drugs to the inner ear, Eur. J. Pharm. Sci. 39 (2010) 110
115. Available from: https://doi.org/10.1016/j.ejps.2009.11.003. [127] G. Kratz, C. Arnander, J. Swedenborg, M. Back, C. Falk, I. Gouda, et al., Heparinchitosan complexes stimulate wound healing in human skin, Scand. J. Plast. Reconstr. Surg. Hand Surg. 31 (1997) 119
123. Available from: https://doi.org/ 10.3109/02844319709085478. [128] M. Ishihara, K. Nakanishi, K. Ono, M. Sato, M. Kikuchi, Y. Saito, et al., Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process, Biomaterials 23 (2002) 833
840. Available from: https://doi.org/ 10.1016/S0142-9612(01)00189-2. [129] A.K. Azad, N. Sermsintham, S. Chandrkrachang, W.F. Stevens, Chitosan membrane as a wound-healing dressing: characterization and clinical application, J. Biomed. Mater. Res. Part B Appl. Biomater. (2004). Available from: https://doi.org/10.1002/ jbm.b.30000. [130] C. Shi, Y. Zhu, X. Ran, M. Wang, Y. Su, T. Cheng, Therapeutic potential of chitosan and its derivatives in regenerative medicine 1 1 this work was supported by “973” programs on severe trauma (no. 1999054205 and no. 2005cb522605) from the Ministry of Science and Technology of China, J. Surg. Res. 133 (2006) 185
192. Available from: https://doi.org/10.1016/j.jss.2005.12.013. [131] Y. Hayashi, S. Yamada, K. Yanagi Guchi, Z. Koyama, T. Ikeda, Chitosan and fish collagen as biomaterials for regenerative medicine, Adv. Food Nutr. Res. (2012). Available from: https://doi.org/10.1016/B978-0-12-416003-3.00006-8. [132] A. Lahiji, A. Sohrabi, D.S. Hungerford, C.G. Frondoza, Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes, J. Biomed. Mater. Res. (2000). Available from: https://doi.org/10.1002/1097-4636 (20000915)51:4 , 586::AID-JBM6 . 3.0.CO;2-S.

Handbook of Chitin and Chitosan

230

7. Fundamentals of chitosan for biomedical applications

[133] S.S. Silva, E.G. Popa, M.E. Gomes, M. Cerqueira, A.P. Marques, S.G. Caridade, et al., An investigation of the potential application of chitosan/aloe-based membranes for regenerative medicine, Acta Biomater. 9 (2013) 6790
6797. Available from: https:// doi.org/10.1016/j.actbio.2013.02.027. [134] M. Prabaharan, J.F. Mano, Chitosan-based particles as controlled drug delivery systems, Drug Deliv. J. Deliv. Target. Ther. Agents 12 (2005) 41
57. Available from: https://doi.org/10.1080/10717540590889781. [135] A. Masotti, F. Bordi, G. Ortaggi, F. Marino, C. Palocci, Nanotechnology (2008) 19. Available from: https://doi.org/10.1088/0957-4484/19/05/055302. ¨ . Langel, X. Zou, Chitosan [136] M. Dowaidar, H. Nasser Abdelhamid, M. Ha¨llbrink, U enhances gene delivery of oligonucleotide complexes with magnetic nanoparticles
cell-penetrating peptide, J. Biomater. Appl. 33 (2018) 392
401. Available from: https://doi.org/10.1177/0885328218796623. [137] E. Baghdan, S.R. Pinnapireddy, B. Strehlow, K.H. Engelhardt, J. Scha¨fer, U. Bakowsky, Lipid coated chitosan-DNA nanoparticles for enhanced gene delivery, Int. J. Pharm 535 (2018) 473
479. Available from: https://doi.org/10.1016/j.ijpharm.2017.11.045. [138] D. Wu, Y. Zhang, X. Xu, T. Guo, D. Xie, R. Zhu, et al., RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration, Acta Biomater. 72 (2018) 266
277. Available from: https://doi.org/10.1016/j.actbio.2018.03.030. [139] B. Javan, F. Atyabi, M. Shahbazi, Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal cancer gene therapy, Life Sci. 202 (2018) 140
151. Available from: https://doi.org/10.1016/j.lfs.2018.04.011. [140] T. Liu, S. Chen, S. Zhang, X. Wu, P. Wu, B. Miao, et al., Transferrin-functionalized chitosan-graft-poly(L-lysine) dendrons as a high-efficiency gene delivery carrier for nasopharyngeal carcinoma therapy, J. Mater. Chem. B 6 (2018) 4314
4325. Available from: https://doi.org/10.1039/C8TB00489G. [141] S. Jaiswal, P.K. Dutta, S. Kumar, J. Koh, S. Pandey, Methyl methacrylate modified chitosan: synthesis, characterization and application in drug and gene delivery, Carbohydr. Polym. 211 (2019) 109
117. Available from: https://doi.org/10.1016/j. carbpol.2019.01.104. [142] C. Shan, H. Yang, D. Han, Q. Zhang, A. Ivaska, L. Niu, Graphene/AuNPs/chitosan nanocomposites film for glucose biosensing, Biosens. Bioelectron. 25 (2010) 1070
1074. Available from: https://doi.org/10.1016/j.bios.2009.09.024. [143] Q. Zhou, Q. Xie, Y. Fu, Z. Su, X. Jia, S. Yao, Electrodeposition of carbon nanotubes— chitosan—glucose oxidase biosensing composite films triggered by reduction of p-benzoquinone or H2O2, J. Phys. Chem. B 111 (2007) 11276
11284. Available from: https://doi.org/10.1021/jp073884i. [144] H.N. Abdelhamid, H.F. Wu, Selective biosensing of Staphylococcus aureus using chitosan quantum dots, Spectrochim. Acta Part A Mol. Biomol. Spectrosc 188 (2018) 50
56. Available from: https://doi.org/10.1016/j.saa.2017.06.047. [145] C.M. Lee, H.J. Jeong, S.L. Kim, E.M. Kim, D.W. Kim, S.T. Lim, et al., SPION-loaded chitosan-linoleic acid nanoparticles to target hepatocytes, Int. J. Pharm 371 (2009) 163
169. Available from: https://doi.org/10.1016/j.ijpharm.2008.12.021.

Handbook of Chitin and Chitosan

C H A P T E R

8 Electrospun chitosan materials and their potential use as scaffolds for bone and cartilage tissue engineering Adriana Herna´ndez-Rangel1, Gina Prado-Prone2, Joseline J. Hidalgo-Moyle3, Phaedra SilvaBermudez4 and Keiko Shirai1 1

Biotechnology Department, Laboratory of Biopolymers and Pilot Plant of Bioprocessing of Agro-Industrial and Food By-Products, Autonomous Metropolitan University, Mexico City, Mexico, 2Postgraduate Studies and Research Division, Faculty of Dentistry, National Autonomous University of Mexico, Mexico City, Mexico, 3Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico, 4 Tissue Engineering, Cellular Therapy and Regenerative Medicine Unit, National Institute of Rehabilitation “Luis Guillermo Ibarra Ibarra”, Mexico City, Mexico

O U T L I N E 8.1 Introduction 8.1.1 Bone physiology 8.1.2 Cartilage physiology

232 233 237

8.2 Electrospinning 8.2.1 Electrospinning parameters 8.2.2 Electrospinning for tissue engineering scaffolds

241 242 244

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00008-X

231

© 2020 Elsevier Inc. All rights reserved.

232

8. Electrospun chitosan materials and their potential use as scaffolds

8.3 Chitosan 8.3.1 Chitosan physicochemical properties 8.3.2 Chitosan cellular interactions 8.3.3 Chitosan electrospinning

249 250 252 253

8.4 Regulatory issues of electrospinning scaffolds

255

8.5 Chitosan electrospun materials for bone tissue engineering 8.5.1 Electrospun chitosan scaffolds 8.5.2 Electrospun chitosan
inorganic particles composite scaffolds

256 257 262

8.6 Chitosan electrospun materials for cartilage tissue engineering

266

Acknowledgments

269

References

269

8.1 Introduction Electrospinning is a technique widely used for the preparation of materials for several purposes, including tissue engineering. In this approach, the chitosan solution or melted polymer with medium viscosity is applied by a syringe into the electric field, forming a Taylor cone at the tip of the needle. The droplets exit the syringe and are deposited on the collector when the electrical voltage exceeds the surface tensile strength of the Taylor cone, and the solvent is evaporated during the jet flight. Several parameters affect the size and shape of the electrospun nanofiber such as polymer type, solvent, structures, and process parameters that include electrical potential, flow rate, polymer concentration, and the distance from the nozzle [1]. Electrospun materials have been used as scaffolds owing to their ability to mimic the extracellular matrix (ECM), thus sustaining cells. Besides drugs, growth factors and additives can be loaded. In this regard, the electrospun scaffolds are promising materials to promote osteogenesis and mineralization of the bone [2]. Thus, electrospun scaffolds can be intended to repair bone defects that have occurred due to trauma, congenital defects, accidents, osteoporosis, and arthritis. Bone is an active tissue of the body with the ability to regenerate and heal when subjected to minor injuries. A bone defect is defined as the absence of bone tissue in the region in which it was normally situated [1]. Bone defects are classified as primary and secondary. The former are mainly caused by fractures, osteoclastic tumors, and gun shots, while the latter are formed as a result of bone resection,

Handbook of Chitin and Chitosan

8.1 Introduction

233

infection, and due to nonunions that do not heal naturally and may be caused by severe fractures, smoking, and the use of antiinflammatory and anticoagulant drugs [3]. Bone plays an important role in the body because it acts as a structural framework. Additionally, it provides mechanical strength, regulates blood pH, and adjusts the level of calcium and phosphate. Therefore chitosan electrospun scaffolds should provide suitable structural, mechanical, and functional properties for the adhesion, growth, and differentiation of the cells and the ability to adjust their rate of degradation with bone healing rate for its reconstruction [3].

8.1.1 Bone physiology Bones constitute one of the main systems of the organism: the skeletal system, and together with connective tissues, such as cartilage, ligaments, and tendons, they provide support and structure to the human body. Bone is a multifunctional tissue that not only provides stiffness and toughness to the body to resist deformation upon impact of high loading, but it is also a dynamic and metabolically active tissue that supplies the mechanochemical and physicochemical cues needed to maintain cellular and functional-response tissue homeostasis. Additionally, bones are located in strategic places of the organism to protect vital organs in areas such as the thorax and head [4]. 8.1.1.1 Bone structure and composition In order to achieve its multiple functions, bone is a complex hierarchically organized tissue in the nano- and microscopic scales. Its ECM is constituted of two main phases: the organic phase (30% of the ECM), and the inorganic phase (70% of the ECM) [5]. The organic phase contains type I collagen fibers, proteoglycans, noncollagenous proteins, such as osteocalcin, osteopontin, etc., and different growth factors. This combined-proteins composition shapes the organic matrix within the bone. The collagen fibers and their structure guide the deposition of the mineral content of the bone tissue, providing a nucleation center for the deposition of phosphate and calcium ions during the mineralization process of the bone formation, and acting as a template for hydroxyapatite deposition [6,7]. Regarding the inorganic phase of the bone’s ECM, it contains minerals in the form of nanocrystals, such as hydroxyapatite, calcium, and phosphates. Together, the organic and the inorganic phases constitute a mineralized matrix in which the chemical properties and the mineral structure dictate the

Handbook of Chitin and Chitosan

234

8. Electrospun chitosan materials and their potential use as scaffolds

mechanical and biological properties of the bone, giving it strength in compression [7,8]. According to the organization of the bone tissue, there are two kinds of macroscopic structures in adult human bones: cortical/compact bone and trabecular/spongy bone. Cortical bone is dense because the mineralized part of its ECM is organized in concentric lamellae forming rings around channels called Haversian system or osteons, which extend longitudinally and are used by blood vessels, the lymphatic system, and nerves to spread throughout the bone (Fig. 8.1). Unlike compact bone, trabecular/spongy bone has irregular interstitial sheets of ECM that form a network of plates called trabeculae. These irregular structures leave gaps that are filled with bone marrow (Fig. 8.1); nevertheless, both types of bone architectures allow the exchange of nutrients, the maintenance of the cellular activity, and the dynamic modulation of tissue [9,10]. The organic and inorganic components that make up the ECM of bone represent 98% of the total mass of the bone. The remaining 2% is made up of bone cells that play an important role in bone remodeling, formation and repair. These bone cells are classified into two main types: osteoblasts and osteoclasts. Bone-forming osteoblasts are originated from mesenchymal stem cells (MSC) and are responsible for synthesizing and secreting large amounts of ECM proteins, such as collagen I and proteoglycans, with the ability to bind calcium. The ECM produced by osteoblasts in the absence of mineral components is known as osteoid [11]. On the other hand, osteoclasts are responsible for the resorption of bone. In the event of tissue damage, osteoclasts are recruited to the affected area to initiate bone resorption by decalcifying the ECM, allowing the degradation of its components [12]. This means that bone can undergo Macrolevel

Nanolevel Collagen fibers

Trabecular bone Hydroxyapatite crystals Cortical bone

FIGURE 8.1

Schematic representation of hierarchical bone structure. Scheme created with Biorender.com.

Handbook of Chitin and Chitosan

8.1 Introduction

235

remodeling processes in order to maintain its shape, size, and woven structure while adapting to mechanical stimuli [13]. Moreover, bone remodeling also works to control the physiological levels of calcium and phosphorus in the human body, ensuring a continuous exchange/equilibrium between the removal of old bone and the formation of new bone. The latter is able to exchange ions more easily with the extracellular fluid in comparison to old bone. Thus, bone remodeling is a mechanism regulated by bone resorption and formation processes that are carried out by osteoclasts and osteoblasts, respectively. The action of bone cells is coupled in a spatiotemporal way, regulating the quantity, quality, and mechanical integrity of the skeleton during adult life. Otherwise, any imbalance between these two processes (resorption
formation of bone) might result in bone defects that lead to fractures or to the loss of the bone capacity for regeneration [14]. 8.1.1.2 Bone defects The intrinsic capacity of bone for self-regeneration is largely due to the ability of bone cells to sense in real-time their functional environment. This capacity is required for monitoring and maintenance of the structurally intact bone tissue. The balance between bone resorption and formation of new bone should be constantly regulated to preserve the mechanical strength of the tissue, and to maintain the bone architecture after fracture healing [15]. An imbalance in bone turnover can lead to changes in the composition of the tissue and, consequently, to the loss of bone mass, affecting its mechanical properties and making it susceptible to fracture. Apart from trauma, other common causes of bone defects are metabolic disorders, such as diabetes, surgical resection of bone tumors, and chronic inflammation due to severe injuries or bacterial infections, such as in periodontitis [15
18]. In this respect, when bone injuries are big enough to constitute a critical size defect (defects of length that exceeds two or three times the diameter of the bone and that will not self-heal during the lifetime of the patient), then the natural capacity of bone for self-regeneration is not sufficient to repair the defect, and surgical planned reconstruction involving filling, augmenting, or fixture intervention is needed to reestablish the functionality and structure of the tissue. Critical size bone defects have been traditionally treated by autologous or heterologous bone tissue implants [19]. Although bone grafts have been considered the gold standard in the treatment of critical size bone defects, it normally involves double surgical intervention, and the availability of autologous bone grafts is largely restricted, mainly due to the morbidity of the extraction site. Bone heterologous grafts are also difficult to obtain, mainly due to a non-appropriate culture of organ donation;

Handbook of Chitin and Chitosan

236

8. Electrospun chitosan materials and their potential use as scaffolds

heterologous grafts might also present other drawbacks such as potential infection transmission or immunological rejection [20
22]. 8.1.1.3 Scaffolds for bone tissue regeneration In the last years, the demand for bone grafts has been constantly ramping up [23], and thus, different strategies have arisen with the purpose of developing synthetic bone grafts, scaffolds, or implantable fixture devices for consolidating, augmenting, or fixing critical size bone defects and allowing appropriate bone healing. Among those strategies is the use of metals, such as iron, cobalt, or titanium, which are generally used to develop permanent implants to “fill” or fix bone defects. Nevertheless, metallic materials can present corrosion and tissue infection problems, and their higher mechanical properties, in comparison to those of native bone tissue, might lead to stress shielding failures [22,23]. Ceramic materials have also been used to develop permanent synthetic bone grafts, scaffolds, or implantable fixture devices; however, they are not stable enough due to their susceptibility to fracture under high loads such as those normally exerted on the skeletal tissue of the body [24
27]. Moreover, bone defects treatment requires not only development of scaffolds with appropriate mechanical and structural properties, but development of temporary degradable graft substitutes with osteoinductive and osteoconductive properties (Table 8.1) that allow (1) proliferation of bone cells; (2) cell maintenance and differentiation towards osteogenic lineage [29
32]; and (3) scaffold degradation while new bone tissue is formed, mediating the scaffold complete replacement by natural tissue, and eliminating the need for further invasive surgery to remove the scaffold or fixative/augmentation device after bone tissue healing [28].

TABLE 8.1 Osteoinductive and osteoconductive properties of materials. Term

Definition

Osteoinductive

Implies the capability of recruiting immature cells and their stimulation to be differentiated into mature cells (osteoblasts and osteoclasts) to form new bone. In a bone-healing situation, the bone-healing rate mainly depends on osteoinduction [28].

Osteoconductive

Refers to the capability of any material to allow or induce bone growth on its surface. Bone growth on a surface depends on the correct function of differentiated bone cells. These cells might originate from preexisting osteoblasts or preosteoblasts that were activated by trauma or they might originate from recruited undifferentiated cells by osteoinduction. Hence, osteoconduction depends to a fairly large extent on osteoinduction [28].

Handbook of Chitin and Chitosan

8.1 Introduction

237

Current advances in the field of biomaterials, nanomedicine, and tissue engineering represent a great promise for developing alternative bone graft substitutes to replace the traditional autologous bone grafts. The bone tissue engineering approach involves the use of scaffolding biomaterials, along with cells and biochemical factors, with suitable characteristics to regulate bone cells’ functions and increase bone ECM formation; finally aiming to accelerate healing time and enhance functionality of repaired tissue [33]. To develop functional scaffolds able to improve bone tissue regeneration and bone defects repair, it is desirable that the scaffolds mimic the properties of the bone ECM as closely as possible. However, one of the most challenging aspects about obtaining appropriate scaffolds for bone regeneration is to mimic the complex structural and compositional characteristics of the bone ECM. In this respect, researchers are looking for novel biomaterials to create scaffolds that can provide such characteristics; for example, polymer-based nanomaterials or nanocomposites with adequate mechanical and biological properties to positively and actively mediate cell behavior [28,32,34
37]. In fact, the structure and composition of bone tissue is considered a nanocomposite from the point of view of materials science. In terms of their mechanical properties, scaffolds must show adequate mechanical properties for surgical handling and resisting the forces exerted by the body to the skeletal system. Scaffolds should also be porous with porous interconnectivity to provide space for cell infiltration, neovascularization, and nutrients and waste transference [38,39].

8.1.2 Cartilage physiology Articular cartilage is a highly specialized connective tissue that covers the surface of bones in the diarthroidal joints [40
42], and it has very complex biomechanical functions. It allows frictionless movement of joints due to its lubricated surface, and protects joint bones from mechanical damage due to its capacity to resist high cyclic compressive and shear loads through efficient load-bearing distribution [40,43
45]. Articular cartilage is mainly composed of a dense ECM with chondrocytes sparsely distributed among it. Chondrocytes are the only cell type present in articular cartilage and are responsible for synthesizing and maintaining the homeostatic balance of cartilage tissue ECM [41,45]. 8.1.2.1 Cartilage structure and composition The ECM of articular cartilage is a dense, highly hydrated matrix (water content . 65% of ECM total volume). It primarily consists of collagen Type II (15%
22% of total ECM wet volume and around 90% of ECM dry weight) and highly hydrophilic proteoglycans (4%
7% of ECM total volume), which are composed of long polysaccharide chains

Handbook of Chitin and Chitosan

238

8. Electrospun chitosan materials and their potential use as scaffolds

known as glycosaminoglycans (GAGs) that are covalently bound to core proteins [40,41,45]. Cartilage is able to support high loads, primarily, due to the frictional resistance of water movement through the pores of the dense hydrated gel structure of its ECM, which is accounted for by its proteoglycan content [40,45]. Cartilage tissue can be divided into four zones of different thickness, and with different structure and composition: (1) the superficial zone distributes loads over a large surface and is formed by dense collagen fibrils oriented parallel to the joint surface, fewer flattened chondrocytes of ellipsoid-shaped, and higher water content; (2) the transitional or intermediate zone deals with shear loads and anchors the superficial and deep zones of the cartilage, it possess larger collagen fibers, high proteoglycan content, and low density spherical chondrocytes with higher ECM production; (3) the deep zone provides resistance to compressive forces and consists of tightly packed abundant collagen fibers perpendicularly oriented to the joint surface, high proteoglycan content, vertically stacked active chondrocytes, and lower water content; (4) the calcified final zone anchors cartilage to the bone, forming a transition zone from the soft cartilage to the stiff subchondral bone [45,46]. While the three first layers of cartilage consist of collagen Type II, GAGs, chondrocytes, and water, this last zone mainly consists of collagen type I and X (bone-like ECM composition), and has a cellular content that is a transition from chondrocytes to osteoblast and osteoclast, such as hypertrophic chondrocytes surrounded by a bone-like ECM [46]. Finally, it is important to emphasize that chondrocytes undergo several stages of maturation along the different layers of cartilage maintaining the organization and homeostasis of the cartilage tissue [41]. 8.1.2.2 Cartilage defects Cartilage defects are the most common joint disease, and can be caused by different reasons such as trauma, age-related degeneration, chronic inflammatory pathologies, or degenerative joint diseases, such as osteoarthritis [47] that induce inflammatory processes that change chondrocyte cells’ phenotype, causing an imbalance in cartilage homeostasis, secretion of proinflammatory cytokines and matrix metalloproteinases, and the consequent loss of cartilage ECM components [44,47]. Symptoms of cartilage injuries typically include, among others, swelling, pain, biomechanical instability, and joint locking and stiffness. These symptoms can significantly impact the patient’s quality of life because they limit their ability to play sports, work, or perform daily activities, as they reduce mobility and independence. Cartilage injuries are common in all age groups, but unfortunately they are frequently diagnosed in young active populations [48]. Incidence reports indicated that  63% of diagnostic knee arthroscopies were performed as a

Handbook of Chitin and Chitosan

8.1 Introduction

239

follow-up to knee pain reports in patients with a median age of 39 years, and evidenced chondral lesions [49
51]. Cartilage defects are one of the most significant orthopedic problems and represent a serious economic burden for the health systems [52]. Unfortunately, cartilage tissue has a very poor self-repair capacity. In skeletal mature adults, cartilage tissue possesses no vascular networks, and it is mainly nurtured by passive diffusion from synovial fluid [43]. Also, chondrocytes are highly organized and scarcely distributed among the dense ECM of cartilage tissue which restricts their migration to any injured site. Restricted chondrocytes migration and lack of blood supply in cartilage tissue account for the poor regenerative capacity of cartilage [41,43,45,50]. Cartilage lesions can be broadly classified into two main categories: partial thickness and full-thickness defects. Partial thickness defects imply damage of the first layers of the cartilage, while full-thickness defects involve damage that goes up to the subchondral bone [48]. Partial thickness defects do not have access to the bone marrow-derived MSCs that are available upon damage of the vascularized subchondral bone (full-thickness defects); and consequently, they have very limited capacity for self-repair [41]. On the other hand, full-thickness lesions might spontaneously heal due to their access to bone marrow-derived MSCs; however, fibrocartilage tissue is formed, instead of cartilage, as a repair mechanism in these cases [53,54]. Fibrocartilage tissue has poorer mechanical and biological properties than articular cartilage, ultimately resulting in fibrocartilage degradation over time and cartilage defects appearing again [41,48]. Moreover, cartilage defects that go untreated, even superficial ones, can easily progress to larger defects over time. Cartilage lesions damage the capacity of chondrocytes to maintain the balance of the ECM composition, leading to a decreased proteoglycan concentration, increased hydration, and altered fibrillar organization of collagen. This imbalance in the ECM composition modifies forces transmission and distribution which damages adjacent healthy chondrocytes (defect progression), finally resulting in osteoarthritis development [40,55]. Osteoarthritis is a progressive joint disease that causes cartilage degeneration and bone overgrowth ultimately ending in joint function loss. Osteoarthritis affects over 10% of the population over 60 years old, and 44.7% of affected people will develop a disability over time [44,54]. 8.1.2.3 Treatment of cartilage defects Due to the poor self-repair capacity of cartilage and the easy progression, and the prevalence of significant symptoms of cartilage lesions, it is quite necessary to develop long-term effective treatments for the repair of cartilage defects. Current treatments can be classified into two

Handbook of Chitin and Chitosan

240

8. Electrospun chitosan materials and their potential use as scaffolds

main categories: nonsurgical and surgical treatments. Nonsurgical or palliative treatments can be used in the case of small focal lesions to decrease the symptoms and delay the evolution of the lesion. Nonsurgical treatment might involve rest, physiotherapy, weight loss, muscle strengthening, analgesic and antiinflammatory therapy, and other medications systemically delivered or locally injected such as glucosamine, chondroitin, corticosteroids, hyaluronate, platelet rich plasma, and rapamycin [47,55,56]. Nevertheless, failure of nonsurgical treatments to decrease lesions symptoms within 4
6 months is an indication for surgical treatment [55]. Knee surgical treatments have an annual incidence in the United States of  300,000 as of 2010 [57] and can be grouped into three main classes depending on the final objective of the surgical treatment: (1) partial replacement or total joint arthroplasty, (2) debridement arthroscopy, and (3) regenerative-intended surgeries. Arthroplasty is intended to replace the damaged parts, or the total, of the joint using partial or total joint prostheses. On the other hand, arthroscopy debridement includes, but is not limited to, articular cavity flush and removal of loose bodies, relieving pain in the short term, but with controversial efficacy about improvement in the long term [47]. Regenerative-intended surgical treatments for cartilage lesions include but are not limited to: 1. Microfractures performed in the damaged area of the articular cartilage to gain access to the subchondral tissue. Upon exposure of the subchondral bone marrow, a blood clot is created to allow bone marrow-derived MSCs to migrate and reach the lesion site. Then, MSCs can repair the lesion by expressing antiinflammatory molecules and differentiating into chondrocyte phenotype cells [47,58]. This method can deliver repair in the medium term, but it has been shown to provoke the formation of fibrocartilage tissue instead of cartilage tissue, which in the long term affects the biological performance of the articular cartilage again [47]. 2. Mosaicoplasty (or autologous osteochondral transplantation) was first applied in 1997, and it consists of removing grafts of healthy osteochondral tissue from nonload-bearing zones of the joint and transplanting them to the cartilage defect zone [47]. This technique has shown a certain degree of success, but it also has a risk of longterm failure due to a lack of graft integration or fibrocartilage differentiation, and the drawback of potential donor site morbidity. 3. Autologous chondrocytes implantation (ACI) was first applied in 1994 in a study conducted by Brittberg et al. [59]. Since then, mid- and long-term positive results have been reported [47]; however, this technique also presents drawbacks such as a second incision needed to

Handbook of Chitin and Chitosan

8.2 Electrospinning

241

obtain a periosteal patch, hypertrophy in the repaired lesion area, and loss of implanted chondrocyte cells [60]. Therefore different improvements to the original ACI technique have been developed (second and third generation ACI) such as the use of substitute membranes to the periosteal patch or the use of different scaffolds (material-mediated ACI) to serve as three-dimensional (3D) cell carriers to avoid cell leakage [47]. As stated by Kyriacos et al. [52], unfortunately, consistently successful or widely acceptable cartilage replacement treatments do not exist today. Thus there is still a need to develop improved regenerative cartilage treatments. Following the principles of tissue engineering, the development of scaffolds with mechanical, structural, and chemical properties that resemble the complex biomechanical structure of cartilage ECM is essential to prevent further mechanical damage to remnant healthy cartilage around the site of the lesion, stopping chondrocyte cells differentiation (one of the main problems of chondrocytes culture in monolayer) and functioning as a 3D microenvironment to guide and enhance cartilage regeneration [40,46]. Scaffolds can be used as (1) complex hierarchical acellular matrices to direct the oriented and organized growth of chondrocytes and to enhance the ECM deposition in the sites of the lesion [61,62]; (2) matrices to develop in vitro cartilage-like tissues that can be lately implanted in the lesion sites; or (3) cellular constructs to carry chondrocytes or other cell types with potential for cartilage repair, such as bone marrowor adipose tissue-derived MSCs, to the lesion sites [47]. Different materials and designs have been used to develop such scaffolds and very good reviews about this have been published [40,41,45]. Among the different materials that have been used are polysaccharides, such as chitosan, that resemble the GAGs composition of the cartilage ECM. GAGsbased materials have been proven to promote chondrogenesis in vitro and to result in high osmotic pressure and, consequently, in hydrogels/ materials with high water content that enables mechanical force transduction as well as nutrient and waste exchange [63,64].

8.2 Electrospinning Electrospinning is a versatile and relatively simple method that uses electrostatic forces to produce polymeric-based fibrillar scaffolds with fiber diameters ranging from tens of nanometers to few micrometers. It was in 1934 when inventor A. Formhals registered in the United States the first patent describing a process and an apparatus that used electric charges to spin synthetic polymer fibers [65]. Using this apparatus,

Handbook of Chitin and Chitosan

242

8. Electrospun chitosan materials and their potential use as scaffolds

Formhals was able to spin polymer filaments of cellulose acetate using ethylene glycol as the solvent. Despite this remarkable invention, the procedure was not utilized with great success, and the work in the field focused on the fundamental understanding of the physical phenomena supporting the electrospinning process. It was not until recent years that electrospinning started attracting interest from both industry and academia due to its unique ability to produce continuous ultrafine fibers of different polymer materials, such as synthetic polymers, natural polymers, polymer blends, and polymer-based composites.

8.2.1 Electrospinning parameters The electrospinning setup consists of three basic components (Fig. 8.2): a high-voltage power supply (  0
30 kV DC), a capillary tube with a spinneret (most often a metallic needle is used), and a metallic collector that is normally grounded. The spinneret is generally connected to a syringe, which is used to supply the polymer solution through the capillary tube at a constant rate using a syringe pump. Nevertheless, gravitational forces or pressurized gas can also be used for supplying the polymer solution through the capillary tube forming a pendant drop at the spinneret. An electrospinning system can have two main different configurations: (1) a horizontal configuration (Fig. 8.3A), where the polymer solution injection rate is governed by hydrostatic pressure using the syringe pump; and (2) a vertical configuration (Fig. 8.3B), where normally gravitational force drives the dispensing of the polymer solution [66]. To produce polymer fibers, the needle is connected to a high-voltage supply. When a high voltage is applied between the spinneret and the collector, then the drop of polymer solution at the nozzle of the spinneret Metallic collector

Polymer solution

Capillary tube with a spinneret Syringe pump High-voltage power supply

FIGURE 8.2 Schematic diagram of a typical electrospinning system.

Handbook of Chitin and Chitosan

8.2 Electrospinning

243

FIGURE 8.3 Schematic diagram of (A) horizontal and (B) vertical configurations used for electrospinning.

becomes statically charged with induced charges uniformly distributed on the surface of the drop. As the high-voltage increases, accumulation of charge on the drop’s surface causes a conic distortion of its shape, which is known as the Taylor cone (Fig. 8.3A and B). When the voltage is strong enough, the repulsive electrostatic potential on the drop overcomes the energy associated with surface tension, and a charged jet of polymer solution is ejected from the tip of the Taylor cone toward the metallic collector. While the polymer solution jet accelerates toward the collector, the solvent evaporates, resulting in a continuous mat of dry polymer fibers, ranging from micrometers to nanometers in diameter, being deposited on the collector. Different collector configurations can be used depending on the application intended for the electrospun scaffold. The most frequently employed are the stationary and rotating collector plates. Stationary plates are used to form unwoven mats of fibers in a random orientation (Fig. 8.3), while rotating collectors can be used to electrospin mats with fibers aligned along the rotation axis (Fig. 8.4). A common aim of the different electrospinning setups is to create very high surface area to volume ratio fibers; hence, fibers should have a uniform average diameter in the whole electrospun mat. Formation of bead-defects in electrospun fibers is counterproductive because beads have a higher volume to surface area ratio than continuous defect-free fibers. Morphologically, the diameter and orientation of individual fibers can be varied by controlling the electrospinning parameters such as the applied voltage, the polymer flow rate, and the spinneret to collector distance. Furthermore, properties of polymer solution such as concentration, viscosity, and conductivity, and ambient parameters, such as humidity and temperature, also influence fibers characteristics. There are many factors simultaneously influencing fibers’ morphology, and they are all closely correlated; thus changes in any one of them can

Handbook of Chitin and Chitosan

244

8. Electrospun chitosan materials and their potential use as scaffolds

FIGURE 8.4 Schematic diagram of an electrospinning setup with a rotating collector plate used to produce aligned fibers.

affect the others. Some of the most important parameters and their effects on fibers’ morphology are listed in Table 8.2.

8.2.2 Electrospinning for tissue engineering scaffolds In recent years, electrospinning has been widely used in the field of tissue engineering to fabricate scaffolds by using a variety of natural and synthetic polymers. The wide varieties of polymer materials that can be used and combined in electrospinning, and the possibility of forming polymer
ceramic/metallic composite materials are the main advantages of the electrospinning technique for the development of tissue engineering scaffolds. Biocompatibility and biodegradability are the most important requirements for any material used to develop tissue engineering scaffolds, because they will be in intimate contact with the biological medium upon implantation and must not exert any undesirable immune reaction. It is also expected that scaffolds will be degraded and replaced by newly generated tissue. Finally, an appropriate choice of biomaterials is required to meet the mechanical properties and the degradation rate specifically required for the tissue whose regeneration is intended to be enhanced through the use of the scaffold. Biocompatible and biodegradable synthetic polymers, such as polylactic acid (PLA), polyglycolic acid (PGA), or polycaprolactone (PCL), have been widely used as materials for electrospun scaffolds due to their mechanical properties. Natural polymers, such as collagen, gelatin, alginate, and chitosan, have been also widely used because, normally, they enhance cell adhesion and proliferation. The number of scientific papers published related to electrospinning has been constantly

Handbook of Chitin and Chitosan

245

8.2 Electrospinning

TABLE 8.2

Effects of basic electrospinning parameters on the morphology of fibers.

Parameter

Effect

References

Applied voltage

There is an optimal range of applied voltage for each polymer
solvent system. In general, if the strength of the applied voltage does not overcome the solutions surface tension, the polymer jet cannot be formed. If it overcomes the surface tension but it is too weak, it will produce bead-defects in the fibers. As the strength of the applied voltage increases, the average fiber diameter increases. This is attributed to the increase in the mass flow rate relative to the increase in the electrostatic force.

[67
71]

Polymer solution flow rate

If flow rate is insufficient to replace the solution ejected as the jet, the Taylor cone at the spinneret cannot be maintained. As the solution flow rate increases, the average fiber diameter and pore size increase as well. However, higher flow rates lead to the formation of flattened fibers instead of fibers with cylindrical shape, because they cannot dry completely before reaching the collector.

[67,69
71]

Spinneret
collector distance

Spinneret
collector distance must be enough to allow an adequate drying of the fibers prior to reaching the collector, but close enough to allow fibers to arrive at the collector. In general, average fiber diameter decreases with increasing spinneret
collector distance.

[69
71]

Polymer concentration

Polymer concentration determines solution’s spinnability and influences both the viscosity and the surface tension of the solution. If the solution is too diluted (low viscosity), then the polymer fibers are going to break up into droplets upon electrospinning (electrostatic spraying). On the other hand, if the solution is too concentrated (high viscosity), it is going to be difficult to control the solution flow rate through the capillary, and fibers will not be formed.

[67,68,71
73]

Surface tension

If the surface tension of the solution is too high, the Taylor cone will eject individual droplets (electrostatic spraying) instead of a continuous jet. The electric field needs to overcome the surface tension in terms of energy to produce a continuous solution jet. Thus an optimal balance between surface tension and electric field energy is needed for the formation of a cylindrical continuous jet.

[74]

Process

Solution

(Continued)

Handbook of Chitin and Chitosan

246

8. Electrospun chitosan materials and their potential use as scaffolds

TABLE 8.2 (Continued) Parameter

Effect

References

Viscosity

Viscosity of solution depends on polymer concentration, molecular weight of polymer, and ambient temperature. A nonuniform viscosity leads to the formation of beads in the fibers due to a nonuniform distribution of polymer within the solvent. To produce uniform fibers, solution viscosity should be constantly maintained.

[70]

Conductivity

Conductivity is affected by ion mobility, polarity of molecules, and charge density in the solution, which in turn can be affected by solution viscosity. The jet of highly conductive solutions is subjected to a greater tensile force in the presence of the electric field in comparison to the jet of a low conductivity solution. If the solution is not sufficiently conductive to be carried from the spinneret to the collector, fibers cannot be formed.

[75]

Humidity

Ambient humidity during the electrospinning process can affect each solution differently, depending on the hydrophilicity of the polymer solution. Nevertheless, the electrospinning process in a very humid environment is one of the methods to produce porous fibers because tiny droplets of water can precipitate into the solution jet to generate phase separation. These water droplets form tiny pores in the solidified fiber after solvent evaporation. Nevertheless, humidity can also cause a slower evaporation rate of the solvent leading to an increase in the fibers diameter.

[76]

Temperature

Solvent evaporation rate rises as ambient temperature increases, drying the solution faster and leaving less time for fibers’ elongation. In general, solution viscosity decreases as ambient temperature increases; thus solutions require less energy to flow, allowing the polymer jet to elongate more and become thinner while it is traveling from the spinneret to the collector.

[73]

Ambient

growing in the last 10 years (Fig. 8.5). Among these papers, hundreds of documents are related to the use of electrospinning for the development of tissue-engineered scaffolds (Fig. 8.5). Tissue engineering uses different scaffolds to provide temporary support for the cells to repair or regenerate tissues that have been

Handbook of Chitin and Chitosan

8.2 Electrospinning

247

FIGURE 8.5 Annual number of publications dedicated to “electrospinning” (all documents) and to “electrospinning for tissue engineering” (electrospinning documents related to tissue engineering). Source: ISI Web of Sciences 2019.

damage by disease, injury, or congenital defects. Scaffolds produced by electrospinning typically result in 3D flat mats with interconnected porosity that are constituted by randomly oriented fibers. Thus their morphology mimics or resemble the morphology of the ECM of different native tissues; mainly the morphology of collagen-based ECM. Since microenvironment (morphology) influences cell behavior, electrospun fibers with a morphological microstructure similar to that of the ECM fibrils can provide a more conducive environment for positively controlling cell behavior. For instance, Guarino et al. [77] showed that fibrillar PCL and PCL/gelatin electrospun mats enhanced the viability, adhesion, and spreading of human MSC (hMSC) with respect to hMSC cultured on nonfibrillar PCL and PCL/gelatin films developed by solvent casting. Another example is the influence of electrospun fibrillar mats on cell differentiation. MSC therapy represents a promising treatment for many diseases; however, the lack of control of cell differentiation remains a major issue. In this sense, fiber diameter of electrospun mats can positively influence cell differentiation. Some reports have shown that polyurethane (PU)-based electrospun fibers with diameters of 200
400 nm significantly promoted osteogenic and chondrogenic differentiation of hMSC, in comparison with fibers with diameters in the range of 600
800 nm and 1.4
1.6 μm [78].

Handbook of Chitin and Chitosan

248

8. Electrospun chitosan materials and their potential use as scaffolds

Various electrospun fibrillar systems have been also developed for local delivery of therapeutic agents (drugs, nanoparticles, biomolecules, proteins, and growth factors) [79
83]. The micro- and nanodiameter of single electrospun fibers and the overall fibrillar structure of electrospun mats allow the control of local drug release profiles; therefore achieving a high therapeutic agent release at the site of lesion and resulting in enhanced therapeutic efficacy and minimization of side effects. For example, a multilayered nanofiber mesh loaded with two different drugs for sequential control of dual drug release was developed by adjusting morphological features of the electrospun layered meshes, such as their fiber diameter, mesh thickness, and hydrophobicity, and mesh layering out [79]. Some drug-loaded nanoparticles have also been embedded in electrospun fibers to retard and control the release rate of therapeutic drugs. Lai et al. [81] embedded EGF-, bFGF-, PDGF-, and VEGF-loaded gelatin nanoparticles into electrospun collagen
hyaluronic acid nanofibers for chronic wound healing, showing that controlled release of the growth factors (early release of EGF and bFGF, and later release of PDGF and VEGF) accelerated epithelialization and vasculature sprouting and induced blood vessel maturation in in vivo tests with diabetic rats. Electrospun mats made of stimuli-responsive polymers can also be used to release encapsulated therapeutic agents as a response to local environment stimuli, such as temperature, light, electric and magnetic fields, pH, and enzyme concentration [83]. The surface area and porosity of electrospun fibers have been shown to be more convenient for faster delivery of therapeutic agents, in comparison to the morphological properties of solvent casted or spin-coated films, making electrospun fibrillar mats more suitable for stimuli-responsive local drug delivery systems. For example, the slight variation in pH between healthy tissues (  7.4) and solid tumors (6.5
7.2) could be used for controlling pH stimuli-responsive systems to provide spatiotemporal control of therapeutic agent release at the tumor site. In this regard, Xie et al. [84] developed a pH-responsive system based on PCL electrospun nanofibers coated with the protein polydopamine that were able to release anticancer drug DOX at low pH values, significantly killing large numbers of cancer cells [85]. The development of scaffolds with aligned fibers is also possible by changing the collector design in the electrospinning setup. The development of aligned fibers is also an efficient way of exerting contactguidance response in cells to control certain cell functions, such as directed motility. Despite electrospinning being widely used for developing tissue engineering scaffolds (mainly due to its ability to fabricate fibrillar structures resembling the ECM), the development of 3D nonplanar

Handbook of Chitin and Chitosan

8.3 Chitosan

249

electrospun fibrous structures with controlled geometric shapes is still an important challenge for the tissue engineering of structures such as bone or cartilage. Some 3D fibrillar macrostructures have been fabricated by modifying the electrospinning process, for instance: (1) replacing the conventional two-dimensional (2D) flat collector with 3D collectors as templates for producing 3D structures with different size, shape, pattern, and structure [86
90]; (2) using liquid-assisted collectors such as ionized water or water vortex for obtaining 3D controlled structures with larger pore size than 2D mats [91
93]; and (3) using multilayer electrospinning for controlling fiber diameter, porosity, thickness, and the composition of each layer, as well as the number of layers [81,94,95].

8.3 Chitosan Chitosan (CS) is a linear polysaccharide composed of randomly distributed β-(1
4) D-glucosamine and N-acetyl D-glucosamine units (Fig. 8.6). This semicrystalline polymer is not extensively found as such in nature; however, it can be obtained from the highly available natural polymer, chitin [96,97]. Chitin is the second most abundant biopolymer after cellulose, and it is mainly present in the exoskeleton of crustaceans (shrimps, lobsters, crabs, etc.) and insects, but it is also found in the cell walls of fungi and bacteria [97]. After chemical manipulation, chitin can be transformed into its soluble form CS. Alkali treatment of chitin produces partial deacetylation, thus increasing the availability of amino groups along the chain [97]. The amino content is called degree of deacetylation (DD), and chitin must have a DD of at least 50% to be considered CS [98]; therefore DD of CS ranges from 50% to 100%. Molecular weight (MW), DD, and crystallinity of CS are defined by its chitin origin and purification process [98,99]. Thus CS can be obtained in a large variety of MW, crystallinities, and DD, depending on the desired application.

FIGURE 8.6 Chemical structure of chitosan.

Handbook of Chitin and Chitosan

250

8. Electrospun chitosan materials and their potential use as scaffolds

CS chemical composition plays a major role in its final properties. CS possesses a polycationic nature due to the presence of hydroxyl (OH) and amino (NH2) functional groups in each glucosamine residue, whose amino groups undergo protonation in dilute acid solutions. The deacetylated portions of the CS molecule have a positive charge at pH # 6, which allows CS solubility and processing under mild conditions, as well as its functionalization to develop a wide range of CS-based materials [100
104]. Versatility of CS makes it a promising biopolymer for applications in different fields, such as wastewater treatment [105], the food industry [106], drug delivery systems [107], cosmetics [108], wound dressings [109], and tissue engineering [110,111]. Materials for tissue engineering applications must meet a number of characteristics in order to have an adequate performance, such as biocompatibility, biodegradability, appropriate swelling capacity, suitable degradation rate, adequate morphology to support cell adhesion and proliferation, and mechanical stability [102,112,113]. In the particular case of CS, the DD is one of the main parameters to control since it dictates CS physicochemical properties, such as crystallinity, degradation rate, swelling capacity, and mechanical behavior. Moreover, DD has also been reported to affect cell interactions with CS-based materials [96]. A number of authors have pointed out that the DD might also influence adhesion, migration, and proliferation of different cell types [114
118]. Thus CS’ physicochemical behavior and cellular responses are intimately related to and tailored by its DD and MW, and these two factors should be carefully taken into account when using CS for biological-related applications. Depending on the chitin source and deacetylation procedure, CS with DD of 50%
100% and MW ranging from 10 to 1000 kDa can be obtained [99,119].

8.3.1 Chitosan physicochemical properties In general, CS mechanical properties, such as tensile strength and elastic modulus, are improved by increasing DD. For example, Wenling et al. [111] prepared CS films using CS with similar MW (  800 kDa) but different DD ranging from 70% to 95%, and observed that tensile strength and elastic modulus exhibited a three-fold increment for CS with 95% DD in comparison to CS with 70% DD. Nunthanid et al. [120] prepared CS films combining CS with different DD and MW. They used low-MW (50
60 kDA) and high-MW (800
1000 kDa) CS either with 80% or 100% DD and found that both factors were related to the films’ mechanical properties. Films’ tensile strength was the highest (62 MPa) for CS with high DD and high MW; on the contrary, films prepared from CS with 100% DD but low MW resulted in tensile strength

Handbook of Chitin and Chitosan

8.3 Chitosan

251

(37 MPa) reduction [120]. Similar results were found by Liu et al. [121], who reported that increasing DD improved the tensile strength of CS
gelatin composite films, where CS with 93% DD resulted in films with twofold higher tensile strength than films prepared using CS with 76% DD. Moreover, it was proved that MW of CS also contributed to the films’ mechanical performance as tensile strength was 1.2 times higher for films prepared from CS with MW of 550 kDA, in comparison to films prepared with CS with MW 5 200 kDa. The effect of higher DD resulting in higher tensile strength has been explained as a result of an increase in CS crystallinity with DD. As DD increases, less large acetyl groups and more small amino groups are available, resulting in a more regular and denser packing of the CS polymer chains [120,121]. On the other hand, higher MW has been related to a higher entanglement of the CS chains, also resulting in higher interchain attraction. Swelling is defined as the amount of water that a biomaterial can absorb; consequently, it is an important indicator of its behavior in a biological environment upon contact with body fluids. It has been demonstrated that initial biopolymer swelling stimulates cell attachment and proliferation in 3D as well as allowing nutrients’ diffusion. CSbased materials rapidly swell in contact with water or biological fluids due to its hydrophilic nature, which is also dependent on the DD of CS. The hydrophilic nature is a measure of the proportion of hydrophilic (amino and hydroxyl) groups present in the CS molecules. It could seem that the higher the content of amino groups in CS, the higher the swelling index of CS materials; however, it has been shown that increasing DD reduces the swelling index of CS films. Maganti et al. [122] obtained different CS scaffolds using CS with two different DD and found that water uptake was 20% greater for the scaffolds prepared from CS with 85% DD than for the scaffolds prepared with CS with 95% DD. Wenling et al. [114] also observed that swelling capacity diminished by increasing DD, showing that CS with 70% DD resulted in films with swelling percentage 30% higher than films prepared from CS with 95% DD. Similarly, Trung et al. [123] observed that films prepared from CS with 75% DD exhibited 30% higher water absorption than those films obtained from CS with 87% and 96% DD. This phenomenon can be explained as a result of the higher degree of CS crystallinity with increasing DD, despite the content of amino groups in the CS molecules. A polymer’s crystallinity is related to its chemical uniformity; therefore, the closer to 100% DD of CS, the higher the chemical uniformity and crystallinity of CS due to higher intermolecular bonds. In turn, the higher crystallinity (higher intermolecular bonds) prevents water from entering the crystal regions of the material, which decreases the swelling capacity of high DD CS scaffolds, in comparison to small DD CS scaffolds.

Handbook of Chitin and Chitosan

252

8. Electrospun chitosan materials and their potential use as scaffolds

Another important characteristic of scaffolds intended for tissue engineering is their biodegradability because scaffolds are expected to degrade over time as the new tissue is conformally developing. CS with high DD and MW exhibits a lower degradation rate due to closer chain packing and higher crystallinity than CS with low DD and MW. In in vivo environments, CS chains break down due to lysozyme action, which hydrolyses the acetyl residues present in the CS molecule; hence degradation rate decreases as DD increases. Maganti et al. [122] reported that scaffolds made up of CS with 95% DD exhibited a slightly lower degradation rate by lysozyme when compared to scaffolds prepared from CS with 85% DD, and showed that the latter scaffolds exhibited 35% mass loss, in comparison to the 25% mass loss exhibited by the scaffolds prepared from CS with 95% DD after 28 days of incubation. Degradation is indicated by changes in MW of CS, and not in total mass loss, and tends to be inversely proportional to DD [115]. This can be explained by the higher amount of H-bonding and crystallinity of CS with higher DD, which limit enzymatic attack and help to stabilize CS molecules, in comparison to CS with low DD. Finally, it is also important to mention that degradation products of CS are also biocompatible and noncytotoxic.

8.3.2 Chitosan cellular interactions Some authors claim that CS biocompatibility can be ascribed to its positively charged free amino groups because cells can interact with these groups to easily attach and grow on the surface of CS-based materials [124]. Thus cell adhesion and proliferation on CS-based materials also seems to change with DD of CS. Sun et al. [117] developed CS materials using CS with the same MW but different DD (74%, 84%, and 94%) to study the effects of DD on corneal keratocytes adhesion, spreading, and morphology. In particular, when adhered to films prepared from CS with DD of 74%, keratocytes appeared to be fibroblastic and elongated in a spindle-like shape, exhibiting the loss of their characteristic dendritic morphology. Furthermore, expression of integrin β (a cellmatrix adhesion molecule) was significantly upregulated on keratocytes adhered to CS films with higher DD, supporting favorable attachment of corneal keratocytes on these films. In a similar way, Foster et al. [118] investigated the influence of DD on the growth of olfactory ensheathing cells and found that cell proliferation linearly grew as DD increased from 72% to 85%; however, even though CS films with DD ,75% were cytocompatible, these induced cellular stress. Additionally, both studies remarked that improvement in cell spreading and proliferation was due to the increase of substrate stiffness and crystallinity, which was linearly

Handbook of Chitin and Chitosan

8.3 Chitosan

253

and proportionally correlated to DD. Chou et al. [125] also suggested that surface properties of CS coatings affected cell proliferation and showed that cell proliferation hiked when decreasing surface roughness but increasing stiffness. This stiffness-induced cell proliferation was ascribed to an increase of integrin signaling through focal adhesion complexes. On the other hand, Yuan et al. [115] coated titanium coupons with CS with different DD (81% or 92%) and evaluated cytocompatibility using osteosarcoma cells. This resulted in no significant differences in cell growth. In the same way, Mao et al. [126] did not identify any significant differences in L929 fibroblast cells attachments on CS films prepared from either CS with 75%, 83%, or 90% DD. Maganti et al. [122] also had difficulties delineating differences in the morphology of preosteoblast cells on scaffolds obtained from CS with 85% or 95% DD. Contradictory results in biological and physicochemical performances of CS-based materials are common in literature. Leaving aside the experimental performances and setups, a full correlation between DD, MW, and CS properties has not been achieved so far since there are other parameters, like mineral and protein content, that might be also important in determining CS properties [124]. However, many studies are vague concerning this information. In addition, changing CS batch, even from the same manufacturer, can also cause different results, meaning that a deeper knowledge of CS source, processing conditions, and validation of MW, DD, and proximal analysis are needed to achieve a better understanding of CS materials. Besides the intrinsic characteristics of CS scaffolds, morphology, and structure are also important parameters for scaffold manipulation and cell response. Nevertheless, it is important to emphasize that CS can be processed into different morphologies, such as films [109], nano- and microparticles [127], hydrogels [128], or fibers [129], to target particular tissue engineering applications.

8.3.3 Chitosan electrospinning As mentioned before, electrospinning is a technique widely used to develop scaffolds intended for tissue engineering, mainly because of its relative simplicity and capability to produce scaffolds with fibrillar morphologies that resemble the morphological structure of collagenbased ECM. Furthermore, electrospinning also allows the combination of different polymers and the formation of polymer-based composites, which increase its versatility, and consequently its potential as a technique to develop tissue engineering scaffolds. Nevertheless, this technique presents several parameters affecting fibers’ formation and

Handbook of Chitin and Chitosan

254

8. Electrospun chitosan materials and their potential use as scaffolds

homogeneity that have to be taken into consideration when designing electrospun scaffolds. Among those parameters, polymer solution properties such as viscosity, homogeneity, and conductivity are very important for electrospinning success [83,130]. Accordingly, solvent choice is a critical factor to determine polymer solution spinnability. Solvents should allow homogeneous dissolution of all polymeric components and appropriate jet drying to produce stable dry fibers in the collector. Thus volatile organic solvents, or mixtures of them, are normally chosen as electrospinning solvents [131]. Consequently, electrospinning of pristine CS is a challenging task due to its poor solubility in common organic solvents. On the other hand, polymer concentration must be such that polymer chains become entangled but within a solution viscosity range that allows solution pumping and the formation of fibers [132]. For CS-only solutions, it is difficult to achieve these conditions because a low CS fraction in solution does not allow appropriate polymer chains entanglement, and they form beads instead of continuous fibers. On the other hand, increasing CS concentration allows CS chains entanglement, but significantly raises solution viscosity at a fast pace, and then overcoming the cohesive forces in high-concentration CS-only solutions (appropriate viscosity for chain entanglement) becomes a challenge and fibers cannot be formed. In order to successfully produce CS-only scaffolds by electrospinning, researchers have focused on the use of concentrated acetic acid solutions. Geng et al. [133] found that increasing acetic acid concentration changed CS fibers morphology from beads to uniform fibers, stating that CS dissolution in 90% aqueous acetic acid was optimal to produce homogeneous, defect-free CS electrospun fibers. Similar results have been reported by other authors [134,135]. The use of other solvents, like triflouroacetic acid [136] and dichloromethane [137], has also been proposed; nonetheless, the toxicities of these solvents are significant and might not be convenient for their particular use in the tissue engineering field. Another strategy for improving CS-only solutions spinnability is to modify the CS backbone to produce CS derivatives, such as hexanoyl [138] and carboxymethyl
CS [139], with modified solubility. Hexanoyl
CS is soluble in organic solvents such as chloroform, whilst carboximethyl
CS becomes water soluble [138,139]. In another attempt to improve CS solubility in common organic volatile solvents, Min et al. [140] proposed to first electrospin chitin by dissolving it in 1,1,1,3,3,3hexafluoro-2-propanol (HFIP), followed by the alkali treatment (deacetylation) of the electrospun fibers to finally achieve CS-only fibrillar mats. Due to the difficulties of producing pristine CS electrospun scaffolds, electrospinning of CS-based scaffolds by combining CS with other

Handbook of Chitin and Chitosan

8.4 Regulatory issues of electrospinning scaffolds

255

synthetic or natural polymers is preferred. CS blends with polyethylene oxide (PEO) [141
143], polyvinyl pyrrolidone (PVP) [144], polyvinyl alcohol (PVA) [145,146], PLA [147,148], PCL [149,150], collagen [151,152], and silk fibroin [153] have been fabricated to produce uniform-fibers electrospun mats. Spinnability of these blends is more feasible than those of pure CS, and the obtained mats are more valuable than pristine CS electrospun fibrillar mats because the mechanical, biocompatible, biodegradable, antibacterial, and other properties of the fibrillar mats can be tuned by tailoring the polymer blend composition.

8.4 Regulatory issues of electrospinning scaffolds Chitosan has been recognized as safe (GRAS) and approved for dietary use in Italy, Japan, and Finland [154]. Nevertheless, according to EU legislation, chitosan is still being evaluated on their toxicity and biocompatibility. In this regard, Kean and Thanou [154] analyzed several works on chitosan toxicity. They claimed that highly deacetylated chitosans with low MW and low protein and mineral impurities do not display negative effects on cell viability in up to 5 mg/mL concentrations, and they are also well tolerated in vivo. While less deacetylated chitosans may stimulate the immune system and enhance reactivity against tumors, there are many chitosan derivatives with potential in medical applications, but most of them still require further toxicological assessment. In addition, there is a concern regarding chitosan’s interactions with other molecules in biological applications, such as endotoxins [155]. Medical devices or combination products are regulated by the FDA and its Center for Devices and Radiological Health and are classified based on the risk involved into three classes, from Class I (the least risk) up to Class III (the highest risk). Topical chitosan wound dressings that do not contain a drug or biologic agent were considered Class I. Thus, currently, chitosan is approved for this application, as well as for cartilage repairing formulation, for example, hemostatic wound dressings for external use (ChitoGauzeXR K102546), which is a gauze coated with chitosan [156]. Another reported use of chitosan is as an absorption enhancing agent in the nasal drug delivery formulations technology for the delivery of peptides, proteins, and small hydrophilic drugs. A chitosan-based formulation for nasal administration of morphine (Rylomine) is in phase 2 (UK and EU) and phase 3 (US) of clinical trials and will be shortly released on the market [157]. Notwithstanding, these products should comply with special controls if they fall into Class II. In this regard, wound dressings containing drugs have been considered combination products, which are defined

Handbook of Chitin and Chitosan

256

8. Electrospun chitosan materials and their potential use as scaffolds

as a product composed of two or more regulated components that are physically, chemically, or otherwise combined or mixed and are produced as a single entity. These are required to indicate the intended use, dosage form, strength, route of administration, and significant change in dose. There are no absorbable hemostatic devices containing chitosan that have been approved since these devices are for internal use and considered Class III and need to demonstrate safety and effectiveness established by clinical studies [155]. And, of course, chitosan as a combined product or the completed chitosan-containing medical device should be tested for safety and toxicological effects using current guidelines, such as ASTM and ISO10993 [158
161]. Electrospinning has been widely used in tissue engineering in which the toxicity of the electrospun material can be related not only to the polymer but also on to the solvent used. In this regard, the biopolymers, such as chitosan, can be used in aqueous solutions, avoiding leaving solvent residues in the material. Chitosan is readily soluble in an aqueous solution of acetic and formic acids that are recognized as safe in food applications, such as organic acids that are considered of lower risk to human health by the FDA [156].

8.5 Chitosan electrospun materials for bone tissue engineering As was mentioned in the bone physiology section, bone is a highly vascularized tissue that forms the skeletal system, providing support and structure to the human body and playing an important role in protecting vital organs from mechanical impact [162]. Bone tissue is composed of different types of cells, including osteoblasts and osteoclasts, embedded in an ECM that consists of (1) an organic phase composed by 90% collagen Type I fibers and 5% noncollagenous proteins [163], and (2) an inorganic phase that is mostly composed of calcium phosphate minerals in the form of crystalline hydroxyapatite [5,164]. Bone injuries can be produced by different causes, such as traumatic injuries, tumor resections, nonunion fractures, and dental infections [165,166]. Bone tissue possesses an intrinsic capacity for selfregeneration due to its continuous remodeling capability [167]. However, critical-sized bone defects cannot be healed through the normal bone remodeling process, and surgical intervention is needed to achieve defect repair and bone regeneration [168,169]. Orthopedic implants, bone grafts, and bone cement are among the available options for treating critical size bone defects [22], where the use of autologous grafts is the gold standard. Nevertheless, there are important challenges associated with the use of autologous grafts such as low availability of donor tissue, donor site morbidity, and double surgical intervention.

Handbook of Chitin and Chitosan

8.5 Chitosan electrospun materials for bone tissue engineering

257

Current advances in the fields of biomaterials, nanomedicine, and tissue engineering represent great promise for developing alternative temporary bone grafts substitutes based on novel biodegradable scaffolding materials capable of regulating bone cells functions and bone ECM deposition to accelerate healing time and enhance the functionality of novo-forming tissue [33]. Many materials have been used in the fabrication of bone tissue engineering scaffolds such as ceramics, natural and synthetic polymers, and composite materials. Most of them are chosen based on their capability to resemble the properties of the native ECM. For example, synthetic biodegradable polymers, such as PCL, PLA, and PGL [170], provide favorable mechanical and degradation properties that are suitable for modulating their macro- and micromorphology by different fabrication techniques and possess fair reproducibility for large-scale production. However, their physicochemical characteristics generate insufficient biological recognition and do not induce appropriate biological signaling for enhancing cell adhesion, proliferation, migration, or differentiation. On the other hand, natural polymers, such as collagen, gelatin, silk fibroin, hyaluronic acid, alginate, or chitosan, resemble the chemical composition of the organic phase of the bone ECM, displaying an active and adequate biological response. Nevertheless, mechanical properties of natural polymers are commonly smaller than those required to develop bone grafts, which are subjected to high mechanical forces in the body [171]. Then, one of the most effective approaches for simultaneously improving mechanical and osteoinductive properties of scaffolds is the use of natural-synthetic polymer blends added or not with inorganic particles (that can work as reinforcement fillers or mineralization improving compounds), aiming to combine the properties of different components (composites) to result in mechanically, chemically, and biologically more favorable scaffolds for bone tissue engineering [172].

8.5.1 Electrospun chitosan scaffolds Among natural polymers, CS has been widely used alone or in combination with different materials (polymers or ceramics) to develop tissue engineering scaffold materials. CS is a linear polysaccharide obtained from the partial deacetylation of one of the most abundant natural resources in the world, chitin [96]. CS provides specific structural and biochemical properties for enhancing adhesion and proliferation of different cell types and the differentiation and mineralization (accelerating bone regeneration) of osteoblasts. Moreover, the DD and MW of CS can be tuned to tailor its physicochemical and biological properties according to the required characteristics for different tissue engineering

Handbook of Chitin and Chitosan

258

8. Electrospun chitosan materials and their potential use as scaffolds

scaffolds [98]. Additionally, CS is a polysaccharide rich in hydroxyl (
OH) and amino (
NH2) functional groups, which facilitates its chemical modification and the addition of side groups, peptides, amino acids, etc. for the biological functionalization of CS-based scaffolds [98]. Several fabrication techniques such as melt-molding, freeze-drying, salt-leaching, 3D-printing, and phase separation have been used for developing CS and CS-based scaffolds [173]. These techniques generally achieve fabrication of highly porous structures but fail to mimic the fibrillar structure of the ECM of bone tissue [174]. Electrospinning allows fabrication of fibrillar scaffolds with random or aligned oriented fibers ranging from nano- to some micrometers that resemble the native morphology of the ECM [175]. Furthermore, electrospun fibrillar scaffolds possess unique properties such as high surface area to volume ratio, interconnected porosity, mechanical stability, permeability, and potential for embedding particles into the fibers (metallic, polymeric or ceramic micro- and nanoparticles), as well as obtaining fibers with core
shell structure by coaxial electrospinning or coating techniques [176]. In addition, some studies have reported CS-only electrospun scaffolds intended for tissue engineering applications (Table 8.3). However, their use has not yet been widely explored due to the different challenges associated with the electrospinning of CS-only solutions [129]. Mainly, the reasons are that the CS amount required to get CS-only solutions with appropriate chain entanglement for electrospinning produces cationic solutions with high viscosity, and consequently with high surface tension, which makes it difficult for the electric field to overcome the cohesive forces and form fibers during the electrospinning process [132]. Nevertheless, blend solutions of CS with different fiber forming agents (mainly easily spinnable synthetic polymers) have resulted in the successful generation of appropriate electrospinning solutions with high CS content (Table 8.3). For example, PEO added to CS solutions (CTS:PEO; 95:5) can form hydrogen bonds with CS chains, reducing the polyelectrolyte effect and enhancing the spinnability as a direct result of decreasing surface tension [185]. The use of solvents such as dimethyl sulfoxide (DMSO) also decrease CTS-PEO solutions viscosity and further facilitate the electrospinning process [177,185]. Scaffolds with the ability to control the release rate of biochemical cues, such as growth factors, have also emerged as a significant alternative to improve the biological response of bone tissue. Bone morphogenetic protein (BMP) is an osteoconductive protein originally identified in demineralized bone. BMP facilitates the healing of bone defects without the need of bone grafts. Particularly, bone morphogenetic protein-2 (BMP-2) induces healing in segmental bone defects. Nie et al. [184] loaded BMP-2 plasmid into fibrous electrospun polymer matrices as a controlled release dosage system for in vivo gene transfection. DNA
CS nanoparticles were obtained by static attraction and then encapsulated into electrospun fibrillar PLGA-HA

Handbook of Chitin and Chitosan

TABLE 8.3 Electrospun chitosan-based scaffolds intended for bone tissue engineering. Material

Electrospinning parameters

Significant results

Reference

Voltage (kV)

Flow rate (mL/h)

Needlecollector distance (cm)

CS/PEO with mesoporous silica NPS

16

1.0

15

AcAc (3%)/DMSO (10:1, w/w)

Cytocompatible to MC3T3 cells, promoted osteoblastic maturation and allowed mineralization

[177]

Cellulose acetate coated with CS and phosvitin

16




20

Acetone-N/ DMAc (2:1, w/w)

Excellent cytocompatibility to MC3T3 cells after mineral deposition (by incubation in SBF). Good cell growth, attachment and spreading on films

[178]

CS/PEO/BG

6

0.4

10

AcAc/water (80/20 volume ratio)

Induced hydroxyl carbonate apatite formation on its surface and allowed differentiation of hMSC

[179]

PCL-HA/CS/PCL-HA (layer by layer)

20




30

TFA/ ChloroformMethanol (75/25 v/v)

Increased cell growth and ALP activity of SaOs-2 osteosarcoma cells. Also provided cell attachment points

[180]

CS/PEO/silica particles

25

0.4

11

AcAc (0.5 M).

Promoted attachment and proliferation of boneforming 7F2. Also promoted nucleation of apatite crystals (by immersion in SBF)

[181]

CS/HA

17




12

TFA/DCM (70/30 % vol)

Increased the ALP expression of rat osteosarcoma UMR-106 cells. Formed nanosized hydroxyapatite crystals on its surface

[182]

Components

Solvent

(Continued)

TABLE 8.3

(Continued)

Material

Electrospinning parameters Needlecollector distance (cm)

Solvent

Significant results

Reference

Components

Voltage (kV)

Flow rate (mL/h)

Fe3O4/CS/PVA

20

0.3

10

AcAc (2%)

Cytocompatible to MG-63 cells and allowed their proliferation

[183]

HAp/PLGA loaded with CS NPS containing plasmid DNA sequence of BMP-2

10

5.0

15

DCM

Sustained release of CTS-DNA nanoparticles, enabled high transfection, and expression level of BMP-2, improving their bioactivity in bone defect of nude mice

[184]

CS/PEO Random and aligned fibers

17.8
22

1.0

15

AcAc:DMSO (ratio 10:1)

Fibers with both orientations permitted the adhesion and infiltration of hES-MP and MG63 cells. Besides, calcium deposition was similar on both fibers’ orientations cultured with hESMPs

[185]

CS neutralized

25

0.8

12

TFA:DCM (ratio 70:30)

CTS electrospun stable fibers were successfully obtained, showing the effectiveness of the electrospinning conditions and neutralization processes. They were used to mechanically reinforce microfibers of CTS/ PBS intended for BTE

[186]

CS/PLA

25

0.5

15

DCM, DMSO

Effectively mineralized with HA by (SBF) immersion

[187]

PHBV/CS/HA

13

1.0

10
15

HFP (8%), TFA:DCM (3:1]

Improved cell proliferation, mineral deposition and ALP activity of human fetal osteoblasts

[188]

CS/nano-HA

13




12

AcAc (2%)

Surface showed apatite-forming ability (bioactivity) in SBF

[189]

AcAc, acetic acid; ALP, alkaline phosphatase; BG, bioactive glass; BMP-2, bone morphogenetic protein-2; CS, chitosan; DCM, dichloromethane; DMAc, N-dimethylacetamide; DMSO, dimethyl sulfoxide; Fe3O4, magnetite; HA, hydroxyapatite; HFP, 1,1,1,3,3,3-hexafluoro-2-propanol; NPS, nanoparticles; PCL, polycaprolactone; PEO, poly(ethylene) oxide; PHBV, poly-3-hydroxybutyrate-co-3-hydroxyvalerate; PLA, poly(lactic acid); PVA, polyvinyl alcohol; PLGA, poly(lacticco-glycolic acid); SBF, simulated body fluid; TFA, trifluoroacetic acid.

262

8. Electrospun chitosan materials and their potential use as scaffolds

scaffolds. The PLGA/HA scaffolds loaded with the DNA
CS nanoparticles were implanted in bone segmental defects of nude mice, and the results revealed that the bioactivity of the BMP-2 plasmid, which was constantly released from the scaffold, enabled high transfection, improved formation of new bone, and enhanced healing of segmental defect [184].

8.5.2 Electrospun chitosan
inorganic particles composite scaffolds On the other hand, mechanical strength of pure-CS scaffolds is not enough for bone load-bearing applications. Nevertheless, mechanical properties of CS can also be improved by combining it with mechanically stronger materials such as synthetic polymers or by using reinforcement agents such as ceramic nanoparticles. Table 8.4 resumes the mechanical properties of different CS-containing electrospun scaffolds. CS mixtures with synthetic polymers, such as PEO, PCL, PVA, and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), reinforced or not with metallic or ceramic nanoparticles, have resulted in appropriate solutions for successful electrospinning of fibrillar CS-containing scaffolds with adequate mechanical properties for bone tissue engineering. The improvement of mechanical properties of CS
synthetic polymer(s) scaffolds regarding the constituent raw materials has been ascribed to the molecular interactions among polymers in the blend. For instance, it has been reported that hydrogen bonds formed between CS chains and PEO molecules in CS:PEO (95:5) electrospun scaffolds conferred them with sufficient mechanical stability to be handled. Appropriate interactions between cells and scaffolds are essential for bone regeneration, and cell
scaffold interactions are mainly regulated by the surface properties of the scaffold such as chemical composition, hydrophilic/hydrophobic character, and roughness. One of the main requisites for scaffolds intended for bone tissue engineering is the ability for promoting mineralization on their surface. In this respect, bioglasses (bioactive glasses, BG) are ceramic compounds that have the ability to initiate mineralization upon immersion in physiological fluids. Thus one of the most extensively used strategies to modify bone tissue engineering scaffolds is the incorporation of bioactive glasses such as phosphorus, calcium, silica, or carbonate compounds [190]. Moreover, BG incorporation can also improve the scaffolds mechanical properties [179], apart from improving their bioactivity. Functionalization of scaffolds surfaces by BG incorporation is based on their ability to promote deposition of calcium ions, which later induces crystallization of the apatite layer that mimics the chemical and structural composition of the inorganic phase of the bone ECM.

Handbook of Chitin and Chitosan

263

8.5 Chitosan electrospun materials for bone tissue engineering

TABLE 8.4

Mechanical parameters of different electrospun scaffolds containing CS.

Electrospun scaffold

Tensile strength (MPa)

Strain at break(%)

Young’s modulus (MPa)

References

PHBV/CS (90:10, w/w)

3.75 6 0.49

14.03 6 1.39

105.15 6 10.35

[188]

PHBV/CS/HA4 (85.5:9.5:5, w/w/w)

3.55 6 0.22

4.40 6 0.08

104.94 6 6.76

[188]

PHBV/CS/HA8 (81:9:10, w/w/w)

4.19 6 0.19

6.66 6 0.64

101.93 6 4.52

[188]

CS

0.45 6 0.01

40.09 6 2.50

0.66 6 0.02

[180]

CS/HA

0.32 6 0.13

73.12 6 2.64

0.44 6 0.01

[180]

PCL/CS

1.02 6 0.01

18.26 6 2.60

4.56 6 0.00

[180]

PCL/CS/HA (5 wt.%)

1.20 6 0.05

74.44 6 2.00

1.85 6 0.02

[180]

PCL
CS
PCL (layer-bylayer)

5.92 6 1.29

25.00 6 2.40

34.65 6 4.27

[180]

PCL/HA(5 wt.%)-CS-PCL/ HA(5 wt.%)(layer-by-layer)

14.37 6 2.69

15.50 6 0.42

70.90 6 9.80

[180]

CS/PVA

3.9 6 1.4

38.5 6 3.4

48.8 6 3.6

[183]

CS/PVA/Fe3O4 NPS (1 wt.%)

3.9 6 1.3

38.1 6 2.3

52.0 6 3.9

[183]

CS/PVA/Fe3O4 NPS (3 wt.%)

3.8 6 1.1

37.5 6 4.1

56.9 6 2.6

[183]

CS/PVA/Fe3O4 NPS (5 wt.%)

3.6 6 1.3

36.6 6 3.7

58.4 6 3.7

[183]

CS/PEO

1.58 6 0.2

2.5




[80]

CS/PEO/BG

3.01 6 0.15

4.0




[179]

BG, bioactive glass; CS, chitosan; Fe3O4, magnetite; HA, hydroxyapatite; NPS, nanoparticles; PCL, polycaprolactone; PEO, poly(ethylene) oxide; PHBV, poly-3-hydroxybutyrate-co-3-hydroxyvalerate; PVA, polyvinyl alcohol.

Bioactive mineralization-inducing molecules such as BG can be embedded into the electrospun fibers by adding them to the electrospinning solutions or can be used to coat the electrospun fibers after electrospinning [191]. Either way, the surface of the fibers is functionalized by enhancing the stiffness and roughness of the scaffold and promoting an osteoinductive response in bone cells. Talebian et al. [179] reported that the incorporation of BG particles (synthesized by sol
gel and with a final composition of SiO2 49.15 mol%, CaO 25.80 mol%, Na2O 23.33 mol %, and P2O5 1.73 mol%) into CS
PEO blend solutions resulted in electrospun nanofibers with hydrophilic character and increased surface roughness, and also improved the tensile strength of the scaffolds, in comparison to CS
PEO electrospun nanofibers without BG. The surface

Handbook of Chitin and Chitosan

264

8. Electrospun chitosan materials and their potential use as scaffolds

characteristics of the CS
PEO
BG scaffolds enhanced the activity of alkaline phosphatase (ALP) during the process of hMSC differentiation toward bone lineage cells [179]. On the same topic, Toskas et al. [171] developed electrospun nanofibers composed of CS
PEO and bioactive crystalline SiO2 particles synthesized by sol
gel that resulted in a selfassembly core
shell fiber structure with the silica component on the surface. Silicate ions in the fibers became silanol groups that effectively promoted the nucleation of apatite crystals on the CS
PEO
SiO2 scaffold’s surface upon immersion in simulated body fluid (SBF) for 7 days. It is important to mention that immersion in SBF is a technique commonly used to study scaffolds potential bioactivity (mineralization) in vitro, because it can induce hydroxyapatatite (HA) precipitation on the surface of the fibers [192]. In the study by Toskas et al. [171], SiO2 particles also formed hydrogen bonds with the CS
PEO compound, and the epoxy groups of silanol on the fibers surface formed covalent bonds with the amine groups of CS, which protect the fibers’ core components from fast decomposition, improve tensile strength of the scaffolds, and generate surface roughness [181]. The scaffolds were able to maintain the cell culture of hMSC and mouse osteoblasts 7F2, which presented extended cell morphology upon culture on the fibers. In addition, the use of SiO2 bioactive particles favored the nucleation of calcium and phosphorus ions leading to mineralization of the nanofibers, which presented crystals similar to HA after 14 days of culture. Mesoporous silica nanoparticles (MSiNps) are other bioactive ceramics extensively used to develop scaffolds for bone tissue engineering. MSiNps present a honeycomb-like structure with high pore volume and controllable pore size. The porous nature of MSiNps allows their mechanical interlocking with polymers, forming strong bonds that can reduce the interfacial adhesion problems commonly observed with other reinforcement materials. Li et al. [177] developed nanofibrous MSiNps
CS scaffolds, showing that MSiNps incorporation into CS nanofibers weakened the mechanical strength of the composite scaffold, making it brittle but increased the scaffold’s modulus of elasticity and surface roughness. Nevertheless, MSiNps
CS composite materials were mechanically stable enough for biodegradability, handling, and capable of maintaining mouse osteoblasts’ viability and increasing their ALP activity, which suggests that MSiNps
CS scaffolds allowed the maturation of osteocytes [177]. CS
nanoHA electrospun scaffolds developed by Shanhnavazi et al. also showed significant bioactivity after immersion in SBF [189]. Formation of HA nanoparticles on the scaffolds surface was a consequence of precipitation, nucleation, and growth of calcium phosphate during SBF incubation [189]. In a study by Sasmazel et al. [180], functionalization of scaffolds surface was performed by means of HA particles’

Handbook of Chitin and Chitosan

8.5 Chitosan electrospun materials for bone tissue engineering

265

TABLE 8.5 Electrospinning conditions for development of CS electrospun scaffolds intended for cartilage repair. Polymer blend

Electrospinning conditions

CS/PEO

20
24 kV

Solvent used

Cell line tested

Reference

Chondrocyte cell line (HTB-94)

[196]

17
20 cm

Acetic acid Triton X100

PCL/CS

18
30 kV

Acetic acid

MSC

[197]

PCL/CMC

0.1
1.0 mL/h

Formic acid

Acetic acid Triton X100

MSC

[198]

150 mm Rotational speed 100 rpm PCL grate- coated with CTS/PEO fibers

20 kV 20 cm 2 cm/min 10 rpm

PHB/CS

9, 13 kV 0.5 mL/h 7 and 14 cm

TFA

Rabbit chondrocytes

[199]

CS/SF

25 kV 1.5 mL/h 12
18 cm

Acetic acid

Rabbits chondrocytes

[200]

CS/PVA/CaCO3

17 kV 10 μL/min 15 cm

Acetic acid

Chondrocyte cell line (ATDC5)

[201]

CS/PEO

12 and 29 cm 592
1779 rpm

Acetic acid

Canine chondrocytes

[202]

CS

18 kV 0.06 mL/min

HFIP/ methylene chloride

Bovine chondrocytes

[203]

P3HB/CS/MWNTs

20 kV 0.5 mL/h 20 cm

TFA

Rabbit chondrocytes

[204]

incorporation, developing a multilayered, PCL-HA/CS/PCL-HA, 3D electrospun scaffold. This combination resulted in the improvement of mechanical properties by increasing the elastic modulus and the tensile strength of the scaffolds; however, elongation of the scaffolds decreased. Incorporation of HA into the scaffolds generated a suitable hydrophilic environment for adhesion and proliferation of

Handbook of Chitin and Chitosan

266

8. Electrospun chitosan materials and their potential use as scaffolds

SaOs-2 osteosarcoma cells, increasing the ALP activity of these cells after 14 days of culture [180]. In general, bioactive molecules have been incorporated into the electrospun nanofibers by adding them to the electrospinning solutions. However, surface coating strategies have also been used to improve electrospun scaffolds’ bioactivity. For instance, apatite-coated surface modifications have been used for promoting fixation/integration between the scaffold and the surrounding bone to promote bone regeneration [193]. Liang et al. [178] reported the functionalization of electrospun nanofibers for bone tissue engineering by alternatively coating cellulose electrospun fibers with positively charged CS and negatively charged phosvitin (PV) via layer-by-layer coating. Morever, in vitro biomimetic mineralization of the cellulose
CS
PV scaffolds resulted in nanofibers coated with HA crystals that increased the diameter of the nanofibers to up to 2 μm. In addition, both compounds, CS and PV, improved apatite minerals nucleation on the fibers creating a homogeneous mineral coating that favored MC3T3-E1 cells’ adhesion and viability [178]. Mineralization with HA crystals on the surface of electrospun CS
PLA nanofibers via direct SBF immersion has also been reported [187]. It is also important to mention that CS nanofibers with neither coatings nor incorporation of BG likewise have the ability to be coated with calcium hydroxide layers upon immersion in SBF, which encourages HA formation on their surface which favors their biocompatibility and osteoblasts’ metabolic activity [182]. Rawlinson et al. [185] assessed the bioactivity of randomly oriented CS electrospun fibers and aligned electrospun CS fibers by quantifying total calcium deposition on the fibers after culturing with hES-MPs cells. Cellular viability and mineral deposition response indicated that randomly orientated fibers promoted the proliferation of osteoblastic cells and allowed the deposition of ECM [185].

8.6 Chitosan electrospun materials for cartilage tissue engineering Different CS-based electrospun scaffolds for cartilage tissue engineering have been developed due to its beneficial properties. In the case of cartilage, the use of CS as a biomaterial represents a structural similarity to that of the GAGs found in the native cartilage ECM [194], which are known to play a significant role in stimulating cartilage chondrogenesis. CS can also support chondrocytes’ attachment and proliferation by maintaining its morphology and preserving the expression of the specific proteins of chondrocytes [195]. Scaffold structure also plays a major

Handbook of Chitin and Chitosan

8.6 Chitosan electrospun materials for cartilage tissue engineering

267

role in cartilage tissue engineering since its characteristics may influence cell attachment, infiltration, and proliferation on the material. In this sense, electrospun scaffolds are gaining attention because they consist of fibrillar arrangements that mimic the structure of the natural ECM of cartilage. Thus fabrication of CS-based electrospun scaffolds for cartilage repair seems quite promising. The main cell sources for cartilage tissue engineering are chondrocytes and MSC, and consequently research efforts have been focused on developing scaffolds capable of maintaining chondrocytes’ morphology and phenotype or stimulating the differentiation of MSC from chondrocytes. Chondrogenesis is associated with several marker molecules, typically including collagen type II, SOX9, GAGs, and aggrecan, which help in constructing the cartilaginous ECM. As previously mentioned, electrospinning CS itself represents a challenge, which is why different electrospinning conditions and solvents have been tested, and most studies report the electrospinning of CS blends with other synthetic or natural polymers. Table 8.5 summarizes different electrospinning conditions used for development of CScontaining scaffolds intended for cartilage repair [196
204]. Shim et al. [203] prepared bare CS electrospun nanofibers by dissolving CS in a mix of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and methylene chloride. The complete scaffold consisted of a nanofibrous matrix of CS electrospun onto a predefined microfibrous CS mesh. Then, a 3D scaffold was produced by rolling the double-layered nano-/microfibrous membrane. In order to compare the influence of CS scaffold architecture on chondrocytes’ behavior, chondrocyte cells were seeded on CS nanofibrous membranes, microfibrous membranes, and flat films as a control surface. The CS nanofibrous surface substantially facilitated cellular attachment and proliferation and prevented phenotypic changes of chondrocytes when compared with chondrocytes cultured on CS microfibrous membranes or flat films. In order to produce electrospun fibers with high CS content, the addition of PEO is commonly used to reduce the viscosity of the CS solutions so that the solution is spinnable at high polymer concentrations [196]. Subramanian et al. [202] obtained electrospun CS mats formed by oriented submicron fibers and tested their tensile properties and biocompatibility with chondrocytes. To achieve this, PEO was added to a CS solution dissolved in acetic acid, and a rotating collector was used to produce fibrous structures with a certain degree of alignment. Scaffolds exhibited fiber diameters of 0.3 μm and chondrocyte cell viability of 69%. In a similar way, Bhattarai et al. [196] obtained electrospun CS by adding PEO in a ratio of 9 to 1 of the electrospinning solution. To obtain better fibrous structures at high CS:PEO ratio, Triton X100 was introduced into the solution as a nonionic surfactant. Triton addition decreased surface tension, which greatly improved

Handbook of Chitin and Chitosan

268

8. Electrospun chitosan materials and their potential use as scaffolds

solution spinnability. Finally, dimethyl sulfoxide (DMSO) was introduced into the solution as a cosolvent to improve processing conditions and increase fiber yields. Synthesized nanofibrous membranes retained good structural integrity in water and exhibited better adhesion of chondrocytes than their solvent casted films counterpart. In another study, Ainola et al. [198] obtained a PCL porous support coated with an electrospun CS
PEO nanofibrillar sheet in different CS: PEO mass ratios. CS:PEO mass ratios of 80:20 and 60:40 worked best in these experiments. Ainola et al. [198] also added Triton X100 to the electrospinning solution, which almost completely eliminated the formation of beads in the fibers. MSC were seeded on the CS
PEO materials, evidencing that the produced nanofibrillar membranes stimulated chondrocyte differentiation as confirmed by cell’s expression of chondrogenic markers, SOX9, RunX2, and collagen type II. Lack of chondrogenic cell differentiation was absent when MSCs were seeded on regular culture tissue plates. Even though CS nanofibers are achievable, most of the research preferred to blend CTS with other polymers. For cartilage purposes, polymers like silk fibroin [205], PVA [201], PCL [197], polyhydroxybutyrate (PHB) [206], and 3-hydroxybutyrate (P3HB) [204] have been used as electrospinning biomaterials. Sadeghi et al. [206] blended PHB with CS to produce more hydrophilic electrospun fibrous scaffolds as the addition of CS to PHB increased the hydrophilicity of PHB. SEM images showed that on pure PHB scaffolds, chondrocyte cells attached to the fibers but did not spread well. However, on CS
PHB scaffolds, chondrocytes attached, spread, and slightly penetrated the polymer fibrillar matrix, demonstrating that CS addition was beneficial for cell culture. Alemi et al. [197] produced PCL
CS and PCL
carboxy methyl chitosan (CMC) electrospun nanofibers. Both scaffolds were then modified by surface plasma treatment to enhance cell attachment and proliferation. MSCs were seeded on the scaffolds and MSC differentiation to cartilage cells phenotype was corroborated after 21 days of culture, through positive expression of SOX9 and collagen type II genes as well as through collagen type II protein positive expression. Liu et al. [205] produced a bilayered fibrillar scaffold by electrospinning a bottom layer of CS
HA and a top layer of CS
silk fibroin to achieve a bone-like and a chondral-like layer, respectively. After 21 days of chondrocytes culture on the chondral-like layer, chondrocytes’ expression of collagen type II and GAGs increased, providing evidence of proliferation and phenotype preservation of chondrocytes. The addition of components in the nanometric scale to CS blends has also been explored, for instance, Sambudi et al. [201] mixed CS with PVA to improve CS processability into electrospun fibers. Additionally, they incorporated CaCO3 nanoparticles to increase the mechanical

Handbook of Chitin and Chitosan

References

269

properties of the fibrillar membranes. Proliferation of ATDC5 cells was tested revealing that CS
PVA
CaCO3 scaffolds with 4 wt.% CaCO3 provided the most suitable environment for cell growth in comparison to CS
PVA fibers with no CaCO3. Mirmusavi et al. [204] added multiwalled carbon nanotubes (MWNTs) into P3HB
CS polymer solutions and fabricated nanofibrous membranes electrospun on silk microfibrillar substrates, and found that higher hydrophilicity and surface area of the scaffolds containing MWNTs enhanced chondrocyte cell attachment. It was also stated that the presence of CS in the scaffolds led to a better environment for the culture of chondrocytes, as evidenced by the results obtained at 7 days of culture.

Acknowledgments The authors would like to thank Consejo Nacional de Ciencia y Tecnologı´a (CONACyT). Miss Claudia H. Barrera is greatly acknowledged for diligent editing and proofreading of this paper.

References [1] M. Janmohammadi, M.S. Nourbakhsh, Electrospun polycaprolactone scaffolds for tissue engineering: a review, Int. J. Polym. Mater. Polym. Biomater. (2018) 1
13. [2] S. Pina, V.P. Ribeiro, C.F. Marques, F.R. Maia, T.H. Silva, R.L. Reis, et al., Scaffolding strategies for tissue engineering and regenerative medicine applications, Materials 12 (1) (2019) 1824. [3] S. Ranganathan, K. Balagangadharan, N. Selvamurugan, Chitosan and gelatin-based electrospun fibers for bone tissue engineering, Int. J. Biol. Macromol. 133 (2019) 354
364. [4] J.A. Gasser, M. Kneissel, Bone physiology and biology digital integrated mobility solutions and pharmacological treatments for tendon, muscle and cartilage regeneration view project PTH agonist for osteoporosis therapy view project, in: S.Y. Smith, A. Varela, R. Samadfam (Eds.), Bone Toxicology, Springer, Switzerland, 2017, pp. 27
94. [5] T.M. Post, S.C.L.M. Cremers, T. Kerbusch, M. Danhof, Bone physiology, disease and treatment: towards disease system analysis in osteoporosis, Clin. Pharmacokinet. 49 (2) (2010) 89
118. [6] X. Yang, Z. Li, Influence of hydroxyapatite and BMP-2 on bioactivity and bone tissue formation ability of electrospun PLLA nanofibers, J. Appl. Polym. Sci. 132 (28) (2015). [7] L. Imbert, S. Gourion-Arsiquaud, E. Villarreal-Ramirez, L. Spevak, H. Taleb, M.C.H. van der Meulen, et al., Dynamic structure and composition of bone investigated by nanoscale infrared spectroscopy, PLoS One 13 (9) (2018). [8] Y. Bala, E. Seeman, Bone’s material constituents and their contribution to bone strength in health, disease, and treatment, Calcif. Tissue Int. 97 (3) (2015) 308
326. [9] A.L. Boskey, P.G. Robey, The composition of bone, in: C.J. Rosen (Ed.), Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, eighth ed., WileyBlackwell, New Jersey, 2013. [10] N. Reznikov, R. Shahar, S. Weiner, Bone hierarchical structure in three dimensions, Acta Biomater. 10 (9) (2014) 3815
3826.

Handbook of Chitin and Chitosan

270

8. Electrospun chitosan materials and their potential use as scaffolds

[11] S.L. Dallas, M. Prideaux, L.F. Bonewald, The osteocyte: an endocrine cell. . .and more, Endocr. Rev. 34 (5) (2013) 658
690. [12] X. Feng, S.L. Teitelbaum, Osteoclasts: new insights, Bone Res. 1 (1) (2013) 11
26. [13] B. Clarke, Normal bone anatomy and physiology, Clin. J. Am. Soc. Nephrol. 3 (Suppl. 3) (2008) 131
139. [14] S. Birajdar, M. Sharp, S. Patole, Metabolic bone disease of prematurity, in: S. Patole (Ed.), Nutrition for the Preterm Neonate: A Clinical Perspective, Springer, Dordrecht, 2013, pp. 115
134. [15] M. Rauner, N. Stein, L.C. Hofbauer, Basics of bone biology, in: P. Pietschmann (Ed.), Principles of Osteoimmunology: Molecular Mechanisms and Clinical Applications, Springer, Switzerland, 2012, pp. 1
30. [16] E. Brett, M.T. Longaker, J. Flacco, C. Blackshear, D.C. Wan, Biomimetics of bone implants: the regenerative road, BioRes. Open. Access. 6 (1) (2017) 1
6. [17] A. Sculean, D. Nikolidakis, G. Nikou, A. Ivanovic, I.L.C. Chapple, A stavropoulos biomaterials for promoting periodontal regeneration in human intrabony defects: a systematic review, Periodontol. 2000. 68 (1) (2015) 182
216. [18] E. Jimi, S. Hirata, K. Osawa, M. Terashita, C. Kitamura, H. Fukushima, The current and future therapies of bone regeneration to repair bone defects, Int. J. Dent. (2012) 1
7. [19] A. Wiese, H.C. Pape, Bone defects caused by high-energy injuries, bone loss, infected nonunions, and nonunions, Orthop. Clin. North. Am. 41 (1) (2010). [20] Y. Fillingham, J. Jacobs, Bone grafts and their substitutes, Bone Joint J. 98-B (1) (2016) 6
9. [21] D. Tang, R.S. Tare, L.Y. Yang, D.F. Williams, K.L. Ou, R.O.C. Oreffo, Biofabrication of bone tissue: approaches, challenges and translation for bone regeneration, Biomaterials 83 (2016) 363
382. [22] A. Hagen, V. Gorenoi, M.P. Scho¨nermark, Bone graft substitutes for the treatment of traumatic fractures of the extremities, GMS Health Technol. Assess. 8 (2012). [23] K.S. Griffin, K.M. Davis, T.O. McKinley, J.O. Anglen, T.M.G. Chu, J.D. Boerckel, et al., Evolution of bone grafting: bone grafts and tissue engineering strategies for vascularized bone regeneration, Clin. Rev. Bone Miner. Metab. 13 (4) (2015). [24] M. Rai, A.P. Ingle, S. Medici, Biomedical Applications of Metals, Springer, Switzerland, 2018. [25] J.J. Jacobs, R.M. Urban, N.J. Hallab, Corrosion and biocompatibility of orthopedic implants, in: M.J. Yaszemski, D.J. Trantolo, K. Lewandrowski, V. Hasirci, D.E. Altobelli, D.L. Wise (Eds.), Biomaterials in Orthopedics, Marcel Dekker, New York, 2013, pp. 63
92. [26] Q. Chen, C. Zhu, G.A. Thouas, Progress and challenges in biomaterials used for bone tissue engineering: bioactive glasses and elastomeric composites, Prog. Biomater. 1 (1) (2012) 1
22. [27] S. Dorozhkin, Medical application of calcium orthophosphate bioceramics, BIO 1 (2011) 1
51. [28] J. Albrektsson, Osteoinduction, osteoconduction and osseointegration, Eur. Spine J. 10 (2001) S96
S101. [29] E. Denes, G. Barrie`re, E. Poli, G. Le´veˆque, Commentary: bioceramics and scaffolds: a winning combination for tissue engineering, Front. Bioeng. Biotechnol. 5 (2017) 15. [30] L.L. Hench, Bioceramics: from concept to clinic, J. Am. Ceram. Soc. 74 (7) (1991) 1487
1510. [31] V. Martin, A. Bettencourt, Bone regeneration: biomaterials as local delivery systems with improved osteoinductive properties, Mater. Sci. Eng. 82 (2018) 363
371. [32] Z. Tang, X. Li, Y. Tan, H. Fan, X. Zhang, The material and biological characteristics of osteoinductive calcium phosphate ceramics, Regen. Biomater. 5 (1) (2018) 43
59.

Handbook of Chitin and Chitosan

References

271

[33] R. Shi, Y. Huang, C. Ma, C. Wu, W. Tian, Current advances for bone regeneration based on tissue engineering strategies, Front. Med. 13 (2) (2019) 160
188. [34] A. Hasan, B. ByambaA, M. Morshed, M.I. Cheikh, R.A. Shakoor, T. Mustafy, et al., Advances in osteobiologic materials for bone substitutes, J. Tissue Eng. Regen. Med. 12 (6) (2018) 1448
1468. [35] T. Gong, J. Xie, J. Liao, T. Zhang, S. Lin, Y. Lin, Nanomaterials and bone regeneration, Bone Res. 3 (1) (2015). [36] L.M. Cross, A. Thakur, N.A. Jalili, M. Detamore, A.K. Gaharwar, Nanoengineered biomaterials for repair and regeneration of orthopedic tissue interfaces, Acta Biomater. 42 (2016) 2
17. [37] R.E. McMahon, L. Wang, R. Skoracki, A.B. Mathur, Development of nanomaterials for bone repair and regeneration, J. Biomed. Mater. Res. Part B Appl. Biomater. 101 (2) (2013) 387
397. [38] S. Wu, X. Liu, K.W.K. Yeung, C. Liu, X. Yang, Biomimetic porous scaffolds for bone tissue engineering, Mater. Sci. Eng. R: Rep. 80 (2014) 1
36. [39] C. Ning, Biomaterials for bone tissue engineering, in: D.G. Poitout (Ed.), Biomechanics and Biomaterials in Orthopedics, second ed., Springer, London, 2016, pp. 35
58. [40] A.K. Dewan, M.A. Gibson, J.H. Elisseeff, M.E. TricE, Evolution of autologous chondrocyte repair and comparison to other cartilage repair techniques, BioMed. Res. Int. (2014) 1
11. [41] T.F. LaPorta, A. Richter, N.A. Sgaglione, D.A. Grande, Clinical relevance of scaffolds for cartilage engineering, Orthop. Clin. 43 (2) (2012) 245
254. [42] S.R. Goldring, M.B. Goldring, Changes in the osteochondral unit during osteoarthritis: structure, function and cartilage
bone crosstalk, Nat. Rev. Rheumatol. 12 (11) (2016) 632
644. [43] G.M. Salzmann, P. Niemeyer, A. Hochrein, M.J. Stoddart, P. Angele, Articular cartilage repair of the knee in children and adolescents, Orthop. J. Sports Med. 6 (3) (2018) 1
12. [44] Z.H. Deng, Y.S. Li, X. Gao, G.H. Lei, J. Huard, Bone morphogenetic proteins for articular cartilage regeneration, Osteoarthr. Cartil. 26 (9) (2018) 1153
1161. [45] R.M. Jeuken, A.K. Roth, J.R.W. Peters, C.C. van Donkelaar, J.C. Thies, L.W. van Rhijn, et al., Polymers in cartilage defect repair of the knee: current status and future prospects, Polymers 8 (6) (2016) 1
30. [46] K. Atesok, M.N. Doral, J. Karlsson, K.A. Egol, L.M. Jazrawi, P.G. Coelho, et al., Multilayer scaffolds in orthopaedic tissue engineering, Knee Surg. Sport. Traumatol. Arthrosc. 24 (7) (2016) 2365
2373. [47] M. Wang, Z. Yuan, N. Ma, C. Hao, W. Guo, G. Zou, et al., Advances and prospects in stem cells for cartilage regeneration, Stem Cell Int. (2017) 1
16. [48] J. Julin, E. Ja¨msen, T. Puolakka, Y.T. Konttinen, T. Moilanen, Younger age increases the risk of early prosthesis failure following primary total knee replacement for osteoarthritis. A follow-up study of 32,019 total knee replacements in the Finnish Arthroplasty Register, Acta Orthop. 81 (4) (2010) 413
419. ˚ roøen, S. Løken, S. Heir, E. Alvik, A. Ekeland, O.G. Granlund, et al., Articular [49] A. A cartilage lesions in 993 consecutive knee arthroscopies, Am. J. Sports Med. 32 (1) (2004) 211
215. [50] W. Widuchowski, J. Widuchowski, T. Trzaska, Articular cartilage defects: study of 25,124 knee arthroscopies, Knee 14 (3) (2007) 177
182. [51] K. Hjelle, E. Solheim, T. Strand, R. Muri, M. Brittberg, Articular cartilage defects in 1,000 knee arthroscopies, Arthroscopy 18 (7) (2002) 730
734. [52] K.A. Athanasiou, D.J. Responte, W.E. Brown, J.C. Hu, Harnessing biomechanics to develop cartilage regeneration strategies, J. Biomech. Eng. 137 (2) (2015).

Handbook of Chitin and Chitosan

272

8. Electrospun chitosan materials and their potential use as scaffolds

[53] A. Guermazi, H. Alizai, M.D. Crema, S. Trattnig, R.R. Regatte, F.W. Roemer, Compositional MRI techniques for evaluation of cartilage degeneration in osteoarthritis, Osteoarthr. Cartil. 23 (10) (2015) 1639
1653. [54] M.D. Crema, M.C. Nevitt, A. Guermazi, D.T. Felson, K. Wang, J.A. Lynch, et al., Progression of cartilage damage and meniscal pathology over 30 months is associated with an increase in radiographic tibiofemoral joint space narrowing in persons with knee OA—the MOST study, Osteoarthr. Cartil. 22 (10) (2014) 1743
1747. [55] J.R. Giuliani, A. Pickett, Cell-based chondral restoration, Curr. Rev. Musculoskelet. Med. 8 (4) (2015) 436
442. [56] T.A.E. Ahmed, M.T. Hincke, Strategies for articular cartilage lesion repair and functional restoration, Tissue Eng. 16 (3) (2010) 305
329. [57] F. McCormick, J.D. Harris, G.D. Abrams, R. Frank, A. Gupta, K. Hussey, et al., Trends in the surgical treatment of articular cartilage lesions in the United States: an analysis of a large private-payer database over a period of 8 years, Arthroscopy 30 (2) (2014) 222
226. [58] R. Mundi, A. Bedi, L. Chow, S. Crouch, N. Simunovic, E. Sibilsky, et al., Cartilage restoration of the knee, Am. J. Sports Med. 44 (7) (2016) 1888
1895. [59] M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, L. Peterson, Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, N. Engl. J. Med. 331 (14) (1994) 889
895. [60] C.B. Foldager, J. Farr, A.H. Gomoll, Patients scheduled for chondrocyte implantation treatment with MACI have larger defects than those enrolled in clinical trials, Cartilage 7 (2) (2016) 140
148. [61] G. Filardo, E. Kon, A. Roffi, A. Di Martino, M. Marcacci, Scaffold-based repair for cartilage healing: a systematic review and technical note, Arthroscopy 29 (1) (2013) 174
186. [62] S. Fischer, A. Kisser, Single-step scaffold-based cartilage repair in the knee: a systematic review, J. Orthop. 13 (4) (2016) 246
253. [63] J. Guo, G.W. Jourdian, D.K. Maccallum, Culture and growth characteristics of chondrocytes encapsulated in alginate beads, Connect. Tissue Res. 19 (2
4) (1989) 277
297. [64] V.F. Sechriest, Y.J. Miao, C. Niyibizi, A. Westerhausen-Larson, H.W. Matthew, C.H. Evans, et al., GAG-augmented polysaccharide hydrogel: a novel biocompatible and biodegradable material to support chondrogenesis, J. Biomed. Mater. Res. 49 (4) (2000) 534
541. [65] A.I. Formhals, Process and apparatus for preparing artificial threads, US Patent No. 1,975,504, 1934. [66] D. Rodoplu, M. Mutlu, Effects of electrospinning setup and process parameters on nanofiber morphology intended for the modification of quartz crystal microbalance surfaces, J. Eng. Fibers Fabr. 7 (2) (2012) 118
123. [67] N. Okutan, P. Terzi, F. Altay, Affecting parameters on electrospinning process and characterization of electrospun gelatin nano fibers, Food Hydrocoll. 39 (C) (2014) 19
26. [68] J.M. Deitzel, J. Kleinmeyer, D. Harris, N.C.B. Tan, The effect of processing variables on the morphology of electrospun nanofibers and textiles, Polymer 42 (1) (2001) 261
272. [69] S. Zahmatkesh, S. Mojtaba, M. Ebrahim, E. Dabiri, Synthesis of ZnO/In2 O3 composite nanofibers by co-electrospinning: a comprehensive parametric investigating the process, Ceram. Int. 45 (2) (2019) 2530
2541. [70] A. Haider, S. Haider, I. Kang, A comprehensive review summarizing the effect of electrospinning parameters and potential applications of nanofibers in biomedical and biotechnology, Arab. J. Chem. 11 (8) (2018) 1165
1188.

Handbook of Chitin and Chitosan

References

273

[71] T. Grothe, J. Brikmann, H. Meissner, A. Ehrmann, Influence of solution and spinning parameters on nanofiber mat creation of poly (ethylene oxide) by needleless electrospinning, Medˇziagotyra 23 (4) (2017) 342
349. [72] B. Tarus, N. Fadel, A. Al-oufy, M. El-messiry, Effect of polymer concentration on the morphology and mechanical characteristics of electrospun cellulose acetate and poly (vinyl chloride) nanofiber mats, Alex. Eng. J. 55 (3) (2016) 2975
2984. [73] L. Yijun, D. Qinghua, J. Riguang, Effects of processing variables on the morphology and diameter of electrospun poly (amino acid ester) phosphazene nanofibers, J. Wuhan. Univ. Technol.-Mater 27 (2) (2012) 207
211. [74] A. Jaworek, A. Krupa, M. Lackowski, A.T. Sobczyk, T. Czech, S. Ramakrishna, et al., Nanocomposite fabric formation by electrospinning and electrospraying technologies, J. Electrost. 67 (2-3) (2009) 435
438. [75] J. Du, X. Zhang, Role of polymer
salt
solvent interactions in the electrospinning of polyacrylonitrile/iron acetylacetonate, J. Appl. Polym. Sci. 109 (5) (2008) 2935
2941. [76] E.S. Medeiros, L.H.C. Mattoso, R.D. Offeman, D.F. Wood, W.J. Orts, Effect of relative humidity on the morphology of electrospun polymer fibers, Can. J. Chem. 86 (6) (2008) 590
599. [77] V. Guarino, M. Alvarez-Perez, V. Cirillo, L. Ambrosio, HMSC interaction with PCL and PCL/gelatin platforms: a comparative study on films and electrospun membranes, J. Bioact. Compat. Polym. 26 (2) (2011) 144
160. [78] Y.C. Kuo, S.C. Hung, S.H. Hsu, The effect of elastic biodegradable polyurethane electrospun nanofibers on the differentiation of mesenchymal stem cells, Colloids Surf. 122 (2014) 4141
4422. [79] T. Okuda, K. Tominaga, S. Kidoaki, Time-programmed dual release formulation by multilayered drug-loaded nanofiber meshes, J. Control. Release 143 (2) (2010) 258
264. [80] G. Prado-Prone, P. Silva-Bermudez, A. Almaguer-Flores, J.A. Garcı´a-Macedo, V.I. Garcı´a, S.E. Rodil, et al., Enhanced antibacterial nanocomposite mats by coaxial electrospinning of polycaprolactone fibers loaded with Zn-based nanoparticles, Nanomedicine 14 (5) (2018) 1695
1706. [81] H.J. Lai, C.H. Kuan, H.C. Wu, J.C. Tsai, T.M. Chen, D.J. Hsieh, et al., Tailored design of electrospun composite nanofibers with staged release of multiple angiogenic growth factors for chronic wound healing, Acta Biomater. 10 (10) (2014) 4156
4166. [82] A. Jain, L.S. Duvvuri, S. Farah, N. Beyth, A.J. Domb, W. Khan, Antimicrobial polymers, Adv. Healthc. Mater. 3 (12) (2014) 1969
1985. [83] T. Jiang, E.J. Carbone, K.W.H. Lo, C.T. Laurencin, Electrospinning of polymer nanofibers for tissue regeneration, Prog. Polym. Sci. 46 (C) (2015) 1
24. [84] T. Fee, S. Surianarayanan, C. Downs, Y. Zhou, J. Berry, Nanofiber alignment regulates NIH3T3 cell orientation and cytoskeletal gene expression on electrospun PCL 1 gelatin nanofibers, PLoS ONE 11 (5) (2016) 1
12. [85] J. Jiang, J. Xie, B. Ma, D.E. Bartlett, A. Xu, C.H. Wang, Mussel-inspired protein-mediated surface functionalization of electrospun nanofibers for pH-responsive drug delivery, Acta Biomater. 10 (3) (2014) 1324
1332. [86] F. Chen, Q.L. Tang, Y.J. Zhu, K.W. Wang, M.L. Zhang, W.Y. Zhai, et al., Hydroxyapatite nanorods/poly(vinyl pyrolidone) composite nanofibers, arrays and three-dimensional fabrics: electrospun preparation and transformation to hydroxyapatite nanostructures, Acta Biomater. 6 (8) (2010) 3013
3020. [87] M.R. Badrossamay, H.A. McIlwee, J.A. Goss, K.K. Parker, Nanofiber assembly by rotary jet-spinning, Nano Lett. 10 (6) (2010) 2257
2261. [88] P. Uttayarat, A. Perets, M. Li, P. Pimton, S.J. Stachelek, I. Alferiev, et al., Micropatterning of three-dimensional electrospun polyurethane vascular grafts, Acta Biomater. 6 (11) (2010) 4229
4237.

Handbook of Chitin and Chitosan

274

8. Electrospun chitosan materials and their potential use as scaffolds

[89] L. Moroni, R. Schotel, D. Hamann, J.R. De Wijn, C.A. Van Blitterswijk, 3D fiberdeposited electrospun integrated scaffolds enhance cartilage tissue formation, Adv. Funct. Mater. 18 (1) (2008) 53
60. [90] D. Meng, M. Erol, A.R. Boccaccini, Processing technologies for 3D nanostructured tissue engineering scaffolds, Adv. Eng. Mater. 12 (9) (2010) B467
B487. [91] S. Hong, G. Kim, Fabrication of size-controlled three-dimensional structures consisting of electrohydrodynamically produced polycaprolactone micro/nanofibers, Appl. Phys. A 103 (4) (2011) 1009
1014. [92] Y. Yokoyama, S. Hattori, C. Yoshikawa, Y. Yasuda, H. Koyama, T. Takato, et al., Novel wet electrospinning system for fabrication of spongiform nanofiber 3-dimensional fabric, Mater. Lett. 63 (3
10) (2009) 754
756. [93] T. Kasuga, A. Obata, H. Maeda, Y. Ota, X. Yao, K. Oribe, Siloxane-poly(lactic acid)vaterite composites with 3D cotton-like structure, J. Mater. Sci. Mater. Med. 23 (10) (2012) 2349
2357. [94] S.B. Orr, A. Chainani, K.J. Hippensteel, A. Kishan, C. Gilchrist, N.W. Garrigues, et al., Aligned multilayered electrospun scaffolds for rotator cuff tendon tissue engineering, Acta Biomater. 24 (2015) 117
126. [95] S. Soliman, S. Pagliari, A. Rinaldi, G. Forte, R. Fiaccavento, F. Pagliari, et al., Multiscale three-dimensional scaffolds for soft tissue engineering via multimodal electrospinning, Acta Biomater. 6 (4) (2010) 1227
1237. [96] H. El Knidri, R. Belaabed, A. Addaou, A. Laajeb, A. Lahsini, Extraction, chemical modification and characterization of chitin and chitosan, Int. J. Biol. Macromol. 120 (Pt A) (2018) 1181
1189. ¨ zogul, J.M. Regenstein, Industrial applications of crustacean by[97] I. Hamed, F. O products (chitin, chitosan, and chitooligosaccharides): a review, Trends Food Sci. Technol. 48 (C) (2016) 40
50. [98] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. Structure, properties and applications, Mar. Drugs 13 (3) (2015) 1133
1174. [99] I. Aranaz, M. Mengibar, R. Harris, B. Miralles, N. Acosta, L. Calderon, et al., Role of physicochemical properties of chitin and chitosan on their functionality, Curr. Chem. Biol. 8 (1) (2014) 27
42. [100] K. Saekhor, W. Udomsinprasert, S. Honsawek, W. Tachaboonyakiat, Preparation of an injectable modified chitosan-based hydrogel approaching for bone tissue engineering, Int. J. Biol. Macromol. 123 (2018) 167
173. [101] S.K. Shukla, A.K. Mishra, O.A. Arotiba, B.B. Mamba, Chitosan-based nanomaterials: a state-of-the-art review, Int. J. Biol. Macromol. 59 (2013) 46
58. [102] F. Croisier, C. Je´roˆme, Chitosan-based biomaterials for tissue engineering, Eur. Polym. J. 49 (4) (2013) 780
792. [103] S.J.K. Francis, H.W.T. Matthew, Application of chitosan based polysaccharide biomaterials in cartilage tissue engineering, Biomaterials. 21 (2000) 2589
2598. [104] K. Kalantari, A.M. Afifi, H. Jahangirian, T.J. Webster, Biomedical applications of chitosan electrospun nanofibers as a green polymer
review, Carbohydr. Polym. 207 (2019) 588
600. [105] J. Desbrie`res, E. Guibal, Chitosan for wastewater treatment, Polym. Int. 67 (1) (2018) 7
14. [106] V. Manigandan, R. Karthik, S. Ramachandran, S. Rajagopal, Chitosan applications in food industry, Biopolym. Food Des. 20 (2018) 469
491. [107] A. Ali, S. Ahmed, A review on chitosan and its nanocomposites in drug delivery, Int. J. Biol. Macromol. 109 (2018) 273
286. [108] I. Aranaz, N. Acosta, C. Civera, B. Elorza, J. Mingo, C. Castro, et al., Cosmetics and cosmeceutical applications of chitin, chitosan and their derivatives, Polymers 10 (2) (2018) 213.

Handbook of Chitin and Chitosan

References

275

[109] S.P. Miguel, A.F. Moreira, I.J. Correia, Chitosan based-asymmetric membranes for wound healing: a review, Int. J. Biol. Macromol. 127 (2019) 460
475. [110] M. Ahmad, K. Manzoor, S. Singh, S. Ikram, Chitosan centered bionanocomposites for medical specialty and curative applications: a review, Int. J. Pharm. 529 (1
2) (2017) 200
217. [111] M. Rodrı´guez-Va´zquez, B. Vega-Ruiz, R. Ramos-Zu´n˜iga, D.A. Saldan˜a-Koppel, L.F. Quin˜ones-Olvera, Chitosan and its potential use as a scaffold for tissue engineering in regenerative medicine, BioMed. Res. Int. (2015) 1
15. [112] D. Vukajlovic, J. Parker, O. Bretcanu, K. Novakovic, Chitosan based polymer/bioglass composites for tissue engineering applications, Mater. Sci. Eng. 96 (2019) 955
967. [113] R. Jayakumar, K.P. Chennazhi, S. Srinivasan, S.V. Nair, T. Furuike, H. Tamura, Chitin scaffolds in tissue engineering, Int. J. Mol. Sci. 12 (3) (2011) 1876
1887. [114] W. Cao, D. Jing, J. Li, Y. Gong, N. Zhao, X. Zhang, Effects of the degree of deacetylation on the physicochemical properties and Schwann cell affinity of chitosan films, J. Biomater. Appl. 20 (2) (2005) 157
177. [115] Y. Yuan, B.M. Chesnutt, W.O. Haggard, J.D. Bumgardner, Deacetylation of chitosan: material characterization and in vitro evaluation via albumin adsorption and preosteoblastic cell cultures, Materials 4 (8) (2011) 1399
1416. [116] H.W. Chang, Y.S. Lin, Y.D. Tsai, M.L. Tsai, Effects of chitosan characteristics on the physicochemical properties, antibacterial activity, and cytotoxicity of chitosan/2glycerophosphate/ nanosilver hydrogels, J. Appl. Polym. Sci. 127 (1) (2013) 169
176. [117] C.C. Sun, S.F. Chou, J.Y. Lai, C.H. Cho, C.H. Lee, Dependence of corneal keratocyte adhesion, spreading, and integrin β1 expression on deacetylated chitosan coating, Mater. Sci. Eng. 63 (2016) 222
230. [118] L.J.R. Foster, S. Ho, J. Hook, M. Basuki, H. Marc¸al, Chitosan as a biomaterial: influence of degree of deacetylation on its physiochemical, material and biological properties, PLoS One 10 (8) (2015) 1
22. [119] J. Jiao, J. Huang, Z. Zhang, Hydrogels based on chitosan in tissue regeneration: how do they work? A mini review, Appl. Polym. Sci. 136 (13) (2019) 1
9. [120] J. Nunthanid, S. Puttipipatkhachorn, K. Yamamoto, G.E. Peck, Drug physical state and drug-polymer interaction on drug release from chitosan matrix films, J. Control. Release 27 (2) (2001) 143
157. [121] Z. Liu, X. Ge, Y. Lu, S. Dong, Y. Zhao, M. Zeng, Effects of chitosan molecular weight and degree of deacetylation on the properties of gelatine-based films, Food Hydrocoll. 26 (1) (2012) 311
317. [122] N. Maganti, P.K.C. Venkat Surya, W.W. Thein-Han, T.C. Pesacreta, R.D.K. Misra, Structure-process-property relationship of biomimetic chitosan-based nanocomposite scaffolds for tissue engineering: biological, physico-chemical, and mechanical functions, Adv. Eng. Mater. 13 (3) (2011) 108
122. [123] T.S. Trung, W.W. Thein-Han, N.T. Qui, C.H. Ng, W.F. Stevens, Functional characteristics of shrimp chitosan and its membranes as affected by the degree of deacetylation, Bioresour. Technol. 97 (4) (2006) 659
663. [124] A. Abarrategi, Y. Lo´piz-Morales, V. Ramos, A. Civantos, L. Lo´pez-Du´rn, F. Marco, et al., Chitosan scaffolds for osteochondral tissue regeneration, J. Biomed. Mater. Res. 95 (4) (2010) 1132
1141. [125] S.F. Chou, J.Y. Lai, C.H. Cho, C.H. Lee, Relationships between surface roughness/ stiffness of chitosan coatings and fabrication of corneal keratocyte spheroids: effect of degree of deacetylation, Colloids Surf. 142 (2016) 105
113. [126] J. Mao, L. Zhao, K. De Yao, Q. Shang, G. Yang, Y. Cao, Study of novel chitosangelatin artificial skin in vitro, J. Biomed. Mater. Res. Part A 64 (2) (2003) 301
308.

Handbook of Chitin and Chitosan

276

8. Electrospun chitosan materials and their potential use as scaffolds

[127] J.J. Wang, Z.W. Zeng, R.Z. Xiao, T. Xie, G.L. Zhou, X.R. Zhan, et al., Recent advances of chitosan nanoparticles as drug carriers, Int. J. Nanomed. 6 (2011) 765
774. [128] M.C.G. Pella´, M.K. Lima-Teno´rio, E.T. Teno´rio-Neto, M.R. Guilherme, E.C. Muniz, A.F. Rubira, Chitosan-based hydrogels: from preparation to biomedical applications, Carbohydr. Polym. 196 (2018) 233
245. [129] S.B. Qasim, M.S. Zafar, S. Najeeb, Z. Khurshid, A.H. Shah, S. Husain, et al., Electrospinning of chitosan-based solutions for tissue engineering and regenerative medicine, Int. J. Mol. Sci. 19 (2) (2018) 407. [130] B.S. de Farias, T.R. Sant’Anna Cadaval Junior, L.A. de Almeida Pinto, Chitosanfunctionalized nanofibers: a comprehensive review on challenges and prospects for food applications, Int. J. Biol. Macromol. 123 (C) (2019) 210
220. [131] S. Khorshidi, A. Solouk, H. Mirzadeh, S. Mazinani, J.M. Lagaron, S. Sharifi, et al., A review of key challenges of electrospun scaffolds for tissue-engineering applications, J. Tissue Eng. Regen. Med. 10 (9) (2016) 715
738. [132] M.Z. Elsabee, H.F. Naguib, R.E. Morsi, Chitosan based nanofibers, review, Mater. Sci. Eng. 32 (7) (2012) 1711
1726. [133] X. Geng, O.H. Kwon, J. Jang, Electrospinning of chitosan dissolved in concentrated acetic acid solution, Biomaterials 26 (27) (2005) 5427
5432. [134] S. De Vrieze, P. Westbroek, T. Van Camp, L. Van Langenhove, Electrospinning of chitosan nanofibrous structures: feasibility study, J. Mater. Sci. 42 (19) (2007) 8029
8034. [135] H. Homayoni, S.A.H. Ravandi, M. Valizadeh, Electrospinning of chitosan nanofibers: processing optimization, Carbohydr. Polym. 77 (3) (2009) 656
661. [136] K. Ohkawa, D. Cha, H. Kim, A. Nishida, H. Yamamoto, Electrospinning of chitosan, Macromol. Rapid Commun. 25 (18) (2004) 1600
1605. [137] V. Sencadas, D.M. Correia, A. Areias, G. Botelho, A.M. Fonseca, I.C. Neves, et al., Determination of the parameters affecting electrospun chitosan fiber size distribution and morphology, Carbohydr. Polym. 87 (2) (2012) 1295
1301. [138] A. Neamnark, R. Rujiravanit, P. Supaphol, Electrospinning of hexanoyl chitosan, Carbohydr. Polym. 66 (3) (2006) 298
305. [139] J. Du, Y.L. Hsieh, Nanofibrous membranes from aqueous electrospinning of carboxymethyl chitosan, Nanotechnology 19 (12) (2008). [140] B.M. Min, S.W. Lee, J.N. Lim, Y. You, T.S. Lee, P.H. Kang, et al., Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nanofibers, Polymer 45 (21) (2004) 7137
7142. [141] S.G. Kuntzler, J.A.V. Costa, M.G. de Morais, Development of electrospun nanofibers containing chitosan/PEO blend and phenolic compounds with antibacterial activity, Int. J. Biol. Macromol. 117 (2018) 800
806. [142] I. Kohsari, Z. Shariatinia, S.M. Pourmortazavi, Antibacterial electrospun chitosanpolyethylene oxide nanocomposite mats containing ZIF-8 nanoparticles, Int. J. Biol. Macromol. 91 (2016) 778
788. [143] J. Song, S.J.A. Remmers, J. Shao, E. Kolwijck, X.F. Walboomers, J.A. Jansen, et al., Antibacterial effects of electrospun chitosan/poly(ethylene oxide) nanofibrous membranes loaded with chlorhexidine and silver, Nanomedicine 12 (5) (2016) 1357
1364. [144] J.T. Yeh, C.L. Chen, K.S. Huang, Y.H. Nien, J.L. Chen, P.Z. Huang, Synthesis, characterization, and application of PVP/chitosan blended polymers, Int. J. Biol. Macromol. 101 (2) (2006) 885
891. [145] P. Das, N. Ojah, R. Kandimalla, K. Mohan, D. Gogoi, S.K. Dolui, et al., Surface modification of electrospun PVA/chitosan nanofibers by dielectric barrier discharge plasma at atmospheric pressure and studies of their mechanical properties and biocompatibility, Int. J. Biol. Macromol. 114 (2018) 1026
1032.

Handbook of Chitin and Chitosan

References

277

[146] Z. Cui, Z. Zheng, L. Lin, J. Si, Q. Wang, X. Peng, et al., Electrospinning and crosslinking of polyvinyl alcohol/chitosan composite nanofiber for transdermal drug delivery, Adv. Polym. Technol. 37 (6) (2018) 1917
1928. [147] S. Ramesh, L. Lungaro, D. Tsikritsis, E. Weflen, I.V. Rivero, A.P.D. Elfick, Fabrication and evaluation of poly(lactic acid), chitosan, and tricalcium phosphate biocomposites for guided bone regeneration, J. Appl. Polym. Sci. 135 (39) (2018) 1
10. [148] Y. Liu, S. Wang, R. Zhang, Composite poly(lactic acid)/chitosan nanofibrous scaffolds for cardiac tissue engineering, Int. J. Biol. Macromol. 103 (2017) 1130
1137. ˆ .M. Moraes, Electrospun multilayer chito[149] R.B. Trinca, C.B. Westin, J.A.F. da Silva, A san scaffolds as potential wound dressings for skin lesions, Eur. Polym. J. 88 (C.) (2017) 161
170. [150] S.R. Gomes, G. Rodrigues, G.G. Martins, M.A. Roberto, M. Mafra, C.M.R. Henriques, et al., In vitro and in vivo evaluation of electrospun nanofibers of PCL, chitosan and gelatin: a comparative study, Mater. Sci. Eng. C. 46 (2015) 348
358. [151] Z. Chen, X. Mo, C. He, H. Wang, Intermolecular interactions in electrospun collagen-chitosan complex nanofibers, Carbohydr. Polym. 72 (3) (2008) 410
418. [152] Z. Chen, X. Mo, F. Qing, Electrospinning of collagen-chitosan complex, Mater. Lett. 61 (16) (2007) 3490
3494. [153] J.P. Chen, S.H. Chen, G.J. Lai, Preparation and characterization of biomimetic silk fibroin/chitosan composite nanofibers by electrospinning for osteoblasts culture, Nanoscale Res. Lett. 7 (1) (2012) 1
11. [154] T.J. Kean, M. Thanou, Toxicological properties of chitosan and derivatives for biopharmaceutical applications, in: B. Sarmento, J. das Neves (Eds.), Chitosan-Based Systems for Biopharmaceuticals, first ed., Wiley, West Sussex, 2012, pp. 451
461. [155] M. Dornish, S.D. Kaplan, S.R. Arepalli, Regulatory status of chitosan and derivatives, in: B. Sarmento, J. das Neves (Eds.), Chitosan-Based Systems for Biopharmaceuticals, first ed., Wiley, West Sussex, 2012, pp. 463
482. [156] Food and Drug Administration, Federal Agency of the United States Department of Health and Human Services. ,https://www.accessdata.fda.gov/cdrh_docs/pdf10/ K102546.pdf. (accessed 21.07.19). [157] B. Bellich, I. D’Agostino, S. Semeraro, A. Gamini, A. Cesa`ro, “The good, the bad and the ugly” of chitosans, Mar. Drugs 14 (5) (2016) 99. [158] Standard guide for characterization and testing of chitosan salts as starting materials intended for use in biomedical and tissue-engineered medical product applications. ,http://www.astm.org/Standards/F2103.hml., 2001 (accessed 08.07.11). [159] Standard practice for selecting generic biological test methods for materials and devices. ASTM International,West Conshohocken, PA, 2010. Available from ,http://www.astm.org/Standards/F748.html., 2010 (accessed 08.07.11). [160] Biological evaluation of medical devices. International Organization for Standardization, Geneva, Switzerland. Available from ,http://www.iso.org. (accessed 07.07.11). [161] L. Ricaurte, M.X. Quintanilla-Carvajal, Use of electrospinning technique to produce nanofibres for food industries: a perspective from regulations to characterisations, Trends Food Sci. Technol. 85 (1) (2019) 92
106. [162] F.F. Safadi, M.F. Barbe, S.M. Abdelmagid, M.C. Rico, R.A. Aswad, J. Litvin, et al., Bone structure, development and bone biology, in: J.S. Khurana (Ed.), Bone Pathology, second ed., Humana Press, New Jersey, 2009, pp. 1
50. [163] X. Wang, Cortical bone mechanics and composition: effects of age and gender, in: M.J. Silva (Ed.), Skeletal Aging and Osteoporosis: Biomechanics and Mechanobiology, Springer, Berlin, 2011, pp. 53
85. [164] M. Mohammadi, S.A. Mousavi Shaegh, M. Alibolandi, M.H. Ebrahimzadeh, A. Tamayol, M.R. Jaafari, et al., Micro and nanotechnologies for bone regeneration: recent advances and emerging designs, J. Control. Rel. 274 (C) (2018) 35
55.

Handbook of Chitin and Chitosan

278

8. Electrospun chitosan materials and their potential use as scaffolds

[165] W.G. Paprosky, M.S. Bradford, T.I. Younger, Classification of bone defects in failed prosthese, Chir. Organi Mov. 79 (4) (1994) 285
291. [166] R. Dimitriou, E. Jones, D. McGonagle, P.V. Giannoudis, Bone regeneration: current concepts and future directions, BMC Med. 9 (1) (2011) 66. [167] R. Bartl, C. Bartl, The Osteoporosis Manual: Prevention, Diagnosis and Management, Springer International Publishing, Switzerland, 2019 (Chapter 4). [168] G.P. Spyros, K.T. Georgios, K.B. Efthimia, G.P. Athanasios, Segmental bone defects: from cellular and molecular pathways to the development of novel biological treatments, J. Cell. Mol. Med. 14 (11) (2010) 2561
2569. [169] J.A. Buza, T. Einhorn, Bone healing in 2016, Clin. Cases Miner. Bone Metab. 13 (2) (2016) 101
105. [170] C. Langford, N. Cameron, Materials for tissue engineering and 3D cell culture, in: N. Bruns, A.F.M. Kilbinger (Eds.), Bio-inspired Polymers, Royal Society of Chemistry, Cambridge, 2016, pp. 460
489. [171] G. Iviglia, S. Kargozar, F. Baino, Biomaterials, current strategies, and novel nanotechnological approaches for periodontal regeneration, J. Funct. Biomater. 10 (1) (2019) 3. [172] L. Zou, Y. Zhang, X. Liu, J. Chen, Q. Zhang, Biomimetic mineralization on natural and synthetic polymers to prepare hybrid scaffolds for bone tissue engineering, Colloids Surf. B: Biointerfaces 178 (2019) 222
229. [173] A. Muxika, A. Etxabide, J. Uranga, P. Guerrero, K. de la Caba, Chitosan as a bioactive polymer: processing, properties and applications, Int. J. Biol. Macromol. 105 (Pt 2) (2017) 1358
1368. [174] M.B. Lyles, J.C. Hu, V.G. Varanasi, J.O. Hollinger, K.A. Athanasiou, Bone tissue engineering, in: S. Nukavarapu, J. Freeman, C. Laurencin (Eds.), Regenerative Engineering of Musculoskeletal Tissues and Interfaces, Woodhead Publishing, Cambridge, 2015, pp. 97
134. [175] S.L. Moradi, A. Golchin, Z. Hajishafieeha, M.M. Khani, A. Ardeshirylajimi, Bone tissue engineering: adult stem cells in combination with electrospun nanofibrous scaffolds, J. Cell. Physiol. 233 (10) (2018) 6509
6522. [176] A. Repanas, S. Andriopoulou, B. Glasmacher, The significance of electrospinning as a method to create fibrous scaffolds for biomedical engineering and drug delivery applications, J. Drug Deliv. Sci. Technol. 31 (C) (2016) 137
146. [177] K. Li, H. Sun, H. Sui, Y. Zhang, H. Liang, X. Wu, et al., Composite mesoporous silica nanoparticle/chitosan nanofibers for bone tissue engineering, RSC Adv. 5 (23) (2015) 1754
17549. [178] H. Liang, F. Sheng, B. Zhou, Y. Pei, B. Li, J. Li, Phosphoprotein/chitosan electrospun nanofibrous scaffold for biomineralization, Int. J. Biol. Macromol. 102 (C) (2017) 218
224. [179] S. Talebian, M. Mehrali, S. Mohan, H.R. Balaji Raghavendran, M. Mehrali, H.M. Khanlou, et al., Chitosan (PEO)/bioactive glass hybrid nanofibers for bone tissue engineering, RSC Adv. 4 (90) (2014) 49144
49152. [180] H.T. Sasmazel, Novel hybrid scaffolds for the cultivation of osteoblast cells, Int. J. Biol. Macromol. 49 (4) (2011) 838
846. [181] G. Toskas, C. Cherif, R.D. Hund, E. Laourine, B. Mahltig, A. Fahmi, et al., Chitosan (PEO)/silica hybrid nanofibers as a potential biomaterial for bone regeneration, Carbohydr. Polym. 94 (2) (2013) 713
722. [182] D.V.H. Thien, S.W. Hsiao, M.H. Ho, C.H. Li, J.L. Shih, Electrospun chitosan/ hydroxyapatite nanofibers for bone tissue engineering, J. Mater. Sci. 48 (4) (2013) 1640
1645. [183] Y. Wei, X. Zhang, Y. Song, B. Han, X. Hu, X. Wang, et al., Magnetic biodegradable Fe3O4/CS/PVA nanofibrous membranes for bone regeneration, Biomed. Mater. 6 (5) (2011).

Handbook of Chitin and Chitosan

References

279

[184] H. Nie, M. Ho, C.K. Wang, C.H. Wang, Y. Fu, Biomaterials BMP-2 plasmid loaded PLGA / HAp composite scaffolds for treatment of bone defects in nude mice, Biomaterials 30 (5) (2009) 892
901. [185] A. Rawlinson, I. Ur, Potential of electrospun chitosan fibers as a surface layer in functionally graded GTR membrane for periodontal regeneration, Dental Mater. 33 (1) (2016) 71
83. [186] R.L. Reis, N.M. Neves, E.D. Pinho, A. Martins, J.V. Arau, Degradable particulate composite reinforced with nanofibres for biomedical applications, Acta Biomater. 5 (4) (2009) 1104
1114. [187] Y. Lee, S. Fu, C. Lin, Preparation of HA-mineralized CS/PLA composites nanofiber GBR Mats, in: 1st Global Conference on Biomedical Engineering and 9th Asian-Pacific Conference on Medical and Biological Engineering, Vol. 47, 2014, pp. 28
31. [188] S. Zhang, M.P. Prabhakaran, X. Qin, S. Ramakrishna, Biocomposite scaffolds for bone regeneration: role of chitosan and hydroxyapatite within poly-3-hydroxybutyrate-co-3-hydroxyvalerate on mechanical properties and in vitro evaluation, J. Mech. Behav. Biomed. Mater. 51 (C) (2015) 88
98. [189] M. Shanhnavazi, M. Ketabi, R. Fekrazad, F. Moztarzadeh, Fabrication of chitosannano hydroxyapatite scaffold for dental tissue engineering, Key Eng. Mater. 720 (2016) 223
227. [190] M. Vaahtio, T. Peltola, T. Hentunen, H. Yla¨nen, S. Areva, J. Wolke, et al., The properties of biomimetically processed calcium phosphate on bioactive ceramics and their response on bone cells, J. Mater. Sci. Mater. Med. 17 (11) (2006) 1113
1125. [191] J.V. Araujo, P.P. Carvalho, S.M. Best, Electrospinning of bioinspired polymer scaffolds, Adv. Exp. Med. Biol. 881 (2015) 33
53. [192] T. Kokubo, S. Yamaguchi, Simulated body fluid and the novel bioactive materials derived from it, J. Biomed. Mater. Res. Part A 107 (5) (2019) 968
977. ´ .J. Leite, R.A. Pires, J.F. Mano, R.L. Reis, Bioactive composites rein[193] A.A. Barros, A forced with inorganic glasses and glass
ceramics for tissue engineering applications, in: B. Ben-Nissan (Ed.), Advances in Calcium Phosphate Biomaterials, Springer, Berlin, 2014, pp. 331
353. [194] G.R. Ragetly, D.J. Griffon, H.B. Lee, L.P. Fredericks, W. Gordon-Evans, Y.S. Chung, Effect of chitosan scaffold microstructure on mesenchymal stem cell chondrogenesis, Acta Biomater. 6 (4) (2010) 1430
1436. [195] M.A. Shamekhi, H. Mirzadeh, H. Mahdavi, A. Rabiee, D. Mohebbi-Kalhori, M. Baghaban Eslaminejad, Graphene oxide containing chitosan scaffolds for cartilage tissue engineering, Int. J. Biol. Macromol. 127 (2019) 396
405. [196] N. Bhattarai, D. Edmondson, O. Veiseh, F.A. Matsen, M. Zhang, Electrospun chitosan-based nanofibers and their cellular compatibility, Biomaterials 26 (31) (2005) 6176
6184. [197] P.S. Alemi, S.A. Atyabi, F. Sharifi, M. Mohamadali, S. Irani, H. Bakhshi, et al., Synergistic effect of pressure cold atmospheric plasma and carboxymethyl chitosan to mesenchymal stem cell differentiation on PCL/CMC nanofibers for cartilage tissue engineering, Polym. Adv. Technol. 30 (6) (2019) 1356
1364. [198] M. Ainola, W. Tomaszewski, B. Ostrowska, E. Wesolowska, H.D. Wagner, W. Swieszkowski, et al., A bioactive hybrid three-dimensional tissue-engineering construct for cartilage repair, J. Biomater. Appl. 30 (6) (2016) 873
885. [199] C. Ning, L. Li, S. Logsetty, S. Ghanbar, M. Guo, W. Ens, et al., Enhanced antibacterial activity of new “composite” biocides with both N-chloramine and quaternary ammonium moieties, RSC Adv. 5 (114) (2015) 93877
93887.

Handbook of Chitin and Chitosan

280

8. Electrospun chitosan materials and their potential use as scaffolds

[200] Z. Liu, X. Yuan, M. Liu, G. Fernandes, Y. Zhang, S. Yang, et al., Antimicrobial peptide combined with BMP2-modified mesenchymal stem cells promotes calvarial repair in an osteolytic model, Mol. Ther. 26 (1) (2018) 199
207. [201] N.S. Sambudi, M. Sathyamurthy, G.M. Lee, S.B. Park, Electrospun chitosan/poly (vinyl alcohol) reinforced with CaCO3 nanoparticles with enhanced mechanical properties and biocompatibility for cartilage tissue engineering, Compos. Sci. Technol. 106 (C) (2015) 76
84. [202] A. Subramanian, D. Vu, G.F. Larsen, H.Y. Lin, Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering, J. Biomater. Sci. Polym. Ed. 16 (7) (2005) 861
873. [203] K.S. In, H.S. Won, Y.L. Sang, H.L. Sang, J.H. Seong, C.L. Myung, et al., Chitosan nano-/microfibrous double-layered membrane with rolled-up three-dimensional structures for chondrocyte cultivation, J. Biomed. Mater. Res. Part. A 90 (2) (2009) 595
602. [204] M.H. Mirmusavi, P. Zadehnajar, D. Semnani, S. Karbasi, F. Fekrat, F. Heidari, Evaluation of physical, mechanical and biological properties of poly 3hydroxybutyrate-chitosan-multiwalled carbon nanotube/silk nano-micro composite scaffold for cartilage tissue engineering applications, Int. J. Biol. Macromol. 132 (2019) 822
835. [205] J. Liu, Q. Fang, X. Yu, Y. Wan, B. Xiao, Chitosan-based nanofibrous membrane unit with gradient compositional and structural features for mimicking calcified layer in osteochondral matrix, Int. J. Mol. Sci. 19 (8) (2018) 2330. [206] D. Sadeghi, S. Karbasi, S. Razavi, S. Mohammadi, M.A. Shokrgozar, S. Bonakdar, Electrospun poly(hydroxybutyrate)/chitosan blend fibrous scaffolds for cartilage tissue engineering, J. Appl. Polym. Sci. 133 (47) (2016) 1
9.

Handbook of Chitin and Chitosan

C H A P T E R

9 Injectable polymeric gels based on chitosan and chitin for biomedical applications Cong Xie1,2,3, Wei Huang3, Weiqing Sun3 and Xulin Jiang1 1

Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan, P.R. China, 2 Non-power Nuclear Technology Research and Development Center, Hubei University of Science and Technology, Xianning, P.R. China, 3Hangzhou Singclean Medical Products Co., Ltd., Hangzhou, P.R. China

O U T L I N E 9.1 Introduction

282

9.2 Injectable polymeric gels based on chitosan and chitin 9.2.1 Thermosensitive physical hydrogels 9.2.2 Chemical hydrogels

283 283 289

9.3 Biomedical applications 9.3.1 Potential use of injectable hydrogels 9.3.2 Clinical trial and human applications

294 294 295

9.4 Conclusions and future perspectives

298

Acknowledgment

299

References

299

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00009-1

281

© 2020 Elsevier Inc. All rights reserved.

282

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

9.1 Introduction Hydrogels, as a kind of hydrophilic compound (natural or synthesized polymers), have gained widespread attention among researchers, due to their three-dimensional (3D) cross-linked structure that can keep swelling without dissolving in water and can provide a microenvironment similar to the extracellular matrix (ECM) [1
3]. With the development of biomedicine, hydrogels have been applied widely as drug/cell carriers, in tissue engineering, and so on [4,5]. Recently, injectable hydrogels have become a research hotspot because of their special properties [6,7]. Before injection, hydrogel precursors exhibit a fluidity that facilitates the inclusion of a drug, cell, or protein, according to demand, by simple mixing. After injection, they can immediately fill the irregular defects and can be cross-linked in situ quickly under external physical or chemical stimulation. This minimally invasive treatment can significantly reduce patient trauma and complications. In addition, the mild gelation conditions of these injectable hydrogels combined with high water content, 3D porous framework, and good biocompatibility can maintain the biological activity of the inclusions, which benefit cell growth and tissue repair/regeneration around the implant position. Therefore injectable hydrogels have great clinical application value and huge development potential. In brief, injectable hydrogels can be divided into physical hydrogels and chemical hydrogels. Physical injectable hydrogels are formed via secondary bonding such as hydrogen bonding, van der Waals attraction, coordination, host
guest interaction, or π
π stacking interactions between macromolecular chains. According to the specific fabrication method, these hydrogels exhibit special responses, for example, being temperature-responsive, pressure-responsive, pH-responsive, or ionresponsive to achieve a sol
gel transition. Different from physical injectable hydrogels, chemical ones are cross-linked by covalent bonding, formed mainly by photocross-linking, Schiff base cross-linking, Michael additions-mediated, enzymatic cross-linking, or a click chemistry reaction. There are a lot of research papers on and reviews of injectable hydrogels that have been published and they are focused mainly on the biomaterials, including hyaluronic acid (HA) [8], collagen [9], polyvinyl alcohol [10], and polyethylene glycol (PEG) [11]. However, a few injectable hydrogels based on chitin have been reported. Chitin and its derivatives, such as chitosan, are nontoxic, natural polysaccharides that contain hydroxyl groups and amino groups on the side chains, thus providing reaction sites for many side group attachments. Hence, these modified chitins and chitosans could be endowed with useful sol
gel transition behavior under physiological conditions for biomedical applications. This review provides an overview of

Handbook of Chitin and Chitosan

9.2 Injectable polymeric gels based on chitosan and chitin

283

the progress of research into the injectable polymeric gels based on chitosan and chitin, and the focus is their biomedical applications for drug/cell delivery, tissue engineering, prevention of postoperative adhesion, etc. Finally, perspectives on future injectable hydrogels based on chitosan and chitin are also discussed.

9.2 Injectable polymeric gels based on chitosan and chitin 9.2.1 Thermosensitive physical hydrogels 9.2.1.1 Injectable chitosan/beta glycerophosphate hydrogels (CS/β-GP hydrogels) Due to the protonation of amino groups in side chains, chitosan is in general only soluble in acid aqueous solution at a pKa range of 5.5
6.5. Neutralization or alkalization of chitosan aqueous solutions to a pH value above 6.2 will lead to the formation of a hydrated gel-like precipitate [12]. Chenite et al. first reported thermally sensitive neutral chitosan solutions by adding β-GP at pH values ranging from 6.8 to 7.2, which can be free-flowing liquid below room temperature for encapsulating living cells and bioactive molecules homogeneously [13]. Interestingly, these CS/β-GP systems exhibit thermal in situ phase transition behavior from sol to gel when the temperature reaches 37 C (body temperature). Due to thermosensitive characteristics, in situ-forming CS/β-GP hydrogels are widely investigated as potential biomaterials for many medical applications. Hoemann et al. repaired cartilage tissue of rabbits by implantation of in situ solidified CS/β-GP hydrogels mixed with autologous whole blood [14]. Combined with drilling, the therapeutic effect of cartilage regeneration was more ideal. They also implanted in situ formed CS/β-GP hydrogels in aged rabbit knees [15]. These subchondral thermogels have contributed to hyaline cartilage regeneration from microdrill holes. Fatimi et al. developed a CS/β-GP hydrogel by adding a sclerosing agent called sodium tetradecyl sulfate [16]. These mixed systems were injectable and showed faster gelation time and better mechanical and sclerosing performances than those of pure CS/β-GP thermogels, which were suitable for treatment of endoleaks. Li et al. modified chitosan by grafting humanlike collagen (HLC) covalently [17]. These CS-HLC/β-GP hydrogels exhibited quick sol
gel transition behavior at body temperature and the compression module of CS-HLC/β-GP hydrogels was stronger than that of unmodified CS/β-GP hydrogels. Thus CS-HLC/β-GP thermogels were ideal candidates for use in soft tissue defect filling. Huang et al. developed CS/β-GP/glycerol-based injectable hydrogels and their thermosensitive behavior could serve as an overheating signal to trace and prevent

Handbook of Chitin and Chitosan

284

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

overheating, which is a side effect of focused ultrasound therapy [18]. Wang et al. used CS/β-GP hydrogels for the hydrophobic drug naproxen delivery [19]. Naproxen nanoparticles were obtained by mixing biblock amphiphilic copolymer monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) with naproxen and they were loaded with chitosan followed by cross-linking with β-GP. Interestingly, CS/β-GP/naproxen systems displayed a thermosensitive behavior suitable for injection and exhibited time-dependent release characteristics. Besides, CS/β-GP hydrogels used in the clinic are summarized in Section 9.3.2. 9.2.1.2 Hydroxybutyl chitosan Hydroxybutyl chitosan (HBC) is fabricated by the reaction between hydroxyl mainly at C-6 of sugar moieties and 1,2-epoxybutane. Hydroxybutyl groups can endow HBC hydrogel with good hydrophilia and obvious thermosensitive behavior. Therefore HBC is becoming one of the promising injectable thermogels for many biomedical applications. Wei et al. synthesized HBC heterogeneously by alkalinized chitosan dispersion in an isopropanol/water mixed solution at room temperature [20]. The obtained HBC solution showed reversible thermosensitive sol
gel transition behavior at 20 C. When HBC solution was injected in mouse, the in situ-forming hydrogel displayed good biodegradability, biocompatibility, and effectiveness in preventing adhesions in vivo. Therefore HBC hydrogels have a promising potential for postoperative adhesion prevention. In addition, Cai et al. developed a method to prepare HBC homogeneously in KOH/LiOH/urea aqueous solutions [21]. The obtained HBC polymers exhibited good solubility in water. And those with a degree of hydroxybutyl molar substitution above 1.25 showed reversible thermoprecipitation behavior instead of sol
gel transition in aqueous solutions with the precipitation temperature from 52 C to 61 C. The authors attributed this behavior to the more uniform distribution of hydroxybutyl groups along the polymer chain in homogeneous HBC synthesis compared with that in heterogeneous HBC synthesis. The reported HBCs discussed below were synthesized in heterogeneous conditions, which showed reversible thermosensitive sol
gel transition suitable for injection. Chen’s research group used thermosensitive HBC hydrogels as a promising embolic agent for the occlusion of selected blood vessels [22]. When HBC solutions were injected into rat renal arteries, hydrogels were formed in situ quickly, and these could attach onto blood vessel inner walls tightly. HBC hydrogels easily generated moderate coagulation owing to the aggregation of red blood cells round them, which facilitated the fixation of the hydrogel in spite of blood flow. Chen’s research group also used thermosensitive HBC hydrogels to encapsulate doxorubicine hydrochloride (DOX•HCl, anticancer agent) [23]. These DOX•HCl loaded

Handbook of Chitin and Chitosan

9.2 Injectable polymeric gels based on chitosan and chitin

285

hydrogels exhibited quick gel transition behavior at 37 C. The results showed that HBC hydrogels exhibited slow release rates and could reduce the viability of 4T-1 cells dramatically. Consequently, these thermosensitive HBC hydrogels have a potential value for drug delivery as injectable hydrogels. In addition, Chen’s research group modified HBC by grafting deoxycholic acid moieties to form a new kind of thermoresponsive amphilic polysaccharide (DAHBC), which could self-assemble into spherical nanoparticles in water [24]. The lower critical solution temperature (LCST) of these polysaccharides could be adjusted to 38 C, which could provide a suitable temperature for hyperthermia therapy. The drug release rate of doxorubicin-loaded DAHBC was accelerated when the temperature exceeded LCST and this special behavior has potential application value in the field of chemotherapy and hyperthermia for drug nanocarriers. Chen’s research group also used thermoresponsive HBC hydroges to load insulin [25]. Burst release phenomena were not observed in all the HBC hydrogels within 48 h and the cumulative insulin released slowly increased with time in the absence or presence of lysozyme. More important, the presence of the lysozyme promoted the release of the loaded insulin in HBC hydrogels due to the enzymatic hydrolysis of HBS. For example, only 38% of insulin was released without using lysozyme after 48 h, while 80% of the insulin was released at the same time in the presence of lysozyme. This work provides a method for sustained drug release by using HBC hydrogel. Chen’s research group has further modified HBC by using succinic anhydride to obtain thermo/pH double response hydrogels (NSHBC) [26]. Similar to HBC, NSHBC solutions also could transform into a gel state at body temperature for the encapsulation of bovine serum albumin. After 24 h, 93.7% bovine serum albumin was released from the NSHBC hydrogels at pH 7.4 (in phosphate buffer saline), whereas only 24.6% bovine serum albumin was released from the NSHBC hydrogels at pH 3.0. Thus NSHBC hydrogels exhibited good pH sensitivity drug release characteristics for biomedical applications in oral drug delivery. Furthermore, Chen’s research group used thermosensitive HBC hydrogels for 3D culture of human umbilical vein endothelial cells [27]. The reversible sol
gel transition behavior of HBC solution occurred at 26 C. The interconnected porous structure of HBC hydrogels ranging from 50 μm to 250 μm could mimic cell growth environments, facilitating the transfer of nutrients and metabolites to promote human umbilical vein endothelial cells proliferation. Chen’s research group further prepared these thermoresponsive HBC hydrogels with different formulations of solvent (Dulbecco’s modified eagle’s medium and phosphate buffered saline, DMEM/PBS) to optimize the human umbilical vein endothelial cells growth environment. The gelation temperature of HBC could be

Handbook of Chitin and Chitosan

286

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

tuned from 10 C to 30 C by changing the DMEM ratio. They showed the best cell viability for the HUVECs encapsulated in HBC-70 hydrogel (DMEM/PBS, 70:30 [v/v]). Leong et al. reported HBC as an injectable hydrogel to load at 4 C and culture human mesenchymal stem cells and intervertebral disk cells at 37 C [28]. Cell proliferation in HBC gel maintained good activity by the detection of metabolic activity, genetic analysis of synthesized mRNA, and histological staining of human mesenchymal stem cell and disk cell cultures. This work indicated the potential value of HBC hydrogel as an injectable cell carrier for the reconstruction of degenerated disk. 9.2.1.3 Other thermosensitive modified chitosan Pure positively charged chitosans in acid aqueous solution show no sol
gel transition behaviors until they are mixed with polyanion compounds such as β-glycerophosphate. Besides, there exist other anionic compounds that can endow chitosans with obvious thermosensitive behaviors by electrostatic interaction. For example, Li et al. reported a thermoresponsive chitosan-based hydrogel by mixing hydroxyapatite [29]. Interestingly, Na2CO3 was a coagulant to construct the chitosanbased thermogel, which can promote the sol
gel transition of the chitosan/hydroxyapatite hybrid hydrogel within 9 min. When the mixed solutions were injected subcutaneously into the abdomen and dorsum of rats, thermogels were in situ formed quickly within a beanlike shape and retained for a long time without swelling and inflammation. These chitosan/hydroxyapatite/Na2CO3 hydrogels would be the potentially ideal candidates for bone tissue engineering. Jiang et al. synthesized a water-soluble hydroxypropyl chitosan homogeneously by the reaction between the alkali chitosan solution and propylene oxide [30]. The degree of molar hydroxypropyl substitution of the prepared hydroxypropyl chitosan was 2.47 and this polymer dissolved in water showed reversible sol-to-gel phase transition behavior at 37 C. Thus these thermosensitive HPC hydrogels could be used as injectable biometerials in many biomedical fields. 9.2.1.4 Thermosensitive modified chitin Compared to chitosan, chitin is more extensively distributed as a kind of natural polymer and it is present in the ECM of many living organism [31]. However, the strong hydrogen bonding between chitin molecules makes it hard to dissolve in most common solvents including water, which limits its application in many biomedical fields. Therefore it is essential to functionalize chitin to improve its solubility. It is worth noting that chitin-based polymers (i.e., degree of acetylation of chitin above 50%) degrade faster than chitosan-based derivatives (degree of

Handbook of Chitin and Chitosan

9.2 Injectable polymeric gels based on chitosan and chitin

287

acetylation of chitosan is below 50%) under in vivo conditions [32,33], implying that chitin-based biomaterials may be harmless to animals and humans. Fortunately, researchers [34,35] have found that chitin can be dissolved in NaOH/urea aqueous solution at low temperature. In this system, many kinds of water-soluble chitin derivatives can be homogeneously synthesized. Interestingly, some of these modified chitins show obvious sol
gel transition behavior, which can form in situ thermogels by injection. For example, our group synthesized carboxymethyl chitin (CMCH) homogeneously in aqueous NaOH/urea solution and adjusted the degree of substitution of CMCH easily in the range of 0.035
0.74 [36]. When the total degree of substitution reached 0.4, the CMCH with a degree of Naceylation above 0.85 could be dissolved in pure water. Interestingly, some CMCH solutions showed fast thermosensitive sol-to-gel phase transition characteristics at body temperature and the gelation time of CMCH thermogels could be controlled by varying the degree of substitution, which is beneficial for embedding cells below 37 C and in situ-forming hydrogel at body temperature. Then we mixed COS-7 cells with CHCH solution at 4 C and the COS-7 cell loaded CMCH hydrogels were in situ formed at 37 C distributed uniformly [37]. In these hydrogels, COS-7 cell self-assembled to 3D multicellular spheroids exhibiting high viability and proliferation. Therefore the CMCH hydrogels are promising for the application of 3D cell culture. In addition, our group synthesized a series of hydroxypropyl chitins (HPCHs) homogeneously in aqueous NaOH/urea solution [38]. Degree of N-deacetylation of HPCHs was less than 30% and the obtained HPCH solutions showed temperature-dependent reversible sol
gel transformation behaviors with LCST below 37 C. Thus HPCH hydrogels can be used in many biomedical fields, for example, as drug/cell carriers. Li et al. also fabricated a series of thermosensitive HPCHs with different degrees of hydroxypropyl molar substitution (DS) in aqueous NaOH solution [39]. However, the degree of N-acetylation of HPCH was not reported there. Ding et al. modified chitins with acrylamide to obtain thermoresponsive chitin derivatives (ACH) [40]. The sol
gel transition temperature of ACH could be easily tuned by changing the degree of substitution of acrylamide, ACH concentration, pH, and the presence of anions or cations. For example, an ACH solution could form a hydrogel at 37 C and return to solution at 4 C. Our group seeded chondrocytes in HPCH solution and then the mixtures were injected subcutaneously in the nude mice [41]. These chondrocyte-containing HPCH hydrogels were in situ formed and degraded gradually at the injection site. After several weeks, cartilage-like tissue was produced, indicating that HPCH thermogels were useful candidate scaffolds for the repair of cartilage. In addition, the thermosensitive HPCH hydrogel were more effective in preventing postoperative adhesion in the rabbit model than

Handbook of Chitin and Chitosan

288

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

the control carboxymethyl chitosan hydrogel, one of the commercially available postoperative adhesion prevention products [42]. In addition, the thermosensitive injectable HPCH hydrogel can load the complex of salmon calcitonin and HA, achieving long-term sustained salmon calcitonin release (28 days) with considerable structure stability, resulting in enhanced osteogenesis and hypocalcemic effects [43]. We also examined the efficiency of the thermosensitive HPCH for mesenchymal stem cell delivery and the immune system regulation [44]. The mesenchymal stem cells incorporated in the HPCH hydrogel acted not only as a stem cell source for osteogenesis, but also as a paracrine source to promote tissue repair and regeneration through increasing M2 macrophage polarization at the injury site. These findings demonstrate the potential of mesenchymal stem cell-loaded thermosensitive HPCH hydrogel combined with a 3D-printed poly(ε-caprolactone)/nanohydroxyapatite scaffold as a novel therapeutic strategy for promoting tissue regeneration. Sun et al. [45] investigated the feasibility of printing human induced pluripotent stem cells in a primed state using thermosensitive HPCH hydrogel as a new type of bioink by extrusion-based 3D bioprinting technology. Four advantages of the HPCH-based bioink over common bioinks are summarized: first, low solute concentrations such as 2% (w/v) are sufficient for in situ formation of cross-linking HPCH thermogel and achieving stable 3D constructs at 37 C. Second, thermosensitive HPCH was cross-linked physically by the formation of hydrogen bonds without additional ions or other cross-linkers. Third, the storage modulus of the HPCH solution at 15 C was very small (around 4 Pa) and changed very little before the temperature reached the gel point, which is very helpful for reducing the damage to cells during printing. More importantly, the thermosensitive features of HPCH are suitable as a basic bioink material to improve printing efficiency and formability with limited cell damage for rapid printing, leaving space for other bioactive additives. In order to further improve the mechanical properties of thermosensitive HPCH hydrogels, we modified HPCH with glycidyl methacrylate to obtain a new photocross-linkable hydroxypropyl chitin-based hydrogel (GM-HPCH) [46]. Similar to HPCH, GM-HPCH solution also showed reversible thermosensitive sol
gel transition behaviors under physiological conditions until it was irradiated by UV light. All these GM-HPCH systems showed much higher mechanical strength than the only physically cross-linked HPCH hydrogels. In addition, photocrosslinking GM-HPCH thermogels were noncytotoxic. Therefore these biodegradable and photocross-linkable GM-HPCH thermogels may be very useful in tissue engineering. N-acetylation of chitosan derivatives under mild conditions is another way to synthesize chitin derivatives. For example, Huh research group synthesized a water-soluble glycol chitin by N-acetylation of

Handbook of Chitin and Chitosan

9.2 Injectable polymeric gels based on chitosan and chitin

289

glycol chitosan in methanol/water solution at room temperature [47]. The obtained glycol chitin aqueous solutions showed fast and reversible sol
gel transition behaviors in response to temperature. The gelation temperature could be tuned from 23 C to 72 C by changing glycol chitin concentration, degree of N-acetylation, and salt concentration. DOX could be embedded into the hydrogels by a simple mixing process and the DOX-loaded hydrogels exhibited sustained release over 13 days, indicating that the thermosensitive glycol chitin hydrogels are promising candidates as injectable biomaterials for drug delivery. Huh’s research group also used glycol chitins as the scaffolds to promote odontogenic differentiation of human dental pulp cells [48]. Enamel matrix derivatives functioning as growth factors were embedded in human dental pulp cells containing glycol chitin hydrogel scaffolds. Compared to nonenamel matrix derivatives loaded hydrogel scaffolds, the expression of odontogenic markers including dentin matrix protein1, dentin sialophosphoprotein, and osteopontin messenger RNA were upregulated notably. In addition, Huh’s research group used the thermosensitive glycol chitins to encapsulate P4 (a female steroidal hormone) for vaginal application [49]. Glycol chitin-P4 hydrogels showed mucosal adhesion, which endowed these materials with extended local residence times and the desired controlled release of P4.

9.2.2 Chemical hydrogels Physical injectable hydrogels based on chitosan or chitin are the ideal biocompatible and biodegradable natural polymers that can be applied in many medical fields. However, hydrogels formed in situ only by physical cross-linking often erode and degrade quickly in vivo because of their weak mechanical strength, which limits their potential applications [50]. Therefore many researchers have focused on the development of injectable hydrogels, which are formed in situ only by chemical cross-linking or by a combination of physical cross-linking with many chemical reactions. 9.2.2.1 Photocross-linking Photocross-linking hydrogels are formed in a few seconds to a minute or more by the cross-linking reactions between vinyl groups on polymer chains and free radicals that are obtained by the decomposition of photoinitiators under ultraviolet (UV) or visible light irradiation [51,52]. These photocross-linking products have many advantages, such as in situ cross-linkable, morphology controllable, rapid curing rate, minimal heat generation, and mild reaction conditions, and they have been widely used in many biomedical fields [53].

Handbook of Chitin and Chitosan

290

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

Gao et al. synthesized a series of water-soluble methacryloyloxy ethyl carboxyethyl chitosans by the Michael addition reaction between the amino groups of chitosan and the acrylate double bond of ethylene glycol acrylate methacrylate [54]. A concentrated precursor solution containing many methacrylate groups was irradiated by UV light for up to 15 min in the presence of D-2959 photoinitiator, and a cross-linked hydrogel was in situ formed. Li et al. synthesized N-methacyloyl chitosans by chemoselective N-acylation of chitosan with methacrylic anhydride [55]. The solubility of N-methacyloyl chitosan in water increased 2.21-fold with the substitution degree increasing from 10.9% to 28.4% and N-methacyloyl chitosan solutions could convert to gel rapidly under the skin of the mouse without skin injury by low-dose UV irradiation in the presence of I2959 photoinitiator. Cho et al. prepared methacrylated hexanoyl glycol chitosans by N-hexanoylation and N-methacrylation of glycol chitosans [50]. These chitosan derivatives with degree of N-methacrylation below 0.08 could be dissolved in water and the obtained solutions displayed reversible thermosensitive sol
gel phase transition behaviors. Under UV irradiation for 5
30 min, these solutions could transform into chemically cross-linked hydrogels in the presence of I2959. Cui et al. mixed glycol chitosan and glycidyl methacrylate to prepare photocross-linkable methacrylated glycol chitosans, which could form in situ nanocomposite hydrogels by introducing twodimensional montmorillonite with intercalation chemistry under visible blue lighting in the presence of the riboflavin photoinitiator [56]. These nanocomposite hydrogels can promote native cell infiltration, proliferation, and in situ differentiation without any growth factors, small molecular drugs, or genes. A photocross-linkable hydroxypropyl chitin-based hydrogel was prepared using glycidyl methacrylate by our group [46]. We also functionalized CMCH by grafting methacrylate groups to fabricate the photocross-linkable methacrylated CMCH [57]. Colorless methacrylated CMCH aqueous solutions in the presence of initiators (Irgacure 2959) were exposed to UV light and the transparent hydrogels were produced quickly. However, photocross-linking hydrogels are not without drawbacks. Photoinitiators are required for the in situ formation of all the photocross-link hydrogels and whether these photoinitiators are nontoxic to organisms remains controversial. Furthermore, irradiation could introduce damage to normal cells and tissues in most living bodies [58]. Additionally, cross-linking efficiency may be low due to the poor penetration of UV or visible light when photocross-linkable polymeric precursor solutions are implanted deeply.

Handbook of Chitin and Chitosan

9.2 Injectable polymeric gels based on chitosan and chitin

291

9.2.2.2 Schiff base cross-linking Injectable hydrogels can be obtained through Schiff base reactions between amino groups and carbonyl groups under physiological conditions, which have potential application prospects in many biomedical fields. However, traditional chemical cross-linkers, such as glutaraldehyde, are limited in some applications due to their potential toxicity to living cells, instability of protein in the hydrogel preparation process, and loss of biological activity after administration into the body [59]. Recently, good biocompatible materials, including chitosan derivatives grafted with carbonyl groups, are becoming a research hot topic as green cross-linkers (Table 9.1). It can be seen in Table 9.1 that amino-containing components of the above Schiff base cross-linking hydrogels are chitosan and chitosan derivatives such as quaternized chitosan, N-succinyl chitosan, carboxymethyl chitosan, carboxyethyl chitosan, and glycol chitosan. Carbonylcontaining components of these in situ-forming hydrogels are natural polysaccharide derivatives, PEG derivatives, and so on. Gelation time and physical properties of these hydrogels can be controlled by the ratio of the amine and carbonyl groups. Enhancing the amount of amino [80] or carbonyl groups [72] in Schiff base reaction systems can further improve the mechanical properties of the in situ-forming hydrogels in some cases. However, these manipulations often bring unexpected problems. For example, excessive cross-linking could make the hydrogel brittle and too much unreacted amino or carbonyl groups remaining in the Schiff base cross-linking hydrogels may be harmful to the living organisms. 9.2.2.3 Michael additions Hydrogels in situ cross-linked by Michael addition reaction have received much attention due to their high selectivity. Hydrogels based on chitosans or chitosan derivatives can be easily formed in situ by Michael-type addition reaction under physiological conditions and the gelation time of these hydrogels is dependent on the pH and the concentration of the polymer precursor solutions [81]. Teng et al. designed an injectable hydrogel by Michael-type addition reaction between thiolmodified chitosan and PEG diacrylate [82]. Gelation times from 25 to 55 min could be controlled by both of the polymer concentrations, temperature, and the content of free thiol groups. Yu et al. designed a new injectable hydrogel through Michael-type addition between thiolated glycol chitosan and oligo(acryloyl carbonate)-b-poly(ethylene glycol)-boligo(acryloyl carbonate) triblock copolymers [83]. The gelation times (from 10 s to 17 min) depended on concentrations of both polymer, the solution pH, and degree of thiol group substitution. In addition,

Handbook of Chitin and Chitosan

292

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

TABLE 9.1 Schiff base cross-linking hydrogels and their applications in biomedical fields. Schiff base cross-linking hydrogel component Amino groups Chitosan

Carbonyl groups Oxidized dextran

Applications Deliver hydrophobic and hydrophilic drugs simultaneously for cancer treatment [60]; delivery of retinal progenitor cells [61]

Oxidized konjac glucomannan

Wound healing [62]

Oxidized alginate

Tumor therapy and bone regeneration [63]

Quaternized

Benzaldehyde-terminated

Joints, skin, wound healing [64]

chitosan

PluronicF127 (PF127-CHO)

N-succinyl

Oxidized carboxymethyl cellulose

Delivery of protein drugs [59]

Aldehyde hyaluronic acid

Adipose tissue regeneration [65]

Oxidized alginate

Breast tumor treatment [66]

Dialdehyde starch

Tissue engineering and cartilage repair [67]

Aldehyde-functionalized chitosan

Vaginal delivery of therapeutic agents [68]

chitosan

Carboxymethyl

Oxidized dextran

chitosan

Drug delivery and tissue engineering, Burn wound healing and skin regeneration [69]

Oxidized carboxymethyl cellulose

Embolizing agent for different interventional radiology therapies/pH-sensitive drug delivery and wound dressing [70]

Aldehyde hyaluronic acid

Postoperative adhesion prevention [71]

Oxidation cholesterol starch

Drug delivery [72]

Oxidized chondroitin sulfate

Drug/cell delivery system in cartilage tissue engineering [73]

Oxidized alginate

Drug delivery and tissue engineering [74]

Carboxyethyl

Dibenzaldehyde-terminated poly

Hepatocellular carcinoma therapy [75]

chitosan

(ethylene glycol) Dextran-graft-aniline tetramer-

Myoblast cell therapy and muscle regeneration [76]

graft-4-formylbenzoic acid

Glycol chitosan

Aldehyde hyaluronic acid

pH-responsive drug release [77]

Oxidized alginate

Drug delivery and tissue engineering [78]

Oxidized dextran

Delivery of protein drugs [79]

Oxidized hyaluronate

Chondrocyte encapsulation [80]

Handbook of Chitin and Chitosan

9.2 Injectable polymeric gels based on chitosan and chitin

293

Mohrman et al. functionalized chitosan with 2-iminothiolane to synthesize a water-soluble polymer [84]. Once mixed with maleimideterminated PEG, the precursor solutions in physiological conditions could transform into hydrogels quickly, which were cross-linked by Michael addition reactions. Chen et al. fabricated a biocompatible multiple cross-linking hydrogel system by combining thermosensitive characteristics, disulfide bond formation, and Michael addition based on thiolated chitosan, β-glycerophosphate, and PEG diacrylate [85]. Gelation times (from 1 to 22 min) of these composite hydrogels could be tuned by varying the molecular weight of PEG diacrylate.

9.2.2.4 Enzymatic cross-linking Recently, enzymatically cross-linked hydrogels have been becoming more and more popular, because enzymatic reactions with high chemoselectivity can occur in mild physiological conditions [86]. Jin et al. synthesized chitosan-g-glycolic acid/phloretic acid by first grafting glycolic acid to chitosan and subsequent conjugating phloretic acid [87]. These chitosan derivative solutions in phosphate buffered saline could transform into hydrogels within 250 s by enzymatic cross-linking with horseradish peroxidase (HRP) and H2O2. Sakai et al. synthesized a chitosan with phenolic hydroxyl groups by grafting chitosan with 3-(phydroxyphenyl) propionic acid [88]. These chitosan derivatives could be dissolved in neutral conditions and in situ gelled using HRP as a catalyst and H2O2 as an oxidant. Gohil et al. grafted glycol chitosan with 3-(4-hydroxyphenyl) propionic acid to synthesize a phenol-conjugated polymer [89]. Irrespective of the polymer concentration, the gelation times of the obtained solutions were less than 1 min in the presence of HRP/H2O2. Our group synthesized a cross-linkable tyramine-modified CMCH, resulting in an injectable crosslinked in situ-forming hydrogel by using HRP/H2O2 catalyst system [90]. Mishra et al. prepared a carboxymethyl chitosan/gelatin/nanohydroxyapatite composite. The composite solutions could in situ form hydrogels in the presence of tyrosinase/p-cresol [91]. Jin et al. designed chitosan-based hydrogels in situ cross-linked through tyrosinase and HRP, respectively [92]. Tyrosinase mediated a slower gelation process than HRP, but the rabbit chondrocytes encapsulated in tyrosinase-crosslinked hydrogels remained much more viable than those in HRP-crosslinked hydrogels, indicating that tyrosinase-cross-linked hydrogels displayed better cytobiocompatibility than HPR-cross-linked hydrogels. In addition, Wang et al. prepared an N-palmitoyl chitosan hydrogel crosslinked by another enzyme called urease [93]. In addition, various enzymes (transglutaminase, lysyloxidase, etc.) have also been used as catalysts of covalent cross-linking for the

Handbook of Chitin and Chitosan

294

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

preparation of hydrogels [81]. However, high enzyme production costs limit their promotion and application in some biomedical applications. 9.2.2.5 Click chemistry In 2001 Sharp et al. first proposed the concept of “click” chemistry, that is the most reliable and practical chemical reactions to connect a diversity of structures [94]. Owing to the advantages of high yield, rapid reactivity, high chemoselectivity, and good regiospecificity under physiological conditions, these reactions can be used to prepare chemical crosslinking hydrogels for many biomedical applications [95]. Cu-catalyzed azide-alkyne addition [96] is a classic click reaction. However, the potential toxicity of metal catalysts may limit their use in biomedical applications. Up to now, many bioclick reactions have been reported, such as, azide-alkyne cycloaddition [97,98], thiol-ene/yne reaction [99,100], Diels
Alder reaction [101], and so on. Combined with easy chemical or enzymatic degradation in vivo, the in situ preparation of hydrogels based on chitosan or chitin by bioclick reactions are becoming more and more popular. For example, Truong et al. prepared an injectable chitosan/PEG hydrogel by copper-free azide-alkyne click chemistry [102]. Matsumoto et al. used chitosan conjugated with maleimide and thiol-terminated PEG to obtain a hydrogel by thiol-ene click chemistry [103]. Yang et al. obtained an injectable hydrogel through thiol-ene click reaction between thiolated chitosan and alkynyl β-cyclodextrin [104]. Li et al. designed a double cross-linked hydrogel through Diels
Alder click reaction and ferric-catechol coordination bonding by using catechol-modified N-(furfural) chitosan and dimaleimide PEG [105]. In addition, our group prepared a degradable and injectable hydrogel via another spontaneous amino-yne click reaction between carboxymethyl chitosan and alkyne groups of dipropiolate ester of PEG under physiological conditions [106].

9.3 Biomedical applications 9.3.1 Potential use of injectable hydrogels As mentioned in the above sections, injectable hydrogels based on chitosan, chitin, or their derivatives have been extensively studied in vitro and these experimental results could be extended to further animal research. If these injectable hydrogels have been proven to be feasible technologically in animals, they will be used ultimately in clinical trials. The local injection of drugs and bioactive molecules has become an indispensable part of routine medical activities spanning from imaging and treatment to anesthesia [107]. In situ forming hydrogels that enable a number of traditional treatments to become less invasive compared to

Handbook of Chitin and Chitosan

9.3 Biomedical applications

295

surgery implantation have emerged as one of the most important potential uses, that is, as carriers for drugs/cells and bioactive molecules, supporting immunoregulation, bioadhesion, and tissue repair/regeneration [108]. But in most cases, the diameters of the loaded drug molecules are much smaller than the mesh sizes of hydrogels, and burst release is inevitable within a short period of time due to the free diffusion of drug molecules in the highly permeable nature of the hydrogel matrix. Until now, adjusting the polymer concentration and cross-linking density are the feasible strategies to optimize the hydrogel network structure. However, more sophisticated strategies such as the use of interpenetrating polymer networks are limited [109], because the formation of these interpenetrating polymer networks requires immersing a preprepared hydrogel network in a solution of a second monomer and polymerizing initiators and this manipulation is typically not suitable for injection. Injectable hydrogels that have emerged as tissue bioadhesives also have the potential for surgical assistance. For example, injectable hydrogel-based bioadhesives, such as TISSEEL (Baxter, fibringlue) [110], have been used in many surgical procedures. Studies have reported that hydrogels based on chitosan, chitin, or their derivatives are very biocompatible, but they are very rarely applied in surgical applications because of poor hydrogel matrix cohesion and hydrogel
tissue interfacial adhesion. More importantly, whether the degradation of these hydrogels match the tissues/organs regeneration window in vivo cannot be traced in real time. Although it is still a major technical challenge, the development of injectable hydrogels based on chitosan, chitin, or their derivatives with excellent wet adhesion and toughness is of great importance for various clinical applications.

9.3.2 Clinical trial and human applications Only a few injectable hydrogels based on chitosan and chitin have been tried in clinic. For example, investigational medical device coded KIO014, an innovative chitosan-based biomaterial (ClinicalTrials.gov Identifier: NCT03679208, Kiomed Pharma, Belgium) was intended to be used in patients for the symptomatic treatment of knee osteoarthritis by intraarticular injection. In addition, some injectable hydrogels based on chitosan and chitin have been used in human applications in some countries such as China (Table 9.2). As can been seen in Table 9.2, four kinds of class III medical devices based on carboxymethyl chitosan and one based on CMCH have been used for preventing postoperative adhesion. Also class III medical devices, CHITOGEL, based on CMCH have been used for treatment of osteoarthritis. In addition, class II medical devices named YICHUANGNING, based on carboxymethyl chitosan, and AmPoSa, based on CMCH, have been used for wound repair. It is worth noting that all these medical devices

Handbook of Chitin and Chitosan

296

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

TABLE 9.2 Examples of hydrogels based on chitosan and chitin approved for clinical use in China. China registration

Applications

Trademarks

Prevention of

SUCHONGNING

Main

certificate for medical

composition

device number

Carboxymethyl

20173640568

chitosan

postoperative

Corporations Shijiazhuang Yishengtang Medical Supplies Co., Ltd.

adhesion SEPTIM

20153641074

Shandong Saikesaisi Biological Technology Co., Ltd.

XINKEDA

20153140101

Hangzhou Xiehe Medical Supplies Co., Ltd.

NIANTINGNING

20173643097

Yantai Wanli Medical Supplies Co., Ltd.

CHITOGEL

Carboxymethyl

20143642114

chitin

Shanghai Qisheng Biological Preparation Co., Ltd.

Wound repair

YICHUANGNING

carboxymethyl

20162640052 (Hebei

Shijiazhuang

chitosan

Province)

Yishengtang Medical Supplies Co., Ltd.

AmPoSa

Carboxymethyl

20172641732

Shenzhen Brightway

chitin

(Guangdong Province)

Novel Bio-material Tech. Co. Ltd.

Treatment of osteoarthritis

CHITOGEL

Carboxymethyl

20173640026

chitin

Shanghai Qisheng Biological Preparation Co., Ltd.

have been approved by the National Medical Products Administration of the People’s Republic of China (NMPA). BST-CarGel, approved as a medical device in several countries worldwide, including Australia, Canada, and most of Europe (but not yet by the FDA or NMPA), can be in situ formed by injecting the mixed solutions of chitosan and β-glycerophosphate into the joint following a microfracture procedure. Combined with arthroscopical technology, BST-CarGel loaded autologous whole blood can be used to treat damaged cartilage in synovial joints such as the knee, hip, ankle, and shoulder. Once implanted, it acts as a scaffold, adhering to the cartilage surface to stabilize the blood clot while new cartilage is regenerated. Many clinical trials are listed in Table 9.3.

Handbook of Chitin and Chitosan

297

9.3 Biomedical applications

TABLE 9.3

Examples of clinical trials for injectable BST-CarGel.

Treatment

ClinicalTrials.

Patient

position

gov Identifier

number

Knee

NCT00314236

80 patients

Evaluation time 12 months

Conclusions Compared to microfracture treatment

from 18 and

alone, BST-CarGel treatment shows

55 years

greater lesion filling and superior repair tissue quality [111]

Not

Not

mentioned

mentioned

12 months

The effect of BST-CarGel microfracture treatment by arthroscopic technique is better than the one by open surgery [112]

NCT00314236

38 patients

13 months

BST-CarGel treatment are analyzed by osteochondral biopsy [113]

from 18 and 55 years Not

39 patients

13 months

mentioned

Microfracture with BST-CarGel showed regenerated tissue effectively consistent with a chondroinduction mechanism [114]

NCT01246895

80 patients

5 years

BST-CarGel exhibits an effective midterm cartilage repair treatment [115]

from 18 and 55 years Not

Not

Construct a

Establish the economic value of BST-

mentioned

mentioned

decision tree with

CarGel vs. microfracture alone from

a 20-year time-

the societal perspective, using

horizon

Germany as the reference market [116]

Talus

Not

Not

From 9 to 12

Superior mechanical properties and

mentioned

mentioned

months

blood clot stability leads to good quality repair tissue in the short term [117]

Hip

Not

Not

mentioned

mentioned

12 months

Expanding clinical research from knee

Not

37 patients

mentioned

with a mean

patients with large cartilage defect

age of 36

sizes [119]

to hip [118] 13 months

Showing satisfactory safety, even for

years 24 months

BST-CarGel with microfracture

Not

13 patients

mentioned

from 18 and

showed good repair quality with

50 years

specific cartilage sequences [120]

Handbook of Chitin and Chitosan

298

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

As can be seen in Table 9.3, clinical investigations of injectable BSTCarGel are mainly focused on the treatment of knee, talus, and hip chondral lesions. Chondral lesions in these positions affect people’s daily activities directly and the precursor solution of chitosan/β-glycerophosphate can be easily injected in the knee, talus, and hip with minimal invasive surgery. Combined with microfracture treatment and arthroscopic technique, BST-CarGel leads to good quality tissue repair with superior mechanical properties and blood clot stability. In the short term, the treatment of cartilage damage is good. However, there is still a lack of long-term clinical treatment data. Fortunately, Restrepo et al. constructed a decision tree with a 20-year time-horizon, in which undesirable clinical events were inferred following initial surgery [116]. What’s more, BST-CarGel will be approved in more countries, for example, by FDA and NMPA in the United States and China, respectively, in the near future.

9.4 Conclusions and future perspectives In this review, we summarized a serial of injectable hydrogels based on chitosan, chitin, or their derivatives and all the polymer networks are biocompatible and biodegradable. These biomaterials can be divided into physical hydrogels and chemical hydrogels. Each hydrogel system has its own advantages and disadvantages in biomedical applications for drug/cell delivery, tissue engineering, postoperative adhesion prevention, and so on. Injectable hydrogels prepared by physical cross-linking can be easily formed without the need for reactive chemicals, but these thermosensitive hydrogels have poor stability and mechanical properties in vivo. Injectable hydrogels prepared by chemical cross-linking have good mechanical properties, but their use in vivo is limited because reactive chemical cross-linking agents may be cytotoxic. Only a few injectable hydrogels based on chitosan, carboxymethyl chitosan, and CMCH have been applied in clinic trials or approved for clinical treatments. More injectable hydrogels based on chitosan and chitin have shown promising potential and will be used in future human applications. However, some issues should be noted from translational research and development. One challenge is the quality of chitosan or chitin. To be specific, chitosan or chitin that is suitable for further modification needs to be fully characterized, including parameters such as degree of deacetylation, molecular weight, and purity (i.e., exclusion of protein, minerals, and endotoxin content). Additionally, the high costs associated with the preparation of medical-grade injectable hydrogels need to be overcome.

Handbook of Chitin and Chitosan

References

299

Acknowledgment This research was financially supported by the National Natural Science Foundation of China (21674083 and 21875168), Hubei Provincial Natural Science Foundation of China (2019CFB239), and the Natural Science Foundation of Jiangsu Province of China (BK20151249).

References [1] Y.-B. Zhang, J.-K. Yu, K.-X. Ren, J.-L. Zuo, J.-X. Ding, X.-S. Chen, Thermosensitive hydrogels as scaffolds for cartilage tissue engineering, Biomacromolecules 20 (2019) 1478
1492. [2] S. Saravanan, S. Vimalraj, P. Thanikaivelan, S. Banudevi, G. Manivasagam, A review on injectable chitosan/beta glycerophosphate hydrogels for bone tissue regeneration, Int. J. Biol. Macromol. 121 (2019) 38
54. [3] Y.P. Singh, J.C. Moses, N. Bhardwaj, B.B. Mandal, Injectable hydrogels: a new paradigm for osteochondral tissue engineering, J. Mater. Chem. B 6 (2018) 5499
5529. [4] F. Ullah, M.B.H. Othman, F. Javed, Z. Ahmad, H.M. Akil, Classification, processing and application of hydrogels: a review, Mater. Sci. Eng. C-Mater. Biol. Appl. 57 (2015) 414
433. [5] A.S. Hoffman, Hydrogels for biomedical applications, Adv. Drug. Deliv. Rev. 64 (2012) 18
23. [6] S. Naahidi, M. Jafari, M. Logan, Y.-J. Wang, Y.-F. Yuan, H. Bae, et al., Biocompatibility of hydrogel-based scaffolds for tissue engineering applications, Biotechnol. Adv. 35 (2017) 530
544. [7] B. Chang, N. Ahuja, C. Ma, X.-H. Liu, Injectable scaffolds: preparation and application in dental and craniofacial regeneration, Mater. Sci. Eng. R-Rep. 111 (2017) 1
26. [8] A. Ehsanipour, T. Nguyen, T. Aboufadel, M. Sathialingam, P. Cox, W.-K. Xiao, et al., Injectable, hyaluronic acid-based scaffolds with macroporous architecture for gene delivery, Cell. Mol. Bioeng. 12 (2019) 399
413. [9] Z.-H. Lu, S.-J. Liu, Y.-G. Le, Z.-N. Qin, M.-W. He, F.-B. Xu, et al., An injectable collagengenipin-carbon dot hydrogel combined with photodynamic therapy to enhance chondrogenesis, Biomaterials 218 (2019). UNSP 119190. [10] M.-J. Liu, X.-X. Lu, L. Gao, S.-T. Wang, Y.-P. Huo, Z.-N. Chen, Polyvinyl alcoholbased thermogel with tunable gelation and self-healing property, Macromol. Chem. Phys. 219 (2018) 1800162. [11] N. Jommanee, C. Chanthad, K. Manokruang, Preparation of injectable hydrogels from temperature and pH responsive grafted chitosan with tuned gelation temperature suitable for tumor acidic environment, Carbohydr. Polym. 198 (2018) 486
494. [12] H.-Y. Zhou, L.-J. Jiang, P.-P. Cao, J.-B. Li, X.-G. Chen, Glycerophosphate-based chitosan thermosensitive hydrogels and their biomedical applications, Carbohydr. Polym. 117 (2015) 524
536. [13] A. Chenite, C. Chaput, D. Wang, C. Combes, M.D. Buschmann, C.D. Hoemann, et al., Novel injectable neutral solutions of chitosan form biodegradable gels in situ, Biomaterials 21 (2000) 2155
2161. [14] C.D. Hoemann, J. Sun, M.D. McKee, A. Chevrier, E. Rossomacha, G.-E. Rivard, et al., Chitosan-glycerol phosphate/blood implants elicit hyaline cartilage repair integrated with porous subchondral bone in microdrilled rabbit defects, Osteoarthr. Cartil. 15 (2007) 78
89. [15] J. Guzman-Morales, C.-H. Lafantaisie-Favreau, G. Chen, C.D. Hoemann, Subchondral chitosan/blood implant-guided bone plate resorption and woven bone repair is coupled to hyaline cartilage regeneration from microdrill holes in aged rabbit knees, Osteoarthr. Cartil. 22 (2014) 323
333.

Handbook of Chitin and Chitosan

300

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

[16] A. Fatimi, P. Chabrot, S. Berrahmoune, J.-M. Coutu, G. Soulez, S. Lerouge, A new injectable radiopaque chitosan-based sclerosing embolizing hydrogel for endovascular therapies, Acta Biomater. 8 (2012) 2712
2721. [17] X. Li, D.-D. Fan, X.-X. Ma, C.-H. Zhu, Y.-N. Luo, B.-W. Liu, et al., A novel injectable pH/temperature sensitive CS-HLC/β-GP hydrogel: the gelation mechanism and its properties, Soft Mater. 12 (2013) 1
11. [18] C.-L. Huang, Y.-B. Chen, Y.-L. Lo, Y.-H. Lin, Development of chitosan/beta-glycerophosphate/glycerol hydrogel as a thermosensitive coupling agent, Carbohydr. Polym. 147 (2016) 409
414. [19] Y. Wang, X.-L. Pang, J. Luo, Q. Wen, Z.-X. Wu, Q.-X. Ding, et al., Naproxen nanoparticle-loaded thermosensitive chitosan hydrogel for prevention of postoperative adhesions, ACS Biomater. Sci. Eng. 5 (2019) 1580
1588. [20] C.-Z. Wei, C.-L. Hou, Q.-S. Gu, L.-X. Jiang, B. Zhu, A.-L. Sheng, A thermosensitive chitosan-based hydrogel barrier for post-operative adhesions’ prevention, Biomaterials 30 (2009) 5534
5540. [21] Y. Cai, J.-F. Cao, C. Xu, J.-P. Zhou, Thermo-responsive behaviors and bioactivities of hydroxybutyl chitosans prepared in alkali/urea aqueous solutions, Carbohydr. Polym. 215 (2019) 90
98. [22] G.-H. Sun, C. Feng, C.-Q. Jiang, T.-T. Zhang, Z.-X. Bao, Y.-J. Zuo, et al., Thermoresponsive hydroxybutyl chitosan hydrogel as artery intervention embolic agent for hemorrhage control, Int. J. Biol. Macromol. 105 (2017) 566
574. [23] Q.-Q. Wang, M. Kong, Y. An, Y. Liu, J.-J. Li, X. Zhou, et al., Hydroxybutyl chitosan thermo-sensitive hydrogel: a potential drug delivery system, J. Mater. Sci. 48 (2013) 5614
5623. [24] K.-K. Yang, T.-T. Gao, Z.-X. Bao, J. Su, X.-G. Chen, Preparation and characterization of a novel thermosensitive nanoparticle for drug delivery in combined hyperthermia and chemotherapy, J. Mater. Chem. B 1 (2013) 6442
6448. [25] T.-T. Gao, M. Kong, X.-J. Cheng, G.-X. Xia, Y.-Y. Gao, X.-G. Chen, et al., A thermosensitive chitosan-based hydrogel for controlled release of insulin, Front. Mater. Sci. 8 (2014) 142
149. [26] X.-Y. Bai, Z.-X. Bao, S.-C. Bi, Y. Li, X.-P. Yu, S.-H. Hu, et al., Chitosan-based thermo/ pH double sensitive hydrogel for controlled drug delivery, Macromol. Biosci. 18 (2018) 1700305. [27] Y.-N. Wei, Q.-Q. Wang, T.-T. Gao, M. Kong, K.-K. Yang, Y. An, et al., 3D culture of human umbilical vein endothelial cells with reversible thermosensitive hydroxybutyl chitosan hydrogel, J. Mater. Sci.-Mater. Med. 24 (2013) 1781
1787. [28] J.M. Dang, D.D.N. Sun, Y. Shin-Ya, A.N. Sieber, J.P. Kostuik, K.W. Leong, Temperature-responsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells, Biomaterials 27 (2006) 406
418. [29] F.-F. Li, Y. Liu, Y. Ding, Q.-F. Xie, A new injectable in situ forming hydroxyapatite and thermosensitive chitosan gel promoted by Na2CO3, Soft Matter 10 (2014) 2292
2303. [30] Z.-Q. Jiang, Z.-K. Wang, K. Zhang, Y.-L. Li, Q.-L. Hu, Preparation and properties of thermo-responsive hydroxyl-propyl chitosan, J. Mater. Sci. Eng. 31 (2013) 687
696. [31] A. Anitha, S. Sowmya, P.T.S. Kumar, S. Deepthi, K.P. Chennazhi, H. Ehrlich, et al., Chitin and chitosan in selected biomedical applications, Prog. Polym. Sci. 39 (2014) 1644
1667. [32] D.-R. Ren, H.-F. Yi, W. Wang, X.-J. Ma, The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation, Carbohydr. Res. 340 (2005) 2403
2410. [33] K. Tomihata, Y. Ikada, In vitro and in vivo degradation of films of chitin and its deacetylated derivatives, Biomaterials 18 (1997) 567
575.

Handbook of Chitin and Chitosan

References

301

[34] Y. Fang, B. Duan, A. Lu, M.-L. Liu, H.-L. Liu, X.-J. Xu, et al., Intermolecular interaction and the extended wormlike chain conformation of chitin in NaOH/urea aqueous solution, Biomacromolecules 16 (2015) 1410
1417. [35] B. Duan, X. Zheng, Z.-X. Xia, X.-L. Fan, L. Guo, J.-F. Liu, et al., Highly biocompatible nanofibrous microspheres self-assembled from chitin in NaOH/urea aqueous solution as cell carriers, Angew. Chem. 54 (2015) 5152
5156. [36] H. Liu, Q.-Z. Yang, L.-N. Zhang, R.-X. Zhuo, X.-L. Jiang, Synthesis of carboxymethyl chitin in aqueous solution and its thermo- and pH-sensitive behaviors, Carbohydr. Polym. 137 (2016) 600
607. [37] H. Liu, J. Liu, C. Qi, Y.-P. Fang, L.-N. Zhang, R.-X. Zhuo, et al., Thermosensitive injectable in-situ forming carboxymethyl chitin hydrogel for three-dimensional cell culture, Acta Biomater. 35 (2016) 228
237. [38] X.-L. Jiang, H. Liu, Q.-Z. Yang, R.-X. Zhuo, Method for homogeneously preparing hydroxypropyl modified chitin with low degree of deacetylation, Chin. Pat. (2016). ZL2014101708718. [39] C. Li, J.-X. Hou, J.-J. Gu, Q.-Y. Han, Y. Guan, Y.-J. Zhang, Synthesis and thermal gelation of hydroxypropyl chitin, RSC Adv. 5 (2015) 39677
39685. [40] F.-Y. Ding, Z. Tang, B.-B. Ding, Y. Xiong, J. Cai, H.-B. Deng, et al., Tunable thermosensitive behavior of multiple responsive chitin, J. Mater. Chem. B 2 (2014) 3050
3056. [41] X.-L. Jiang, H. Liu, G.-D. Zhou, Y.-P. Feng, W. Fang, X. Long, et al., Thermosensitive injectable modified chitin hydrogel for cell delivery, J. Control. Release 259 (2017) e161
e162. [42] J. Jiang, Q. Liu, S.-H. Huang, Z.-W. Yuan, Z.-X. Xiang, B. Xiong, Postoperative adhesion prevention with a thermosensitive injectable in situ forming modified chitin hydrogel, Med. J. Wuhan. Univ. 39 (2018) 605
609. [43] P. Yu, J. Xie, Y. Chen, J.-M. Liu, Y.-P. Liu, B. Bi, et al., A thermo-sensitive injectable hydroxypropyl chitin hydrogel for sustained salmon calcitonin release with enhanced osteogenesis and hypocalcemic effects, J. Mater. Chem. B 8 (2020) 270
281. [44] X.-F. Ji, X. Yuan, L.-M. Ma, B. Bi, H. Zhu, Z.-H. Lei, et al., Mesenchymal stem cell-loaded thermosensitive hydroxypropyl chitin hydrogel combined with a three-dimensionalprinted poly(ε-caprolactone)/nano-hydroxyapatite scaffold to repair bone defects via osteogenesis, angiogenesis and immunomodulation, Theranostics 10 (2020) 725
740. [45] Y. Li, X.-L. Jiang, L. Li, Z.-N. Chen, G. Gao, R. Yao, et al., 3D printing human induced pluripotent stem cells with novel hydroxypropyl chitin bioink: scalable expansion and uniform aggregation, Biofabrication 10 (2018) 044101. [46] M. Yuan, B. Bi, J.-C. Huang, R.-X. Zhuo, X.-L. Jiang, Thermosensitive and photocrosslinkable hydroxypropyl chitin-based hydrogels for biomedical applications, Carbohydr. Polym. 192 (2018) 10
18. [47] Z.-Z. Li, S. Cho, I.C. Kwon, M.M. Jana´t-Amsbury, K.M. Huh, Preparation and characterization of glycol chitin as a new thermogelling polymer for biomedical applications, Carbohydr. Polym. 92 (2013) 2267
2275. [48] S.-J. Park, Z.-Z. Li, I.-N. Hwang, K.M. Huh, K.-S. Min, Glycol chitin-based thermoresponsive hydrogel scaffold supplemented with enamel matrix derivative promotes odontogenic differentiation of human dental pulp cells, J. Endod. 39 (2013) 1001
1007. [49] A.A. Almomen, S. Cho, Z.-Z. Li, K.M. Huh, C.M. Peterson, M.M. Jana´t-Amsbury, A thermosensitive glycol chitin hydrogel for the vaginal delivery of progesterone, J. Control. Release 213 (2015) e74
e75. [50] I.S. Cho, M.O. Cho, Z.-Z. Li, M. Nurunnabi, S.Y. Park, S.-W. Kang, et al., Synthesis and characterization of a new photo-cross-linkable glycol chitosan thermogel for biomedical applications, Carbohydr. Polym. 144 (2016) 59
67.

Handbook of Chitin and Chitosan

302

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

[51] I. Mironi-Harpaz, D.Y. Wang, S. Venkatraman, D. Seliktar, Photopolymerization of cell-encapsulating hydrogels: cross-linking efficiency versus cytotoxicity, Acta Biomater. 8 (2012) 1838
1848. [52] L.-Y. Xu, N. Sheybani, W.A. Yeudall, H. Yang, The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application, Biomater. Sci. 3 (2015) 250
255. [53] J.L. Ifkovits, J.A. Burdick, Review: photopolymerizable and degradable biomaterials for tissue engineering applications, Tissue Eng. 13 (2007) 2369
2385. [54] X.-Y. Gao, Y.-S. Zhou, G.-P. Ma, S.-Q. Shi, D.-Z. Yang, F.-M. Lu, et al., A watersoluble photocross-linkable chitosan derivative prepared by Michael-addition reaction as a precursor for injectable hydrogel, Carbohydr. Polym. 79 (2010) 507
512. [55] B.-Q. Li, L. Wang, F. Xu, X.-M. Gang, U. Demirci, D.-Q. Wei, et al., Hydrosoluble, UV-cross-linkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo, Acta Biomater. 22 (2015) 59
69. [56] Z.-K. Cui, S. Kim, J.J. Baljon, B.M. Wu, T. Aghaloo, M. Lee, Microporous methacrylated glycol chitosan-montmorillonite nanocomposite hydrogel for bone tissue engineering, Nat. Commun. 10 (2019) 3523. [57] W.-T. Kang, B. Bi, R.-X. Zhuo, X.-L. Jiang, Photocross-linked methacrylated carboxymethyl chitin hydrogels with tunable degradation and mechanical behavior, Carbohydr. Polym. 160 (2017) 18
25. [58] U.P. Kappes, D. Luo, M. Potter, K. Schulmeister, T.M. Runger, Short- and long-wave UV light (UVB and UVA) induce similar mutations in human skin cells, J. Investig. Dermatol. 126 (2006) 667
675. [59] S.-Y. Lu¨, M.-Z. Liu, B.-L. Ni, An injectable oxidized carboxymethylcellulose/N-succinyl-chitosan hydrogel system for protein delivery, Chem. Eng. J. 160 (2010) 779
787. [60] N.-N. Gao, S.-Y. Lu¨, C.-M. Gao, X.-G. Wang, X.-B. Xu, X. Bai, et al., Injectable shellcross-linked F127 micelle/hydrogel composites with pH and redox sensitivity for combined release of anticancer drugs, Chem. Eng. J. 287 (2016) 20
29. [61] F. Jiang, Z.-M. Tang, Y.-H. Zhang, Y.-H. Ju, H.-Q. Gao, N. Sun, et al., Enhanced proliferation and differentiation of retinal progenitor cells through a self-healing injectable hydrogel, Biomater. Sci. 7 (2019) 2335
2347. [62] H.-L. Chen, J.-W. Cheng, L.-X. Ran, K. Yu, B.-T. Lu, G.-Q. Lan, et al., An injectable self-healing hydrogel with adhesive and antibacterial properties effectively promotes wound healing, Carbohydr. Polym. 201 (2018) 522
531. [63] S.-Y. Luo, J. Wu, Z.-R. Jia, P.-F. Tang, J. Sheng, C.-M. Xie, et al., An injectable, bifunctional hydrogel with photothermal effects for tumor therapy and bone regeneration, Macromol. Biosci. 19 (2019) 1900047. [64] J. Qu, X. Zhao, Y.-P. Liang, T.-L. Zhang, P.X. Ma, B.-L. Guo, Antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility as wound dressing for joints skin wound healing, Biomaterials 183 (2018) 185
199. [65] H.-P. Tan, J.P. Rubin, K.G. Marra, Injectable in situ forming biodegradable chitosanhyaluronic acid based hydrogels for adipose tissue regeneration, Organogenesis 6 (2010) 173
180. [66] J.-B. Shi, W. Guobao, H.-L. Chen, W. Zhong, X.-Z. Qiu, M.M.Q. Xing, Schiff based injectable hydrogel for in situ pH-triggered delivery of doxorubicin for breast tumor treatment, Polym. Chem. 5 (2014) 6180
6189. [67] E.A. Kamoun, N-succinyl chitosan-dialdehyde starch hybrid hydrogels for biomedical applications, J. Adv. Res. 7 (2016) 69
77. [68] E. Jalalvandi, A. Shavandi, In situ-forming and pH-responsive hydrogel based on chitosan for vaginal delivery of therapeutic agents, J. Mater. Sci. Mater. Med. 29 (2018) 158.

Handbook of Chitin and Chitosan

References

303

[69] L.-C. Li, J. Ge, P.X. Ma, B.-L. Guo, Injectable conducting interpenetrating polymer network hydrogels from gelatin-graft-polyaniline and oxidized dextran with enhanced mechanical properties, RSC Adv. 5 (2015) 92490
92498. [70] L.-H. Weng, N. Rostambeigi, N.D. Zantek, P. Rostamzadeh, M. Bravo, J. Carey, et al., An in situ forming biodegradable hydrogel-based embolic agent for interventional therapies, Acta Biomater. 9 (2013) 8182
8191. [71] L. Li, N. Wang, X. Jin, R. Deng, S.-H. Nie, L. Sun, et al., Biodegradable and injectable in situ cross-linking chitosan-hyaluronic acid based hydrogels for postoperative adhesion prevention, Biomaterials 35 (2014) 3903
3917. [72] Y.-L. Li, Y. Tan, K. Xu, C.G. Lu, X.-C. Liang, P.-X. Wang, In situ cross-linkable hydrogels formed from modified starch and O-carboxymethyl chitosan, RSC Adv. 5 (2015) 30303
30309. [73] M. Fan, Y. Ma, H.-P. Tan, Y. Jia, S.-Y. Zou, S.-X. Guo, et al., Covalent and injectable chitosan-chondroitin sulfate hydrogels embedded with chitosan microspheres for drug delivery and tissue engineering, Mater. Sci. Eng. C-Mater. Biol. Appl. 71 (2017) 67
74. [74] B.-W. Ren, X.-Y. Chen, S.-K. Du, Y. Ma, H.-N. Chen, G.-L. Yuan, et al., Injectable polysaccharide hydrogel embedded with hydroxyapatite and calcium carbonate for drug delivery and bone tissue engineering, Int. J. Biol. Macromol. 118 (2018) 1257
1266. [75] J. Qu, X. Zhao, P.X. Ma, B.-L. Guo, pH-responsive self-healing injectable hydrogel based on N-carboxyethyl chitosan for hepatocellular carcinoma therapy, Acta Biomater. 58 (2017) 168
180. [76] B.-L. Guo, J. Qu, X. Zhao, M.-Y. Zhang, Degradable conductive self-healing hydrogels based on dextran-graft-tetraaniline and N-carboxyethyl chitosan as injectable carriers for myoblast cell therapy and muscle regeneration, Acta Biomater. 84 (2019) 180
193. [77] C. Qian, T.-B. Zhang, J. Gravesande, C. Baysah, X.-Y. Song, J.-F. Xing, Injectable and self-healing polysaccharide-based hydrogel for pH-responsive drug release, Int. J. Biol. Macromol. 123 (2019) 140
148. [78] X.-Y. Chen, M. Fan, H.-P. Tan, B.-W. Ren, G.-L. Yuan, Y. Jia, et al., Magnetic and selfhealing chitosan-alginate hydrogel encapsulated gelatin microspheres via covalent cross-linking for drug delivery, Mater. Sci. Eng. C-Mater. Biol. Appl. 101 (2019) 619
629. [79] I.S. Cho, T. Ooya, An injectable and self-healing hydrogel for spatiotemporal protein release via fragmentation after passing through needles, J. Biomater. Sci. Polym. Ed. 29 (2018) 145
159. [80] D.Y. Kim, H. Park, S.W. Kim, J.W. Lee, K.Y. Lee, Injectable hydrogels prepared from partially oxidized hyaluronate and glycol chitosan for chondrocyte encapsulation, Carbohydr. Polym. 157 (2017) 1281
1287. [81] J.-A. Yang, J. Yeom, B.W. Hwang, A.S. Hoffman, S.K. Hahn, In situ-forming injectable hydrogels for regenerative medicine, Prog. Polym. Sci. 39 (2014) 1973
1986. [82] D.-Y. Teng, Z.-M. Wu, X.-G. Zhang, Y.-X. Wang, C. Zheng, Z. Wang, et al., Synthesis and characterization of in situ cross-linked hydrogel based on self-assembly of thiolmodified chitosan with PEG diacrylate using Michael type addition, Polymer 51 (2010) 639
646. [83] Y.-X. Yu, C. Deng, F.-H. Meng, Q. Shi, J. Feijen, Z.-Y. Zhong, Novel injectable biodegradable glycol chitosan-based hydrogels cross-linked by Michael-type addition reaction with oligo(acryloyl carbonate)-b-poly(ethylene glycol)-b-oligo(acryloyl carbonate) copolymers, J. Biomed. Mater. Research. Part A 99 (2011) 316
326.

Handbook of Chitin and Chitosan

304

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

[84] A.E. Mohrman, M. Farrag, R.K. Grimm, N.D. Leipzig, Evaluation of in situ gelling chitosan-PEG copolymer for use in the spinal cord, J. Biomater. Appl. 33 (2018) 435
446. [85] C. Chen, L. Wang, L.-D. Deng, R.-J. Hu, A.-J. Dong, Performance optimization of injectable chitosan hydrogel by combining physical and chemical triple crosslinking structure, J. Biomed. Mater. Research. Part. A 101 (2013) 684
693. [86] L.S. Moreira Teixeira, J. Feijen, C.A. van Blitterswijk, P.J. Dijkstra, M. Karperien, Enzyme-catalyzed cross-linkable hydrogels: emerging strategies for tissue engineering, Biomaterials 33 (2012) 1281
1290. [87] R. Jin, L.S. Moreira Teixeira, P.J. Dijkstra, M. Karperien, C.A. van Blitterswijk, Z.Y. Zhong, et al., Injectable chitosan-based hydrogels for cartilage tissue engineering, Biomaterials 30 (2009) 2544
2551. [88] S. Sakai, Y. Yamada, T. Zenke, K. Kawakami, Novel chitosan derivative soluble at neutral pH and in-situ gellable via peroxidase-catalyzed enzymatic reaction, J. Mater. Chem. B 19 (2009) 230
235. [89] S.V. Gohil, S.B. Brittain, H.-M. Kan, H. Drissi, D.W. Rowe, L.S. Nair, Evaluation of enzymatically cross-linked injectable glycol chitosan hydrogel, J. Mater. Chem. B 3 (2015) 5511
5522. [90] B. Bi, H. Liu, W.-T. Kang, R.-X. Zhuo, X.-L. Jiang, An injectable enzymatically crosslinked tyramine-modified carboxymethyl chitin hydrogel for biomedical applications, Colloids Surf. B: Biointerfaces 175 (2019) 614
624. [91] D. Mishra, B. Bhunia, I. Banerjee, P. Datta, S. Dhara, T.K. Maiti, Enzymatically crosslinked carboxymethyl
chitosan/gelatin/nano-hydroxyapatite injectable gels for in situ bone tissue engineering application, Mater. Sci. Eng. C. 31 (2011) 1295
1304. [92] R. Jin, C. Lin, A.-N. Cao, Enzyme-mediated fast injectable hydrogels based on chitosan
glycolic acid/tyrosine: preparation, characterization, and chondrocyte culture, Polym. Chem. 5 (2014) 391
398. [93] H.-J. Wang, L.-Y. Jia, L.-Z. Cong, H.-X. Yu, X. Wang, Enzymatically mediated, physiologically triggered N-palmitoyl chitosan hydrogels with temporally modulated high injectability, Colloids Surf. A: Physicochem. Eng. Asp. 582 (2019) 123940. [94] H.C. Kolb, M.G. Finn, K.B. Sharpless, Click chemistry: diverse chemical function from a few good reactions, Angew. Chem.-Int. Ed. 40 (2001) 2004
2021. [95] K.S. Anseth, H.-A. Klok, Click chemistry in biomaterials, nanomedicine, and drug delivery, Biomacromolecules 17 (2016) 1
3. [96] R.T. Chen, S. Marchesan, R.A. Evans, K.E. Styan, G.K. Such, A. Postma, et al., Photoinitiated alkyne-azide click and radical cross-linking reactions for the patterning of PEG hydrogels, Biomacromolecules 13 (2012) 889
895. [97] J. Christoffersson, C. Aronsson, M. Jury, R. Selegard, D. Aili, C.-F. Mandenius, Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device, Biofabrication 11 (2018) 015013. [98] X.-Y. Wang, Z.-H. Li, T. Shi, P. Zhao, K.-K. An, C. Lin, et al., Injectable dextran hydrogels fabricated by metal-free click chemistry for cartilage tissue engineering, Mater. Sci. Eng. C-Mater. Biol. Appl. 73 (2017) 21
30. [99] B. Yan, H.-W. Zhou, J.-L. Lai, Z.-W. Wang, C.-Y. Luo, H.-B. Liu, et al., Pluronic F127 gels fabricated by thiol-ene click chemistry: preparation, gelation dynamics, swelling behaviors and mechanical properties, Polym. Bull. 76 (2019) 6049
6061. [100] Y.-L. Xu, H.-J. Xu, X.-S. Jiang, J. Yin, Versatile functionalization of the micropatterned hydrogel of hyperbranched poly(ether amine) based on “thiol-yne” chemistry, Adv. Funct. Mater. 24 (2014) 1679
1686. [101] H.S. Abandansari, M.H. Ghanian, F. Varzideh, E. Mahmoudi, S. Rajabi, P. Taheri, et al., In situ formation of interpenetrating polymer network using sequential thermal and click cross-linking for enhanced retention of transplanted cells, Biomaterials 170 (2018) 12
25.

Handbook of Chitin and Chitosan

References

305

[102] V.X. Truong, M.P. Ablett, H.T.J. Gilbert, J. Bowen, S.M. Richardson, J.A. Hoyland, et al., In situ-forming robust chitosan-poly(ethylene glycol) hydrogels prepared by copper-free azide
alkyne click reaction for tissue engineering, Biomater. Sci. 2 (2014) 167
175. [103] M. Matsumoto, W. Udomsinprasert, P. Laengee, S. Honsawek, K. Patarakul, S. Chirachanchai, A water-based chitosan-maleimide precursor for bioconjugation: an example of a rapid pathway for an in situ injectable adhesive gel, Macromol. Rapid Commun. 37 (2016) 1618
1622. [104] N. Yang, Y. Wang, Q.-S. Zhang, L. Chen, Y.-P. Zhao, In situ formation of poly (thiolated chitosan-co-alkylated β-cyclodextrin) hydrogels using click cross-linking for sustained drug release, J. Mater. Sci. 54 (2018) 1677
1691. [105] S.-B. Li, L. Wang, X.-M. Yu, C.-L. Wang, Z.-Y. Wang, Synthesis and characterization of a novel double cross-linked hydrogel based on Diels-Alder click reaction and coordination bonding, Mater. Sci. Eng. C. 82 (2018) 299
309. [106] J.-C. Huang, X.-L. Jiang, Injectable and degradable pH-responsive hydrogels via spontaneous amino
yne click reaction, ACS Appl. Mater. Interfaces 10 (2017) 361
370. [107] H. Zhou, C.-Y. Liang, Z. Wei, Y.-J. Bai, S.B. Bhaduri, T.J. Webster, et al., Injectable biomaterials for translational medicine, Mater. Today 28 (2019) 81
97. [108] Y.-L. Li, J. Rodrigues, H. Tomas, Injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications, Chem. Soc. Rev. 41 (2012) 2193
2221. [109] M.A. Haque, T. Kurokawa, J.-P. Gong, Super tough double network hydrogels and their application as biomaterials, Polymer 53 (2012) 1805
1822. [110] D.H. Sierra, Fibrin sealant adhesive systems: a review of their chemistry, material properties and clinical applications, J. Biomater. Appl. 7 (1993) 309
352. [111] W.D. Stanish, R. McCormack, F. Forriol, N. Mohtadi, S. Pelet, J. Desnoyers, et al., Novel scaffold-based BST-CarGel treatment results in superior cartilage repair compared with microfracture in a randomized controlled trial, J. Bone Jt. Surg. Am. 95 (2013) 1640
1650. [112] M.R. Steinwachs, B. Waibl, M. Mumme, Arthroscopic treatment of cartilage lesions with microfracture and BST-CarGel, Arthrosc. Techn. 3 (2014) e399
e402. [113] S. Methot, A. Changoor, N. Tran-Khanh, C.D. Hoemann, W.D. Stanish, A. Restrepo, et al., Osteochondral biopsy analysis demonstrates that BST-CarGel treatment improves structural and cellular characteristics of cartilage repair tissue compared with microfracture, Cartilage 7 (2016) 16
28. [114] C.D. Hoemann, N. Tran-Khanh, A. Chevrier, G.-P. Chen, V. Lascau-Coman, C. Mathieu, et al., Chondroinduction is the main cartilage repair response to microfracture and microfracture with BST-CarGel: results as shown by ICRS-II histological scoring and a novel zonal collagen type scoring method of human clinical biopsy specimens, Am. J. Sports Med. 43 (2015) 2469
2480. [115] M.S. Shive, W.D. Stanish, R. McCormack, F. Forriol, N. Mohtadi, S. Pelet, et al., BSTCarGel treatment maintains cartilage repair superiority over microfracture at 5 years in a multicenter randomized controlled trial, Cartilage 6 (2015) 62
72. [116] J. Frappier, W. Stanish, M. Brittberg, M. Steinwachs, L. Crowe, D. Castelo, et al., Economic evaluation of BST-CarGel as an adjunct to microfracture vs microfracture alone in knee cartilage surgery, J. Med. Econ. 17 (2014) 266
278. [117] J. Vila´, Y. Rico, A. Dalmau, F. Javier Chaques, J. Asuncion, Treatment of osteochondral lesions of the talus with bone marrow stimulation and chitosan-glycerol phosphate/blood implants (BST-CarGel), Arthrosc. Tech. 4 (2015) e663
e667. [118] M. Tey, J. Mas, X. Pelfort, J. Carles Monllau, Arthroscopic treatment of hip chondral defects with bone marrow stimulation and BST-CarGel, Arthrosc. Tech. 4 (2015) e29
e33.

Handbook of Chitin and Chitosan

306

9. Injectable polymeric gels based on chitosan and chitin for biomedical applications

[119] C. Rhee, E. Amar, M. Glazebrook, C. Coday, I.H. Wong, Safety profile and shortterm outcomes of BST-CarGel as an adjunct to microfracture for the treatment of chondral lesions of the hip, Orthop. J. Sports Med. 6 (2018). 2325967118789871. [120] M. Tahoun, T.A. Shehata, I. Ormazabal, J. Mas, J. Sanz, M. Tey Pons, Results of arthroscopic treatment of chondral delamination in femoroacetabular impingement with bone marrow stimulation and BST-CarGel, Sicot-J 3 (2017). UNSP 51.

Handbook of Chitin and Chitosan

C H A P T E R

10 Preparation and application of biomimetic and bioinspired membranes based on chitosan Laxmi Gond, Preeti Pradhan and Anjali Bajpai Department of Chemistry, Government Science College, Jabalpur, India

O U T L I N E 10.1 Introduction

308

10.2 Biomimetic and bioinspired membranes 10.2.1 Preparation of biomimetic and bioinspired membranes

308 308

10.3 Applications of biomimetic and bioinspired membranes 10.3.1 Wound dressing: basic requirements 10.3.2 Biomimetics in wound closure 10.3.3 Regenerative medicine or tissue engineering 10.3.4 Biomimetic sensor-based membranes

310 311 313 314 315

10.4 Chitosan 10.4.1 Biomedical applications of chitosan 10.4.2 Application of chitosan membranes 10.4.3 Chitosan membranes in wound dressing 10.4.4 Molecularly imprinted membranes from chitosan as biosensors 10.4.5 Asymmetric membranes as wound dressings 10.4.6 Application of chitosan-based membranes for guided bone regeneration tissue engineering

316 318 318 323 326 326

References

331

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00010-8

307

327

© 2020 Elsevier Inc. All rights reserved.

308

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

10.1 Introduction Over the last few decades, sustainable technology has evolved as a green, pervasive technology that focuses on the development of materials for medical, food packaging, and environmental applications. Researchers have adopted a “learn from nature” philosophy, as emulating an already proven methodology increases the prospects of successful development of an analogous synthetic material or process. As a result, research focused on the design and development of biomimetic and bioinspired materials has come to the forefront in materials science, chemistry, and chemical engineering [1]. In this context, biopolymers, such as cellulose, starch, chitin, chitosan (CH), wool, silk, gelatin, and collagen, are deemed promising thanks to their better biocompatibility in comparison with synthetic materials. However, biomedical applications also require the material to exhibit a certain level of mechanical performance. These innate challenges of natural biopolymers have manifested in a surge of interest in biohybrid and biomimetic materials research.

10.2 Biomimetic and bioinspired membranes Biomimetic membrane refers to the imitation of a biological membrane in terms of its composition, structure, formation process, and functions. In situ self-assembly of amphiphilic phospholipid and functional proteins forms the asymmetric and hierarchical bilayer structure of a biological membrane, which also ensures the environmental stability of cell, the participation in multiple interactions of different cells, the efficient conversion of energies, signal transduction between cells, and conversion and transmembrane transport of substances. The highly complex structure of a biological membrane comprising lipids and proteins is difficult to emulate in its entirety. Hence, simplified synthetic models comprising key components and/or features of biological membrane are being developed [2]. Biomimetic and bioinspired membranes find application similar to those of the existing synthetic membranes. The hierarchical structures, ability for controlled selective transport and stability/resistance further extend their applications with regards to sustainable resources, environment, and energy aspects. Some of their key applications are in water treatment, clean energy, carbon capture, and health care [3].

10.2.1 Preparation of biomimetic and bioinspired membranes Biological systems exhibit a synergy between mutually and dynamically connected, intelligent combinations of several parallel functional processes. For example, the structural designs of the lotus leaf and

Handbook of Chitin and Chitosan

10.2 Biomimetic and bioinspired membranes

309

gecko foot have inspired the fabrication of advanced functional materials such as superhydrophobic [4
6] and superadhesive [7,8] surfaces. Self-assembly in biological systems has been identified as a practical tool that can also be used for the fabrication of advanced materials. The techniques of supramolecular chemistry, namely, layer-by-layer (LbL) adsorption [9,10], Langmuir
Blodgett (LB) method [11,12], sol
gel fabrication [13,14], and template synthesis [15
18] can be used for material fabrication based on self-assembly. Layered assemblies of mesoporous materials [19,20] based on hierarchical structural designs is also an attractive approach for performing advanced functions. The choice of raw material (natural polymers or biocompatible synthetic polymers) and gelation technique dictate the design of tissuemimicking hydrogels for a specific application. Bioinspiration is a useful concept for hydrogel design for manipulating physical, mechanical, and biological behaviors by taking advantage of naturally occurring chemical and physical architectures. Depending on the polymer nature and/ or the solvent, common structuring methods used to induce structuring within hydrogels at different length scales are freeze- or vacuumcasting, LbL deposition, templating, 3D-printing, and self-assembly [21]. Progress in the construction of nanostructured biohybrid materials has been reported, with a focus on material preparation and applications resulting from synergistic assembly of biopolymers and the inorganic material [22]. Possible supports for biomaterial immobilization can be classified as (1) porous solids, (2) micro- or nanoparticulated solids, and (3) soft networks. Construction of bioinorganic hybrids or biohybrids with welldefined nanostructures is possible by the bottom-up self-assembly process from well-designed units. Three strategies are usually employed (Fig. 10.1): 1. Sol
gel process: to entrap a large number of organic or biological moieties in matrices derived from precursor monomers. The hybrid materials involve preformed natural or synthetic inorganic solids to act as host for organic moieties. 2. Intercalation of organic moieties between the lamellae of 2D solids or embedded in 3D nano- or mesoporous structures: this involves host
guest interaction through (1) electrostatic interactions, (2) hydrogen bonding and water bridges, (3) ion
dipole coordination, (4) proton and electron transfer processes, or (5) van der Waals forces. 3. Grafting of organic moieties: functionalization of inorganic solids through covalent bonding with organic moieties. Occasionally, a combination of two of these routes is required to provide the hybrid materials with the desired structural and functional properties.

Handbook of Chitin and Chitosan

310

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

FIGURE 10.1 Schematic presentation of strategies employed for development of biohybrid materials (A) entrapment in matrices generated by sol
gel, (B) intercalation in 2D solids or inclusion in 3D-solids (C) grafting on inorganic solid surface.

Wear resistance of soft materials can be controlled and improved by internal structuring. Choice of raw material (natural polymers or biocompatible synthetic polymers) and the gelation technique helps in the design of tissue-mimicking hydrogels for a specific application. To manipulate physical, mechanical, and biological behaviors of biohybrids bioinspiration, that is, mimicking chemical and physical architectures of natural biomaterials, is taken into consideration. The common structuring methods used are (1) freeze- or vacuum-casting, (2) LbL deposition, (3) templating, (4) 3D-printing, and (5) self-assembly depending on the nature of polymer and/or the solvent [23,24].

10.3 Applications of biomimetic and bioinspired membranes The prerequisites for clinical applications of biomimetic and bioinspired membranes are noncytotoxicity, biocompatibility, bioactivity (provide surface for cell proliferation), reabsorption (biodegradation),

Handbook of Chitin and Chitosan

10.3 Applications of biomimetic and bioinspired membranes

311

and mechanical stability. Biomimetic membranes find the most useful application in the biomedical field, especially for wound dressing.

10.3.1 Wound dressing: basic requirements Skin injuries can be caused by physical and chemical damage. Skin, the first line of defense against environmental assaults, is the biggest organ in vertebrates, constituting approximately one-tenth of the body mass [25,26]. This complex three-layered structure (epidermis, dermis, and hypodermis) is the outermost barrier that also gives support to blood vessels and nerves, regulates body temperature, prevents dehydration, and protects visceral organs from microbial, mechanical, and chemical harm [27]. It also performs immune surveillance and sensory detection [28,29]. It is intrinsically self-renewable under normal physiological conditions [30]. If the skin is damaged, a complex and painful wound-healing process begins. Wound healing is the biological route for growth and tissue regeneration. A series of interreliant stages involves a variety of cellular and matrix components to restore the integrity of damaged tissue and replacement of lost tissue [31]. This complex and dynamic regenerative process comprises (1) hemostasis, (2) inflammation, (3) proliferation, and (4) remodeling [32] (Fig. 10.2). During the healing process, the wound should be covered to minimize damage and risk of infection and to promote restoration of integrity of tissue.

FIGURE 10.2 Schematic representation of the three main stages of skin wound-healing process: inflammation, proliferation, and remodeling.

Handbook of Chitin and Chitosan

312

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

One of the leading causes of death for severely injured persons is hemorrhage, mainly on account of primitive first aid. Prehospital care generally involves the use of gauze dressings, direct pressure, and tourniquets (compression bandage) to stop bleeding [33]. New techniques and devices that control hemorrhage are being developed [34]. An effective bleeding control in aortic injury equivalent to sutured repair is fibrin dressing [35]. However, there is a high risk of disease transmission due to the involvement of blood components derived from human sources. An ideal dressing should protect the wound from physical damage and the invasion of microorganisms. If not protected, wounds may be colonized by multiple species of microorganisms resulting in a polymicrobial population that can extend to around 100 M microbes/g of skin within 48 h [36,37]. Chronic wounds due to microbial colonization are observed in diabetic foot ulcers, pressure ulcers, and venous leg ulcers [38]. Diabetic foot ulcers constitute a major challenge worldwide [39]. Wound dressing should be comfortable, nontoxic, nonallergenic, nonadherent, nonirritant, compatible with topical therapeutic agents, compliant, and durable. The dressing should allow gaseous exchange and maintain a moist environment at the wound interface, remove excess exudates, and should not retard or inhibit any stage of the woundhealing process [40]. It should be easily removed without trauma and preferably made from a readily available biomaterial that possesses antimicrobial properties and requires minimal processing. Frequently, the skin wound healing results in a fibrotic scar with limited functional restoration. Various types of skin grafts have been applied as gold standard treatment to promote scarless wound healing. However, skin grafts have limitations, such as shortage of donor sites, morbid donor sites, pain, and a risk of infection. It is highly dependent on the biochemical interactions of host cells and tissues, with the surrounding microenvironment created by effective skin graft materials, to be able to recruit host cells and promote their proliferation and differentiation. Tissue engineering approaches have been investigated for regenerating skin with a support of biomaterials (e.g., film or hydrogel), growth factors (GFs) and antibacterial drugs by stimulating endogenous or exogenous cells. Dressing formulations with functional, biocompatible, and renewable feed stocks (e.g., polysaccharides), use of chemically stable antimicrobial agents of relatively low cost and well tolerated by patients are also fascinating. A wide range of wound dressings made from synthetic and natural materials, including polyurethane (e.g., PolyMems) or polysaccharides such as starch (e.g., Iodosorbs), respectively, is commercially available. A variety of wound dressings is required for the management of different types of wounds, considering the complexity of the wound-healing process [41,42].

Handbook of Chitin and Chitosan

10.3 Applications of biomimetic and bioinspired membranes

313

Categories of wound dressing are (1) biologic (e.g., Alloskin and pigskin), (2) synthetic, and (3) biologic
synthetic. Biologic dressings have limited supplies, high antigenicity, poor adhesiveness, and risk of cross contamination. Synthetic dressings carry almost no risk of pathogen transmission, have a long shelf life, and induce minimal inflammatory reaction. Biologic
synthetic dressings are bilayered and consist of high polymer and biological materials. Morgado et al. summarized the techniques typically applied in the development of wound dressings [43]. The following intrinsic properties need to be evaluated for the use of any material as a wound dressing: (1) morphological, (2) swelling in water and hydrophilicity, (3) water vapor transmission rate and oxygen permeation, (4) elasticity and tensile strength, (5) antibacterial activity, (6) in vitro drug release profiles, and (7) in vitro and in vivo cytotoxicity. Properties required for an ideal wound dressing are: • • • • • • •

Forms barrier against infection Encourages natural blood clotting Blocks nerve endings to reduce pain Absorbs fluids from inflammation Provides scaffold for cell growth Strengthens new tissue Minimizes scarring

10.3.2 Biomimetics in wound closure Tissue incisions are closed mechanically to induce healing after surgical operations. The most commonly used wound closure technique is suturing, which is time-consuming and requires delicate skills so as to preclude secondary tissue damage or an immune response. Tissue adhesives or medical sealants are good alternatives. These should have sufficient adhesive strength upon coming in contact with biological fluids and minimum dispersion into the body fluids besides low cytotoxicity and good biocompatibility. Synthetic tissue adhesives (cyanoacrylates and resorcinol/formaldehyde) possess favorable underwater adhesion strength but may be cytotoxic or generate heat while curing. Protein-based adhesives (fibrins and gelatins) have good biocompatibility but low wet adhesive strength. The problems with the current bioadhesives can be overcome by understanding the wound-healing mechanism of tunicates. The tough and often translucent body armor (tunic) of tunicates (sessile marine organisms, see Fig. 10.3) is mainly composed of a highly crystalline cellulose nanofiber called tunicin and proteins, some peptides (tunichrome) containing 3,4-dihydroxyphenylalanine (DOPA) with a catechol moiety, and 3,4,5-trihydroxyphenylalanine (TOPA) with a pyrogallol moiety. Tunicin mechanically supports the tunic. The key adhesion functionality

Handbook of Chitin and Chitosan

314

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

FIGURE 10.3 Tunicate. Source: From https://en.wikipedia.org/wiki/Tunicate#/media/File: Bluebell_tunicates_Nick_Hobgood.jpg

is provided by pyrogallol group of TOPA. Pyrogallol is oxidized at the pH of seawater (8.2) and forms covalent cross-links with tunicin, TOPA, and proteins. It also forms tris-complexes with vanadium in the tunicate’s blood. These pyrogallol-mediated bonds seal the torn tunic and control bleeding [44,45]. The limitation of biomaterial-based adhesives for medical applications is mainly due to their poor wet adhesion strength owing to hydrationinduced softening and dissolution. Oh et al. [46] mimicked the woundhealing process found in tunicates. They fabricated a tunicate-mimetic hydrogel adhesive based on a chitin nanofiber/gallic acid (a pyrogallol acid) composite. The nanofibrous structures and pyrogallol groupmediated cross-linking improved resistance to dissolution and increased cohesion strength of the hydrogel in a wet condition. The tunicate-mimetic adhesives showed higher adhesion strength between fully hydrated skin tissues than did fibrin glue and mussel-35 mimetic adhesives.

10.3.3 Regenerative medicine or tissue engineering Cartilage tissue engineering is an important topic in biomedical science for the articular cartilage treatment of the skeletal system. It

Handbook of Chitin and Chitosan

10.3 Applications of biomimetic and bioinspired membranes

315

involves the isolation of articular chondrocytes or their precursor cells to be used for regeneration or replacement of damaged tissues. Biocompatible matrix, biofilm, fibers, sponges, hydrogels, or scaffold that closely mimic the natural extracellular matrix (ECM) are advantageous to favor cell attachment, proliferation, migration, and new tissue formation. The biomaterials are seeded with these cells for cultivation in vitro and allowed to proliferate. Histochemical, biochemical, and biomechanical properties of the resulting material should be identical to native cartilage. The artificially generated tissue is implanted into the damaged joint [47,48]. For the success of oral and orthopedic surgery, two key processes are necessary (1) healing and (2) new bone formation. The growth of connective tissue occurs at a greater rate than that of bone at bone defect sites resulting in the invasion of fibrous tissue, which creates a barrier to prevent the growth of new bone. This invasion leads to anatomical aberrations and loss of bone function, thus generating a need for additional surgery to remove the overgrown tissue. A well-established option that promotes osteogenesis or bone augmentation is guided bone regeneration (GBR) using a barrier membrane to protect the lesion against connective tissue invasion. Consequently, researchers aim to design bioactive membranes that not only function as barriers but also perform biological activities to stimulate in situ regeneration in the bone defect site [49].

10.3.4 Biomimetic sensor-based membranes It is an important challenge to produce artificial systems, which are able to mimic the recognition mechanisms of living systems. Molecular imprinting contributed significantly in this direction, whereby selective molecular recognition sites are introduced into a polymer to confer biomimetic properties. The potential applications of these systems are medical diagnostics, affinity separations, catalysis, drug delivery, etc. Biosensing systems, developed by molecularly imprinted membranes and integrated with a transducer component, are used as biomimetic recognition elements as alternatives to traditional bioassay methods for environmental, food, and clinical uses [50]. Three essential components are required for the production of molecularly imprinted polymers (MIPs): 1. target molecule (or template), which forms prepolymerization complexes with polymerizable monomer; 2. functional monomer, which has chemical and shape complementarity to the template and will polymerize to a polymer matrix; and

Handbook of Chitin and Chitosan

316

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

3. cross-linker, a multifunctional molecule to interact with specific functional groups present on a polymeric chain to form a rigid polymer matrix to maintain the location of the functional groups for binding the template. Finally, the template is removed from the polymeric matrix for the generation of specific recognition sites. The molecular imprinting process is represented in Fig. 10.4.

10.4 Chitosan Chitin, a long-chain polymer of N-acetylglucosamine, is the second most abundant natural polymer after cellulose produced annually by biosynthesis. It is a mucopolysaccharide and a common constituent of the exoskeleton in crustaceans, mollusks, and insects. Chitin has received less attention than cellulose, primarily due to its inertness. However, CH, usually obtained by alkaline deacetylation of chitin, is a relatively reactive compound. CH consists of β-(1 - 4)-linked

FIGURE 10.4 Schematic presentation of the molecular imprinting process.

Handbook of Chitin and Chitosan

10.4 Chitosan

FIGURE 10.5

317

Structures of (A) chitin and (B) CH.

2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose units. The structures of chitin and CH are similar to cellulose (Fig. 10.5). CH is soluble in dilute aqueous acetic, lactic, malic, formic, and succinic acids. The peculiar properties of CH arise from the presence of primary amines along its backbone. Chemically modified CH with improved properties is especially interesting since modification keeps the original physicochemical and biochemical properties with no change in the fundamental skeleton of CH. Chemical modification affords a wide range of derivatives with specific end user applications. A variety of chemical modifications, such as oligomerization, alkylation, acylation, quternization, hydroxyalkylation, carboxyalkylation, thiolation, sulfation, phosphorylation, enzymatic modifications, and graft copolymerization, have been reported. Some assorted modifications include CH hybrids with sugars, cyclodextrin, dendrimers, and crown ethers, resulting in interesting multifunctional macromolecules. [51] CH and its modified derivatives can be processed in various forms, as presented schematically in Fig. 10.6. CH is polycationic at pH 6 and readily interacts with negatively charged species, namely fatty acids, bile acids, proteins, anionic polysaccharides (such as alginate and carrageenan), and phospholipids. CH the

FIGURE 10.6

Various forms of processed CH.

Handbook of Chitin and Chitosan

318

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

ability to chelate metal ions [52]. It exhibits fungicidal properties [53]. Its nontoxic, biodegradable, and biocompatible properties make it useful for diverse applications in biomedicine, membranes, drug delivery systems, hydrogels, water treatment, food packaging, etc. [54,55]. CH has been found to be a good biosorbent for the removal of dissolved hazardous organic and inorganic materials from aqueous solutions [56]. Recent reports on CH membranes for water treatment and other miscellaneous applications are summarized in Table 10.1. Some of the food packaging applications of CH films are shown in Table 10.2.

10.4.1 Biomedical applications of chitosan The unique properties of CH, such as biodegradability, nontoxicity, nonallergenic nature, antibacterial effect, and biocompatibility, have made it useful for biomedical materials. Some of the biomedical applications of CH are presented in Fig. 10.7. The mechanism of degradation of CH in the body is an important factor in the design of a biomaterial. Depending on the intended use of the device, different preparation methods have been used to create CH that degrades over the course of days, weeks, or months. This control requires selection of the appropriate deacetylation degree and molecular weight. High deacetylation degree tends to have a slower degradation rate as compared to lower deacetylation degree because of close chain packing through hydrogen bonding. Further, pre- and postprocessing methods can control the degradation to match the functional requirements for implants [84]. Kalantari et al. reviewed mechanisms of degradation for CH implants [85]. CH stimulates hemostasis and accelerates tissue regeneration, hence it is suitable for wound healing. CH is biodegradable and is metabolized by certain human enzymes, such as lysozyme [86]. Structural similarities to glycosaminoglycans (GAG) and its hydrophilic nature make CH an attractive material for tissue engineering scaffolds. N-acetylglucosamine occurs in hyaluronic acid, an extracellular macromolecule that is important in wound repair [87].

10.4.2 Application of chitosan membranes CH membranes find potential application in tissue engineering, food preservation, wastewater purification, environmental protection, fuel cell, and separation technology [55,88
90]. Though, compatibility of CH is appreciable, the poor mechanical and thermal properties of biodegradable membranes prepared from CH restrict its widespread application. To improve the mechanical property, cross-linking, blending, and the addition of reinforcing agents is being explored. A reinforcement technique has

Handbook of Chitin and Chitosan

319

10.4 Chitosan

TABLE 10.1 Recent reports on chitosan membranes for water treatment and some miscellaneous applications. S. no.

Materials/process

Product

Application

References

1.

CH/poly(ethylene oxide) (PEO) nanofibers fabricated at different CH:PEO weight ratio by electrospinning process

Mesoporous-high surface area CH/ PEO nanofibrous membrane

Maximum adsorption capacity for Cu(II), Zn(II), and Pb(II) ions were found to be 120, 117, and 108 mg/g, respectively.

[57]

2.

Chitosan nanofibers membrane by electrospinning process membranes were grafted with diethylenetriamine via stepwise route

Amine grafted CH nanofibrous membrane

Enhanced metal ions removal by batch technique to synthetic solutions of Cu(II) and Pb(II) ions

[56]

3.

CH interfacially polymerized with trimesoyl chloride monomer on the surface of sulfonated polyethersulfone support layer

Hydrophilic CHbased thin active layer membrane

Low cost and effective adsorbent for removal of dye, heavy metal ions, and other contaminants from water and wastewater

[58]

4.

CH/PVA membrane modified by addition of some amine group to the membrane structure utilizing polyethyleneimine

CH/PVA adsorptive membrane

Heavy metal ions adsorption capacity of 112.13, 86.08, and 75.5 mg/g for Cd21, Cu21, and Ni21, respectively at 25 C and pH 6.

[59]

5.

Effect of degree of deacetylation on property and adsorption capacity of CH/PVA electrospun membrane

Electrospun CH/ PVA nanofibrous membrane

Removal of methyl orange, Fe(III) and Cr(VI) Ions

[60]

6.

La31 incorporated chitosan biopolymeric matrix membrane prepared by casting method

La@CH biopolymeric membrane

Adsorption capacity of 76.6 and 62.6 mg/ g for phosphate and nitrate ions from aqueous solution

[61]

(Continued)

Handbook of Chitin and Chitosan

320

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

TABLE 10.1

(Continued)

S. no.

Materials/process

Product

Application

References

7.

CH/PVA/zeolite nanofibrous membrane fabricated by electrospinning process

CH/PVA/zeolite electrospun composite nanofibrous membrane

Adsorption capacity of the membrane for methyl orange 153 mg/g

[62]

8.

Pure CH nanofibrous membranes prepared by electrospinning

CH nanofibrous membranes with average fiber diameter of 86 6 18, 114 6 17, 164 6 28 nm

Dye removal adsorption capacity of 1377 mg/g

[63]

9.

CH/PVA/TiO2 electrospun nanofibrous membrane

Degradation of methyl orange and congo red

Adsorption capacity for congo red and methyl orange 131 and 314 mg/g, respectively

[64]

10.

Quaternized Ntrimethyl CH (TMC) synthesized from CH via reductive alkylation and methylation

TMC incorporated polyethersulfone polymer membranes

Enhanced antifungal activity for drinking water treatment

[65]

11.

Hydrolyzed electrospinning polyacrylonitrile nanofiber membrane and covalently grafted CH functionalized with quaternary amine (i.e., glycidyl trimethyl ammonium chloride)

Quaternized CH nanofiber membrane

Microfiltration membrane to effectively disinfect E. coli

[66]

12.

A polyelectrolyte complex prepared from CH and λ-carrageenan using a layer-by-layer deposition of polyion solutions on a plated nonporous support CH and λ-Carrageenan

Complex film (25
30 μm thick)

Multilayer membrane for the pervaporation separation of aqueous ethanol solutions

[67]

(Continued)

Handbook of Chitin and Chitosan

321

10.4 Chitosan

TABLE 10.1

(Continued)

S. no.

Materials/process

Product

Application

References

13.

Sulfonated polyaniline, nano silica as inorganic filler and sulfuric acid as an ionic cross-linking agent in CH nanocomposites

Ionic cross-linked CH-based hybrid nanocomposites

Best overall performance as a polymer electrolyte membrane for fuel cells

[68]

14.

Solution casting strategy using twodimensional exfoliated molybdenum disulfide (E-MoS2) nanosheets

CH-based proton exchange membranes

Alternate to Nafion in direct methanol fuel cells

[69]

15.

Hybrid CH/calcium aluminosilicate nanocomposite membranes doped with (3, 5, and 7 mol%) Al2O3 nanoparticles

CH/calcium aluminosilicate nanocomposite membranes

To capture CO2 gas at lower and moderate temperatures as support for CO2 sensor

[70]

received great attention due to its simplicity and effectiveness. Cellulosic fibers at the nanoscale are attractive for reinforcement of CH to produce environment-friendly composite films with improved physical properties, which have wide application and potential in biomedical, packaging, and water treatment fields. The preparation and properties of CH
cellulose blends and CH bionanocomposites for different applications were summarized in a review [91]. Chitin whisker, a kind of nanoscale crystallite, is prepared by removing the amorphous phase in chitin. It is an emerging nanofiller for synthetic and natural polymers [92
94]. Generally a simple blend method is used to prepare chitin whisker-reinforced materials. Ma and coworkers [95] combined the reinforcement of chitin whiskers and the hierarchical structure of natural materials to prepare multilayered CH membranes, resulting in 2.5 times increase in tensile strength and a sharp increase in elongation at break compared with neat CH membrane. This is a promising material for wound dressing, mulching film in agriculture, and conglutination prevention after surgery. CH-based scaffolds have been obtained by surface modifications, blending, composite formation, and drug-loading. An overview was

Handbook of Chitin and Chitosan

322

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

TABLE 10.2

Chitosan films for food packaging.

S. no.

Materials/process

Product

Application

References

1.

CH films prepared by casting method after neutralization with NaOH solution

Fresh-keeping CH films for chilled meat

Effectively inhibit the oxidative deterioration and the growth of spoilage microorganisms

[71]

2.

CH incorporated with mango leaf extract

CH antioxidant films

Active packaging films for food preservation

[72]

3.

CH-based film production for food technology

Biocompatible and biodegradable CH films

A review summarizing the advancements made in the last 5
7 years in the field of CH films

[73]

4.

Antimicrobial CH films with acetic acid and propionic acid and glycerol as plasticizer

Organic acid incorporated CH films

Suitable as wraps for increasing storage stability of dried fish

[74]

5.

Tailoring physicochemical properties of CH films by varying drying temperature

CH films

Films dried at lower temperatures showed superior juice retention capacity and superior preservation effect on chilled meat

[75]

6.

Chitosan lactate impregnated as an antimicrobial additive into low-density polyethylene (LDPE)

CH incorporated LDPE films

Significant extension of red color, shelf life of sliced red meats

[76]

7.

Uniformly embedding 1, 3, and 5% CH (w/w) in low-density polyethylene matrix using maleic anhydride grafted LDPE as a compatible agent

LDPE/CH antimicrobial films

Packaging material for chilled storage of Tilapia (Oreochromis mossambicus)

[77]

8.

Embedding nanochitosan (0.5%, 1% and 2%) in a polylactic acid matrix, polyethylene glycol (cross-linking agent) and PVA (plasticizer)

Polylactic acid/ CH composite films

Packaging of Indian white prawn (Fenneropenaeus indicus)

[78]

(Continued)

Handbook of Chitin and Chitosan

323

10.4 Chitosan

TABLE 10.2

(Continued)

S. no.

Materials/process

Product

Application

References

9.

CH films incorporated with apricot kernel essential oil (a major agricultural seed waste, containing oleic acid and N-methyl-2pyrrolidone)

Active food packaging CH Films

Excellent antimicrobial and antioxidant properties as compared to neat chitosan films

[79]

10.

Films prepared by mixing the corn starch solution and CH chitosan solution (1:1) by casting

Active packaging corn starch/CH films

Active packaging films for food and pharmaceutical applications

[80]

11.

CH film incorporated with tea polyphenol (TP)

Packaging film for extending the shelf life of pork meat patties

Microbiological shelf life extension of 6 days

[81]

12.

Films obtained by casting high-molecularweight CH with sunflower oil

CH
sunflower oil edible films

Applied to the surface of pork meat hamburgers. Chitosan-based films increased the metmyoglobin (MtMb) content. Incorporation of sunflower oil to the chitosan matrix led to a reduction in MtMb

[82]

13.

Polylactic acid, tea polyphenol, and CH prepared by stretch film method

Composite membranes

Potential for application in food packaging

[83]

presented on the inherent properties of CH, their modification, and their use in biomedical engineering, particularly with regard to antiinflammatory activity and wound healing [96].

10.4.3 Chitosan membranes in wound dressing Chitin and CH are excellent dressing materials for the treatment of wounds and burns on the basis of their high molecular mass, high charge density, and mucoadhesive properties. CH is a favored choice

Handbook of Chitin and Chitosan

324

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

FIGURE 10.7 Possible biomedical applications of CH.

out of the available natural polymers due to its adhesive nature, antifungal and bactericidal character, and permeability to oxygen. The high durability, good biocompatibility, low toxicity, liquid absorption, and antibacterial nature help to accelerate wound healing [97]. Easy fabrication by casting-evaporation from an acetic solution of CH and good stability has drawn special attention for wound-healing applications. To further enhance the stability and mechanical properties of the membranes chemical cross-linkers, for example, glutaraldehyde, carbodiimide, and genipin, are applied. The drawback is the intrinsic cytotoxicity of the cross-linker and the solubility to use these approaches for wound healing. Furthermore, serious deformations like shrink and crimp occur upon secondary drying of membrane. Moreover, additives such as drugs may be washed away from the membrane during alkaline neutralization. Thus it is desired to enhance the chemical resistance, maintain the hydrophilicity, and long-term biological degradation. The use of glycerol as a plasticizer in CH matrices has demonstrated improvements in the mechanical properties of CH membranes. The use of CH floccules suspension is recommended in place of the acetic solution. The CH floccule is prepared by neutralization of acid solution with aqueous alkali under stirring until the occurrence of a milky precipitate. Ma and coworkers [98] reported the preparation of CH membranes from flocculence by the addition of glycerol. They examined the in vitro effects of glycerol content on morphology, tensile strength, water vapor permeability, biodegradation, static contact angle, and water absorption. The water-soluble drug tetracycline hydrochloride and the waterinsoluble drug silver sulfadiazine were incorporated in the matrix. Cumulative drug release and antibacterial activity against E. coli and S. aureus were evaluated. Jayakumar and coworkers [99] reviewed the progress of chitin and CH-based fibrous materials, hydrogels, membranes, scaffolds, and sponges in wound dressing. A variety of polymers, that is, alginate,

Handbook of Chitin and Chitosan

10.4 Chitosan

325

hyaluronic acid, polyethylene glycol diacrylate, poly(vinyl alcohol) (PVA), γ-poly(glutamic acid), and 2-hydroxyethyl methacrylate, were used to prepare chitin and CH-based composite membranes to achieve the desired properties for wound-healing applications. Ag/ZnO-incorporated CH membranes may prove to be potential wound dressings due to their lower cytotoxicity and antibacterial activity than the traditional materials. Chitin and CH scaffolds/sponges could be promising candidates for wound dressing on the basis of improved antibacterial activity, cell attachment ability, and oxygen permeability. An occlusive dressing for wound management could be prepared from chitin and CH-based hydrogels owing to their ability to accelerate wound contraction and healing, since the moisture permeability of the hydrogels prevents the heavy accumulation of fluid in exuding wounds. The versatility, functionality, and efficacy of chitin and its derivatives to improve appearance and functionality of regenerated tissue proved a valid reason for application as per the modern clinical approach. Ahmed and Ikram [96] extensively reviewed the research efforts in the field of CH-based scaffolds, including surface modifications, the fabrication of CH-based blends, CH-based composite scaffolds, and drugloaded scaffolds. An overview of the key features of the inherent properties of CH, its modification, and its use in biomedical engineering, particularly toward antiinflammatory and wound-healing applications was presented. CH/collagen membrane could be used to hasten wound healing and induce cell migration and proliferation. Polypropylene-NIPAAmcollagen-CH membrane is a temperature-sensitive material that leads to automatic release of the dressing material upon healing of wound [100,101]. CH hydrogel-coated Dacron grafts cross-linked by ultraviolet light irradiation exhibited a resistance against E. coli in vitro and in vivo [102]. The biocompatible carboxyethyl CH/PVA nanofibers were prepared by electrospinning of aqueous solution as fibrous mats for wounds, and they were shown to be good in promoting the L929 cell attachment and proliferation [103]. An antibiotic material can be loaded through binding for the development of bacterial-resistant prosthetic counterparts [96]. Improved water uptake ability, antibacterial activity, and wound closure was exhibited by a composite sponge of curcumin/CH/gelatin prepared at various ratios of CH and gelatin. A higher content of gelatin in the composite sponge led to release at a faster rate up to 240 min. These composite sponges enhanced the formation of collagen and wound closure in vivo to improve wound-healing activity [104]. Polyelectrolyte complexes (PECs) are formed by mixing oppositely charged polyelectrolytes in solution. PECs are stabilized by electrostatic

Handbook of Chitin and Chitosan

326

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

and secondary forces (intra- and intermolecular associations) between polymer networks. Thus it is advantageous that the use of toxic crosslinkers (glutaraldehyde, epichlorohydrin, genipin, etc.) commonly used to create hydrogels can be avoided. CH
alginate PEC exerts positive effects in the wound-healing process in rats, being capable of modulating the inflammatory phase and stimulating fibroplasia and collagenesis [105,106]. Rodrigues et al. [41] investigated CH
alginate PEC production by preparation conditions to improve therapeutic efficacy. Recently developed innovative techniques were used to fabricate the highly sustainable artificial nacre-like films. Yao et al. fabricated nacre-like CH layered double hydroxide hybrid films by sequential dipping coating and the LBL technique, which had tensile strength of up to 160 MPa [107,108]. Artificial nacre-like CH
montmorillonite (MTM) bionanocomposite films were fabricated by self-assembly of CH
MTM hybrid building blocks by a simple process. The CH molecules were adhered to exfoliated MTM nanosheets by strong electrostatic and hydrogen-bonding interactions to yield hybrid building blocks, which could be dispersed in distilled water and then aligned to a nacre-like lamellar microstructure by vacuum-filtration or waterevaporation induced self-assembly [109].

10.4.4 Molecularly imprinted membranes from chitosan as biosensors Biomimetic membranes based on MIMs have been successfully developed by several researchers using the phase inversion imprinting method introduced by Kobayashi, which is now one of the most commonly utilized procedures in this field [50]. Ma et al. [110] reported the preparation of CH MIM for recognizing naringin in aqueous media. An acetyl cholinester liposome bioreactors
CH multilayer nanocomposite film was developed as a novel acetylcholinesterase biosensor for the detection of organophosphates pesticides [111]. Creatininase, creatinase, and sarcosine oxidase were immobilized on iron oxide nanoparticles/CH-g-polyaniline composite film for the construction of an amperometric creatinine biosensor [112]. Chen et al. [113] reported the preparation of an electrochemical sensor based on CH MIM film for the detection of urea, which is an indicator of abnormal human physiological conditions.

10.4.5 Asymmetric membranes as wound dressings Asymmetric membranes have captured great attention due to their structural similarity with the architecture of the native skin [114]. Asymmetric

Handbook of Chitin and Chitosan

10.4 Chitosan

327

membranes are composed of a dense outer layer mimicking the epidermis of skin, which protects from external physical, chemical, and bacterial agents and controls gaseous exchanges. The interior layers of these membranes have a porous structure, which absorbs the exudates and supports cell adhesion and proliferation. Techniques for the production of asymmetric membranes are wet and dry/wet phase inversion, supercritical CO2-assisted phase inversion, electrospinning, and bioprinting. CH is one of the most explored biomaterials for the preparation of asymmetric membranes due to its inherent capacity to promote wound healing. Morgado et al. [115] reviewed different wound dressings composed of CH-based asymmetric membranes in addition to the general properties and production techniques of asymmetric membranes.

10.4.6 Application of chitosan-based membranes for guided bone regeneration tissue engineering Bone defects can be restored through the use of membrane made out of biocompatible materials using the GBR method. At present materials widely used for GBR are nondegradable, such as expanded Teflon, which makes a secondary surgery necessary to remove the membrane when bone is restored. Therefore to avoid secondary surgery biodegradable/bioresorbable materials are sought. The biodegradability, biocompatibility, and superior bone affinity of CH show its potential as a GBR membrane. CH has a structure similar to GAG, one of the principal components of the natural cartilage specific ECM, hence it has better cell attachment and proliferation with respect to osteoblasts than fibroblasts. The GBR membranes should have an interconnected pore structure with a suitable pore size to achieve a high transferring density of bone cells to a new tissue. Chen et al. [116] fabricated porous calcium phosphate/CH membranes. They compared the adhesion, proliferation, and differentiation of osteoblast cells on these membranes with different calcium phosphates. In vivo studies of calcium phosphate/CH hybrid membranes using rat as an animal model demonstrated that 3-week postsurgery, new bone generation up to 57% of the original bone defect area was enhanced. Li et al. [117] compared the effect on GBR by CH/collagen membranes with a standard collagen membrane. They found no statistically significant difference on the basis of histological observation and histomorphometrical analysis. However, the CH/collagen membranes revealed statistically higher vertical bone gain than the untreated group, especially the membrane with more CH material. They recommended that CH/collagen membranes would give better remarkable bone regeneration in conjunction with the use of bone grafting materials.

Handbook of Chitin and Chitosan

TABLE 10.3 Recent reports on chitosan membranes for biomedical applications. S. No.

Materials/process

Product

Application

References

1.

CH and CH-blend electrospun nanofibers

Functionalized nanofibers

Drug delivery, tissue engineering scaffolds, wound dressings, and antibacterial coatings

[85]

2.

Rod-like chitin whisker used as a filler to reinforce the CH composite membranes prepared by castingevaporation method

Chitin whisker-reinforced CH membrane

Promising material for packaging and wound dressing

[123]

3.

Supercritical carbon dioxide (scCO2)assisted phase inversion method to prepare (PVA/CH) asymmetrical membranes

PVA/CH asymmetrical membranes

Highly controlled morphology toward the ideal skin wound dressing

[115]

4.

PVA/CH membranes containing (s)ibuprofen-β-cyclodextrins

Ibuprofen loaded PVA/CH membranes

Avoided scab formation and an excessive inflammation, enabling an earlier skin healing

[124]

5.

Blended membranes composed of CH and Aloe vera gel prepared by solvent casting and cross-linked with genipin

CH/Aloe-based membranes

Genipin cross-linking enhanced the stability and mechanical properties of the blended membranes, potential as active wound dressings

[125]

6.

Antibacterial activity and cellular response of chitosan/Aloe vera-based membranes. In vitro assays demonstrated that these blended membranes have good cell compatibility with primary human dermal fibroblasts

CH/Aloe vera-based membranes

Promising wound dressing materials

[126]

7.

Incorporation of Aloe vera gel in different proportions into film-forming chitosan solution

Chitosan
Aloe vera gel blend films

Improved physicochemical properties

[127]

8.

CH/Aloe/curcumin-loaded poly(lacticco-glycolic acid) (PLGA) μS prepared by an ultrasound emulsification technology and a tape-casting processing.

Curcumin-encapsulated (PLGA) embedded CH/Aloe composite film

Enhanced antiinflammatory characteristics and repair capability for wound healing and skin regeneration

[128]

9.

Electrospinning processing of xanthanCH curcuminnanofibers

Encapsulation and delivery system of curcumin

Oral delivery applications of poorly water-soluble compounds at the gastrointestinal tract

[129]

10.

CH/alginate polyelectrolyte complex incorporated with polyhexamethylenebiguanide chloride

CH/alginate membranes

Wound dressings with antimicrobial activity against P. aeruginosa and S. aureus

[106]

11.

Salicylaldehyde as a monoaldehyde cross-linker with CH to form hydrogel membrane in presence of TiO2 NPs through casting technique

Schiff bases from CH and salicylaldehyde/TiO2 nanocomposite membrane

Effective antimicrobial activity toward S. aureus and P. aeruginosa, full eradication of bacteria

[130]

12.

Elucidation of the effect of proteins and inorganic ions on the antibacterial properties

Silver nanoparticles incorporated into CH-based nanofibrous membranes

Enhanced antibacterial efficacy and the wound healing ability in vivo.

[131]

13.

Diatomaceous earth, or diatomite, a natural silica material incorporated novel chitosan-based composite membranes

Diatomite-reinforced chitosan composite membrane biocomposite organic/inorganic biomaterial

Bone tissue engineering applications suggested based on in vitro cytotoxicity and cellular activities including cell proliferation and cell differentiation studies.

[132]

14.

CH-doped with polyethylene oxide (PEO) (ratio 95:5) was prepared for electrospinning

Electrospun chitosan (CH) membranes with a low or high degree of fiber orientation for Guided tissue regeneration (GTR) membrane

Surface layer would act as seal to prevent junctional epithelium from falling into the defect site and hence maintain space for bone regeneration

[49]

(Continued)

TABLE 10.3 (Continued) S. No.

Materials/process

Product

Application

References

15.

CH incorporating particles of biphasic calcium phosphate, zinc oxide, and copper oxide in polyethylene glycol matrix

Composite membranes of CH
polyethylene glycol matrix

Applications in tissue regeneration and antibacterial coatings of implants were assessed

[133]

16.

Carboxymethylation of CH as a hydrophilic modification

Carboxymethyl CH (CMC)

Antimicrobial, anticancer, antitumor, antioxidant, and antifungal biological activities useful in various areas like wound healing, tissue engineering, drug/enzyme delivery, bioimaging and cosmetics

[134]

17.

Gelatin
CH, Titania And Hydroxyapatite, UV radiation was used as a nontoxic cross-linking agent to improve the thermophysical/ mechanical characteristics and to control the biodegradability of the nanocomposed membrane

Gelatin
chitosan polymeric membrane which contains hydroxyapatite and titania nanoparticles as two very welldocumented osteoconductive materials

The in vitro biocompatibility of the new nanocomposite was evaluated by cell adhesion and proliferation assays. osteoconductive ability was determined by an alkaline phosphatase production assay using mouse embryonic fibroblast (MEF) cells

[135]

18.

Diatomaceous earth, or diatomite, a natural silica material incorporated novel chitosan-based composite membranes

Diatomite-reinforced chitosan composite membrane biocomposite organic/inorganic biomaterial

Bone tissue engineering applications suggested based on in vitro cytotoxicity and cellular activities including cell proliferation and cell differentiation studies

[132]

19.

Bovine hydroxyapatite, rabbit collagen type 1 (Col1)and shrimp CH scaffold was fabricated using thermally induced phase separation technique crosslinked by either glutaraldehyde, dehydrothermal treatment, irradiation or 2-hydroxyethyl methacrylate

Biocompatible porous scaffolds

Histological and radiological observations indicated the restoration of defected bone

[136]

References

331

Faivre et al. demonstrated that the generation of bioinspired microstructures in physical CH hydrogels dramatically controls their lubrication and wear resistance. CH hydrogels are excellent for 3D-chondrocyte growth and proliferation, hence they are especially suitable for cartilage repair for cartilage substitutes [21]. Pectin/CH blend membranes were prepared by the solvent evaporation method, and they exhibited cytocompatibility allowing them to be exploited as scaffolds for tissue engineering. These membranes have similar structures to ECM. It was shown that a pectin/CH membrane promoted attachment, adhesion, and proliferation of human adiposederived stem cells (ADSC), and hence could be a promising candidate for wound healing and tissue engineering [118]. Collagen, GAG, chondroitin, and hyaluronic acid are the major constituents of eggshell membrane (ESM), hence they are particularly suitable biomaterials for connective tissue repair in tissue engineering applications [119]. Silk fibroin/CH thin film combines the properties of these biomaterials to promote osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells [120]. Adali et al. synthesized hydrogels from CH, silk fibroin, and ESM and successfully carried out an assessment for cartilage tissue engineering applications, namely the cell proliferation activity of normal human articular chondrocyte-knee cells [121]. Sharma and coworkers reported the development of CH-based hydrogels using different concentrations of cross-linker. The hydrogels exhibited good tissue adhesive properties along with antimicrobial activity against E. coli, K. pneumonia, S. aureus, C. albicans, and M. gypseum bacteria. The adhesive strength of hydrogel was found to be 14 KPa. Tissue adhesive applications were successfully validated on Drosophila (Oregon-R) tissues [122]. Some of the studies involving biomedical applications of CH are summarized in Table 10.3.

References [1] J. Zhao, X. Zhao, Z. Jiang, Z. Li, X. Fan, J. Zhu, et al., Biomimetic and bioinspired membranes: preparation and application, Prog. Polym. Sci. 39 (9) (2014) 1668
1720. [2] E. Drioli, L. Giorno (Eds.), Encyclopedia of Membranes, Springer-Verlag Berlin Heidelberg, 2014. Available from: http://doi.org/10.1007/978-3-642-408724_1281-6. [3] A. Abdelrasoul, H. Doan and A. Lohi, Biomimetic and Bioinspired Membranes for New Frontiers in Sustainable Water Treatment Technology, Open access peerreviewed Monograph, IntechOpen, (2017) https://doi.org/10.5772/65691 [4] X.J. Feng, L. Jiang, Design and creation of superwetting/antiwetting surfaces, Adv. Mater. 18 (23) (2006) 3063
3078. Available from: https://doi.org/10.1002/ adma.200501961.

Handbook of Chitin and Chitosan

332

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

[5] X.-M. Li, D. Reinhoudt, M. Crego-Calama, What do we need for a superhydrophobic surface? A review on the recent progress in the preparation of superhydrophobic surfaces, Chem. Soc. Rev. 36 (8) (2007) 1350
1368. [6] T. Nakanishi, T. Michinobu, K. Yoshida, N. Shirahata, K. Ariga, H. Mohwald, et al., Nanocarbon superhydrophobic surfaces created from fullerene based hierarchical supramolecular assemblies, Adv. Mater. 20 (3) (2008) 443
446. Available from: https://doi.org/10.1002/adma.200701537. [7] H. Lee, B.P. Lee, P.B. Messersmith, A reversible wet/dry adhesive inspired by mussels and geckos, Nature 448 (2007) 338
341. Available from: https://doi.org/ 10.1038/nature05968. [8] L. Qu, L. Dai, M. Stone, Z. Xia, Z.L. Wang, Carbon nanotube arrays with strong shear binding-on and easy normal lifting-off, Science 322 (5899) (2008) 238
242. Available from: https://doi.org/10.1126/science.1159503. [9] K. Ariga, J.P. Hill, Q. Ji, Layer-by-layer assembly as a versatile bottom-up nanofabrication technique for exploratory research and realistic application, Phys. Chem. Chem. Phys. 9 (2007) 2319
2340. Available from: https://doi.org/10.1039/B700410A. [10] K. Ariga, J.P. Hill, Q. Ji, Biomaterials and biofunctionality in layered macromolecular assemblies, Macromol. Biosci. 8 (2008) 981
990. [11] S. Acharaya, A. Shundo, J.P. Hill, K. Ariga, Langmuir films of unusual components, J. Nanosci. Nanotechnol. 9 (2009) 3
18. [12] S. Acharya, J.P. Hill, K. Ariga, Soft Langmuir
Blodgett technique for hard nanomaterials, Adv. Mater. 21 (29) (2009) 2959
2981. [13] R. Gupta, N.K. Chaudhury, Biosens: entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects, Bioelectron 22 (2007) 2387
2399. [14] S.W. Boettcher, J. Fan, C.-K. Tsung, Q. Shi, G.D. Stucky, Harnessing the sol-gel process for the assembly of non-silicate mesostructured oxide materials, Acc. Chem. Res. 40 (2007) 784
792. [15] H.P. Hentze, M. Antonietti, Template synthesis of porous organic polymers, Curr. Opin. Solid State Mater. Sci 5 (2001) 343
353. [16] Y. Wang, A.S. Angelatos, F. Caruso, Template synthesis of nanostructured materials via layer-by-layer assembly, Chem. Mater. 20 (2008) 848
858. [17] A. Vinu, T. Mori, K. Ariga, New families of mesoporous materials, Sci. Technol. Adv. Mater. 7 (2006) 753
771. [18] K. Ariga, A. Vinu, J.P. Hill, T. Mori, Coordination chemistry and supramolecular chemistry in mesoporous nanospace, Coord. Chem. Rev 251 (2007) 2562
2591. [19] Q. Ji, M. Miyahara, J.P. Hill, S. Acharya, A. Vinu, S.B. Yoon, et al., Stimuli-free automodulated material release from mesoporous nanocompartment films, J. Am. Chem. Soc. 130 (2008) 2376
2377. [20] K. Ariga, A. Vinu, Q. Ji, O. Ohmori, J.P. Hill, S. Acharya, et al., A layered mesoporous carbon sensor based on nanopore-filling cooperative adsorption in the liquid phase, Angew. Chem. Int. Ed 47 (2008) 7254
7257. [21] J. Faivre, G. Sudre, A. Montembault, S. Benayoun, X. Banquy, T. Delair, et al., Bioinspired microstructures of chitosan hydrogel provide enhanced wear protection, Soft Matter. 14 (11) (2018) 2068
2076. [22] E.R. Hitzky, M. Darder, P. Aranda, K. Ariga, Advances in biomimetic and nanostructured biohybrid materials, Adv. Mater. 22 (2010) 323
336. [23] M. Liu, X. Zeng, C. Ma, H. Yi, Z. Ali, X. Mou, et al., Injectable hydrogels for cartilage and bone tissue engineering, Bone Res. 5 (2017) 17014. Available from: https://doi. org/10.1038/boneres.2017.14. [24] S.E. Naleway, M.M. Porter, J. McKittrick, M.A. Meyers, Structural design elements in biological materials: application to bioinspiration, Adv. Mater. 27 (2015) 5455
5476.

Handbook of Chitin and Chitosan

References

333

[25] M. Balasubramani, T.R. Kumar, M. Babu, Skin substitutes: a review, Burns 27 (5) (2001) 534
544. [26] A.D. Metcalfe, M.W.J. Ferguson, Bioengineering skin using mechanisms of regeneration and repair, Biomaterials 28 (2007) 5100
5113. [27] S. Bo¨ttcher-Haberzeth, T. Biedermann, E. Reichmann, Tissue engineering of skin, Burns. 36 (2010) 450
460. [28] D.L. Sachs, J.J. Voorhees, Age-reversing drugs and devices in dermatology, Clin. Pharmacol. Ther. 89 (1) (2010) 34
43. Available from: https://doi.org/10.1038/ clpt.2010.259. [29] R.A. Clark, K. Ghosh, M.G. Tonnesen, Tissue engineering for cutaneous wounds, J. Invest. Dermatol. 127 (5) (2007) 1018
1029. Available from: https://doi.org/10.1038/ sj.jid.5700715. [30] L. Yildirimer, N.T. Thanh, A.M. Seifalian, Skin regeneration scaffolds: a multimodal bottom-up approach, Trends Biotechnol. 30 (12) (2012) 638
648. Available from: https://doi.org/10.1016/j.tibtech.2012.08.004. [31] J.S. Boateng, K.H. Matthews, H.N. Stevens, G.M. Eccleston, Wound healing dressings and drug delivery systems: a review, J. Pharm. Sci. 97 (2008) 2892
2923. [32] H. Sinno, S. Prakash, Complements and the wound healing cascade: an updated review, Plast. Surg. Int. (2013) 1
7. Available from: https://doi.org/10.1155/2013/ 146764. Article ID 146764. [33] B.L. Bennett, L.F. Littlejohn, B.S. Kheirabadi, F.K. Butler, R.S. Kotwal, M.A. Dubick, et al., Management of external hemorrhage in tactical combat casualty care: chitosanbased hemostatic gauze dressings—TCCC guidelines-change 13-05, J. Spec. Oper. Med. 14 (3) (2014) 40
57. [34] T. Peng, Biomaterials for hemorrhage control, trends biomater, Artif. Organs. 24 (1) (2010) 27
68. [35] B.S. Kheirabadi, E.M. Acheson, R. Deguzman, J.M. Crissey, A.V. Delgado, S.J. Estep, et al., The potential utility of fibrin sealant dressing in repair of vascular injury in swine, J. Trauma 62 (1) (2007) 94
103. [36] S. Bertesteanu, S. Triaridis, M. Stankovic, V. Lazar, M.C. Chifiriuc, M. Vladf, et al., Polymicrobial wound infections: pathophysiology and current therapeutic approaches, Int. J. Pharm. 463 (2014) 119
126. Available from: https://doi.org/ 10.1016/j.ijpharm.2013.12.012. [37] W.-K. Loke, S.-K. Lau, L.L. Yong, E. Khor, C.K. Sum, Wound dressing with sustained anti-microbial capability, J. Biomed. Mater. Res. 53 (2000) 8
17. [38] E.A.R. Domingues, P.C.P. da Costa, M.H.B. de Moraes Lopes, I. Monteiro, N.M.C. Alexandre, et al., Pain prevalence, sociodemographic and clinical features in patients with chronic ulcers, J. Tissue Viability 25 (3) (2016) 180
184. Available from: https:// doi.org/10.1016/j.jtv.2016.03.005. [39] J.R.W. Brownrigg, J. Apelqvist, K. Bakker, N.C. Schaper, R.J. Hinchliffe, Evidencebased management of PAD & the diabetic foot, Eur. J. Vasc. Endovasc. Surg. 45 (6) (2013) 673
681. Available from: https://doi.org/10.1016/j.ejvs.2013.02.014. [40] G.C. Gurtner, S. Werner, Y. Barrandon, M.T. Longaker, Wound repair and regeneration, Nature 453 (2008) 314
321. [41] A.P. Rodrigues, E.M.S. Sanchez, A.C.D. Costa, A.M. Moraes, The influence of preparation conditions on the characteristics of chitosan alginate dressings for skin lesions, J. Appl. Polym. Sci. 109 (2008) 2703
2710. [42] C. Alemdaroglu, Z. Degim, N. C ¸ elebi, F. Zor, S. Oztu¨rk, D. Erdogan, An investigation on burn wound healing in rats with chitosan gel formulation containing epidermal growth factor, Burns 32 (3) (2006) 319
327. Available from: https://doi.org/10.1016/ j.burns.2005.10.015.

Handbook of Chitin and Chitosan

334

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

[43] P.I. Morgado, A. Aguiar-Ricardo, I.J. Correia, Asymmetric membranes as ideal wound dressings: an overview on production methods, structure, properties and performance relationship, J. Membrane Sci. 490 (2015) 139
151. Available from: https:// doi.org/10.1016/j.memsci.2015.04.064. [44] M. Cai, M. Sugumaran, W.E. Robinson, The crosslinking and antimicrobial properties of tunichrome, Comp. Biochem. Physiol. B-Biochem. Mol. Biol. 151 (2008) 110
117. [45] E. Bayer, G. Schiefer, D. Waidelich, S. Scippa, M. De Vicentiis, Structure of the tunichrome of tunicates and its role in concentrating vanadium, Angew. Chem. Int. Ed 31 (1992) 52
54. [46] D.X. Oh, S. Kim, D. Lee, D.S. Hwang, Tunicate-mimetic nanofibrous hydrogel adhesive with improved wet adhesion, Acta Biomater. 20 (2015) 104
112. [47] J.S. Temenoff, A.G. Mikos, Review: tissue engineering for regeneration of articular cartilage, Biomaterials 21 (2000) 431
440. Available from: https://doi.org/10.1016/ S0142-9612(99)00213-6. [48] Z. Cao, C. Dou, S. Dong, Scaffolding biomaterials for cartilage regeneration, J. Nanomater (2014) 8. Available from: https://doi.org/10.1155/2014/489128. Article ID 489128. [49] S.B. Qasim, S. Najeeb, R.M. Delaine-Smith, A. Rawlinson, I. Ur Rehman, Potential of electrospun chitosan fibers as a surface layer in functionally graded GTR membrane for periodontal regeneration, Dent. Mater. 33 (1) (2017) 71
83. Available from: https://doi.org/10.1016/j.dental.2016.10.003. [50] C. Algieri, E. Drioli, L. Guzzo, L. Donato, Bio-mimetic sensors based on molecularly imprinted membranes, Sensors 14 (8) (2014) 13863
13912. Available from: https:// doi.org/10.3390/s140813863. [51] V.K. Mourya, N.N. Inamdar, Chitosan modifications and applications: opportunities galore, React. Funct. Polym. 68 (6) (2008) 1013
1051. Available from: https://doi. org/10.1016/j.reactfunctpolym.2008.03.002. [52] M. Ahmad, S. Ahmed, B. l Swami, S. Ikram, Adsorption of heavy metal ions: role of chitosan and cellulose for water treatment, Int. J. Pharmacogn. 2 (6) (2015) 280
289. E- ISSN: 2348-3962, P-ISSN: 2394-5583. [53] K. Xing, Y. Xing, Y. Liu, Y. Zhang, X. Shen, X. Li, et al., Fungicidal effect of chitosan via inducing membrane disturbance against Ceratocystis fimbriata, Carbohydr. Polym. 192 (2018) 95
103. [54] H. Honarkar, M. Barikani, Applications of biopolymers I: chitosan, Monatsh. Chem. 140 (2009) 1403
1420. Available from: https://doi.org/10.1007/s00706-009-0197-4. [55] D. Xu, S. Hein, K. Wang, Chitosan membrane in separation applications, Mater. Sci. Technol. 24 (9) (2008) 1076
1087. Available from: https://doi.org/10.1179/ 174328408X341762. Biomaterials. [56] S. Haider, F.A.A. Ali, A. Haider, W.A. Al-Masry, Y. Al-Zeghayer, Novel route for amine grafting to chitosan electrospun nanofibers membrane for the removal of copper and lead ions from aqueous medium, Carbohydr. Polym. 199 (2018) 406
414. [57] M.I. Shariful, S.B. Sharif, J.J.L. Lee, U. Habiba, B.C. Ang, M.A. Amalina, Adsorption of divalent heavy metal ion by mesoporous-high surface area chitosan/poly (ethylene oxide) nanofibrous membrane, Carbohydr. Polym. 157 (2017) 57
64. [58] A. Shakeri, H. Salehi, M. Rastgar, Chitosan-based thin active layer membrane for forward osmosis desalination, Carbohydr. Polym. 174 (2017) 658
668. [59] N. Sahebjamee, M. Soltanieh, S.M. Mousavi, A. Heydarinasab, Removal of Cu21, Cd21 and Ni21 ions from aqueous solution using a novel chitosan/polyvinyl alcohol adsorptive membrane, Carbohydr. Polym. 210 (2019) 264
273. [60] U. Habiba, T.A. Siddique, S. Talebian, J.J.L. Lee, A. Salleh, B.C. Ang, et al., Effect of deacetylation on property of electrospun chitosan/PVA nanofibrous membrane and

Handbook of Chitin and Chitosan

References

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

335

removal of methyl orange, Fe(III) and Cr(VI) ions, Carbohydr. Polym. 177 (2017) 32
39. P. Karthikeyan, H.A.T. Banu, S. Meenakshi, Removal of phosphate and nitrate ions from aqueous solution using La31 incorporated chitosan biopolymeric matrix membrane, Int. J. Biol. Macromol. 124 (2019) 492
504. U. Habiba, T.A. Siddique, J.J.L. Lee, T.C. Joo, B.C. Ang, A.M. Afifi, Adsorption study of methyl orange by chitosan/polyvinyl alcohol/zeolite electrospun composite nanofibrous membrane, Carbohydr. Polym. 191 (2018) 79
85. C. Li, T. Lou, X. Yan, Y.Z. Long, G. Cui, X. Wang, Fabrication of pure chitosan nanofibrous membranes as effective absorbent for dye removal, Int. J. Biol. Macromol. 106 (2018) 768
774. Available from: https://doi.org/10.1016/j.ijbiomac.2017.08.072. U. Habiba, J.J.L. Lee, T.C. Joo, B.C. Ang, A.M. Afifi, Degradation of methyl orange and congo red by using chitosan/polyvinyl alcohol/TiO2 electrospun nanofibrous membrane, Int. J. Biol. Macromol. 131 (2019) 821
827. A. Tabriz, M.A.U.R. Alvi, M.B.K. Niazi, M. Batool, M.F. Bhatti, A.L. Khan, et al., Quaternized trimethyl functionalized chitosan based antifungal membranes for drinking water treatment, Carbohydr. Polym. 207 (2019) 17
25. W.Y. Cheah, P.L. Show, I.S. Ng, G.Y. Lin, C.Y. Chiu, Y.K. Chang, Antibacterial activity of quaternized chitosan modified nanofiber membrane, Int. J. Biol. Macromol. 126 (2019) 569
577. S.V. Kononova, A.V. Volod’ko, V.A. Petrova, E.V. Kruchinina, Y.G. Baklagina, E.A. Chusovitin, et al., Pervaporation multilayer membranes based on a polyelectrolyte complex of λ-carrageenan and chitosan, Carbohydr. Polym. 181 (2018) 86
92. V. Vijayakumar, D. Khastgir, Hybrid composite membranes of chitosan/sulfonated polyaniline/silica as polymer electrolyte membrane for fuel cells, Carbohydr. Polym. 179 (2018) 152
163. K. Divya, D. Rana, S. Alwarappan, M.S.S.A. Saraswathi, A. Nagendran, Investigating the usefulness of chitosan based proton exchange membranes tailored with exfoliated molybdenum disulfide nanosheets for clean energy applications, Carbohydr. Polym. 208 (2019) 504
512. A.B. Abou Hammad, A.M. Elnahrawy, A.M. Youssef, A.M. Youssef, Sol gel synthesis of hybrid chitosan/calcium aluminosilicate nanocomposite membranes and its application as support for CO2 sensor, Int. J. Biol. Macromol. 125 (2019) 503
509. W. Chang, F. Liu, H.R. Sharif, Z. Huang, H.D. Goff, F. Zhong, Preparation of chitosan films by neutralization for improving their preservation effects on chilled meat, Food Hydrocoll 90 (2019) 50
61. K. Rambabu, G. Bharath, F. Banat, P.L. Show, H.H. Cocoletzi, Mango leaf extract, incorporated chitosan antioxidant film for active food packaging, Int. J. Biol. Macromol. 126 (2019) 1234
1243. M. Mujtaba, R.E. Morsi, G. Kerch, M.Z. Elsabee, M. Kaya, J. Labidi, et al., Current advancements in chitosan-based film production for food technology: a review, Int. J. Biol. Macromol. 121 (2019) 889
904. Available from: https://doi.org/10.1016/j. ijbiomac.2018.10.109. S. Vimaladevi, S.K. Panda, K.A.M. Xavier, J. Bindu, Packaging performance of organic acid incorporated chitosan films on dried anchovy (Stolephorusindicus), Carbohydr. Polym. 127 (2015) 189
194. F. Liu, W. Chang, M. Chen, F. Xu, J. Ma, F. Zhong, Tailoring physicochemical properties of chitosan films and their protective effects on meat by varying drying temperature, Carbohydr. Polym. 212 (2019) 150
159. S. Park, K.S. Marsh, P. Dawson, Application of chitosan-incorporated LDPE film to sliced fresh red meats for shelf life extension, Meat Sci. 85 (3) (2010) 493
499.

Handbook of Chitin and Chitosan

336

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

[77] K.V. Reesha, S.K. Panda, J. Bindu, T.O. Varghese, Development and characterization of an LDPE/chitosan composite antimicrobial film for chilled fish storage, Int. J. Biol. Macromol. 79 (2015) 934
942. [78] P.E. Fathima, S.K. Panda, P.M. Ashraf, T.O. Varghese, J. Bindu, Polylactic acid/chitosan films for packaging of Indian white prawn (Fenneropenaeus indicus), Int. J. Biol. Macromol. 117 (2018) 1002
1010. Available from: https://doi.org/10.1016/j. ijbiomac.2018.05.214. [79] R. Priyadarshi, Sauraj, B. Kumar, F. Deeba, A. Kulshreshtha, Y.S. Negi, Chitosan films incorporated with Apricot (Prunusarmeniaca) kernel essential oil as active food packaging material, Food Hydrocoll. 85 (2018) 158
166. [80] L. Ren, X. Yan, J. Zhou, J. Tong, X. Su, Influence of chitosan concentration on mechanical and barrier properties of corn starch/chitosan films, Int. J. Biol. Macromol. 105 (3) (2017) 1636
1643. [81] Y.Y. Qin, J.Y. Yang, H.B. Lu, S.S. Wang, J. Yang, X.C. Yang, et al., Effect of chitosan film incorporated with tea polyphenol on quality and shelf life of pork meat patties, Int. J. Biol. Macromol. 61 (2013) 312
316. [82] M. Vargas, A. Albors, A. Chiralt, Application of chitosan-sunflower oil edible films to pork meat hamburgers, Procedia Food Sci 1 (2011) 39
43. [83] J. Ye, S. Wang, W. Lan, W. Qin, Y. Liu, Preparation and properties of polylactic acidtea polyphenol-chitosan composite membranes, Int. J. Biol. Macromol. 117 (2018) 632
639. [84] A. Di Martino, M. Sittinger, M.V. Risbud, Chitosan: a versatile biopolymer for orthopaedic tissue-engineering, Biomaterials 26 (30) (2005) 5983
5990. [85] K. Kalantari, A.M. Afifi, H. Jahangirian, T.J. Webster, Biomedical applications of chitosan electrospun nanofibers as a green polymer—review, Carbohydr. Polym. 207 (2019) 588
600. Available from: https://doi.org/10.1016/j.carbpol.2018.12.011. [86] R.A.A. Muzzarelli, Chitosan-based dietary foods, Carbohydr. Polym. 29 (1996) 309
316. [87] L.C. Keong, A.S. Halim, In vitro models in biocompatibility assessment for biomedicalgrade chitosan derivatives in wound management, Int. J. Mol. Sci. 10 (2009) 1300
1313. [88] H.S. Blair, J. Guthrie, T.K. Law, P. Turkington, Chitosan and modified chitosan membranes I: preparation and characterisation, J. Appl. Polym. Sci. 33 (2) (1987) 641
656. Available from: https://doi.org/10.1002/app.1987.070330226. [89] C. Clasen, T. Wilhelms, W.M. Kulicke, Formation and characterization of chitosan membranes, Biomacromolecules 7 (11) (2006) 3210
3222. Available from: https:// doi.org/10.1021/bm060486x. [90] L. Cui, S. Gao, X. Song, L. Huang, H. Dong, J. Liu, et al., Preparation and characterization of chitosan membranes, RSC Adv. 8 (2018) 28433. Available from: https://doi. org/10.1039/c8ra05526b. [91] H.P.S. Abdul Khalil, K.S. Chaturbhuj, A.S. Adnan, M.R. NurulFazita, M.I. Syakir, Y. Davoudpour, et al., A review on chitosan-cellulose blends and nanocellulose reinforced chitosan biocomposites: properties and their applications, Carbohydr. Polym. 150 (2016) 216
226. [92] Y.-L. Ji, P.S. Wolfea, I.A. Rodriguez, G.L. Bowlin, Preparation of chitin nanofibril/ polycaprolactone nanocomposite from a nonaqueous medium suspension, Carbohydr. Polym. 87 (2012) 2313
2319. [93] A.J. Uddin, M. Fujie, S. Sembo, Y. Gotoh, Outstanding reinforcing effect of highly oriented chitin whiskers in PVA nanocomposites, Carbohydr. Polym. 87 (2012) 799
805. [94] A. Watthanaphanit, P. Supaphol, H. Tamura, S. Tokura, R. Rujiravanit, Fabrication, structure, and properties of chitin whisker-reinforced alginate nanocomposite fibers, J. Appl. Polym. Sci. 110 (2008) 890
899.

Handbook of Chitin and Chitosan

References

337

[95] B. Ma, A. Qin, X. Li, X. Zhao, C. He, Bioinspired design and chitin whisker reinforced chitosan membrane, Mater. Lett. 120 (2014) 82
85. Available from: https:// doi.org/10.1016/j.matlet.2014.01.015. [96] S. Ahmed, S. Ikram, Chitosan based scaffolds and their applications in wound healing, Achiev. Life Sci. 10 (1) (2016) 27
37. Available from: https://doi.org/10.1016/j. als.2016.04.001. [97] A. Rasool, S. Ata, A. Islam, Stimuli responsive biopolymer (chitosan) based blend hydrogels for wound healing application, Carbohydr. Polym. 203 (2019) 423
429. Available from: https://doi.org/10.1016/j.carbpol.2018.09.083. [98] Y. Ma, L. Xin, H. Tan, M. Fan, J. Li, Y. Jia, et al., Chitosan membrane dressings toughened by glycerol to load antibacterial drugs for wound healing, Mater. Sci. Eng. 81 (2017) 522
531. and references there in. [99] R. Jayakumar, M. Prabaharan, P.T. Sudheesh Kumar, S.V. Nair, H. Tamura, Biomaterials based on chitin and chitosan in wound dressing applications, Biotechnol. Adv. 29 (2011) 322
337. [100] K.S. Chen, Y.A. Ku, C.H. Lee, H.R. Lin, F.H. Lin, T.M. Chen, Immobilization of chitosan gel with cross-linking reagent on PNIPAAm/gel/PP non-woven composite surface, Mater. Sci. Eng. 25 (2005) 472
478. [101] S.P. Chen, G.Z. Wu, H.Y. Zeng, Preparation of high antimicrobial activity thiourea chitosan
Ag1 complex, Carbohydr. Polym. 60 (2005) 33
38. [102] M. Fujita, M. Kinoshita, M. Ishihara, Y. Kanatani, Y. Morimoto, M. Simizu, et al., Inhibition of vascular prosthetic graft infection using a photocrosslinkable chitosan hydrogel, J. Surg. Res. 121 (2004) 135
140. [103] Y. Zhou, D. Yang, X. Chen, Q. Xu, F. Lu, J. Nie, Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration, Biomacromolecules 9 (2008) 349
354. [104] V.C. Nguyen, V.B. Nguyen, M.-F. Hsieh, Curcumin-loaded chitosan/gelatin composite sponge for wound healing application, Int. J. Polym. Sci. (2013) 7. Available from: https://doi.org/10.1155/2013/106570. Article ID 106570. [105] G.F. Caetano, M.A.C. Frade, T.A.M. Andrade, M.N. Leite, C.Z. Bueno, A.M. Moraes, et al., Chitosan-alginate membranes accelerate wound healing, J. Biomed. Mater. Res. Part B Appl. Biomater. 103 (5) (2015) 1013
1022. ˆ .M. Moraes, Influence of the incorporation of the antimicrobial agent [106] C.Z. Bueno, A polyhexamethylenebiguanide on the properties of dense and porous chitosanalginate membranes, Mater. Sci. Eng. 93 (2018) 671
678. Available from: https:// doi.org/10.1016/j.msec.2018.07.076. and references therein. [107] H.B. Yao, H.Y. Fang, Z.H. Tan, L.H. Wu, S.H. Yu, Biologically inspired, strong, transparent and functional layered organic- inorganic hybrid films, Angew. Chem. Int. Ed. 122 (2010) 2186
2191. [108] H.B. Yao, H.Y. Fang, Z.H. Tan, L.H. Wu, S.H. Yu, Biologically inspired, strong, transparent and functional layered organic- inorganic hybrid films, Angew. Chem. Int. Ed. Engl. 49 (12) (2010) 2140
2145. [109] H.-B. Yao, Z.-H. Tan, H.-Y. Fang, S.-H. Yu, Artificial nacre-like bionanocomposite films from the self-assembly of chitosan
montmorillonite hybrid building blocks, Angew. Chem. Int. Ed. 49 (52) (2010) 10127
10131. [110] X. Ma, R. Chen, X. Zheng, H. Youn, Z. Chen, Preparation of molecularly imprinted CS membrane for recognizing naringin in aqueous media, Polym. Bull. 66 (2011) 853
863. [111] H. Guan, F. Zhang, J. Yu, D. Chi, The based on liposome bioreactors
chitosan nano composite film for detection of organophosphates pesticides, Food Res. Int. 49 (2012) 15
21.

Handbook of Chitin and Chitosan

338

10. Preparation and application of biomimetic and bioinspired membranes based on chitosan

[112] S. Yadav, R. Devi, P. Bhar, S. Singhla, C.S. Pundir, Immobilization of creatininase, creatinase and sarcosine oxidase on iron oxide nanoparticles/chitosan-g-polyaniline modified Pt electrode for detection of creatinine, Enzym. Microb. Technol. 50 (2012) 247
254. [113] Y.P. Chen, B. Liu, H.T. Lian, X.Y. Sun, Preparation and application of urea electrochemical sensor based on chitosan molecularly imprinted films, Electroanalysis 23 (2011) 1454
1461. [114] S.P. Miguel, A.F. Moreira, I.J. Correia, Chitosan based-asymmetric membranes for wound healing a review, Int. J. Biol. Macromol. 127 (2019) 460
475. [115] P.I. Morgado, P.F. Lisboa, M.P. Ribeiro, S.P. Miguel, P.C. Simo˜es, I.J. Correia, et al., Poly(vinyl alcohol)/chitosan asymmetrical membranes: highly controlled morphology toward the ideal wound dressing, J. Membrane Sci. 469 (2014) 262
271. ISSN 0376-7388. [116] Y.H. Chen, H.Y. Tai, E. Fu, T.M. Don, Guided bone regeneration activity of different calcium phosphate/chitosan hybrid membranes, Int. J. Biol. Macromol. 126 (2019) 159
169. Available from: https://doi.org/10.1016/j.ijbiomac.2018.12.199. and references therein. [117] X. Li, X. Wang, T. Zhao, B. Gao, Y. Miao, D. Zhang, et al., Guided bone regeneration using chitosan/collagen membranes in dog dehiscence-type defect model, J. Oral Maxillofac. Surg. 72 (2) (2014). Available from: https://doi.org/10.1016/j. joms.2013.09.042. 304.e1
304.e14. [118] J.G. Martins, S.E.A. Camargo, T.T. Bishop, K.C. Popat, M.J. Kipper, A.F. Martins, Pectin-chitosan membrane scaffold imparts controlled stem cell adhesion and proliferation, Carbohyd. Polym. 197 (2018) 47
56. ISSN 0144-8617. [119] K.J. Ruff, D.P. DeVore, M.D. Leu, M.A. Robinson, Eggshell membrane: a possible new natural therapeutic for joint and connective tissue disorders: results from two open-label human clinical studies, Clin. Interv. Aging 4 (2009) 235
240. [120] W. Li, J. He, F.L. He, Y.L. Liu, Y.Y. Liu, Y.J. Ye, et al., Silk fibroin/chitosan thin film promotes osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells, J. Biomater. Appl. 32 (9) (2018) 1164
1173. [121] T. Adali, R. Kalkan, L. Karimizarandi, The chondrocyte cell proliferation of a chitosan/silk fibroin/egg shell membrane hydrogels, Int. J. Biol. Macromol. 124 (2019) 541
547. [122] S. Sharma, R. Kumar, P. Kumari, R.N. Kharwar, A.K. Yadav, S. Saripella, Mechanically magnified chitosan based hydrogel as tissue adhesive and antimicrobial candidate, Int. J. Biol. Macromol. 125 (2019) 109
115. Available from: https://doi.org/10.1016/j. ijbiomac.2018.12.018. [123] B. Ma, A. Qin, X. Li, X. Zhao, C. He, Structure and properties of chitin whisker reinforced chitosan membranes, Int. J. Biol. Macromol. 64 (2014) 341
346. [124] P.I. Morgado, S.P. Miguel, I.J. Correia, A.A. Ricardo, Ibuprofen loaded PVA/chitosan membranes: a highly efficient strategy towards an improved skin wound healing, Carbohydr. Polym. 159 (2017) 136
145. [125] S.S. Silva, S.G. Caridade, J.F. Mano, R.L. Reis, Effect of crosslinking in chitosan/aloe vera-based membranes for biomedical applications, Carbohydr. Polym. 98 (1) (2013) 581
588. [126] S.S. Silva, E.G. Popa, M.E. Gomes, M. Cerqueira, A.P. Marques, S.G. Caridade, et al., An investigation of the potential application of chitosan/aloe-based membranes for regenerative medicine, Acta Biomater. 9 (6) (2013) 6790
6797. [127] S.K. Abras, M.H. Azizi, Z. Hamidy, Mechanical, physicochemical and color properties of chitosan based-films as a function of Aloe veragel incorporation, Carbohydr. Polym. 87 (2012) 2058
2062.

Handbook of Chitin and Chitosan

References

339

[128] X. Liu, L. You, S. Tarafder, L. Zou, Z. Fang, J. Chen, et al., Curcumin-releasing chitosan/aloe membrane for skin regeneration, Chem. Eng. J. 359 (2019) 1111
1119. [129] A. Faralli, E. Shekarforoush, F. Ajalloueian, A.C. Mendes, I.S. Chronakis, In vitro permeability enhancement of curcumin across Caco-2 cells monolayers using electrospun xanthan-chitosan nanofibers, Carbohydr. Polym. 206 (2019) 38
47. [130] A.S. Montaser, A.R. Wassel, O.N. Al-Shaye’a, Synthesis, characterization and antimicrobial activity of Schiff bases from chitosan and salicylaldehyde/TiO2 nanocomposite membrane, Int. J. Biol. Macromol. 124 (2019) 802
809. ISSN 0141-8130. [131] J. Shao, B. Wang, J. Li, J.A. Jansen, X.F. Walboomers, F. Yang, Antibacterial effect and wound healing ability of silver nanoparticles incorporation into chitosan-based nanofibrous membranes, Mater. Sci. Eng. 98 (2019) 1053
1063. [132] S. Tamburaci, F. Tihminlioglu, Diatomite reinforced chitosan composite membrane as potential scaffold for guided bone regeneration, Mater. Sci. Eng. 80 (2017) 222
231. [133] I.C. Pereira, A.S. Duarte, A.S. Neto, J.M.F. Ferreira, Chitosan and polyethylene glycol based membranes with antibacterial properties for tissue regeneration, Mater. Sci. Eng. 96 (2019) 606
615. ISSN 0928-4931. [134] Z. Shariatinia, Carboxymethyl chitosan: properties and biomedical applications, Adv. Colloid Interface Sci. 263 (2019) 131
194. [135] C.A. Acevedo, Y. Olguı´n, M. Bricen˜o, J.C. Forero, N. Osses, P.D. Caldero´n, et al., Design of a biodegradable UV-irradiated gelatin-chitosan/nanocomposed membrane with osteogenic ability for application in bone regeneration, Mater. Sci. Eng. 99 (2019) 875
886. [136] M.S. Rahman, M.M. Rana, L.S. Spitzhorn, N. Akhtar, M.Z. Hasan, N. Choudhury, et al., Fabrication of biocompatible porous scaffolds basedon hydroxyapatite/collagen/chitosan composite for restoration of defected maxillofacial mandible bone, Prog. Biomater (2019) 1
18. Available from: https://doi.org/10.1007/s40204-0190113-x.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

11 Chitin, chitosan, marine to market G.M. Oyatogun1, T.A. Esan2, E.I. Akpan3, S.O. Adeosun4, A.P.I. Popoola5, B.I. Imasogie1, W.O. Soboyejo6, A.A. Afonja1, S.A. Ibitoye1, V.D. Abere7, A.O. Oyatogun1, K.M. Oluwasegun1, I.E. Akinwole1 and K.J. Akinluwade8 1

Department of Materials Science and Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria, 2Department of Restorative Dentistry, Obafemi Awolowo University, Ile-Ife, Nigeria, 3Institute for Composite Materials, Technical University, Kaiserslautern, Germany, 4Department of Metallurgical and Materials Engineering, University of Lagos, Akoka, Nigeria, 5Deparment of Chemical, Metallurgical and Materials Engineering, Tshwane University of Technology, Pretoria, South Africa, 6Faculty of Engineering, Wisconsin Polytechnic Institute, Menomonie, WI, United States, 7Department of Mineral Processing, National Metallurgical Development Centre, Jos, Nigeria, 8Department of Research and Development, Prototype Engineering Development Institute (National Agency for Science and Engineering Infrastructure, NASENI), Ilesa, Nigeria

O U T L I N E 11.1 Introduction

342

11.2 Origin and sources of chitin and chitosan 11.3 Synthesis of chitin and chitosan

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00011-X

341

343

345

© 2020 Elsevier Inc. All rights reserved.

342

11. Chitin, chitosan, marine to market

11.3.1 Synthesis of chitin 345 11.3.2 Synthesis of chitosan 350 11.3.3 Synthesis of derivatives of chitin and chitosan

352

11.4 Properties of chitin and chitosan 354 11.4.1 Physicochemical properties of chitin and chitosan 354 11.4.2 Biological properties of chitosan 357 11.5 Potential applications of chitin and chitosan 11.5.1 Biomedical application of chitosan 360 11.5.2 Industrial applications of chitosan 362 11.6 Economic potential of chitin and chitosan 11.7 Conclusion References

359

367

370

370

Further reading

381

11.1 Introduction Chitin is the second most abundant natural amino polysaccharide on earth, after cellulose [1
3]. It is a nitrogen-modified, high-molecularweight, linear homopolysaccharide that consists of repeated units of N-acetyl-D-glucosamine, bound together in β-(1
4)-N-acetyl-D-glucosamine bonds [2]. Chitin has a chemical formula of (C8H13O5N)n, hence it is considered to be a complex polysaccharide, whose structure resembles that of cellulose but with one hydroxyl group on each monomer replaced by an acetyl amine group, as shown in Fig. 11.1A. There are three different allomorphs of chitin. These are α-chitin, β-chitin, and γ-chitin. The main differences between the three are the degree of hydration, the size of the unit cell, and the number of chitin chains per unit cell [3,4]. The most abundant polymorph is α-chitin, which has a tightly compacted orthorhombic cell, formed by alternating sheets of antiparallel chains [3]. This polymorph occurs in fungal and yeast cell walls, in krill, lobster, and crab tendons, in crustacean shells, and in insect cuticles. The isomorph β-chitin has a monoclinic unit cell, with polysaccharide chains attached in a parallel manner, and is found in association with proteins in squid pens [4]. γ-Chitin is more of a combination of both α-chitin and β-chitin structures rather than a third polymorph [3]. The compact structure of chitin limits its reactivity and solubility in most solvents, which consequently limit its application. This has led to several chemical modifications

Handbook of Chitin and Chitosan

11.2 Origin and sources of chitin and chitosan

FIGURE 11.1

343

Structure of cellulose and chitin [6].

of chitin in an attempt to produce more soluble and reactive derivatives, one of which is chitosan [2
9]. Chitosan is a natural carbohydrate polymer obtained from the deacetylation of chitin under alkaline conditions [1,3,5]. It has a linear chain chemical structure that consists of β-(1,4)-linked 2-acetamino-2-deoxy-β-D-glucopyranose with 2-amino-2-deoxy-β-D-glucopyranose (see Fig. 11.1B). Chitosan is characterized by the presence of three functional groups: an amino group along with primary and secondary hydroxyl groups situated at the C-2, C-3, and C-6 positions, respectively [2,4,7]. Chitosan has been found to be more reactive than chitin. It is biocompatible, biodegradable, nontoxic, and possesses good antimicrobial characteristics, good film-forming ability, good chelation, and absorption properties. Consequently, chitosan has found diverse applications in fields such as environmental remediation, biomedical engineering, water engineering, biotechnology, food processing, cosmetics industries, textile industry, paper industry, agriculture, and photography [1,2,8
20]. Chitin and chitosan are therefore natural and abundant polymers with extensive structural possibilities for chemical and physical modifications [7
9]. These often culminate in novel properties that meet diverse functional requirements, consequently facilitating the versatile applications of chitin and chitosan [1,2,8,9,12
16]. Shrimp, crab, squid, lobster, insect cuticle, fungi, and yeast are the best naturally occurring sources of chitin. This chapter discussed the isolation of chitin from various marine-based sources. It also reviewed the synthesis of chitosan and the diverse applications of these versatile biopolymers.

11.2 Origin and sources of chitin and chitosan Chitin is a polysaccharide found in the exoskeleton of crustaceans, such as shrimp, crab, lobster, crawfish shells, and other marine zooplankton species such as cnidarian, foraminifera, and molluscs [16,17,20
29]. Similarly, marine gastropods such as sea shells, cone snails, coral, and cowry shells, have been reported to be other main sources of chitin [3,25
27,29
34]. Insect’s wings and fungi cell walls

Handbook of Chitin and Chitosan

344

11. Chitin, chitosan, marine to market

FIGURE 11.2 Some marine based sources of chitin and chitosan.

are also reported to contain chitin [5,21]. Fig. 11.2 shows some of the marine-based sources of chitin and chitosan. The chitinous solid waste fraction of the average Indian landing of shellfish was observed to range from 60,000 to 80,000 tons [23]. It has also been reported that dry prawn waste contained 23% chitin, while dried squilla contained 15% chitin [5,23,28,29]. Similarly, Asford et al. reported that chitin represents 14%
27% and 13%
15% of the dry weight of shrimp and crab processing wastes, respectively [28]. Consequently, commercial chitins are usually isolated from marine crustaceans, because a large amount of waste is available as a by-product during food processing. According to Ibrahim et al. shrimp consists of about 45% raw material used by seafood processing industry, while about 30%
40% by weight, which constitutes the exoskeleton (shells), is discarded as waste [28]. If not properly disposed of, this may pose a threat to the environment. The conversion of this waste to useful and economically viable materials, such as chitin and chitosan, will maximize the economic potential of this waste and minimize its threat as a potential source of environmental pollution [29]. Dutta et al. affirmed that chitin and chitosan, which are waste products of the crabbing and shrimp canning industry, are natural resources waiting for a market [2]. They also reported that in 1973 over 150,000 Mt of chitin was produced from

Handbook of Chitin and Chitosan

11.3 Synthesis of chitin and chitosan

345

waste from shellfish, krill, clams, oysters, squid, and fungi in the United States alone [2]. The innovative use of this renewable product to produce economically valuable and sustainable materials will therefore facilitate the conversion of waste to wealth, which could open up an untapped market.

11.3 Synthesis of chitin and chitosan The exoskeletons of arthropods, crustaceans, marine zooplankton, and marine gastropods are composed of chitin, which is found as a constituent of a complex network of proteins and calcium carbonate deposits that form the rigid shell [35]. Consequently, chitin isolation from these sources require the removal of two major constituents of the shell, that is, proteins by deproteinization and inorganic calcium carbonate by demineralization [3,35]. Most often, this is followed by decolorization to remove some of the residual pigments [16,17]. Many methods of chitin isolation have been documented, although no standard method has been adopted. Both deproteinization and demineralization could be carried out using chemical or enzymatic treatments in which the order of the two steps may be reversed [3]. Microbial fermentation has also been employed to isolate chitin; in this case however deproteinization and demineralization steps are carried out simultaneously [3]. The chitin residue is then converted to chitosan by the standard deacetylation process. This section presents an in-depth review of the techniques used for chitin isolation and chitosan production, with an emphasis on chitin from marine-based sources.

11.3.1 Synthesis of chitin The biosynthesis of chitin is catalyzed by the enzyme chitin synthase, the pivotal enzyme in the chitin synthesis pathway that exists in every chitin-synthesizing organism [22]. The enzyme utilizes uridine-diphosphate-N-acetylglucosamine (UDP-N-acetylglucosamine; UDP-GlcNAc), as the activated sugar donor to form the chitin polymer and remains bound to the growing polymer chains as it undergoes addition polymerization, that is, sequential addition of single GlcNAc units to the nonreducing end of the extending chain [21,22,30]. The linear polymer chain spontaneously assembles into microfibrils of varying diameter and length [22,30]. After polymerization, these are transported to the extracellular space where they are embedded in a matrix of protein, calcium carbonate, and phosphate [21]. Consequently, chitin is widely found naturally in marine invertebrates, insects, fungi, yeast, and other chitinsynthesizing organisms.

Handbook of Chitin and Chitosan

346

11. Chitin, chitosan, marine to market

Chitin was first synthesized by Professor Henri Braconnot in 1811, who isolated it from mushrooms and named it Fungine [31]. Subsequently, in 1823 Antoine Odier synthesized chitin from beetle cuticles and named it chitin after the Greek word chiton, meaning a coat of mail [31]. Since then, global attention has been focused on the isolation of chitin, and extensive work on chitin isolation from various chitin-synthesizing organisms has been documented. In this chapter, attention is focused on the isolation of chitin from marine chitin-synthesizing organisms, since these have been reported as the most abundant sources of chitin [29,30,33]. 11.3.1.1 Synthesis of chitin by chemical method Synthesis of chitin by chemical procedure is the most common method used to isolate chitin. Numerous studies have documented the successful isolation of chitin from crustacean shells by chemical processes [33,34,36
47]. This process comprises deproteinization, demineralization, and decolorization. Demineralization involves the removal of inorganic matters, such as calcium carbonate, by acid treatment, while deproteinization entails the extraction of protein in an alkaline medium and decolorization consists of the removal of pigments by chemical reagents to achieve a colorless product. Fig. 11.3 is a schematic flow chart of process of isolation of chitin from marine-based exoskeleton.

FIGURE 11.3 Process of chemical isolation of chitin.

Handbook of Chitin and Chitosan

11.3 Synthesis of chitin and chitosan

347

11.3.1.1.1 Deproteinization

Deproteinization entails the removal of the protein content of the shell. According to Younes and Rinaudo, a wide range of alkaline solutions, such as sodium hydroxide (NaOH), sodium carbonate (Na2CO3), sodium hydrogen carbonate (NaHCO3), potassium hydroxide (KOH), potassium carbonate (K2CO3), calcium hydroxide [Ca(OH)2], sodium sulfite (Na2SO3), sodium hydrogen sulfite (NaHSO3), calcium hydrogen sulfite [Ca(HSO3)2], trisodium phosphate (Na3PO4), and sodium sulfide (Na2S), have been studied as deproteinization reagents under different reaction conditions [3]. These authors however reported that NaOH was found to be the preferred reagent when applied at a concentration ranging from 0.125 to 5.0 M, at varying temperature (up to 160 C) and duration, from a few minutes up to a few days [3]. Subsequently, chemical deproteinization is often accomplished by reacting the shells with an aqueous alkaline solution of NaOH [30]. In some of the reported work, deproteinization was carried out after demineralization, hence the order in which any of these two processes is carried out during chitin isolation is not fixed [16,17,30]. 11.3.1.1.2 Demineralization

Demineralization entails the removal of minerals, specifically calcium carbonate (CaCO3), by acid treatment. It is usually carried out using any of the following acids: hydrochloric acid (HCl), nitric acid (HNO3), sulfuric acid (H2SO4), or acetic acid (CH3COOH). The conventional demineralization process is usually carried out using dilute hydrochloric acid [3]. During demineralization, the decomposition of calcium carbonate into the water-soluble calcium salts with the release of carbon dioxide, as shown in Eq. 11.1, occurred. Simultaneously, other mineral matters in chitin-bearing shells are also removed by this reaction to give soluble salts, 2HCl 1 CaCO3 -CaCl2 1 H2 O 1 CO2 m

(11.1)

which are then separated by filtration to obtain chitin residue. Extensive washing of the residue is carried out using deionized water. This is followed by acidimetric titration: to ensure that the solid residue is chitin [3]. According to Younes and Rinaudo, the demineralization process is often empirical and found to vary with the degree of mineralization of shell, extraction time, temperature, particle size, acid concentration, and solute to solvent ratio [3]. These authors reported that using HCl demineralization could be achieved in 2
3 h under stirring. The reaction time was however found to be dependent on preparation methods [3]. Longer demineralization time was found to result in a slight drop in ash content along with polymer degradation [3]. In addition, Truong et al. reported that carrying out demineralization at high temperature accelerates the demineralization reaction [48]. Furthermore, to have a complete reaction and ensure total

Handbook of Chitin and Chitosan

348

11. Chitin, chitosan, marine to market

removal of all the minerals, it was suggested that acid intake should be greater than the stoichiometric amount of minerals, or that acid with a higher concentration be utilized [37,38]. Marquis-Duval, noted that the decisive factor in demineralization is the contact area between the chitin matrix and the solvent [49]. It was therefore established that high temperatures, longer incubations, high acid concentrations, and particle size affect the final physicochemical properties of the resulting chitin [3]. Consequently, development of different demineralization processes evolved with varying demineralization process parameters to enhance the efficiency of the process [34,39,40]. For example, Horowitz et al. and Synowiecki et al. carried out demineralization at room temperature using 90% formic acid and 22% HCl, respectively [42,43]. Foster et al. however stated that these drastic treatments may result in polymer degradation as the molecular weight (Mw) and acetylation degree was lowered [44]. Other researchers therefore considered the use of mild acid to prevent this [45,46]. For example, Austin et al. carried out demineralization using ethylene-diamine-tetracetic acid (EDTA) [45]. Similarly, Brine and Austin made use of acetic acid in their work [46] while Peniston and Johnson studied the sulfurous acid process [47]. 11.3.1.1.3 Decolorization

Decolorization is carried out to remove natural pigment existing in chitin. This is often accomplished by the addition of acetone to chitin residue under reflux condition for a period of time [30,50]. Teli and Sheikh however accomplished this using potassium permanganate (KMnO4) and oxalic acid [16]. Although the most documented method for isolating chitin from crustacean shells is the chemical method, the use of enzymatic hydrolysis for deproteinization [51] and microorganisms for both demineralization and deproteinization has been also reported [51,52]. The chemical extraction method was however reported to have higher yields and to produce higher purity chitin when compared to biological extraction [52]. The next section will give a brief review of chitin isolation by biological methods. 11.3.1.2 Synthesis of chitin by biological method Several works on the synthesis of chitin by biological methods have been reported [3,53
58]. Bahasan et al. studied the isolation of chitin from the shrimp shell waste using microbes isolated from fermented milk and bread [53]. These authors concluded that application of microorganisms for the extraction of chitin from the shrimp shell waste could be an alternative to the traditional chemical methods. According to Kaur and Dhillon, the chemical methods of chitin extraction utilize harsh chemicals at elevated temperatures for a prolonged time, which often affect the

Handbook of Chitin and Chitosan

11.3 Synthesis of chitin and chitosan

349

physicochemical properties of the isolated chitin and constitute a serious environmental hazard [58]. These authors declared that green extraction methods are increasingly gaining popularity due to their environmentfriendly nature, though not currently exploited to its maximum potential on the commercial level [58]. They therefore advocated for this novel method of chitin synthesis, stating that microorganisms-mediated fermentation processes are easy to handle, simple, fast, can be optimized by controlling the process parameters, while the use of ambient temperature and negligible solvent consumption would result in reduced adverse environmental impact and costs [58]. Adour et al. extracted chitin from the teguments of white shrimp, Parapenaeus longirostris, by means of Lactobacillus helveticus grown on different media for comparison. The media comprised date juice waste and glucose. The authors extracted the chitin by inoculating the shrimp shells with a suspension of L. helveticus strain, Milano, in a fermentor [54]. It was observed that within the pH range of 8.5
9.0 and at a temperature of 30 C, the maximum deproteinization and demineralization obtained were 76% and 53%, for the 80 and 300 g/L glucose, respectively. Increasing the processing temperature from 30 C to 35 C was noted to result in a 60% increase in demineralization level. The use of date juice, as an alternative to the use of glucose, however resulted in low demineralization, with the highest demineralization obtained being 44%. Similarly, increasing temperature and total sugar content were found to enhance the deproteinization process [54]. According to Healy et al. the conventional, harsh, chemical method of chitin isolation is extremely hazardous, energy consuming, and not ecofriendly because it employs high concentrations of mineral acid and alkali [55]. They therefore investigated the potential of the isolation of chitin from prawn shell by a fermentation process. This was claimed to be a multiproduct process, which would eliminate waste and generate revenue. The authors carried out anaerobic fermentations of prawn shell waste in a benchtop, stirred tank bioreactor, from which they obtained various components of the fermentation products. They reported that the unique microbial mixture used in the fermentation of prawn shell waste resulted in the production of a purified chitin with calcium removal as high as 93.8%. They also reported the production of by-products, which consisted of a pigmented liquor containing peptides and amino acids [55]. In a similar study, biological treatment of prawn waste for chitin production was investigated by Aytekin and Elibol [56]. These authors fermented prawn shell waste using two different microorganisms, Lactococcus lactis and Teredinobacter turnirae, in order to extract chitin from prawn waste in the presence of different glucose concentrations [56]. They applied different strategies in the course of the study. In one set of experiments, both the bacteria were inoculated individually while in cocultivation experiments three different strategies were

Handbook of Chitin and Chitosan

350

11. Chitin, chitosan, marine to market

applied. The first consisted of simultaneous inoculation of the bacteria, the second entailed starting the fermentation with a protease producer and inoculating T. turnirae at the end of the fourth day of cultivation while the third strategy was the reverse of the second, that is, first Lactococcus lactis and then T. turnirae inoculations were carried out. They reported that the individually cultured approach resulted in the efficient removal of inorganic mineral matter when cultured with L. lactis while the T. turnirae cultured gave better deproteinization. The author also reported that cofermentation of both bacteria using three different protocols resulted in the highest yield, 95.5%, when Teredinobacter turnirae was first inoculated [56]. They however affirmed that the extraction of chitin by biological treatment, though environmentally friendly, was incomplete when compared to that of chemical method. Furthermore, Cremades et al. obtained carotenoproteins and chitin from crawfish by a combined process that was based on flotation (sedimentation) and in situ lactic acid production [57]. The chitin isolated by this process was reported to be of high quality, comparable to that available commercially for medical and nutritional uses. In conclusion, the isolation of chitin by biological methods, though not as efficient as the chemical method, resulted in the production of high-quality chitin, under environment-friendly conditions. The biological treatment of shell waste for chitin extraction may therefore be considered as an alternative to the chemical method.

11.3.2 Synthesis of chitosan Chitosan is mostly obtained by deacetylation of chitin using alkaline hydrolysis or an enzymatic method [30,57
68]. Certain bacteria and fungi have been reported to enable the enzymatic deacetylation of chitin [61]. These deacetylases have been isolated from various types of fungi, namely Mucor rouxii, Aspergillus nidulans, and Colletotrichum lindemuthianium. The activity of these deacetylases is however reported to be severely limited by the insolubility of the chitin substrate [61]. According to Younes and Rinaudo, chemical methods are used extensively for the commercial synthesis of chitosan due to its low production cost and the ability to adapt it for mass production [3]. Furthermore, it was suggested that either acids or alkalis may be used to deacetylate chitin, the use of acid was however reported to result in the destruction of the chain because of the susceptibility of the glycosidic bonds to acid, hence the alkali deacetylation process is often used to obtain chitosan from chitin [32]. Generally, the different alkaline hydrolysis methods may be broadly classified into two main categories. These are the heterogeneous deacetylation of solid chitin and the homogeneous deacetylation of preswollen chitin under reduced pressure in an aqueous medium [60]. In both processes the deacetylation reaction involves the use of concentrated alkali solutions and

Handbook of Chitin and Chitosan

11.3 Synthesis of chitin and chitosan

351

long processing times, which may vary from 1 to 80 h [60,61]. The preferred industrial method is however the heterogeneous deacetylation, which involves a preferential reaction in the amorphous regions of the polymer while leaving, almost intact, the intractable crystalline native regions in the parent chitin [60]. Homogeneous deacetylation, on the other hand, entails the hydrolysis of preswollen chitin using moderately concentrated alkali (13% w/w) at 25 C
40 C for 12
24 h [60]. The quality of chitosan obtained was found to be dependent on the source of chitin and the isolation process [67]. It has been reported that deacetylation of chitin results in the degradation of the polymeric chain [68]. Harsh reaction conditions have also been established to damage the crystallinity of chitosan [69]. Consequently, the reaction conditions must be controlled when preparing chitosan [68,70]. The use of heterogeneous conditions with NaOH 75% (w/v) and a temperature of 110 C have been established to result in the deacetylation of chitin with minimal degradation [70]. The degree of deacetylation (DD) in both processes was found to be dependent on the concentration of the alkali, previous treatment, particle size, and density of chitin [68]. Several studies on chitosan synthesis with variation in processing parameters have been carried out and documented [13,17,33,50]. Toan investigated the effects of varying alkaline concentration on deacetylation of chitin obtained from shrimp shell using four different concentrations of sodium hydroxide, NaOH (30%, 40%, 50%, 60%) at 65 C with a solid to solvent ratio of 1:10 (w/v) for 20 h [62]. He reported that although 60% NaOH treatment yielded the highest deacetylated chitosan with maximum solubility, 50% NaOH treatment could be used to get high-quality chitosan of 79.57% DD and 97.02% solubility [62]. Al-Sagheer et al. investigated the effects of deacetylation time on the Mw and crystallinity of synthesized chitosan [30]. They converted chitin to chitosan using two methods of deacetylation. The first was the standard deacetylation method, which involved treating chitin with 45% concentration of NaOH in 1 g:15 mL solid to solvent ratio at 110 C. The second method was the microwave method. This consisted of a mixture of chitin in a 45% concentration of NaOH in a conical flask covered tightly with cotton under microwave radiation. They reported that a microwave technique was able to reduce deacetylation time from 8 h to approximately 15 min and that it resulted in the synthesis of chitosan with a higher Mw and crystallinity [30]. Based on the aforementioned, several alternative processing methods have been developed to reduce processing times and to also minimalize quantities and concentration of alkali needed for deacetylation. Some documented processes include the use of successive alkali treatments using thiophenol in DMSO [63], thermomechanical process using a cascade reactor operated under low alkali concentration [64], flash treatment under saturated steam [65], and the use of microwave dielectric heating [66].

Handbook of Chitin and Chitosan

352

11. Chitin, chitosan, marine to market

The commercial production of chitin and chitosan has already commenced in some parts of the world, for example, the United States, Japan, Poland, India, Australia, and Norway. Global attention has also been focused on chitin and chitosan in order to tailor and impart the required functionalities in an attempt to maximize their effectiveness and expand their scope of applications [1,2,12
16,32,33,35,43
45,50,51,60].

11.3.3 Synthesis of derivatives of chitin and chitosan The reactivity of the primary amino group and the primary and secondary hydroxyl side groups result in possibilities for chemical modifications and the formation of various derivatives, with different structure and properties, resulting in versatile applications [71
74]. For example, the introduction of alkyl or carboxymethyl groups to the chitin or chitosan structure was reported to drastically increase the solubility of chitin and chitosan at neutral and alkaline pH, while substitution with carboxyl groups has been reported to yield polymers with polyampholytic properties [73,74]. Furthermore, derivatives of chitin and chitosan have shown promise for metal ions adsorption. They have also found diverse applications in drug delivery, tissue engineering, and wound healing. Chitin, chitosan, and their derivatives are widely researched as antimicrobial agents and as components in cosmetics and food [71
73]. According to Kim et al., derivatives of chitin may be classified into two categories. These authors reported that in each case the N-acetyl groups were removed while the exposed amino function reacts either with acyl chlorides, or anhydrides to give the group NHCOR, or is modified by reductive amination to NHCH2COOH [72]. According to the authors, derivatives of both types are formed by reactions with bior polyfunctional reagents that result in the formation of highly reactive derivatives with great potential [72]. Research attention has therefore been focused on the chemical modification of chitin and chitosan because of its great potential, which is yet to be fully exploited. Some of the useful derivatives of chitin and chitosan are produced by cross-linking, graft copolymerization, complexation, chemical modifications, and blending [71
77]. Glycol chitin, a partially o-hydroxyethylated chitin, was the first derivative of chitin of practical importance synthesized [75]. Some of the recent work on chitin and chitosan derivatives are discussed below. Kurita [18] was among those that investigated the effects of chemical modifications of chitosan on its solubility. The author treated fully deacetylated chitosan with phthalic anhydride in dimethylformamide to give N-phthaloyl-chitosan. It was reported that the new derivative was readily soluble in polar organic solvents [18]. In the same vein Sashiwa

Handbook of Chitin and Chitosan

11.3 Synthesis of chitin and chitosan

353

et al. [76] successfully synthesized dendronized chitosan
sialic acid hybrids using convergent grafting of preassembled dendrons built on gallic acid and tri(ethylene glycol) backbone. The new derivatives were found to be soluble in water. It was also confirmed that the water solubility of the novel derivatives was further enhanced by N-succinylation of the remaining amine functionality [76]. Baba et al. [73] synthesized methylthiocarbamoyl and phenylthiocarbamoyl chitosan derivatives and examined their selectivity toward metal ions from aqueous ammonium nitrate solution. The authors reported that the adsorption of copper(II) on the synthesized derivative occurred at low pH by four units more than that of original cross-linked chitosan and phenylthiocarbamide. They also noted that selective adsorption of copper(II) by the derivative is greater than that of iron (III), indicating that the new derivative will be an effective selective adsorbent of copper ions [73]. Similarly, Zhang et al. fabricated chitosan microparticles with 251.22 mg/g of adsorption capacity for Cd(II), higher than most of the counterparts, by chemical coprecipitation, spray drying, and Michael addition reaction, without any cross-linker participation [77]. The authors suggested that this might serve as a promising adsorbent for the recycling of contaminated water. The synthesis of chitosan hydrogels by direct grafting of D,L-lactic and/or glycolic acid onto chitosan in the absence of catalysts was carried out by Qu et al. [78]. They demonstrated that a stronger interaction existed between water and chitosan chains after grafting lactic and/or glycolic acid. According to the authors, the side chains aggregated to form physical cross-linking, which resulted in pH-sensitive chitosan hydrogels that are considered potentially useful for biomedical applications [78]. Recent work has also reported the potential of gadolinium neutran capture therapy by chitosan nanoparticles [79,80]. The authors demonstrated that the novel gadolinium-loaded nanoparticles are potentially suitable for intratumoral injection into solid tumor [80]. Research focused on chitin and its derivatives has therefore resulted in the production of polymers with extensive structural possibilities that culminated in novel properties and meet diverse functional requirements, thus expanding the scope of applications [1,2,4,6,12
16,18
20,72
117]. Several reports demonstrated that the development and applications of these functional derivatives facilitated the development of various products such as hydrogels [81], membranes [82,83], nanofibers [84
86], beads [87], micro/nanoparticles [79,80,88,91], scaffolds [81,89
93], and sponges [94,95] with broad applications. According to Kumar, the potential and traditional applications of chitin, chitosan, and their derivatives are estimated to be above 200 [19]. It can therefore be affirmed that chitin and its derivatives are indeed an expanded market in waiting.

Handbook of Chitin and Chitosan

354

11. Chitin, chitosan, marine to market

11.4 Properties of chitin and chitosan Chitin contains a high content of GlcNAc units, hence it is found to be insoluble in water and most organic solvents [7]. However, when the degree of N-acetylation, which is defined as the average number of N-Acetyl-D-glucosamine units per 100 monomers, is less than 50%, chitin becomes soluble in aqueous acidic solutions (pH , 6.0) and is called chitosan [113]. Chitosan therefore comprises a group of fully and partially deacetylated chitin [19]. The effects of degree of acetylation (DA) on the solubility, conformation, and dimensions of chitosan chains in aqueous media have been extensively studied [118
122]. The general laws of chitosan behavior in aqueous solutions was proposed by Schatz et al. [120]. According to these authors chitosan exhibits the highest structural charge density and displays polyelectrolyte behavior related to long-distance intra- and intermolecular electrostatic interactions responsible for chain expansion, high solubility, and ionic condensation at DA below 20%. They also affirmed that hydrophilic and hydrophobic interactions are progressively counterbalanced when the values of DA are in the range of 20%
50%. DA above 50% was however found to result in electrostatic interactions that are essentially short-distance interactions, rather than hydrophobic interactions, due to the increase in the acetyl group content [120]. Similarly, Sandford [123] and Aranaz et al. [60] affirmed that chitosan with DA values from 0% to 30% has the optimum biological properties, hence it is the most useful for biomedical applications. Currently, research attention is focused on chitosan and its derivatives because of their extensive array of properties that make them versatile [1
3,8
21]. According to Aranaz et al. [60] most of the characteristic properties of chitosan are strictly related to its averaged Mw and the high content of glucosamine residues containing primary amino groups. The presence of the amino (
NH2) groups had been found to enhance the polymer’s reactivity and made it considerably more versatile than cellulose. It has also facilitated the production of functional derivatives of chitosan by chemical modifications, graft reactions, and ionic interactions [98,104,111]. This has further enhanced the properties of the polymer and expanded its range of application. This section discusses extensively some of the physiochemical and biological properties of chitin, chitosan, and its derivatives.

11.4.1 Physicochemical properties of chitin and chitosan Chitin is a polymorphic substance that occurs in three different crystalline structure: α-, β-, and γ-chitin [1,51]. The most abundant polymorph of the three is the α-chitin that occurs in the exoskeleton of

Handbook of Chitin and Chitosan

11.4 Properties of chitin and chitosan

FIGURE 11.4 (c) γ-Chitin [63].

355

Three polymorphic configurations of Chitin (a) α-chitin, (b) ß-chitin and

crustaceans, such as lobsters and crabs, and other marine-based invertebrates [3,108]. Fungal, yeast cell walls, and arthropods are other main sources of α-chitin, while β-chitin on the other hand is found in association with proteins in squid pens [4,108]. These show that different polymorphic forms, with different physiochemical properties, are associated with different chitin sources. According to Khor [108] and Jang et al. [109], the differences between the three polymorphs are due to the arrangement of chains in the crystalline regions. In the α form, all chains exhibit an antiparallel orientation while in the β form the chains are arranged in a parallel manner, see Fig. 11.4A and B. In the γ form sets of two parallel strands alternate with single antiparallel strands (Fig. 11.4C). This shows that γ-chitin is composed of both α- and β-chitin [108]. Several authors including Muzzarelli et al. [110] reported that the crystal structure of β-chitin lacks hydrogen bonds along the b axis [3,4,110]. The susceptibility of β-chitin to intracrystalline swelling, acid hydrolysis, and loss of scarcely crystalline fractions was ascribed to this lack of intrasheet hydrogen bonds [3,4,110]. β-Chitin is therefore more reactive and shows a higher affinity for solvents than α-chitin. Synowiecki and Al-Khateeb [111] reported that the DD may range from 30% to 95%, depending on the source and processing technique. The crystallinity of the semicrystalline polymer has been found to be dependent on the DD [114,115]. Rinaudo [12] affirmed that the origin of chitin influences not only its crystallinity and purity but also its polymer chain arrangement and consequently its properties. Consequently, the influence of chitin source on the structure and subsequently properties of chitin and chitosan cannot be overemphasized. Due to these differences in the properties of chitin polymorphs, chitin from different sources possess different properties [3,109,110]. Knowledge of the source will therefore facilitate a good understanding of the crystalline

Handbook of Chitin and Chitosan

356

11. Chitin, chitosan, marine to market

structure, which is crucial for comprehending the structure, properties, and application relationships of chitin and its derivatives. Apart from the sources of chitin, the processes and conditions under which they are prepared affect the physicochemical properties of chitosan [12,14,17,18,30,46,60,111,112]. Extensive studies have shown that the Mw and, subsequently, the DA are dependent on both processing methods and conditions [60,100,102,105
108]. Generally, commercial chitins are prepared by a chemical method. It was however noted that when deproteinization preceded demineralization, a collapsed chitin, that is, the loss of its native structure, was isolated. On the other hand, when demineralization occurred before deproteinization, compacted chitin, in which the native chain and fibrous structures are intact and stabilized, was obtained [96,97]. Furthermore, the use of harsh chemicals for the decolorization of chitin was also observed to result in the distortion of the chitin structure [96,97]. Research focused on varying processing parameters to facilitate the production of chitin and subsequently chitosan with desirable properties has been extensively documented [46,60
76,102,105,107]. The physicochemical properties of chitin and chitosan have been found to be influenced by the variation in Mw [106]. The variation in the Mw of chitin/chitosan is extensive and was reported to range from several to more than thousands of kDa [102
104]. Based on this, the polymer was categorized into three categories. These are: 1. low-molecular-weight chitosan (LMWC); 2. medium-molecular-weight chitosan (MMWC); and 3. high-molecular-weight chitosan (HMWC). Several works have been carried out to investigate the effects of increasing Mw on the properties of chitin and chitosan [104
107]. Khan and Peh [107] reported that the density of chitosan and its solution increased with decreasing Mw. Similarly, solubility in water, permeation into cell nucleus, antioxidant activity, and biodegradability were reported to increase with decreasing Mw [60,100]. Conversely, the viscosity of chitosan solution and adsorption to fat droplets were reported to be enhanced by increasing Mw [60,107]. However, the effect of Mw on pH, surface tension, and conductivity of chitosan solutions was found to be unpredictable [107]. Apart from the influence of averaged Mw on the physiochemical properties of chitin and chitosan, characteristic properties of chitosan were also observed to be related to the high content of glucosamine residues containing primary amino groups. Its cationic properties, such as solubility, biodegradability, and absorption of its substrates, have been ascribed to the amount of protonated amino groups in the polymer chain and thus on its DD [114
116]. The cationic nature of chitosan was also observed to facilitate electrostatic interactions with anionic glycosaminoglycan and

Handbook of Chitin and Chitosan

11.4 Properties of chitin and chitosan

357

proteoglycans [103]. The high density of positive charges of chitosan has been claimed to be responsible for the versatile nature of chitosan, considering that it facilitates the formation of different derivatives, which promotes the utilization of chitosan in diverse applications [107,114,116,117,124
127]. Moreover, the presence of the hydroxyls and amino groups, which act as electron donors, has been established to enhance chelating ability for many transitional metals [8,34]. Metal binding by chitosan takes place under acidic or near neutral pH. The amino groups of chitosan and hydroxyl groups are protonated and interact with metal anions by electrostatic attraction under acidic conditions and adsorption at pH close to neutral. The chelating process depends on the pH, ion type, composition of the solution, and the chitosan’s DD and Mw [116]. Jung and Zhao [128] studied the chelating ability on ferrous ions of chitosan with a wide range of Mw and observed the highest values for 4
5 kDa chitosan, while 280
300 kDa chitosan did not exhibit this property.

11.4.2 Biological properties of chitosan In addition to the various physicochemical properties, chitin, chitosan, and its derivatives have been shown to possess diverse biological properties that are directly related to their physicochemical properties [3,60,101
108,116,125
127]. Indeed, the reactive amino and hydroxyl groups confer on chitosan important biological properties such as antitumor, antimicrobial, antiinflammatory, bone regeneration, human hemostasis, antioxidant, hypocholesterolemic, antihypertensive, prebiotic, and ion-binding activities. Most of these biological properties are dependent on the extent of acetylation, Mw of the chitosan, its polydispersity, crystallinity, and the distribution of GlcNAc and GlcN units along the polymeric chain [107,116,118
123,127
135]. Extensive studies, investigating the effects of Mw on the antimicrobial properties of chitosan and its derivatives, have been documented [3,101
103,125
128]. Sudarshan et al. [125] stated that low Mw chitosan could easily penetrate the cell wall of bacteria, interact with DNA, and as a result inhibit synthesis of mRNA and DNA transcription. It has also been shown that HMWC could interact with the cell surface and consequently alter cell permeability [126]. Lim and Hudson [101] ascribed the antimicrobial activity of chitosan to its polycationic nature that resulted in its interactions with predominantly anionic components with the subsequent change in permeability. These interactions have been associated with the death of cells by induced leakage of intracellular components [20,101,136]. Similarly, chitosan has been reported to adsorb electronegative substrate in the cell of microbe proteins, thereby disrupting the physiological activities of the microorganism, subsequently leading to death of cells [102].

Handbook of Chitin and Chitosan

358

11. Chitin, chitosan, marine to market

It has been demonstrated that the DD greatly influences the positive charge density of chitosan [103]. Higher DD was found to result in higher positive charge density when compared with that of moderate, or low DD. Subsequently, chitosan with higher DD may result in stronger antibacterial activity when compared to that of lower DD [103]. Similarly, the pH of the system has been found to influence its antimicrobial activities [3]. Younes and Rinaudo [3] reported that absorption of chitosan by bacteria occurs more at lower pH, as this results in an increase in chitosan positive ionic charge. The authors therefore affirmed that low DA, low Mw, and low pH offered higher antibacterial efficiency [3]. The effects of Mw on antioxidant activities have been extensively studied [130
133]. Anraku et al. [130] demonstrated that LMWC had higher antioxidant activity when compared to the HMWC. In the same vein, Chang et al. [131] established that chitosan with a low Mw significantly increased antioxidant scavenging activity against 2,2-diphenyl-1picrylhydrazyl (DPPH) radicals. Chien et al. [132] produced LMWC (12 kDa) that exhibited stronger scavenging activity toward DPPH radicals and higher ferrous ion chelating activity when compared with MMWC and HMWC (95 and 318 kDa, respectively). Tomida et al. [133] studied the relationship between antioxidant properties and Mw of chitosan. The authors also affirmed that LMWC has impressive antioxidant properties, which were ascribed to the ability of the polymer to scavenge hydroxyl radicals and reduce cupric acid ions. Furthermore, Anraku et al. [135] investigated the effect of HMWC supplementation in normal volunteers and reported that treatment with chitosan for 8 weeks produced a decrease in oxidized to reduced albumin ratio and an increase in total plasma antioxidant activity. These authors also reported that a significant decrease in total cholesterol levels and atherogenic index and an increase in levels of high density lipoprotein were observed. With these findings they demonstrated the antioxidative potential of HMWC in the systemic circulation in humans and suggested that HMWC significantly reduced the levels of prooxidants, such as cholesterol and uremic toxins, in the gastrointestinal tract, thus inhibiting the subsequent development of oxidative stress in the systemic circulation in humans [135]. Indeed, a vast amount of work has been documented on the study of the antifungal nature of chitin and chitosan [137
144]. Allan and Hadwiger [137] confirm the ability of chitosan to induce the in vitro growth of a number of fungi, excluding Zygomycytes that have chitosan as a component of their cell walls. According to Chen et al. [138] direct applications of chitosan film to the colony-forming Rhodotorula rubra and Penicillium notatum inhibit the growth of the organisms. Jung et al. [139] studied the effects of chitosan graft copolymers on Candida albicans, Trichophyton rubrum, and Trichophyton violaceum. The authors observed that

Handbook of Chitin and Chitosan

11.5 Potential applications of chitin and chitosan

359

the number and type of grafted chains, as well as the pH, substantially influenced the activities of these fungi. In the same vein, extensive studies investigating the antifungal activity of chitosan derivatives were documented by Rabea et al. [140]. They carried out a radial hyphal growth bioassay of Botrytis cinerea and Pyricularia grisea to assess the fungicidal activity of 24 new derivatives of chitosan (i.e., N-alkyl, N-benzylchitosans) and reported that all the derivatives are better fungicides than native chitosan. These authors further established that N-dodecylchitosan, N-(p-isopropylbenzyl) chitosan, and N-(2,6-dichlorobenzyl) chitosan were the most active against B. cinerea while the N-(m-nitrobenzyl) chitosan were the most against P. grisea. A similar work carried out by Zhong et al. [141] using 12 types of new hydroxylbenzenesulfonilanide derivatives of chitosan, carboxymethyl chitosan, and chitosan sulfate reported that all the derivatives displayed stronger antifungal properties than the original materials. Nutritional studies on chitosan show that it is able to reduce serum cholesterol, have excellent lipid binding properties, and exercise a hypocholesterolamic effect on animals [142
144]. Chitosan is not digested in the gastrointestinal tract by the digestive enzymes, hence it acts as a dietary fiber in humans and animals [142,143]. According to Lian et al. [144] LMWC is a good dietary fiber that acts as a fat blocker, though it increases the intestinal excretion of essential fatty acids and decreases absorption of fat-soluble vitamins and minerals. HMWC (150
600 kDa) was however considered a more effective dietary fiber with none of the side effects associated with LMWC [144]. Some other biological properties that facilitate its versatile applications include biocompatibility, biodegradability, immunogenicity, wound-healing activity, low-toxicity, bioactivity, and its mucoadhesive properties [33,114,116,145,146]. The positive surface charge of this biomaterial and its biocompatibility enable it to effectively support cell growth, while the hydrophilic surface facilitates adhesion, proliferation, and differentiation of cells [146]. According to Kumirska et al. [116] the properties of chitin/chitosan are interrelated and many times the polymer could elicit conflicting biological response such as obtained when would-healing properties and permeation enhancement properties are inevitably linked to others such as mucoadhesion and cytotoxicity [116]. The authors suggested that research efforts investigating these conflicting biological responses be intensified to facilitate the production of safe and efficient chitin and chitosan-based biomedical products [116].

11.5 Potential applications of chitin and chitosan Chitin and its deacetylated form chitosan are established marine biopolymers that have found diverse industrial and biomedical applications

Handbook of Chitin and Chitosan

360

11. Chitin, chitosan, marine to market

[12
21]. The potential applications of chitin and chitosan depend on their physicochemical and biological properties [116]. These outstanding properties have resulted in the versatile applications of the biopolymer in diverse areas including pharmaceutics, gene delivery, tissue engineering, ophthalmology, cosmetics, water treatment, textiles, and the paper industry [1
3,12
16,19,32,51,75,85,89
93,102]. This section discussed some of the diverse applications of chitin/chitosan and its derivatives and also identifies opportunities to develop value-added products from this marine-based biopolymer.

11.5.1 Biomedical application of chitosan The marine biopolymer and its derivatives have attracted considerable research interest for biomedical applications based on their outstanding biological attributes, such as biocompatibility, biodegradability, biosafety, and nontoxicity. Chitin/chitosan has also been found to be hemostatic, fungistatic, bacteriostatic, spermicidal, anticholestermic, and anticarcinogenic, which further expand its range of biomedical applications [12,13,19,33,114,116,145
181]. Additionally, the polycationic surface of the biopolymer has resulted in its capability to form inter- and intramolecular hydrogen bonding, which has also facilitated its use in the development of novel biomedical products [3,12,13,19,99]. 11.5.1.1 Wound dressing/wound healing Wound dressings are used to prevent the infection of wounded skin. Purified chitin-based wound dressings have been found to prevent bacteria infiltration, enhance dermal regeneration, and accelerate wound healing [87,107]. Other attributes such as good strength, flexibility, and its bioabsorbable properties facilitated chitin/chitosan’s utilization as surgical threads and wound-dressing materials [117,149,150]. Fibers made of chitin/chitosan have been shown to be useful for the production of absorbable sutures and wound-dressing materials [117,149,150]. Unlike other absorbable sutures chitin/chitosan-based sutures have been found to resist attack in bile, urine, and pancreatic juice [149]. It has also been reported to accelerate wound-healing in spray, gel, and gauze [107,151,152]. Le et al. confirmed that wound dressings made of chitin and chitosan fibers accelerated the healing of wounds by about 75% [149]. Moreover, chitin/chitosan has been used as a coating on normal biomedical materials to enhance the wound-healing process. Sutures made from materials such as standard silk and catgut when coated with regenerated chitin/chitosan were reported to show enhanced wound-healing activities [149].

Handbook of Chitin and Chitosan

11.5 Potential applications of chitin and chitosan

361

11.5.1.2 Burn treatment/artificial skin graft Artificial skin graft has been identified as another likely application of chitosan in the medical and cosmetic industries [2,8,107]. According to Dutta et al. [2] chitin/chitosan is a promising candidate for burn treatment. The authors ascribed this to the fact that chitosan can form tough, water-absorbent, biocompatible films directly on the burn. They noted that the use of chitosan for burn treatment allows excellent oxygen permeability, which is important to prevent oxygen-deprivation of injured tissues. They further stated that chitosan films have the ability to absorb water and are naturally degraded by body enzymes, and thus there is no need for removal of the wound dressing [2]. The use of nonantigenic membrane that may serve as a biodegradable template for the synthesis of neodermal tissue has been the focus of research on the design of artificial skin applicable for long-term chronic use [2,179]. Due to similar structural characteristics to glycosamino glycans, the suitability of chitosan for the development of this substratum for skin replacement such as may be required in brain-scalp damage, plastic skin surgery, etc. has been investigated [179,180]. 11.5.1.3 Tissue engineering Tissue engineering is another thriving area of biomedical application of chitin/chitosan and its derivatives. The development and manipulation of laboratory-grown cells, tissues, or organs that would replace or support the function of defective or injured parts of the body would facilitate the revolution of current technology in the repair, support, or replacement of organs or tissues that have lost their function [2]. The current methods used include the use of transplants, implants, or surgical reconstruction, all of which are fraught with diverse limitations. Most implant materials, although biocompatible, may lack the ability to meet the long-term mechanical, geometrical, and functional requirements of the body, hence the development of artificial tissues that can mimic the natural ones by combining with modulated cells with different types of scaffolding materials offers a better option [2,174]. The use of chitin/chitosan as scaffolds has been reported to have an acceleratory effect on the tissue engineering processes owing to the polycationic nature that enhances the adsorption of cells on the polymer. Consequently, research efforts have been focused on the use of chitin/chitosan as a scaffolding material in tissue engineering. It has been demonstrated that chitosan can be easily processed into porous scaffolds, films, and beads [175], while Kast et al. [176] affirmed that chitosan
thioglycolic acid (chitosan-TGA) conjugate is a promising candidate as scaffolding material in tissue engineering. Similarly, Zhang and Zhang [177] synthesized and characterized microporous chitosan/calcium phosphate

Handbook of Chitin and Chitosan

362

11. Chitin, chitosan, marine to market

composite scaffolds for tissue engineering application. The authors showed that the chitosan was able to provide a scaffold form while the bioactivity of the calcium phosphates encouraged osteoblast attachment and strengthened the scaffold, resulting in the development of a stronger, bioactive, and biodegradable scaffold [177]. In the same vein, Madihally and Matthew attempted to develop procedures for synthesizing many porous chitosan scaffolds [178]. They were able to prepare bulk, planar, and tubular scaffolds that could be used to develop different types of engineered tissue that may be applied in tissue engineering of nerves and blood vessels as a template for cells. Furthermore, Prasitsilp et al. [179] investigated the effects of DD of chitosan on its in vitro cellular responses and reported that cells are more readily attached to more highly deacetylated chitosan. 11.5.1.4 Drug delivery Drug delivery is another important area where chitin/chitosan is finding a broad range of applications. The development of drug delivery systems, such as nanoparticles, hydrogels, microspheres, films, and tablets, using this important marine biopolymer has been extensively researched and documented [106,125,126,147,148,157
160]. The pharmaceutical applications include nasal, ocular, oral, parenteral, and transdermal drug delivery. Chitosan has also been shown to be a suitable material for efficient nonviral gene therapy [157,161]. In addition, chitin/chitosan has been found to inhibit tumor cell growth [162
167] and studies on targeted delivery of drugs to tumor tissues have demonstrated the potential for applications in cancer therapy and diagnosis [168,169]. 11.5.1.5 Ophthalmology Characteristics required of an ideal contact lens, such as optical clarity, mechanical stability, sufficient optical correction, gas permeability, wettability, and immunological compatibility, are possessed by chitin/ chitosan, thus making it an ideal candidate for ophthalmological applications [2,170,181]. Contact lenses made from partially depolymerized and purified squid pen chitosan that are clear, tough, and possess other required properties, such as modulus, tensile strength, elongation, and oxygen permeability, have been reported [2]. The antimicrobial and wound-healing properties, coupled with excellent film-forming capability, were also claimed to be responsible for the suitability of the marine biopolymer for the development of ocular bandage lens [170,181].

11.5.2 Industrial applications of chitosan Chitin and chitosan possess very interesting physiochemical and biological properties which facilitate their utilization in the manufacturing

Handbook of Chitin and Chitosan

11.5 Potential applications of chitin and chitosan

363

of a vast array of widely different products. Some of the diverse array of industrial applications of chitin/chitosan and its derivatives include water engineering, biotechnology, food processing, cosmetics industries, textile industry, paper industry, agriculture, photography, and electronics [1
3,12
16,21,27
30]. Different arrays of properties are required by different industrial applications, most of which are met by the diverse array of properties of this important marine-based biopolymer [2]. This section gives an overview of some of the important industrial applications of chitin/chitosan and its derivatives. 11.5.2.1 Water engineering Water pollution caused by chemical contamination of water from a wide range of toxic products, such as metals, aromatic molecules, and dyes, constitutes a serious environmental problem [60]. Research into solutions to this hazard is therefore critical to forestall the potential animal and human toxicity that may result from this pollution. The use of chitin and chitosan to remove water pollutants has been extensively researched and documented [20,60,182
191]. Due to its polycationic nature, chitin/chitosan can be used as a flocculating agent, and it can also act as a chelating agent and heavy metals trapper [2]. Guibal et al. [184,185] examined the effect of chitosan properties on the adsorption of metals, dyes, and organic compounds. Similarly, the use of chitin/chitosan-based material for the removal of anionic dyes was extensively reviewed and documented by Crini and Badot [182]. The coagulation
flocculation process and the adsorption process were reported to depend on the DD. Saha et al. affirmed that chitosan with higher DD has a higher efficiency for the adsorption of an azo dye [191]. When compared to chitin, chitosan was found to be more efficient in the removal of metal ions [184,188], polychlorinated biphenyls (PCBs) [189], and anionic dyes [183]. Conversely, chitin was found to be more efficient than chitosan in the removal of polycyclic aromatic hydrocarbons from petrochemical wastewater [190]. Chitosan has been used as adsorbent, coagulant, and bactericide in the treatment of aquaculture wastewater [192
194]. Weltroswki et al. [193] used chitosan N-benzyl sulfonate derivatives as sorbents for the removal of metal ions in acidic medium. Considerable amounts of the world’s production of chitin and chitosan and derivatives were reported to be used in wastewater treatment [195]. 11.5.2.2 Agriculture The threat posed by antimicrobial-resistant pathogenic organisms to human and animal health has led to the need to investigate the development of natural products that exhibit unique, superior, cost-effective, and safe microbicidial action [170]. It has been reported that chitin/

Handbook of Chitin and Chitosan

364

11. Chitin, chitosan, marine to market

chitosan possesses antimicrobial activity against a number of Gramnegative and Gram-positive bacteria [72,125,153,171]. Cuero [155] demonstrated that chitin and chitosan from crustacean sources exhibited antifungal activity against a large number of human pathogenic fungi. The author however noted that the biopolymer acts more quickly on fungi than on bacteria. Furthermore, chitosan was reported to have a higher antifungal activity than chitin, although it is found to be less effective against fungi that have chitin or chitosan components in their cell walls [137]. These natural antibacterial and antifungal characteristics have been widely explored in the development of commercial disinfectant products [136
141,153,154] and have also facilitated its agrochemical applications [156]. Extensive work has been carried out on the use of chitosan coating as a protective barrier to extend the shelf life of many fruits and vegetables [196
201]. Chien et al. [199] demonstrated that coating with low Mw chitosan retarded ripening, water loss, and decay of sliced red pitayas. The authors affirm that the coated samples exhibited greater antifungal resistance than thiabendazole [199]. In addition to this the utilization of chitin/chitosan as foliage spray to induce disease resistance and increase quality and production of plants has been reported [2,12,60]. The successful applications of the marine biopolymer in crops such as rice, palm, corn, cassava, and many other tropical fruits to inhibit infection has been reported [2,60]. Similarly, several works have been carried out on the applications of chitosan to induce resistance to disease in animals and for the enhancement of the shelf life of animal products [2,12,60]. Jeon et al. [200] compared the preservative efficacy of different viscosity chitosan in coated herring and Atlantic cod and demonstrated the potential of chitosan as a preservative coating in reducing or preventing moisture loss, lipid oxidation, and microbial growth of the fish. It was reported that higherviscosity (360 cP) chitosan exerted a better preservative effect in both fish model systems [200]. In a similar study, presoaking of fish fillets with a high-DD chitosan solution was found to extend its shelf life from 5 to 9 days [201]. Investigation into enhancing the shelf life of eggs has shown that chitosan offers a protective barrier against the transfer of carbon dioxide and moisture through the eggshell [202]. Kim and No [202] also reported that this barrier was effective in keeping a high Haugh unit and yolk index because it was able to prevent diffusion of water from the albumen. Furthermore, chitin/chitosan has been incorporated into animal feed for fish and shrimps, as feed coating, and also used as a supplement in the drinking water of poultry, cattle, and pigs to prevent infection [2,12,60]. The use of chitosan as fertilizer and pesticide have been documented [203]. Hadwiger reported that when absorbed by a plant, chitosan activates and improves its natural defense

Handbook of Chitin and Chitosan

11.5 Potential applications of chitin and chitosan

365

mechanism by influencing the biochemistry of plant cells. He further demonstrated that the application of this chitosan-based solution enhances seed germination, plant growth, and yields. It may therefore be concluded that the applications of chitin/chitosan in agriculture are versatile and crucial to meet the food demands of the ever-increasing population. 11.5.2.3 Food processing Due to its outstanding attributes chitin/chitosan and its derivatives have found a wide range of unique applications in the food industry [20]. According to Aranaz et al. [60] microcrystalline chitin (MCC), which shows good emulsifying properties, superior thickening, and gelling activity, has found application in the stabilization of food [60]. Due to the antimicrobial action against food spoilage microorganisms and antioxidant properties, the use of chitin/chitosan in the protection of food from microbial deterioration has been widely documented [60,196
201]. Edible, semipermeable chitosan films and coatings have been reported to not only retard ripening, water loss, and reduce decay [196
201] but also to create a controlled atmosphere, such as used in storage, at a lower cost. This also results in the preserved food’s flavor and color being maintained and not altered by the preservatives, as is usually the case with other edible coatings [2,60]. It has also been reported that chitin/chitosan exhibits anticholesterolemic properties, that is, cholesterol-binding capacity, which facilitate its application as a dietary agent in multiple nutritional supplement products [2,60,204
206]. Dutta et al. [2] suggested that chitin/chitosan can be used as a nonabsorbable carrier for highly concentrated food ingredients such as food dyes and nutrients. 11.5.2.4 Cosmetics industries Chitin/chitosan and its derivatives have found use in three main areas of cosmetics: hair care, skin care, and oral care [2]. Owing to the fact that chitin/chitosan and hair carry opposite electrical charges—chitosan positive and hair negative—they readily complement each other. According to Dutta et al. [2] a clear solution that contains chitosan forms a clear, elastic film on hair, that enhance the softness, smoothness, and mechanical strength of hair. Therefore chitin/chitosan is found in hair products such as shampoos, rinses, permanent wave agents, hair colorants, styling lotions, hair sprays, and hair tonics [2]. The physicochemical and biological attributes of chitosan and its derivatives facilitate its use in the development of skincare products. The Mw of most chitosan products is so high that they cannot penetrate the skin, hence chitosan has found use in the production of skin moisturizer and will be a costeffective replacement for hyaluronic acid that hitherto has been used as

Handbook of Chitin and Chitosan

366

11. Chitin, chitosan, marine to market

a skin moisturizer [2,207,208]. Consequently, chitin/chitosan and its derivatives are found in cosmetics products such as creams, lotions, antiaging cosmetics, nail enamel, nail lacquers, foundation, eye shadow, lipstick, and cleansing materials [2,207,208]. The antimicrobial and antifungal attributes of chitin/chitosan and its derivatives facilitate their application in the production of oral care products such as toothpaste, mouthwashes, and chewing gum; their components in the products result in fresh breath and prevent the formation of plaque and tooth decay [2]. 11.5.2.5 Photography Chitin/chitosan has found important applications in photography due to its resistance to abrasion, optical characteristics, and filmforming ability [8]. According to Muzzarelli chitosan can easily release silver complexes, used in photography, so that it can easily penetrate from one film layer to another by the diffusion transfer reversal process [8]. Dutta et al. [2] reported the use of chitosan as a fixing agent for the acid dyes in gelatin and that it also acts as an aid to improve diffusion in color photography. 11.5.2.6 Chitosan gel for light-emitting device The use of dyes containing chitosan gels as a potential components in lasers and other light-emitting devices (LEDs) has been reported [1,2]. According to Dutta et al. [2] the doping process utilizes dyes such as porphyrin compounds that resemble the heme groups in blood. According to the authors, research on porphyrins and other dyes, such as fluorinated coumarin and rhodamine B, for transparent thin films are ongoing [2]. 11.5.2.7 Textile industry Chitin/chitosan has also found application in the textile industry [2]. Incorporation of chitin/chitosan fibers into both woven and nonwoven fabric has been reported to result in fabrics that can control odor and prevent microbial growth [2]. According to Kulkarni et al. [209], “the fusion of conventional structural textile materials with advanced properties given by ‘smart’ functional finishing technology offers a wide range of high added-value product options.” Consequently, the authors were able to devise an innovative strategy for functional finishing of textile materials that is based on the incorporation of a thin layer of surface-modifying systems (SMS), in the form of stimuli-sensitive microgels or hydrogels to produce textiles with smart functional finishing that is responsive to both temperature and pH [209]. Campos et al. [210] investigated the effect of pretreatment of cotton with chitosan in natural dyeing. The authors

Handbook of Chitin and Chitosan

11.6 Economic potential of chitin and chitosan

367

reported that fabrics pretreated with a natural mordant have better color strength than fabrics which have not been pretreated. Thus it can be concluded that chitin/chitosan and its derivatives are a vital material that is required in all the phases of production in the textile industry. 11.5.2.8 Paper industry Cellulose constitutes the main raw materials for paper manufacturing. Due to the structural similarities between cellulose and chitosan, it is currently being used in the manufacturing of paper in the place of cellulose [1,2]. According to Dutta et al. [1] chitosan utilization in paper making also saves chemical additives, increases output, and results in the production of paper with better quality [1]. The use of the marinebased biopolymer in paper recycling results in the production of strengthened recycled paper while at the same time reducing environmental degradation. Chitosan has therefore found application in the paper industry in the manufacturing of products such as toilet paper, wrapping paper, and cardboard used for packaging.

11.6 Economic potential of chitin and chitosan Seafood processing industries produce hundreds of thousands of tons of shell waste that is discarded on a regular basis. Thailand is one of the major producers and exporters of shrimp and other marine products worldwide, and deals with over 600,000 metric tons of marine products containing chitinous materials, such as shrimp, crab, and squid, annually [128,211]. Production of this vast amount of seafood will result in enormous waste generation. Disposal of this waste is not only costly but may result in a serious environmental threat as a result of leaching of toxins into the environment. Conversion of this waste to wealth will therefore facilitate the production of useful products such as chitin, chitosan, and other derivatives, which will result in value additions. Furthermore, the versatile nature of chitin/chitosan and its derivatives has resulted in the expanded scope of applications of the polymer. Consequently, processing of the seafood waste will culminate in socioeconomic advancement. In addition, the environmental challenges related to the use of petroleum-based products, such as synthetic plastics, has resulted in an intensive research drive toward the use of materials derived from renewable natural resources, such as chitin and chitosan, that are biodegradable. This will enhance the maintenance of global environmental sustainability by reducing the reliance on fossil fuels. The sustainable

Handbook of Chitin and Chitosan

368

11. Chitin, chitosan, marine to market

and cost-effective production of the hitherto petroleum-based products from chitin will also improve the economy. According to Hayes, the first patent on chitosan production was introduced in the 1920s and today there are several hundred patents on the production of chitin and its derivatives along with their applications [212]. Large-scale production of the biopolymer was confirmed to have started approximately two decades ago but its growth was hindered due to several factors, such as unavailability of a large and reliable supply of raw material, inconsistent quality, the presence of pollutants such as heavy metals, ash, and other foreign materials and finally the high production cost [212]. Presently, marine-based sources of raw material for chitin and chitosan production, that is, by-products of seafood processing industry, are readily available. These include marine zooplankton and exoskeletons of crustaceans such as crab shell, lobster shell, shrimps, and prawns [3,27
30,211]. Another untapped source of chitin is fungal mycelia [69]. The seafood industries however constitute the main sources of raw materials for the commercial production of chitin, chitosan, and its derivatives [19,28,30]. Since these wastes are relatively inexpensive, the production of chitosan on a large scale from this renewable bioresource was reported to be economically feasible [128]. The extraction of chitin from crustacean shells has been commercialized in the last couple of decades in different parts of the world. These include Europe, Latin America, North America, Asia, Middle East, and Africa [128,211]. An improved production process has also facilitated the production of high-purity chitin and chitosan and consequently has expanded the scope of biomedical applications [1
3]. Recent advances in chitin and chitosan research has dramatically increased their application in various fields and spurred the development of new products, with more potential applications either in the incubation phase or yet to be discovered [1
3,108,124,128]. The application of chitin/chitosan in agriculture, water treatment, textile industry, paper industry, cosmetics, and biomedicine has been documented, some of which have been extensively discussed in the previous section [1
3,108,124,128]. The emerging applications of chitin and chitosan have resulted in increasing demand for chitin and its derivatives by these end-use industries. Furthermore, with considerable research efforts being directed toward developing safe and efficient chitin/chitosan-based products, the expansion of the scope of applications and subsequently demand will be increased. Industries such as medicine, water treatment plant, food industry, beverage industry, pharmaceutical companies, bioplastics, cosmetics industry, textile industry, paper manufacturing, and agriculture constitute some of the end-users that have also fueled the market globally. This has made the global market

Handbook of Chitin and Chitosan

11.6 Economic potential of chitin and chitosan

369

of chitin, chitosan, and their derivatives extremely attractive. Based on the Global Industry Analysts Inc. report the global demand for chitin in 2015 was above 60,000 ton, while its global production was around 28,000 ton [213]. In 2015 the global chitosan market was reported to be US$1,205 million [213]. In 2016 the global chitin and chitosan market was reported to be US$2 billion and was forecast to reach US$4.2 billion by 2021, at a compound annual growth rate (CAGR) of 15.4%. The report estimated that the global market for chitin derivatives, including chitosan, should reach US$6.38 billion by 2024 [213] growth over the forecast. The forecasted increase in CAGR within this period was ascribed to its rising application in various end-use industries. The Asia-Pacific was reported to hold the highest share of the global market, that is, two-thirds share, while the European market is less developed [213]. Of the different grades of chitin/chitosan, the industrial grade constitutes the most widely used grade. Some of these diverse industrial applications include water treatment, food and beverage processing, agrochemicals, the cosmetics industry, and the biomedical and pharmaceuticals industry. The increasing demand for chitosan in these diverse areas resulted in the growth of the industrial grade segment of the chitosan market and may be responsible for the estimated increase in CAGR. Of the industrial segment, the water treatment segment is projected to have the largest share of the chitosan market. This was attributed to ongoing rapid industrialization, the increasing inflow of wastewater from various industries and an increase in the scarcity of freshwater, which has led to an increase in the global demand for treated recycled water [214
216]. The Asia-Pacific region has been projected to further fuel the growth of the industrial grade chitosan market due to the ongoing rapid industrialization in China and India [216]. According to Prasad, China chitosan market is projected to grow at the highest CAGR, during the forecast period, followed by Vietnam and India [205]. Prasad stated that countries such as Thailand, Indonesia, and Malaysia, which have emerged as manufacturing hubs for different industries, might also fuel the growth of the Asia-Pacific chitosan market [216]. Meanwhile, due to the strong pharmaceutical sector in North America, the region is projected to witness gains close to 18% in the coming years [214]. These extremely attractive global chitosan market may further be enhanced by extensive research and development initiatives to ascertain other future novel applications of the marine biopolymer [214]. According to Pulidindi and Pandey, the chitosan market size for the cosmetics and toiletries segment is projected to witness gains closer to 18.5% in the coming years [214]. This was attributed to its application in the manufacture of antiaging creams, shampoos, styling gels, hair sprays, and hair colorants. Due to its antiplaque and antidecay properties, it has also found application in the manufacture of toothpastes,

Handbook of Chitin and Chitosan

370

11. Chitin, chitosan, marine to market

mouthwashes, and chewing gums, which will further expand the chitosan market size for the cosmetics segment [214].

11.7 Conclusion Indeed, the journey of chitin/chitosan from the marine to the enduser industry and finally the consumer can be described as viable and a highly lucrative emerging market.

References [1] P.K. Dutta, M.N.V. Ravikumar, J. Dutta, Chitin and chitosan for versatile applications, JMS Polym. Rev. C42 (2002) (2002) 307. [2] P.K. Dutta, J. Dutta, V.S. Tripathi, Chitin and chitosan: chemistry, properties and applications, J. Sci. Ind. Res. 63 (2004) 20
31. [3] S. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. Structure, properties and applications, Mar. Drugs 13 (2015) 1133
1174. [4] S.S. Kim, Y.M. Lee, C.S. Cho, Synthesis and properties of semi-interpenetrating polymer networks composed of β-chitin and poly(ethylene glycol) macromer, Polymer 36 (1995) 4497. [5] D. Wanule, J. Balkhande, P. Ratnakar, A.N. Kulkarni, C.S. Bhowate, Extraction and FTIR analysis of chitosan from Amarican cockroach, periplaneta, Americana, Int. J. Eng. Sci. Innov. Tech. (IJEIT) 3 (2014) 299
304. [6] M.N.V. Ravikumar, Chitin and chitosan fibres: a review, Bull. Mater. Sci. 22 (5) (1999) 905
915. [7] K. Kurita, Controlled functionalization of the polysaccharide chitin, Prog. Polym. Sci 26 (2001) 1921
1971. [8] R.A.A. Muzzarelli, Some modified chitosans and their niche applications, in: R.A.A. Muzzarelli, M.G. Peter (Eds.), Chitin Handbook, European Chitin Society, Pergamon Press, Oxford, 1997, pp. 47
52. [9] K.D. Yao, F. Zhao, F. Li, Y.J. Yin, Chitosan-based gels, in: M. Schwartz (Ed.), Encyclopedia of Smart Materials, Vol 1, John Wiley, New York, 2002, pp. 182
190. [10] D.V. Luyen, V. Rossbach, Mixed esters of chitin, J. Appl. Polym. Sci. 55 (1995) 679. [11] N. Kubota, Permeability properties of chitosan-transition metal complex membranes, J. Appl. Polym. Sci. 64 (1997) 819. [12] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (603) (2006) 32. [13] M. Rinaudo, Physical properties of chitosan and derivatives in sol and gel states, in: B. Sarmento, J. das Neves (Eds.), In Chitosan-Based Systems for Biopharmaceuticals: Delivery, Targeting and Polymer Therapeutics, John Wiley & Sons, Chichester, UK, 2012, pp. 23
44. [14] M. Rinaudo, Main properties and current applications of some polysaccharides as biomaterials, Polym. Int. 57 (2008) 397
430. [15] M. Rinaudo, Materials based on chitin and chitosan, in: S. Kabasci (Ed.), Bio-Based Plastics: Materials and Applications, John Wiley and Sons, Chichester, UK, 2014, pp. 63
80. [16] M.D. Teli, J. Sheikh, Extraction of chitosan from shrimp shells waste and application in antibacterial finishing of bamboo rayon, Int. J. Biol. Macromol. 50 (2012) 1195
1200.

Handbook of Chitin and Chitosan

References

371

[17] M.M. Islam, S.M. Masum, M.A.I. Molla, M.M. Rahman, A.A. Shaikh, S.K. Roy, Preparation of chitosan from shrimp shell and investigation of its properties, Int. J. Basic Appl. Sci. 11 (1) (2011) 116
130. [18] K. Kurita, Chemistry and application of chitin and chitosan, Polym. Degrad. Stab. (59)(1998) 117. [19] M.N.V.R. Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (1) (2000) 1
27. [20] F. Shahidi, R. Abuzaytoun, Chitin, chitosan, and co-products: chemistry, production, applications, and health effects, Adv. Food Nutr. Res. (49) (2005) 93
135. [21] R.N. Tharanathan, F.S. Kittur, Chitin—the undisputed biomolecule of great potential, Crit. Rev. Food Sci. Nutr. (43) (2003) 61
87. [22] H. Merzendorfer, L. Zimoch, Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases, J. Exp. Biol. 206 (Pt 24) (2003) 4393
4412 [PubMed: 14610026]. [23] P. Madhavan, K.G.R. Nair, Proceedings of the First International Conference on Chitin and Chitosan, MIT, Cambridge, 1978, pp. 88
102. [24] F. Nessa, S.M. Masum, M. Asaduzzaman, S.K. Roy, M.M. Hossain, M.S. Jahan, A process for the preparation of chitin and chitosan from prawn shell waste, Bangladesh J. Sci. Ind. Res. 45 (4) (2010) 323
330. [25] I.E. Akinwole, G. Alebiowu, G.M. Oyatogun, D.V. Abere, K.M. Oluwasegun, A.O. Oyatogun, et al., Synthesis and characterization of cowry and crab shells based chitosan for drug delivery, Bioceram. Dev. Appl. (8) (2018) 107. Available from: https:// doi.org/10.4172/2090-5025.1000107. [26] K.J. Akinluwade, G.M. Oyatogun, G. Alebiowu, A.P.I. Popoola, Morphological analysis of chitosan-acacia gum nanoparticles loaded and unloaded with cisplatin, Eur. J. Pharm. Med. Res. 5 (6) (2018) 162
166. [27] N.A. Ashford, D. Hattis, A.E. Murray, Industrial prospects for chitin and protein from shellfish wastes, MIT Sea Grant Report MISG 77-3, MIT, Cambridge, MA, 1977. [28] H.M. Ibrahim, M.F. Salama, H.A. El-Banna, Shrimp’s waste: chemical composition nutritional value and utilization, Nahrung (43) (1991) 418
423. [29] S. Subasinghe, The Development of Crustacean and Mollusk Industries, Ampnag Press, Malaysia, 1995, p. 27. [30] F.A. Al-Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Extration and characterization of chitin and chitosan from marine sources in Arabian Gulf, Carbohydr. Polym. 77 (1) (2009) 410
419. [31] R.A.A. Muzzarelli, R. Jeniaux, G.W. Gooday, Chitin in Nature and Technology, Plenum Publishing Corporation, New York, 1986. [32] I. Aranaz, D. Elieh-Ali-Komi, M.R. Hamblin, Chitin and chitosan: production and application of versatile biomedical nanomaterials, Int. J. Adv. Res. (Indore) 4 (3) (2016) 411
427. [33] D. Zvezdova, Synthesis and characterization of chitosan from marine sources in Black sea, in: HAУЧHИ ТРУДОВE HA РУCEHCKИЯ УHИВEРCИТEТ—2010, том 49, серия 9.1, 2010, pp. 65
69. [34] R.A.A. Muzzarelli, F. Tanfani, M. Emanuelli, S. Gentile, The chelation of cupric ions by chitosan membranes [Callinectes sapidus, blue crab shell], J. Appl. Biochem. (2) (1980) 380
389. [35] S.A. Anicuta, L. Dobre, M. Stroescu, L. Jipa, Fourier Transformer Infrared (FTIR) spectroscopy for characterization of antimicrobal films containing chitosan, Analele ´ Universit˘aNii din Oradea Fascicula: Ecotoxicologie, Zootehnie s¸ i Tehnologii de Industrie Alimentar˘a, 2010, pp. 1234
1240. [36] N. Acosta, C. Jimenez, V. Borau, A. Heras, Extraction and characterization of chitin from crustaceans, Biomass Bioenergy. (5) (1995) 145
153.

Handbook of Chitin and Chitosan

372

11. Chitin, chitosan, marine to market

[37] E.L. Johnson, Q.P. Peniston, Utilization of shellfish waste for chitin and chitosan production, in: R.E. Martin, G.J. Flick, C.E. Hebard, D.R. Ward (Eds.), Chemistry and Biochemistry of Marine Food Products, 1982, AVI Publishing Co, Westport, CT, USA, 1982, p. 415. Chapter 19. [38] F. Shahidi, J. Synowiecki, Isolation and characterization of nutrients and value-added products from snow crab (Chifroeceles opilio) and shrimp (Panda-111sb orealis) processing discards, J. Agric. Food Chem. (39) (1991) 1527
1532. [39] R.H. Hackman, Studies on chitin. I. Enzymatic degradation of chitin and chitin esters, Aust. J. Biol. Sci. (7) (1954) 168
178. [40] R.H. Hakman, M. Goldberg, Light-scattering and infrared-spectrophotometric studies of chitin and chitin derivatives, Carbohydr. Res. (38) (1974) 35
45. [41] G.G. Anderson, N. de Pablo, C. Romo, Antartic krill (Euphausia superba) as a source of chitin and chitosan, in: R.A.A. Muzzarelli, E.R. Priser (Eds.), Proceedings of First International Conference on Chitin and Chitosan, 1978, Mit Sea Grant Program: Cambridge, MA, 1978, pp. 54
63. [42] S.T. Horowitz, S. Roseman, H.J. Blumental, Preparation of glucosamine oligosaccharides. 1. Separation, J. Am. Chem. Soc. (79) (1957) 5046
5049. [43] J. Synowiecki, Z.E. Sikorski, M. Naczk, Immobilization of invertase on krill chitin, Biotechnol. Bioeng. (23) (1981) 231
233. [44] A.B. Foster, J.M. Webber, Chitin, Adv. Carbohydr. Chem. 15 (1960) 371
393. [45] P.R. Austin, C.J. Brine, J.E. Castle, J.P. Zikakis, Chitin: facets of research, Science (212) (1981) 749
753. [46] C.J. Brine, P.R. Austin, Chitin variability with species and method of preparation, Comp. Biochem. Physiol. (69B) (1981) 283
286. [47] Q.P. Peniston, E.L. Lohnson, Process for demineralization of crustacea shells. U.S. Patent 4,066,735, January 3, 1978. [48] T. Truong, R. Hausler, F. Monette, P. Niquette, Fishery industrial waste valorization for the transformation of chitosan by hydrothermo-chemical method, Rev. Sci. Eau (20) (2007) 253
262. [49] F.O. Marquis-Duval, Isolation et valorisation des constituants de la carapace de la crevette nordique. Ph.D. Dissertation, Laval University, Quebec, Canada, 2008. [50] H. Mirzadeh, N. Yaghobi, S. Amanpour, H. Ahmadi, M.A. Mohagheghi, F. Hormozi, Preparation of chitosan derived from shrimp’s shell Persian gulf as blood hemostasis agent, Iran. Polym. J. 11 (1) (2002) 63
68. [51] N.M. Alves, J.F. Mano, Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications, Int. J. Biol. Macromol. (43) (2008) 401
414. ISSN 0141-8130. [52] K. Shirai, D. Palella, Y. Castro, I. Guerrero-Legarreta, G. Saucedo-Castaneda, S. Huerta-Ochoa, et al., Characterization of chitins from lactic acid fermentation of prawn wastes, in: R.H. Chen, H.C. Chen (Eds.), Advances in Chitin Science, Volume III, Elsevier, Taiwan, 1998, pp. 103
110. [53] S.H.O. Bahasan, S. Sathianeson Satheesh, M.A. Mohammad Abdulaziz Ba-akdah, Extraction of chitin from the shell wastes of two shrimp species fenneropenaeus semisulcatus and fenneropenaeus indicus using microorganisms, J. Aquat. Food Prod. Technol. 26 (4) (2017) 390
405. [54] L. Adour, W. Arbia, A. Amrane, N. Mameri, Combined use of waste materials— recovery of chitin from shrimp shells by lactic acid fermentation supplemented with date juice waste or glucose, J. Chem. Technol. Biotechnol. (83) (2008) 1664
1669. [55] M. Healy, A. Green, A. Healy, Bioprocessing of marine crustacean shell waste, J. Eng. Life Sci. 23 (2
3) (2003) 151
160. [56] O. Aytekin, M. Elibol, Cocultivation of Lactococcus lactis and Teredinobacter turnirae for biological chitin extraction from prawn waste, Bioproc. Biosyst. Eng. (33) (2010) 393
399.

Handbook of Chitin and Chitosan

References

373

[57] O. Cremades, E. Ponce, R. Corpas, J.F. Gutie´rrez, M. Jover, M.C. Alvarez-Ossorio, et al., Processing of crawfish (Procambarus clarkii) for the preparation of carotenoproteins and chitin, J. Agric. Food Chem. 49 (11) (2001) 5468
5470. [58] S. Kaur, G.S. Dhillon, Recent trends in biological extraction of chitin from marine shell wastes: a review, Crit. Rev. Biotechnonol. 35 (1) (2015) 44
61. [59] K.L. Morley, G. Chauve, R. Kazlauskas, C. Dupont, F. Shareck, R.H. Marchessault, Acetyl xylan esterase-catalyzed deacetylation of chitin and chitosan, Carbohydr. Polym. (63) (2006) 310
315. [60] I. Aranaz, M. Mengı´bar, R. Harris, I. Pan˜os, B. Miralles, N. Acosta, et al., Functional characterization of chitin and chitosan, Curr. Chem. Biol. (3) (2009) 203
230. [61] G.W. Gooday, J.I. Prosser, K. Hillman, M.G. Cross, Mineralization of chitin in an estuarine sediment: the importance of the chitosan pathway, Biochem. Syst. Ecol. 19 (5) (1991) 395
400. [62] N.V. Toan, Production of chitin and chitosan from partially autolyzed shrimp shell materials, Open Biomater. J. (1) (2009) 21
24. [63] A. Domard, M. Rinadudo, Preparation and characterization of fully deacetylated chitosan. I, J. Biol. Macromol. (5) (1983) 49
52. [64] A. Pelletier, I. Lemire, J. Sygusch, E. Chornet, R.P. Overend, Chitin/chitosan transformation by thermo-machano-chemical treatment including characterization by enzymatic depolymerization, Biotechnol. Bioeng. (36) (1990) 310
315. [65] B. Focher, P.L. Beltrane, A. Naggi, G. Torri, Alkaline N-deacetylation of chitin enhanced by flash treatments. Reaction kinetics and structure modifications, Carbohydr. Polym. (12) (1990) 405
418. [66] F.M. Goycoolea, I. Higuera-Ciapara, J.L.J. Hernandez, K.D. Garcı´a, Preparation of chitosan from squid (Loligo spp) pen by a microwave-accelerated thermochemical process, in: A. Domard, G.A.F. Roberts, K.M. Va˚rum (Eds.), Advances in Chitin Science, Jacques Andre´, Lyon, 1998, pp. 78
83. [67] G. Galed, B. Miralles, I. Pan˜os, A. Santiago, A.N. Heras, Deacetylation and depolymerization reactions of chitin/chitosan: influence of the source of chitin, Carbohydr. Polym. 62 (4) (2005) 316
320. [68] P.R. Rege, L.H. Block, Chitosan processing: influence of process parameters during acidic and alkaline hydrolysis and effect of the processing sequence on the resultant chitosan’s properties, Carbohydr. Res. 321 (3
4) (1999) 235
245. [69] M. Sukwattanasinitt, H. Zhu, H. Sashiwa, S. Aiba, Utilization of commercial nonchitinase enzymes from fungi for preparation of 2-acetamido-2-deoxy--glucose from [beta]-chitin, Carbohydr. Res. 337 (2) (2002) 133
137. [70] G. Galed, E. Diaz, A. Heras, Conditions of N-deacetylation on chitosan production from alpha chitin, Nat. Prod. Commun. 3 (4) (2008) 543
550. [71] V. Zargar, M. Asghari, A. Dashti, A review on chitin and chitosan polymers: structure, chemistry, solubility, derivatives, and applications, ChemBioEng. Rev. 2 (3) (2015) 204
226. [72] C.H. Kim, J.W. Choi, H.J. Chun, K.S. Choi, Synthesis of chitosan derivatives with quaternary ammonium salt and their antibacterial activity, Polym. Bull. (38) (1997) 387. [73] Y. Baba, H. Noma, R. Nakayama, Y. Matsushita, Preparation of chitosan derivatives containing methylthiocarbamoyl and phenylthiocarbamoyl groups and their selective adsorption of copper (II) over iron (III), Anal. Sci. 18 (3) (2002) 359
361. [74] M. Sugimoto, M. Morimoto, H. Sashiwa, H. Saimoto, Y. Shigemasa, Preparation and characterization of water-soluble chitin and chitosan derivatives, Carbohydr. Polym. 36 (1) (1998) 49
59. [75] Q. Li, E.T. Dunn, E.W. Grandmaison, M.F.A. Goosen, in: M.F.A. Goosen (Ed.), Applications and Properties of Chitosan, Applications of Chitin and Chitosan, Technomic Publishing Company, Lancaster, 1997, pp. 3
29.

Handbook of Chitin and Chitosan

374

11. Chitin, chitosan, marine to market

[76] H. Sashiwa, Y. Shigemasa, R. Roy, Chemical modification of chitosan: synthesis of dendronized chitosan-sialic acid hybrid using convergent grafting of preassembled dendrons built on gallic acid and tri(ethylene glycol) backbone, Macromolocules (2001) (2001) 3905. [77] H. Zhang, Q. Dang, C. Liu, D. Yub, Y. Wang, X. Pu, et al., Fabrication of methyl acrylate and tetraethylenepentamine grafted magnetic chitosan microparticles for capture of Cd(II) from aqueous solutions, J. Hazard. Mater. (366) (2019) 346
357. [78] X. Qu, A. Wirsen, A.C. Albertsson, Effect of lactic/glycolic acid side chains on the thermal degradation kinetics of chitosan derivatives, Polymer (41) (2001) 4841. [79] H. Tokumitsu, J. Hiratsuka, Y. Sakurai, T. Kobayashi, H. Ichikawa, Y. Fukumori, Gadolinium neutron-capture therapy using novel gadopentetic acid-chitosan complex nanoparticles: in vivo growth suppression of experimental melanoma solid tumor, Cancer Lett. (150) (2000) 177. [80] H. Tokumitsu, H. Ichikawa, Y. Fukumori, Chitosangadopentetic acid complex nanoparticles for gadolinium neutron-capture therapy of cancer: preparation by novel emulsiondroplet coalescence technique and characterization, Pharm. Res. (16) (1999) 1830. [81] H. Tamura, T. Furuike, S.V. Nair, R. Jayakumar, Biomedical applications of chitin hydrogel membranes and scaffolds, Carbohydr. Polym. (84) (2011) 820
824. [82] H. Nagahama, T. Kashiki, N. Nwe, R. Jayakumar, T. Furuike, H. Tamura, Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications, Carbohydr. Polym. (73) (2008) 456
463. [83] H. Nagahama, N. Nwe, R. Jayakumar, S. Koiwa, T. Furuike, H. Tamura, Novel biodegradable chitin membranes for tissue engineering applications, Carbohydr. Polym. (73) (2008) 295
302. [84] K. Azuma, S. Ifuku, T. Osaki, Y. Okamoto, S. Minami, Preparation and biomedical applications of chitin and chitosan nanofibers, J. Biomed. Nanotechnol. (10) (2014) 2891
2920. [85] Y. Fan, T. Saito, A. Isogai, Preparation of chitin nanofibers from squid pen beta-chitin by simple mechanical treatment under acid conditions, Biomacromolecules (9) (2008) 1919
1923. [86] S. Ifuku, M. Nogi, K. Abe, M. Yoshioka, M. Morimoto, H. Saimoto, et al., Preparation of chitin nanofibers with a uniform width as alpha-chitin from crab shells, Biomacromolecules (10) (2009) 1584
1588. [87] N.L.B.M. Yusof, L.Y. Lim, E. Khor, Preparation and characterization of chitin beads as a wound dressing precursor flexible chitin films as potential wound-dressing materials: wound model studies, J. Biomed. Mater. Res. (54) (2001) 59
68. 23. [88] A. Anitha, N. Deepa, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Development of mucoadhesive thiolated chitosan nanoparticles for biomedical applications, Carbohydr. Polym. (83) (2010) 66
73. [89] M. Peter, Sudheesh, P.T. Kumar, N.S. Binulal, S.V. Nair, H. Tamura, et al., Development of novel chitin/nano bioactive glass ceramic nanocomposite scaffolds for tissue engineering applications, Carbohydr. Polym. (78) (2009) 926
931. [90] M. Peter, N.S. Binulol, S. Soumya, S.V. Nair, H. Tamura, R. Jayakumar, Nanocomposite scaffolds of bioactive glass ceramic nanoparticles disseminated chitosan matrix for tissue engineering applications, Carbohydr. Polym. (79) (2010) 284
289. [91] C.A. Ibekwe, G.M. Oyatogun, T.A. Esan, K.M. Oluwasegun, Synthesis and characterization of chitosan/gum arabic nanoparticles for bone regeneration, Am. J. Mater. Sci. Eng. 5 (1) (2017) 28
36. [92] M. Prabaharan, R. Jayakumar, Chitosan-graft-β-cyclodextrin scaffolds with controlled drug release capability for tissue engineering applications, Int. J. Biol. Macromol. (44) (2009) 320
325.

Handbook of Chitin and Chitosan

References

375

[93] Y. Maeda, R. Jayakumar, H. Nagahama, T. Furuike, H. Tamura, Synthesis, characterization and bioactivity studies of novel β-chitin scaffolds for tissue-engineering applications, Int. J. Biol. Macromol. (42) (2008) 463
467. [94] K. Muramatsu, S. Masuda, Y. Yoshihara, Fujisawa, A. In vitro degradation behavior of freeze-dried carboxymethyl-chitin sponges processed by vacuum-heating and gamma irradiation, Polym. Degrad. Stab. (81) (2003) 327
332. [95] A. Portero, D. Teijeiro-Osorio, M.J. Alonso, C. Remunan-Lopez, Development of chitosan sponges for buccal administration of insulin, Carbohydr. Polym. (68) (2007) 617
625. [96] M.L. Bade, Structure and isolation of native animal chitins, in: M.F.A. Goosen (Ed.), Applications of Chitin and Chitosan, Technomic Publishing, Lancaster, 1997, pp. 253
277. [97] K. Kurita, T. Sannan, Y. Iwakura, Studies on chitin, evidence for formation of block and random copolymers of N-acetyl-d-glucosamine and d-glucosamine by heteroand homogeneous hydrolyses, Makromol. Chem. (178) (1997) 3197
3202. [98] B. Klaykruayat, K. Siralertmukul, K. Srikulkit, Chemical modification of chitosan with cationic hyperbranched dendritic polyamidoamine and its antimicrobial activity on cotton fabric, Carbohydr. Polym. 80 (1) (2010) 197
207. [99] K. Tomihata, Y. Ikada, In vitro and in vivo degradation of films of chitin and its deacetylated derivatives, Biomaterials 18 (7) (1997) 567
575. [100] H. Zhang, S.H. Neau, In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation, Biomaterials 22 (12) (2001). 1653
165. [101] S.H. Lim, S.M. Hudson, Synthesis and antimicrobial activity of a water-soluble chitosan derivative with a fiber-reactive group, Carbohydr. Res. 339 (2) (2004) 313
319. [102] L.Y. Zheng, J.F. Zhu, Study on antimicrobial activity of chitosan with different molecular weights, Carbohydr. Polym. 54 (4) (2003) 527
530. [103] M. Kong, X.G. Chen, K. Xing, H.J. Park, Antimicrobial properties of chitosan and mode of action: a state of the art review, Int. J. Food Microbiol. 144 (1) (2010) 51
63. [104] K.V. Harish-Prashanth, R.N. Tharanathan, Chitin/chitosan: modifications and their unlimited application potential—an overview, Trends Food Sci. Technol. (18) (2007) 117
131. pISSN 0924-2244. [105] S.B. Lin, Y.C. Lin, H.H. Chen, Low molecular weight chitosan prepared with the aid of cellulase, lysozyme and chitinase: characterisation and antibacterial activity, Food Chem. (116) (2009) 47
53. ISSN 0308-8146. [106] Y. Sun, F. Cui, K. Shi, J. Wang, M. Niu, R. Ma, The effect of chitosan molecular weight on the characteristics of spray-dried methotrexate-loaded chitosan microspheres for nasal administration, Drug Dev. Ind. Pharm. (35) (2009) 379
386. ISSN 0363-9045. [107] T.A. Khan, K.K. Peh, Influence of chitosan molecular weight on its physical properties, Int. Med. J. 2 (1) (2003). ISSN 1823-4631. [108] E. Khor, Chitin: fulfilling a biomaterials promise, Biomaterials 23 (18) (2001) 3913
3915. [109] M.K. Jang, B.G. Kong, Y.I. Jeong, C.H. Lee, J.W. Nah, Physicochemical characterization of α-chitin, β-chitin and γ-chitin separated from natural resources, J. Polym. Sci. A1 (42) (2004) 3423
3432. [110] R.A.A. Muzzarelli, M. El-Mehtedi, M. Mattioli-Belmonte, Emerging biomedical applications of nano-chitins and nano-chitosans obtained via advanced eco-friendly technologies from marine resources, Mar. Drugs (12) (2014) 5468
5502. [111] J. Synowiecki, N.A. Al-Khateeb, Production, properties and some new applications of chitin and its derivatives, Crit. Rev. Food Sci. Nutr. (43) (2003). 145-17. [112] I.F.M. Rumengan, E. Suryanto, R. Modaso, S. Wullur, T.E. Tallei, D. Limbong, Structural characteristics of chitin and chitosan isolated from the biomass of cultivated rtifer, Brachionus rotundiformis, Int. J. Fish. Aquat. Sci. 3 (1) (2014) 12
18.

Handbook of Chitin and Chitosan

376

11. Chitin, chitosan, marine to market

[113] C.K.S. Pillai, W. Paul, C.P. Sharma, Chitin and chitosan polymers: chemistry, solubility and fiber formation, Prog. Polym. Sci. (34) (2009) 641
678. [114] A. Costa-Pinto, R. Reis, N.M. Neves, Scaffolds based bone tissue engineering: the role of chitosan, Tissue Eng. Part B 17 (5) (2011) 1
18. [115] M. Rodriguez-vazquez, B. Vega-Ruiz, R. Ramos-Zuniga, D.A. Saldana-Koppel, L.F. Quinones-Olvera, Chitosan and its potential use as a scaffold for tissue engineering in regenerative medicine, BioMed Res. Int. 2015 (2015) 1
15. [116] J. Kumirska, M.X. Weinhold, J. Tho¨ming, P. Stepnowski, Biomedical activity of chitin/chitosan based materials-influence of physicochemical properties apart from molecular weight and degree of N-acetylation, Polymers (3) (2011) 1875
1901. [117] S.M. Hudson, C. Smith, Polysaccharide: chitin and chitosan: chemistry and technology of their use as structural materials, in: D.L. Kaplan (Ed.), Biopolymers from Renewable Resources, Springer-Verlag, New York, 1998, pp. 96
118. [118] G. Berth, H. Dautzenberg, The degree of acetylation of chitosans and its effect on the chain conformation in aqueous solution, Carbohydr. Polym. (47) (2002) 39
51. ISSN 0144-8617. [119] G. Berth, H. Dautzenberg, M.G. Peter, Physico-chemical characterization of chitosans varying in degree of acetylation, Carbohydr. Polym. (36) (1998) 205
216. ISSN 0144-8617. [120] C. Schatz, C. Viton, T. Delair, C. Pichot, A. Domard, Typical physicochemical behaviors of chitosan in aqueous solution, Biomacromolecules (4) (2003) 641
648. ISSN 1525-7797. [121] P. Sorlier, A. Denuziere, C. Viton, A. Domard, Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan, Biomacromolecules (2) (2001) 765
772. ISSN 1525-7797. [122] P. Sorlier, C. Viton, A. Domard, Relation between solution properties and degree of acetylation of chitosan: role of aging, Biomacromolecules (3) (2002) 1336
1342. pISSN 1525-7797. [123] P. Sandford, Chitosan: commercial uses and potential applications, in: E. SkjakBraek, T. Anthonsen, P. Standorf (Eds.), Chitin and Chitosan: Sources Chemistry, Biochemistry, Physical Properties and Applications, Elsevier Applied Science, London, 1989, pp. 51
69. [124] G. Gomez d’Ayala, M. Malinconico, P. Laurienzo, Marine derived polysaccharides for biomedical applications: chemical modification approaches, Molecules 2008 (13) (2008) 2069
2106. [125] N.R. Sudarshan, D.G. Hoover, D. Knorr, Antibacterial action of chitosan, Food Biotechnol. (6) (1992) 257
272. [126] S. Leuba, P. Stossel, Chitosan and other polyamines: antifungal activity and interaction with biological membranes, in: R.A.A. Muzzarelli, C. Jeuniaux, C. Gooday (Eds.), Chitin in Nature and Technology, Plenum Press, New York, 1985, p. 217. [127] I. Younes, S. Sellimi, M. Rinaudo, K. Jellouli, M. Nasri, Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities, Int. J. Food Microbiol. (185) (2014) 57
63. [128] J. Jung, Y. Zhao, Comparison in antioxidant action between α-chitosan and β-chitosan at a wide range of molecular weight and chitosan concentration, Bioorg. Med. Chem. (20) (2012) 2905
2911. [129] E.I. Rabea, M.E.T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Chitosan as antimicrobial agent: applications and mode of action, Biomacromolecules (4) (2003) 1457
1465. [130] M. Anraku, A. Michihara, T. Yasufuku, K. Akasaki, D. Tsuchiya, H. Nishio, et al., The antioxidative and antilipidemic effects of different molecular weight chitosans in metabolic syndrome model rats, Biol. Pharmaceut. Bull. 33 (12) (2010) 1994
1998.

Handbook of Chitin and Chitosan

References

377

[131] S.H. Chang, C.H. Wu, G.J. Tsai, Effects of chitosan molecular weight on its antioxidant and antimutagenic properties, Carbohydr. Polym. (181) (2018) 1026
1032. [132] P.J. Chien, F. Sheu, W.T. Huang, M.S. Su, Effect of molecular weight of chitosans on their antioxidative activities in apple juice, Food Chem. (102) (2007) 1192
1198. [133] W. Xie, P. Xu, Q. Liu, Antioxidant activity of watersoluble chitosan derivatives, Bioorg. Med. Chem. Lett. (11) (2001) 1699
1701. [134] H. Tomida, T. Fujii, N. Furutani, A. Michihara, T. Yasufuku, K. Akasaki, et al., Antioxidant properties of some different molecular weight chitosans, Carbohydr. Res. (344) (2009) 1690
1696. [135] M. Anraku, T. Fujii, Y. Kondo, E. Kojima, T. Hata, N. Tabuchi, et al., Antioxidant properties of high molecular weight dietary chitosan in vitro and in vivo, Carbohydr. Polym. (83) (2011) 501
505. [136] I.M. Helander, E.L. Nurmiaho-Lassila, R. Ahvenainen, J. Rhoades, S. Roller, Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria, Int. J. Food Microbiol. (71) (2001) 235
244. ISSN 0168-1605. [137] C. Allan, L.A. Hadwiger, The fungicidal effect of chitosan on fungi of varying cell wall composition, Exp. Mycol. (3) (1979) 285
287. ISSN 0147-5975. [138] M.C. Chen, G.H.C. Yeh, B.H. Chiang, Antimicrobial and physicochemical properties of methylcellulose and chitosan films containing a preservative, J. Food Process. Preserv. (20) (1996) 379
390. ISSN 01458892. [139] B.O. Jung, C.H. Kim, K.S. Choi, Y.M. Lee, J.J. Kim, Preparation of amphiphilic chitosan and their antimicrobial activities, J. Appl. Polym. Sci. (72) (1999) 1713
1719. ISSN 1097-4628. [140] E.I. Rabea, M.T. El Badawy, T.M. Rogge, C.V. Stevens, M. Ho¨fte, W. Steurbaut, et al., Insecticidal and fungicidal activity of new synthesized chitosan derivatives, Pest Manag. Sci. (61) (2005) 951
960. pISSN 1526-498X. [141] Z. Zhong, R. Chen, R. Xing, X. Chen, S. Liu, Z. Guo, et al., Synthesis and antifungal properties of sulfanilamide derivatives of chitosan, Carbohydr. Res. (342) (2007) 2390
2395. ISSN 0008-6215. [142] I. Ikeda, Y. Tomari, M. Sugano, Interrelated effects of dietary fiber and fat on lymphatic cholesterol and triglyceride absorption in rats, J. Nutr. (119) (1989) 1383
1387. [143] C.M. Gallaher, J. Munion, R. Hesslink, J. Wise, D. Gallaher, Cholesterol reduction by glucomannan and chitosan is mediated by changes in cholesterol absorption and bile acid and fat excretion in rats, J. Nutr. (130) (2000) 2753
2759. [144] C.M. Lian, K.L. Chen, K.C. Chan, Effect of high molecular weight chitosan on nutrient digestibility, blood lipids and fecal contents in rats, in: R.H. Chen, H. Chen (Eds.), Advances in Chitosan Science, Vol. 3, National Taiwan Ocean University, Taiwan, 1999, pp. 299
306. [145] P. He, S.S. Davis, L. Illum, In vitro evaluation of the mucoadhesive properties of chitosan microspheres, Int. J. Pharm. (166) (1998) 75
88. pISSN 0378-5173. [146] J. Mota, N. Yu, S.G. Caridade, G.M. Luz, E.M. Gomes, R.L. Reis, et al., Chitosan/bioactive glass nanoparticle composite membranes for periodontal regeneration, Acta Biomater. (8) (2012) 4173
4180. [147] C. Saikia, P. Gogoi, T.K. Maji, Chitosan: a promising biopolymer in drug delivery applications, J. Mol. Genet. Med. (S4) (2015) 006. Available from: https://doi.org/ 10.4172/1747-0862.S4-006. [148] M.N.V. RaviKumar, R.A.A. Muzzarelli, C. Muzzarelli, H.A. Sashiwa, J. Domb, Chitosan chemistry and pharmaceutical perspectives, Chem. Rev. (104) (2004) 6017
6084. [149] Y. Le, S.C. Anand, A.R. Horrocks, Recent developments in fibres and materials for wound management, Indian J. Fibre Text. Res. (22) (1997) 337.

Handbook of Chitin and Chitosan

378

11. Chitin, chitosan, marine to market

[150] K. Murakami, H. Aoki, S. Nakamura, S. Nakamura, M. Takikawa, M. Hanzawa, et al., Hydrogel blends of chitin/chitosan, fucoidan and alginate as healingimpaired wound dressings, Biomaterials (31) (2010) 83
90. [151] N.L. Yusof, A. Wee, L.Y. Lim, E. Khor, Flexible chitin films as potential wounddressing materials: wound model studies, J. Biomed. Mater. Res. Part A (66A) (2003) 224
232. [152] P. Wongpanit, N. Sanchavanakit, P. Pavasant, P. Supaphol, S. Tokura, R. Rujiravanit, Preparation and characterization of microwave-treated carboxymethylchitin and carboxymethylchitosan films for potential use in wound care application, Macromol. Biosci. 5 (2005) 1001
1012. [153] Y.C. Chung, H.L. Wang, Y.M. Chen, S.L. Li, Effect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens, Biores. Technol. (88) (2003) 179
184. [154] Z. Limam, S. Selmi, S. Sadok, A. El Abed, Extraction and characterization of chitin and chitosan from crustacean by-products: biological and physicochemical properties, Afr. J. Biotechnol. 10 (4) (2011) 640
647. [155] R.G. Cuero, Antimicrobial action of exogenous chitosan, EXS (87) (1999) 315
333. [156] S.L. Wang, J.R. Huang, Microbial reclamation of shellfish wastes for the production of chitinases, Enzyme Microb. Tech. (28) (2001) 376
382. [157] R. Jayakumar, R.L. Reis, J.F. Mano, Phosphorous containing chitosan beads for controlled oral drug delivery, J. Bioact. Compat. Polym. (21) (2006) 327
340. [158] N. Acosta, I. Aranaz, C. Peniche, A. Heras, Tramadol release from a delivery system based on alginate-chitosan microcapsules, Macromol. Biosci (3) (2003) 546
551. [159] C. Peniche, W. Arguelles-Monal, H. Peniche, N. Acosta, Chitosan: an attractive biocompatible polymer for microencapsulation, Macromol. Biosci. (3) (2003) 511
520. [160] L. Illum, N.F. Farraj, S.S. Davis, Chitosan as novel nasal delivery system for peptides drugs, Pharm. Res. (11) (1994) 1186
1189. [161] S. Mao, W. Sun, T. Kissel, Chitosan-based formulations for delivery of DNA and siRNA, Adv. Drug Deliv. Rev. (62) (2010) 12
27. [162] A. Tokoro, N. Tatewaki, K. Suzuki, T. Mikami, S. Suzuki, M. Suzuki, Growth inhibitory effect of hexa-N-acetylchitohexaose and chitohexaos and Meth-A solid tumor, Chem. Pharm. Bull. (Tokyo) (36) (1998) 784
790. [163] J. Murata, I. Saiki, S. Nishimura, N. Nishi, S. Tokura, I. Azuma, Inhibitory effect of chitin heparinoids on the lung metastasis of B16-BL6 melanoma, Jpn. J. Cancer Res. (80) (1986) 866
872. [164] M. Hasegawa, K. Yagi, S. Iwakawa, M. Hirai, Chitosan induces apoptosis via caspase-3 activation in bladder tumor cells, Jpn. J. Cancer Res (92) (2001) 459
466. [165] L. Qi, Z. Xu, M. Chen, In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles, Eur. J. Cancer (43) (2007) 184
193. [166] M. Guminska, J. Ignacak, E. Wojcik, In vitro inhibitory effect of chitosan and its degradation products on energy metabolism in Ehrlich ascites tumour cells (EAT), Polish J. Pharmacol. (48) (1996) 495
501. [167] S.Y. Lin, H.Y. Chan, F.H. Shen, M.H. Chen, Y.J. Wang, C.K. Yu, Chitosan prevents the development of AOM-induced aberrant crypt foci in mice and suppressed the proliferation of AGS cells by inhibiting DNA synthesis, J. Cell Biochem. (100) (2007) 1573
1580. [168] C.R. Dass, P.F. Choong, The use of chitosan formulations in cancer therapy, J. Microencapsul (25) (2008) 275
279. [169] W.R. Chen, R.L. Adams, R. Carubelli, R.E. Nordquist, Laser-photosensitizer assisted immunotherapy: a novel modality for cancer treatment, Cancer Lett. (115) (1997) 25
30. [170] M.L. Markey, L.M. Bowman, V.M. Bergamini, Contact lenses made of chitosan, in: G. Skjak-Braek, T. Anthonsen, P. Sandford (Eds.), Chitin and Chitosan: Source,

Handbook of Chitin and Chitosan

References

[171]

[172] [173] [174]

[175]

[176] [177]

[178] [179]

[180] [181]

[182] [183] [184] [185]

[186]

[187]

[188]

[189]

379

Chemistry, Biochemistry, Physical Properties and Applications, Elsevier Applied Science, London and New York, 1989, pp. 713
717. M. Anand, M. Maruthupandy, S. Prasanna kumar, S. Ravikumar, N. Jeyaraj, R. Kumaran, Antimicrobial potential of chitosan from marine biowastes—a wealth from waste approach, J. Chitin Chitosan Sci. 3 (2) (2015) 1
4. Z.S. Jia, D.F. Shen, W.L. Xu, Synthesis and antibacterial activities of quaternary ammonium salt of chitosan, Carbohydr. Res. 333 (1) (2001) 2001. H. Liu, Y.M. Du, J.H. Yang, H.Y. Zhu, Structural characterization and antimicrobial activity of chitosan/betaine derivative complex, Carbohydr. Poly. (55) (2004) 291
297. J. Dutta, P.K. Dutta, Tissue Engineering: an Emergent Process for Development of Bio-products, Industrial Products Finder, Business Press, Mumbai, India, 2003, pp. 246
253. August 2003. C. Jarry, C. Chaput, A. Chenite, M.A. Renaud, M. Buschmann, J.C. Leroux, Effects of steam sterilization on thermogelling chitosan-based gels, J. Biomed. Mater. Res (58) (2001) 127
132. C.E. Kast, W. Frick, U. Losert, A.B. Schnurch, Chitosanthioglycolic acid conjugate: a new scaffold material for tissue engineering, Int. J. Pharm. (256) (2003) 183
189. Y. Zhang, M. Zhang, Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering, J. Biomed. Mater. Res. (55) (2001) 304
307. S.V. Madihally, H.W.T. Matthew, Porous chitosan scaffolds for tissue engineering, Biomaterials (20) (1999) 1133
1337. M. Prasitslip, R. Jenwithisuk, K. Kongsuwan, N. Damrongchai, P. Watts, Cellular responses to chitosan in vitro: the importance of deacetylation, J. Mater. Sci. Mater. Med. (11) (2000) 773. M. Mucha, Rheological characteristics of semi-dilute chitosan solutions, Macromol. Chem. Phys. (198) (1997) 471
475. H. Jing, W. Su, S. Caracci, T.G. Bunning, T. Cooper, W. Adams, Optical waveguiding and morphology of chitosan thin films, J. Appl. Polym. Sci. (61) (1996) 1163
1169. G. Crini, Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment, Prog. Polym. Sci. 30 (1) (2005) 38
70. H.K. No, S.P. Meyers, Application of chitosan for treatment of wastewaters, Rev. Environ. Contam. Toxicol. (163) (2000) 1
28. E. Guibal, Interactions of metal ions with chitosan-based sorbents: a review, Sep. Purif. Technol. 38 (1) (2004) 43
74. E. Guibal, M. Van-Vooren, B.A. Dempsey, J.A. Roussy, Review of the use of chitosan for the removal of particulate and dissolved contaminants, Sep. Sci. Technol. 41 (11) (2006) 2487
2514. J. Roussy, M. Van-Vooren, B.A. Dempsey, E. Guibal, Influence of chitosan characteristics on the coagulation and the flocculation of bentonite suspensions, Water Res. 39 (14) (2005) 3247
3258. G. Crini, P. Badot, Application of chitosan, a natural aminopolysaccharide, for dye removal from aqueous solutions by adsorption processes using batch studies: a review of recent literature, Prog. Polym. Sci (33) (2008) 399
447. R. Muzzarelli, O. Tuberni, Chitin and chitosan as chromatographic supports and adsorbents for collection of metal ions from organic and aqueous solutions and seawater, Talanta (71) (1969) 1571
1577. J.P. Thome´, Y. Van-Daele, Adsorption of polychlorinated biphenyls (PCB) on chitosan and application to decontaminated of polluted stream waters, in: R. A. A. Muzzarelli, C. Jeuniaux, G. W. Gooday (Eds.), Proceedings of the Third International Conference on Chitin and Chitosan, Senigallia, 1986, pp. 551
554.

Handbook of Chitin and Chitosan

380

11. Chitin, chitosan, marine to market

[190] R. Crisafully, M.A.L. Milhome, R.M. Cavalcante, E.R. Silveira, D. De Keufeleire, R.F. Nascimiento, Removal of some polycyclic aromatic hydrocarbons from petrochemical wastewater using low-cost adsorbents of natural origin, Biores. Technol. (99) (2008) 4515
4519. [191] T.K. Saha, S. Karmaker, H. Ichikawa, Y. Fukumori, Mechanisms and kinetics of trisodium 2-hydroxy-1,1-azonaphthalene-3,4,6-trisulfonate adsorption onto chitosan, J. Colloid. Interf. Sci. (286) (2005) 433
439. [192] Y.C. Chung, Y.H. Li, C.C. Chen, Pollutant removal from aquaculture wastewater using the biopolymer chitosan at different molecular weights, Environ. Sci. Health A (40) (2005) 1775
1790. [193] M. Weltrowski, B. Martel, M. Morcellet, Chitosan N-benzyl sulfonate derivatives as sorbents for removal of metal ions in an acidic medium, J. Appl. Poly. Sci. (59) (1996) 647
654. [194] K.D. Bhavani, P.K. Dutta, Physico-chemical adsorption properties on chitosan for dyehouse effluent, Am. Dyestuff. Rep. (88) (1999) 53
58. [195] T.R. Sridhari, P.K. Dutta, Synthesis and characterization of maleilated chitosan for dye house effluent, Indian J. Chem. Tech. (7) (2000) 198
201. [196] A. El Ghaouth, J. Arul, C. Wilson, N. Benhamou, Biochemical and cytochemical aspects of the interactions of chitosan and Botrytis cinerea in bell pepper fruit, Postharvest Biol. Technol. (12) (1997) 183
194. [197] P. Herna´ndez-Mun˜oz, E. Almenar, M. Ocio, R. Gavara, Effect of calcium dips and chitosan coatings on postharvest life of strawberries (Fragaria x ananassa), Postharvest Biol. Technol. 39 (3) (2006) 247
253. [198] C. Ribeiro, A. Vicente, J. Teixeira, C. Miranda, Optimization of edible coating composition to retard strawberry fruit senescence, Postharvest Biol. Technol. 44 (1) (2007) 63
70. [199] P.J. Chien, F. Sheu, H.R. Lin, Quality assessment of low molecular weight chitosan coating on sliced red pitayas, J. Food Eng. 79 (2) (2007) 736
740. [200] Y. Jeon, J. Kamil, F. Shahidi, Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod, J. Agric. Food Chem. 50 (18) (2002) 5167
5178. [201] G. Tsai, W. Su, H. Chen, C. Pan, Antimicrobial activity of shrimp chitin and chitosan from different treatments and applications of fish preservation, Fish. Sci. (68) (2002) 170
177. [202] S. Kim, H. No, W. Prinyawiwatkul, Effect of molecular weight, type of chitosan, and chitosan solution pH on the shelf-life and quality of coated eggs, J. Food Sci. 72 (1) (2007) S044
S048. [203] L.A. Hadwiger, Multiple effects of chitosan on plant systems: solid science or hype, Plant Sci. 208 (2013) 42
49. [204] J. Liu, J. Zhang, W. Xia, Hypocholesterolaemic effects of different chitosan samples in vitro and in vivo, Food Chem. 107 (1) (2008) 419
425. [205] L. Zeng, C. Qin, W. Wang, W. Chi, W. Li, Absorption and distribution of chitosan in mice after oral administration, Carbohydr. Polym. 71 (3) (2008) 435
440. [206] M. Sumiyoshi, Y. Kimura, Low molecular weight chitosan inhibits obesity induced by feeding a high-fat diet long-term in mice, J. Pharm. Pharmacol. (58) (2006) 201
207. [207] P. Morgantim, Chitin nanofibrils and their derivatives as cosmeceuticals, in: S.K. Kim (Ed.), Chitin, Chitosan, Oligosaccharides and Their Derivatives: Biological Activities and Application, CRC Press, New York, 2010, pp. 531
542. [208] P. Morganti, P. Palombo, M. Palombo, G. Fabrizi, A. Cardillo, F. Svolacchia, et al., A phosphatidylcholine hyaluronic acid chitin
nanofibrils complex for a fast skin remodeling and a rejuvenating look, Clin. Cosmet. Investig. Dermatol. (5) (2012) 213
220.

Handbook of Chitin and Chitosan

Further reading

381

[209] A. Kulkarni, A. Tourrette, M.M.C.G. Warmoeskerken, D. Jocic, Microgel-based surface modifying system for stimuli-responsive functional finishing of cotton, Carbohydr. Polym. 82 (4) (2010) 1306
1314. ISSN 0144-8617. [210] J. Campos, P. Dı´az-Garcı´a, I. Montava, M. Bonet-Aracil, E. Bou-Belda, Chitosan pretreatment for cotton dyeing with black tea, IOP Conf. Series: Mater. Sci. Eng. 254 (2017) 112001. Available from: https://doi.org/10.1088/1757-899X/254/11/112001. [211] A.K. Singla, M. Chawla, Chitosan: some pharmaceutical and biological aspects—an update, J. Pharm. Pharmacol. (53) (2001) 1047
1067. [212] M. Hayes, Chitin, chitosan and their derivatives from marine rest raw materials: potential food and pharmaceutical applications, in: M. Hayes (Ed.), Marine Bioactive Compounds, Springer, Boston, MA, 2012, pp. 115
128. [213] Global Industry Analysts Inc., Chitin and chitosan derivatives market report—2015, ,https://www.alliedmarketresearch.com/chitosan-market.. [214] K. Pulidindi, H. Pandey, Potential, price trends, competitive market share and forecast, 2018
2024, Global Industry Size Report, Chitosan Market Analysis 2018
2024, ,https://www.gminsights.com/industry-analysis/chitosan-market., 2016. [215] Future Market Insights Report, Chitin Market: Global Industry Analysis (2012
2016) and Opportunity Assessment (2017
2027), ,https://www.futuremarketinsights.com/reports/chitin-market.. [216] E. Prasad, Chitosan market by grade (industrial, food, and pharmaceutical), application (water treatment, food and beverages, cosmetics, medical and pharmaceuticals, and agrochemicals), and region (Asia Pacific, North America, Europe, Row)— Global Forecast to 2022, ,https://www.reportsnreports.com/reports/1467340chitosan-market-by-grade-industrial-food-and-pharmaceutical-application-watertreatment-food., 2017.

Further reading [1] M.N. Horst, A.N. Walker, E. Klar, The pathway of crustacean chitin synthesis, in: M. N. Horst, J.A. Freeman (Eds.), The Crustacean Integument: Morphology and Biochemistry, CRC, Boca Raton, FL, 1993, pp. 113
149. [2] M.S. Hossain, A. Iqbal, Production and characterization of chitosan from shrimp waste, J. Bangladesh Agri. Univ. 12 (1) (2014) 153
160. [3] Z. Sheikh, S. Najeeb, Z. Khurshid, V. Verma, H. Rashid, M. Glogauer, Biodegradable materials for bone repair and tissue engineering applications, Materials (8) (2015) 2953
2993. Available from: www.mdpi.com/journal/Materials.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

12 Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications Jafar M. Milani and Mohammad Hossein Naeli Department of Food Science and Technology, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

O U T L I N E 12.1 Introduction

384

12.2 Application of chitin- and chitosan-based oleogels

386

12.3 Preparation method and mechanism of chitin- and chitosan-based oleogels 387 12.4 Chemical and physical modifications of chitin and chitosan 12.4.1 Acylation reactions 12.4.2 NCO-functionalization reaction of chitosan and chitin 12.4.3 Chitin-based polyurethanes synthesis 12.4.4 Mixing of chitin with different types of surfactants

389 390 393 396 396

12.5 Rheological properties of chitin- and chitosan-based oleogels 12.5.1 Influence of chemical modification on rheological properties of the oleogels

397

12.6 Thermogravimetric properties of chitin- and chitosan-based oleogels 12.6.1 Effect of chemical modification on thermogravimetric characteristics of the oleogels

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00012-1

383

399 400 401

© 2020 Elsevier Inc. All rights reserved.

384

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

12.7 Thermal behavior (differential scanning calorimetry thermogram) of chitin- and chitosan-based oleogels 402 12.7.1 Influence of chemical modification on thermal behavior of the oleogels 402 References

404

12.1 Introduction Nowadays, there is a general tendency for the replacement of nonrenewable materials with the natural degradable ones in the production of a wide range of industrial products in order to minimize environmental risks caused by industrial waste products. The important point is that, if the industry successfully achieves biodegradable and environmentfriendly products, their production will grow rapidly. Even consumers are willing to pay a higher price for trying these new products which are completely or partially biodegradable. Based on the statistics, in 2004 and 2005, about 2.4 billion gallons of lubricants were produced worldwide. For lubricants, it is estimated that more than 10 million tons of engine, industrial, and hydraulic oil and lubricating greases are released to the environment every year. The negative effects of this release are considerable on the environment, because releasing even a small amount of these products can prevent the growth of plants and pose a serious threat to aquatic animals [1]. Most of the available lubricants contain petroleum products and their release to the environment is worrisome. Over 60% of lubricants used in the United States are released into the environment [2]. The major role of grease in a rolling bearing is to create a matrix which reduces friction and increases lubrication and the life of equipment by separating two layers. The main advantages of grease compared to oil are as follows: • • • •

Ease of use due to consistency High endurance Inherent sealing action Protection against corrosion and low friction (if the bearing is correctly covered).

In recent years, the achievement of lubricating greases based on 100% degradable compounds has been introduced as a very complex problem. This problem is still not resolved as it is difficult to find natural environment-friendly substitutes with appropriate features which are useful for common thickeners (such as metal soaps, or urea). The major

Handbook of Chitin and Chitosan

12.1 Introduction

385

difference between lubricating greases and liquid lubricants lies in their rheological behaviors which are basically dependent on the type of thickener. The appropriate rheological properties of lubricating greases with a solid or semisolid structure makes them appropriate for the applications, in which liquids cannot perform well. Therefore fluctuations with temperature, loads, vibrations, and ambient conditions can be reasonably tolerated using these products. Other advantages of the matrix of lubricating greases on the surface of metals are the attraction of pollutants such as particles and water without reducing their lubricating properties. The rheological behaviors and functional properties of lubricating greases depend on their compounds and production conditions [3]. Vegetable oil can be used as an environment-friendly lubricant considering properties such as good lubricity, adhering to metal surfaces, weak viscosity
temperature dependence, nontoxicity, and high biological degradability. Nevertheless, these compounds have limitations such as weak oxidation stability, low lubricating properties at low temperature, and a small range of viscosity, which can be decreased using some additives. In fact, the most important factor limiting the use of vegetable oils as lubricants is their liquid form, lack of texture, and poor rheological behavior. Therefore the use of these compounds as lubricants is possible only when appropriate structure and texture are created in them using modification processes. Due to these disadvantages, vegetable oils contribute to only 0.1% of the lubricant market [2,4]. Oleogelation is a novel technique for oil structuring and solidification. It refers to the use of a compound for creating gel in an organic liquid and is a new method actively studied in the past decade [5]. Semisolid systems, called organogels, can be created by adding organogelators to organic solutions. If the organic solution used is vegetable oil, the resulting structured system is called an oleogel. In fact, an oleogel results from trapping organic solutions such as edible oil in a threedimensional matrix which is usually thermally reversible. The mechanism of the formation of organogels’ gel matrix in organic solutions is similar to hydraulic solutions (hydrogel). As a result, hydrogen bonds, van der Waals bonds between long alkyl chains, and electrostatic forces form an integrated structured and, eventually, organogel [6]. Based on general classification, oleogelator compounds are divided into two basic groups: lipid- and biopolymer-based. Lipid-based oleogelators all produce thermoreversible gels and are therefore inappropriate for lubricating applications. On the other hand, biopolymer-based oleogelators often product thermoresistant oleogels [7]. Chitin and chitosan are biopolymer-based oleogelators which have recently received more attention. Chitosan is a chitin derivative produced by its N-deacetylation, often in basic conditions. Nevertheless, this N-deacetylation is usually not performed completely and only when the degree of

Handbook of Chitin and Chitosan

386

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

deacetylation reaches over 50% can chitosan can be obtained. After cellulose, chitosan is the most abundant natural biopolymer in the world. It can be extracted from fungi through basic or acidic treatment. However, the main method for obtaining chitosan is the deacetylation of chitin, basic deacetylation with a strongly basic solution, or thermochemical treatment to remove the acetyl group from chitin [8]. Chitin and chitosan have interesting properties such as an environment-friendly nature and biodegradability, and their degradation products are nontoxic, safe, and noncarcinogenic. This has made them interesting biological compounds [9]. In recent years, chitin- and chitosan-based oleogels in vegetable oils have been introduced as one of the most attractive structured sources. Below, the most important potential and actual applications of these oleogels, preparation methods, modification techniques, and their rheological and thermal behaviors are discussed.

12.2 Application of chitin- and chitosan-based oleogels Among the organogelators, biopolymers have received more attention due to their good commercial access, relatively mass production, lower cost, and higher purity. In general, polymer-based vegetable oil oleogels have diverse food and industrial applications. The most important application for chitin- and chitosan-based oleogels is as a biodegradable alternative to lubricating greases. Another important point is that, although FDA approved chitosan as a food additive in 1993, its use for the production of edible oleogels has not yet been evaluated. Considerable efforts have been made to produce lubricating greases using gellifiers and biopolymers in vegetable oils to obtain a desirable gel-like structure. Generally speaking, rheological and thermal properties similar to those of common metal soap-based greases have been reported. However, some limitations in terms of physical properties (under quiescent conditions) and mechanical properties (under operational conditions) have been demonstrated. Vegetable oils with high oleic content are good candidates for substituting common mineral oiland synthetic ester-based lubricants. In addition to being biodegradable and nontoxic, vegetable oils have other advantages such as very low volatility due to the high molecular weight of triglyceride, good capability of adhering to metal surfaces using their polar ester groups, and desirable lubricating capability. Moreover, vegetable oils have a high solubility for polar pollutants [2]. Lubrication greases are usually structured suspensions containing a thickener compound, often made of lithium, calcium, sodium, aluminum, or barium fatty-acid soaps in mineral or synthetic oils. Moreover, some additives are usually added to improve the properties of lubricating

Handbook of Chitin and Chitosan

12.3 Preparation method and mechanism of chitin- and chitosan-based oleogels

387

greases. Studies on the structure of lubricating greases using electron and atomic force microscopy show that these thickeners form a complex 3D matrix which traps oil and leads to the special rheological properties of grease. The main difference between lubricating greases and other lubricants is their rheological properties. In lubricant industries, it is the special consistency of the grease that makes it more efficient than liquids in most applications. In addition, this thickener is added to prevent the decrease in lubricant in the operational conditions, but it should not cause a significant increase in resistance to the movement of layers. In the past, the lubricating performance of grease was attributed to its oil phase. However, based on microscopic techniques, there are small soap particles on contact surfaces which demonstrate the more important role of thickener in the lubricating process. There are higher expectations of lubricating greases in operation conditions, the most important of which include: • resistance against high and low temperatures (that is, in applications near furnaces and freezers) • resistance to high pressures • tolerance of undesirable environments with high humidity, powder materials, or oxidative materials The first step for producing environment-friendly greases is to replace vegetable oils with mineral oil, which is the main component of lubricant formulation (with 70%
95% share in the formulation). Nevertheless, a grease is completely biodegradable only when the thickener agents are biodegradable in addition to the oil phase. Chitin- and chitosan-based oleogels are the most successful green lubricating greases [10]. For the first time, Sa´nchez et al. [11,12] attempted to use chitin, chitosan, and their acylated derivatives as thickener agents in vegetable oils (castor and soybean oil). Based on their results, the use of acylated chitosan expresses rheological properties very similar to common lubricating greases. In total, chitosan is more efficient than chitin. Nevertheless, chitin- and chitosan-based oleogels have higher thermal stability than common chitosan-based formulations. Also, in general, most of the chitin- and chitosan-based oleogels express very poor mechanical stability in rolling elements. Further efforts have been made by Chen et al. [13], Gallego et al. [14], and Sa´nchez et al. [1] for the use of chitin- and chitosan-based oleogels as biodegradable lubricating greases [1,13,14].

12.3 Preparation method and mechanism of chitin- and chitosan-based oleogels Generally speaking, the physical properties and appearance of oils and fats directly depend on the type of fatty acids and their distribution in

Handbook of Chitin and Chitosan

388

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

triglyceride molecules. A high level of saturation produces a substance with high melting temperature and a solid-like behavior at room temperature; this substance is usually referred to as fat. The nonsaturated nature produces a substance with a lower melting point which expresses a liquid-like behavior at room temperature; this substance is known as an oil [15]. In the process of fat structuring, first, triglycerides which have a higher melting point begin the aggregation and creation of crystal structures at the molecule scale. Then, upon further aggregation of triglycerides at the nanoscale, layered structures called lamella (crystal nanotubes) are formed. As these structures aggregate, crystal clusters are produced, and with their aggregation, flocs of fat at the microlevel are formed. Eventually, upon the aggregation of fat flocs, the fat mass structure is produced which is trapped in the voids between flocs of fat with a low melting point (liquid), resulting in solid-like gels [16]. There are also other methods in addition to the common method, that is, increasing the saturation to form a structure in the lipid system. New methods for structuring in lipid systems are as follows: creating structured emulsions, interesterification, and oleogelogation [17]. In contrast to fats, in oleogelogation the liquid phase is trapped in a matrix not made of triglyceride. Supermolecular assembly is the main mechanism for structuring organic liquids, similar to the mechanism of the effect of lost lipid-structure oleogels. However, oleogels can also be produced using macromolecules (like polymer gels). Biopolymer-based oleogels are made of polymers which create a 3D matrix through physical forces, for example, hydrogen bonds, van der Waals bonds, and electrostatic interactions, thus trapping organic solvents. Only a few studies have been focused on the behavior of polymer oleogels. These studies show that the semisolid nature is usually created by polymer
polymer and polymer
solvent interactions through hydrogen bonds and electrostatic interactions [18]. Hydrophobic polymer organogelators can gellify liquid oil through a direct route (dissolving in oil), while hydrophilic and semihydrophilic polymers can perform this by an indirect method. First, they produce an emulsion and, then, eliminate water. This method is called an emulsiontemplated method, which was first developed by Romoscanu and Mezzanga [19] using proteins [19,20]. To produce hydrophobic polymerbased oleogels, initially, we need to dissolve polymers in the solvent, which occurs only at a temperature higher than the glass transition. At the glass transition temperature, molecules receive the energy necessary for distancing from one another and polymers become viscous, elastic, and flexible. In this step, the polymer connects to the solvent, such that after cooling, the gel system is created as the result of the solvent being trapped in the polymer’s 3D matrix. The polymer
polymer, polymer
solvent, and solvent
solvent interactions are mainly responsible for the production of oil gel. Studies show that hydrogen bonds between chitosan fibers belong to polymer
polymer interactions with a slight effect on polymer
solvent

Handbook of Chitin and Chitosan

12.4 Chemical and physical modifications of chitin and chitosan

389

interactions. Solvent
solvent interactions are bipolar/bipolar or van der Waals. Results show that the functional group of glycerol has a slight effect in creating electrostatic interactions and forming oleogels. However, acyl chains of triglycerides can highly affect the gel formation process through electrostatic bonds. The level of effect of acyl chains in triglycerides depends on the molar volume of the solvent. The higher the content of nonsaturated fatty acid, the greater their effectiveness in increasing the separation of polymer fibers and reducing the connection points in the polymer matrix due to their larger volume. In these conditions, the solvent interacts more with polymers [21]. Chitin and chitosan belong to hydrophobic oleogelators. Chitosan oleogel is produced by directly dispersing polymer in oil and heating (80 C
100 C) and then cooling. The concentration of chitosan for creating oleogels is rather high (about 20%). This is one of the reasons for reducing its application in the food industry [22]. Fig. 12.1A shows the chemical structure of chitin, which includes acetamido-2-deoxy-D-glucopyranose-2 units (Fig. 12.1B). Chitin is, in fact, similar to ethylcellulose, with the difference that, in chitin, the acetamido group is in position C-2, instead of a hydroxyl or ethyl ester group. Hydroxyl groups (OH) of polymers generally create hydrogen bonds, keeping polymers together, and the carboxylic acid groups in oil can probably establish an electrostatic bond with the hydroxyl groups in the polymer. Chitin is a highly crystal substance with strong hydrogen bonds and is dissolved in hydraulic systems with difficulty. The deacetylated form of chitin (i.e., chitosan; Fig. 12.2) has received more attention for producing oleogel due to its better biological and physiochemical properties [18].

12.4 Chemical and physical modifications of chitin and chitosan Similar to what is observed in the case of cellulose, chitin and chitosan have low processing capability and productivity without chemical modifications. Nevertheless, chemical changes in chitosan, especially N-alkylation, N-acylation, O-acylation, and N-carboxyalkylation, improve its solubility in organic solvents. These modifications can increase the range of application of these biopolymers [1]. Among the various techniques for the modification of chitin and chitosan, acylation is the most efficient. Below, the most important physical and chemical modification processes of chitin and chitosan are mentioned. Sa´nchez et al. observed that acylated chitosans have better rheological and tribological properties than common forms. In fact, the acylation of chitosan is an interesting chemical tool for reducing the polarity of chitosan and increasing its solvency [12].

Handbook of Chitin and Chitosan

390

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

OH O HO

NHAc

HO O

NHAc

OH O

O HO

HO O

NHAc

OH

NHAc

OH

O HO OH

O

HO O

NHAc

NHAc O OH

Chemical structure of chitin OH O O

HO HN

C

CH3

O

FIGURE 12.1 Chemical structure of chitin and its building units [18].

FIGURE 12.2 Chemical structure of chitin and chitosan [23].

12.4.1 Acylation reactions Despite all its positive features, due to its complex crystal structure, chitosan is not easily solved in hydraulic (at pH 5 7) and organic media. Still, under some controlled chemical reactions and by replacing some amino groups with functional groups, the solubility of chitosan can be improved. So far, extensive effort has been made to improve its solubility in hydraulic media. For instance, various forms of water-soluble chitosan derivatives have been produced using heterocyclic aldehydes [24,25], alkylation [26,27], quaternary [28,29], and carboxyl methyl [30] reactions. Acylation is the most efficient technique for improving the solubility of chitosan in organic solvents. In this technique, by introducing acyl groups into the chemical structure of chitosan, its nonpolarity is increased, thereby increasing its solubility in organic media. The degree

Handbook of Chitin and Chitosan

12.4 Chemical and physical modifications of chitin and chitosan

391

of substitution of acyl groups in chitosan can be controlled by adjusting the moral ratio of agents containing acyl groups to the reactive group of chitosan (
NH2,
OH). The first attempts for increasing chitosan’s solubility in organic solvents were made by [31] through acylation of chitosan by hexanonyl, decanoyl, and lauroyl chlorides. They used elemental analyses, including IR, H-NMR, C-NMR, and GPC, to examine the chemical structures of these polymers. Results revealed the degree of substitution of four per monosaccharide cycle. These acylated chitosans had excellent solubility in organic solutions such as chloroform, benzene, pyredence, and THF, and produced transparent solutions. Replacing hexanoyl, decanoyl, and lauroyl respectively creates H-, D-, and L-chitosans. Products of the acylation of chitosan in a medium containing pyridine/ chloroform are depicted in Fig. 12.3. In another study Ma et al. [32] synthesized organic-soluble acylated chitosan from the reaction of chitin with stearoyl chloride. Based on their results, using 1H NMR, the products had a degree of substitution of 1.8
2.8. Acylated chitosan in this study had excellent solubility in an organic solvent, including acetone, pyridine, benzene, and dichloromethane at 25 C. Products of the acylation of chitosan in a medium containing triethylamine/acetone are depicted in Fig. 12.4. 12.4.1.1 N-acylation The N-acylation of chitosan is actually a specific acylation in NH2 groups. In this reaction, acyl groups replace the NH2 groups of chitosan. N-acylation of chitosan with various fatty acid chlorides (C6
C16) was first performed in a hydraulic medium by Le Tien et al. [33]. They examined the potential of using acylated products for hydrophobic matrices for controlled drug release. Their method included the following six steps:

FIGURE 12.3 Synthetic production of acylated chitosan in a medium containing pyridine/chloroform [31].

Handbook of Chitin and Chitosan

392

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

6

64

HOH2C

HOH2C

4

O 5

3

O

44

2

5

O NH2

14 34

1

Tricthylamine/acctone

NH

IIO

HO

CI

R

24

4

O

x

1-x CH3

6

64

ROCOH2C

ROCOH2C

4

O 5 3

RCOO

O

44

2

24

54

O

14 34

1

N(COR)2

RCOO x

NCOR O CH3

1-x

R=(CH2)16CH3

FIGURE 12.4 Synthetic production of acylated chitosan in a medium containing triethylamine/acetone [32].

1. Preparing a mixture of chitosan in acetic acid hydraulic solution and stirring for 24 h to ensure the complete solution of chitosan 2. Increasing mixture pH by adding NaOH and vigorous stirring 3. Adding acyl chloride to the mixture and continuing stirring for 6 h (during which the pH is gradually decreased) 4. Neutralizing the interaction mixture (pH 5 7) and sedimentation using acetone 5. Collecting the sediments using filtration and washing them with methanol 6. Drying the products using acetone until achieving a homogeneous powder Based on their findings, the best mechanical properties and drug release properties belonged to palmitoyl chitosan (substitution degree of 40%
50%). Sa´nchez et al. also attempted to N-acylate chitosan by decanoyl, lauroyl, palmitoyl, and stearoyl chlorides in a hydraulic medium. They reported that oleogels based on chitosan N-acylated with decanoyl chloride expressed a rheological behavior similar to traditional metallic soap-based lubricating greases. A schematic view of the N-acylation of chitosan is presented in Fig. 12.5. 12.4.1.2 N- and O-acylation In N- and O-acylation of chitosan, both NH2 and OH groups are randomly acylated. During this reaction, the polarity of chitosan is reduced

Handbook of Chitin and Chitosan

12.4 Chemical and physical modifications of chitin and chitosan

FIGURE 12.5

393

A schematic view of N-acylation of chitosan [1].

much more than in N-acylation, thereby increasing its solubility in organic solvents. This reaction was first designed by [32] and included the following five steps: 1. Soaking and solving chitosan in the triethylamine solution and acetone for 6 h 2. Adding acyl chloride solved in acetone to the mixture and heating to 90 C for 6 h 3. Filtering the reaction mixture and separating acetone sediments 4. Washing the sediments with methanol 5. Drying sediments in the oven at 40 C According to Sa´nchez et al., oleogels based on chitosan N- and O-acylated with palmitoyl chloride express a rheological behavior similar to traditional metallic soap-based lubricating greases. A schematic view of the N- and O-acylation of chitosan is presented in Fig. 12.6.

12.4.2 NCO-functionalization reaction of chitosan and chitin Both chitin and chitosan have two types of the reactive group which can establish hydrogenized bonds and keep polymer fibers together: free amine groups in deacylated units and hydroxyl groups on C3 and C6 carbons in acylated or deacylated units. These reactive sites enable the connection of many functional reactive molecules [34]. One of these molecules can bond with 1,6-hexamethylenediisocyanate (HMDI). HMDI is commercially available and is generally used as a strong cross-linker for
OH or
NH2 groups, because it has two reactive isocynanate groups (
N 5 C 5 O).
OH groups of chitin and chitosan can react with
NCO groups to create a urethane linkage (
NH-C(O)-O
) due to the transfer of a proton from
OH groups to nitrogen atoms of
NCO.

Handbook of Chitin and Chitosan

394

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

FIGURE 12.6 A schematic view of N- and O-acylation of chitosan [1].

HMDI can also react with deacylated amino groups in biopolymers. The products of the reaction between chitosan and HMDI were previously used in various applications, such as molecular carriers in biomedical and environmental studies [35], as a support material for Pd catalysts used in hydrogen reactions [36], as a solid support for Cu and Pd catalysts in attracting cholesterol [37], and attracting gold [38]. Gallego et al. [14] incorporated reactive isocyanate groups into chitosan and chitin through HMDI reaction with chitosan and chitin. They used NCO-functionalized chitosan and chitin to produce oleogel in castor oil. The components of isocyanate were introduced to improve the role of biopolymers as reductive agents in castor oil. The level of
NCO groups in the final polymer is the criterion for determining its potential as a proper thickener. Therefore they chose two laboratory settings for preparing modified chitosan polymers: 1. In the first condition, the amount of
NCO was half the amount needed for reaction with all deacylated
OH and
NH groups in chitosan. In other words, the molar ratio of
NCO to deacylated
OH and
NH groups in chitosan was 0.5/1 equiv. 2. In the second condition, the amount of
NCO was one-fourth of the amount needed for reaction with all deacylated
OH and
NH groups in chitosan. In other words, the molar ratio of
NCO to chitosan was 0.25/1 equiv. Moreover, this condition was the only choice for modifying chitin (Fig. 12.7). The use of two
NCO/biopolymer ratios allowed for controlling the process of functionalization and obtaining polymers with different numbers of free
OH and
NCO groups. Fig. 12.8 presents a schematic view of the NCO-functionalization reaction of chitin and chitosan and reaction with ricinoleic fatty acid during the preparation of oleogel [14].

Handbook of Chitin and Chitosan

12.4 Chemical and physical modifications of chitin and chitosan

395

FIGURE 12.7 The reaction of chitosan and chitin with hexamethylene diisocyanate (HMDI) [14].

FIGURE 12.8 NCO-functionalization reaction of chitin and chitosan and reaction with ricinoleic fatty acid during the preparation of oleogel [14].

Studies show that NCO-functionalized chitosan- and chitin-based oleogels express a rheological behavior similar to that of common lubricating greases. These oleogels were stable physically and did not become two-phased in the storage period. They also showed further thermal stability than common lubricating greases [14,39].

Handbook of Chitin and Chitosan

396

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

FIGURE 12.9 Schematic representation of TRCPUs composed of IPDI, PEG, and chitin [13].

12.4.3 Chitin-based polyurethanes synthesis Chitin-based polyurethane (TRCPU) synthesis allows the production of chitins which can produce temperature-dependent sol-to-sol oleogels. Chen et al. [13] synthesized chitin-based polyurethane copolymer by introducing isophorone diisocyanate and polyethylene glycol (PEG) into the chitin structure. Soft segments of the prepolymer originating from PEG give elastomeric and hydrophilic properties to TRCPU. The hard segments of chitin and IPDI, which contain highly polar urethane linkages, offer lipophilic properties to TRCPU in the polar organic solvent. A schematic view of TRCPU synthesis is illustrated in Fig. 12.9.

12.4.4 Mixing of chitin with different types of surfactants Chitin and its derivatives have little ability to produce strong and stable oleogels on their own. Due to the onset of chitin aggregation, oleogels based on them become two phases only several days after production. In fact, sedimentation of chitin in vegetable oils prevents the formation of a

Handbook of Chitin and Chitosan

12.5 Rheological properties of chitin- and chitosan-based oleogels

397

stable gel matrix due to aggregation. Although weak interactions, for example, bipolar
bipolar interactions and hydrogen bonds, may exist in chitin, these interactions are not strong enough to create a strong stable system. Therefore complementary additives must be used to compensate for this problem. Nikiforidis et al. examined the effect of three types of surfactant, phosphatidylcholine, enzymatically modified phosphatidylcholine, and sorbitan monostearate, on the dispersion of raw chitin and chitin nanocrystals and the resulting oleogel. Surfactants have an amphiphilic nature and their hydrophilic sites can connect to the chitin surface, while they extend their hydrophobic sites in the continuous phase of oil. In this way, the dispersion of chitin can be improved via hydrophilic chitin
chitin interactions and aggregation can be minimized. In addition, surfactants act as a plasticizer. Plasticizers reduce the friction between polymers and their rigidity, and therefore change mechanical properties in oleogel systems as well. Phosphatidylcholine unmodified at pH 5 7 is electrically neutral, but its enzymatic modified derivative has a negative load due to its phosphate group. In addition to the difference in electric load, due to the removal of hydrophilic choline group, the hydrophobicity of the modified derivative was higher than that of the nonmodified one. Therefore unmodified phosphatidylcholine has further potential for establishing hydrophobic links with the solvent in addition to being able to have hydrophilic interactions with chitin, and thus is a more effective plasticizer. Results of studies on their microstructures by SEM showed that the addition of surfactant allows for the observation of long chitin fibers in SEM images. Accordingly, they hypothesized that the addition of surfactants helps the dispersibility of polysaccharides. When phosphatidylcholine and chitin in combination are added to a hydrophobic solvent (triglyceride), first, hydrophilic reactions are performed. It is possible that further strong hydrogen bonds are formed and relatively dipole
dipole reactions are gradually performed. Based on these interactions, phosphatidylcholine molecules are probably inhibited on chitin fibers and their fatty acid chains create a lipophilic surface which infiltrates the hydrophobic solvent.

12.5 Rheological properties of chitin- and chitosan-based oleogels Small deformation dynamic oscillatory testing offers valuable data on the structure of crystal matrix in fats and gel structure in oleogels. The main rheological parameters of a viscoelastic substance measured in these tests include: • Storage modulus (G’), which is a solid-like behavior index • Loss modulus, which presents an estimate of fluid-like behavior

Handbook of Chitin and Chitosan

398

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

These first step in these tests is to perform a strain sweep test to determine the linear viscoelastic region (LVE). In this region, G’ and G’’ are constant, and deformation in the structure is reversible (small deformation). Examination of the viscoelastic behavior using a frequency sweep test and temperature sweep test is reliable only when it is performed in LVE. In general, vegetable oils have a Newtonian rheological behavior, meaning that their viscosity is only a function of temperature and does not depend on the shear rate. However, fats express a weak viscoelastic gel-like rheological behavior. By increasing temperature and, consequently, decreasing solid fat content (SFC), we observe a decrease in the viscoelastic character of fats, such that at the melting point, this substance has a Newtonian behavior [40]. Oleogelation leads to a shift in the Newtonian behavior of oil to viscoelastic behavior by creating a structure in oil. Examination of the rheological behavior of chitin- and chitosan-based oleogels by oscillatory shear measurements show their solid-like gel nature. Generally speaking, substances with such a character have higher G’ than G’’ almost all throughout the research period. When examining the rheological behavior of these substances, we find a term called the plateau region. In this region, G’ is always more than G’’ in the studied frequency range. G’ is increased slightly with frequency, while G’’ shows a specific minimum. Such a gel-like property is due to hydrogen bonds between molecules, dipole
dipole reactions (between hydroxyl and amine groups) in chitin and chitosan chains, and polar groups of triglyceride molecules (especially the carbonyl group). In fact, these interactions trap oil in the structure of 3D gel matrix and lead to the long-term physical stability of these oleogels [1,12,14,39]. Elasticity (G’) of these gels is increased by increasing the chitin and chitosan ratio. On the other hand, the lower the molecular weight of chitosan, the higher the concentration of biopolymer required for producing a stable dispersion, preventing phase separation, and producing a homogeneous appearance. A minimum of 35% of low-molecularweight chitosan is necessary for obtaining an oleogel with appropriate physical stability. Moreover, the type of oil used affects the level of elasticity. For instance, oleogels containing castor oil have much higher G’ than those containing soybean oil. This is because of the presence of hydroxyl groups in ricinoleic acid in castor oil, which increases the polar sites and, as a result, hydrogen connections between the polymer and fatty acid [12]. Regarding the effect of the type of fatty acid on the rheological behavior of these oleogels, the strange point is that, contrary to expectations, saturated fatty acids create poorer gels than nonsaturated fatty acids. This is because fatty acids, which are linear, lead to less separation in polymer chains than the nonsaturated types, thereby having

Handbook of Chitin and Chitosan

12.5 Rheological properties of chitin- and chitosan-based oleogels

399

difficulty in infiltrating the gel matrix and being trapped. In addition, another reason may be less polarity of saturated fatty acids, causing a weaker connection between them and polymers [21]. Chitosan-based oleogels are much more elastic than the types produced based on chitin (even up to 10 times more). This is due to the higher degree of deacetylation of chitosan which leads to further hydrogen bonds and a stronger oleogel [12].

12.5.1 Influence of chemical modification on rheological properties of the oleogels Sanchez et al. reported that acylation decreases both modules of G’ and G’’ in chitin- and chitosan-based oleogels. The reduction in these modules means a reduction in the complex modulus (G*) which is an index for total hardness. The less the total hardness in a substance, the poorer its viscoelastic behavior would be. In fact, it seems that acylations weaken the gel matrix. They also reported that the higher the degree of deacylation, the more severe the reduction in these rheological parameters would be. Still, it seems that the most important advantage of using acylated chitosan is increasing its solubility in oil which reduces polarity, and thus produces a more stable and homogeneous oleogel over time. These researchers have reported that oleogels containing chitosan lose their gel-like behavior at moderate temperature and become highly liquid substances at 50 C. This causes a serious limitation to the use of their products for use in lubricating greases at high temperatures. However, based on their results, the rheological behavior of oleogels containing common chitin and chitosan is less affected by temperature (5 C
130 C). This group of oleogels is highly affected by temperature up to 100 C, but at higher temperatures, a more severe reduction in G’ and G’’ is observed which may be due to oil separation. Nevertheless, contrary to previous studies, Sanchez et al. synthesized N-acylated chitosan and N- and O-acylated chitosan with higher mechanical stability and proper thermal stability. During the frequency sweep test, the values of linear viscoelastic functions based on castor oil and chitosan N-acylated with decanoyl chloride or chitosan N- and O-acylated with palmitoyl chloride oleogels were quite similar to those of commercial lubricating greases. They reported that, by reducing the length of the carbon chain in the acyl group, both G’ and G’’ modules were significantly reduced. Based on the results reported by Gallego et al. [39], the higher the degree of isocyanate-functionalization in chitin and chitosan, the higher the modulus of G’ and G’’ would become (i.e., a stronger oleogel is produced). This has been attributed to further cross-links in these oleogels.

Handbook of Chitin and Chitosan

400

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

Nikiforidis et al. reported that adding surfactants such as phosphatidylcholine, enzymatically modified phosphatidylcholine, and sorbitan monostearate to chitin and chitosan reduced aggregation, thereby forming stable oleogels (with a G’ module between 102 and 106 Pa). Temperature sensitivity depends on the type of surfactant used. Oleogels containing phosphatidylcholine and sorbitan monostearate showed structural changes over heating, while those with modified phosphatidylcholine showed no change in G’ up to 90 C.

12.6 Thermogravimetric properties of chitin- and chitosanbased oleogels Thermogravimetric analysis (TGA) is a thermal analysis method, in which the sample mass is measured over time and with temperature variation. In this analysis, valuable data on physical phenomena, phase transitions, absorption, adsorption, and desorption are offered. Moreover, chemical phenomena such as chemisorptions, thermal decomposition, and solid
gas reactions can also be studied. Data extracted from TGA can be presented in a diagram with mass or the percentage of primary mass on the y-axis and temperature or time on the x-axis. The TGA technique can be used to study the properties of substances through the analysis of characteristic decomposition pattern. This is a very useful technique for studying polymeric materials, including thermoplastics, thermosets, elastomers, composites, plastic films, fibers, coatings, and paints [41]. TGA thermograms show that the thermal decomposition of oleogels based on chitin, chitosan, or their derivatives occurs in two main steps. The first is the degradation of polymers, while the second, which is considerable, is the thermal degradation of the oil phase (castor oil). In the case of chitin-based oleogel, thermal decomposition of biopolymers and castor oil occurs at similar temperatures; as a result, only one main thermal event is clearly identified, that is, one major peak in the weight loss versus temperature derivative function (Fig. 12.10). The first temperature, at which the sample starts losing weight, is called Tonset and the temperature, at which maximum weight loss occurs, is called Tmax. Studies show that chitin-based oleogels have the highest values of Tonset and Tmax among those containing chitosan or acylated derivatives and can thus be considered as the most stable chemical formula [12]. Based on the findings of [14], thermal degradation of nonfunctionalized chitosan and chitin in a nitrogen atmosphere occurs in one general step. For chitin, this thermal event occurs in the 343 C
405 C range due to the degradation of saccharides molecule, including dehydration of saccharide cycles and degradation of both acylated and deacylated

Handbook of Chitin and Chitosan

12.6 Thermogravimetric properties of chitin- and chitosan-based oleogels

401

FIGURE 12.10 TGA thermograms for (A) selected biopolymers and (B) some of their corresponding oleogel formulations. Weight loss versus temperature plot and its derivative function. A2MMC, acylated medium-molecular-weight chitosan; A2MMC-35, oleogel based on 35% acylated medium-molecular-weight chitosan; CC, chitin; CC-21, oleogel based on 21% chitin; MMC, medium-molecular-weight chitosan; MMC-25, oleogel based on 25% medium-molecular-weight chitosan [12].

chitin units. Based on their results, the decomposition step of chitosan occurs at a lower temperature, showing its lower thermal stability.

12.6.1 Effect of chemical modification on thermogravimetric characteristics of the oleogels Sanchez et al. [12] reported that the acylated derivatives of chitosan had a lower Tonset and Tmax than the common form. However, they offered no reason for this. Similar results were reported [32] for chitosan and its acylated derivative. Sanchez at al. [1] observed that the smaller the length of the acyl chain in N-acylated chitosans, the lower the initial weight loss (Tmax) would become. Moreover, the longer the acyl chain, the higher the thermal stability would be. Oleogels based on N- and

Handbook of Chitin and Chitosan

402

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

O-acylated chitosans have higher thermal stability than those prepared based on N-acylated chitosans. The thermal decomposition of oleogels based on N-acylated chitosan and N- and O-acylated chitosan occurs in three and two general steps, respectively. In all cases, the preliminary reduction in mass in tomographs demonstrates the thermal degradation of biopolymer due to losing acyl groups. In addition, the main reduction in weight is because of the evaporation of the oil phase which overlaps the destruction of nonacylated groups, that is, they occur simultaneously. In the case of functionalized chitin and chitosan, the inclusion of HMDI components to the polymer structure reduces their thermal stability. Increasing the
NCO content leads to a more severe reduction in oleogels’ thermal stability [14].

12.7 Thermal behavior (differential scanning calorimetry thermogram) of chitin- and chitosan-based oleogels Differential scanning calorimetry (DSC) measures the differences in heat flow into a substance and a reference as a function of sample temperature while both are subjected to a controlled temperature program. This method provides access to accurate thermodynamic data as well as information regarding reactivity and phase transformations. According to the classification, calorimetry is a technique for determining the quantity of heat that is either absorbed or released by a substance undergoing a physical or a chemical change. Such a change alters the internal energy of the substance. At constant pressure, the internal energy is known as enthalpy. Data extracted from DSC measurement can be presented in a thermogram with heat flux on the y-axis and temperature or time on the x-axis. There are two different conventions: • Endothermic reactions in the sample shown with a negative peak, such as heat capacity (heating), glass transition, melting, and evaporation • Exothermic reactions in the sample shown with a positive peak, such as heat capacity (cooling), crystallization, oxidation, and curing of polymers [41]

12.7.1 Influence of chemical modification on thermal behavior of the oleogels According to Sanchez et al. [12], samples of medium-molecularweight chitosan and low-molecular-weight chitosan do not show any melting or glass transition event in the temperature range from 280 C

Handbook of Chitin and Chitosan

12.7 Thermal behavior (differential scanning calorimetry thermogram)

403

to 200 C. In this sense, the rheological properties of the final products thickened by these biopolymers should not dramatically change with temperature. On the other hand, the acylated biopolymers’ heat flow curves display two main peaks, which can be related to acylated chitosan structures with different degrees of substitution and, consequently, two different melting temperatures (Fig. 12.11). They reported that the melting temperatures are the same for all acylated chitosan samples with different degrees of substitution, except for the sample of acylated medium-molecular-weight chitosan (with substitution degree of 0.3) with a lower degree of substitution, which displays a first melting peak slightly shifted to a higher temperature (Fig. 12.11). According to their result, the fusion enthalpy (ΔHf) corresponding to the second peak clearly decreases with chitosan molecular weight and increases with the substitution degree. This fact supports the X-ray diffraction results previously reported [32], which suggest that the crystallinity of acylated chitosan increases with the number of long aliphatic chains introduced. Similar results were reported by Sanchez et al. [10]. They also saw three main peaks in the DSC thermogram of the acylated chitosan, and thus three different melting temperatures (213.5 C, 12.8 C, and 182.9 C).

FIGURE 12.11

DSC heat flow curves for medium-molecular-weight chitosan (MMC), low-molecular-weight chitosan (LMC), acylated medium-molecular-weight chitosan (A2MMC, with substitution degree of 0.3), and acylated low-molecular-weight chitosan (A3LMC, with substitution degree of 1.0) samples [12].

Handbook of Chitin and Chitosan

404

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

FIGURE 12.12 DSC thermogram of TRCPU 1.0 in 5 wt.% lithium chloride/N, N-dimethylacetamide between 250 C and 150 C [13].

The main peak at around 183 C corresponds to the chitosan acylated to a relatively low degree. Chen et al. [13] studded the DSC thermogram of TRCPU in the range of 250 C to 160 C (Fig. 12.12). The DSC thermograms can show the curve behavior of the sol-to-gel transition above the phase transformation temperature but prior to degradation. Their thermograms showed a sharp endothermic peak between 82 C and 88 C. They believed that the peak was likely due to the rearrangement of chitin segments as the hydrogen bonds between the hydroxyl groups of TRCPU and carbonyl, and tertiary amine groups of N, N-dimethylacetamide solvent began to break. The second endothermic peak, which appeared at approximately 105 C as a second upshift in the curve, was the result of heat absorption during the gelling process of the partially soluble phase.

References [1] R. Sa´nchez, G. Alonso, C. Valencia, J. Franco, Rheological and TGA study of acylated chitosan gel-like dispersions in castor oil: influence of acyl substituent and acylation protocol, Chem. Eng. Res. Des. 100 (2015) 170
178. [2] S.Z. Erhan, B.K. Sharma, J.M. Perez, Oxidation and low-temperature stability of vegetable oil-based lubricants, Ind. Crop. Prod. 24 (2006) 292
299. [3] L. Hamnelid, Introduction to rheology of lubricating greases, Rheology Lubricating Greases, ELGI, Amsterdam, 2000, pp. 1
20. [4] L. Quinchia, M. Delgado, J. Franco, H. Spikes, C. Gallegos, Low-temperature flow behavior of vegetable oil-based lubricants, Ind. Crop. Prod. 37 (2012) 383
388. [5] A.K. Zetzl, A.G. Marangoni, S. Barbut, Mechanical properties of ethylcellulose oleogels and their potential for saturated fat reduction in frankfurters, Food Funct. 3 (2012) 327
337.

Handbook of Chitin and Chitosan

References

405

[6] N.E. Hughes, A.G. Marangoni, A.J. Wright, M.A. Rogers, J.W. Rush, Potential food applications of edible oil organogels, Trends Food Sci. Technol. 20 (2009) 470
480. [7] L.S.K. Dassanayake, D.R. Kodali, S. Ueno, Formation of oleogels based on edible lipid materials, Curr. Opin. Colloid Interface Sci. 16 (2011) 432
439. [8] E.S. Abdou, K.S. Nagy, M.Z. Elsabee, Extraction and characterization of chitin and chitosan from local sources, Bioresour. Technol. 99 (2008) 1359
1367. [9] M.R. Kumar, R.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. Domb, Chitosan chemistry and pharmaceutical perspectives, Chem. Rev. 104 (2004) 6017
6084. [10] R. Sa´nchez, C. Valencia, J. Franco, Rheological and tribological characterization of new acylated chitosan-based biodegradable lubricating grease: a comparative study with traditional lithium and calcium greases, Tribol. Trans. 57 (2014) 445
454. [11] R. Sa´nchez, J. Franco, M. Delgado, C. Valencia, C. Gallegos, Thermal and mechanical characterization of cellulosic derivatives-based oleogels potentially applicable as biolubricating greases: influence of ethyl cellulose molecular weight, Carbohydr. Polym. 83 (2011) 151
158. [12] R. Sa´nchez, G. Stringari, J. Franco, C. Valencia, C. Gallegos, Use of chitin, chitosan and acylated derivatives as thickener agents of vegetable oils for bio-lubricant applications, Carbohydr. Polym. 85 (2011) 705
714. [13] S.-H. Chen, et al., Synthesis and characterization of thermal-responsive chitin-based polyurethane copolymer as a smart material, Carbohydr. Polym. 88 (2012) 1483
1487. [14] R. Gallego, J. Arteaga, C. Valencia, J. Franco, Isocyanate-functionalized chitin and chitosan as gelling agents of castor oil, Molecules 18 (2013) 6532
6549. [15] M. Davidovich-Pinhas, S. Barbut, A. Marangoni, Development, characterization, and utilization of food-grade polymer oleogels, Annu. Rev. Food Sci. Technol. 7 (2016) 65
91. [16] A.R. Patel, K. Dewettinck, Current update on the influence of minor lipid components, shear and presence of interfaces on fat crystallization, Curr. Opin. Food Sci. 3 (2015) 65
70. [17] A.G. Marangoni, N. Garti, Edible Oleogels: Structure and Health Implications, Elsevier Science, 2015. [18] C.V. Nikiforidis, E. Scholten, Polymer organogelation with chitin and chitin nanocrystals, RSC Adv. 5 (2015) 37789
37799. [19] A.I. Romoscanu, R. Mezzenga, Emulsion-templated fully reversible protein-in-oil gels, Langmuir 22 (2006) 7812
7818. [20] Z. Meng, K. Qi, Y. Guo, Y. Wang, Y. Liu, Macro-micro structure characterization and molecular properties of emulsion-templated polysaccharide oleogels, Food Hydrocoll. 77, 2018, 17
29. [21] T. Laredo, S. Barbut, A.G. Marangoni, Molecular interactions of polymer oleogelation, Soft Mater 7 (2011) 2734
2743. [22] K. Sato, Crystallization of Lipids: Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals, John Wiley & Sons, 2018. [23] Berezina N. (2016) Production and application of chitin. Phys. Sci. Rev. 1, 1
8. [24] F.A. Tirkistani, Thermal analysis of chitosan modified by cyclic oxygenated compounds, Polym. Degrad. Stab. 61 (1998) 161
164. [25] F.A. Tirkistani, Thermal analysis of some chitosan Schiff bases, Polym. Degrad. Stab. 60 (1998) 67
70. [26] S.-H. Lim, S.M. Hudson, Synthesis and antimicrobial activity of a water-soluble chitosan derivative with a fiber-reactive group, Carbohydr. Res. 339 (2004) 313
319. [27] H. Sashiwa, N. Yamamori, Y. Ichinose, J. Sunamoto, S.-i Aiba, Michael reaction of chitosan with various acryl reagents in water, Biomacromolecules 4 (2003) 1250
1254.

Handbook of Chitin and Chitosan

406

12. Chitin- and chitosan-based oleogels: rheological and thermal behavior modifications

[28] M. Ignatova, N. Manolova, I. Rashkov, Novel antibacterial fibers of quaternized chitosan and poly (vinyl pyrrolidone) prepared by electrospinning, Eur. Polym. J. 43 (2007) 1112
1122. [29] J.-i Murata, Y. Ohya, T. Ouchi, Possibility of application of quaternary chitosan having pendant galactose residues as gene delivery tool, Carbohydr. Polym. 29 (1996) 69
74. [30] B. Sreedhar, Y. Aparna, M. Sairam, N. Hebalkar, Preparation and characterization of HAP/carboxymethyl chitosan nanocomposites, J. Appl. Polym. Sci. 105 (2007) 928
934. [31] Z. Zong, Y. Kimura, M. Takahashi, H. Yamane, Characterization of chemical and solid-state structures of acylated, Chitosans Polym. 41 (2000) 899
906. [32] G. Ma, D. Yang, J.F. Kennedy, J. Nie, Synthesize and characterization of organicsoluble acylated chitosan, Carbohydr. Polym. 75 (2009) 390
394. [33] C. Le Tien, M. Lacroix, P. Ispas-Szabo, M.-A. Mateescu, N-acylated chitosan: hydrophobic matrices for controlled drug release, J. Control. Release. 93 (2003) 1
13. [34] J. Filipovi´c-Grˇci´c, D. Voinovich, M. Moneghini, M. Be´cirevi´c-La´can, L. Magarotto, I. Jalˇsenjak, Chitosan microspheres with hydrocortisone and hydrocortisone
hydroxypropylβ-cyclodextrin inclusion complex, Eur. J. Pharm. Sci. 9 (2000) 373
379. [35] M. Prabaharan, J. Mano, Chitosan derivatives bearing cyclodextrin cavities novel adsorbent matrices, Carbohydr. Polym. 63 (2006) 153
166. [36] S. Schu¨ßler, N. Blaubach, A. Stolle, G. Cravotto, B. Ondruschka, Application of a cross-linked Pd
chitosan catalyst in liquid-phase-hydrogenation using molecular hydrogen, Appl. Catal. A: Gen. 445 (2012) 231
238. [37] S.-H. Chiu, T.-W. Chung, R. Giridhar, W.-T. Wu, Immobilization of β-cyclodextrin in chitosan beads for separation of cholesterol from egg yolk, Food Res. Int. 37 (2004) 217
223. [38] M.L. Arrascue, H.M. Garcia, O. Horna, E. Guibal, Gold sorption on chitosan derivatives, Hydrometallurgy 71 (2003) 191
200. [39] R. Gallego, M. Gonza´lez, J. Arteaga, C. Valencia, J. Franco, Influence of functionalization degree on the rheological properties of isocyanate-functionalized chitin-and chitosan-based chemical oleogels for lubricant applications, Polymers 6 (2014) 1929
1947. [40] M.H. Naeli, J. Farmani, A. Zargaraan, Rheological and physicochemical modification of trans-free blends of palm stearin and soybean oil by chemical interesterification, J. Food Process. Eng. 40 (2017). [41] R.B. Prime, H.E. Bair, S. Vyazovkin, P.K. Gallagher, A. Riga, Thermogravimetric analysis (TGA), in: J.D. Menczel, R.B. Prime (Eds.), Thermal Analysis of Polymers: Fundamentals and Applications, Wiley Online Liberary, 2009, pp. 241
317.

Handbook of Chitin and Chitosan

C H A P T E R

13 Chitosan as biomaterial in drug delivery and tissue engineering Poliana Pollizello Lopes, Eduardo Hiromitsu Tanabe and Daniel Assumpc¸a˜o Bertuol Chemical Engineering Department, Federal University of Santa Maria— UFSM, Santa Maria, RS, Brazil

O U T L I N E 13.1 Introduction

408

13.2 General aspects of chitosan and functional features 13.2.1 Source of chitosan 13.2.2 Chitosan structure 13.2.3 Physicochemical properties of chitosan 13.2.4 Biological properties of chitosan

408 408 409 410 411

13.3 Chitosan-based therapeutic systems 13.3.1 Ocular drug delivery 13.3.2 Oral drug delivery 13.3.3 Transdermal drug delivery 13.3.4 Local administration

413 413 415 417 418

13.4 Chitosan as tissue supporting material 13.4.1 Application in bone and cartilage tissue engineering 13.4.2 Application in skin and liver tissue engineering

419 420 422

13.5 Conclusion

424

References

424

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00013-3

407

© 2020 Elsevier Inc. All rights reserved.

408

13. Chitosan as biomaterial in drug delivery and tissue engineering

13.1 Introduction Chitosan is a natural linear biopolyaminosacharide derived by the alkaline deacetylation of chitin, which is the second most abundant biopolymer after cellulose [1,2]. Chitin is synthesized by an enormous number of living organisms, among which are insects (cuticles), crustaceans (skeletons), and fungi cell walls, whereas chitosan occurs naturally in some fungi [3,4]. The physicochemical characteristics of chitosan can influence their functional properties such as solubility, chemical reactivity, and biological activities [5]. The deacetylation degree (DD) and the molecular weight are the main parameters that affect strongly many physicochemical and biological properties of this biopolymer [6]. Chitosan exhibits excellent antimicrobial activity against bacteria, fungi, and viruses [7]. Several possibilities have been described for its antimicrobial activity. The widely accepted hypothesis is that the positively charged protonated amino groups of the polymer interact with negatively charged surface components of many microorganisms. In general, chitosan causes extensive alterations to the cell surface, leading to the leakage of intracellular substances, resulting in cell death [8
10]. Among natural/biobased polymers, chitosan is of great interest due to its inherent properties, such as biocompatibility, biodegradability, low toxicity, mucoadhesion, stimuli response, and hemostatic nature [11
13]. Based on these features, chitosan and its derivatives are being successfully investigated for drug delivery and tissue engineering applications [14
18]. This chapter reports the most recent works on chitosan-based systems that have been developed to meet the specific requirements of ocular, oral, and transdermal drug delivery vehicles, as well as the emerging strategies proposed that are considering this polymer as a tissue supporting material that can stimulate regeneration in different parts of the human body like bone, cartilage, and skin.

13.2 General aspects of chitosan and functional features 13.2.1 Source of chitosan Crustacean shell waste consists of chitin (15%
30%), proteins (20%
40%), calcium and magnesium salts (30%
50%), and lipids (0%
14%). These proportions vary with the species and the season [1,2,19]. In contrast with shell mollusks (a less pure source of chitin), the squid skeleton contains 40% chitin, which is nearly free of calcium salts [2]. Moreover, the cell wall of fungus is composed about 1%
40%

Handbook of Chitin and Chitosan

13.2 General aspects of chitosan and functional features

409

of chitin. The main components of particular classes of fungi are zygomycetes (chitin/chitosan), chytridiomycetes (chitin/β-glucan), ascomycetes (chitin/mannan/β-glucan), and basidiomycetes (chitin/β-glucan) [20,21]. Usually, the zygomycetes have the highest amounts of chitin in their cell walls compared with other fungi classes [22]. Insect cuticles can contain 2%
64%, especially in diptera and butterfly which can have the highest chitin content [5]. In order to separate chitin from crustacean shells, chemical processing steps such as demineralization and deproteinization are applied using strong acids and bases to remove minerals and proteins, respectively [1,20,23]. The demineralization is carried out using HCl to eliminate calcium carbonate (0.25
1 M HCl) and the deproteinization is performed using sodium hydroxide (1 M NaOH at 70 C for 1 h) [23]. Finally, the resulting chitin is deacetylated in 40%
45% sodium hydroxide at 110 C
120 C for 1
3 h [23,24]. Biological treatments can offer an alternative way to extract chitin from crustacean shells. For demineralization, lactic acid produced by bacteria react with the calcium carbonate component in the biomass waste resulting in the formation of calcium lactate, which can be precipitated and removed by washing [5]. For the deproteinization, proteases from bacteria will eliminate proteins [25]. The processing of the fungal wastes from Aspergillus niger, Mucor rouxii, and Streptomyces consists of an alkali treatment that yields chitosan
glucan complexes [19]. The chitin obtained from fungal sources has the valuable feature of having a lower mineral content than chitin obtained from marine products [21].

13.2.2 Chitosan structure Chitosan is a linear, semicrystalline polysaccharide that contains copolymers of D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units) linked by β-(1,4) glycosidic bonds [25,26]. Chitosan is composed of less than 20% of b-(1,4)-2-acetamido-D-glucopyranose and more than 80% of b-(1,4)-2-amino-D-glucopyranose [27]. It is obtained by deacetylation (alkaline conditions or enzymatic hydrolysis) of chitin, a polysaccharide widely distributed in nature [28]. The DD of chitosan (indication of the number of amino groups along the chains) is generally defined as the glucosamine/N-acetyl glucosamine ratio [25]. Chitosan DD usually varies between 60% and 100%, while its molecular weight typically ranges from 300 to 1000 kDa, depending on the source and preparation [28]. Chitosan DD has been found to influence its physical and chemical properties and its biological activities [23]. Chitosan structure has three types of reactive functional groups, an amino/acetamido group as well as both primary and secondary

Handbook of Chitin and Chitosan

410

13. Chitosan as biomaterial in drug delivery and tissue engineering

hydroxyl groups at the C-2, C-3, and C-6 positions, respectively. The amino group contents are the main reason for the differences in their structures and physicochemical properties [29].

13.2.3 Physicochemical properties of chitosan Chitosan is a very attractive compound because of the diversity that is linked to its chemical structure. The presence of hydroxyl (OH) and amino (NH2) groups provides the flexibility for preparing molecularly imprinted polymers and for structural modification [6]. These amino groups make a good chelator, capable of binding to a variety of metal ions and can adsorb dye anions by electrostatic attraction. [6]. In addition, chitosan with protonated amino groups can subsequently form ionic complexes with a wide variety of natural or synthetic anionic species, such as lipids, proteins, DNA, and some negatively charged synthetic polymers, such as poly(acrylic acid) [28]. The solubility of chitosan depends on its biological origin, molecular weight, and degree of deacetylation [1]. Chitosan is water-insoluble and highly viscous in dilute acidic solutions. It is soluble in aqueous acids owing to the presence of free amino groups [27]. Some organic acids, such as formic, acetic, lactic, pyruvic, and oxalic acids, are usually employed for dissolution. Mineral acids such as hydrochloric and nitric acids also can be used for chitosan solutions, but phosphoric and sulfuric acids are not suitable [19]. The chemical modifications of molecular structure of chitosan can enhance their solubility in solutions. Phosphorylation, quaternization, and carboxymethylation of chitosan have been employed as chemical modification methods that significantly improve the solubility of this polymer in different solvents at ambient conditions [6]. To measure the solubility of chitosan, it is dissolved in a 1% acid solution and subjected to centrifugation. The undissolved solid mass is separated, dried in an oven, and weighed. The solubility data of the samples can be calculated using the Eq. (13.1) [1]. %solubility 5



Initial weight of tube 1 chitosan 2 Final weight of tube 1 chitosan

Initial weight of tube 1 chitosan 2 Initial weight of tube

3 100 (13.1) The moisture content in chitosan can be obtained by employing the gravimetric method [1]. The water mass can be determined by drying the sample to constant weight for 24 h at 105 C and measuring the

Handbook of Chitin and Chitosan

13.2 General aspects of chitosan and functional features

411

sample before and after drying [1,30]. The water mass is the difference between the mass of the wet and oven dry samples. Moisture content can be calculated using the Eq. (13.2) [1]:

wet weight g 2 dry weight g
3 100 (13.2) %moisture content 5 wet weight g The ash content can be determined using a muffle furnace preheated at 600 C for 6 h. The crucibles were allowed to cool in the furnace to ,200 C and then placed into desiccator [1,31]. The ash content can be calculated using the Eq. (13.3) [31]:
weight of residue g
3 100 (13.3) %ash content 5 sample weight g The molecular weight of chitosan is one parameter that can be applied as a quality standard. For a low-molecular-weight, chitosan is suitably used as an antibacterial, antioxidant, and antitumor substance. Chitosan having a medium molecular weight can have a higher anticholesterol activity than the chitosan with high molecular weight. [1]. The viscosity average molecular weight of chitosan (Mv) can be determined using Mark
Houwink Eq. (13.4) [30,32]: ½η 5 KMαv

(13.4)

where [η] denotes the intrinsic viscosity, K and α are constants which values depend on the nature of the polymer and the solvent, as well as the temperature. Values of K and α can be calculated by 1.64 3 10
30 3 DD14 (mL/g) and 1.02 3 1022 3 DD 11.82, respectively, where DD is the degree of deacetylation of chitosan expressed as the percentage [30,32]. The chitosan DD can be determined using a Fourier transform infrared instrument with frequency of 4000
400 cm21. This parameter can be calculated using the following expression [1,33]:   A1655 100 3 (13.5) DDð%Þ 5 100 2 1:33 A3450 where A1655 and A3450 were the absorbance at 1655 cm21 of the amide-I band and 3450 cm21 of the hydroxyl band. The factor “1.33” denoted the value of the ratio of A1655 /A3450 for fully N-acetylated chitosan.

13.2.4 Biological properties of chitosan Chitosan have attracted considerable interests because of their biological activities, biodegradability, nontoxicity against humans, and potential of

Handbook of Chitin and Chitosan

412

13. Chitosan as biomaterial in drug delivery and tissue engineering

applications in food, pharmaceutical, agricultural, wound dressing, and environmental industries [10,27]. Furthermore, chitosan also can inhibit microbial growth by the chelation of nutrients and essential metals. In addition, chitosan can form a polymer membrane on the surface which prevents nutrients from entering the cell or acts as an oxygen barrier which can inhibit the growth of aerobic bacteria [8]. According to literature, chitosan antimicrobial activity is dependent on several factors, such as its molecular weight (Mw), degree of deacetylation, type of microorganism, and on environmental parameters, such as pH, temperature, and salinity [34,35]. According to the literature, it is generally agreed that chitosan with higher DD exhibits greater antimicrobial activity because of the higher number of free amino groups in the polymer backbone [36]. Despite this, some controversial data were reported in the literature. Younes et al. investigated the antibacterial and antifungal effect of 15 different chitosans with well-defined characteristics in terms of DD and Mw. The authors concluded that the antibacterial activity of chitosan increases with the lower the degree acetylation and the lower the pH. Antibacterial activity was further enhanced for Gram-negative bacteria with decreasing Mw, whereas the opposite effect was observed with the Gram-positive [37]. The degree of substitution on the amino groups is the other structural factor affecting the positive charge density. This intrinsic factor is directly linked to the hydrophilic/hydrophobic character of chitosan. N-modification on chitosan through alkylation, acylation, saccharization, quaternarization, and metallization is a common strategy to change the hydrophilicity/hydrophobicity of the polymer and also to improve the antibacterial activity [36]. Carboxymethyl chitosan (CMC), alkylsulfonated chitosan, and sulfate or sulfonate chitosan modifications can enhance the antimicrobial effectiveness [34,38]. The combined antimicrobial effect of chitosan and metals has been used to prepare novel materials with improved microbicidal properties. According to the literature, it is suggested that the chitosan
silver, chitosan
zinc, chitosan
copper, chitosan
gold, and chitosantitanium dioxide complexes showed higher antimicrobial activity compared with chitosan itself [9,39]. Chitosan can also be used as an antioxidant agent. This polysaccharide has attracted great interest because it can scavenge or prevent the production of reactive oxygen species and activate a battery of detoxifying proteins. According to the scientists, the antioxidant action can be likely caused by the presence of free amino groups and hydroxyl groups in the chitosan backbone [29,34].

Handbook of Chitin and Chitosan

13.3 Chitosan-based therapeutic systems

413

13.3 Chitosan-based therapeutic systems Chitosan is a singular material as previously mentioned, its intrinsic properties are so unique and remarkable that it has attracted a wide variety of academic research for pharmaceutical and medical applications, especially in the field of controlled drug delivery [14,16,40
44]. A therapeutic system can be defined by the approach for transporting a suitable pharmaceutical substance to the site of action within the body in order to safely achieve the desired therapeutic effect. This process is heavily connected with the dosage form and route of administration, ensuring that the active drug is available at the target place for the correct time and duration [13,45]. Considering the concept of delivery systems, drugs may be introduced directly to the organ affected by disease (local) or delivered by systemic routes, where it reaches the bloodstream, so that the entire body is affected [46
48]. Conventional dosage forms normally use multiple daily dosing of drugs for the treatment of medical disorders. Most of the therapeutic content is immediate released after administration and often it can cause unacceptable side effects due to wide swings in concentration of medication in plasma [49,50]. However, the controlledrelease technology creates some control of the drug in the body, generating the desired environment with minimal side effects and prolonged efficacy [13]. Thus the most recent work on chitosan-based therapeutic systems have been specifically on anatomical routes, such as ocular, oral, transdermal, and other local administrations, and will be reported below.

13.3.1 Ocular drug delivery The treatment of ophthalmic disease encounters many challenges with regard to poor ocular bioavailability. The eye is a small and complex organ composed of multiple layers of biological barriers, which protect the internal ocular structures and tissue from the external environment [42,51]. Thus it is problematic to maintain an effective drug concentration at the target place for an appropriate period of time. In fact, less than 5% of the drug applied penetrates the cornea and reaches the intraocular tissue requiring a frequent dosing regimen [43,52]. This limitation can be overcome by a nanoparticle-based drug carrier system. Owing to their reduced dimensions, chitosan nanoparticles are capable of passing through biological barriers in vivo and delivering drugs to the lesion site to enhance efficacy [51,53].

Handbook of Chitin and Chitosan

414

13. Chitosan as biomaterial in drug delivery and tissue engineering

The potential of chitosan
dextran sulfate nanoparticles (CDNs) as a candidate system for ocular drug delivery, considering lutein as a model lipophilic drug, was reported by Chaiyasan [43]. The entrapment efficiency of lutein was found to be in the range of 60%
76%. Luteinloaded CDNs (LCDNs) possess a positive surface charge (146 mV) indicating a potential mucoadhesiveness, which can be attributed to electrostatic interactions between a positively charged CS and a negatively charged mucin. This could be useful to prolong the contact time of a drug delivery system in the mucosa and improve the treatment efficacy of lutein and/or other drugs. Nanoparticles of thiolated chitosan with sodium alginate have shown an effective encapsulating capacity with high mucoadhesive properties and the results suggest an improved delivery of drugs into human corneal epithelium cells in vitro and cornea in vivo (studies performed in rat corneas) [53]. Chitosan was proven to be well tolerated by the ocular surface after topical administration of polymer solutions regardless of the DD (between 53% and 87%), concentration, and molecular weight [54]. New approaches have been developed to enhance the low corneal drug absorption by prolonging the residence time between the formulations and the ocular tissue and reducing systemic side effects. Chitosan has the ability to form thermosensitive hydrogels when cross-linked with β-glycerophosphate (β-GP) [55
58]. These hydrogels have shown to sustain the in vitro release of many compounds depending on their molecular weight and the presence of lysozyme in the release media over a period of several hours to a month [59]. Gelatin molecules have been recently added to the CS/β-GP formulation to form new carrier systems [55,57]. When introducing 1% of gelatin, the gelation time was shortened and the gel strength improved at physiological temperature. Ferulic acid can be introduced in CS/gelatin/β-GP systems to treat oxidative damage to the cornea. The developed hydrogels showed potential to decrease the inflammation-level and apoptosis of the corneal epithelial cells under oxidative stress and to promote the corneal wound healing in a rabbit corneal alkali burn model [55]. The potential of chitosan
gelatin-based hydrogel to deliver latanoprost via a subconjunctival route for the treatment of glaucoma has been demonstrated [57]. Prolonged sustained release was observed in vitro for at least 28 days and no cytotoxic effect was verified on human corneal epithelial cells. In vivo studies indicate a steady drug concentration was observed in the aqueous humor, no hemolytic effect, and no signs of inflammation at the end of the study (60 days). A rabbit model of glaucoma demonstrated latanoprost-loaded hydrogel successfully decreased the ocular hypertension. Moreover, other chitosan-based gels demonstrated further excellent biocompatibility [58], low toxicity toward

Handbook of Chitin and Chitosan

13.3 Chitosan-based therapeutic systems

415

human retinal pigmented epithelium cells [60], and ability in glaucoma treatment [56]. Mucoadhesive chitosan films of a single application to be placed in the conjunctival sac have been successfully used as a carrier for ocular delivery, revealing the absence of cytotoxic effects and significant suppression of interleukin-2 secretion in concanavalin A-stimulated jurkat T cells [42]. Timolol maleate has also been combined with chitosan to produce films of single administration, that are equally effective in reducing intraocular pressure in rabbits as twice a day instillation of commercial ophthalmic timolol maleate solution, being safe and efficient in the treatment and prevention of glaucoma [61]. More recently, cross-linked chitosan/polyvinyl alcohol (PVA) film loaded 5-fluorouracil was investigated [62]. According to the results, almost all the drug was diffused from the cross-linked Ch/PVA films after 60 h and no toxic action was observed on the eye structure of chinchilla rabbits, leading to a lack of inflammatory reaction.

13.3.2 Oral drug delivery The oral route is the most preferred route for the administration of drugs, due to its improved convenience and patient compliance [63]. The buccal mucosa is a barrier, providing protection to underlying tissue, but is more permeable than other alternative routes such as the skin [64]. The excellent blood supply, ability to recover from stress of damage, and permeability profile make the buccal cavity an attractive site for local and systemic drug delivery [65]. The structure of chitosan is important in determining its mucoadhesive properties since it binds to the mucosal epithelium via ionic bonds between its amino group and sialic acid residues on mucosa [66,67]. Moreover, the polymer protects loaded drug against acidic denaturation and enzymatic degradation in the gastrointestinal tract and it prolongs the intestinal residence time of therapeutics [68]. Recently the development has been reported of dual chitosan/albumincoated alginate/dextran sulfate nanoparticles for enhanced oral delivery of insulin with clinical potential for the therapy of type 1 diabetes mellitus [69]. The oral insulin delivery would be a noninvasive alternative that is much more comfortable than the standard treatment which consists of repeated subcutaneous injections [70]. The developed nanoparticles showed a higher permeability and bioavailability of insulin in comparison with the uncoated system. Sung et al. [71] showed that chitosan/poly(γ-glutamic acid)/insulin nanoparticles infiltrate into the mucus layer and transiently open the tight junctions located between epithelial cells in the small intestine. These pHresponsive nanoparticles become less stable and degrade releasing encapsulated insulin which permeates through the opened paracellular pathway

Handbook of Chitin and Chitosan

416

13. Chitosan as biomaterial in drug delivery and tissue engineering

and moves into the systemic circulation. Furthermore, a complex coacervation method was used to prepare pH-sensitive insulin nanoparticles using Eudragit and chitosan with different molecular weights [72]. The results indicated that insulin release rates are increased by increasing the molecular weight of chitosan. In turn, it was also demonstrated in a type 1 diabetes rat model that the chitosan/tripolyphosphate/insulin smaller nanoparticles (45 nm and 115 nm) control the blood glucose level through oral administration more effectively than the larger particles (240 nm) [73]. Considering type 2 diabetes, chitosan-modified polylactic-co-glycolic acid nanoparticles improved the hypoglycemic effect of tolbutamide, avoiding the gastrointestinal reaction [74]. Dong and coworkers [75] have reported the development of polymer
lipid hybrid nanoparticles for oral drug delivery. For the preparation of these nanoparticles, chitosan was selected as the polymer, glyceryl monooleate as lipid, and enoxaparin as the drug model. Enoxaparin is a low-molecular-weight heparin, an anticoagulant medication, indicated to treat and prevent deep vein thrombosis and pulmonary embolism. In this study it was determined that hybrid nanoparticles with the optimal composition and greatest stability enhanced the oral bioavailability of drug. In order to facilitate oral bioavailability of enoxaparin, chitosan was combined with alginate and in vitro and in vivo tests to assess their drug delivery ability were conducted [76]. From the results, it is evident that the system can have anticoagulant activity, being more than 75% enoxaparin permeated across the intestinal epithelium and suggesting around 60% reduction in thrombus formation in rat venous thrombosis model. Another promising strategy employed graphene quantum dots as a cross-linker for chitosan beads, which were immersed in sodium salicylate solution and then encapsulated with carboxymethylcellulose hydrogel beads [77]. This system can provide better protection of the drug against stomachic degradation due to the synergistic effects of biopolymers used, resulting in a safe and efficient carrier reaching the colon as intact as possible. In alternative to pure chitosan, graft copolymers of CMC have been prepared and combined with alginate, aiming to produce a pH-sensitive hydrogel suitable for site-specific protein drug delivery in the intestine [78]. Bovine serum albumin was chosen as a model of protein drug to assess the potential of hydrogel. The burst release of the protein was slightly decreased in the harsh acidic environment (pH 1.2), while the release at pH 7.4 was improved. CMC/β-cyclodextrin nanoparticles were also investigated and ibuprofen, a nonsteroidal antiinflammatory drug, was loaded with an entrapment efficiency of 93.25% [79]. The release rate of therapeutic agent from the CMC/β-cyclodextrin was slower than chitosan/β-cyclodextrin (control) in a simulated gastric medium (pH 1.2), and the inverted trend was verified in a simulated

Handbook of Chitin and Chitosan

13.3 Chitosan-based therapeutic systems

417

intestinal medium (pH 6.8). These results suggested that CMC-based nanoparticles were more suitable for the oral delivery of hydrophobic drugs compared with the control. CMC, a water-soluble derivative of chitosan, has emerged as an interesting biopolymer for the development of new drug delivery systems [80
82]. More recently, chitosan-modified poly(mPEGMA-co-MAA) nanoparticles were developed to improve the bioavailability of ibuprofen [83]. Poly (mPEGMA-co-MAA) is a copolymer of methoxy poly(ethylene glycol) methacrylate-co-poly(methylacrylic acid) synthesized via radical polymerization. The in vitro release profiles suggested that the ibuprofen-loaded nanoparticles released the drug in pH 7.4 buffer in a continuous manner. High viability of 293 T cells indicated that the nanoparticles were nontoxic and animal experiments showed that they had obvious antifebrile effects. According to the results, these novel nanoparticles could reduce the dosing frequency of ibuprofen, and improve its bioavailability.

13.3.3 Transdermal drug delivery Transdermal drug delivery has offered great benefits ever since it came into existence, including noninvasiveness, prolonged therapeutic effect, reduced side effects, decreased dose frequency, improved bioavailability by circumvention of hepatic first-pass metabolism, and better patient compliance [84
86]. It is known that the skin is the largest organ in the body, thus transdermal delivery is a more selective and targeted delivery method, since the large exposed area of the tissue facilitates the absorption of therapeutic compounds that cannot be easily administered orally [87]. In view of this, many approaches have been accomplished to enhance drug permeation through skin, a multilayered structure, for use in transdermal drug delivery [88
92]. Bigucci et al. [93] have demonstrated the feasibility of using chitosan/ hyaluronan films for the delivery of thiocolchicoside, a muscle relaxant with antiinflammatory and analgesic effects. This approach confirmed that the selection of a suitable polymeric weight ratio and appropriate preparative conditions allows the modulation of film functional properties to achieve the effective release of drug. Moreover, it indicates the importance of polyelectrolyte complexes as vehicles to develop flexible dosage forms able to allow minimal dosage and frequency, characterized by minimal impact on lifestyle with easy and reliable administration. A particular fabrication process, electrospinning, has attracted attention for producing transdermal drug delivery [11,94
96]. However, chitosan is hardly electrospinnable because a polymeric acidic solution is a cationic polyelectrolyte. To overcome the lack of stability of the individual electrospun nanofibers, chitosan is blended with an easily electrospinnable

Handbook of Chitin and Chitosan

418

13. Chitosan as biomaterial in drug delivery and tissue engineering

polymer. Mendes and coworkers [95] have fabricated uniform and homogeneous hybrid chitosan/phospholipid nanofibers, which have similar metabolic activity comparative to the cells seeded on a tissue culture plate (control). The release of curcumin, diclofenac, and vitamin B12, as model drugs, from hybrid nanofibers was investigated. Drug release studies revealed that the release of vitamin B12 is higher compared with the other two model drugs. The drug-loaded PVA/chitosan (CS) composite nanofibers were produced and cross-linked through glutaraldehyde [95]. The results revealed that the cross-linked network structure could effectively reduce the drug release rate and the burst effect of ampicillin sodium from the PVA/CS composite nanofibers. Microemulsion and nanoemulsion formulations are also promising approaches to deliver drugs through skin. Alkilani et al. [97] studied nanoemulsion-loaded film as a transdermal delivery vehicle for carvedilol, an antihypertensive agent. This medication acts as a nonselective betaadrenergic receptor blocker and an alpha-adrenergic receptor blocker [84]. Drug-loading efficiency of the control and nanoemulsion-based films were 77% and 95.2%, respectively. The drug release process was performed using a synthetic membrane Strat-M that closely correlates to human skin, resulting in the delivery of 123.5 μg/cm2 and 45.0 μg/cm2 carvedilol from developed film and control after 24 h. Accordingly, it should be noted that the nanoemulsion promotes more efficient transport of drug across the skin. It is known that the stratum corneum is a protective barrier of skin. In order to enhance drug permeation other studies have been proposed to improve medication diffusion to deeper skin layers. Curcumin-loaded chitosan nanoparticles were prepared using an ionic gelation method; their interaction and pathway through the skin were tracked by confocal laser scanning microscopy [98]. It was verified that the appendageal route is the main route of penetration of the nanoparticles and the hair follicles their main localization. It is believed that the hair follicles play a fundamental role in skin permeation thanks to its peculiar anatomy. Additionally, curcumin could be observed through the whole depth of the stratum corneum, indicating the potential of these nanoparticles. Pirfenidone is one of the pyridine family components with antiinflammatory and antifibrotic effects used for the treatment of idiopathic pulmonary fibrosis. Chitosan
sodium alginate nanogel carriers containing this pharmacological agent were developed for transdermal application, in order to minimize some side effects caused after oral administration [92].

13.3.4 Local administration In orthopedic procedures bacterial infection is one of the most serious complications to the patient. The excessive use of antibiotics can cause

Handbook of Chitin and Chitosan

13.4 Chitosan as tissue supporting material

419

bacterial resistance, which makes treatment with traditional systemic antibiotics exceedingly difficult. Local delivery of antibiotics offers a better result to common prophylactic techniques, since it allows to reach high antibiotic concentration directly at the implant site [99,100]. Ordikhani et al. [101] have investigated chitosan/laponite films with antibiotic-eluting capacity (local drug delivery) on implants. These nanocomposite coatings were prepared on the surface of titanium foils by an electrophoretic deposition technique. A controlled vancomycin release was achieved and measured for 4 weeks. The biological tests indicated that the gradual dissolution of laponite particles could be effective on the cell viability and alkaline phosphatase activity of MG-63 osteoblast-like cells. In a similar approach, chitosan/graphene oxide films containing vancomycin were used to functionalize the surfaces of orthopedic implants [102]. The study showed that the nanocomposites exhibited a suitable bactericidal potential, almost no Gram-positive bacteria (Staphylococcus aureus) survived at the drug concentration .0.5 g/L. However, a slight cytotoxicity was verified for higher concentrations than 30 wt% graphene oxide. Another therapeutic system for the local delivery of antibiotic to bone was proposed by Dorati and coworkers [103]. A biodegradable moldable or injectable scaffold based on chitosan chloride cross-linked with glycerol 2-phosphate disodium salt, bovine bone granules, and gentamicin-loaded PLGA-PEG microparticles demonstrated a high bactericidal action in the first 4 h. The results prove that the investigated material is useful as a three-dimensional (3D) bone regenerative scaffold and antibiotic delivery system.

13.4 Chitosan as tissue supporting material The term “tissue engineering” was first defined in the 1990s by Langer and Vacantiin [104,105]. It is considered an interdisciplinary field, in which the main purpose is to develop biological substitutes able to restore, maintain, or improve biofunction, thus promoting the formation of new viable tissue [80]. It is known that the human body has the natural ability to self-repair and regenerate from disease or injure. Despite this, capacity is limited by the degree of damage and loss of function of tissues [106]. Thus the strategy of tissue engineering is to develop new biological therapeutics to assist and accelerate the regenerative process to form a natural tissue. In the future, this approach can cure patients with revivable organ failure, overcoming the lack of tissue donors and the problems of transplant rejection due to immune repulsion of the body to the transplanted organ [80].

Handbook of Chitin and Chitosan

420

13. Chitosan as biomaterial in drug delivery and tissue engineering

Over the past 30 years, there have been many papers published in this area of literature. In this chapter will be discussing some of the progress that has been made in the field of tissue engineering, based on chitosan as a tissue supporting material, which can stimulate regeneration in different parts of the human body, such as bone, cartilage, skin, and liver.

13.4.1 Application in bone and cartilage tissue engineering Apart from the properties previously mentioned, the ease of molding into porous scaffolds through various simple procedures and the minimal impact on recipients due to the biodegradability, biocompatibility and antimicrobial activity make chitosan a common choice for tissue engineering applications [11,18,107,108]. The appropriate mechanical properties are critical to the success of the implanted material. It is essential to maintain the biostability of the scaffold, which depends on the parameters such as strength, elasticity, and material degradation rate, after its implantation for the reconstruction of tissue [109]. Chitosan is structurally similar to various glycosaminoglycans found in articular cartilage and bone [110]. However, pure polymer lacks the mechanical performance particularly necessary for bone repair. Hence, it has been used in combination with different fillers, blending with other polymers or other biomaterials in attempts to regenerate hard tissues in the body [81,107,111]. In this context, that is, aiming to overcome the mechanical limitations of the polymer, researchers [112] have produced and characterized chitosan/alginate/nanohydroxyapatite scaffolds. The compressive strength and the elastic modulus improved with increasing nano-HAp content in the material, reaching values of 0.68 MPa and 13.4 MPa. The porosity ranged from 78% to 84% and was independent of ceramic content. The addition of calcium phosphate also improved the differentiation and mineralization of the MC3T3-E1 cells on the scaffolds. More recently, a similar formulation based on chitosan/alginate/ hydroxyapatite/nanocrystalline cellulose was also produced [113]. The average pore size attained up to 230 μm, a maximum value of porosity was 93.6%, and compressive yield strength was 0.54 MPa. It was observed that the Osteoblast MG63 cells grew inside the scaffold pores. Jahan, Mekhail, and Tabrizian [110] investigated the inclusion of hydroxyapatite and beta-tricalcium phosphate into the chitosan sponges obtained by a new technique that they named “all-in-onestep” procedure. For the formation of soft scaffold, adenosine diphosphate was used as the anionic cross-linker. The presence of a ceramic

Handbook of Chitin and Chitosan

13.4 Chitosan as tissue supporting material

421

filler provided an increase in biomineralization, as can be seen for CS50HA and CS25TCP, considering calcium quantification by the alizarin red method, and it maintaining the overall porosity at higher than 80% for all the composites. Following a different approach, injectable hydrogels offer a wide range of applications and can be used to fill bone defects in nonloadbearing sites, through minimally invasive procedures. They are remarkable platforms and act as a carriers for delivering therapeutic agents and cells. Several studies have demonstrated the feasibility of using thermosensitive hydrogels based on chitosan/β-glycerophosphate in treating bone defects [111]. In tissue engineering the regeneration of complex tissues or organ systems is also desired. It was observed that a spatially controlled gene delivery system in the bilayer integrated scaffolds could induce MSCs (mesenchymal stem cells) in each layer to differentiate into chondrocytes and osteoblasts in vitro, involving simultaneous regeneration of subchondral bone and articular cartilage in a rabbit knee osteochondral defect model [114]. Traumas, diseases, and sport accidents often cause articular cartilage injury. The self-healing ability of this tissue is limited due to the lack of blood vessels and poor supply of repair cells (chondrocytes) [115,116]. Chitosan has been proven to create a favorable chondrogenic microenvironment, as it can resemble the extracellular matrices of cartilage tissues comprising various glycosaminoglycan and collagen. In view of this, chitosan and derivatives are being used to build supports with potential for application in articular cartilage engineering [117
119]. Aiming to obtain photocross-linkable hydrogels, chitosan was chemically modified to incorporate highly reactive vinyl groups on the side chain. Maleilated chitosan/methacrylated silk fibroin (MCS/MSF) hydrogels were prepared by a photopolymerization process under UV light irradiation. When the MSF content was 0.1%, the developed material exhibited compressive modulus values, which were compatible with the range of articular cartilage. Cell-culture studies demonstrated that hydrogels with transforming growth factor beta were biocompatible to mouse articular chondrocytes and possess suitable cell supportive property [115]. With the intent of producing a formulation to treat cartilage damage, Singh and Pramanik [120] fabricated scaffolds constituted by a combination of chitosan and micronsized nonmulberry silk fiber. The silk fibers improve the mechanical property of the scaffold and also facilitate human mesenchymal stem cell (hMSCs) attachment, growth, as well as extracellular matrix deposition. Immunocytochemistry studies revealed an increase in collagen type II and aggrecan expression over CH/SFF scaffold in comparison to chitosan scaffold. Within this strategy, the same group has also

Handbook of Chitin and Chitosan

422

13. Chitosan as biomaterial in drug delivery and tissue engineering

investigated the effect of glucosamine components on properties of silk fibroin
chitosan scaffold [121]. The positive influence of glucosamine on improving the cell supportive property of material was observed with no significant change in its physicochemical properties. This indicates the higher chondrogenic potential of developed composite scaffold for cartilage tissue engineering. In an attempt to promote a fresh approach in injectable hydrogels to develop cartilage-like material, chitosan-beta glycerophosphate-hydroxyethyl cellulose (CH-GP-HEC) hydrogel was designed [122]. These CH-GP-HEC have been successfully used to deliver insulin, which may be considered an appropriate growth factor to improve cartilage regeneration. Furthermore, MSCs demonstrated very good survival and proliferative rates within hydrogel during the 28-day investigation.

13.4.2 Application in skin and liver tissue engineering Chitosan is an abundant, nontoxic, biodegradable, and biocompatible material. Due to its physicochemical characteristics, chitosan has been used in several areas, such as paper production, pharmaceutical and medical applications, biotechnology, cosmetics, food processing, agriculture, and wastewater treatment [17,123]. Due to the characteristics related to biocompatibility and microbacterial activity presented by chitosan, this material has been studied in several applications in the biomedical area. Among the different areas of application in biomedical engineering, it is worth mentioning its application in the engineering of liver and skin tissues. Chen et al. [124] developed and tested a bioinspired multifunctional hybrid hydrogel with self-healing capability. This hybrid hydrogel is constituted by a four-armed benzaldehyde-terminated polyethylene glycol and dodecyl-modified chitosan. The self-healing ability of the hydrogel occurs through the reversible Schiff base between the benzaldehyde and amino groups in the polymeric compounds. The dodecyl tails are anchored in the lipid bilayer of the membrane cell, resulting in remarkable tissue adhesion, besides the improvement of other properties. The results obtained in an in vivo study showed that acute tissue injuries such as vessel and liver bleedings can be rapidly repaired. In addition, this hybrid hydrogel can also be applied in the healing process of chronically infected cutaneous wounds. This hybrid hydrogel also has antiinfective features due to the coordinated effects of the dodecyl bacterial anchoring capacity and the bacterial killing capacities of chitosan. Zhou et al. [125] studied the effect of camptothecin (CPT), on the inhibition of mouse hepatic cancer. CPT is a new chemotherapy drug that has antitumor activity, however, its utilization is difficult due to its

Handbook of Chitin and Chitosan

13.4 Chitosan as tissue supporting material

423

extreme water insolubility. They chose a water-soluble quaternized cationic chitosan derivative, N-trimethyl chitosan (TMC), a nonabsorbable and nontoxic polymer to encapsulate CPT, named it CPT
TMC, and evaluated its antitumor and antimetastatic effects on the high lymphogenous metastatic mouse model of hepatic cancer. TMC increases the permeation of hydrophilic macromolecules through the mucosae, thus facilitating paracellular drug transport. The obtained data demonstrated that CPT
TMC has the ability to inhibit the growth and metastasis of mouse hepatic cancer and it may become a novel and potentially effective therapeutic strategy against human advanced hepatic cancer without conspicuous systemic toxic effects. Sun et al. [126] developed an in vivo system of magnetic drug delivery with chitosan nanoparticles for magnetic resonance imaging (MRI) monitored photodynamic therapy. Magnetic nanoparticles formed by a magnetic core, such as iron oxide, and a biocompatible polymeric shell constitute an efficient drug delivery system. The particles, after encapsulating the drugs, are directed to the exact place where the treatment is to be performed by externally located magnetic steering. In addition, magnetic nanoparticles have other important characteristics, such as visibility for MRI imaging and nanoparticle tracking. In this study, the localization of the nanoparticles in the skin and in the hepatic tissue was significantly lower than in the tumor tissue. Therefore problems related to photosensitivity and hepatotoxicity can be attenuated. These results indicate that the technique has the potential to improve the administration of a large number of traditional drugs that are effective but hepatotoxic. Amin et al. [127] synthesized a new composite from chitosan/ hydroxyapatite and glycopolymer grafted onto a Cloisite 30B clay surface (CS/HAP/clay-Gh) in aqueous media via a solvent casting and evaporation method. This study aimed to develop a new material that can be used as support for the growth of cells for applications in regenerative medicine and tissue engineering. The cytotoxicity of the resulting tri-component composites was tested against a liver carcinoma cell line and a normal human skin fibroblast cell line, among others. The obtained results demonstrated that the obtained tricomponent composite is a highly powerful kind of biomaterial. In addition, the composite showed improved thermal stability and no significant toxicity in all cell lines tested. Therefore this composite can be used as a suitable material for the delivery of bioactive compounds as well as tissue engineering. Lih et al. [128] developed a methodology for the in situ formation of enzyme-activated hydrogels. These hydrogels were obtained using chitosan as a tissue adhesive material for hemostasis and wound healing. Chitosan has been used as a material for the preparation of wound dressings due to its excellent adhesive properties, hemostatic activity,

Handbook of Chitin and Chitosan

424

13. Chitosan as biomaterial in drug delivery and tissue engineering

low toxicity, biodegradability, and antiinfective activity. Chitosan is a cationic polysaccharide and its adhesive properties are mainly based on ionic interactions with tissues or layers of mucus. In order to obtain the hydrogels, the chitosan was grafted with tyramine-modified poly(ethylene glycol) (PEG) and the tyramines were cross-linked by horseradish peroxidase and H2O2. Enzymatic cross-linking allowed the water-soluble chitosan to rapidly form hydrogels, which stably adhered to the wound site for a desired period of time. The hemostatic and adhesive properties of the hydrogels, as well as the wound-healing capability, were evaluated in vivo. The results demonstrate that the mechanical strength and the adhesion capacity of the hydrogels can be adjusted using different concentrations of H2O2. In this way, hydrogels can be obtained with specific characteristics for use in different medical applications. When applied to a rat liver defect or to an incision in rat skin, the hydrogels presented excellent hemostatic properties and healing effects.

13.5 Conclusion After providing a brief introduction of the main characteristics of chitosan, this chapter presented an overview of the general aspects of chitosan and its main functional features. The main sources of chitosan as well as its structural, physicochemical, and biological properties are also presented. Subsequently, a review is presented on chitosan-based therapeutic systems, where different applications of drug delivery in addition to local administration are presented. Finally, in the last section of this chapter we present a review on chitosan as a tissue-supporting material, where we have focused on a description of chitosan application in bone, cartilage, skin, and liver tissue engineering. Therefore it is concluded that chitosan is a very versatile material, which can be used for different biomedical applications. The unique properties of chitosan make this biopolymer an important option for the development of new materials, thus having a promising future for its use in tissue engineering and drug delivery.

References [1] S. Kumari, H.K. Annamareddy, S. Abanti, K. Rath, Physicochemical properties and characterization of chitosan synthesized from fish scales, crab and shrimp shells, Int. J. Biol. Macromol. 104 (2017) 1697
1705. [2] M. Anraku, J.M. Gebicki, D. Iohara, H. Tomida, K. Uekama, T. Maruyama, et al., Antioxidant activities of chitosans and its derivatives in in vitro and in vivo studies, Carbohydr. Polym. 199 (2018) 141
149.

Handbook of Chitin and Chitosan

References

425

[3] H. Mittal, S.S. Ray, B.S. Kaith, et al., Recent progress in the structural modification of chitosan for applications in diversified biomedical fields, Eur. Polym. J. 109 (2018) 402
434. [4] H. Hamedi, S. Moradi, S.M. Hudson, A.E. Tonelli, Chitosan based hydrogels and their applications for drug delivery in wound dressings: a review, Carbohydr. Polym. 199 (2018) 445
460. [5] W. Arbia, L. Arbia, L. Adour, A. Amrane, Chitin extraction from crustacean shells using biological methods
a review, Food Technol. Biotechnol 51 (2013) 12
25. [6] H. El Knidri, R. Belaabed, A. Addaou, A. Laajeb, A. Lahsini, Extraction, chemical modification and characterization of chitin and chitosan, Int. J. Biol. Macromol. 120 (2018) 1181
1189. [7] K. Xing, T.J. Li, Y.F. Liu, J. Zhang, Y. Zhang, X.Q. Shen, et al., Antifungal and eliciting properties of chitosan against Ceratocystis fimbriata in sweet potato, Food Chem. 268 (2018) 188
195. [8] M. Hosseinnejad, S.M. Jafari, Evaluation of different factors affecting antimicrobial properties of chitosan, Int. J. Biol. Macromol. 85 (2016) 467
475. [9] Z. Ma, A. Garrido-Maestu, K.C. Jeong, Application, mode of action, and in vivo activity of chitosan and its micro- and nanoparticles as antimicrobial agents: a review, Carbohydr. Polym. 176 (2017) 257
265. [10] A. Verlee, S. Mincke, C.V. Stevens, Recent developments in antibacterial and antifungal chitosan and its derivatives, Carbohydr. Polym. 164 (2017) 268
283. [11] S. Ahmed, A. Annu, J. Ali, et al., A review on chitosan centred scaffolds and their applications in tissue engineering, Int. J. Biol. Macromol. 116 (2018) 849
862. [12] I. Aranaz, R. Harris, F. Navarro-Garcı´a, A. Heras, N. Acosta, Chitosan based films as supports for dual antimicrobial release, Carbohydr. Polym. 146 (2016) 402
410. [13] P.P. Lopes, N.B. Barroca, B.J. Daniel-da-Silva, Ana Luı´sa, Leite Ferreira, Application of chitosan based materials for drug delivery, in: L.A. de A.P. Guilherme Luiz Dotto, Sergio Paulo Campana-Filho (Eds.), Chitosan Based Mater: Its Applications, Bentham Science, Sharjah, 2017, pp. 181
248. [14] L. Hu, Y. Sun, Y. Wu, Advances in chitosan-based drug delivery vehicles, Nanoscale 5 (2013) 3103
3111. [15] J.J. Wang, Z.W. Zeng, R.Z. Xiao, T. Xie, G.L. Zhou, X.R. Zhan, et al., Recent advances of chitosan nanoparticles as drug carriers, Int. J. Nanomedicine 6 (2011) 765
774. [16] A. Bernkop-Schnu¨rch, S. Du¨nnhaupt, Chitosan-based drug delivery systems, Eur. J. Pharm. Biopharm. 81 (2012) 463
469. [17] S. Islam, M.A.R. Bhuiyan, M.N. Islam, Chitin and chitosan: structure, properties and applications in biomedical engineering, J. Polym. Environ 25 (2017) 854
866. [18] M. Rodrı´guez-va´zquez, B. Vega-ruiz, R. Ramos-zu´n˜iga, D.A. Saldan˜a-koppel, L.F. Quin˜ones-olvera, Chitosan and its potential use as a scaffold for tissue engineering in regenerative medicine, Biomed Res. Int. (2015) 1
15. [19] R. Singh, K. Shitiz, A. Singh, Chitin and chitosan: biopolymers for wound management, Int. Wound J. 14 (2017) 1276
1289. [20] V. Ghormade, E.K. Pathan, M.V. Deshpande, Can fungi compete with marine sources for chitosan production? Int. J. Biol. Macromol. 104 (2017) 1415
1421. [21] L.-F. Zhu, J.-S. Li, J. Mai, M.-W. Chang, Ultrasound-assisted synthesis of chitosan from fungal precursors for biomedical applications, Chem. Eng. J. 357 (2018) 498
507. [22] O.M. Darwesh, Y.Y. Sultan, M.M. Seif, D.A. Marrez, Bio-evaluation of crustacean and fungal nano-chitosan for applying as food ingredient, Toxicol Rep. 5 (2018) 348
356. [23] F.A. Al Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf, Carbohydr. Polym. 77 (2) (2009) 410
419.

Handbook of Chitin and Chitosan

426

13. Chitosan as biomaterial in drug delivery and tissue engineering

[24] P.-Y. Chen, A. Yu-Min Lin, J. McKittrick, M. Andre´ Meyers, Structure and mechanical properties of crab exoskeletons, Acta Biomaterialia 4 (2008) 587
596. ¨ zogul, J.M. Regenstein, Industrial applications of crustacean by[25] I. Hamed, F. O products (chitin, chitosan, and chitooligosaccharides): a review, Trends Food Sci. Technol. 48 (2016) 40
50. [26] T.M. Ways, W. Lau, V. Khutoryanskiy, T.M.M. Ways, W.M. Lau, V.V. Khutoryanskiy, Chitosan and its derivatives for application in mucoadhesive drug delivery systems, Polymers (Basel), 10, 2018, p. 267. [27] P. Zou, X. Yang, J. Wang, Y. Li, H. Yu, Y. Zhang, et al., Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides, Food Chem. 190 (2016) 1174
1181. [28] F. Croisier, C. Je´roˆme, Chitosan-based biomaterials for tissue engineering, Eur. Polym. J. 49 (2013) 780
792. [29] D.-H. Ngo, T.-S. Vo, D.-N. Ngo, K.-H. Kang, J.-Y. Je, H. Nguyen-Duc Pham, et al., Biological effects of chitosan and its derivatives, Food Hydrocoll. 51 (2015) 200
216. [30] A. Kucukgulmez, M. Celik, Y. Yanar, D. Sen, H. Polat, A.E. Kadak, Physicochemical characterization of chitosan extracted from Metapenaeus stebbingi shells, Food Chem. 126 (2011) 1144
1148. [31] V. Mohanasrinivasan, M. Mishra, J.S. Paliwal, S.K. Singh, E. Selvarajan, V. Suganthi, et al., Studies on heavy metal removal efficiency and antibacterial activity of chitosan prepared from shrimp shell waste, 3 Biotech 4 (2014) 167
175. [32] Q.Z. Wang, X.G. Chen, N. Liu, S.X. Wang, C.S. Liu, X.H. Meng, et al., Protonation constants of chitosan with different molecular weight and degree of deacetylation, Carbohydr. Polym. 65 (2006) 194
201. [33] T. Ahmad Khan, K. Khiang Peh, H. Seng Ch, Reporting degree of deacetylation values of chitosan: the influence of analytical methods, J. Pharm. Pharmaceut. Sci. 5 (3) (2002) 205
212. [34] S. Dimassi, N. Tabary, F. Chai, N. Blanchemain, B. Martel, Sulfonated and sulfated chitosan derivatives for biomedical applications: a review, Carbohydr. Polym. 202 (2018) 382
396. [35] B.S. Shahid-ul-Islam, Recent advances in chitosan polysaccharide and its derivatives in antimicrobial modification of textile materials, Int. J. Biol. Macromol. 121 (2019) 905
912. [36] D.R. Perinelli, L. Fagioli, R. Campana, J.K.W. Lam, W. Baffone, G.F. Palmieri, et al., Chitosan-based nanosystems and their exploited antimicrobial activity, Eur. J. Pharm. Sci. 117 (2018) 8
20. [37] I. Younes, S. Sellimi, M. Rinaudo, K. Jellouli, M. Nasri, Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities, Int. J. Food Microbiol. 185 (2014) 57
63. [38] Z. Shariatinia, Carboxymethyl chitosan: properties and biomedical applications, Int. J. Biol. Macromol. 120 (2018) 1406
1419. [39] N.M. Abu-Elala, H.O. Abubakr, M.S. Khattab, S.H. Mohamed, M.A. El-Hady E.R.A. Ghandour, et al., Aquatic environmental risk assessment of chitosan/silver, copper and carbon nanotube nanocomposites as antimicrobial agents, Int. J. Biol. Macromol. 113 (2018) 1105
1115. [40] Y. Yang, S. Wang, Y. Wang, X. Wang, Q. Wang, M. Chen, Advances in self-assembled chitosan nanomaterials for drug delivery, Biotechnol. Adv. 32 (2014) 1301
1316. [41] S. Mura, J. Nicolas, P. Couvreur, Stimuli-responsive nanocarriers for drug delivery, Nat. Mater. 12 (2013) 991
1003. [42] K. Hermans, D. Van den Plas, S. Kerimova, R. Carleer, P. Adriaensens, W. Weyenberg, et al., Development and characterization of mucoadhesive chitosan films for ophthalmic delivery of cyclosporine A, Int. J. Pharm. 472 (2014) 10
19.

Handbook of Chitin and Chitosan

References

427

[43] W. Chaiyasan, S.P. Srinivas, W. Tiyaboonchai, Crosslinked chitosan-dextran sulfate nanoparticle for improved topical ocular drug delivery, Mol. Vis. 21 (2015) 1224
1234. [44] K. Delmar, H. Bianco-Peled, Composite chitosan hydrogels for extended release of hydrophobic drugs, Carbohydr. Polym. 136 (2016) 570
580. [45] R. Riva, H. Ragelle, A. des Rieux, N. Duhem, C. Je´roˆme, V. Pre´at, et al., Chitosan and chitosan derivatives in drug delivery and tissue engineering, Chitosan Biomater I.I, Springer, 2011, pp. 19
44. [46] J.B. Wolinsky, Y.L. Colson, M.W. Grinstaff, Local drug delivery strategies for cancer treatment: gels, nanoparticles, polymeric films, rods, and wafers, J. Control. Release 159 (2012) 14
26. [47] S.P. Noel, H. Courtney, J.D. Bumgardner, W.O. Haggard, Chitosan films: a potential local drug delivery system for antibiotics, Clin. Orthop. Relat. Res. 466 (2008) 1377
1382. [48] A. Trapani, S. Di Gioia, N. Ditaranto, N. Cioffi, F.M. Goycoolea, A. Carbone, et al., Systemic heparin delivery by the pulmonary route using chitosan and glycol chitosan nanoparticles, Int. J. Pharm. 447 (2013) 115
123. [49] N. Gao, X.J. Li, Controlled drug delivery using microfluidic devices, Microfluid. Devices Biomedical Applications, Woodhead Publishing, 2013, pp. 167
184. [50] N. Martinho, C. Damge´, C.P. Reis, Recent advances in drug delivery systems, J. Biomater. Nanobiotechnol. (2011) 510
526. [51] S. Liu, L. Jones, F.X. Gu, Nanomaterials for ocular drug delivery, Macromol. Biosci. 12 (2012) 608
620. [52] H.R. Lin, P.C. Chang, Novel pluronic-chitosan micelle as an ocular delivery system, J. Biomed. Mater. Res. Part B Appl. Biomater. 101 B (2013) 689
699. [53] X. Zhu, M. Su, S. Tang, L. Wang, X. Liang, F. Meng, et al., Synthesis of thiolated chitosan and preparation nanoparticles with sodium alginate for ocular drug delivery, Mol. Vis. 18 (2012) 1973
1982. [54] O. Felt, P. Furrer, J.M. Mayer, B. Plazonnet, P. Buri, R. Gurny, Topical use of chitosan in ophthalmology: tolerance assessment and evaluation of precorneal retention, Int. J. Pharm 180 (1999) 185
193. [55] C.Y. Tsai, L.C. Woung, J.C. Yen, P.C. Tseng, S.H. Chiou, Y.J. Sung, et al., Thermosensitive chitosan-based hydrogels for sustained release of ferulic acid on corneal wound healing, Carbohydr. Polym. 135 (2016) 308
315. [56] M.H. Hsiao, S.H. Chiou, M. Larsson, K.H. Hung, Y.L. Wang, C.J.L. Liu, et al., A temperature-induced and shear-reversible assembly of latanoprost-loaded amphiphilic chitosan colloids: characterization and in vivo glaucoma treatment, Acta Biomater. 10 (2014) 3188
3196. [57] Y.H. Cheng, K.H. Hung, T.H. Tsai, C.J. Lee, R.Y. Ku, A.W.H. Chiu, et al., Sustained delivery of latanoprost by thermosensitive chitosan-gelatin-based hydrogel for controlling ocular hypertension, Acta Biomater. 10 (2014) 4360
4366. [58] Y. Chien, Y. Liao, D. Liu, H. Lin, S. Chen, H. Chen, et al., Biomaterials corneal repair by human corneal keratocyte-reprogrammed iPSCs and amphiphatic carboxymethylhexanoyl chitosan hydrogel, Biomaterials 33 (2012) 8003
8016. [59] M. Rahmati, A. Samadikuchaksaraei, M. Mozafari, Insight into the interactive effects of β-glycerophosphate molecules on thermosensitive chitosan-based hydrogels, Bioinspir. Biomim. Nanobiomater. 5 (2016) 67
73. [60] M. Hsiao, M. Larsson, A. Larsson, H. Evenbratt, Y.Y. Chen, Y.Y. Chen, et al., Design and characterization of a novel amphiphilic chitosan nanocapsule-based thermo-gelling biogel with sustained in vivo release of the hydrophilic anti-epilepsy drug ethosuximide, J. Control. Release 161 (2012) 942
948.

Handbook of Chitin and Chitosan

428

13. Chitosan as biomaterial in drug delivery and tissue engineering

[61] G.D.O. Fulgeˆncio, F.A.B. Viana, R.R. Ribeiro, M.I. Yoshida, A.G. Faraco, A.D.S. Cunha-Ju´nior, New mucoadhesive chitosan film for ophthalmic drug delivery of Timolol Maleate: in vivo evaluation, J. Ocul. Pharmacol. Ther. 28 (2012) 350
358. [62] A. Baiyrkhanova, A. Ismailova, T. Botabekova, E. Enin, Y. Semenova, Crosslinked chitosan/PVA film, suturated with 5-fluorouracil for the prevention of proliferative vitreoretinopathy, Int. J. Drug Deliv. Technol. 6 (2016) 47
51. [63] S. Kumar, S.K. Gupta, P.K. Sharma, A review on recent trends in oral drug deliveryfast dissolving formulation technology, Adv. Biol. Res. 6 (1) (2012) 6
13. [64] M. Montenegro-Nicolini, J.O. Morales, Overview and future potential of buccal mucoadhesive films as drug delivery systems for biologics, AAPS PharmSciTech (2016) 1
12. [65] V. Hearnden, V. Sankar, K. Hull, D.V. Juras, M. Greenberg, A.R. Kerr, et al., New developments and opportunities in oral mucosal drug delivery for local and systemic disease, Adv. Drug Deliv. Rev. 64 (2012) 16
28. [66] E. Russo, F. Selmin, S. Baldassari, C.G.M. Gennari, G. Caviglioli, F. Cilurzo, et al., A focus on mucoadhesive polymers and their application in buccal dosage forms, J. Drug Deliv. Sci. Technol. 32 (2016) 113
125. [67] S. Mansuri, P. Kesharwani, K. Jain, R.K. Tekade, N.K. Jain, Mucoadhesion: a promising approach in drug delivery system, React. Funct. Polym. 100 (2016) 151
172. [68] K. Chaturvedi, K. Ganguly, M.N. Nadagouda, T.M. Aminabhavi, Polymeric hydrogels for oral insulin delivery, J. Control. Release 165 (2013) 129
138. [69] M. Lopes, N. Shrestha, A. Correia, M.-A. Shahbazi, B. Sarmento, J. Hirvonen, et al., Dual chitosan/albumin-coated alginate/dextran sulfate nanoparticles for enhanced oral delivery of insulin, J. Control. Release 232 (2016) 29
41. [70] G. Sharma, A.R. Sharma, J.-S. Nam, G.P.C. Doss, S.-S. Lee, C. Chakraborty, Nanoparticle based insulin delivery system: the next generation efficient therapy for type 1 diabetes, J. Nanobiotechnol. 13 (2015) 74. [71] H. Sung, K. Sonaje, Z. Liao, L.-W. Hsu, E.-Y. Chuang, pH-responsive nanoparticles shelled with chitosan for oral delivery of insulin: from mechanism to therapeutic applications, Acc. Chem. Res. 45 (2012) 619
629. [72] M. Jelvehgari, P. Zakeri-Milani, M.R. Siahi-Shadbad, B.D. Loveymi, A. Nokhodchi, Z. Azari, et al., Development of pH-sensitive insulin nanoparticles using Eudragit L100-55 and chitosan with different molecular weights, AAPS PharmSciTech 11 (2010) 1237
1242. [73] Z. He, J.L. Santos, H. Tian, H. Huang, Y. Hu, L. Liu, et al., Scalable fabrication of size-controlled chitosan nanoparticles for oral delivery of insulin, Biomaterials 130 (2017) 28
41. [74] Y. Shi, J. xue, L. Jia, Q. Du, J. Niu, D. Zhang, Surface-modified PLGA nanoparticles with chitosan for oral delivery of tolbutamide, Colloids Surf. B Biointerfaces 161 (2018) 67
72. [75] W. Dong, X. Wang, C. Liu, X. Zhang, X. Zhang, X. Chen, et al., Chitosan based polymer-lipid hybrid nanoparticles for oral delivery of enoxaparin, Int. J. Pharm. 547 (2018) 499
505. [76] A.P. Bagre, K. Jain, N.K. Jain, Alginate coated chitosan core shell nanoparticles for oral delivery of enoxaparin: in vitro and in vivo assessment, Int. J. Pharm. 456 (2013) 31
40. [77] S. Javanbakht, A. Shaabani, Encapsulation of graphene quantum dot-crosslinked chitosan by carboxymethylcellulose hydrogel beads as a pH-responsive bio-nanocomposite for the oral delivery agent, Int. J. Biol. Macromol. 123 (2019) 389
397. [78] J. Yang, J. Chen, D. Pan, Y. Wan, Z. Wang, PH-sensitive interpenetrating network hydrogels based on chitosan derivatives and alginate for oral drug delivery, Carbohydr. Polym. 92 (2013) 719
725.

Handbook of Chitin and Chitosan

References

429

[79] J. Ji, S. Hao, W. Liu, J. Zhang, D. Wu, Y. Xu, Preparation and evaluation of Ocarboxymethyl chitosan/cyclodextrin nanoparticles as hydrophobic drug delivery carriers, Polym. Bull. 67 (2011) 1201
1213. [80] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, The implications of recent advances in carboxymethyl chitosan based targeted drug delivery and tissue engineering applications, J. Control. Release 186 (2014) 54
87. [81] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, Biomedical applications of carboxymethyl chitosans, Carbohydr. Polym. 91 (2013) 452
466. [82] M.-C.C. Chen, F.-L.L. Mi, Z.-X.X. Liao, C.-W.W. Hsiao, K. Sonaje, M.-F.F. Chung, et al., Recent advances in chitosan-based nanoparticles for oral delivery of macromolecules, Adv. Drug Deliv. Rev. 65 (2013) 865
879. [83] Y. Shi, J. Xue, S. Xu, Y. You, X.Q. Yan, X. Zhao, et al., Polyelectrolyte complex nanoparticles based on chitosan and methoxy poly(ethylene glycol) methacrylate-co-poly (methylacrylic acid) for oral delivery of ibuprofen, Colloids Surf. B Biointerfaces 165 (2018) 235
242. [84] A. Ahad, F.I. Al-Jenoobi, A.M. Al-Mohizea, N. Akhtar, M. Raish, M. Aqil, Systemic delivery of β-blockers via transdermal route for hypertension, Saudi Pharm. J. 23 (2015) 587
602. [85] A. Ahad, A.A. Al-Saleh, N. Akhtar, A.M. Al-Mohizea, F.I. Al-Jenoobi, Transdermal delivery of antidiabetic drugs: formulation and delivery strategies, Drug Discov. Today 20 (2015) 1217
1227. [86] J.I. Lim, W.-K. Lee, Preparation of macroporous chitosan patch using co-solvent as a transdermal drug-delivery system, J. Porous Mater. 21 (2014) 783
788. [87] M. Amjadi, S. Sheykhansari, B.J. Nelson, M. Sitti, Recent advances in wearable transdermal delivery systems, Adv. Mater. 30 (2018) 1704530. [88] I. Takeuchi, T. Takeshita, T. Suzuki, K. Makino, Iontophoretic transdermal delivery using chitosan-coated PLGA nanoparticles for positively charged drugs, Colloids Surf. B Biointerfaces 160 (2017) 520
526. [89] E. Larran˜eta, R.E.M. Lutton, A.D. Woolfson, R.F. Donnelly, Microneedle arrays as transdermal and intradermal drug delivery systems: materials science, manufacture and commercial development, Mater. Sci. Eng. R Reports 104 (2016) 1
32. [90] S. Jana, S. Manna, A.K. Nayak, K.K. Sen, S.K. Basu, Carbopol gel containing chitosanegg albumin nanoparticles for transdermal aceclofenac delivery, Colloids Surf. B Biointerfaces 114 (2014) 36
44. ¨ zsoy, Optimization and characterization of chitosan [91] A. Can, M. Erdal, S. Gu¨ngo¨r, Y. O films for transdermal delivery of ondansetron, Molecules 18 (2013) 5455
5471. [92] M. Abnoos, M. Mohseni, S.A.J. Mousavi, K. Ashtari, R. Ilka, B. Mehravi, Chitosanalginate nano-carrier for transdermal delivery of pirfenidone in idiopathic pulmonary fibrosis, Int. J. Biol. Macromol. 118 (2018) 1319
1325. [93] F. Bigucci, A. Abruzzo, B. Saladini, M.C. Gallucci, T. Cerchiara, B. Luppi, Development and characterization of chitosan/hyaluronan film for transdermal delivery of thiocolchicoside, Carbohydr. Polym. 130 (2015) 32
40. [94] Z. Cui, Z. Zheng, L. Lin, J. Si, Q. Wang, X. Peng, et al., Electrospinning and crosslinking of polyvinyl alcohol/chitosan composite nanofiber for transdermal drug delivery, Adv. Polym. Technol. 37 (2018) 1917
1928. [95] A.C. Mendes, C. Gorzelanny, N. Halter, S.W. Schneider, I.S. Chronakis, Hybrid electrospun chitosan-phospholipids nanofibers for transdermal drug delivery, Int. J. Pharm. 510 (2016) 48
56. [96] B. Ardeshirzadeh, N.A. Anaraki, M. Irani, L.R. Rad, S. Shamshiri, Controlled release of doxorubicin from electrospun PEO/chitosan/graphene oxide nanocomposite nanofibrous scaffolds, Mater. Sci. Eng. C 48 (2015) 384
390.

Handbook of Chitin and Chitosan

430

13. Chitosan as biomaterial in drug delivery and tissue engineering

[97] A. Zaid Alkilani, R. Hamed, S. Al-Marabeh, A. Kamal, R. Abu-Huwaij, I. Hamad, Nanoemulsion-based film formulation for transdermal delivery of carvedilol, J. Drug Deliv. Sci. Technol. 46 (2018) 122
128. [98] S.M. Abdel-Hafez, R.M. Hathout, O.A. Sammour, Tracking the transdermal penetration pathways of optimized curcumin-loaded chitosan nanoparticles via confocal laser scanning microscopy, Int. J. Biol. Macromol. 108 (2018) 753
764. [99] J. Slane, J. Vivanco, W. Rose, H.-L. Ploeg, M. Squire, Mechanical, material, and antimicrobial properties of acrylic bone cement impregnated with silver nanoparticles, Mater. Sci. Eng. C 48 (2015) 188
196. ¨ hman, H. Engqvist, C. Persson, W. Xia, Enhanced drug deliv[100] T. Qin, A. Lo´pez, C. O ery of antibiotic-loaded acrylic bone cements using calcium phosphate spheres, J. Appl. Biomater. Funct. Mater. 13 (2015). 0
0. [101] F. Ordikhani, M. Dehghani, A. Simchi, Antibiotic-loaded chitosan
Laponite films for local drug delivery by titanium implants: cell proliferation and drug release studies, J. Mater. Sci. Mater. Med. 26 (2015) 269. [102] F. Ordikhani, M. Ramezani Farani, M. Dehghani, E. Tamjid, A. Simchi, Physicochemical and biological properties of electrodeposited graphene oxide/chitosan films with drug-eluting capacity, Carbon N. Y. 84 (2015) 91
102. [103] R. Dorati, A. De Trizio, I. Genta, A. Merelli, T. Modena, B. Conti, Formulation and in vitro characterization of a composite biodegradable scaffold as antibiotic delivery system and regenerative device for bone, J. Drug Deliv. Sci. Technol. 35 (2016) 124
133. [104] S. Stratton, N.B. Shelke, K. Hoshino, S. Rudraiah, S.G. Kumbar, Bioactive polymeric scaffolds for tissue engineering, Bioact. Mater. 1 (2016) 93
108. [105] R. Langer, J.P. Vacanti, Tissue engineering, Science 260 (1993) 920
926. [106] F.-M. Chen, X. Liu, Advancing biomaterials of human origin for tissue engineering, Prog. Polym. Sci. 53 (2016) 86
168. [107] S.M. Ahsan, M. Thomas, K.K. Reddy, S.G. Sooraparaju, A. Asthana, I. Bhatnagar, Chitosan as biomaterial in drug delivery and tissue engineering, Int. J. Biol. Macromol. 110 (2018) 97
109. [108] S. Preethi Soundarya, A. Haritha Menon, S. Viji Chandran, N. Selvamurugan, Bone tissue engineering: scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques, Int. J. Biol. Macromol. 119 (2018) 1228
1239. [109] B. Dhandayuthapani, Y. Yoshida, T. Maekawa, D.S. Kumar, Polymeric scaffolds in tissue engineering application: a review, Int. J. Polym. Sci. (2011) 1
19. [110] K. Jahan, M. Mekhail, M. Tabrizian, One-step fabrication of apatite-chitosan scaffold as a potential injectable construct for bone tissue engineering, Carbohydr. Polym. 203 (2019) 60
70. [111] S. Saravanan, S. Vimalraj, P. Thanikaivelan, S. Banudevi, G. Manivasagam, A review on injectable chitosan/beta glycerophosphate hydrogels for bone tissue regeneration, Int. J. Biol. Macromol. 121 (2019) 38
54. [112] H.-L. Kim, G.-Y. Jung, J.-H. Yoon, J.-S. Han, Y.-J. Park, D.-G. Kim, et al., Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering, Mater. Sci. Eng. C 54 (2015) 20
25. [113] T.I. Shaheen, A.S. Montaser, S. Li, Effect of cellulose nanocrystals on scaffolds comprising chitosan, alginate and hydroxyapatite for bone tissue engineering, Int. J. Biol. Macromol. 121 (2019) 814
821. [114] J. Chen, H. Chen, P. Li, H. Diao, S. Zhu, L. Dong, et al., Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds, Biomaterials 32 (2011) 4793
4805.

Handbook of Chitin and Chitosan

References

431

[115] Y. Zhou, K. Liang, S. Zhao, C. Zhang, J. Li, H. Yang, et al., Photopolymerized maleilated chitosan/methacrylated silk fibroin micro/nanocomposite hydrogels as potential scaffolds for cartilage tissue engineering, Int. J. Biol. Macromol. 108 (2018) 383
390. [116] F. Li, V.X. Truong, P. Fisch, C. Levinson, V. Glattauer, M. Zenobi-Wong, et al., Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells, Acta Biomater. 77 (2018) 48
62. [117] R. Muzzarelli, M. El Mehtedi, C. Bottegoni, A. Aquili, A. Gigante, R.A.A. Muzzarelli, et al., Genipin-crosslinked chitosan gels and scaffolds for tissue engineering and regeneration of cartilage and bone, Mar. Drugs 13 (2015) 7314
7338. [118] L. Li, F. Yu, L. Zheng, R. Wang, W. Yan, Z. Wang, et al., Natural hydrogels for cartilage regeneration: modification, preparation and application, J. Orthop. Transl. (2018). [119] A.R. Armiento, M.J. Stoddart, M. Alini, D. Eglin, Biomaterials for articular cartilage tissue engineering: learning from biology, Acta Biomater. 65 (2018) 1
20. [120] B.N. Singh, K. Pramanik, Fabrication and evaluation of non-mulberry silk fibroin fiber reinforced chitosan based porous composite scaffold for cartilage tissue engineering, Tissue Cell 55 (2018) 83
90. [121] V. Vishwanath, K. Pramanik, A. Biswas, Development of a novel glucosamine/silk fibroin
chitosan blend porous scaffold for cartilage tissue engineering applications, Iran. Polym. J. 26 (2017) 11
19. [122] H. Naderi-Meshkin, K. Andreas, M.M. Matin, M. Sittinger, H.R. Bidkhori, N. Ahmadiankia, et al., Chitosan-based injectable hydrogel as a promising in situ forming scaffold for cartilage tissue engineering, Cell Biol. Int. 38 (2014) 72
84. [123] G. Kummer, C. Schonhart, M.G. Fernandes, G.L. Dotto, A.L. Missio, D.A. Bertuol, et al., Development of nanofibers composed of chitosan/nylon 6 and tannin/nylon 6 for effective adsorption of Cr(VI), J. Polym. Environ. 26 (2018) 4073
4084. [124] G. Chen, Y. Yu, X. Wu, G. Wang, J. Ren, Y. Zhao, Bioinspired multifunctional hybrid hydrogel promotes wound healing, Adv. Funct. Mater. 28 (2018) 1
10. [125] L. Zhou, X. Li, X. Chen, Z. Li, X. Liu, S. Zhou, et al., In vivo antitumor and antimetastatic activities of camptothecin encapsulated with N-trimethyl chitosan in a preclinical mouse model of liver cancer, Cancer Lett. 297 (2010) 56
64. [126] Y. Sun, Z.L. Chen, X.X. Yang, P. Huang, X.P. Zhou, X.X. Du, Magnetic chitosan nanoparticles as a drug delivery system for targeting photodynamic therapy, Nanotechnology 20 (2009). [127] A. Amin, H. Kandil, H.M. Awad, M.N. Ismail, Preparation and characterization of chitosan
hydroxyapatite
glycopolymer/cloisite 30B nanocomposite for biomedical applications, Polym. Bull. 72 (2015) 1497
1513. [128] E. Lih, J.S. Lee, K.M. Park, K.D. Park, Rapidly curable chitosan-PEG hydrogels as tissue adhesives for hemostasis and wound healing, Acta Biomater. 8 (2012) 3261
3269.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

14 Biomedical applications carboxymethyl chitosans Shanta Biswas, Tanvir Ahmed, Md. Minhajul Islam, Md. Sazedul Islam and Mohammed Mizanur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

O U T L I N E 14.1 Introduction

434

14.2 Processing of chitosan

435

14.3 Properties and limitations of chitosan in the field of biomedical applications 436 14.4 Preparative methods of various carboxymethyl chitosan 14.4.1 Synthesis of O-carboxymethyl chitosan 14.4.2 Preparation of N-carboxymethyl chitosan 14.4.3 Preparation of N,O-carboxymethyl chitosan 14.4.4 Preparation of N,N-dicarboxymethyl chitosan 14.4.5 Derivatives of carboxymethyl chitosan

437 438 439 439 439 440

14.5 Properties of carboxymethyl chitosan 14.5.1 Physicochemical properties 14.5.2 Biological properties

441 441 443

14.6 Application of carboxymethyl chitosan 14.6.1 Biomedical applications 14.6.2 Other applications

445 445 461

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00014-5

433

© 2020 Elsevier Inc. All rights reserved.

434

14. Biomedical applications carboxymethyl chitosans

14.7 Conclusion

463

References

463

14.1 Introduction Recently a water soluble derivative of chitosan has come into the spotlight of the researchers due to its very attractive physical and chemical properties. And the water-soluble derivative is carboxymethyl chitosan (CMC). Chitosan is a partially deacetylated derivative of chitin, which is one of the most abundant carbohydrates in nature. It is a cationic copolymer of glucosamine and N-acetylglucosamine, mostly extracted from the exoskeleton of crustaceans [1,2]. Chitosan is nontoxic, biodegradable, biocompatible, etc. and therefore it can be applied in various fields like water treatment [3], food industry [4], pharmaceuticals [5], drug delivery [6], and so on. However, the applications of chitosan have some restrictions, such as its insolubility in neutral or alkaline pH. The solubility is possible only in acidic aqueous solutions below pH 6.5 (below the pKa of chitosan). Furthermore, another limitation of chitosan in the preparation of sustained release systems is the rapid adsorption of water and a higher swelling degree in aqueous environments, which can lead to rapid drug release [7]. This abnormal solubility can be improved by depolymerization and its chemical modifications. For this chemical modification, reactive amino, primary hydroxyl, and secondary hydroxyl groups can be used [8] under mild reaction conditions to alter its properties such as solubility, controlled polymer
drug interaction etc. Moreover, the modification resulted in enhanced loading capacity, improved bulk properties of drug delivery systems, and controlled drug release rate by the matrix [9]. In general, four different types of CMC molecule can be formed from chitosan molecules based on the appropriate reaction conditions and temperature. For the last few decades, there has been a significant increase in the number of articles reporting the use of water-soluble derivatives of chitosan and their applications in biomedical fields. In particular, there has been a considerable increase in the applications of CMC in drug delivery [10], gene therapy [11], wound healing [12], biosensors [13], etc. The derivative of chitosan is considered exciting due to its physical, chemical, and biological properties, such as high viscosity, low toxicity, superior bio/mucoadhesive properties, biocompatibility, ability to form gels, etc. [14]. This chapter aims in summarizing the recent developments in this prominent field of biomedical application of CMC. The first two

Handbook of Chitin and Chitosan

14.2 Processing of chitosan

435

sections of the chapter will briefly summarize the extraction process of chitosan, its properties, and limitations. The next two sections will cover the chemical modification of chitosan leading to CMC (with structure), as well as types, and properties of CMC will also be discussed. The final section of this chapter highlights some recent developments and fieldspecific uses of CMC in biomedical sector.

14.2 Processing of chitosan Chitosan is a natural amino-polysaccharide derived from chitin. Chitin is the second most abundant organic material in nature after cellulose-the most abundant natural polymer [2]. Chitin is found in the shells of crustaceans like crab and shrimp which is converted into chitosan by different methods. Chitosan has become immensely popular due to its properties such as biocompatibility, biodegradability, nontoxicity, and ability to chelate metal ions [1]. In general, chitosan is obtained from chitin by two types of extraction processes, one is chemical and the other is a biological method. The chemical method utilizes strong acids and bases to dissolve calcium carbonates and proteins, respectively. Though the chemical methods have exhibited some disadvantages, though the short extraction time still makes it very attractive. In the chemical method, chitosan extraction can be carried out in three steps, such as demineralization, deproteinization, and deacetylation [3]. Another step is sometimes added to eliminate pigments like astaxanthin and β-carotene. This step is called decolorization and solvents like sodium hypochlorite, acetone, and hydrogen peroxide are used [15]. The first major processing step, demineralization, is carried out in dilute hydrochloric acid solution. The purpose of this step is to eliminate calcium carbonate and calcium chloride. These two compounds are present as the main inorganic components of the exoskeleton of crustaceans. During the digestion reaction, carbon dioxide gas is emitted as a result of decomposition of these compounds. The obtained materials are then filtered, washed to neutrality with distilled water and then dried in an oven. Then the second major step, deproteinization, is carried out by subjecting the previously obtained material to alkaline treatment using dilute sodium hydroxide solution to remove proteins. This is then filtrated, washed several times with deionized water to remove the excess sodium hydroxide and dried in an oven. The product obtained is designated as purified chitin. The final step, deacetylation, is carried out to convert chitin to chitosan by removal of acetyl groups. This is done by subjecting purified chitin to treatment with concentrated sodium or potassium hydroxide solution at elevated temperature. After the reaction, the material

Handbook of Chitin and Chitosan

436

14. Biomedical applications carboxymethyl chitosans

produced is washed several times with distilled water until neutrality and then dried in an oven overnight. On the other hand, biological methods offer a milder alternative way to extract chitosan from chitin. Lactic acid and proteases produced by bacteria have been used for demineralization and deproteination steps, respectively. Sedaghat et al. reported a chitosan extraction process from shrimp wastes using a biological method. Both the demineralization and deproteinization steps of shrimp shells were carried out using lactic acid fermentation by Pseudomonas aeruginosa bacterium for 4 and 6 days. Chemical deacetylation was achieved by treatment of extracted chitin with 50% of sodium hydroxide [16]. Characteristics of extracted chitosan mainly depended on the extraction method and the source from which chitin was isolated. Since different procedures were applied, the quality of chitosan differed greatly. In the chemical methods, the use of high temperature tended to increase the deacetylation degree and thereafter the solubility of the chitosan. Though in this method thermal degradation of chitosan and breakage of molecular chain were reported which resulted in a decrease molecular weight (MW). All the more so, the viscosity of chitosan was dependent on the initial treatment conditions applied during chitosan extraction. It was reported that the use of hydrochloric acid at high concentrations adversely affected chitosan viscosity. Moreover, viscosity of chitosan tended to decrease with increased demineralization time [17]. As a result, it has been difficult to prepare high-quality chitosan with a deacetylation degree greater than 90% without avoiding chain degradation. For these reasons, some novel chitosan extraction methods have been considered, such as electrochemical, photochemical, sonochemical, and microwave synthesis methods [18]. El Knidri et al. reported an extraction method by microwave irradiation. All the major steps-the demineralization, deprotenation, and deacetylation—were carried out under microwave irradiation. The proteins were removed using microwave technology, then the deacetylation of chitin was also carried out by microwave irradiation [19]. Partial use of microwaves for the preparation of chitosan was reported by many other researchers [20,21]. The microwave technology was used by Mahdy Samar et al. at the end of the process of the extraction of chitosan, specifically in the deacetylation step of chitin [22].

14.3 Properties and limitations of chitosan in the field of biomedical applications Polysaccharides have attracted massive interest due to possessing intriguing properties which have applicability in diverse fields ranging

Handbook of Chitin and Chitosan

14.4 Preparative methods of various carboxymethyl chitosan

437

from environmental remediation to biomedical applications [23]. Chitosan like other polysaccharides possesses properties like biocompatibility, biodegradability, nontoxicity, which make it an enticing candidate in the field of biomedical products. Besides nontoxicity and biocompatibility, chitosan also exhibits other properties like osteoconductivity, osteoinductivity, and a structural similarity to bone, as well as excellent mechanical strength and cost-effectiveness [24]. Chitosan showed a very promising characteristic which has opened the door for new therapeutic uses. It exhibited degradation in vivo by several enzymes, mainly by lysozyme (a nonspecific protease present in all mammalian tissues). Moreover, these degradation products are nontoxic oligosaccharides which can be then excreted or incorporated to glycosaminoglycans and glycoproteins [25]. These properties make chitosan suitable for numerous clinical use. On top of these, other properties of chitosan like low immunogenicity, controlled release behavior, mucoadhesiveness, and structural functionality have been useful for chemical modifications to prepare new functional materials for the application in drug and gene delivery and bioimaging [26]. Chitosan-based hydrogels and wound-healing bandages showed excellent potential in the field of medicine. Even more, chitosan has become massively popular for its use as a matrix molecule for drug delivery and has been used in the area of dentistry with impressive results. Furthermore, chitosan has also exhibited the capacity to enhance drug penetration by opening the tight junctions between epithelial cells. All these properties present chitosan as an ideal candidate for use in the preparation of new materials for biomedical applications. The biggest disadvantage in chitosan’s applicability is its limited solubility in different solvents. Chitosan is insoluble in water, and only soluble in acetic acid [27]. As a result, derivatives of chitosan have been prepared to overcome the problem. One such derivative is CMC. It is a derivative of chitosan obtained by the etherification of alkaline chitosan with monochloroacetic acid. CMC has a higher solubility than chitosan; therefore it is more readily applicable for use in various fields [28]. In the next sections a detail of this derivative of chitosan will be discussed.

14.4 Preparative methods of various carboxymethyl chitosan The main aim of this section is to briefly discuss the synthesis methods of CMC and their important derivatives suitable for biomedical applications. Synthesis of different types of CMCs is dependent on various factors like reaction medium, chemical routes, degree of substitution (DS), degree of deacetylation (DD), degree of acetylation (DA), MW,

Handbook of Chitin and Chitosan

438

14. Biomedical applications carboxymethyl chitosans

FIGURE 14.1 Preparative methods of different types of CMCs [29].

and structure. Fig. 14.1 shows the preparative methods for four different types of CMCs.

14.4.1 Synthesis of O-carboxymethyl chitosan O-CMC is known to be an amphiprotic ether derivative that contains
COOH groups and
NH2 groups in the molecule, among the other water-soluble derivatives of CMCs. A strongly alkaline solution medium is used for the synthesis of O-CMC. By suspending the chitosan in sodium hydroxide in a reaction vessel with isopropanol as solvent, and stirring the alkaline solution at room temperature for 1 h, OCMC is prepared. Monochloroacetic acid dissolved in isopropanol is then added dropwise for 30 min to the reaction mixture. The entire mixture is kept at 55 C to react for 4 h. The solid is finally filtered and

Handbook of Chitin and Chitosan

14.4 Preparative methods of various carboxymethyl chitosan

439

washed with ethyl alcohol and vacuum dried. Water solubility of O-CMC is dependent on the preparation condition and degree of carboxymethylation, which is experimentally demonstrated by Chen et al. [30]. Moreover, applications for O-CMCs for immobilization of enzymes of clinical significance were demonstrated by Xu et al. [31].

14.4.2 Preparation of N-carboxymethyl chitosan N-CMC synthesis occurs in a slightly acidic medium and the preparation process involves the reaction of a free amino group of chitosan with glyoxylic acid to give soluble aldimine and then to reduce it with sodium borohydride. The reaction does not require either warming or cooling. The ratio of acetyl, carboxymethyl, and free amino groups is determined by the choice of chitosan (in terms of DA and MW) and the amount of glyoxylic acid. The advantage of this reaction is that it does not require heating [32].

14.4.3 Preparation of N,O-carboxymethyl chitosan N,O-CMC is a carboxymethyl substituted derivative in some of the glucosamine units of the chitosan,s amino and primary hydroxyl sites. It can be prepared by using chitosan and aqueous sodium hydroxide solution in a flask with stirring facility and reflux condenser. Isopropyl alcohol and chloroacetic acid were added into the flask subsequently and the reaction medium was maintained 65 C for 4 h. The mixture was filtered and washed with alcohol at 80 wt% until the pH became neutral. The product was then vacuum dried [33]. Attractive characteristics like moisture retention, gel-forming ability, biocompatibility, increased water solubility, and enhanced microbial activity make N,O-CMC a good contender for various biomedical applications [33,34].

14.4.4 Preparation of N,N-dicarboxymethyl chitosan Several important factors govern the synthesis of N,N-dicarboxymethylCMC (N,N-di-CMC). The factors include the concentration of chitosan, water, glacial acetic acid, glyoxylic acid, and sodium borohydride. In order to synthesize N,N-di-CMC, 27 g of glacial acetic acid is added and stirred for 20 min at a fixed concentration of chitosan (30 g) suspended in demineralized water (3 L). Hereafter glyoxylic acid is added (178 mL 50% v/v corresponding to 119 g of glyoxylic acid) and the molar ratio of amine/ glyoxylic acid is set to 1:9 at pH 2
3. As a final point, sodium borohydride (90 g) in water (2.5 L) is supplied as a 3.6% solution to the reaction vessel using a peristaltic pump (1.2 mL/min). The N,N-di-CMC prepared by this

Handbook of Chitin and Chitosan

440

14. Biomedical applications carboxymethyl chitosans

method exhibits good film-forming ability, good chelation ability, and excellent osteoinductive properties with calcium phosphate [35].

14.4.5 Derivatives of carboxymethyl chitosan 14.4.5.1 N-carboxybutyl chitosan N-carboxybutyl chitosan (NCBC) is a widely used chitosan derivative, and is an amphoteric polymer and soluble in acidic, neutral, and basic conditions. It can be synthesized by reacting chitosan with levulinic acid in the presence of a reducing agent (sodium borohydride) [36]. The reaction tends to form NCBC or 5-methyl pyrrolidinone chitosan (5-MPCs), a cyclic product, depending on the chemical conditions [37]. So it is very important to control the reaction medium effectively as it can produce 5-MPC, which is only soluble in acidic condition like pure chitosan. Several properties of NCBC like water solubility, good film-forming capacity, and antimicrobial activity make it favorable to use in different applications like wound management, production of skin care products, tissue expanders, and regeneration of cutaneous tissues [36,38,39]. 14.4.5.2 N-carboxyethyl chitosan By using 3-halopropionic acids in mild alkaline media (pH 8
9) with NaHCO3, the N-carboxyethyl chitosan (NCEC) derivative can be prepared. Shigemasa et al. [40] reported the first synthesis of 1-carboxyethyl chitosan from chitin and chitosan by a reaction with 2-chloropropionic acid. Correspondingly, N-(2-carboxyethyl) chitosans were prepared by a reaction of low-MW chitosan with a low DA and 3-halopropionic acids under mild alkaline media (pH 8
9, NaHCO3) at 60 C [41]. Characteristics of NCEC like water solubility in a wide pH range, better biocompatibility, excellent antioxidative property, and antimutagenic activity made it very useful in various biomedical applications [42
44]. 14.4.5.3 N-succinyl chitosan N-succinyl chitosan (NSC) is a chitosan derivative, which can be obtained by the introduction of succinyl groups into the chitosan N-terminal of the glucosamine units. By varying the concentration of succinic anhydride in the reaction medium, a degree of succinylation can be modified. NSC can be prepared by dissolving chitosan in acetic acid solution into a flask and by dropwise adding of succinic acid which is dissolved in acetone into that flask [45,46]. NSC has a wide range of biomedical applications; mostly in drug delivery applications, tissue engineering, wound dressing, and cosmetics production [47
49].

Handbook of Chitin and Chitosan

14.5 Properties of carboxymethyl chitosan

441

14.4.5.4 O-succinyl chitosan O-succinyl chitosan (OSC) can be obtained from phthalimide chitosan. Phthalimide chitosan can be prepared by dissolving chitosan in a mixture of phthalic anhydride and dimethylformamide (DMF) solution and heating the solution to 130 C in inert conditions with the help of stirring. Using phthalimide chitosan, DMF solution, succinic anhydride, and pyridine, OSC can be synthesized [50]. This water-soluble chitosan derivative has several biomedical applications, like the other derivatives discussed earlier. The applications include, drug delivery, wound dressing and production of skin care products [50,51].

14.5 Properties of carboxymethyl chitosan This section will briefly discuss the improved physical, chemical, and biological properties of CMC. In general improved solubility both in acidic and basic solution, high viscosity, low toxicity, superior bio/ mucoadhesive properties, biocompatibility, and ability to form gels has enabled the wide application of CMC in the field of biomedical applications.

14.5.1 Physicochemical properties 14.5.1.1 Solubility and aggregation properties A remarkably important characteristic of CMC is its solubility in water. Due to carboxymethylation, the solubility of CMC in aqueous solution increased significantly compared to chitosan, while imparting novel functionalities. A considerable number of researches have been conducted to understand the solubility behaviour of CMC and its derivatives. RJN Hjerde et al. [52] reported that, if the degree of substitution (DS) of carboxymethylation exceeds 60% then CMC could be dissolved in any aqueous solution with any range of pH. Although in an other research conducted by Chen et al. [30] the critical value of DS to make CMC soluble in water, was reported in the range of 0.4
0.45 and different solubility values for CMC were also included. While studying the solubility studies of O-CMC by varying the DS value, Chen et al. [53] found that the water solubility of CMCs is closely related to changing reaction conditions and the degree of carboxymethylation. Their study showed that CMCs prepared at temperatures from 0 C to 10 C were soluble in water, but CMC prepared between 20 C and 60 C were insoluble in water at near neutral pH. Thus CMC’s water insolubility varied with the DS at different pHs. Both fraction of carboxymethylation and insolubility in water at lower pHs

Handbook of Chitin and Chitosan

442

14. Biomedical applications carboxymethyl chitosans

increased with increase in reaction temperature. But a decrease in the fraction of carboxymethylation and increase in insolubility in water at higher pHs could be found with the increment of the ratio of water/ isopropanol in the reaction solvent media. The insoluble portion can either be due to the aggregation of highly acetylated chain segments or the formation of amide following thermal drying. Another important characteristic of CMC is its aggregation property and this behavior can be found in dilute aqueous solution of O-CMC, as explained by Zhu et al. [54]. The research indicated that the H-bonding between water and the polymer and the presence of COO2 on the O-CMC chain are the two combined driving forces that are responsible for the solubility of O-CMC in water. On the other hand, the intermolecular H-bonding of O-CMC and the electrostatic repulsion between them are the key forces responsible for the aggregation of O-CMC in solution. A similar study was carried out to justify the effects of acid, pH, and ionic concentrations on deoxycholate chitosan and CMC aggregation behaviors in aqueous systems by Pang et al. [55]. Their study showed that the alteration of both hydrophobic groups and hydrophilic groups affected the rheological properties of the polymers, although it was found that ionic strength did not affect the self aggregation of CMC and the parent chitosan. Felicio et al. [56] described the spherical aggregation behavior by N-CMC. The authors confirmed this behavior by static and dynamic light scattering and atomic force microscopy images. The critical aggregation concentration of N-CMC was observed to be 1.0 mg/mL with a DS value of 10% to 60%. In other research by Thanou et al. [57] it was revealed that CMC with 87% to 90% DS has a polyampholyte (zwitterionic) character, which allows the construction of clear gels or solutions at neutral and alkaline pH values depending on the concentration of polymers, but aggregates under acidic conditions. 14.5.1.2 Moisture retention and absorption properties For its possible use in cosmetics and clinical medicine and other biomedical applications, CMC’s moisture retention properties have received considerable attention. Moreover, researchers have found that the CMC seemed to be more suitable for moisture retention than other chitosan derivatives [58]. CMC seems to be more suitable for the preparation of hyaluronan-like materials, which is known for its excellent moisture retention properties, among many chitosan derivatives [32]. Muzzarelli [59] revealed that in terms of moisture retention capacity a 0.25% CMC aqueous solution was comparable to a 20% aqueous solution of propylene glycol, and the viscosity was nearly equal to that of hyaluronic acid, a compound with known excellent moisture retention properties. Chen et al. [30,60] have experimentally demonstrated and established a relationship not only for water solubility of O-CMC, pH,

Handbook of Chitin and Chitosan

14.5 Properties of carboxymethyl chitosan

443

temperature, and fraction of carboxymethylation but also demonstrated the relationship between the moisture absorption and moisture retention by CMC with its molecular structure, DD, and DS. So, in summary, CMC’s moisture retention properties have led to its industrial production as a hydrating agent for skin care. 14.5.1.3 Chelating and sorption properties Several factors are responsible for the excellent adsorption properties that have been shown by CMCs. The factors includes the presence of a large number of hydroxyl and amino groups and the high hydrophilicity of chitosan, and the flexible structure of the polymer chain which enables the chitosan to adopt the suitable configuration for complexation with metal ions [61]. For the interpretation of the absorption mechanisms of different metal ions, two hypotheses have been proposed by Nieto et al. [62]. Their works suggested that the metal ions are bound to several amine groups from the same chain or from different chains by means of inter- or intramolecular complexation in the bridge model, while in the pendant model they are bound to an amine group. An earlier research by Muzzarelli et al. [63] showing the chelation of N-CMC with metal transition ions, where the addition of N-CMC to N-CMC solutions (0.2
0.5 mM) produced an immediate insolubilization of N-CMC-metal ion chelates. Sun et al. [64] studied the adsorption properties of CMC and cross-linked CMC resin with Cu(II) as template. Their study revealed that template CMC resin can adsorb Cu(II) ions selectively from a mixed solution containing three types of metal ions. Moreover, the reusability of template CMC resin made them more efficient for further use as the adsorption capacity for Cu(II) ions after reuse for 10 times. In another study by Wang et al. [65] it was found that the adsorption of Fe(III) ions by using CMC hydrogels are very fast due to the chelation of Fe(III) ions with amino, hydroxyl, and carboxyl groups of CMC.

14.5.2 Biological properties 14.5.2.1 Antimicrobial activities Chitosan and its derivative CMC show a great ability to inhibit different types of bacteria and fungi. To demonstrate the antibacterial activity of chitosan and CMC several mechanisms have been proposed by many researchers. As per one mechanism, the polycationic character of chitosan obstructs the negatively charged deposits of macromolecules at the cell surface and modifies cell permeability. The other mechanism is to bind cationic chitosan to DNA to inhibit RNA synthesis. Liu et al. [66] described the increased order of antibacterial activity of chitosan and various CMCs against

Handbook of Chitin and Chitosan

444

14. Biomedical applications carboxymethyl chitosans

Escherichia coli and the order is N,O-CMC , chitosan , O-CMC. A reduced number of protonated amino groups in N,O-CMC is the cause of the lower antibacterial activity in N,O-CMC. On the other hand, enhanced antimicrobial activity can be found in O-CMC as substitution occurs only in
OH group in O-CMC. Several other factors are also responsible for the antimicrobial activity of CMC. MW, DD, pH of the reaction medium, and concentration in solution governs the antibacterial activity of CMC. Sun et al. [64] established a relationship between the structure and the antimicrobial activity of quaternized CMC. The DS of CMC was observed to have little effect on its antimicrobial activity, while the increase in its quaternization DS or the reduction of its MW increases the antimicrobial activity of quaternized CMC. A more recent study by Anitha et al. [67] reported the antibacterial activity of chitosan, O-CMC, and N,O-CMC nanoparticles against Staphylococcus aureus experimentally. The results have shown that chitosan nanoparticles have less antibacterial activity than N,O-CMC and O-CMC nanoparticles. The detailed report is discussed in Section 14.6 of this chapter. 14.5.2.2 Antioxidant properties The antioxidant activity of chitosan and its derivatives has shown that active hydroxyl and amino groups in the polymer chains can participate in free radical scavenging and have contributed to antioxidant activity. The content of active hydroxyl, amino, amido groups, and MW in their polymer chains affects the antioxidant activity of chitosan and derivatives like CMC [68
70]. Due to the partial loosening of intermolecular and intramolecular hydrogen bonds, the antioxidant activity of CMC increases with the decrease in MW. Through oxidative degradation, low-molecular-weight CMC were prepared by Sun et al. [68] and their scavenging activity was evaluated. The results showed that CMC had a better scavenging activity for superoxide anion with lower MW. The Schiff bases of CMC, alternatively, showed no improvement in antioxidant activity due to the destruction of part of the hydrogen bonds, the formation of new hydrogen bonds, and the change of NH2 to C~N shown by Guo et al. [71]. 14.5.2.3 Antifungal properties Due to their valuable biological functions such as antibiofilm activities, chitosan and its derivatives are attractive as antifungal materials and can inhibit candidiasis, which is responsible for creating infections in immunocompromised persons and other animals [72,73]. In addition, CMC has been found with noticeable antifungal activity than chitosan [74]. CMC was shown to inhibit the formation of bacterial biofilms by the dual aggregation mechanism, including flocculation at bridging and load neutralization [75]. Due to its antifungal capacity, CMC was used

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

445

as a gauze-coating material and it was shown that the antifungal activities of the CMC-coated gauze measured by the diameter of growth inhibition were 0.30 cm higher than the gauze coated with chitosan (0.12 cm) [28]. Although most candidiasis is associated with Candida albicans, several non C. albicans species have also been isolated in patients. The inhibition effect of CMC on single and mixed species in biofilms of nonalbicans Candida species, has recently been evaluated, and it has been found that CMC can inhibit both Candida planktonic growth and adhesion [76]. 14.5.2.4 Apoptosis inhibitory activities The potential value of chitosan in cancer treatment was illustrated by the study of its growth inhibitory effect on human bladder tumor cells 5637, examined by colorimetric test WST-1 and cell counting [77]. However, the protection of rabbit chondrocytes from interleukin1β-induced apoptosis was detected with CMC (DS 5 0.91 and MW 5 3.9 3 104 Da) [78]. The apoptosis inhibitory effects of CMC were suggested to be due to the protection of mitochondrial function, the decrease in nitric oxide levels, and reactive oxygen species. The suggestion was based on other actions observed with CMC in chondrocytes treated with interleukin-1β, such as partial restoration of mitochondrial membrane potential and ATP, a reduction in the production of nitric oxide by a reduction in the inducible expression of nitric oxide synthatase mRNA, and the scavenging of reactive oxygen species. In addition, CMC significantly suppressed the mRNA expressions of matrix metalloproteinase-1,3 in osteoarthritic cartilage and reduced cartilage degradation severity [79,80].

14.6 Application of carboxymethyl chitosan 14.6.1 Biomedical applications 14.6.1.1 Wound healing In wound healing applications, the major concern is the good interaction and positive response of fibroblasts with biomaterials. Results from different researches proved that CMC is biocompatible and therefore promoting the growth of fibroblast cells. Not only CMC but also its biocomposites with other polymers and complexes with metals have been investigated for their potency to be used in wound healing applications. Sikai et al. investigated the effects of CMC on wound healing by evaluating its influence on cytokines secretion of fibroblasts and macrophages (in vitro) and thereby determining transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-1, and interleukin(IL)-6 in

Handbook of Chitin and Chitosan

446

14. Biomedical applications carboxymethyl chitosans

wounds in vivo [81]. In vitro results showed that both the proliferation of fibroblasts and phagocytosis ability of macrophages were significantly enhanced by CMC treatment. From the in vivo results, it was found that CMC significantly accelerated the wound healing of seconddegree burns created in rats. TGF-β1, IL-6, and MMP-1 contents at the wound area were significantly increased by treatment of CMC, which speed up the healing process. CMC also reduced scar formation by regulating collagen secretion. The effects of MW of CMC on wound healing were also studied and CMC with lower MW was found to be more effective at promoting and accelerating cell proliferation. The researchers concluded that CMC was able to activate macrophages, accelerate the growth of fibroblasts, and significantly affect the secretion of a series of cytokines which ultimately led to accelerated wound healing. In another research, CMC membrane was applied on second-degree burn wounds and a number of factors, like TGF-β1, interleukin-8 (IL-8) protein levels, tumor necrosis factor (TNF)-α, and Smad3 gene expression were measured to investigate the healing mechanism of CMC [82]. The membranes were implanted in both musculature and subcutaneous tissue to observe the degradability and it was found that the lysozyme concentrations in the adjacent environment controlled the degradation behavior of CMC membrane. The result of TGF- β1, IL-8 protein levels, TNF-α, and Smad3 gene expression measurement revealed the pathway of the accelerated wound healing ability of CMC, which consisted of its ability to induce the expression of inflammatory cytokines which triggered the wound healing cascade and activated downstream proteins such as Smad3. CMC
metal complex like O,N-CMC
zinc complex can be used in topical formulations for the treatment of skin infections and wound healing as it possesses good antimicrobial activity [83]. This complex was evaluated for antimicrobial activities against S. aureus and E. coli in vitro and then compared with the activity of chitosan
zinc complex. O,N-CMC
zinc complex showed better in vitro antimicrobial activity than chitosan
zinc complex against the above mentioned bacteria. The reason for this better activity was the enhanced solubility of O,NCMC
zinc complex in aqueous solution which led to higher diffusion, and a higher concentration was available at the site of action. A hydrogel was prepared from radiation cross-linked CMC/gelatin by Huang et al. and investigated in vivo for its biodegradability, biocompatibility, and effects on wound healing [84]. Formation of granulation tissue and the reepithelialization process were also observed. Gelatin was used in this formulation to stabilize the structure of hydrogel and to prolong its degradation time. To evaluate the degradation and integration behavior in vivo, the hydrogels of different ratio of CMC to gelatin were implanted into a full thickness cutaneous wound

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

447

FIGURE 14.2 Macroscopic observation of the wounds treated by radiation cross-linked CMC/gelatin hydrogel (A, B, C) with respect to blank control (D, E, F) at days 3, 9, and 15. Reprinted with permission from X. Huang, Y. Zhang, X. Zhang, L. Xu, X. Chen, S. Wei, Influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing, Mater. Sci. Eng. C. 33 (8) (2013) 4816
4824.

model created in Sprague
Dawley rats. It was found that the rate of degradation depended on the ratio of CMC and gelatin, and hydrogels containing a higher proportion of CMC led to a faster degradation. The in vivo studies revealed that the hydrogel was biocompatible with mild inflammatory response by the host cell and had a positive effect on promoting cell proliferation and neovascularization. It was also observed that the full thickness cutaneous wound healing was accelerated with thicker granulation tissue (Fig. 14.2) and earlier reepithelialization as the CMC/gelatin hydrogel facilitated fibroblast proliferation, neovascularization, and collagen synthesis. 14.6.1.2 Drug delivery Extensive research has been carried out to investigate CMC in both in vitro and in vivo conditions for its potential to be used in controlled drug delivery system. CMC has the potential to become a good drug carrier because it has the properties like good water solubility, biodegradability, biocompatibility, antibacterial and antifungal activity, and can promote permeability. In a study, resveratrol-loaded CMC nanoparticles were prepared by emulsion cross-linking and the release profile was studied in vitro and in vivo [85]. The in vitro drug release profile, carried out in simulated gastric fluid, showed that the cumulative release percentage of resveratrol from resveratrol-loaded CMC nanoparticles was higher than that of resveratrol raw powder. It was also found that the release profile of the drug from the nanoparticles were divided into two phases, the initial burst release up to 1 h due to the diffusion of loosely bound drug in the surface of nanoparticles, followed by a sustained release of about 79.2% in 68 h. An in vivo study revealed that the bioavailability of the

Handbook of Chitin and Chitosan

448

14. Biomedical applications carboxymethyl chitosans

encapsulated drug was increased by 3.516 times with respect to resveratrol raw powder. This CMC nanoparticles-based drug delivery system also improved the water solubility of resveratrol and prolonged its duration of action. An antimetabolite and antineoplastic drug 6-Mercaptopurine (6-MP) was attached to CMC by hydrophobic modification of CMC, which formed self-assembly nanoparticles in aqueous solution [86]. The system was turned to become stimuli-responsive by adding glutathione (GSH)-sensitive disulfide linker between CMC and 6-MP. Consequently, the release profile of 6-MP was found to depend on the GSH concentration. In vitro drug release profile showed that 6-MP was stable or had a little release in the media containing micromolar GSH, but a fast release was observed at millimolar concentration levels of GSH. Besides, the pH of the release medium and drug content in the matrix also affected the release behavior. Injectable hydrogels with pH and temperature sensitivity can be prepared by grafting poly(N-isopropyl acrylamide (PNIPAm)) and glycidyl methacrylate to CMC followed by UV cross-linking. These hydrogels were cytocompatible and investigated as localized drug carriers for anticancer and antiinflammatory drugs. 5-Fluorouracil and diclofenac sodium were used as model drugs, which were encapsulated into the hydrogels in situ before photocross-linking. From the in vitro drug release experiment, it was found that the PNIPAm grafting percentage, pH, and temperature of the release medium controlled the release of drug molecules from hydrogel matrix. The release rate of both of the drugs increased with the increase in temperature. In the case of diclofenac sodium, the release percentage was about 27% at pH 2.1 for 24 h, whereas it was 89% at pH 7.4 for 24 h, which indicated that these hydrogels were able to protect the drug in a medium of low pH (e.g., stomach) and substantially release them at a higher pH system (such as intestine) [87]. An amphiphilic polymeric micelle of CMC-quercetin conjugate was synthesized for oral delivery of an anticancer drug, paclitaxel (PTX) [88]. The drug was encapsulated in the micelle with the aim of enhancing its oral bioavailability by increasing its water solubility and bypassing the P-gp drug efflux pumps. The PTX-loaded micelle showed a sustained-release profile of the drug in simulated gastrointestinal fluids (pH 1.2/pH 6.8) and PBS (pH 7.4) in vitro. Significant improvement in the effective permeability of PTX in the duodenum and jejunum was observed during the in situ intestinal absorption experiments. In vivo pharmacokinetic studies showed that the drug-loaded conjugate significantly increased the oral absorption of PTX. Moreover, the antitumor efficacy against murine hepatic carcinoma tumor xenograft models was highest in the case of PTX-loaded CMC
quercetin conjugate with

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

449

negligible toxicity. Such results indicated that CMC
quercetin conjugate could be a potential oral drug carrier for water-insoluble anticancer drugs. Doxorubicin (DOX) incorporated nanoparticles composed of poly (ethylene glycol)-grafted CMC were prepared by ion-complex formation and the antitumor activity of these nanoparticles were tested against DOX-resistant C6 glioma cells in vitro [89]. The release of DOX was found to be pH dependent and the release was faster at acidic pH than neutral or basic pH. As the cells were DOX resistant, the drug was not able to penetrate into cells and did not effectively inhibit cell proliferation. However, DOX-loaded nanoparticles penetrated into cells and effectively inhibited cell proliferation which was confirmed by fluorescence (FL) microscopy. There are many other CMC-based systems which have been investigated for their application in drug delivery. A list of recent works is given in Table 14.1. 14.6.1.3 Tissue engineering and regenerative medicine Tissue engineering research is usually based on the construction of biomaterials like porous scaffolds that can promote cell attachment, proliferation, and differentiation. CMC has emerged as a potential candidate for tissue engineering application because it is biodegradable, biocompatible and accelerates cell growth and tissue formation while minimizing inflammatory reactions and toxic degradation products. An injectable hydrogel was developed from alginate and O-CMC and loaded with fibrin nanoparticles for adipose tissue engineering because frequent replacement of adipose tissue is often required during the reconstruction of damaged soft tissues [107]. Human adipose-derived stem cells were cultured on prepared hydrogel scaffold to study cell attachment, viability, proliferation, and differentiation. It was found that fibrin nanoparticles-loaded scaffold promoted cell adhesion and proliferation. Better cell differentiation was also confirmed by the oil red O staining technique. CMC coated with hydroxyapatite was investigated to discover its ability to promote cell differentiation of MC3T3 osteoblasts and human bone marrow stem cells [108]. A comparison was also performed between the coated and noncoated types of CMC. Both coated and noncoated CMC were biocompatible and found to support cell attachment, proliferation, and differentiation of the osteoblasts and bone marrow stem cells. However, these processes were substantially improved with coated CMC, especially in osteoblast and bone marrow stem cell differentiation. Agarwal et al. reported a gelatin
CMC based scaffold that could be utilized as a matrix for dermal tissue engineering applications [109]. A

Handbook of Chitin and Chitosan

450

14. Biomedical applications carboxymethyl chitosans

TABLE 14.1 Various carboxymethyl chitosan-based systems investigated in recent years for drug delivery application. Sl. no.

Drug carrier

Form of the carrier

Loaded drug

References

1.

N,O-Carboxymethyl chitosan

Nanoparticles, hydrogel

5-Flourouracil

[90,91]

2.

Carboxymethyl chitosan

Hydrogel

Theophylline

[92]

3.

Thiolated carboxymethyl chitosan-graft-cyclodextrin

Nanoparticles

Albendazole

[93]

4.

O-Carboxymethyl chitosan

Nanoparticles

Curcumin, tetracycline, metformin

[94
96]

5.

Carboxymethyl chitosan-tethered lipid vesicles

Nanoblanket

Paclitaxel

[97]

6.

Chitosan/O-Carboxymethyl chitosan

Nanoparticles

Doxorubicin hydrochloride

[98]

7.

Thiolated carboxymethyl chitosan

Nanoparticles

Methotrexate

[99]

8.

O-Carboxymethyl chitosan/poly (acrylic acid)

Beads

Rabeprazole sodium

[100]

9.

Galactosylated O-Carboxymethyl chitosan-graftstearic acid

Nanoparticles

Doxorubicin

[101]

10.

O-Carboxymethyl Chitosan/ Fucoidan

Nanoparticles

Curcumin

[102]

11.

Folic acid conjugated Ocarboxymethyl chitosan

Nanoparticles

Methotrexate

[103]

12.

O-Carboxymethyl chitosan/ cyclodextrin

Nanoparticles

Ibuprofen

[104]

13.

Oleoyl-carboxymethyl chitosan

Nanoparticles

Rifampicin

[105]

14.

Calcium phosphate/ carboxymethyl chitosan

Nanoparticles

Doxorubicin hydrochloride

[106]

number of scaffolds were prepared by varying the CMC and gelatin ratio and all the formulations were found to be highly biocompatible and support adhesion, spreading, growth, and proliferation of 3T3 mouse fibroblasts. However, hydrogels with higher CMC in the formulations resulted enhanced cell proliferation. In addition, these scaffolds were also able to release drugs and proteins in a sustained manner which was also helpful in tissue engineering applications.

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

451

In other research, nanohydroxyapatite was incorporated in an enzymatically cross-linked carboxymethyl
chitosan/gelatin matrix to obtain injectable gels that were investigated for their applicability in bone tissue engineering applications [110]. The prepared gel was susceptible to tyrosinase/p-cresol-mediated in situ gelling at physiological temperature without inflammation at the site of implantation which revealed its nonimmunogenic nature. Results from the in vitro study of osteoblast cell proliferation and differentiation showed that the gels were cytocompatible and supportive toward the growth and proliferation of murine primary osteoblasts. In vivo injectibility and gelation study in murine model revealed that the formulations were capable of producing stable in situ gels (Fig. 14.3) and the stability of the in situ formed gels were dependent on the degree of cross-linking and carboxymethyl
chitosan concentration. These results clearly indicated the potential of tyrosinase/p-cresol cross-linked carboxymethyl
chitosan/gelatin/nanohydroxyapatite injectable gels in treating irregular small bone defects with minimal clinical invasion as well as for bone cell delivery.

FIGURE 14.3 Macroscopic image of postmortem mouse showing the location and texture of carboxymethyl
chitosan/gelatin/nanohydroxyapatite injectable gel after 24 h of implantation. Reprinted with permission from D. Mishra, B. Bhunia, I. Banerjee, P. Datta, S. Dhara, T.K.Maiti, Enzymatically crosslinked carboxymethyl
chitosan/gelatin/nano-hydroxyapatite injectable gels for in situ bone tissue engineering application, Mater. Sci. Eng. C. 31 (7) (2011) 1295
1304.

Handbook of Chitin and Chitosan

452

14. Biomedical applications carboxymethyl chitosans

14.6.1.4 Targeted drug delivery CMC is water soluble containing
COOH and
NH2 groups where the targeting ligands can be attached which ultimately leads to the development of the targeted drug delivery system. Folic acid (FA)modified CMC (FCC) can be a good candidate for receptor targeted drug delivery application. FA was conjugated to amino groups of CMC by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)mediated reaction [111]. FCC was able to form self-assembled nanoparticles by undergoing intra- and/or intermolecular interaction between FA moieties in aqueous solution. An anticancer drug, DOX was loaded into nanoparticles to study its in vitro release profiles. It was found that the nanoparticles showed initial burst release of drug followed by a sustained release up to 7 days. In vitro antitumor activity showed that DOX was released in a continuous way from the nanoparticles (inside the cells) without losing its activity. Moreover, DOX-loaded FCC showed increased inhibitory activity because the cell uptake efficiency of FCC nanoparticles were higher due to a specific interaction with Hela cells containing overexpressed folate receptors via ligand
receptor recognition. A similar result was obtained in another study where confocal microscopy and flow cytometric analysis revealed that folate receptor-mediated targeting system significantly enhanced the cellular uptake of the folate-modified CMC nanoparticles and thus facilitated apoptosis of cancer cells (HeLa, B16F1) [112]. Glycyrrhizin-modified O-CMC nanoparticles were prepared by Shi et al. and investigated for hepatocellular carcinoma-targeted delivery of PTX [113]. PTX was loaded into the nanoparticles with maximum encapsulation efficiency of 83.7% and its release showed an initial burst release followed by slower and sustained release. Biodistribution results revealed that glycyrrhizin-modified O-CMC nanoparticles enhanced the accumulation of PTX in hepatic tumor tissues and the targeted delivery of PTX to hepatoma carcinoma cells, which resulted in remarkable enhancement of in vitro cytotoxicity and in vivo tumor inhibition compared to PTX injection and unmodified O-CMC nanoparticles. Transferrin receptor-mediated targeting is also possible with CMC because transferrin receptors are found to be overexpressed in malignant cells. In a study, transferrin-conjugated lauric acid-O-CMC micelles were prepared and loaded with an anticancer drug paclitaxel (PTX) for its site-specific targeted delivery [114]. In vitro cytotoxicity and hemolytic potential analysis showed that the prepared micelles were safe for intravenous administration as they possessed very low hemolytic activity. The results of anticancer activity, confocal laser scanning microscopy, and flow cytometry analysis confirmed that transferrin-modified lauric acid-O-CMC micelles were quickly taken up by cancer cells via

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

453

receptor-mediated endocytosis facilitated by the transferrin ligand of micelles and the overexpressed transferrin receptors present on the surface of the cancer cells. There are also some other targeting ligands which can be attached to CMC to achieve site-specific targeted drug delivery, for example, octreotide to target somatostatin receptors [115] and cetuximab to target epidermal growth factor receptors overexpressed in cancer cells [116]. Moreover, magnetic nanoparticles like iron oxide nanoparticles can be incorporated into CMC and utilized in targeted cancer diagnostic and therapy [117]. 14.6.1.5 Gene therapy In a study conducted by Anitha et al. CMC derivatives were combined with curcumin forming curcumin-loaded O-CMC nanoparticles which aimed to evaluate the ability of O-CMC as a carrier for hydrophobic drugs in cancer drug delivery applications. CMC is widely known for its diversified biomedical applications because of its biodegradability and nontoxicity, whereas curcumin is rich with many efficient biological properties. The in vitro drug release profile was studied and the cytotoxicity data revealed that these nanoparticles were nontoxic to normal healthy cells and toxic to cancer cells [94]. In a separate study Xie et al. synthesized a pH-sensitive polymer of PEG-grafted CMC (PEG-CMCS) nanoparticles with calcium phosphate (CaP) for siRNA delivery. The practical application of this nanoparticle on tumor-containing mice through intravenous injection showed an accumulation of the particles in the targeted tumor region by EPR effect. This ultimately led to inhibition of the growth of the tumor via silencing hTERT expression and inducing cell apoptosis in HepG2 tumor xenograft [118]. 14.6.1.6 Antibacterial agents Nanogels prepared from CMC and PVA have been reported as agents capable of capturing or isolating bacteria and fungi from the aquatic environment. Several analytical methods were applied to investigate the various properties of the nanogels of numerous compositions relevant to the final application. Beside antimicrobial property, these nanogels were observed with satisfying pH sensitivity, swelling behavior, and improved surface property. It was reported that after 2 h of swelling, upto 500% of water can be absorbed. In case of antibacterial properties this gel is capable of functioning against two types of bacteria E. coli (G 2 ) and S. aureus (G 1 ), whereas two types of fungi can be affected by the prepared nanogels, namely Aspergillus flavus and C. albicans [119].

Handbook of Chitin and Chitosan

454

14. Biomedical applications carboxymethyl chitosans

In the case of application, CMC can be transformed into various usable forms like film, gel, or fiber [120] accompanied by other polymers. Besides these, it is also possible to graft polymer onto CMC under different reaction condition. Such a phenomenon was described by Sabaa et al. where poly(N-vinyl imidazole) (PNVI) was grafted onto CMC in aqueous solution using potassium persulphate (KPS) as initiator. Successful grafting was confirmed by various analytical techniques and the antimicrobial activity of these grafted products were also evaluated. It was observed that the grafted product performed as a better antimicrobial agent compared to pristine CMC. Chitosan, CMC, and their grafted copolymers lowered the viable cell counts of S. aureus and E. coli. The antibacterial activities of these derivatives against E. coli are stronger than S. aureus. Also grafting of vinyl imidazole onto chitosan and CMC was successful in inhibiting fungal growth. The increase in grafting percentage leads to higher inhibition of fungal growth as well as the reaction condition needed for grafting is also satisfactory and controllable [121]. From the very beginning of modern civilization, textile materials used in hospitals and hotels have experienced the possibility of crossinfection or transmission of diseases caused by microorganisms. One of the possible solutions to this problem could be imparting antimicrobial properties to textile materials either chemically or physically, by incorporating functional agents onto fibers or fabrics. In searching for this kind of material, Shafei et al. reported that a bionanocomposite prepared from CMC and ZnO particles can be used as a finishing agent for cotton fabric for microbial and UV radiation protection. Finished cotton fabric gives excellent antibacterial properties against Gram-positive and Gram-negative bacteria which increased with an increase in the composite concentration and resulted a good UV protection which also increased with an increase in the curing temperature. Other than using the composite material in the living body as an antimicrobial agent, these researchers found an innovative way to incorporate the composite material as an antimicrobial agent with some easier production steps. The optimum reaction proceeding at 50 C results in the formation of smaller nanoparticles with respect to the reaction carried out in water at 25 C or 90 C. In both cases, the nanoparticles appear to be nearly spherical with a narrow size range. The mean sizes of ZnO and CMC particles were  28 nm and  100 nm, respectively [122]. 14.6.1.7 Hydrogels for medicinal use For the last few decades, hydrogels have become a very prominent medium for drug delivery in living organisms. Hydrogels fabricated from naturally occurring biodegradable materials appeal to the users because of their low toxicity, low cost, and availability. In a continuation

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

455

of this theme, Li et al. developed a covalently cross-linked hydrogel composed of N,O-CMC and oxidized alginate, intended for drug delivery application. Several in vitro tests like cytocompatibility, acute cytotoxicity, subcutaneous injection test, skin irritation test, and hemolysis were performed and showed promising results for them to be used for drug delivery purposes. In vitro cytocompatibility tests showed that the developed hydrogel exhibited good cytocompatibility against NH3T3 cells after a 3-day incubation. The prepared hydrogel showed a negative cytotoxicity result as well as negative cutaneous reaction within 72 h of subcutaneous injection. Moreover, slow degradation and adsorption within the time as well as 0% hemolysis ratio makes it more convenient for further application within the living body. All these results strongly suggest that this CMC-based hydrogel can be considered as a promising agent for drug delivery [123]. In a recent study, UV radiation was applied for the fabrication of hydrogel from CMC associated by N-isopropyl acrylamide and glycidyl methacrylate for efficient grafting. The prepared hydrogel is observed with pH and temperature sensitivity for application as a carrier for anticancer and antiinflammatory drugs. The pH and temperatureresponsive nature of the hydrogel was evaluated by releasing anticancer and anti-inflammatory drugs and for this purpose two different drugs, namely 5-fluorouracil (anticancer) and diclofenac sodium (anti-inflammatory), were selected by the author. The detail of this study was provided in Section 14.6.1.2 [87]. Another variation of the CMC based hydrogel is available in the bead form and which is also injectable. This advanced form of hydrogels were introduced by Luo et al. In this case, solvent controlled hydrogel beads were fabricated from CMC by chemical modification. Glutaraldehyde was used as cross-linking agent for further hydrogel integrity as well as introducing substantial morphology prior to drying. In this case solvent is given equal importance along with other components which enables a satisfactory release of the hydrophobic drugs or nutrients. For instance, the aqueous-30% alcoholic binary solvent is used for obtaining smooth surfaces of the spherical beads. Enhanced hydrophobicity, reduced surface charge, interchain H-bonding, and higher chain entanglement were reported to be responsible for spherical beads formation. The prepared hydrogels are considered to be a better media for vitamin D3 encapsulation by 96.9%. Besides in vitro release of the hydrophobic model nutrient from the hydrogel was studied in a simulated gastrointestinal condition which results in a sustained release of the drug or nutrient. Furthermore, the effects of freeze-dried and room temperature dried beads were investigated in terms of swelling behaviors and release properties in simulated gastric and intestinal fluid. It was observed that, in gastric fluid media of pH 1.2, burst

Handbook of Chitin and Chitosan

456

14. Biomedical applications carboxymethyl chitosans

swelling was recorded with two times the swelling ratio in room temperature dried beads, whereas it was 10 times in freeze-dried beads within 2 h. Similarly, higher sustained swelling ratio was found in the case of freeze-dried beads compared to room temperature dried ones in intestinal fluid after 6 h. However, an irregular swelling nature was observed for freeze-dried hydrogels in intestinal fluid (pH 7.4). This encapsulation efficiency and sustained release profile have made this hydrogel a promising medium not only for drug delivery but also for enzyme immobilization and protein encapsulation. However, further investigation is required to use the material in living cells [124]. In another study, water soluble N,O-CMC (NOCMC) was applied by Lin et al. to deliver site-specific protein drug (bovine serum albumin was used as a model drug) in the intestine. Similar to the previous study, the bead form of the alginate-blended NOCMC was preferred, where the complex alginate
NOCMC mixture was turned to microencapsulated beads by dropping it into a Ca21 ion solution. This bead is capable of restoring the bioactivity of the delivered protein drug, which is due to the procedures applied in a neutral aqueous medium. It was also recorded that increasing the concentration of alginate
NOCMC in the hydrogel increases the effective cross-linking density of the beads, allowing it to capture the maximum amount of drug (up to 77%). This scenario is observed at pH 7.4, which is the pH of the intestine, whereas a neutral scenario is experienced in the case of the acidic solution (pH 5 1.2). It was observed that at pH 1.2, increasing the concentration of alginate or NOCMC doesn’t show any positive impact on the drug encapsulation as like at pH 7.4. Based on the swelling nature of the hydrogel beads an alginate-to-NOCMC weight ratio of 1:1 was preferred. A similar pattern was observedfor swelling behavior as for drug encapsulation at pH 7.4 and 1.2. By increasing the total concentration of alginate-NOCMC, a large swelling force created by the electrostatic repulsion between the ionized acid groups (
COO2), the swelling ratios of test beads increased significantly (20.0
40.0) at pH 7.4. In acidic environment (pH 1.2), the swelling ratios of all test groups were approximately the same. The retention of drug into the hydrogel beads at pH 1.2 was facilitated by rinsing the beads with acetone, which really slows down the bovine serum albumine (BSA) release (below 15%) into this simulated acidic environment. At pH 7.4 the amounts of BSA released was increased significantly (B 80%) as compared to those released at pH 1.2. Considering these facts, this polymeric hydrogel beads can be recommended for oral protein drug delivery, as it could perform satisfactorily in the various regions of the intestine [125]. In a different study, a temperature and pH sensitive semi-interpenetrating polymer networks (IPN) hydrogel was fabricated using CMC as one of the major ingredients. Besides this element, poly(N-isopropylacrylamide) was

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

457

also added as the second important raw material, and N,N0 methylenebisacrylamide was preferred as an active cross-linking agent. To find the optimum composition of the raw materials, hydrogels based on various compositions were fabricated and also investigated accordingly. The swelling characteristics of these hydrogels as a function of pH and temperature (at distinct compositions) were evaluated. Like many other hydrogelbased delivery systems, this is also based on the oral route of administration. Other than protein, nutrients, vitamin, or typical drugs, this hydrogel is best suited for enzyme delivery. As a model enzyme, coenzyme A (CoA) was selected. Considering the pH sensitivity, CoA release of 22.6% was recorded at pH 2.1, whereas 89.1% was found at solution pH of 7.4. At both pHs, the temperature was kept constant at 37 C. Comparing the release profile of CoA, the authors investigated a higher release when the temperature was 37 C compared to 25 C, while maintaining the pH at 7.4. However, increasing the CMC content in the hydrogel acceleratedthe release of the captured enzyme at 25 C. All the data studied revealed that this semi-IPN hydrogel can be proposed as a promising media for pH
temperature-responsive orally administered drug delivery system [126]. A radiation-induced cross-linked hydrogel was prepared by using AgNO3 solution, CMC, and gelatin solution to be applied as a woundhealing material. The porous structure exhibited antibacterial property on E. coli along with a capacity to absorb 62 to 108 times of deionized water to its dry weight. It is also stated by the authors that the enhanced antibacterial property and increased particle size could be found by increasing the silver particle content in the prepared hydrogel. Besides introducing the antibacterial property, it is also found that this silver particle is responsible for antiinflammatory property shown by the hydrogel. Compressive strength of the hydrogel is another important factor to be considered for the use of the material for wound-covering purposes. This fabricated hydrogel is observed with a compressive modulus of 44 to 56 kPa, which is considered to be better suited for the desired application. Furthermore, a porous three-dimensional network structure with smooth surface was obtained with various silver nanoparticle concentrations, which was confirmed by scanning electron microscopic analysis. Silver particles were not visible in the microscopy images (SEM) due to their small size and this kind of porous structure is desired for the transport of oxygen and promotion of drainage. Focusing on the preparation technique, the radiation induced crosslinking and reduction process results in a stable and homogeneous distribution of nanosilver particles in the hydrogel matrix (Fig. 14.4) [127] For any hydrogel, a porous structure and high swelling ratio are considered as the most common features. Continuing the search for a advanced hydrogel, at the end of the 20th century, scientists developed the new idea of a hydrogel which is able to swell by several hundred

Handbook of Chitin and Chitosan

458

14. Biomedical applications carboxymethyl chitosans

FIGURE 14.4 SEM images of nanosilver/gelatin/CMC composite hydrogels with AgNO3 concentrations of (A) 0 mM, (B) 0.5 mM, (C) 1 mM, (D) 2 mM, (E) 5 mM, and (F) 10 mM [127].

times within a few minutes. This introduced a new dimension in the era of natural hydrogels, named “superporous hydrogels (SPH).” This very first type of SPH is called first-generation SPH, and in general is known as conventional SPH (CSPH). However, this CSPH showed poor mechanical strength and thus researchers kept searching for modified SPH with better mechanical properties. And this developed the idea of second and third-generation SPH. Besides these, a mucoadhesive controlled delivery system for protein drugs (via the gastric intestinal tract) has been developed to enhance the drug absorption process in a sitespecific manner. Owing to their high water affinity and biocompatibility, hydrogels based on mucoadhesive polymers, such as chitosan, alginate, poly(acrylic acid) and its derivatives, and collagen, have attracted the attention of the researchers [128]. As a part of the third-generation hydrogel with mucoadhesive controlled delivery systems, Yen et al. developed a SPH containing poly (acrylic acid-co-acrylamide)/O-CMC IPN. At first, a semi-IPN was developed which further converted into full IPN. The semi-IPN introduced SPH leaving the O-CMC site open for bonding. When glutaraldehyde (GA) is added in this SPH, the nonbonded O-CMC sites get

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

459

attached to this GA forming the final superporous hydrogel for the desired application. In this study, O-CMC was selected for its biocompatibility, good water solubility, and mucoadhesion [128]. SPH-IPNs contained both IPN network and large numbers of pores wherethe cross-linked O-CMC molecules were located on the peripheries of these pores. One of the major properties of any hydrogel is its swelling nature, and in this case this property is dominated by the OCMC content, amount of GA, and cross-linking time. After the fabrication of the final material the compressive strength, tensile strength, swelling behavior, and mucoadhesive force were investigated and the results suggested that the SPH-IPN may be a suitable candidate for a mucosal drug delivery system, especially for effective peroral delivery of peptide and protein drugs. Therefore, as a model protein-based drug, insulin was selected to study the drug-loading capacities of the prepared SPH-IPNs as well as the drug release pattern was also investigated in a simulated intestinal medium and both showed convincing data for the sue of this SPH-IPN in protein drug delivery [128]. Most of the scientific studies use a model drug for analyzing the usability of the prepared CMC based hydrogel for delivering the drug. Vaghani et al. incorporated the drug at the time of the cross-linking operation between CMC and glutaraldehyde. Ornidazole was used as the drug and the hydrogel was proven to be a potential carrier for colon-targeted delivery of this drug. Several analyses proved that the drug remained significantly unchanged on being captured by the hydrogel. However, the swelling behavior of the hydrogel was checked based on three different pH values (1.2, 6.8, and 7.4) and the results concluded that pH 1.2 and 6.8 gives slow and quick swelling, respectively whereas pH 7.4 results in a linear swelling behavior. Based on the results obtained from various analytical data, it was stated by the authors that the prepared hydrogels can be used as promising carriers for colon-specific delivery of ornidazole [129]. 14.6.1.8 Bioimaging As a part of multifunctional properties, nanoparticles offer scope for targeted drug delivery with imaging agents. At present, researchers have considered bioimaging as a very useful and promising technique in targeting, drug delivering, and imaging of many parts of the living cells. Polymeric material is always being considered as a very versatile agent for various routes of application in this sector. Especially in delivering multiple pharmacological agents, specifically to enhance therapeutic effect and overcome drug resistance in cancer treatment, these materials are always the top in demand. Mathew et al. developed a CMC-based system including folic acid (FA), zinc sulfide, and manganese to produce quantum dot nanoparticles. These FA-conjugated CMC-coordinated
manganese-doped zinc

Handbook of Chitin and Chitosan

460

14. Biomedical applications carboxymethyl chitosans

sulfide quantum dot (FA
CMC
ZnS:Mn) nanoparticles can be used for targeting, controlled drug delivery, and also for imaging breast cancer cells. As an anticancer drug 5-Fluorouracil (5-FU) is used. The prepared nanoparticles are recorded as nontoxic to mouse fibroblast L929 cells, and as a part of the cancer cells imaging technique fluorescent microscopy was preferred. In this imaging process, without affecting the metabolic activity and morphology of the supplied drug, a bright and stable luminescence of quantum dots can be used to image the drug carrier at the cancer cells. Considering the importance of CMC in this process it is a water-soluble chitosan derivative in which the
CH2OH group of each monomer substituted by
COOH group and thereby preferred over chitosan. Moreover, at neutral pH values, the absorption efficiency of chitosan is so poor that the researchers tend to develop a derivative which can considerably overcome these restraints. The preparation technique is simple and the drug encapsulation efficiency obtained was 92.08% with CMC to 5-FU ratio 2:1. The in vitro drug release studies showed controlled release of 5-FU from fabricated nanoparticles. In addition, the nanoparticles without drug encapsulation can be used as a potential bioprobe for the delivery of genes and proteins with simultaneous imaging [130]. CMC can be used as a shell material for preparing multifunctional core
shell magnetic nanostructures using Fe3O4 as the magnetic element. Besides this iron compound, several other elements are used for various purposes in the final drug delivery agent. In the core
shell structure Fe3O4
CdTe@SiO2 provides the magnetic and fluorescent property. Then CMC started to form a covalent bond with the silanol group with Fe3O4
CdTe@SiO2. As a model drug DOX is incorporated in the next step. The overall process ends with the shell prepared by CMC and poly(acrylic acid). This multistep fabrication process of the nanocarriers is very crucial for enabling some distinct and diversified properties in the product, such as magnetic targeting and FL properties, colloidal stability and pH-regulated drug release, good biocompatibility, and a cancer therapeutic effect. The fabricated nanocarriers showed an average size of 160 nm, good DOX-loading content (7.06%), and encapsulation efficiency (up to 76.8%), and they also maintained a satisfactory solubility and stability in the physiological environment [131]. 14.6.1.9 Biosensors Throughout recent years many biopolymers and their derivatives have been used to detect many useful or toxic elements in the environment. Continuing this research Tan et al. developed a novel material by using CMC to detect nitric oxide in the human body. They noted that nitric oxide (NO), a free radical molecule possessing very important functions in biological systems, is generated in various tissues from the

Handbook of Chitin and Chitosan

14.6 Application of carboxymethyl chitosan

461

amino acid (L-arginine) by different forms of NO synthase. Tan et al. formulated a novel system, i.e., cadmium telluride quantum dots (CdTe QD)
CMC nanocomposite (CdTe QDs-CMC) NO donors, which contain both diazeniumdiolates and FL probes. CMC is selected as a supporting material due to its water solubility and a great number of secondary amine groups, which are capable of reacting with NO to achieve high NO loading. The most popular NO donor is diazeniumdiolates, which actually represents a class of compounds containing the anionic [N(O)NO]2 functional groups that are typically synthesized by reactions of a nucleophile with NO at elevated pressure. However, some of the diazeniumdiolate carriers are harmful to the human body, thereby limiting their application in the treatment of relevant diseases. This specific group is capable of reacting with metal cations and can uptake the metal cations by a chelating mechanism. Moreover, a flexible polymer chain facilitates the formation of a suitable configuration for capturing metal ions. Focusing on the use of chitosan derivatives in this purpose, this polymeric diazeniumdiolate has proved an effective and reliable source of NO in a physiological environment. It is observed that the secondary amine groups in some chitosan derivatives can act as a nucleophile to which NO is attached. The resultant chitosan
NO complex has the [N(O)NO]2 groups and is capable of releasing NO sustainably. This polymeric diazeniumdiolate has proved to be an effective source of NO in a physiological environment. Finally, the QD
CMC nanocomposite NO donors may have potential biomedical applications under many possible physiological conditions (Fig. 14.5) [132].

14.6.2 Other applications Chitosan is a naturally occurring biopolymer with important biological properties, such as biocompatibility, antifungal, and antibacterial activity, wound-healing ability, anticancer property, anticholesteremic properties, and an immunoenhancing effect. Recently, chitosan nanoparticles have been used for biomedical applications. However, due to the limited solubility of chitosan in water, its water-soluble derivatives are preferred for the above said applications. In a work, the nanoparticles of chitosan and its water-soluble derivatives such as O-CMC and N,OCMC were synthesized and characterized. In addition, the cytotoxicity and antibacterial activity of the prepared nanoparticles were also evaluated for biomedical applications. Nanoparticles of CS, O-CMC, and N, O-CMC were synthesized by simple ionic cross-linking using TPP and CaCl2. The cytotoxicity of the prepared nanoparticles were also determined and the prepared nanoparticles showed less toxicity to breast cancer cells (almost 98% viability was found for breast cancer cells-MCF-7,

Handbook of Chitin and Chitosan

462

14. Biomedical applications carboxymethyl chitosans

FIGURE 14.5 (A) Schematic illustration of fluorescence effect and fluorescence quenching by NO of CdTe QD
CMCS. (B) Fluorescence quenching of the CdTe QD
CMCS nanocomposite NO donors upon the release of NO in a phosphate-buffered saline solution [132].

which was treated with the nanoparticle samples). Antibacterial activities of these CS, O-CMC, and N,O-CMC nanoparticles were also studied with ATCC strains of S. aureus by the MIC method using three different concentrations of each of these three nanoparticles. These studies revealed that CS nanoparticles showed less antibacterial activity compared to O-CMC and N,O-CMC nanoparticles. This antibacterial effect was increased with an increase in concentration; N,O-CMC nanoparticles showed maximum antibacterial activity out of the three and no colonies were found for the last concentration with respect to the control; for O-CMC and CS nanoparticles, a fewer number of colonies were found with respect to the control for the last concentration under investigation [67]. As a protecting and stabilizing agent CMC is mixed with silver nanoparticles (AgNPs) along with polyethylene oxide to convert it into final nanofiber mat form. This mat is made from fiber prepared from the three component mixture (CMC, AgNPs, and CMC) by electrospinning technique. This mat is capable of forming a barrier against different species of pathogenic/nonpathogenic (S. aureus, P. aeruginosa, E. coli, and C. albicans). In addition, the prolonged aging period is responsible for converting the Ag1 ion to Ag0. This mat is suggested for many biological applications such as prolonged antimicrobial wound-dressing materials [133].

Handbook of Chitin and Chitosan

References

463

14.7 Conclusion CMC and CMC-based composites have been considered as important tools to fulfill the demand of modern applications because of their physical and chemical characteristics and the technical feasibility of their synthesis and utilization. Over the years researchers have developed a wide variety of CMC and modified CMC-based hydrogels, nanoparticles, quantum dots, and so on. Because of their controllable nature, they have become attractive materials for numerous fields of application, such as wound healing, drug delivery, tissue engineering, antibacterial activity, gene therapy, biosensors, and bioimaging. However, the different types of CMC fabrication require the correct control of experimental conditions. The ease of fabrication and modification of this specific CMC-based material is increasing due to its range of applications in different sectors; we hope that the current rate of advancements, especially in biomedical fields, will expose a promising scope in developing next generation advanced materials.

References [1] S. Biswas, T.U. Rashid, A.K. Mallik, M. Islam, M.N. Khan, P. Haque, et al., Facile preparation of biocomposite from prawn shell derived chitosan and kaolinite-rich locally available clay, Int. J. Polym. Sci. 2017 (2017). [2] M.M. Islam, M.N. Khan, S. Biswas, T.R. Choudhury, P. Haque, T.U. Rashid, et al., Preparation and characterization of bijoypur clay-crystalline cellulose composite for application as an adsorbent, Adv. Mater. Sci. 2 (3) (2017) 1
7. [3] S. Biswas, M.M. Islam, M.M. Hasan, S.H. Rimu, M.N. Khan, P. Haque, et al., Evaluation of Cr (VI) ion removal from aqueous solution by bio-inspired chitosanclay composite: kinetics and isotherms, Iran. J. Chem. Eng. 15 (4) (2018). [4] E. Poverenov, H. Arnon, Y. Zaicev, V. Bar, O. Danay, B. Horev, et al., Potential of chitosan from mushroom waste to enhance quality and storability of fresh-cut melons, Food Chem. (2018). [5] T.D.A. Senra, S.P. Campana-Filho, J. Desbrie`res, Surfactant-polysaccharide complexes based on quaternized chitosan. Characterization and application to emulsion stability, . Eur. Polym. J. 104 (2018) 128
135. [6] M.S. Islam, P. Haque, T.U. Rashid, M.N. Khan, A.K. Mallik, M.N.I. Khan, et al., Core
shell drug carrier from folate conjugated chitosan obtained from prawn shell for targeted doxorubicin delivery, J. Mater. Sci. Mater. Med. 28 (4) (2017) 55. [7] M. Prabaharan, Chitosan derivatives as promising materials for controlled drug delivery, J. Biomater. Appl. 23 (1) (2008) 5
36. [8] C. Choi, J.-P. Nam, J.-W. Nah, Application of chitosan and chitosan derivatives as biomaterials, J. Ind. Eng. Chem. 33 (2016) 1
10. [9] R. Riva, H. Ragelle, A. des Rieux, N. Duhem, C. Je´roˆme, V. Pre´at, Chitosan and chitosan derivatives in drug delivery and tissue engineering, Chitosan Biomater. II:, Springer, 2011, pp. 19
44. [10] H. Yang, D.H. Bremner, L. Tao, H. Li, J. Hu, L. Zhu, Carboxymethyl chitosanmediated synthesis of hyaluronic acid-targeted graphene oxide for cancer drug delivery, Carbohydr. Polym. 135 (2016) 72
78.

Handbook of Chitin and Chitosan

464

14. Biomedical applications carboxymethyl chitosans

[11] X. Liu, Y. Mo, X. Liu, R. Guo, Y. Zhang, W. Xue, et al., Synthesis, characterisation and preliminary investigation of the haemocompatibility of polyethyleneiminegrafted carboxymethyl chitosan for gene delivery, Mater. Sci. Eng. C. 62 (2016) 173
182. [12] M.A. Zarandi, P. Zahedi, I. Rezaeian, A. Salehpour, M. Gholami, B. Motealleh, Drug release, cell adhesion and wound healing evaluations of electrospun carboxymethyl chitosan/polyethylene oxide nanofibres containing phenytoin sodium and vitamin C, IET Nanobiotechnol. 9 (4) (2015) 191
200. [13] Z. Liu, H. Liu, L. Wang, X. Su, A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles, Anal. Chim. Acta 932 (2016) 88
97. [14] V.K. Mourya, N.N. Inamdar, A. Tiwari, Carboxymethyl chitosan and its applications, Adv. Mater. Lett. 1 (1) (2010) 11
33. [15] S. Kumari, P. Rath, A. Sri Hari Kumar, T.N. Tiwari, Extraction and characterization of chitin and chitosan from fishery waste by chemical method, Environ. Technol. Innov. 3 (2015) 77
85. [16] F. Sedaghat, M. Yousefzadi, H. Toiserkani, S. Najafipour, Bioconversion of shrimp waste Penaeus merguiensis using lactic acid fermentation: an alternative procedure for chemical extraction of chitin and chitosan, Int. J. Biol. Macromol. 104 (2017) 883
888. [17] H. El Knidri, R. Belaabed, A. Addaou, A. Laajeb, A. Lahsini, Extraction, chemical modification and characterization of chitin and chitosan: a review, Int. J. Biol. Macromol. (2018). [18] M.A. Surati, S. Jauhari, K.R. Desai, A brief review: microwave assisted organic reaction, Arch. Appl. Sci. Res. 4 (1) (2012) 645
661. [19] H. El Knidri, R. El Khalfaouy, A. Laajeb, A. Addaou, A. Lahsini, Eco-friendly extraction and characterization of chitin and chitosan from the shrimp shell waste via microwave irradiation, Process. Saf. Environ. Prot. 104 (2016) 395
405. [20] F.A.A. Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf, Carbohydr. Polym. 77 (2) (2009) 410
419. [21] X. Wang, B. Xia, J. Wu, Preparation and characterization of quaternized chitosan under microwave irradiation, J. Macromol. Sci. Part A 47 (9) (2010) 952
956. [22] M. Mahdy Samar, M.H. El-Kalyoubi, M.M. Khalaf, M.M. Abd El-Razik, Physicochemical, functional, antioxidant and antibacterial properties of chitosan extracted from shrimp wastes by microwave technique, Ann. Agric. Sci. 58 (1) (2013) 33
41. [23] M.M. Islam, S. Biswas, M.M. Hasan, P. Haque, S.H. Rimu, M.M. Rahman, Studies of Cr (VI) adsorption on novel jute cellulose-kaolinite clay biocomposite, Desalin. Water Treat. 123 (2018) 265
276. [24] S.M. Ahsan, M. Thomas, K.K. Reddy, S.G. Sooraparaju, A. Asthana, I. Bhatnagar, Chitosan as biomaterial in drug delivery and tissue engineering, Int. J. Biol. Macromol. 110 (2018) 97
109. ´ [25] E. Szymanska, K. Winnicka, Stability of chitosan—a challenge for pharmaceutical and biomedical applications, Mar. Drugs 13 (4) (2015). [26] M. Ahmad, K. Manzoor, S. Ikram, Chitosan based nanocomposites for drug, gene delivery, and bioimaging applications, in: A.A.M. Inamuddin, A. Mohammad (Eds.), Applications of Nanocomposite Materials in Drug Delivery, Woodhead Publishing, 2018, pp. 27
38. [27] D. Zhao, S. Yu, B. Sun, S. Gao, S. Guo, K. Zhao, Biomedical applications of chitosan and its derivative nanoparticles, Polymers 10 (4) (2018). [28] M. Kurniasih, Purwati, T. Cahyati, R.S. Dewi, Carboxymethyl chitosan as an antifungal agent on gauze, Int. J. Biol. Macromol. 119 (2018) 166
171.

Handbook of Chitin and Chitosan

References

465

[29] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, The implications of recent advances in carboxymethyl chitosan based targeted drug delivery and tissue engineering applications, J. Control. Release 186 (2014) 54
87. [30] L. Chen, Y. Du, X. Zeng, Relationships between the molecular structure and moistureabsorption and moisture-retention abilities of carboxymethyl chitosan: II. Effect of degree of deacetylation and carboxymethylation, Carbohydr. Res. 338 (4) (2003) 333
340. [31] Q. Xu, C. Mao, N.-N. Liu, J.-J. Zhu, J. Shen, Immobilization of horseradish peroxidase on O-carboxymethylated chitosan/sol
gel matrix, React. Funct. Polym. 66 (8) (2006) 863
870. [32] L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, Biomedical applications of carboxymethyl chitosans, Carbohydr. Polym. 91 (1) (2013) 452
466. [33] Y. Chen, Y.-f Liu, H.-m Tan, J.-x Jiang, Synthesis and characterization of a novel superabsorbent polymer of N, O-carboxymethyl chitosan graft copolymerized with vinyl monomers, Carbohydr. Polym. 75 (2) (2009) 287
292. [34] L. Wang, A. Wang, Adsorption behaviors of Congo red on the N, O-carboxymethylchitosan/montmorillonite nanocomposite, Chem. Eng. J. 143 (1
3) (2008) 43
50. [35] R.A.A. Muzzarelli, V. Ramos, V. Stanic, B. Dubini, M. Mattioli-Belmonte, G. Tosi, et al., Osteogenesis promoted by calcium phosphate N, N-dicarboxymethyl chitosan, Carbohydr. Polym. 36 (4) (1998) 267
276. [36] R. Muzzarelli, M. Weckx, O. Filippini, C. Lough, Characteristic properties of Ncarboxybutyl chitosan, Carbohydr. Polym. 11 (4) (1989) 307
320. [37] R.A.A. Muzzarelli, P. Ilari, M. Tomasetti, Preparation and characteristic properties of 5-methyl pyrrolidinone chitosan, Carbohydr. Polym. 20 (2) (1993) 99
105. [38] L. Zhao, F. Luo, J.M. Wasikiewicz, H. Mitomo, N. Nagasawa, T. Yagi, et al., Adsorption of humic acid from aqueous solution onto irradiation-crosslinked carboxymethylchitosan, Bioresour. Technol. 99 (6) (2008) 1911
1917. [39] R. Muzzarelli, R. Tarsi, O. Filippini, E. Giovanetti, G. Biagini, P.E. Varaldo, Antimicrobial properties of N-carboxybutyl chitosan, Antimicrob. Agents Chemother. 34 (10) (1990) 2019
2023. [40] Y. Shigemasa, A. Ishida, H. Sashiwa, H. Saimoto, Y. Okamoto, S. Minami, et al., Synthesis of a new chitin derivative,(1-carboxyethyl) chitosan, Chem. Lett. 24 (8) (1995) 623
624. [41] Y.A. Skorik, C.A.R. Gomes, M.T.S.D. Vasconcelos, Y.G. Yatluk, N-(2-Carboxyethyl) chitosans: regioselective synthesis, characterisation and protolytic equilibria, Carbohydr. Res. 338 (3) (2003) 271
276. [42] H. Sashiwa, N. Yamamori, Y. Ichinose, J. Sunamoto, S.-I. Aiba, Michael reaction of chitosan with various acryl reagents in water, Biomacromolecules 4 (5) (2003) 1250
1254. ˇ nanova ˇ ˇ ´ , Lv Kriˇzkova´, Z. Duraˇ [43] G. Kogan, Y.A. Skorik, I. Zit ckova´, C.A.R. Gomes, et al., Antioxidant and antimutagenic activity of N-(2-carboxyethyl) chitosan, Toxicol. Appl. Pharmacol. 201 (3) (2004) 303
310. [44] Y. Zhou, D. Yang, X. Gao, X. Chen, Q. Xu, F. Lu, et al., Semi-interpenetrating polymer network hydrogels based on water-soluble N-carboxylethyl chitosan and photopolymerized poly (2-hydroxyethyl methacrylate), Carbohydr. Polym. 75 (2) (2009) 293
298. [45] Y. Kato, H. Onishi, Y. Machida, N-succinyl-chitosan as a drug carrier: waterinsoluble and water-soluble conjugates, Biomaterials 25 (5) (2004) 907
915. [46] Z. Aiping, C. Tian, Y. Lanhua, W. Hao, L. Ping, Synthesis and characterization of N-succinyl-chitosan and its self-assembly of nanospheres, Carbohydr. Polym. 66 (2) (2006) 274
279. [47] Y.N. Dai, P. Li, J.P. Zhang, A.Q. Wang, Q. Wei, A novel pH sensitive N-succinyl chitosan/alginate hydrogel bead for nifedipine delivery, Biopharm. Drug. Dispos. 29 (3) (2008) 173
184.

Handbook of Chitin and Chitosan

466

14. Biomedical applications carboxymethyl chitosans

[48] Y. Kuroyanagi, A. Shiraishi, Y. Shirasaki, N. Nakakita, Y. Yasutomi, Y. Takano, et al., Development of a new wound dressing with antimicrobial delivery capability, Wound Repair. Regen. 2 (2) (1994) 122
129. [49] M. Izume, The application of chitin and chitosan to cosmetics, Chitin Chitosan Res. 4 (1998) 12
17. [50] C. Zhang, Q. Ping, H. Zhang, J. Shen, Synthesis and characterization of water-soluble O-succinyl-chitosan, Eur. Polym. J. 39 (8) (2003) 1629
1634. [51] W. Sui, Y. Wang, S. Dong, Y. Chen, Preparation and properties of an amphiphilic derivative of succinyl-chitosan, Colloids Surf. A: Physicochem. Eng. Asp. 316 (1
3) (2008) 171
175. [52] R.J.N. Hjerde, K.M. Va˚rum, H. Grasdalen, S. Tokura, O. Smidsrød, Chemical composition of O-(carboxymethyl)-chitins in relation to lysozyme degradation rates, Carbohydr. Polym. 34 (3) (1997) 131
139. [53] X.-G. Chen, H.-J. Park, Chemical characteristics of O-carboxymethyl chitosans related to the preparation conditions, Carbohydr. Polym. 53 (4) (2003) 355
359. [54] A. Zhu, M.B. Chan-Park, S. Dai, L. Li, The aggregation behavior of O-carboxymethylchitosan in dilute aqueous solution, Colloids Surf. B: Biointerfaces 43 (3
4) (2005) 143
149. [55] H.T. Pang, X.G. Chen, H.J. Park, D.S. Cha, J.F. Kennedy, Preparation and rheological properties of deoxycholate-chitosan and carboxymethyl-chitosan in aqueous systems, Carbohydr. Polym. 69 (3) (2007) 419
425. [56] S.G.F. Felicio, M.R. Sierakowski, C.L. de Oliveira Petkowicz, J.L.M. Silveira, A.F. Lubambo, R.A. de Freitas, Spherical aggregates obtained from N-carboxymethylation and acetylation of chitosan, Colloid Polym. Sci. 286 (12) (2008) 1387
1394. [57] M. Thanou, J.C. Verhoef, H.E. Junginger, Oral drug absorption enhancement by chitosan and its derivatives, Adv. Drug. Deliv. Rev. 52 (2) (2001) 117
126. [58] S. Matsumura, H.C. Cheng, M. Minami, S. Yoshikawa, T. Kariyone, Synthesis and water uptake and holding capacity of chitin and chitosan derivatives, J. Jpn. Oil Chem. Soc. 38 (6) (1989) 492
500. [59] R.A.A. Muzzarelli, Carboxymethylated chitins and chitosans, Carbohydr. Polym. 8 (1) (1988) 1
21. [60] L. Chen, Y. Du, H. Wu, L. Xiao, Relationship between molecular structure and moisture-retention ability of carboxymethyl chitin and chitosan, J. Appl. Polym. Sci. 83 (6) (2002) 1233
1241. [61] X. Wang, Y. Du, H. Liu, Preparation, characterization and antimicrobial activity of chitosan
Zn complex, Carbohydr. Polym. 56 (1) (2004) 21
26. [62] J.M. Nieto, C. Peniche-Covas, J. Del Bosque, Preparation and characterization of a chitosan-Fe (III) complex, Carbohydr. Polym. 18 (3) (1992) 221
224. [63] R.A.A. Muzzarelli, F. Tanfani, M. Emanuelli, S. N.- Mariotti, carboxymethylidene) chitosans and N-(carboxymethyl) chitosans: novel chelating polyampholytes obtained from chitosan glyoxylate, Carbohydr. Res. 107 (2) (1982) 199
214. [64] L. Sun, Y. Du, L. Fan, X. Chen, J. Yang, Preparation, characterization and antimicrobial activity of quaternized carboxymethyl chitosan and application as pulp-cap, Polymer 47 (6) (2006) 1796
1804. [65] M. Wang, L. Xu, M. Zhai, J. Peng, J. Li, G. Wei, γ-ray radiation-induced synthesis and Fe (III) ion adsorption of carboxymethylated chitosan hydrogels, Carbohydr. Polym. 74 (3) (2008) 498
503. [66] X. Fei Liu, Y. Lin Guan, D. Zhi Yang, Z. Li, K. De Yao, Antibacterial action of chitosan and carboxymethylated chitosan, J. Appl. Polym. Sci. 79 (7) (2001) 1324
1335. [67] A. Anitha, V.V.D. Rani, R. Krishna, V. Sreeja, N. Selvamurugan, S.V. Nair, et al., Synthesis, characterization, cytotoxicity and antibacterial studies of chitosan,

Handbook of Chitin and Chitosan

References

[68] [69] [70]

[71]

[72] [73] [74]

[75]

[76]

[77] [78]

[79]

[80]

[81] [82]

[83]

[84]

[85]

467

O-carboxymethyl and N, O-carboxymethyl chitosan nanoparticles, Carbohydr. Polym. 78 (4) (2009) 672
677. T. Sun, D. Zhou, J. Xie, F. Mao, Preparation of chitosan oligomers and their antioxidant activity, Eur. Food Res. Technol. 225 (3
4) (2007) 451
456. T. Feng, Y. Du, J. Li, Y. Hu, J.F. Kennedy, Enhancement of antioxidant activity of chitosan by irradiation, Carbohydr. Polym. 73 (1) (2008) 126
132. M. Anraku, M. Kabashima, H. Namura, T. Maruyama, M. Otagiri, J.M. Gebicki, et al., Antioxidant protection of human serum albumin by chitosan, Int. J. Biol. Macromol. 43 (2) (2008) 159
164. Z. Guo, R. Xing, S. Liu, H. Yu, P. Wang, C. Li, et al., The synthesis and antioxidant activity of the Schiff bases of chitosan and carboxymethyl chitosan, Bioorg. Med. Chem. Lett. 15 (20) (2005) 4600
4603. A. Verlee, S. Mincke, C.V. Stevens, Recent developments in antibacterial and antifungal chitosan and its derivatives, Carbohydr. Polym. 164 (2017) 268
283. E. Costa, S. Silva, F. Tavaria, M. Pintado, Antimicrobial and antibiofilm activity of chitosan on the oral pathogen Candida albicans, Pathogens 3 (4) (2014) 908
919. F. Seyfarth, S. Schliemann, P. Elsner, U.C. Hipler, Antifungal effect of high-and lowmolecular-weight chitosan hydrochloride, carboxymethyl chitosan, chitosan oligosaccharide and N-acetyl-D-glucosamine against Candida albicans, Candida krusei and Candida glabrata, Int. J. Pharm. 353 (1
2) (2008) 139
148. Y. Tan, S. Ma, C. Liu, W. Yu, F. Han, Enhancing the stability and antibiofilm activity of DspB by immobilization on carboxymethyl chitosan nanoparticles, Microbiol. Res. 178 (2015) 35
41. Y. Tan, M. Leonhard, D. Moser, B. Schneider-Stickler, Antibiofilm activity of carboxymethyl chitosan on the biofilms of non-Candida albicans Candida species, Carbohydr. Polym. 149 (2016) 77
82. M. Hasegawa, K. Yagi, S. Iwakawa, M. Hirai, Chitosan induces apoptosis via caspase-3 activation in bladder tumor cells, Jpn. J. Canc. Res. 92 (4) (2001) 459
466. Q. Chen, S.-Q. Liu, Y.-M. Du, H. Peng, L.-P. Sun, Carboxymethyl-chitosan protects rabbit chondrocytes from interleukin-1β-induced apoptosis, Eur. J. Pharmacol. 541 (1
2) (2006) 1
8. W. Hongbin, D. Jingyuan, C. Linyun, D. Yuming, Carboxymethylated chitin reduces MMP-1 expression in rabbit ACLT osteoarthritic cartilage, Ann. Rheum. Dis. 63 (4) (2004) 369
372. S.-Q. Liu, B. Qiu, Chen L-y, H. Peng, Y.-M. Du, The effects of carboxymethylated chitosan on metalloproteinase-1, 2 3 and tissue inhibitor of metalloproteinase-1 gene expression in cartilage of experimental osteoarthritis, Rheumatol. Int. 26 (1) (2005) 52
57. S. Peng, W. Liu, B. Han, J. Chang, M. Li, X. Zhi, Effects of carboxymethyl-chitosan on wound healing in vivo and in vitro, J. Ocean. Univ. China 10 (4) (2011) 369
378. J. Chang, W. Liu, B. Han, S. Peng, B. He, Z. Gu, Investigation of the skin repair and healing mechanism of N-carboxymethyl chitosan in second-degree burn wounds, Wound Repair. Regen. 21 (1) (2013) 113
121. R.L. Patale, V.B. Patravale, O, N-carboxymethyl chitosan
zinc complex: A novel chitosan complex with enhanced antimicrobial activity, Carbohydr. Polym. 85 (1) (2011) 105
110. X. Huang, Y. Zhang, X. Zhang, L. Xu, X. Chen, S. Wei, Influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing, Mater. Sci. Eng. C. 33 (8) (2013) 4816
4824. Y. Zu, Y. Zhang, W. Wang, X. Zhao, X. Han, K. Wang, et al., Preparation and in vitro/in vivo evaluation of resveratrol-loaded carboxymethyl chitosan nanoparticles, Drug. Deliv. 23 (3) (2016) 971
981.

Handbook of Chitin and Chitosan

468

14. Biomedical applications carboxymethyl chitosans

[86] H. Zheng, Y. Rao, Y. Yin, X. Xiong, P. Xu, B. Lu, Preparation, characterization, and in vitro drug release behavior of 6-mercaptopurine-carboxymethyl chitosan, Carbohydr. Polym. 83 (4) (2011) 1952
1958. [87] L. Zhang, L. Wang, B. Guo, P.X. Ma, Cytocompatible injectable carboxymethyl chitosan/N-isopropylacrylamide hydrogels for localized drug delivery, Carbohydr. Polym. 103 (2014) 110
118. [88] X. Wang, Y. Chen, F.Z. Dahmani, L. Yin, J. Zhou, J. Yao, Amphiphilic carboxymethyl chitosan-quercetin conjugate with P-gp inhibitory properties for oral delivery of paclitaxel, Biomaterials 35 (26) (2014) 7654
7665. [89] Y.-I. Jeong, S.-G. Jin, I.-Y. Kim, J. Pei, M. Wen, T.-Y. Jung, et al., Doxorubicinincorporated nanoparticles composed of poly (ethylene glycol)-grafted carboxymethyl chitosan and antitumor activity against glioma cells in vitro, Colloids Surf. B: Biointerfaces 79 (1) (2010) 149
155. [90] A. Anitha, K.P. Chennazhi, S.V. Nair, R. Jayakumar, 5-flourouracil loaded N, O-carboxymethyl chitosan nanoparticles as an anticancer nanomedicine for breast cancer, J. Biomed. Nanotechnol. 8 (1) (2012) 29
42. [91] L.-Q. Yang, Y.-Q. Lan, H. Guo, L.-Z. Cheng, J.-Z. Fan, X. Cai, et al., Ophthalmic drug-loaded N, O-carboxymethyl chitosan hydrogels: synthesis, in vitro and in vivo evaluation, Acta Pharmacol. Sin. 31 (12) (2010) 1625. [92] Kumar Singh Yadav H, Shivakumar HG. In vitro and in vivo evaluation of pHsensitive hydrogels of carboxymethyl chitosan for intestinal delivery of theophylline. ISRN Pharm. 2012; 2012. [93] G. Alamdarnejad, A. Sharif, S. Taranejoo, M. Janmaleki, M.R. Kalaee, M. Dadgar, et al., Synthesis and characterization of thiolated carboxymethyl chitosan-graftcyclodextrin nanoparticles as a drug delivery vehicle for albendazole, J. Mater. Sci. Mater. Med. 24 (8) (2013) 1939
1949. [94] A. Anitha, S. Maya, N. Deepa, K.P. Chennazhi, S.V. Nair, H. Tamura, et al., Efficient water soluble O-carboxymethyl chitosan nanocarrier for the delivery of curcumin to cancer cells, Carbohydr. Polym. 83 (2) (2011) 452
461. [95] S. Maya, S. Indulekha, V. Sukhithasri, K.T. Smitha, S.V. Nair, R. Jayakumar, et al., Efficacy of tetracycline encapsulated O-carboxymethyl chitosan nanoparticles against intracellular infections of Staphylococcus aureus, Int. J. Biol. Macromol. 51 (4) (2012) 392
399. [96] K.S. Snima, R. Jayakumar, A.G. Unnikrishnan, S.V. Nair, V.-K. Lakshmanan, O-Carboxymethyl chitosan nanoparticles for metformin delivery to pancreatic cancer cells, Carbohydr. Polym. 89 (3) (2012) 1003
1007. [97] N. Joshi, R. Saha, T. Shanmugam, B. Balakrishnan, P. More, R. Banerjee, Carboxymethyl-chitosan-tethered lipid vesicles: hybrid nanoblanket for oral delivery of paclitaxel, Biomacromolecules 14 (7) (2013) 2272
2282. [98] C. Feng, Z. Wang, C. Jiang, M. Kong, X. Zhou, Y. Li, et al., Chitosan/o-carboxymethyl chitosan nanoparticles for efficient and safe oral anticancer drug delivery: in vitro and in vivo evaluation, Int. J. Pharm. 457 (1) (2013) 158
167. [99] C. Gao, T. Liu, Y. Dang, Z. Yu, W. Wang, J. Guo, et al., pH/redox responsive core cross-linked nanoparticles from thiolated carboxymethyl chitosan for in vitro release study of methotrexate, Carbohydr. Polym. 111 (2014) 964
970. [100] N.A. Gujarathi, B.R. Rane, J.K. Patel, pH sensitive polyelectrolyte complex of O-carboxymethyl chitosan and poly (acrylic acid) cross-linked with calcium for sustained delivery of acid susceptible drugs, Int. J. Pharm. 436 (1
2) (2012) 418
425. [101] H. Guo, D. Zhang, C. Li, L. Jia, G. Liu, L. Hao, et al., Self-assembled nanoparticles based on galactosylated O-carboxymethyl chitosan-graft-stearic acid conjugates for delivery of doxorubicin, Int. J. Pharm. 458 (1) (2013) 31
38. [102] Y.-C. Huang, T.-H. Kuo, O-carboxymethyl chitosan/fucoidan nanoparticles increase cellular curcumin uptake, Food Hydrocoll. 53 (2016) 261
269.

Handbook of Chitin and Chitosan

References

469

[103] J. Ji, D. Wu, L. Liu, J. Chen, Y. Xu, Preparation, evaluation, and in vitro release of folic acid conjugated O-carboxymethyl chitosan nanoparticles loaded with methotrexate, J. Appl. Polym. Sci. 125 (S2) (2012) E208
E215. [104] J. Ji, S. Hao, W. Liu, J. Zhang, D. Wu, Y. Xu, Preparation and evaluation of O-carboxymethyl chitosan/cyclodextrin nanoparticles as hydrophobic drug delivery carriers, Polym. Bull. 67 (7) (2011) 1201
1213. [105] Y. Li, S. Zhang, X. Meng, X. Chen, G. Ren, The preparation and characterization of a novel amphiphilic oleoyl-carboxymethyl chitosan self-assembled nanoparticles, Carbohydr. Polym. 83 (1) (2011) 130
136. [106] J. Wang, B. Chen, D. Zhao, Y. Peng, R.-X. Zhuo, S.-X. Cheng, Peptide decorated calcium phosphate/carboxymethyl chitosan hybrid nanoparticles with improved drug delivery efficiency, Int. J. Pharm. 446 (1
2) (2013) 205
210. [107] D. Jaikumar, K.M. Sajesh, S. Soumya, T.R. Nimal, K.P. Chennazhi, S.V. Nair, et al., Injectable alginate-O-carboxymethyl chitosan/nano fibrin composite hydrogels for adipose tissue engineering, Int. J. Biol. Macromol. 74 (2015) 318
326. [108] R. Budiraharjo, K.G. Neoh, E.T. Kang, Hydroxyapatite-coated carboxymethyl chitosan scaffolds for promoting osteoblast and stem cell differentiation, J. Colloid Interface Sci. 366 (1) (2012) 224
232. [109] T. Agarwal, R. Narayan, S. Maji, S. Behera, S. Kulanthaivel, T.K. Maiti, et al., Gelatin/carboxymethyl chitosan based scaffolds for dermal tissue engineering applications, Int. J. Biol. Macromol. 93 (2016) 1499
1506. [110] D. Mishra, B. Bhunia, I. Banerjee, P. Datta, S. Dhara, T.K. Maiti, Enzymatically crosslinked carboxymethyl
chitosan/gelatin/nano-hydroxyapatite injectable gels for in situ bone tissue engineering application, Mater. Sci. Eng. C. 31 (7) (2011) 1295
1304. [111] Tan Y-l, C.-G. Liu, Preparation and characterization of self-assemblied nanoparticles based on folic acid modified carboxymethyl chitosan, J. Mater. Sci. Mater. Med. 22 (5) (2011) 1213
1220. [112] S.K. Sahu, S.K. Mallick, S. Santra, T.K. Maiti, S.K. Ghosh, P. Pramanik, In vitro evaluation of folic acid modified carboxymethyl chitosan nanoparticles loaded with doxorubicin for targeted delivery, J. Mater. Sci. Mater. Med. 21 (5) (2010) 1587
1597. [113] L. Shi, C. Tang, C. Yin, Glycyrrhizin-modified O-carboxymethyl chitosan nanoparticles as drug vehicles targeting hepatocellular carcinoma, Biomaterials 33 (30) (2012) 7594
7604. [114] J.-P. Nam, S.-C. Park, T.-H. Kim, J.-Y. Jang, C. Choi, M.-K. Jang, et al., Encapsulation of paclitaxel into lauric acid-O-carboxymethyl chitosan-transferrin micelles for hydrophobic drug delivery and site-specific targeted delivery, Int. J. Pharm. 457 (1) (2013) 124
135. [115] A. Zou, M. Huo, Y. Zhang, J. Zhou, X. Yin, C. Yao, et al., Octreotide-modified N-octyl-O, N-carboxymethyl chitosan micelles as potential carriers for targeted antitumor drug delivery, J. Pharm. Sci. 101 (2) (2012) 627
640. [116] S. Maya, L.G. Kumar, B. Sarmento, N.S. Rejinold, D. Menon, S.V. Nair, et al., Cetuximab conjugated O-carboxymethyl chitosan nanoparticles for targeting EGFR overexpressing cancer cells, Carbohydr. Polym. 93 (2) (2013) 661
669. [117] G. Li, L. Cao, Z. Zhou, Z. Chen, Y. Huang, Y. Zhao, Rapamycin loaded magnetic Fe3O4/carboxymethylchitosan nanoparticles as tumor-targeted drug delivery system: Synthesis and in vitro characterization, Colloids Surf. B: Biointerfaces 128 (2015) 379
388. [118] Y. Xie, H. Qiao, Z. Su, M. Chen, Q. Ping, M. Sun, PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy, Biomaterials 35 (27) (2014) 7978
7991. [119] R.K. Farag, R.R. Mohamed, Synthesis and characterization of carboxymethyl chitosan nanogels for swelling studies and antimicrobial activity, Molecules 18 (1) (2012) 190
203.

Handbook of Chitin and Chitosan

470

14. Biomedical applications carboxymethyl chitosans

[120] L. Chen, Y. Du, Z. Tian, L. Sun, Effect of the degree of deacetylation and the substitution of carboxymethyl chitosan on its aggregation behavior, J. Polym. Sci. Part B: Polym. Phys. 43 (3) (2005) 296
305. [121] M.W. Sabaa, N.A. Mohamed, R.R. Mohamed, N.M. Khalil, S.M.A. El Latif, Synthesis, characterization and antimicrobial activity of poly (N-vinyl imidazole) grafted carboxymethyl chitosan, Carbohydr. Polym. 79 (4) (2010) 998
1005. [122] A.E. Shafei, A. Abou-Okeil, ZnO/carboxymethyl chitosan bionano-composite to impart antibacterial and UV protection for cotton fabric, Carbohydr. Polym. 83 (2) (2011) 920
925. [123] X. Li, X. Kong, Z. Zhang, K. Nan, L. Li, X. Wang, et al., Cytotoxicity and biocompatibility evaluation of N, O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application, Int. J. Biol. Macromol. 50 (5) (2012) 1299
1305. [124] Y. Luo, Z. Teng, X. Wang, Q. Wang, Development of carboxymethyl chitosan hydrogel beads in alcohol-aqueous binary solvent for nutrient delivery applications, Food Hydrocoll. 31 (2) (2013) 332
339. [125] Y.-H. Lin, H.-F. Liang, C.-K. Chung, M.-C. Chen, H.-W. Sung, Physically crosslinked alginate/N, O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs, Biomaterials 26 (14) (2005) 2105
2113. [126] B.-L. Guo, Q.-Y. Gao, Preparation and properties of a pH/temperature-responsive carboxymethyl chitosan/poly (N-isopropylacrylamide) semi-IPN hydrogel for oral delivery of drugs, Carbohydr. Res. 342 (16) (2007) 2416
2422. [127] Y. Zhou, Y. Zhao, L. Wang, L. Xu, M. Zhai, S. Wei, Radiation synthesis and characterization of nanosilver/gelatin/carboxymethyl chitosan hydrogel, Radiat. Phys. Chem. 81 (5) (2012) 553
560. [128] L. Yin, L. Fei, F. Cui, C. Tang, C. Yin, Superporous hydrogels containing poly (acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks, Biomaterials 28 (6) (2007) 1258
1266. [129] S.S. Vaghani, M.M. Patel, C.S. Satish, Synthesis and characterization of pH-sensitive hydrogel composed of carboxymethyl chitosan for colon targeted delivery of ornidazole, Carbohydr. Res. 347 (1) (2012) 76
82. [130] M.E. Mathew, J.C. Mohan, K. Manzoor, S.V. Nair, H. Tamura, R. Jayakumar, Folate conjugated carboxymethyl chitosan
manganese doped zinc sulphide nanoparticles for targeted drug delivery and imaging of cancer cells, Carbohydr. Polym. 80 (2) (2010) 442
448. [131] G. Wang, L. Jin, Y. Dong, L. Niu, Y. Liu, F. Ren, et al., Multifunctional Fe 3 O 4
CdTe@ SiO 2
carboxymethyl chitosan drug nanocarriers: synergistic effect towards magnetic targeted drug delivery and cell imaging, N. J. Chem. 38 (2) (2014) 700
708. [132] L. Tan, A. Wan, H. Li, Q. Lu, Novel quantum dots
carboxymethyl chitosan nanocomposite nitric oxide donors capable of detecting release of nitric oxide in situ, Acta Biomater. 8 (10) (2012) 3744
3753. [133] M.M.G. Fouda, M.R. El-Aassar, S.S. Al-Deyab, Antimicrobial activity of carboxymethyl chitosan/polyethylene oxide nanofibers embedded silver nanoparticles, Carbohydr. Polym. 92 (2) (2013) 1012
1017.

Handbook of Chitin and Chitosan

C H A P T E R

15 Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing Simone S. Silva1,2, Joana M. Gomes1,2, Luı´sa C. Rodrigues1,2 and Rui L. Reis1,2,3 1

3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimara˜es, Portugal, 2 ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimara˜es, Portugal, 3The Discoveries Centre for Regenerative and Precision Medicine, Guimara˜es, Portugal

O U T L I N E 15.1 Introduction

472

15.2 Dissolution of chitin and chitosan using ionic liquids

474

15.3 Chitin 15.3.1 Structure 15.3.2 Parameters that affect its dissolution

474 474 475

15.4 Chitosan 15.4.1 Structure 15.4.2 Parameters that affect its dissolution

477 477 478

15.5 Processability of chitin and chitosan via ionic liquids 15.5.1 Chemical modification

479 479

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00015-7

471

© 2020 Elsevier Inc. All rights reserved.

472

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

15.6 Chitin 15.6.1 15.6.2 15.6.3

and chitosan-based architectures Films and hydrogels Nano-microfibers and nano-microparticles Scaffolds, sponges, and beads

484 485 485 485

15.7 Biomedical applications of chitin and chitosan in ionic liquids

489

15.8 Bone repair

490

15.9 Drug and gene delivery

490

15.10 Neuron repair

491

15.11 Final remarks and future trends

491

Acknowledgments

493

Abbreviators

493

References

494

15.1 Introduction Ionic liquids (ILs) have emerged as the most promising alternative solvents and reaction media to enhance the processability of the biomacromolecules namely keratin [1], cellulose [2
4], xantham gum [5], agarose [6], chitin, and chitosan [2,6,7]. The described strategies proposed by many researchers combine two green chemistry principles, namely the use of biorenewable feedstocks (natural polymers) and the environmental solvents (ILs). ILs are salts in the liquid state at ambient or near ambient temperature, where the combination of different cations and anions allow the production of a vast range of available ILs, and tailoring their intrinsic features, namely density, viscosity, ionic conductivity, melting point, polarity, solvation power, hydrophilicity, and hydrophobicity [8]. ILs are recognized as green solvents due to their low vapor pressure, high chemical and thermal stabilities (up to 300 C), nonflammability, high ionic conductivity, wide electrochemical window, and high solvation power [8
10]. Based on these features, several studies have shown the potential of ILs as sustainable platforms for the development of innovative biomaterials. However, despite the advantages of ILs described above, many researchers have pointed out that their high cost is a major concern when considering large-scale applications, thus making their recycling an important issue to guarantee the economic sustainability. Since ILs cannot be readily purified

Handbook of Chitin and Chitosan

473

15.1 Introduction

by distillation, due to their low volatility, the recycling of ILs could be challenging. Therefore simple protocols should be defined, for instance, based on the solubility of ILs in organic solvents. Based on that, some studies already report ILs applied to the dissolution/modification of chitin/chitosan that can be used and recovered for reuse by distilling the aqueous washings of polymer/IL solutions. For instance, 1-allyl-3-methylimidazolium chloride (AMIMCl) used on the acetylation of chitosan could be reused several times using simple vacuum treatment [11], thus reducing costs. In a recent study, Ishii et al. demonstrated that a high deacetylation degree of chitosan (86.7%) could be attained by simple hydrothermal treatment in the 1-butyl-3methylimidazolium acetate (BMIMAc)
chitosan
water system without using caustic alkalis [12]. The proposed approach also allows the recovery and reuse of BMIMAc by evaporation, after collecting the chitosan, confirming that these solutions could be based on recyclable methods and overcome the IL cost problem. The pioneering work on ILs as solvents for polysaccharides was reported by Swatloski et al. in 2002 [13]. Their success on the dissolution of cellulose in ILs stimulated a series of studies on the dissolution of other biopolymers. Particularly for chitin and chitosan, several studies have shown the potential of ILs as sustainable platforms for the production of 2D- and 3D-based chitin and chitosan matrices, namely gels, films, micro/nanoparticles, and sponges. Given the performance of these matrices, they have been proposed as wound dressing, drug delivery, bone repair, and gene delivery systems. Therefore this chapter is focused on the overview of the properties, strategies, and biomedical applications of chitin and chitosan-based matrices processed in different ILs (Fig. 15.1).

Wound dressings

Chitin chitosan Biomedical applications Dissolution chemical modification Processability

Drug delivery

Ionic liquids Bone repair

Gene delivery

FIGURE 15.1 Overview of the strategies and biomedical applications of chitin/ chitosan-based materials prepared in ILs.

Handbook of Chitin and Chitosan

474

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

15.2 Dissolution of chitin and chitosan using ionic liquids It is widely known that chitin and chitosan are not soluble either in water or the common organic solvents due to their compact crystalline structure [14] and the strong inter- and intramolecular hydrogen bonds established between their hydroxyl/amido/carbonyl groups from the glucose units [15]. Just a restricted number of solvents have been capable of promoting their dissolution, however with a high number of drawbacks related to their toxicity, volatility, causticity or costliness and also polymer chain degradation. To avoid this issue, the use of ILs have increased in the last decades due to their unique physicochemical properties and improved solvating power, which allows them to dissolve a variety of organic and inorganic compounds, through the disruption of hydrogen bonding. Numerous ILs, such as alkylimidazolium chloride, alkylimidazolium dimethyl phosphate [16], 1-allyl-3-methyl-imidazolium acetate (AMIMAc) [16], AMIMCl [16], 1-ethyl-3-methyl-imidazolium acetate (EMIMAc) [17,18], 1-ethyl-3-methylimidazolium chloride (EMIMCl) [17], 1-butyl3-methyl-imidazolium chloride (BMIMCl) [16,19,20], BMIMAc [4,16,17,21], 1, 3-dimethylimidazolium dimethylphosphate (MMIM[Me2PO4]) [16], 1-ethyl-3-methylimidazolium dimethyl phosphate (EMIM[Me2PO4]) [16], 1-allyl-3-methylimidazolium bromide (AMIMBr) [22
24], and 1-carboxymethyl-3-methylimidazolium hydrochloride ([IMIM-COOH]Cl) [25], have been reported in literature as capable of dissolving chitin or chitosan. The majority of the reported studies are with reference to acetate and chloride-based ILs, and more specifically to BMIMAc and BMIMCl [4,16,21,24], which are the most commonly used. Envisioning a greener approach in the application of ILs into those biopolymers’ dissolution, Feng et al. proposed the use of (IMIM-COOH) Cl [25]. According to them, the dissolution of chitosan at 50 C for 1 h could allow the retention of its crystalline structure and properties.

15.3 Chitin 15.3.1 Structure Chitin is a structural biopolymer produced by insects and crustaceans in their shells [26], which provides structural integrity and protection to those animals [26,27]. The chitin structure is composed of a long chain made up of β-(1-4)-linked primary units of N-acetyl D-glucosamine [28
30] (Fig. 15.2). In nature, chitin occurs as ordered crystalline microfibrils forming structural components into arthropod exoskeletons or in fungi and yeast cell walls [29,31]. In crustaceans, chitin is found to occur as a fibrous material embedded in a six-stranded protein helix [32].

Handbook of Chitin and Chitosan

475

15.3 Chitin

CH3 O

OH O

O HO

NH HO O

O

NH OH

O CH3

FIGURE 15.2

n

Chitin chemical structure representation.

Depending on its origin, chitin presents two crystalline polymorphic forms, α- and β-chitins, that present stable antiparallel and metastable parallel chain alignments, respectively, promoting the existence of differences in the packing and polarities. Furthermore, α-chitin materials (e.g., from crab and shrimp shells (major sources)) show lower solubilities and reactivity than the β-chitins ones (e.g., from squid pens (minor sources)) [28
30]. This fact is related to the strength of the intermolecular forces, which in β-chitin are much weaker than in α-chitin, leading to the higher reactivity of β-chitin [33
35]. In chitin the degree of acetylation (DA), that is, the ratio of 2-acetamido-2deoxy-D-glucopyranose to 2-amino-2-deoxy-D-glucopyranose structural units, is typically 0.90, indicating the presence of about 5%
15% of amino groups due to deacylation that might occur during chitin extraction [30].

15.3.2 Parameters that affect its dissolution Chitin is a highly crystalline polysaccharide with reduced reactivity due to the strong intra- and intermolecular interactions (hydrogen bonds derived from acetamido groups more specifically between C 5 O and NH groups of the adjacent chitin chains) established between the polymeric chains, which make its processing difficult and are the main obstacle to its dissolution. The exploitation of its dissolution led to the investigation of chitin’s intrinsic features and its ability to influence polysaccharide solubility besides the cationic and anionic composition of the IL. Recent studies have reported the effect of chitin’s features, namely polymorphic form, origin, molecular weight (MW), and DA [16]. Regarding the clarification of the influence of ILs’ composition and polarity in chitin dissolution, it was noted that there is a requirement for a higher polarity and more basic anions such as acetate, due to the higher number of hydrogen bond donors and acceptors [16,36]. The published data proved that acetate ions gave origin to weak conjugate acids capable of interacting with H-bonds of chitin, destroying them and leading to chitin crystal dissolution. It shows that they were considerably more effective than chloride or dimethyl phosphate anions. (Table 15.1).

Handbook of Chitin and Chitosan

TABLE 15.1

Chitin solubilization behavior in imidazolium ILs.

Ionic liquid

Abbreviation

Temperature (K)

Solubility (%wt)

References

1-Allyl-3-methylimidazolium bromide

AMIMBr

373

4.8 (solution)

[4,16,18,22,37
40]

10 (ion gel) 1-Ethyl-3-methylimidazolium acetate

EMIMAc

378

9.0

[4,16,18,22,37
40]

1-Butyl-3-methylimidazolium acetate

BMIMAc

383

6.0

[4,16,18,22,37
41]

1-Allyl-3-methylimidazolium acetate

AMIMAc

383

5.0

[4,16,18,22,37
40]

1-Ethyl-3-methylimidazolium chloride

EMIMCl

378

3.0

[4,16,18,22,37
40]

1-Butyl-3-methylimidazolium chloride

BMIMCl

383

1.0

[4,16,18,20,22,37
40]

1-Butyl-3-methylmidazoium bromide

BMIMBr

373

1.0

[4,16,18,22,37
40]

1-Allyl-3-methylimidazolium chloride

AMIMCl

, 318

0.5

[4,16,18,22,37
40]

Tris(2-hydroxyethyl) methylammonium acetate

[THEMA][Ac]/EDA

RT

0.1

[41]

Tris(2-hydroxyethyl) methyl ammonium methyl sulfate

[THEMA][MeSO3] /EDA

RT

0.1 (partial dissolution)

[41]

Tris(2-hydroxyethyl) methylammonium trifluoro methansulfonate

[THEMA]

RT

0.1 (partial dissolution)

[41]

[CF3SO3]/EDA

1,3-Dimethylimidazolium dimethyl phosphate

[MMIM][Me2PO4]

, 333

1.5

[4,16,18,22,37
40]

1-Ethyl-3-methylimidazolium dimethyl phosphate

[EMIM][Me2PO4]

, 333

1.5

[4,16,18,22,37
40]

15.4 Chitosan

477

Besides the chosen solvent, there are three other factors, directly related to the polymer, that considerably affect chitin solubility, that is, the DA, pH, and chain MW. The degree of deacetylation determines the hydrophilic character of the chitin backbone due to the number of Namino-D-glucosamine units responsible for the hydrophilic nature and positive charge in acidic solution. The circumstance determines if chitin exhibits a transition between hydrophilic and hydrophobic characters due to the rise of hydrogen bonds established by the acetyl groups which are consequently responsible for its crystallinity. From another perspective, the pH also affects chitin solubility. The amino groups of chitin chains, of low deacetylation degree, at acidic pHs (,6.0) capture a proton (H1) from the solution ions acquiring a positive surface charge. Those charged chains cause coulombic repulsion forces which are responsible for resistance to chain aggregation. The influence of this resistance decreases with the increase of the acetylation degree as the domain of H-bonds rises. Considering the MW influence into chitin dissolution, it is noticeable that a reduced MW of the polymeric chains favors their dissolution [16]. This fact is related to a decreased incidence of van der Waals forces in reduced MW chitins, promoting an increase of the free volume between the chains, facilitating the macromolecule solubilization [16,18,42]. In summary, chitin with lower deacetylation degree, MW, and consequently crystallinity is easily dissolved using ILs.

15.4 Chitosan 15.4.1 Structure Chitosan is a biodegradable cationic aminopolysaccharide with a linear distribution of β-(1
4)-linked D-glucosamine (deacetylated monomer) and N-acetyl-D-glucosamine (acetylated monomer) units (Fig. 15.3) in which the glucosamine backbone holds a high number of available amino groups that can be protonated. Obtained by deacetylating chitin, chitosan is available in a range of MWs and degrees of deacetylation. Moreover, the chitosan amino group has a pKa value close to 6.5, being soluble in weakly acidic solutions (e.g., dilute acidic solutions of acetic, citric, lactic, or glutamic acids) where charge density is dependent on the pH and the degree of deacetylation. Therefore, under acidic conditions, the amine groups become protonated, which leads to repulsion between the positively charged macromolecular chains, thus allowing the diffusion of the water molecules and consequently leading the polymer to solvation. However, those systems have drawbacks as acids are corrosive and toxic.

Handbook of Chitin and Chitosan

478

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

OH

OH HO HO

O NH2

O HO

O

OH O HO

O OH NH2

NH2 n

FIGURE 15.3 Chitosan chemical structure representation.

Chitosan properties are mainly affected by two critical structural parameters, which are the degree of deacetylation and the MW. These features, considerably influence the biopolymer’s physical, chemical, and biological properties, such as solubility, biodegradability, and biocompatibility. From a general point of view, similarly to chitin, chitosan is extremely difficult to dissolve in most of the conventional organic solvents, due to the close packing caused by the intra- and intermolecular hydrogen bonds which is the main obstacle for the further application of chitosan. Consequently, it is highly desirable to seek effective and sustainable solvents for chitosan dissolution.

15.4.2 Parameters that affect its dissolution Several studies have been performed over the years to elucidate the impact of influencing factors, such as ILs composition and dissolution conditions, into the chitosan dissolution process. Due to the similarity between chitosan and cellulose structures, the previous knowhow achieved through cellulose processing using ILs has been used as a starting point for chitosan exploitation. Based on that knowledge, some ILs including those with chloride, formate, and acetate as anions, and 1-butyl-3-methylimidazolium (BMIM), 1-allyl-3-methylimidazolium (AMIM), 1,3-dimethylimidazolium (DMIM), and 1-hydrogen-3-methylimidazolium (MIM) as cations, as well as their mixtures, have been studied as possible solvents for chitosan [4,17,43,44]. The anion nature in ILs composition already has proved to have a major influence into chitosan solubility, however, besides the key role played by the anionic structure in the chitosan hydrogen bonds disruption, the role of the ILs cation in chitosan dissolution cannot be neglected and neither can temperature and water content [17]. Sun et al. evaluated the performance of a series of ILs and reported that the chitosan dissolution was improved by imidazolium-based ILs with Ac2 with shorter alkyl chains length, with the [Bmim]Ac IL being the most efficient one by dissolving at 140 C more than 15 g of chitosan per 100 g of

Handbook of Chitin and Chitosan

15.5 Processability of chitin and chitosan via ionic liquids

479

IL. According to those authors the ability to dissolve chitosan follows the order: [BMIM]Ac . [EMIM]Ac; [BMIM]Ac . [HMIM]Ac . [OMIM] Ac and [BMIM]Ac . [BMIM]Ac. Considering the influence of water content it was proved that the presence of water can significantly decrease the solubility of chitosan due to the competitive hydrogen bonds with the chitosan microfibrils, as water molecules can form strong O
H
O hydrogen bonds with Ac2 and weak C
H
O hydrogen bonds with cations [17]. In this study comprising chitosan dissolution into acetate-imidazolium-based ILs, the solubility of chitosan decreases with the increase of water content at temperatures below 110oC, after which the values are probably due to water evaporation. The %majority of the performed studies evaluate the solubility of chitosan in ILs as a function of temperature [25,44], denoting an enhancement of chitosan solubility with an increase of the dissolution temperature (e.g., from 50 C up to 150 C). This influence is mostly related to a change of the transport properties of the ILs and simultaneously to the evaporation of residual water from the system that is considered an antisolvent. So, the employment of ILs with increased thermal stability, such as the imidazolium-based ILs, is advantageous and preferential in chitosan dissolution [25].

15.5 Processability of chitin and chitosan via ionic liquids 15.5.1 Chemical modification As explored earlier, both chitin and chitosan present diverse functional groups into their chains that allow for manipulation of the otherwise insoluble biopolymers and their processing through chemical modification. The straightforward modification of the material’s architecture results in tailored matrices with distinct physicochemical features such as shape, size, morphology/porosity, and specific functionalities [45
47]. Various routes for biopolymers modification have been explored as graft polymerization via atom transfer radical polymerization (ATRP) or chemical derivatizations and modifications. However, there have been only a limited number of studies regard chitin and chitosan exploitation due to the required severe reaction settings [23,42,48] (heterogeneous conditions, prolonged reaction time, high temperatures and hazard solvents) which lead to reduced yield, partial degradation (MW lowering), and lack of reproducibility. To achieve some progress in the manipulation of these biopolymers, the resource to new biotechnological tools may provide breakthrough technologies to set up safer, cleaner, and more efficient industrial manufacturing processes, promoting the exploitation of these abundant biopolymers’ potential.

Handbook of Chitin and Chitosan

480

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

These features have stimulated the use of ILs not only to reduce dissolution times but also to promote homogeneous reaction media, improving the efficiency of organic reactions applying solvents that are less toxic and less corrosive than those presently used. Due to their ability to efficiently dissolve cellulose and other biomaterials, ILs gained attention, being considered a useful and sustainable green biomass chemistry tool in various types of chemical derivatizations and modifications [11], such as acetylation [48], hydrolysis [49], or carboxymethylation [13,50]. Modifying chitin or chitosan chains with different reagents through the removal of one to two hydrogens from the amino group and their replacement with hydrophobic groups promotes a reduction of their inherent crystalline structure and consequently improves their solubility in general organic solvents and thus induces new desired properties [27] (examples Table 15.2). Acyl derivatives have been identified as a useful functional material derived from chitin and chitosan, especially α-chitin where its stable antiparallel chain alignment makes solubilization difficult. Imidazolium-based ILs are homogeneous reaction media in a mild condition that enable the production of α-chitin acetyl derivatives [48], and they are considered excellent catalytic media during graft copolymerization and ATRP [52], as they can reduce the extent of side-reactions in ATRP [66]. The α-chitin acetylation in AMIMBr was explored to synthesize several chitin acylates with different substituents [33]. The efficient acylation of α-chitin using various acyl chlorides was achieved in AMIMBr under homogeneous conditions, resulting in chitin acylates with high substitution degree values, being the same methodology further applied to the synthesis of the chitin macroinitiator for ATRP [42]. Similar results were achieved for chitosan acetylation using AMIMCl [11]. Furthermore, chitin-based composite materials were obtained using the same acylation methodology, followed by graft ATRP. The chitin macroinitiators modified with α-haloalkylacyl groups were graftpolymerized to styrene, a representative monomer for ATRP, to obtain chitin-graft-polystyrene [52] with GPC data indicating that the progress of the graft polymerization occurred in a living manner. Moreover, chitin may also be modified by sol
gel chemistry in the presence of multifunctional ILs, which act as solvents, catalyst, and chemical additives [67]. Chitin-based functional, tailored materials were produced and functionalized through the dissolution of chitin in BMIMAc [68] to produce chitin hybrid beads using the extrusion dipping method. The tetraethylorthosilicate (TEOS)-ethanol-H2O-HCl bath was used to promote the formation of a silica network that will act as a coating of the beads, able to allow penetration until the bulk particle is formed by the chitin molecules in the IL phase, allowing the formation of hydrogen bridge bonds. The produced chitin beads were loaded and

Handbook of Chitin and Chitosan

TABLE 15.2 Material

Application of ionic liquid as reaction media for chemical modification of chitin and chitosan. Ionic liquid/other reagents

Chemical process

Proposed application

References

Chitin

Acetic anhydride in AMIMBr

Acetylation

Not defined

[48]

Chitin-g-PLGA

AMIMBr

Surface initiated graft polymerization

TE

[51]

Chitin-gpolystyrene

AMIMBr

ATRP

Base for the production chitin-based functional materials

[52]

Chitin

Acyl chlorides in AMIMBr

Acylation

Not defined

[33]

Chitosan

CMIMCl

Amidation reaction

Anion adsorbent for wastewater treatment

[53]

Chitosan

BMIMCl

Single electron transfer living radical polymerization

Potential antibacterial application

[54]

Chitosan

BSMIMCF3SO3 MFA, EDC, NHS

Chemical reaction

Ingredients for food preservation and cosmetics

[55]

Chitosan

BMIPF6

Ionic liquid assisted grinding

Photothermal agent for nanomedicine

[56]

Chitosan

AMIMCl

Acetylation

Not defined

[11]

Chitosan

BMIMBr/BMIMCl

Hydrolysis

Not defined

[49]

Chitosan

SFIL 1; SFIL 2; SFIL 3

Hydrolysis

Not defined

[57]

Chitosan

BMIMCl using

O-alkylation

Gene delivery

[58]

N, N’carbonyldiimidazole as a bonding agent (Continued)

TABLE 15.2

(Continued)

Material

Ionic liquid/other reagents

Chitosan Chitosanoxycellulose

Chemical process

Proposed application

References

[C3SO3Hmim]HSO4

Conversion of chitosan to Levulinic acid by catalysis of acidic ILs

Not defined

[59]

AmimCl

Crosslinking reaction

Antimicrobial and biomedical products or water treatment membranes

[60]

SmimHSO4 as the catalyst

Chitosan-g-PCL

EMIMAc

Ring-opening polymerization

TE or drug and gene delivery.

[61]

Chitosan-g-PEI

BMIMAc

Graft polymerization using CDI as a linking agent.

Gene delivery

[62]

Chitosan-Lilial conjugate and chitosan

BMIMCl

Schiff reaction

Drug delivery system

[63]

Chitosan-graftpolycaprolactone (ACS-g-PCL)

BMIMAc

Protection 2 ring-opening graft polymerization 2 deprotection

TE

[64]

Chitosan-glinoleic acid

BMIMAc

Chemical modification of water-soluble chitosan oligosaccharide (COS) in BMIMAc

Drug transduction vectors

[65]

AMIMBr, 1-Allyl-3-mthylimidazolium bromide; AMIMCl, 1-allyl-3-methylimidozolium chloride; ATRP, atom transfer radical polymerization; BMIMAc, 1-butyl-3methylimidazolium acetate; BMIPF6, 1-butyl-3-methylimidazolium hexafluorophosphate; BSMIMCF3SO3, 1-sulfobutyl-3-methylimidazolium trifluoromethanesulfonate; CDI, carbonyldiimidazole; CMIMCl, 1-carboxybutyl-3-methylimidazolium chloride; DMSO, Dimethylsufoxide; DS, degrees of substitution; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; EMIMAc, 1-ethyl-3-methylimidazolium acetate; MFA, monomethyl fumaric acid; NHS, with or without Nhydroxysuccinimide; PCL, polycaprolactone; PEI, Polyethylenimine; SFIL 1, 1-(3-sulfonic acid) propyl pyridinium hydrogen sulfate; SFIL 2, 1-(3-sulfonic acid) propyl pyridinium toluenesulfonate; SFIL 3, 1-(3-sulfonic acid) propyl pyridinium dihydrogen phosphate; SMIMHSO4, 1-sulfobutyl-3-methylimidazoliumhydrogen sulfate; TE, tissue engineering; [C3SO3HMIM]HSO4, 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogensulfate; [HMIM][HSO4], 1-hydrogen-3-methylimidazolium hydrogen sulfate.

15.5 Processability of chitin and chitosan via ionic liquids

483

agglomerated with gellan gum followed by the supercritical assisted agglomeration method drying. ILs may also promote the surface modification of chitin particles, Jaworska et al. reported the effect of six ILs having chloride as anion, five of which were based on the 3-methyl-imidazolium entity and differed in the structure of the 1-alkyl substituent (AMIM, EMIM, BMIM, HMIM, MoMIM) and the sixth had 1-butyl-2,3-dimethyl-imidazolium (BdMIM) as cations [39]. According to the authors, although the solubility of chitin reduced in those imidazolium chloride ILs, its regeneration gave rise to porous architectures, which resulted in the modification of chitin particles’ physical structure [39]. Regarding chitosan, its grafting with aliphatic polyesters was considered a successful strategy to effectively extend chitosan’s applications as a functional biomaterial as it modulates chitosan’s physicochemical properties (e.g., solubility, hydrophilicity/hydrophobicity, drug releasing ability), but also provides unique properties as a result of the combination of the merits of both chitosan and polyesters. Both the functional groups of chitosan (
OH and
NH2) participate in the grafting modification reactions of chitosan using ILs [51,61,64,65,69], significantly increasing grafting efficiency, reaching values as high as 630% [61], which are guaranteed by the homogeneity of the chitosan-IL solution. One of the first reports of a grafted chitosan in IL media were made by Wang et al., who proposed an efficient method for chitosang-polycaprolactone (PCL) synthesis by grafting the PCL onto chitosan using ring-opening polymerization of PCL in EMIMAc as a homogeneous reaction media with stannous octoate (Sn(Oct)2) as the catalyst [61]. The high PCL grafting content achieved with the proposed methodology is attributed to the efficient chitosan solubilization in EMIMAc, that was not verified in dimethylsulfoxide (DMSO), the traditionally used solvent. In parallel, Chen et al. described chitosan grafting with polyethyleneimine (PEI) using BMIMAc [69] and BMIMCl [70], the proposed methodology allowed to achievement of high grafting degrees due to the good solubility of chitosan, enhanced nucleophilicity of amino groups, and stability of the activated complexes in IL media leading to a significant reduction of the reaction times. Another approach to modifying chitosan was inspired by the debundling of single-walled carbon nanotubes (CNTs) and graphene mediated by IL, according to Zhang et al. who proposed the synthesis of molybdenum disulfide (MoS2) nanosheets by an IL-assisted exfoliation grinding method [56]. Chitosan was introduced into the grinding process to promote the physiological stability and biocompatibility of MoS2 nanosheets, fabricated as chitosan-MoS2 nanosheet dispersions in aqueous media.

Handbook of Chitin and Chitosan

484

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

15.6 Chitin and chitosan-based architectures Several 2D- and 3D-based architectures have been processed using chitin/IL and chitosan/IL solutions (see Table 15.2). The production of the matrices starts by the dissolution of chitin or chitosan in a specific IL at moderated high temperatures (80 C
110 C) [20,38,71,72] and is followed by cooling the chitin/IL or chitosan/IL solutions to low temperature (4 C
25 C), promoting the formation of weak gel-like materials (ion gels) and hydrogels [22,72]. By merely soaking these gels in water or ethanol, or applying an adequate processing technique such as electrospinning, supercritical fluid technique or freeze-drying on the biopolymer-IL
based solutions, hydrogels, nano/microspheres, fibers, and sponges can be produced [24]. In the processing of these structures, an IL removal step—total or partial—should be made, considering that toxicity studies have shown that ILs can exhibit a certain level of toxicity [73]. Examples of the chitin- and chitosan-based structures prepared via IL platform can be found in Fig. 15.4 and Table 15.2. Supercritical fluid technology

Extrusion di

ing pp

t spinning We

n tio

Ge ion lat

di Blen ng/ ge la

Chitin and chitiosan in lonic liquind

Sol-gel and supercritial fluid technology

FIGURE 15.4

Schematic illustration of chitin- and chitosan-based materials created from both chitin and chitosan dissolved in ILs and different technologies. (A) Chitin aerogel, (B) chitin/silk fibroin-based microspheres, (C) chitin hydrogel, (D) chitin hybrid scaffold, (E) chitosan/silk fibroin-based hydrogel and (F) chitin fibers.

Handbook of Chitin and Chitosan

15.6 Chitin and chitosan-based architectures

485

15.6.1 Films and hydrogels The ability of chitin and chitosan dissolved in ILs to create films and hydrogels can also be described in the literature. The general procedure involves leaving the solutions standing at room temperature to promote gelation, followed by immersion of the polymer/IL gels in a series of solvents, for example, ethanol, acetone, or isopropanol, to remove ILs and exchange the liquid phase to obtain films or hydrogels. Both chitin and chitosan can be combined with other polysaccharides, proteins, or even inorganic particles using a suitable IL as a common solvent, which has been proposed to mimic the naturally occurring environment of certain tissues, and the produced structures were proposed for skin regeneration. Chitosan/chondroitin sulfate hydrogels prepared in 1-hydrogen-3methylimidazolium hydrogen sulfate, [HMIM][HSO4], achieved excellent stability in the 1.2
10 pH range, considerable swelling abilities, and they were devoid of toxicity toward normal healthy kidney epithelial and epithelial colorectal adenocarcinoma cells [74]. These features suggested that these hydrogels could be applied for many technological purposes, like in medical, pharmaceutical, and environmental fields. Chitosan/silk fibroin hydrogels prepared in BMIMAc, as a common solvent, and Soxhlet extraction with ethanol for IL removal, exhibited viscoelastic behavior, lamellar structure, and rubbery consistency, which are exciting features for biomedical applications, such as cartilage regeneration [75].

15.6.2 Nano-microfibers and nano-microparticles The production of chitin and chitosan-based nanofibers and nanoparticles has been achieved via an IL platform associated with the use of techniques such as electrospinning, wet spinning, and self-assembly (see Fig. 15.4). Studies have suggested that due to the typical high viscosity of ILs and its lack of volatility it has become a challenge to obtain nanofibers by electrospinning using polymer/IL solutions. Therefore a coagulation bath should be used to promote both IL removal and fiber precipitation [76]. For instance, chitosan/cellulose-based nanofibers can be obtained by dissolving both polymers in EMIMAc, and an ethanol bath as cosolvent, where EMIMAc can be removed. In another approach, the interaction of chitosan and surface active ILs promoted the formation of spherical chitosan nanoparticles with controlled size (range 300
600 nm) [77].

15.6.3 Scaffolds, sponges, and beads The application of green chemistry principles, coupling supercritical fluids and ILs, demonstrated the possibility to develop ultralight

Handbook of Chitin and Chitosan

486

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

highly porous chitin structures [71]. According to the authors, chitin was dissolved in BMIMAc at high temperature, and then the solution was gelified at room temperature. Afterward, the IL was removed by supercritical fluid drying (SCF) using Soxhlet extraction, and SCF removal occurs using carbon dioxide/ethanol ratios. The chitin aerogels obtained using the described procedure present porous interconnected structures, large surface area, and low density. A similar approach had been used on other works to produce materials in this particular form [78]. 3D chitin constructs were built through functionalization of chitin microparticles prepared in ILs, followed by the application of a sol
gel methodology that has two functions, namely formation of silica network as a coating in the chitin beads and which acted in the removal of IL from the beads [68]. Further, a supercritical agglomeration method was also applied. Such developed materials have the flexibility to adapt to the shape of the constructs according to defect sites, osteoinductive behavior, and they may also be used as a controlled drug release. Recent trends demonstrated the relevance and efficient use of blending of chitin or chitosan with other polysaccharides or even inorganic particles [hydroxyapatite (HA)] using ILs as a solvent in the production of multifunctional composites. In an integrated approach, chitin was dissolved in BMIMAc, followed by the addition of salt particles (salt leaching methodology) to promote pore formation and/or HA to induce osteoinductive behavior and drying by SCF [79]. The codissolution of cellulose and chitosan in ILs and HA addition also allowed composite formation with excellent antimicrobial activity, ability to deliver growth factors/drugs (from chitosan), and mechanical strength (from cellulose) [80]. Therefore the performances of those matrices for bone tissue engineering were evaluated, and found to be promising. In most of the studies on the production of chitin and chitosanbased architectures cited in this chapter, the ILs were synthesized from imidazolium-based cations and, consequently, an IL removal step should also be considered to reduce the potential toxicity of the resulting matrices. Therefore there have been attempts to develop biodegradable and biocompatible ILs, mainly if biomedical applications are foreseen [54,81
83]. ILs can also be prepared from salts, sugars, amino acids, and biomolecules that exist in nature [84]. The ammonium-based ILs and choline-based ILs are considered the most promising and safer ILs for pharmaceutical and medical applications; since choline is a natural substance with low toxicity that acts as an antimicrobial and a cell signaling agent [85]. Despite the promising use of biocompatible ILs, there are a few studies on their application on chitosan [83]. (Table 15.3).

Handbook of Chitin and Chitosan

487

15.6 Chitin and chitosan-based architectures

TABLE 15.3 Processability of chitin and chitosan in different ionic liquids and their potential biomedical applications. Potential biomedical application

References

Sol
gel process/ supercritical assisted agglomeration method

Bone repair

[68]

BMIMCl

Stepwise regeneration process

Nd

[86]

EMIMAc

Electrospinning

Nd

[36]

Chitin-based artificial hard tissues

BMIMAc

Heat-induced dopamine oxidation

Artificial hard tissues

[87]

Chitin/alginate fibers

EMIMAc

Dry-jet wet spinning

Wound care dressings

[88]

Chitin/cellulose composite gel and films

AMIMBr

gelation

Nd

[2]

Chitin/cellulose blend films

BMIMOPr

Wet-spinning process

Nd

[89]

Chitin/CMC/ HA composite materials

BMIMCl

Blending

Bone regeneration

[90]

Chitin/CMC composite

EMIMAc

Dissolution/ Electrospinning

Nd

[91]

Chitin/HA scaffolds

BMIMAc

Supercritical fluid drying/salt leaching.

Bone tissue engineering

[79]

Chitin-MWNT nanocomposite scaffolds

EMIMAc

Dissolution and the CVD-grown highlyaligned MWNTs

Stem cell growth

[92]

Chitin-MWNT films

EMIMAc

Dissolution and growth MWNTs assisted oxygen plasma treatment

Neuron growth

[93]

Chitin/PVA nanowhiskers

AMIMBr

Gelation/ regeneration

Nd

[72]

Chitin membranes

EMIMAc

Dissolution/ solvent-casting

Nd

[94]

Composition/ matrix

Ionic liquid

Processing methodology

Chitin microparticles

BMIMAc

Chitin nanofibers

BMIMCl

(Continued)

Handbook of Chitin and Chitosan

488

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

TABLE 15.3

(Continued)

Composition/ matrix

Ionic liquid

Processing methodology

Chitosan nanoparticles

[C8mim] [Cl]

Ionic cross-linking or self-assembly

In vivo vaccine delivery and antitumor activity

[77]

Solvent casting

Drug delivery systems

[83]

ChNO3

Solvent casting

Implantable medical devices

[95]

Chitosan fibers

(Gly)Cl

Wet spinning process

Hernioplasty, saturation or wrinkle filling.

[96]

Chitosan/ agarose nanocomposite ionogels

BMIMCl

Sol-gel transition

Biotechnology and biomedical applications

[6]

Chitosan/ cellulose nanofibers

EMIMAc

Electrospinning

Wound repair

[76]

Chitosan/ cellulose porous films

BMIMAc

Dissolution/ gelation

Nd

[97]

Chitosan/ cellulose microspheres

BMIMCl

Water-oil suspension

Enzyme immobilization

[98]

Chitosan/ cellulose/HA composite films

BMIMCl

Co-dissolution

Bone tissue engineering

[80]

Chitosan/CMC blend membrane

BMIMF4

Solution

Nd

[99]

Chitosan/ chondroitin sulfate hydrogels

[Hmim] [HSO4]

Solution

Nd

[74]

Chitosan/silk fibroin hydrogels

BMIMAc

Gelation

Skin tissue engineering

[75]

[C4mim] [C8OSO3] Chitosan doped films

ChCl ChDHP

Potential biomedical application

References

blending method

Blending method

(Continued)

Handbook of Chitin and Chitosan

489

15.7 Biomedical applications of chitin and chitosan in ionic liquids

TABLE 15.3

(Continued)

Composition/ matrix

Ionic liquid

Processing methodology

Chitosan ionic liquid-based mPEG grafted nanocarrier

MIMoxiranCl

Polymerization followed by Schiff base formation reaction

Targetedmultidrug delivery for breast cancer

[100]

Chitosan hybrid hydrogels/films

[C12mpip] [AcSa]

Dissolution/ gelation/solvent casting

Drug carrier systems

[101]

Chitosan/ graphene oxide nanocomposite film

[BMIM] PF6

Dispersion

Dissolution testing of anticancer drugs

[102]

Chitosan/ cellulose hybrid gel

EMIMAc

Dissolution/ϒ-ray irradiation

Biodevices and soft actuators

[103]

Potential biomedical application

References

BMIBF4, 1-n-butyl-3-methylimidazolium tetrafluoroborate; BMIMAc, 1-buthyl-3-methylimidazolium acetate; BMIMOPr, 1-ethyl-3-methylimidazolium propionate; BMIPF6, 1-butyl-3-methylimidazolium hexafluorophosphate; ChCl, choline chloride; ChDHP, choline dihydrogenphosphate; ChNO3, choline nitrate; CMC, carboxymethylcellulose; CVD, chemical vapor deposition; C4MIMC8OSO3, 1-butyl-3methylimidazolium octylsulfate; C8MIMCl, 3-methyl-1-octylimidazolium chloride; EMIMAc, 1-ethyl-3-methylimidazolium acetate; (Gly)Cl, glycine chloride; HA, hydroxyapatite; MIM-oxiranCl, 1-methyl-3-(oxiran-2-ylmethyl)-1H-imidazol-3-ium chloride; PVA, poly(vinyl) alcohol; Nd, not defined; [C12MIMP][AcSa], 1-dodecyl-1-methylpiperidinium acetylsalicylate.

15.7 Biomedical applications of chitin and chitosan in ionic liquids Chitin and chitosan-based biomaterials produced using ILs as solvent and reaction media possess attractive physical and biological performances, suggesting their use predominantly in wound healing, gene delivery, bone regeneration/repair, and drug delivery [75,79,90]. The beneficial effects of chitin and chitosan for wound healing [104] have stimulated many studies involving chitosan/IL-based solution in the production of biomaterials to be used as wound dressings [75,105]. Chitosan/silk fibroin-based hydrogels were developed through the dissolution of both chitosan and silk fibroin in BMIMAc [75]. The biomacromolecules combination played a positive influence on the adhesion and proliferation of human dermal fibroblasts (hDFb) on those hydrogels suggesting their potential to be applied as a wound dressing. Composite materials formed by chitosan, cellulose, and keratin in BMIMCl were developed combining the superior mechanical strength

Handbook of Chitin and Chitosan

490

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

(from cellulose), hemostasis and bactericide action (from chitosan), and controlled release of drugs (from keratin) to be used as a highperformance bandage for treatment of chronic ulcerous wounds of diabetes patients [105]. Moreover, chitin-based fibers were proposed as effective and versatile wound dressings [88]. They were made codissolving chitin and alginate in BMIMAc followed by extrusion of the solution into a coagulation bath. Those fibers were applied on a full-dermal thickness wound model, and after 7 days of the treatment a suitable wound healing response was observed.

15.8 Bone repair Given the properties attributed to chitin/chitosan, it would be expected that a composite containing chitosan or chitin and HA [79,80] may have exciting features for bone regeneration purposes. Composite scaffolds can be produced through the dissolution of chitin [79] or chitosan [80,90] within ILs and blending with HA. The resulting composite materials have, for instance, antimicrobial activity (from chitosan) and are osteoconductive (from HA). Chitin-based biocomposite (ChHA) have been prepared by an integrated strategy using BMIMAc as a solvent for chitin combined with SCF and the addition of salt leaching and HA particles [79]. This approach allowed the formation of a composite system with suitable structural characteristics (porous microstructure with 65%
85% of porosity, and pore sizes in the range of 100
300 μm) and a positive influence on the viability and proliferation of osteoblastlike cells, suggesting that they can be applied in bone tissue engineering purposes.

15.9 Drug and gene delivery The potential use of ILs in pharmaceutical drug delivery and active pharmaceutical formulations has also been exploited due to their unique and tunable properties [106]. In literature, it is possible to find reports of stimuli-responsive drug release devices for intravenous administration can be obtained conjugating hydrophobic drugs with chitosan via Schiff reaction in BMIMCl [63]. Other approaches described the creation of multiresponsive chitosan biomaterials doped with biocompatible and biodegradable ammonium-based ILs, namely choline chloride and choline dihydrogen phosphate [83]. The release of sodium phosphate dexamethasone, an ionic drug from systems which depend on the different ionic interactions that can be established between the chitosan, chitosan/IL, and dexamethasone, suggests that

Handbook of Chitin and Chitosan

15.11 Final remarks and future trends

491

they can be used as electrically modulated drug release systems for iontophoretic applications. The advantage of chitosan-based vectors lies not only in getting away from the cytotoxicity problems that are inherent to most synthetic polymeric vehicles but also on its unique capability for transcellular transport. Besides, the presence of positive charges from amine groups in chitosan enables it to transport plasmid DNA (pDNA) into cells via endocytosis and membrane destability [58]. Moreover, attempts to modify chitosan through grafting with PEI in BMIMAc [62] or the synthesis of O-alkylated chitosan derivatives in BMIMCl [58] has been used to improve its gene transfection performance. It is proposed that the particular properties of the IL solvent should be responsible for the selective alkylation of hydroxyl groups of chitosan without protecting the amino groups of chitosan. Furthermore, an improvement of the solubility of the derivatives in organic solvent could also improve their use in gene delivery tests.

15.10 Neuron repair Chitin can also be combined with the high electrical conductivity of CNTs using EMMIAc as a common solvent [92]. In such a study, the IL promoted both the uniform dispersion of CNTs and the dissolution of chitin, allowing the formation of composite scaffolds that can support cell growth and electrical stimulation. Such materials would be valuable in regenerative medicine as they promote stem cells’ differentiation into a specific lineage [92], and as an implantable electrode for stimulation and repair of neurons [93]. In Fig. 15.5, a large proportion of live mesenchymal stem cells (MSCs) attachment was observed after 3 and 14 days with a negligible number of dead cells on all chitin and MWNT composite films. In a similar approach [93], neurons were able to adhere, attach, and sustain their functional integrity even after 21 days on chitin/CNT composite films subjected to oxygen plasma treatment.

15.11 Final remarks and future trends In the biomedical field, a great deal of effort has been taken to combine natural polymers, with a focus on chitin and chitosan, and ILs to provide derivatives with different shapes via appropriate and highperformance techniques acting as a biotechnological tool. ILs can be useful not only as valuable tools for the dissolution/processing of polymers but also as cost-effective platforms to extract chitin directly from biomass while increasing the quality of the achieved polymer. Despite the

Handbook of Chitin and Chitosan

492

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

3days

(A)

14days

(B) 0.01 weight fraction MWNTs

(C)

(D) 0.03 weight fraction MWNTs

(E)

(F) 0.07 weight fraction MWNTs

(G)

(H) 0.1 weight fraction MWNTs

(I)

(J) Controls

FIGURE 15.5 Live and dead viability assay to test the viability of MSCs on the chitin
MWNT film scaffolds. Cells were seeded on the scaffolds and were stained with the Live/Dead viability stain. Green cells showed the number of live cells and red cells showed dead cells due to the excitation of fluorescent dye (calcein AM) at 490 nm. The images were obtained after 3 days (left column) and 14 days (right column) of cells seeding. Reproduced with permission of ROYAL SOCIETY OF CHEMISTRY from ref. N.Singh, K.K. K. Koziol, J.H. Chen, A.J. Patil, J.W. Gilman, P.C. Trulove, et al. Green. Chem. 15 (2013) 1192
1202.

clear advantages herein discussed there are few literature data on this topic, suggesting that the research on the use of ILs for the processing of these specific natural polymers is still at an early stage. ILs have undoubtedly opened up novel opportunities for the processing of high-

Handbook of Chitin and Chitosan

Abbreviators

493

value-added biomaterials based on chitin and chitosan. Despite the facts, little has been reported regarding their scale-up possibilities and in vitro/in vivo biocompatibility performance, which could limit their biomedical use. More developments on that field are expected in the coming years with the use of biocompatible ILs as a more eco- and biofriendly alternative family of ILs compounds with reduced toxicity.

Acknowledgments The authors especially acknowledge financial support from Portuguese FCT (PD/BD/ 135247/2017, SFRH/BPD/93697/2013). This work is also financially supported by PhD programme in Advanced Therapies for Health (PD/00169/2013), FCT R&D&I projects with references PTDC/BII-BIO/31570/2017, PTDC/CTM-CTM//29813/2017, PTDC/ CTM-BIO/4706/2014-(POCI-01
0145-FEDER-016716), and R&D&I Structured Projects with reference NORTE-01
0145-FDER-000021.

Abbreviators [AMIM] AMIMAc AMIMBr AMIMCl ATRP BdMIM [BMIM] BMIMAc BMIBF4 BMIMBr BMIMCl BMIMOPr BMIPF6 BSMIMCF3SO3 CDI ChCl ChDHP ChHA ChNO3 CMC CMIMCl [C3SO3HMIM]HSO4 C4MIMC8OSO3 C8MIMCl CNTs CVD [C12MPIP][AcSa] DA [DMIM] DMSO DS

1-Allyl-3-methylimidazolium 1-Allyl-3-methylimidazolium acetate 1-Allyl-3-methylimidazolium bromide 1-Allyl-3-methylimidazolium chloride Atom transfer radical polymerization 1-Butyl-2,3-dimethyl-imidazolium cation 1-Butyl-3-methylimidazolium 1-Butyl-3-methylimidazolium acetate 1-n-butyl-3-methylimidazolium tetrafluoroborate; 1-Butyl-3-methylmidazoium bromide 1-Butyl-3-methylimidazolium chloride 1-Ethyl-3-methylimidazolium propionate; 1-Butyl-3-methylimidazolium hexafluorophosphate 1-Sulfobutyl-3-methylimidazolium trifluo-romethanesulfonate Carbonyldiimidazole Choline chloride Choline dihydrogenphosphate Chitin-based biocomposite Choline nitrate Carboxymethylcellulose 1-Carboxybutyl-3-methylimidazolium chloride 1-Methyl-3-(3-sulfopropyl)-imidazolium hydrogensulfate 1-Butyl-3-methylimidazolium octylsulfate 3-Methyl-1-octylimidazolium chloride Carbon nanotubes Chemical vapor deposition 1-Dodecyl-1-methylpiperidinium acetylsalicylate Degree of acetylation 1,3-Dimethylimidazolium Dimethylsulfoxide Degrees of substitution

Handbook of Chitin and Chitosan

494

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

EDC EMIMAc EMIMCl [EMIM][Me2PO4] (Gly)Cl HA hDFb IL [IMIM-COOH]Cl MFA [MIM] MIM-oxiranCl [MMIM][Me2PO4] MoS2 MSCs MW NHS PCL pDNA PEI PVA SCF SFIL 1: 1 SFIL 2: 1 SFIL 3: 1 SMIMHSO4 Sn(Oct)2 TE TEOS [THEMA][Ac]/EDA [THEMA][CF3SO3]/EDA [THEMA][MeSO3]/EDA

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide 1-Ethyl-3-methylimidazolium acetate 1-Ethyl-3-methylimidazolium chloride 1-Ethyl-3-methylimidazolium dimethyl phosphate Glycine chloride Hydroxyapatite Human dermal fibroblasts Ionic liquid 1-Carboxymethyl-3-methylimidazolium hydrochloride Monomethyl fumaric acid 1-Hydrogen-3-methylimidazolium 1-Methyl-3-(oxiran-2-ylmethyl)-1H-imidazol-3-ium chloride 1, 3-Dimethylimidazolium dimethyl phosphate Molybdenum disulfide Mesenchymal stem cells Molecular weight N-Hydroxysuccinimide Polycaprolactone Transport plasmid DNA Polyethylenimine Poly(vinyl) alcohol Supercritical fluid technology (3-Sulfonic acid) propyl pyridinium hydrogen sulfate (3-Sulfonic acid) propyl pyridinium toluenesulfonate (3-Sulfonic acid) propyl pyridinium dihydrogen phosphate 1-Sulfobutyl-3-methylimidazoliumhydrogen sulfate Stannous octoate Tissue engineering Tetraethylorthosilicate Tris(2-hydroxyethyl) methylammonium acetate Tris(2-hydroxyethyl) methylammonium trifluoro methansulfonate Tris(2-hydroxyethyl) methyl ammonium methyl sulfate

References [1] A. Idris, R. Vijayaraghavan, U.A. Rana, D. Fredericks, A.F. Patti, D.R. MacFarlane, Green. Chem. 15 (2013) 525
534. [2] A. Takegawa, M. Murakami, Y. Kaneko, J. Kadokawa, Carbohydr. Polym. 79 (2010) 85
90. [3] J. Zhang, J. Wu, J. Yu, X. Zhang, J. He, J. Zhang, Mater. Chem. Front. 1 (2017) 1273
1290. [4] Y. Wu, T. Sasaki, S. Irie, K. Sakurai, Polymer 49 (2008) 2321
2327. [5] H. Izawa, Y. Kaneko, J.-I. Kadokawa, J. Mater. Chem. 19 (2009) 6969
6972. [6] T.J. Trivedi, K.S. Rao, A. Kumar, Green. Chem. 16 (2014) 320
330. [7] S.S. Silva, J.F. Mano, R.L. Reis, Green. Chem. 19 (2017) 1208
1220. [8] R. Shi, Y. Wang, Sci. Rep. 6 (2016) 19644. [9] J. Hulsbosch, D.E. De Vos, K. Binnemans, R. Ameloot, ACS Sustain. Chem. Eng. 4 (2016) 2917
2931. [10] S.S. Silva, R.L. Reis, Ionic liquids as tools in the production of smart polymeric hydrogels, Polymerized Ionic Liquids, The Royal Society of Chemistry, 2018, pp. 304
318. [11] L. Liu, S. Zhou, B. Wang, F. Xu, R.C. Sun, J. Appl. Polym. Sci. 129 (2013) 28
35.

Handbook of Chitin and Chitosan

References

495

[12] D. Ishii, C. Ohashi, H. Hayashi, Green. Chem. 16 (2014) 1764
1767. [13] R.P. Swatloski, S.K. Spear, J.D. Holbrey, R.D. Rogers, J. Am. Chem. Soc. 124 (2002) 4974
4975. [14] M.-K. Jang, B.-G. Kong, Y.-I. Jeong, C.H. Lee, J.-W. Nah, J. Polym. Sci. Part. A: Polym. Chem. 42 (2004) 3423
3432. [15] H. Tamura, M. Sawada, H. Nagahama, T. Higuchi, S. Tokura, Influence of amide content on the crystal structure of chitin, Holzforschung, De Gruyter, 2006, pp. 480. [16] W.T. Wang, J. Zhu, X.L. Wang, Y. Huang, Y.Z. Wang, J. Macromol. Sci. B 49 (2010) 528
541. [17] X.F. Sun, Q.Q. Tian, Z.M. Xue, Y.W. Zhang, T.C. Mu, RSC Adv. 4 (2014) 30282
30291. [18] Y. Qin, X. Lu, N. Sun, R.D. Rogers, Green. Chem. 12 (2010) 968
971. [19] S. Islam, L. Arnold, R. Padhye, BioMed. Res. Int. 2015 (2015) 874316. [20] H.B. Xie, S.B. Zhang, S.H. Li, Green. Chem. 8 (2006) 630
633. [21] J. Horinaka, Y. Urabayashi, T. Takigawa, M. Ohmae, J. Appl. Polym. Sci. 130 (2013) 2439
2443. [22] K. Prasad, M.-a Murakami, Y. Kaneko, A. Takada, Y. Nakamura, J.-i Kadokawa, Int. J. Biol. Macromol. 45 (2009) 221
225. [23] J. Kadokawa, Green. Sustain. Chem. 3 (2013) 19
25. [24] J.-i Kadokawa, RSC Adv. 5 (2015) 12736
12746. [25] J.X. Feng, H.J. Zang, Q. Yan, M.G. Li, X.Q. Jiang, B.W. Cheng, J. Appl. Polym. Sci. 132 (2015) 41965. [26] R.A.A. Muzzarelli, Carbohydr. Polym. 76 (2009) 167
182. [27] R.A.A. Muzzarelli, Mar. Drugs 9 (2011) 1510
1533. [28] I. Younes, M. Rinaudo, Mar. Drugs 13 (2015) 1133
1174. [29] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603
632. [30] C.K.S. Pillai, W. Paul, C.P. Sharma, Prog. Polym. Sci. 34 (2009) 641
678. [31] D. Raabe, A. Al-Sawalmih, S.B. Yi, H. Fabritius, Acta Biomater. 3 (2007) 882
895. [32] J. Qiu, J. Wang, Chitin in rubber based blends and micro composites, in: P.M. Visakh (Ed.), Rubber Based Bionanocomposites: Preparation, Springer International Publishing, Cham, 2017, pp. 71
107. [33] H. Hirayama, J. Yoshida, K. Yamamoto, J.-i Kadokawa, Carbohydr. Polym. 200 (2018) 567
571. [34] K. Kurita, S. Ishii, K. Tomita, S.-I. Nishimura, K. Shimoda, J. Polym. Sci. Part. A: Polym. Chem. 32 (1994) 1027
1032. [35] L. Reys, S. Silva, J. Oliveira, S. Caridade, J. Mano, T. Silva, et al., Biomed. Mater. 8 (2013) 045002. [36] P.S. Barber, C.S. Griggs, J.R. Bonner, R.D. Rogers, Green. Chem. 15 (2013) 601
607. [37] T. Uto, S. Idenoue, K. Yamamoto, J.-i Kadokawa, Phys. Chem. Chem. Phys. 20 (2018) 20669
20677. [38] M.M. Jaworska, T. Kozlecki, A. Gorak, J. Polym. Eng. 32 (2012) 67
69. [39] M.M. Jaworska, A. Gorak, Mater. Lett. 164 (2016) 341
343. [40] M.M. Jaworska, A. Rak, J. Zdunek, Adv. Mater. Sci. Eng. 2017 (2017) 9. [41] M. Shimo, M. Abe, H. Ohno, ACS Sustain. Chem. Eng. 4 (2016) 3722
3727. [42] J.-i Kadokawa, Int. J. Biol. Macromol. 123 (2019) 732
737. [43] J. Chen, F. Xie, X. Li, L. Chen, Green. Chem. 20 (2018) 4169
4200. [44] Q. Chen, A. Xu, Z. Li, J. Wang, S. Zhang, Green. Chem. 13 (2011) 3446
3452. [45] N.M. Alves, J.F. Mano, Int. J. Biol. Macromol. 43 (2008) 401
414. [46] S.S. Silva, A. Motta, M.R.T. Rodrigues, A.F.M. Pinheiro, M.E. Gomes, J.O.F. Mano, et al., Biomacromolecules 9 (2008) 2764
2774. ´ [47] M.M. Jaworska, I. Ste˛pniak, M. Galinski, D. Kasprzak, D. Binia´s, A. Go´rak, Carbohydr. Polym. 202 (2018) 397
403.

Handbook of Chitin and Chitosan

496 [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70]

[71] [72] [73] [74] [75] [76] [77] [78]

[79]

15. Biomedical exploitation of chitin and chitosan-based matrices via ionic liquid processing

S. Mine, H. Izawa, Y. Kaneko, J. Kadokawa, Carbohydr. Res. 344 (2009) 2263
2265. Z. Zhang, C. Li, Q. Wang, Z.K. Zhao, Carbohydr. Polym. 78 (2009) 685
689. C.F. Liu, R.C. Sun, A.P. Zhang, W.Y. Li, ACS Symp. Ser. 1033 (2009) 287
297. J.-i Kadokawa, T. Setoguchi, K. Yamamoto, Polym. Bull. 70 (2013) 3279
3289. K. Yamamoto, S. Yoshida, S. Mine, J. Kadokawa, Polym. Chem. 4 (2013) 3384
3389. Y. Wei, W. Huang, Y. Zhou, S. Zhang, D. Hua, X. Zhu, Int. J. Biol. Macromol. 62 (2013) 365
369. A. Costa, A. Forte, K. Zalewska, G. Tiago, Z. Petrovski, L.C. Branco, Green. Chem. Lett. Rev. 8 (2015) 8
12. Z. Wang, L. Zheng, C. Li, D. Zhang, Y. Xiao, G. Guan, et al., Carbohydr. Polym. 117 (2015) 973
979. W. Zhang, Y. Wang, D. Zhang, S. Yu, W. Zhu, J. Wang, et al., Nanoscale 7 (2015) 10210
10217. Q. Chen, W. Xiao, L. Zhou, T. Wu, Y. Wu, Polym. Degrad. Stab. 97 (2012) 49
53. H. Chen, S. Cui, Y. Zhao, B. Wang, S. Zhang, H. Chen, et al., Engineering 4 (2012) 114
117. W. Hou, L. Liu, H. Shen, Carbohydr. Polym. 195 (2018) 267
274. Y.H. Zhou, M.Z. Fan, X.L. Luo, L.L. Huang, L.H. Chen, Carbohydr. Polym. 113 (2014) 108
114. Z. Wang, L. Zheng, C. Li, D. Zhang, Y. Xiao, G. Guan, et al., Carbohydr. Polym. 94 (2013) 505
510. H. Chen, S. Cui, Y. Zhao, C. Zhang, S. Zhang, X. Peng, PLoS One 10 (2015) e0121817. D.B. Hua, J.L. Jiang, L.J. Kuang, J. Jiang, W. Zheng, H.J. Liang, Macromolecules 44 (2011) 1298
1302. L. Yang, J. Zhang, J. He, J. Zhang, Z. Gan, Polym. Int. 64 (2015) 1045
1052. Y.Y. Liu, Y. Huang, P.O. Boamah, L. Cao, Q. Zhang, Z.R. Lu, et al., J. Appl. Polym. Sci. (2015) 132. J. Lu, F. Yan, J. Texter, Prog. Polym. Sci. 34 (2009) 431
448. A. Vioux, L. Viau, S. Volland, J. Le Bideau, Cr. Chim. 13 (2010) 242
255. S.S. Silva, A.R.C. Duarte, J.F. Mano, R.L. Reis, Green. Chem. 15 (2013) 3252
3258. H. Chen, S. Cui, Y. Zhao, C. Zhang, S. Zhang, X. Peng, PLoS ONE 10 (2015) 1
17. H.Y. Chen, S.B. Zhang, X.J. Peng, S.H. Cui, Y.N. Zhao, C.M. Zhang, et al., Homogeneous graft of chitosan with polyethylenimine in ion liquid and its application in gene delivery, Acta Poly. Sinica. 8 (2014) 1089
1095. S.S. Silva, A.R.C. Duarte, A.P. Carvalho, J.F. Mano, R.L. Reis, Acta Biomater. 7 (2011) 1166
1172. J.-I. Kadokawa, A. Takegawa, S. Mine, K. Prasad, Carbohydr. Polym. 84 (2011) 1408
1412. D. Zhao, Y. Liao, Z. Zhang, CLEAN.—Soil, Air, Water 35 (2007) 42
48. C.S. Nunes, K.B. Rufato, P.R. Souza, E.A.M.S. de Almeida, M.J.V. da Silva, D.B. Scariot, et al., Carbohydr. Polym. 170 (2017) 99
106. S.S. Silva, T.C. Santos, M.T. Cerqueira, A.P. Marques, L.L. Reys, T.H. Silva, et al., Green. Chem. 14 (2012) 1463
1470. T.-J. Park, Y. Jung, S.-W. Choi, H. Park, H. Kim, E. Kim, et al., Macromol. Res. 19 (2011) 213
215. P. Bharmoria, T. Singh, A. Kumar, J. Colloid Interf. Sci. 407 (2013) 361
369. S. Silva, A. Duarte, J.F. Mano, R.L. Reis, Development of a supercritical assisted particle-agglomeration method for the preparation of bioactive chitin-based matrices, in: 10th International Symposium on Supercritical Fluids, San Francisco, CA, 2012. S.S. Silva, A.R.C. Duarte, J.M. Oliveira, J.F. Mano, R.L. Reis, J. Bioact. Compat. Polym. 28 (2013) 481
491.

Handbook of Chitin and Chitosan

References

497

[80] T.M. Mututuvari, A.L. Harkins, C.D. Tran, J. Biomed. Mater. Res. Part. A 101 (2013) 3266
3277. [81] B.S. Gupta, M. Taha, M.-J. Lee, RSC Adv. 5 (2015) 106764
106773. [82] R.F.M. Frade, A.A. Rosatella, C.S. Marques, L.C. Branco, P.S. Kulkarni, N.M.M. Mateus, et al., Green. Chem. 11 (2009) 1660
1665. [83] A.M.A. Dias, A.R. Cortez, M.M. Barsan, J.B. Santos, C.M.A. Brett, H.C. de Sousa, ACS Sustain. Chem. Eng. 1 (2013) 1480
1492. [84] D.E. Gloria, K. Regina, R.M. Douglas, The development of ionic liquids for biomedical applications? prospects and challenges, Ionic Liquids: From Knowledge to Application, American Chemical Society, 2009, pp. 95
105. [85] J. Pernak, P. Chwała, Eur. J. Med. Chem. 38 (2003) 1035
1042. [86] J. Kadokawa, R. Endo, D. Hatanaka, K. Yamamoto, J. Polym. Environ. 23 (2015) 348
355. [87] D. Oh, S. Shin, C. Lim, D. Hwang, Materials 6 (2013) 3826. [88] J.L. Shamshina, G. Gurau, L.E. Block, L.K. Hansen, C. Dingee, A. Walters, et al., J. Mater. Chem. B 2 (2014) 3924
3936. [89] K. Mundsinger, A. Muller, R. Beyer, F. Hermanutz, M.R. Buchmeiser, Carbohydr. Polym. 131 (2015) 34
40. [90] R.M. Ion, A.A. Poinescu, S.M. Doncea, Key Eng. Mater. 587 (2014) 197
204. [91] J.L. Shamshina, O. Zavgorodnya, H. Choudhary, B. Frye, N. Newbury, R.D. Rogers, ACS Sustain. Chem. Eng. 6 (2018) 14713
14722. [92] N. Singh, K.K.K. Koziol, J.H. Chen, A.J. Patil, J.W. Gilman, P.C. Trulove, et al., Green. Chem. 15 (2013) 1192
1202. [93] N. Singh, J. Chen, K.K. Koziol, K.R. Hallam, D. Janas, A.J. Patil, et al., Nanoscale 8 (2016) 8288
8299. [94] J. Chakravarty, M.F. Rabbi, N. Bach, V. Chalivendra, C.-L. Yang, C.J. Brigham, Carbohydr. Polym. 198 (2018) 443
451. [95] X. Jia, Y. Yang, C. Wang, C. Zhao, R. Vijayaraghavan, D.R. MacFarlane, et al., ACS Appl. Mater. Interfaces 6 (2014) 21110
21117. [96] L. Li, B. Yuan, S. Liu, S. Yu, C. Xie, F. Liu, et al., J. Mater. Chem. 22 (2012) 8585
8593. [97] C. Stefanescu, W.H. Daly, I.I. Negulescu, Carbohydr. Polym. 87 (2012) 435
443. [98] Z. Liu, H. Wang, B. Li, C. Liu, Y. Jiang, G. Yu, et al., J. Mater. Chem. 22 (2012) 15085
15091. [99] C. Quadros, V. Faria, M. Klein, P. Hertz, C. Scheeren, J. Nanotechnol. 2013 (2013). [100] M. Rahimi, V. Shafiei-Irannejad, K.D. Safa, R. Salehi, Carbohydr. Polym. 196 (2018) 299
312. [101] N.V. Sastry, D.K. Singh, P.A. Trivedi, Colloids Surf. A: Physicochem. Eng. Asp. 555 (2018) 668
678. [102] L.K. Shpigun, E.Y. Andryukhina, Electroanalysis 30 (2018) 2356
2365. [103] A. Kimura, N. Nagasawa, M. Taguchi, Polym. Degrad. Stab. 159 (2019) 133
138. [104] K. Azuma, R. Izumi, T. Osaki, S. Ifuku, M. Morimoto, H. Saimoto, et al., J. Funct. Biomater. 6 (2015) 104
142. [105] C.D. Tran, T.M. Mututuvari, Langmuir 31 (2015) 1516
1526. [106] N. Adawiyah, M. Moniruzzaman, S. Hawatulaila, M. Goto, MedChemComm. 7 (2016) 1881
1897.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

16 Chitin and chitosan composites for bone tissue regeneration E.I. Akpan1, O.P. Gbenebor2, S.O. Adeosun2 and Odili Cletus2 1

Institute for Composite Materials, Technical University, Kaiserslautern, Germany, 2Department of Metallurgical and Materials Engineering, University of Lagos, Lagos, Nigeria

O U T L I N E 16.1 Introduction

500

16.2 Bone tissue engineering

501

16.3 Insights to bone biology

504

16.4 Clinical requirements for bone tissue engineering scaffolds 16.4.1 Biofunctionality 16.4.2 Biocompatibility 16.4.3 Bioresorbability or biodegradability 16.4.4 Osteoconductivity 16.4.5 Osteoinductivity 16.4.6 Osteogenicity 16.4.7 Osteointegrity

506 506 507 507 507 509 509 509

16.5 Physical and mechanical requirements for bone tissue replacement 16.5.1 Scaffold architecture 16.5.2 Porosity 16.5.3 Pore size and pore size distribution 16.5.4 Surface properties 16.5.5 Pore shape 16.5.6 Mechanical properties

509 509 510 511 511 511 512

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00016-9

499

© 2020 Elsevier Inc. All rights reserved.

500

16. Chitin and chitosan composites for bone tissue regeneration

16.6 Chitin/chitosan-based materials for bone tissue engineering 16.6.1 Chitin and chitosan for bone tissue engineering 16.6.2 Chitin and chitosan for cartilage regeneration 16.6.3 Chitin and chitosan composites for bone and bone repair

513 513 519 519

16.7 Methods of fabrication of chitin and chitosan scaffolds 16.7.1 Freeze-drying 16.7.2 Freeze gelation 16.7.3 Wet spinning 16.7.4 Electrospinning 16.7.5 Melt spinning 16.7.6 Fiber bonding 16.7.7 Particle leaching 16.7.8 3D printing and bioprinting 16.7.9 Phase separation 16.7.10 Monodispersion foaming

528 528 528 528 532 533 534 535 535 535 537

16.8 The dilemma of chitosan scaffold applicability

537

16.9 Conclusions

540

References

541

16.1 Introduction Chitosan is a derivative of chitin, a biopolymer present in the exoskeleton of crustaceans, fungi, insects, and some algae [1]. Chitin is one of the most abundant polymers in nature. Chitosan is derived from chitin by N-deacetylation using a series of reactions either homogeneously [2
5] or heterogeneously [6
14]. Chitosan is a macromolecule with β (1
4) glycosidic bonds linked D-glucosamine residues with a variable number of randomly located N-acetyl-D-glucosamines. Precisely, chitosan has a fraction of β-(1-4)-D-glucosamine and a fraction of β-(1-4)-N-acetyl-D-glucosamine randomly distributed and in varying proportions dependent on the extraction process. Chitosan is close in structure to glycosaminoglycan which is one of the components of the extracellular matrix that participates in cell to cell adhesion. On the other hand, depolymerization of chitosan produces bioactive materials with antimicrobial properties. It is biocompatible, renewable, nontoxic, nonallergenic, and biodegradable. These qualities make chitosan a very interesting material for biomedical applications. Recent areas of applications of chitosan include gene delivery, drug delivery, enzyme

Handbook of Chitin and Chitosan

16.2 Bone tissue engineering

501

immobilization, wound healing, drug encapsulation, surface modification, bone regeneration, dialysis membrane, and cosmetics [15
21]. Studies have shown that chitosan possesses anticholesteremic, antifungal, antitumor, immunoadjuvant, antithrombogenic, and antibacterial activities. Chitosan displays significant ability to proliferate osteoblasts and mesenchymal cells, showing that it can be used for bone tissue repair and regeneration [22
27]. In this chapter, a state-of-the-art overview of the use of chitosan and chitosan composites in bone tissue engineering (BTE) is presented. The chapter highlights the preparation and properties of innovative chitosan and chitosan composites with respect to BTE applications. Advancements and areas that require extra research attention are systematically presented.

16.2 Bone tissue engineering Bone disease is one of the most prevalent diseases in the world. Statistics have shown that the second most commonly transplanted tissue in the human body is bone. Worldwide four million transplant operations are estimated to occur annually involving the use of bone grafts or bone substitute materials [28]. The most prevalent methods of bone treatment are autologous or allogeneic bone grafting which are prone to disease transmission, interposition of soft tissues, improper fracture fixation, loss of bone, metabolic disturbances, and impairment of blood supply [29]. Studies have shown that scaffolds made of synthetic or natural biomaterials can be used to support the migration, proliferation, and differentiation of allogenic bone cells instead of grafting. Extensive research in the area of scaffolds also show that with adequate design, optimized patterns in terms of biodegradability and bioactivity can be realized making it possible to manufacture scaffolds with streamlined physical, biological, and mechanical properties [30]. The quest for the development of these materials into realizable products for applications gave birth to an interdisciplinary research area called BTE. BTE is an area of research that seeks to prompt functional bone regeneration through the systematic combination of biomaterials, cells, and factor therapy [31,32]. The basic idea is to construct artificial substitute materials for bone regeneration which possess biological functions, by combining basic principles and techniques of engineering and life sciences. Materials for bone tissue regeneration should fulfill some conditions including the following: (1) the cellular source should be nonimmunogenic, nontumorigenic, possess off-the-shelf availability, and express proliferative and osteogenic potential [29,33]; (2) the microstructure should possess interconnected pores throughout almost its entire volume to support cellular growth and ensure sufficient supply; (3) the

Handbook of Chitin and Chitosan

502

16. Chitin and chitosan composites for bone tissue regeneration

material should exhibit adequate load-bearing stability after implantation; and (4) the material should possess the capability for the formation of new bone matrix [34
36]. Over the years, it has been noticed that the combination of two materials for the fabrication of scaffolds can generate a synergistic effect to provide good mechanical strength to the scaffold and facilitate cell adhesion, proliferation, and differentiation. This has driven researchers to focus on the fabrication of scaffolds with multipolymers to mimic the properties of bone extracellular matrix ECM [36
45]. Fig. 16.1 shows the various aspects of BTE [46,47]. The first issue to address in BTE is the procurement of adequate cells. The source of the cell is required to be immediately available and in pertinent quantities, it should have controlled cell proliferation rate, predictable and consistent osteogenic potential, as well as controlled integration into the surrounding tissues. The cell source must also have no immune rejection, no graftversus-host disease, and no tumorigenicity. These conditions can be met when the cells are collected directly from each patient. The major challenges surrounding adequate cell procurement are the issues of sterility, acute setting minimal invasive harvesting procedure, patient variability, difference in patient ages, and histocompatibility. The second aspect of BET of high importance is the development of scaffolds as the substrate carrier. Scaffolds must be able to provide a permissive environment for the bone cells to migrate, differentiate, and deposit bone matrix. Scaffold materials should have specific biochemical, physicochemical, and geometric aspects [48
50]. They should be biocompatible, absorbable, osteoconductive, easy to manufacture and sterilize, and easy to handle.

FIGURE 16.1 Bone engineering.

Handbook of Chitin and Chitosan

16.2 Bone tissue engineering

503

FIGURE 16.2 (A) Hierarchical structural organization of bone. (B) Description of natural bone tissues. Source: (A) Reprinted with permission from Springer Nature J. Henkel, M.A. Woodruff, D.R. Epari, R. Steck, V. Glatt, I.C. Dickinson, et al., Bone Res. 1 (2013) 216
248; (B) Reprinted with permission from Elsevier S. Deepthi, J. Venkatesan, S.K. Kim, J.D. Bumgardner, R. Jayakumar, Int. J. Biol. Macromol. 93 (2016) 1338
1353.

Handbook of Chitin and Chitosan

504

16. Chitin and chitosan composites for bone tissue regeneration

16.3 Insights to bone biology Natural bone is a highly specialized inorganic
organic nanocomposite material consisting of inorganic mineral salts and proteins [51]. The inorganic part is the primary tissue and generally contains calcium hydroxyapatite (HA) [Ca10(PO4)6(OH)2] nanocrystallites (65% dry weight). It is the relatively hard part of the bone and responsible for rigidity. The organic part is mainly made of an elastic protein—collagen (35% dry weight). This is responsible for fracture resistance and helps in cell growth, proliferation, and differentiation. Other organic constituents of the bone are glycosaminoglycans, osteocalcin, osteonectin, bone sialoprotein, and osteopontin (Fig. 16.2A). They form a microenvironment stimulatory to cellular functions [53]. The inorganic part of the bone is relatively strong but brittle. On the other hand, the organic part is flexible with relatively low strength. A combination of the strong inorganic part and the soft organic part is the reason for the toughness of the bone [54
56]. Bone is a natural solid biocomposite, with a distinct hierarchical multiscale structural organization which is responsible for the high strength and fracture toughness. The bone structure is intricately organized such that the nanomineral crystals are embedded within the collagen matrix a well-organized structure which is thought to be the reason for the excellent mechanical properties [57]. Bone tissue can be described in terms of its macrostructure, microstructure, submicrostructure, nanostructure, and subnanostructure (Fig. 16.2B). The macroscopic structure of bone is made of a dense hard cylindrical shell of cortical bone along the shaft of the bone (Fig. 16.3). It gets thinner as the distance is farther from the center of the shaft towards the articular surfaces. Within the cortical bone are increasing amounts of porous trabecular bone (also referred to as cancellous or spongy bone).

FIGURE 16.3

Macroscopic structure of bone. (A) Human femur bone. (B) Osteon; the red ellipse indicates the rough contours of an osteon and corresponds to the position of a cement line. (C) Lamella. (D) Fiber bundle. (E) Mineralized fibril. (F) Mineral particle and collagen molecule. The size scales indicate the typical diameters of the various structural elements of human cortical bone. Source: (A
D, F) Reprinted with permission from Springer Nature P. Fratzl, Nat. Mater. 7 (2008) 610
612.

Handbook of Chitin and Chitosan

16.3 Insights to bone biology

505

Microscopically, mineralized collagen fibers are stacked parallel to form layers in the trabecular struts and dense cortical bone. At nanolevel, the prominent structures are mineral filled collagen fibers. A little farther away from the nanolevel, bone crystals, molecules and noncollagenous organic proteins are visible (Fig. 16.4). Detailed descriptions of bone can be found in literature [47,59
79]. The mechanical properties of bone are dependent on several conditions which should be taken into consideration in the development of alternative materials [52,58,90,313,328,351] for bone regeneration [80
88].

FIGURE 16.4 The microstructure and nanostructure of bone and the nanostructured material used in bone regeneration. (A) At the macroscopic level, bone consists of a dense shell of cortical bone with porous cancellous bone at both ends. (B) Repeating osteon units within cortical bone. In the osteons, 20
30 concentric layers of collagen fibers, called lamellae, are arranged surrounding the central canal, which contains blood vessels and nerves. (C) Collagen fibers (100
2000 nm) are composed of collagen fibrils. The tertiary structure of collagen fibrils includes a 67 nm periodicity and 40 nm gaps between collagen molecules. The HA crystals are embedded in these gaps between collagen molecules and increase the rigidity of the bone. Nanostructures with features of nanopattern (D), nanofibers (E), nanotube (F), nanopores (G), nanospheres (H), and nanocomposites (I) are structural components with a feature size in the nanoscale [33].

Handbook of Chitin and Chitosan

506

16. Chitin and chitosan composites for bone tissue regeneration

16.4 Clinical requirements for bone tissue engineering scaffolds 16.4.1 Biofunctionality The ability of a scaffold to meet its functional requirements for which it was designed at the required period effectively is called biofunctionality. In this case, scaffolds are biofunctional if they have the capacity to restore the functions of the replaced tissues. Characteristics of biofunctional scaffolds are adequate degradability to prevent partial loss of functionality and chemical and mechanical stability for the period which it has been enabled. Most scaffolds are not biofunctional but they can be functionalized using different methods [89]. A few methods that have been used to create biofunctionality are shown in Fig. 16.5.

FIGURE 16.5 Methods of enhancing material biofunctionality. Source: From I.C. Bonzani, et al. Novel materials for bone and cartilage regeneration, Curr. Opin. Chem. Biol., 10 (2006), 568.

Handbook of Chitin and Chitosan

16.4 Clinical requirements for bone tissue engineering scaffolds

507

16.4.2 Biocompatibility The meaning of biocompatibility of materials has been widely debated over the years. A conclusive meaning and mechanisms that are encompassed within the phenomena have not yet been attained. Generally, biocompatibility defines the ability of a biomaterial to sustain cellular activity including molecular signaling systems without provoking or arousing local or adverse effects to the host [91
97]. In a simple sense, materials are biocompatible when they exert the expected beneficial tissue response and clinically relevant performance. The other components of biocompatibility are cytotoxicity, genotoxicity, mutagenicity, carcinogenicity and immunogenicity. Biocompatibility is normally estimated through test animals, histologic and pathologic examinations of neighboring tissues, and host responses such as immunogenic, carcinogenic, and thrombogenic responses [98]. Nevertheless, the interactions between scaffold materials and tissues involve intricate phenomena in which the tissue and the material affect each other conversely. Williams [93] attempted a generic pathway for the biocompatibility of materials (Fig. 16.6).

16.4.3 Bioresorbability or biodegradability Ideally, scaffolds should be able to degrade with time at a controlled and well-defined rate, allowing space for the ingrowth of new bone. The ability of a material to degrade with time in vitro or in vivo at controlled resorption rate in order to create space for new tissue to grow is what is referred to as bioresorbability. It is expected that in the course of application, the rate of degradation matches the rate of growth due to the healing or regeneration process. Bioresorbability is related to biocompatibility in the sense that degradation products should be nontoxic and must be able to get metabolized and eliminated from the body [91].

16.4.4 Osteoconductivity Osteoconductivity is the ability of the bone cells to attach, multiply, and form extracellular matrix on its surface and pores. It is regularly seen in bone healing processes. A surface is said to be osteoconductive if it permits bone growth on its surface or down into pores, channels, or pipes. A great deal of discussion on osteoconduction can be found in Wilson-Hench [99] and Glantz [100]. It is related to biodegradability because in the process the scaffold material must be reabsorbed and allow the growth of the tissue that it was initially supporting.

Handbook of Chitin and Chitosan

FIGURE 16.6 The essential biocompatibility paradigm. Source: Reprinted with permission from Elsevier W. Elshahawy, in: C. Sikalidis (Ed.), Adv. Ceram.—Electr. Magn. Ceram. Bioceram. Ceram. Environ. (2011) 359
378.

16.5 Physical and mechanical requirements for bone tissue replacement

509

16.4.5 Osteoinductivity Osteoinduction is the ability of the scaffolds to induce new bone creation through biomolecular or mechanical stimuli. It implies the employment of immature cells and the stimulation of these cells to develop into preosteoblasts [101]. Osteoinductivity is demonstrated by injecting osteoblast into a soft tissue pouch to monitor if bone formation does occur under normal conditions. This translates that primitive, undifferentiated, and pluripotent cells are stimulated to change into the boneforming cell lineage.

16.4.6 Osteogenicity Osteogenicity is the ability of a scaffold to act as a reservoir for osteoblasts or mesenchymal cells. It is the property of the material that promotes new bone formation on and around the implant [102].

16.4.7 Osteointegrity Osteointegrity is the ability of a scaffold to form strong bonds with neighboring osseous tissue allowing for the continuity of the material and proper load transfer.

16.5 Physical and mechanical requirements for bone tissue replacement 16.5.1 Scaffold architecture Scaffolds are expected to have interconnected pore structure to allow for cellular penetration and diffusion of nutrients. This interconnected structure is usually defined by the pore size (pore diameter), pore distribution, and pore volume. These parameters define the architecture of a scaffold. Several functionalities of biomaterial scaffolds depend on the architecture of the scaffolds including cell metabolic activities, matrix biosynthesis, cell survival, and chondrogenesis. Architectures with high surface area to polymer mass ratio as well as high surface area to volume ratio have been found to permit tissue ingrowth, even cell delivery, and proliferation to high cell density [103
107]. Research has shown that large pore diameter and fine interconnected pores, and low tortuosity pores result in better cell penetration during seeding, ingrowth, and improve liquid uptake. There is a high possibility of controlling scaffold pore architecture using different scaffold fabrication techniques and processes [108
111].

Handbook of Chitin and Chitosan

510

16. Chitin and chitosan composites for bone tissue regeneration

16.5.2 Porosity Porosity is a morphological property of a scaffold defined as the percentage of void space in the scaffold which is independent of the material. Porosity plays a critical role in bone formation both in vitro and in vivo. It has been experimentally confirmed that the ideal porosity for bone tissue scaffolds should be more than 90%. Generally, for in vitro examinations, lower porosity is found to arouse osteogenesis by subduing cell proliferation and driving cell aggregation. On the other hand, higher porosity in vivo results in greater bone ingrowth [112
114]. Several methods have been used to measure porosity of scaffolds including a liquid displacement method, mercury intrusion porosimetry, gravimetry, scanning electron microscopy, and microcomputed tomography (micro-CT) imaging and analysis [115
131]. Fig. 16.7 shows the effect of microporosity on osteogenicity of cells.

FIGURE 16.7 Microporosity as it affects osteogenicity of cells [132].

Handbook of Chitin and Chitosan

16.5 Physical and mechanical requirements for bone tissue replacement

511

16.5.3 Pore size and pore size distribution Pore size and pore size distribution are some of the most important properties of polymeric scaffolds [133
137]. They are known to affect the progression of osteogenesis, cell migration, and diffusion [115
119,125,130,138
146]. Early studies showed that pore size should be in the minimum range of B100 μm to satisfy the requirement of cell size, cell migration, and transport. It was also reported that the minimum pore size required to regenerate mineralized bone is approximately 100 μm [114]. However, pore sizes greater than 300 μm are recommended to enhance the formation of capillaries and new bones. A study has shown that pores in the range of 100
150 and 150
200 μm enhance substantial bone ingrowth, whereas pores in the range of 75
100 μm result in ingrowth of unmineralized osteoid tissue. On the other hand, pores in the range of 10
75 μm only allow the penetration of fibrous tissues [144]. It is suggested that gradients in pore sizes may be useful in the formation of multiple tissues and tissue interfaces. Pore size is largely dependent on the fabrication techniques. In the case of particle leaching, pore size is dependent on the size of the porogens used. Fig. 16.8 shows the effect of pore size on cell growth.

16.5.4 Surface properties Surface properties of scaffolds such surface roughness, topography, surface free energy, surface charge, chemical functionalities, and morphology have direct effect on the performance of scaffolds [132,147
149]. It has been reported that surface properties directly affect tissue formation, mineralization, cell differentiation, proliferation, cell attachment, seeding efficiency, response, and ultimately bone formation [150
155]. Surface energy has also been confirmed to influence protein absorption on scaffolds [156]. Theoretically, a higher surface roughness translates to higher surface area which corresponds to an increase in initial cell attachment and seeding efficiency [157
162]. Cell growth on surfaces is known to follow the initial surface pattern of the material confirming the idea that surface topography will have a substantial effect on cell morphology. Surface properties are to a large extent controlled by microporosity of the bone tissue scaffold.

16.5.5 Pore shape Pore shape or pore geometry is another important parameter that affects the performance of scaffolds. Pore shapes can take several forms including simple cubic unit cells, square, triangular, parallelogram, hexagonal, truncated cuboctahedron, rhombic dodecahedron, and diamond-type unit cells. Bone tissue regeneration is somewhat dependent on pore shape and pore curvature [163,164]. Research has shown that longer and tortuous pores

Handbook of Chitin and Chitosan

512

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.8 Effect of pore size on cell growth in chitosan (A) Chitosan, small pore size, (B) (A) Chitosan, medium pore size, (C) Chitosan, large pore size, [105].

could lead to hindered cell penetration, making bone formation happen primarily on the surface [165]. It has also been shown that parallelogram pore geometry is the best pore geometry regarding cell growth. Studies on the effect of pore shape and geometry on the seeding efficiency, cell migration, alignment, proliferation, differentiation, tissue formation, and mineralization have been reported by several researchers [164,166
174]. The effect of pore shape on the growth of cells can be seen in Fig. 16.9

16.5.6 Mechanical properties Mechanical properties of scaffolds are among the properties directly determining the applicability of porous scaffolds [175]. In bone tissue regeneration, scaffold should be strong enough to allow for surgical processes during implantation and should have sufficient mechanical ability to function till the end of the remodeling. The required

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

513

FIGURE 16.9 Illustrative effect of pore shape on cell growth. (A) New tissue formed in three-dimensional matrix channels. Actin stress fibers are stained with phalloidin-FITC and visualized under a confocal laser scanning microscope. Here, the tissue formation is shown (i
iii) after 21 days and (iv) after 30 days of cell culture in the channels of a (i) triangular, (ii) square, (iii) hexagonal, and (iv) round shape introduced into a HA plate in vitro. (B) Numerical simulation of tissue formation within channels of various shapes: (i) triangular, (ii) square, (iii) hexagonal, and (iv) round. The lines (early time point 1, ongoing times 2 and 3) mark the simulated development of tissue formation due to ongoing culture time, which corresponds closely to the observed development of new tissue formation in vitro. Source: From M. Rumpler, A. Woesz, J. W.C Dunlop, J. T. van Dongen, P. Fratzl, The effect of geometry on three-dimensional tissue growth, J. R. Soc. Interface. 5 (2008) 1173
1180.

mechanical properties in BTE are elastic modulus, Poisson ratio, toughness, shear modulus, tensile strength, and in some cases fatigue and compressive strength. In scaffold tissue engineering, the expectation is to meet the capabilities of native bone to as large extent as possible including mechanical properties [176]. Scaffolds made from biopolymers are reported to possess very poor mechanical properties compared to native bone. However, researchers have also attempted to improve on the mechanical properties of scaffolds [177,178] by altering the structure of the scaffolds and addition of ceramic particles. Attempts to improve mechanical properties of scaffolds also come with the sacrifice of porosity which is very important for vascularization. This underscores the need to balance between mechanical performance and porous architecture of scaffolds [179].

16.6 Chitin/chitosan-based materials for bone tissue engineering 16.6.1 Chitin and chitosan for bone tissue engineering Several studies have been conducted on the use of chitosan and chitosan blends for BTE applications. A study by Huang et al. focused on

Handbook of Chitin and Chitosan

514

16. Chitin and chitosan composites for bone tissue regeneration

characterizing the effect of 3D and 2D blending of gelatin with chitosan on degradation properties, mechanical properties, and cell
matrix interactions [180]. Analyses were performed on human umbilical vein endothelial cells and mouse embryonic fibroblasts to understand the interactions of the cells. Results showed that the addition of gelatin affected the stiffness of the 2D and 3D scaffolds. But the structure showed weak cell adhesion in 2D culture. Wu et al. [181] developed polycaprolactone (PCL)/chitosan blend scaffolds using a particleleaching technique for improved adhesion and growth of osteoblasts with very significant impact on the osteoblastic phenotypes in rats. Blends of chitosan and gelatin were investigated as scaffolds for bone tissue regeneration using chemical cross-linking with glutaraldehyde or genipin by freeze-drying [182]. The scaffolds formed a gel-like interconnected pore structure capable of supporting adhesion, viability, proliferation, and osteogenic differentiation capacity of both preosteoblasts and bone marrow mesenchymal stem cells in vitro. Adhikari et al. [183] also reported the production of chitosan-based scaffolds by incorporation of magnesium gluconate for tissue engineering applications. The scaffolds were found to possess uniform porosity with highly interconnected pores of 50
250 μm size range and remained intact, retaining their original three-dimensional frameworks (Fig. 16.10) during in vitro conditions. Using the lyophilization technique Sajesh et al. [184] fabricated chitosan/polypyrole
alignate scaffolds for bone tissue regeneration applications. Results showed very good cell attachment (Fig. 16.11) suggesting that the developed scaffolds can be used in tissue regeneration applications. Chitosan sponges have been prepared to serve as tissues for bone formation [185]. The prepared chitosan sponges had a porous structure with a 100
200 μm pore diameter which implies that they can allow cell proliferation. Zhang et al. [186] also examined the production of macroporous phosphate
chitosan scaffolds for BTE. Results showed that cultured MG63 cells on the HA scaffolds nested chitosan sponges showed significantly higher alkaline phosphatase level and osteocalcin production compared to the control culture. VandeVord et al. [187] investigated the biocompatibility of chitosan/collagen scaffolds for bone implantation. Histological examination indicates noticeable neutrophil buildup within the implant, which was solved with increasing implantation time. It was also realized that collagen accumulates within the chitosan pore spaces, showing that connective tissue matrix was deposited within the implant. These results confirm that chitosan can be used to form biocompatible scaffolds for bone implantation. Klokkevold et al. [188] evaluated the influence of chitosan on osteoblast differentiation and bone formation in vitro. Results showed that chitosan can differentiate osteoprogenitor cells and should facilitate bone formation. The effect of the

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

515

FIGURE 16.10 SEM images A
C: scaffolds containing 2% (A), 4% (B), and 5% (C) of CS/carboxymethyl chitosan with no magnesium gluconate dihydrate; and D
F: 5% CS/ carboxymethyl chitosan containing relative amounts of 5% (D), 10% (E), and 20% (F) magnesium gluconate dihydrate, respectively. Source: Reprinted with permission from Elsevier U. Adhikari, N.P. Rijal, S. Khanal, D. Pai, J. Sankar, N. Bhattarai, Bioact. Mater. 1 (2016) 132
139.

chitosan nanofiber membranes on bone regeneration in rabbit calvarial defects has been examined by Shin et al. [189]. The membrane was biocompatible, and capable of bone regeneration with no evidence of inflammatory reaction. Growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) have been examined using a blend of chitosan and poly(butylene succinate). Results show that the blend supports the proliferation and osteogenic differentiation of hBMSCs cultured at its surface in vitro [190]. Muzzarelli et al. [191] synthesized methylpyrrolidinone chitosan and examined its potential for bone regeneration and growth. Histological studies showed that chitosan supported the formation of bone tissues and stimulated growth factors leading to the formation of intramembranous bone. The same author also examined the osteoconduction ability of the methylpyrrolidinone chitosan used in dental surgery [192]. Thermo- and pHresponsive chitosan
collagen scaffolds for pulsatile release of bioactive molecules have been fabricated by cross-linking with glutaraldehyde, and coated with poly(N,Nʹ-diethylacrylamide) via the freeze-drying method [193]. Highly porous chitosan scaffolds with controlled microstructure in several tissue-relevant geometries have been prepared by controlled freezing and lyophilization of solutions and gels [194]. Controlling the freezing conditions gave

Handbook of Chitin and Chitosan

516

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.11 SEM image of cell attachment on (A) chitosan, (B) chitosan/PPy
Alg scaffolds, actin/DAPi staining showing cell attachment and distribution on (C) chitosan, (D) chitosan/PPy
Alg scaffolds. Source: Reprinted with permission from Elsevier K.M. Sajesh, R. Jayakumar, S.V. Nair, K.P. Chennazhi, Int. J. Biol. Macromol. 62 (2013) 465
471.

rise to pore diameters in the range of 1
250 μm (Fig. 16.12). These scaffolds can be used in bone tissue applications. Chitosan scaffolds with improved mechanical properties and tunable topography intended for bone tissue application have been fabricated using a novel compression method [178]. Scaffolds with varying pore structures suitable for specific tissue applications can be obtained by varying the processing parameters. Yuan et al. [195] also reported the development of chitosan scaffolds with a tunable structure using the freeze gelation method. Chitosan scaffolds have been produced by freeze gelation with interconnected pores ranging from 60 to 150 μm [196]. ROS 17/2.8 osteoblast-like cells cultured on the scaffolds was found to attach, spread, and proliferate well, indicating potential application in tissue engineering. Chitosan fibers and 3D fiber meshes for tissue engineering applications have been fabricated using the wet spinning method [197]. Results showed that osteoblasts directly cultured over the chitosan fiber mesh had very good morphology and did not inhibit cell proliferation (Fig. 16.13). Porous chitosan scaffolds have been developed using freeze

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

FIGURE 16.12

517

SEM micrographs of porous chitosan microcarriers. Micrographs show low- and high-magnification views of microcarriers formed by various freezing protocols: (A, B) chitosan droplets directly frozen in liquid nitrogen; (C, D) interior views of directly frozen microcarriers; (E, F) chitosan gel beads directly frozen in liquid nitrogen; (G, H) chitosan gel beads directly frozen in methylene chloride at 53 C. Source: Reprinted with permission from Elsevier S.V. Madihally, H.W.T. Matthew, Biomaterials 20 (1999) 1133
1142.

Handbook of Chitin and Chitosan

518

16. Chitin and chitosan composites for bone tissue regeneration

gelation combined with cross-linking processes [198]. Mechanical properties examined showed that the scaffolds are suitable for tissue engineering applications. Textile-like chitosan fibers have been designed, fabricated, and investigated as a potential scaffold for tissue engineering applications [199]. The chitosan fibers were modified by coating with collagen type I. The formulated scaffolds were found to support the attachment, proliferation, and differentiation of osteoblast-like cells suggesting their potential application in tissue engineering of bone. Using a sintering process, chitosan microspheres with pore size, porosity, and mechanical properties suitable for BTE applications have been developed [200]. The study realized microspheres with compressive modulus in the range of human cancellous bone. NanoHA-reinforced chitosan composite hydrogel has been studied for bone tissue repair [201]. Another study on the use of nanohydroxylapatite/chitosan composite for bone defect repair was reported by Zhang et al. [202]. Chitosan microspheres encapsulated with bone morphogenetic have been designed for bone defect repair [203]. Mohamed et al. [204] investigated nanoHA/chitosan
gelatin as scaffolds for BTE. Alignate, chitosan, and polylactic acid (PLA)-HA composite scaffolds have been investigated as a drug release agent for bone repair treatment [205]. Gentile et al. [206] investigated the

FIGURE 16.13 Osteoblast-like cells proliferating over chitosan-based fibers after 7 days of culture. Reprinted with permission from John Wiley and Sons K. Tuzlakoglu, C.M. Alves, J.F. Mano, R.L. Reis, Macromol. Biosci. 4 (2004) 811
819.

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

519

viability of chitosan
gelatin scaffolds engrafted with simvastatin-loaded poly(lactic-co-glycolic acid) (PLGA) micron-sized particles as a drug release scaffolds for bone tissue repair. Chitosan and poly-3-hydroxybutyrate-co-3-hydroxyvalerate, and HA blends have been investigated as scaffolds for bone regeneration [207]. Biomimetic nanofibers of HA, collagen, and chitosan have been produced for bone tissue regeneration [208]. The nanofibers were found to promote osteogenic differentiation and bone regeneration. Jian et al. [209] incorporated PCL and polyethylene glycol into chitosan scaffolds for bone regeneration applications. Pourhaghgouy et al. [210] investigated the bioactivity of freeze cast chitosan bioactive glass composites. Serra et al. [211] also investigated chitosan/gelatin/β-TCP scaffolds for bone tissue regeneration.

16.6.2 Chitin and chitosan for cartilage regeneration Chitin and chitosan and their composites have been investigated for cartilage regeneration. Blends of chitosan/poly(3-caprolactone) with a 3D fiber-mesh structure have been developed and studied as possible support structures for articular cartilage tissue repair [212]. Blends were wet spun with different compositions. Histological studies revealed cartilaginous extracellular matrix in all formulations. Chitosan hydrogel has been investigated for cartilage engineering [213]. Ding et al. studied silk/chitosan scaffolds with excellent cell proliferation for cartilage repair [214]. Chitosan hydrogels with nanostructured bone-like HA was also investigated by Demirtas et al. [215] for bone regeneration. Collagen/chitosan microspheres composite has been investigated for bone regeneration in rabbit [216]. Ragetly et al. [217] also investigated the microstructure of fibrous chitosan scaffolds aimed at cartilage tissue regeneration. Chitosan
gelatin scaffolds have been investigated for cartilage tissue engineering by other authors [218].

16.6.3 Chitin and chitosan composites for bone and bone repair Composites of chitin and chitosan have been investigated for BTE applications. Yu et al. [219] investigated the viability of alginate, chitosan, collagen, and HA composite as a scaffold for bone tissue applications. The composite was found to be highly porous with a core sheath structure and is capable of beneficial cell infiltration and growth. A study by Nazemi et al. [220] on the incorporation of PLGA nanoparticles in chitosan/bioactive glass scaffolds shows that the scaffold has the potential to be used as bioactive bone tissue with localized drug delivery capability. Kucharska et al. [221] fabricated foamed chitosan/β-TCP

Handbook of Chitin and Chitosan

520

16. Chitin and chitosan composites for bone tissue regeneration

scaffolds for BTE. In another study, Zhang and Zhang [222] reinforced chitosan with β-tricalcium phosphate (β-TCP) and calcium phosphate invert glass using a thermally induced phase separation technique. The composites formed a highly macroporous open interconnected pore (Fig. 16.14). The study showed that desirable pore structure, biodegradation rate, and bioactivity can be achieved by controlling the ratio of chitosan and calcium phosphates or β-TCP and the glass. The authors also reported the use of macroporous chitosan scaffolds reinforced by β-TCP and calcium phosphate invert glasses for drug release applications [223] (Fig. 16.15). The study reported a good cellular compatibility between osteoblastic cells and the composite with no apparent difference from pure chitosan. Composites of chitosan fiber calcium phosphate cement have been fabricated for BTE scaffold applications [224]. The in vitro performance was examined with canine bone marrow stem cells. Results indicate that the scaffolds can be used to resolve the problem of relatively low mechanical strength of porous scaffolds for BTE. Zhang and Zhang [225] also investigated the fabrication of 3D macroporous calcium phosphate bioceramics embedded with porous chitosan sponges. Composites with pore diameters between 300 and 600 μm were developed with calcium phosphate bioceramics using a porogen burn-out technique, then the chitosan sponges were formed inside the pores using a freeze-drying process (Fig. 16.16). The sponges formed inside the pores were 100 μm in size which is suitable for bone tissue ingrowth. Osteoblast cells were found to attach to the composite scaffolds and migrated to the pore walls which indicate cell biocompatibility of the scaffolds. Chitosan and HA nanocomposite has been developed as an internal fixation of bone fracture using in situ hybridization [226]. Results showed that HA nanoparticles were well dispersed in the chitosan matrix with chitosan molecules assembled layer-by-layer. Chitosan matrix composites with poly(L-lactic acid) (PLLA) have been examined for bone regenerative potential [227]. Composites were fabricated using a freeze-drying method and cross-linking of aqueous chitosan solution. All the composites prepared in the study showed improved bone-forming capacity by increasing mechanical stability and biocompatibility. There was also a significant increase in osteoinductive effect in addition to the high osteoconducting capacity of the porous chitosan matrices making them useful for bone healing and regeneration. Leroux et al. [228] used chitosan as an adjuvant in bone cements to examine its effect on the injectability of the cement while maintaining the physicochemical properties suitable. The study showed that chitosan on its own improved injectability and setting time and limited the evolution of the cement by maintaining the Octacalucium Phosphate (OCP) phase. Xu et al. [229] studied the change in strength and toughness of

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

521

FIGURE 16.14

SEM photos of (A) pure chitosan scaffolds and chitosan/β-TCP/glass composite scaffolds with weight ratios of (B) 90/10/0, (C) 50/50/0, (D) 30/70/0, (E) 90/ 5/5, and (F) 90/0/10 at an original magnification of 200 3 . Source: Reprinted with permission from John Wiley and Sons Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 55 (2001) 304
312.

calcium phosphate cement when macropores appropriate for cell infiltration and bone ingrowth are created using chitosan. The study revealed that the use of chitosan mesh resulted in a superior synergistic strengthening as opposed to traditional single reinforcing agent. Interconnected macropores (osteoconductive) were formed and provided strength to implants during tissue regeneration. Weeraphat et al. [230] fabricated composites of bioactive glass, polyvinyl alcohol, chitosan, and collagen for BTE. Lima et al. [231] investigated chitosan, silk fibroin, and HA composite for BTE. Ghosh et al. [232] produced ionotropically cross-linked chitosan fibers for enhanced bone

Handbook of Chitin and Chitosan

522

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.15 SEM images of MG63 cells after 5 days of culture on the pure chitosan scaffold (A, B) and chitosan/β-TCP/glass composite scaffolds, with the weight ratio equal to (C, D) 90/10/0, (E, F) 90/5/5, (G, H) 90/0/10. Source: Reprinted with permission from John Wiley and Sons Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 62 (2002) 378
386.

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

523

FIGURE 16.16

Schematic of the process of producing macroporous bioceramics. Source: Reprinted with permission from John Wiley and Sons Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 61 (2002) 1
8.

regeneration. Chitosan, alignate, and nanosilica composites have been fabricated with well-defined porous architecture for bone tissue applications [233]. Martel-Estrada et al. examined the use of chitosan/Mimosa tenuiflora cortex composites for bone regeneration applications [234]. Duarte et al. [235] investigated blends of chitosan and PLA for tissue engineering applications. Composites containing iron oxide, HA, and chitosan have been investigated as scaffolds for bone tissue applications [236]. In an attempt to form a composite similar to human bone a biodegradable composite consisting of HA /chitosan
gelatin network has been formulated and examined [131]. Using a conventional phase separation technique, scaffolds with varying compositions were formulated and examined. The study realized a regular ordered three-dimensional network scaffold with very high porosity (Fig. 16.17). The scaffolds showed improved adhesion, proliferation, and expression on rat calvaria osteoblasts. In another study a new bioactive bone cement was proposed consisting of HA, chitosan powder, and polymethylmethacrylate for vertebroplasty and bone filler applications [237]. Results showed that the formulated bone cement is a suitable replacement for poly(methyl methacrylate) (PMMA) bone cements. Zhao et al. [238] attempted the development of calcium phosphate cement scaffolds with improved resistance to fatigue and fracture, for the delivery of human umbilical cord mesenchymal stem cells using chitosan and polyglactin (Fig. 16.18). The study revealed increase in fatigue resistance and excellent proliferation and viability of the stem cells on the scaffolds. Ang et al. [239] used chitosan HA composites to produce a 3D scaffold with regular and reproducible macropore architecture using a robotic dispensing system. Ge et al. [240] examined the use of chitin/HA composite to fabricate scaffolds with good osteoinductivity, rapid degradation and neovascularization. Composites

Handbook of Chitin and Chitosan

524

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.17 SEM micrographs of HA/CS-gel scaffolds prepared from HA/CS-gel/ acetic acid mixtures with different CS-gel concentrations: (A) CS-gel%: 1.0%, HA/CS-gel: 50/50, 3 40, (B) CS-gel%: 2.5%, HA/CS-gel: 30/70, 3 100. Source: Reprinted with permission from Elsevier P.A.L. Lima, C.X. Resende, G.D.De Almeida Soares, K. Anselme, L.E. Almeida, Mater. Sci. Eng. C 33 (2013) 3389
3395.

were processed into air- and freeze-dried materials and examined. Histological experiments revealed that explants showed bone regeneration with biodegradation of the HA
chitin matrix. In vivo effect of chitosan/HA paste applied on the surface of the tibia after periosteum removal has been examined by Kawakami et al. [241]. The study realized the formation of bone tissue with chondral tissue suggesting osteoconductive properties of the composite paste. Wang et al. [242] suggested the use of phosphorylated chitosan in calcium phosphate cements as a composite scaffold for treatment of bone defects in the radius and tibia. Experiments conducted in vivo showed that the composite was biocompatible, osteoinductive, bioresorbable, and can remodel via the creeping substitution process. Chitosan/TCP composite was also examined as an effective scaffold for the carrier of plateletderived growth factor. The study revealed that the sponge was capable of promoting osseous healing of rat calvarial defects and is therefore suitable for bone regeneration [243]. The effect of hyaluronic acid, calcium sulfate, and chitosan on early bone consolidation in distraction osteogenesis of a canine model was examined by Cho et al. [244]. Results showed that normal cortical bones were developed in the test subjects after 6 weeks of implantation. Another study by Kim et al. [245] on the effect of chitosan on early bone consolidation showed that chitosan can promote osteogeneic progenitor cell recruitment and attachment to facilitate bone formation. Calcium phosphate/chitosan composite has been used as a coating for Ti6Al4V plates to improve the biocompatibility of the apatite coatings [246]. The presence of chitosan influenced the crystallization and

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

525

FIGURE 16.18 SEM of hUCMSC attachment on: (A) CPC control, and (B) CPC
chitosan
fiber scaffold. Cells are designated as “C,” which anchored to CPC in (A), and to the fibers in the scaffold in (B). Cells developed long, cytoplasmic extensions “E,” shown in (C) at a higher magnification, attaching firmly to the fiber in the CPC
chitosan
fiber scaffold. Source: Reprinted with permission from Elsevier L. Leroux, Z. Hatim, M. Fre`che, J. Lacout, Bone 25 (1999) 31S
34S.

formation of calcium phosphate. Moreover, the composite proved favorable for marrow stromal cell attachment in goats compared with coatings without chitosan. In another study, Bumgardner et al. [247] reported the effectiveness of chitosan coating on titanium coupons via silane-glutaraldehyde molecules. Results obtained showed that chitosan has the latency to be used as a biocompatible, bioactive coating for orthopedic and craniofacial implant devices. The authors also reported the contact angle, protein adsorption, and osteoblast precursor cell attachment of chitosan coatings to titanium coupons [248]. Cell growth and function of macroporous chitosan scaffolds filled with HA and calcium phosphate invert glass have been examined [249]. The incorporation of glass into the chitosan matrix resulted in enhanced cell growth, alkaline phosphate activity, and osteocalcin production when compared to pure HA. Self-hardening chitosan composites with HA granules, ZnO, and CaO have been examined for BTE applications [250]. The composite showed mechanical strength comparable to that of

Handbook of Chitin and Chitosan

526

16. Chitin and chitosan composites for bone tissue regeneration

the cancellous bone derived from tibial eminentia. Porous scaffolds composed of fused chitosan/nanoHA microspheres have been examined for bone tissue regeneration [251]. The scaffolds were found to be biocompatible and osteoconductive. Porous HA/chitosan
alginate composite scaffolds have been prepared for BTE [252]. Composites were prepared through in situ coprecipitation and freeze-drying. The composites formed had a 3D porous structure (Fig. 16.19). Results of implantation experiment in mouse skulls showed a strong positive effect on bone formation in vivo in mice (Fig. 16.20). Composites of chitosan and HA, and HA/carbonate apatite compounds have been synthesized and examined for bone substitute applications [253]. Another study examined composites of chitosan and calcium sulfate as a delivery vehicle for osteoblasts [254]. Histological studies showed that the composite is a good delivery system for osteoblasts and a suitable material for bone grafting. Chitosan/silk fibroin nanofiber composites have been fabricated for modulation of osteogenic differentiation and proliferation of human mesenchymal stem cells [255]. Composites were examined by electrospinning and studied for growth and osteogenic differentiation of the stem cells. It was revealed that the composites are suitable for BTE. Mesoporous HA/chitosan composites have been examined for cell adhesion and proliferation [256]. In vitro examination showed good adherence, proliferation, and migration of osteoblast-like cells on the composite surfaces through the pores. In another study, Teimouri et al. [257] combined silk fibroin,

FIGURE 16.19

(A) Photograph of HAp/chitosan
alginate composite scaffolds, and SEM morphology of the composite scaffolds with different HAp contents; (B) 0, (C) 10, (D) 30, (E) 50, and (F) 70 wt.%. Source: Reprinted with permission from Elsevier H.-H. Jin, D.-H. Kim, T.-W. Kim, K.-K. Shin, J.S. Jung, H.-C. Park, et al., Int. J. Biol. Macromol. 51 (2012) 1079
1085.

Handbook of Chitin and Chitosan

16.6 Chitin/chitosan-based materials for bone tissue engineering

527

FIGURE 16.20 Histological analysis of (A) the mouse skull with H&E staining after 8 weeks, (B) the site implanted with control sample, (D) high magnification of (B), (C) the site implanted with HAp/chitosan
alginate composite scaffold, (E) high magnification of (C) (HBT, host bone tissue; NBT, new bone tissue; Ob, osteoblast; Oc, osteoclast; B, bone; Hl, Howship’s lacuna). Source: Reprinted with permission from Elsevier H.-H. Jin, D.-H. Kim, T.-W. Kim, K.-K. Shin, J.S. Jung, H.-C. Park, et al., Int. J. Biol. Macromol. 51 (2012) 1079
1085.

chitosan, and zirconia (nano-ZrO2) using a freeze-drying technique to fabricate a biocomposite scaffold for tissue engineering applications. Chitosan/HA composite scaffolds [258] has been found to show good cellular infiltration capability and delayed degradation profiles. A similar study was reported by Rogina et al. [259] but in this case the HA was in situ precipitated in the chitosan matrix. Blends of chitosan and synthetic aliphatic polyesters (polybutylene succinate, polybutylene

Handbook of Chitin and Chitosan

528

16. Chitin and chitosan composites for bone tissue regeneration

succinate adipate, PCL, and polybutylene terepthalate adipate) and HA have been investigated as scaffolds for BTE [260]. Chitosan composite scaffolds with β-TCP and gelatin were produced in the form of a hybrid polymer network via cross-linking with glutaraldehyde [261]. The scaffolds were found to display a homogeneous 3D porous structure with interconnecting polyangular pores. Biocompatibility evaluations showed that the composite scaffolds have good biocompatibility but can be used only in nonloading bone regeneration. Lee et al. [262] developed a chitosan based material that has been shown to improve wound healing. The study aimed to develop chitosan/TCP sponges as tissue engineering scaffolds for bone formation by three-dimensional osteoblast culture. Muzzarelli et al. [263] reported the use of modified chitosan with covalently linked imidazole groups to stimulate bone formation in an animal model. Results revealed that the modified chitosan was osteoinductive and capable of activating biological functions. A few other studies that have been conducted on chitosan and composite scaffolds for BTE are listed in Table 16.1.

16.7 Methods of fabrication of chitin and chitosan scaffolds 16.7.1 Freeze-drying Freeze-drying is one of the most common methods of producing chitosan scaffolds. It involves freezing the chitosan solution at a low temperature usually between 270 C to 280 C and placing the frozen solution in a low-pressure chamber so that the water goes out by direct sublimation. This stage is called the primary drying. The residual water is removed in a secondary drying stage by desorption [302
311]. Schematic of the freeze-drying process is shown in Fig. 16.21.

16.7.2 Freeze gelation Freeze gelation is a method of producing chitosan scaffolds in which chitosan in solution is frozen to induce phase separation and immersed in a gelation environment at a temperature lower than the freezing point of the solution (Fig. 16.22). The gelled scaffold is then air-dried to remove residual liquid. The method is noted to be energy and time-efficient with less residual solvent, and it is easy to scale up [196,314].

16.7.3 Wet spinning Wet spinning is a fiber production technique that requires the polymer to be soluble in a solvent [315]. An example of wet spinning can be

Handbook of Chitin and Chitosan

529

16.7 Methods of fabrication of chitin and chitosan scaffolds

TABLE 16.1

Research in chitin and chitosan bone tissue materials. Fabrication method

Anticipated application

Chitosan
gelatin/ nanohydroxyapatite
montmorillonite composite

Freeze-drying with ice particulates as porogen

Bone tissue engineering

[264]

2.

Porous titanium filled with chitosan/ hydroxyapatite sponge

Electron beam melting and freeze-drying

Bone repair in loadbearing sites

[265]

3.

Nanocomposite scaffold containing bioactive glass, carbon nanotube and chitosan

Hot press and salt leaching process

Bone tissue regeneration

[266]

4.

Chitosan/bioactive glass

Freeze-drying of solution

Bone tissue regeneration

[267]

5.

Chitosan
gelatin 3D scaffolds

Ice segregation induced selfassembly

Biomaterials

[268]

6.

Tricalcium phosphate
chitosanfucoidan biocomposite scaffold

Freeze-drying technique

Bone-tissue engineering applications

[269]

7.

Chitosan with bioactive glass nanoparticles

Sol
gel process

Bone tissue engineering

[270]

8.

Polypropylene-chitosan nanofibers

Freeze-drying

Tissue scaffolds

[271]

9.

Chitosan loaded with chondroitin 4sulfate or alginate with biomimetic apatite surface layer

Lyophilized in a freeze-dryer.

Bone regeneration

[272]

10.

Arginine-glycine-aspartic acid cell binding peptide conjugated chitosan scaffolds

UV cross linking

Bone tissue engineering

[273]

11.

Scaffolds of silk fibroin and amino polysaccharide chitosan

Freeze-drying

Tissue engineering

[274]

12.

Chitosan
gelatin/ nanohydroxyapatite composite

Solution blending

Bone tissue regeneration

[275]

13.

Chitosan microspheres

Cross-linking using genipin reagent

Protein release

[276]

14.

Amide bonded poly(lactic-co-glycolic acid) (PLGA) and chitosan scaffold

Air drying and freeze-drying

Cartilage regeneration

[277]

S/N

Chitosan-based scaffold composition

1.

Ref.

(Continued)

Handbook of Chitin and Chitosan

530

16. Chitin and chitosan composites for bone tissue regeneration

TABLE 16.1

(Continued) Fabrication method

Anticipated application

Chondrogenic extracellular matrix incorporated collagen/chitosan scaffold

Threedimensional multilayered cocultured system

Cartilage regeneration

[278]

16.

Aligned chitosan-based ultrafine fibers

Jet electrospinning

Tendon tissue regeneration

[279]

17.

Methacrylamide chitosan hydrogel

UV cross-linking

Bone tissue regeneration

[280]

18.

Chitosan
PEO scaffolds

Wet spinning

Bone tissue delivery system

[281]

19.

Chitosan/PLGA microspheres

Sintering

Bone tissue regeneration

[282]

20.

Scaffolds of chitosan, natural nanohydroxyapatite and fucoidan

Freeze-drying

Bone tissue regeneration

[283]

21.

Scaffolds of chitosan, mesoporous wollastonite particles and carboxymethylcellulose

Freeze-drying

Bone tissue regeneration

[284]

22.

Scaffolds of chitosan/hydroxyapatite beta tricalcium phosphate composites

Freeze-drying

Bone tissue regeneration

[285]

23.

Chitosan
micro hydroxyapatite and chitosan
nanohydroxyapatite scaffolds

Lyophilized in freeze-dryer

Bone graft substitute

[286]

24.

Scaffolds of chitosan and keratin nanoparticles from chicken feathers

Freeze-drying

Bone tissue engineering

[23]

25.

3D nanocomposite scaffold of chitosan, gelatin and nanosilica

Lyophilization

Bone tissue engineering

[287]

26.

Hydroxyapatite, chitosan and alignate composite scaffolds

In situ coprecipitation and freezedrying

Bone tissue engineering

[252]

27.

Scaffolds of chitosan/natural hydroxyapatite with chondroitin sulfate and amylopectin

Freeze-drying

Bone graft material

[288]

28.

Composites of chitosan and carbon nanotubes

Freezing and lyophilization

Bone tissue engineering

[289]

S/N

Chitosan-based scaffold composition

15.

Ref.

(Continued)

Handbook of Chitin and Chitosan

531

16.7 Methods of fabrication of chitin and chitosan scaffolds

TABLE 16.1

(Continued) Fabrication method

Anticipated application

Composites of chitosan, nanoscaled silicon dioxide, and zirconia

Freeze-drying

Bone tissue engineering

[290]

30.

Composites of nanozirconia chitin
chitosan

Lyophilization

Tissue engineering scaffold

[291]

31.

Biocomposite scaffold of chitosan, nanohydroxyapatite, nanosilver

Lyophilization in a freeze-dryer followed by ion exchange and reduction to deposit silver nanoparticles

Bone tissue engineering

[292]

32.

Composites of nano-TiO2, chitin
chitosan

Lyophilization

Tissue engineering scaffold

[293]

33.

Composite scaffold containing chitosan, gelatin, alginate and a bioceramic
nanohydroxyapatite

Simple foaming

Bone tissue engineering

[294]

34.

Composites of chitosan
polyvinyl pyrrolidone and bioglass

Foam replication and chemical cross-linking

Bone tissue regeneration

[295]

35.

Composite scaffolds containing nanosized HA, alignate and chitosan.

Freeze-drying

Bone tissue engineering

[296]

36.

Poly(N-isopropylacrylamide) and water soluble chitosan

Precipitation

Cartilage formation

[297]

37.

Hydroxybutyl chitosan

Gelation

Drug delivery

[298]

38.

Chitosan/collagen blend

Freeze-drying

Gene delivery

[299]

39.

Chitosan alignate

Lyophilization

Bone regeneration

[300]

40.

Nanocomposites of hydroxyapatite and chitosan in the presence of polylactic acid

In situ precipitation

Bone tissue engineering

[301]

S/N

Chitosan-based scaffold composition

29.

Ref.

seen in Fig. 16.23. The solution (dope) is extruded through a fine orifice directly into a liquid bath (coagulant), thus the name wet spinning [317]. The method is based on precipitation or coagulation of the polymer drawn through a spinneret into a nonsolvent [318]. Solvents that

Handbook of Chitin and Chitosan

532

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.21 Schematic of freeze-drying process [312]. Source: Adapted from S.L. Levengood, M. Zhang, Chitosan-based scaffolds for bone tissue engineering. J. Mater. Chem. B., 2(21) (2014), 3161
3184.

have been used in wet spinning of chitosan include sodium hydroxide [319], aqueous acetic acid [320], formic acid [321], ionic liquid [316,322], and sodium alginate [323].

16.7.4 Electrospinning Electrospinning is a method of producing fibers ranging from the submicron to nanometers using a high-voltage electrostatic field. In the process, a solution of the polymer is forced through an orifice using a high voltage to form a continuous fiber collected on a plate (Fig. 16.24). Chitosan are not soluble in alkali, water, and some mineral acids. Although chitosan is insoluble in inorganic acids, it is soluble in organic acids, such as dilute aqueous acidic solutions such as acetic, formic, and lactic acids. Because chitosan has free amino groups which make it a polyelectrolyte with positive charge within a pH range of 2
6, it is highly viscous leading to poor electrospinning properties. The large 3D hydrogen bonding network in chitosan prevents free movement of the polymeric chains exposed to the electric

FIGURE 16.22 Freeze gelation experiment. Source: Reprinted with permission from Royal Society of Chemistry S.K.L. Levengood, M. Zhang, J. Mater. Chem. B 2 (2014) 3161.

Handbook of Chitin and Chitosan

16.7 Methods of fabrication of chitin and chitosan scaffolds

533

FIGURE 16.23

Schematic of wet spinning of chitosan. Source: Reprinted with permission from Royal Society of Chemistry L. Li, B. Yuan, S. Liu, S. Yu, C. Xie, F. Liu, et al., J. Mater. Chem. 22 (2012) 8585.

field. Chitosan has been electrospun using acetic acid [324] and chloroform [325].

16.7.5 Melt spinning Melt spinning is a method of fiber formation that is typically applied to thermoplastic polymers which melt under heat and solidifies after cooling. This is very difficult to achieve in biofunctional polymers such as chitosan because it degrades without melting at elevated temperatures. However, the use of melt spinning in the processing of chitosan fibers will overcome the limitations of solution processing. To achieve melt spinning, chitosan is usually blended with another polymer to make it melt spinnable [190,326,327]. Fig. 16.25 shows a schematic of a melt spinning apparatus. The blended polymer is fed into the hopper and with well-determined temperature and screw speed the polymer is spun into fibers.

FIGURE 16.24

Schematic of electrospinning process.

Handbook of Chitin and Chitosan

534

16. Chitin and chitosan composites for bone tissue regeneration

16.7.6 Fiber bonding Fiber bonding is a method used to form interconnected scaffold fiber structure [329
332]. Research has shown that nonbonded fiber meshes possess poor mechanical properties for in vivo tissue regeneration (Fig. 16.26). To offer improved mechanical properties fiber bonding technique was developed to bind the fibers together at the points of intersection. Usually polymer fibers of the intended scaffolds are embedded in a solution of low melting temperature polymer. The solvent is allowed to evaporate so that the fibers are embedded in the matrix of the low melting polymer. Heating the composite above the melting temperature of the low melting polymer causes it to melt and fill all the voids which are not intercrossed in the network structure. This helps to retain the spatial arrangement of the fibers so that with further increase in the temperature the structure does not collapse. To minimize the interfacial energy when the fibers begin to melt, the fibers at the cross-points melt and become “welded” together, leading to the formation of a highly porous material. Finally, a solvent is used to dissolve the unwanted polymer. Although the technique is simple and retains the fibers’ original properties it suffers from lack of control over porosity and pore size, unavailability of suitable solvents, immiscibility of the polymers in the melt state, and the requirement for relative melting temperatures of the polymers involved.

FIGURE 16.25 Schematic of melt spinning apparatus. Source: Reprinted with permission from Royal Society of Chemistry J. Feng, D. Zhang, M. Zhu, et al., J. Mater. Chem. B 5 (2017) 5176
5188.

Handbook of Chitin and Chitosan

16.7 Methods of fabrication of chitin and chitosan scaffolds

535

16.7.7 Particle leaching Particle leaching is one of the most used methods for the formation of scaffolds for biomedical applications. In the process, the polymer is dissolved in a solvent and a porogen (e.g., NaCl) is added and stirred to make it homogeneous. The solvent is removed by evaporation and the cast composite is soaked in water to remove the salt. A porous structure is formed with pore size and distribution controlled by the amount and size of the porogen used [333
335] (Fig. 16.27).

16.7.8 3D printing and bioprinting Three-dimensional printing and bioprinting are novel methods of developing sophisticated scaffolds for tissue engineering. They have the potential of constructing complex cell structures similar in architecture and organization to native cells. 3D printing offers solution to the deficiencies of traditional scaffold fabrication techniques such as poor and limited control over pore size, geometry and interconnectivity. 3D printing offers ability to spatially control scaffold macro-architecture and spatial contents [337]. It fabricates objects using layer-by-layer processing of powder, solid, or liquid materials. It uses computer-aided design model to dictate the structure of the printed object. Patient specific models can be created by converting three-dimensional images of their clinical defects from computer tomography and magnetic resonance imaging into 3D models. A software can then be used to convert these models into codes (G-codes) that the printer can process. The printer physically reproduces models through an additive process. An example of 3D printing is shown in Fig. 16.28. Several methods have been adapted to produce bone tissues including stereolithography, fused deposition modeling, and selective laser sintering (Fig. 16.29). An important 3D printing method is bioprinting. Bioprinting is used to deposit living cells, extracellular matrices, and other biomaterials in user friendly patterns to build complex tissue constructs (Fig. 16.30). Popular bioprinting techniques include inkjet, laser-assisted, microvalve, and extrusion (Fig. 16.31). Several studies have reported the use of 3D printing in the formation of chitosan scaffolds [109,119,339
348].

16.7.9 Phase separation Phase separation is a method of scaffold production in which the conditions (e.g., temperature) of the solution are changed drastically to induce phase separation. The most common phase separation technique is thermally induced phase separation. In temperatureinduced phase separation, chitosan is dissolved in a solvent at room

Handbook of Chitin and Chitosan

536

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.26 Schematic representation of fiber-bonding. Source: Reprinted with permission from John Wiley and Sons A.G. Mikos, Y. Bao, L.G. Cima, D.E. Ingber, J.P. Vacanti, R. Langer, J. Biomed. Mater. Res. 27 (1993) 183
189.

Handbook of Chitin and Chitosan

16.8 The dilemma of chitosan scaffold applicability

FIGURE 16.27

537

Schematic of solvent casting with particle leaching [336].

temperature and cooled (soaking in nitrogen or at low temperatures) (Fig. 16.32). The final matrix is obtained by subsequent lyophilization.

16.7.10 Monodispersion foaming Monodispersion foaming is a method of forming highly monodisperse scaffold foams that utilizes millifluidic technique. In the process a solution of chitosan and air are injected simultaneously and at constant flow rates into two neighboring branches of a T-junction [349,350]. With suitable parameters the gas thread produced is pinched off by the liquid flow in a regular manner, leading to the production of monodisperse bubbles Fig. 16.33 shows the schematic of the process. The efficiency of the process relies highly on the selection of accurate parameters [352
355].

16.8 The dilemma of chitosan scaffold applicability Chitosan is an excellent biomaterial for BTE scaffolds as could be seen in the foregoing sections. However, chitosan possess very poor mechanical properties compared to those of native bone tissues. For example, the average tensile strength of cortical bone is between 100 and 230 MPa

Handbook of Chitin and Chitosan

538

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.28 Schematic diagram of 3D printing [337].

FIGURE 16.29 Common 3D printing techniques: (A) stereolithography; (B) fused deposition modeling; (C) selective laser sintering [28].

Handbook of Chitin and Chitosan

16.8 The dilemma of chitosan scaffold applicability

FIGURE 16.30

539

Illustration of bioprinting technique [338].

[356]. To obtain values in this range, chitosan has been modified by blending and addition of filler particles. However, these modifications affect adversely the biofunctionality. For example addition of ceramic particles in chitosan leads to a significant decrease in porosity of the composites which is detrimental to biofunctionality. Moreover, in an attempt to increase cell growth efficiency, modifications that will alter pore architecture have been utilized. These modifications also negatively affect the mechanical properties. These scenarios create an unsolved dilemma for the development of chitosan scaffolds for BTE applications (Fig. 16.34). A systematic research that will reveal optimum modifications for optimum combination of mechanical properties and biofunctionality should be undertaken. The use of ceramic particles proves useful in mechanical property improvement but affects the pore structure negatively. It may be of scientific relevance to attempt the use of pore activated particles. This will ensure high porosity to be maintained even with very high volume fraction of filler particles.

Handbook of Chitin and Chitosan

540

16. Chitin and chitosan composites for bone tissue regeneration

FIGURE 16.31 Common bioprinting techniques [28].

FIGURE 16.32 Schematic of phase separation.

16.9 Conclusions This chapter provides an overview of the state-of-the art research in the use of chitosan in BTE. The functionality of chitosan in bone tissue applications is dependent on the processing. With appropriate fabrication technique, blend, and addition of particles, chitosan scaffolds with very good

Handbook of Chitin and Chitosan

References

541

FIGURE 16.33 Setup to generate the monodisperse foams. Source: Reprinted with permission from American chemical society A. Testouri, C. Honorez, A. Barillec, D. Langevin, W. Drenckhan, Macromolecules 43 (2010) 6166
6173.

FIGURE 16.34

The paradigm of scaffold applicability.

pore architecture that will promote new bone growth, excellent cell adhesion, and proliferation can be fabricated. Research is needed in the area of improving the mechanical properties of chitosan-based scaffolds while retaining the needed porosity.

References [1] R. Logithkumar, A. Keshavnarayan, S. Dhivya, A. Chawla, S. Saravanan, N. Selvamurugan, Carbohydr. Polym. 151 (2016) 172
188.

Handbook of Chitin and Chitosan

542

16. Chitin and chitosan composites for bone tissue regeneration

[2] M.R. Kasaai, R.K. Mohammad, H. Nmr, C. Nmr, N. Nmr, H. Nmr, et al., J. Agric. Food Chem. 57 (2009) 1667
1676. [3] S. Ichi Aiba, Int. J. Biol. Macromol. 13 (1991) 40
44. [4] H. Sashiwa, H. Saimoto, Y. Shigemasa, S. Tokura, Carbohydr. Res. 242 (1993) 167
172. [5] T.T. Khong, Vietnamese Chitin Raw Material, the Chitin de-N-Acetylation Reaction, and a New Chitosan—Alginate Gelling Concept, Norwegian University of Science and Technology, 2013. [6] J. Bossaer, Water Soluble Chitosan by Heterogeneous Deacetylation of Chitin, Uniiversita¨t Gent, 2011. [7] V. Novikov, I. Konovalova, Y. Kuchina, N. Dolgopyatova, Y. Cherkun Vestnik, MGTU 20 (2017) 515
525. [8] S.P. Campana-Filho, R. Signini, M.B. Cardoso, Int. J. Polym. Mater. Polym. Biomater. 51 (2002) 695
700. [9] B. Bradi´c, D. Bajec, A. Pohar, U. Novak, B. Likozar, React. Chem. Eng. 3 (2018) 920
929. [10] G. Lamarque, G. Chaussard, A. Domard, Biomacromolecules 8 (2007) 1942
1950. [11] A. Wojtasz-Paja˛k, J. Szumilewicz, Prog. Chem. Appl. Chitin Deriv 14 (2019) 15
24. [12] T.H.D. Ngo, D.N. Ngo, Int. J. Biol. Macromol. 104 (2017) 1604
1610. [13] K.L.B. Chang, G. Tsai, J. Lee, W.-R. Fu, Carbohydr. Res. 303 (1997) 327
332. [14] C.J. Jiang, M.Q. Xu, Chem. Eng. Technol. 29 (2006) 511
516. [15] M. Kucharska, M.H. Struszczyk, M. Cichecka, K. Brzoza, Prog. Chem. Appl. Chitin Deriv 2012 (2012) 129
136. [16] A.O. Pereira, D.J. Cartucho, A.S. Duarte, M.H. Gil, A.M.S. Cabrita, J.A. Patrı´cio, et al., Curr. Drug Discov. Technol. 2 (2005) 231
238. ¨ zbac¸s-Turan, C. Aral, L. Kabasakal, M. Keyer-Uysal, J. Akbuǧa, J. Pharm. Pharm. [17] S. O Sci. 6 (2003) 27
32. [18] A. Alishahi, M. Aı¨der, Food Bioprocess Techn 5 (2012) 817
830. [19] K. Ohkawa, K.-I. Minato, G. Kumagai, S. Hayashi, H. Yamamoto, Biomacromolecules 7 (2006) 3291
3294. [20] H. Honarkar, M. Barikani, Monatsh. Chem. 140 (2009) 1403
1420. [21] S.M. Kuo, G.C.-C. Niu, S.J. Chang, C.H. Kuo, M.S. Bair, J. Appl. Polym. Sci. 94 (2004) 2150
2157. [22] I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo, I.Y. Park, B.C. Kim, et al., Biotechnol. Adv. 26 (2008) 1
21. [23] S. Saravanan, D.K. Sameera, A. Moorthi, N. Selvamurugan, Int. J. Biol. Macromol. 62 (2013) 481
486. [24] T. Jiang, R. James, S. G. Kumbar, C. T. Laurencin, Chapter 5 - Chitosan as a Biomaterial: Structure, Properties, and Applications in Tissue Engineering and Drug Delivery, in: S. G. Kumbar, C. T. Laurencin, M. Deng, (eds.) Natural and synthetic biomedical polymers, Elsevier, 2014, pp. 91
113. [25] C. Koushik, S. Saravanan, A. Moorthi, M. Vairamani, R. Niranjan, N. Selvamurugan, Int. J. Biol. Macromol. 54 (2012) 24
29. [26] N. Saranya, S. Saravanan, A. Moorthi, B. Ramyakrishna, N. Selvamurugan, J. Biomed. Nanotechnol. 7 (n.d.) 238
244. [27] A.R. Costa-Pinto, R.L. Reis, N.M. Neves, Tissue Eng. Part B Rev 17 (2011) 331
347. [28] G. Turnbull, J. Clarke, F. Picard, P. Riches, L. Jia, F. Han, et al., Bioact. Mater 3 (2018) 278
314. [29] D. Logeart-Avramoglou, F. Anagnostou, R. Bizios, H. Petite, J. Cell. Mol. Med. 9 (2005) 72
84. [30] D. Puppi, F. Chiellini, A.M. Piras, E. Chiellini, Prog. Polym. Sci. 35 (2010) 403
440.

Handbook of Chitin and Chitosan

References

543

[31] B.H. Atak, B. Buyuk, M. Huysal, S. Isik, M. Senel, W. Metzger, et al., Carbohydr. Polym. 164 (2017) 200
213. [32] D. Tang, R.S. Tare, L.Y. Yang, D.F. Williams, K.L. Ou, R.O.C. Oreffo, Biomaterials 83 (2016) 363
382. [33] T. Gong, J. Xie, J. Liao, T. Zhang, S. Lin, Y. Lin, Bone Res. 3 (2015) 15029. [34] J. Neman, A. Hambrecht, C. Cadry, R. Jandial, Biol. Targets Ther 6 (2012) 47
57. [35] S. Sundelacruz, D.L. Kaplan, Semin. Cell Dev. Biol. 20 (2009) 646
655. [36] p S. Nancy, M. Shilpita, S. Sen, J. Swathi, Tissue Eng. Regen. Med. 4 (2012) 524
531. [37] S. Gupta, S. Kapoor, J. Biosoc. Sci. 45 (2013) 1
11. [38] S.K. Gupta, A.K. Dinda, P.D. Potdar, N.C. Mishra, Mater. Sci. Eng. C 33 (2013) 4032
4038. [39] S. Datta Gupta and R. R. Mazumdar, Inferring causality in networks of WSS processes by pairwise estimation Methods, 2013 Information Theory and Applications Workshop, ITA 2013 - Conference Proceedings, 2013, pp. 264
272. [40] R. Dorati, C. Colonna, I. Genta, A. De Trizio, T. Modena, H. Klo¨ss, et al., Polym. Degrad. Stab. 119 (2015) 151
158. [41] G. Gupta, G.S. Sohi, in: Proceedings of the 44th Annual IEEE/ACM International Symposium Microarchitecture—MICRO-44’11, ACM Press, New York, 2011, p. 59. [42] N.C. Mishra, C.-F. Chou, S. Gautam, A.K. Dinda, P.D. Potdar, J. Mater. Sci. 49 (2013) 1076
1089. [43] C. Sharma, A.K. Dinda, N.C. Mishra, J. Appl. Polym. Sci. 127 (2013) 3228
3241. [44] S. Gautam, C.F. Chou, A.K. Dinda, P.D. Potdar, N.C. Mishra, Mater. Sci. Eng. C 34 (2014) 402
409. [45] C. Sharma, S. Gautam, A.K. Dinda, N.C. Mishra, Adv. Mater. Lett. 2 (2011) 90
99. [46] A. Kumar, S. Mandal, S. Barui, R. Vasireddi, U. Gbureck, M. Gelinsky, et al., Mater. Sci. Eng. R Reports 103 (2016) 1
39. [47] J. Mitra, G. Tripathi, A. Sharma, B. Basu, RSC Adv 3 (2013) 11073
11094. [48] U. Ripamonti, S. Ma, A.H. Reddi, Matrix 12 (1992) 202
212. [49] T. Kohgo, K. Atsumi, Q.-M. Jin, Y. Kuboki, H. Takita, H. Itoh, J. Biomed. Mater. Res. 52 (2002) 841
851. [50] Y. Kuboki, H. Takita, D. Kobayashi, E. Tsuruga, M. Inoue, M. Murata, et al., J. Biomed. Mater. Res. 39 (1998) 190
199. [51] J. Henkel, M.A. Woodruff, D.R. Epari, R. Steck, V. Glatt, I.C. Dickinson, et al., Bone Res. 1 (2013) 216
248. [52] S. Deepthi, J. Venkatesan, S.K. Kim, J.D. Bumgardner, R. Jayakumar, Int. J. Biol. Macromol. 93 (2016) 1338
1353. [53] T.J. Webster and E.S. Ahn, Nanostructured Biomaterials for Tissue Engineering Bone BT - Tissue Engineering II: Basics of Tissue Engineering and Tissue Applications, N. K. Lee and D. Kaplan, Hrsg., Berlin, Heidelberg, Springer Berlin Heidelberg, 2007, pp. 275
308. [54] J.Y. Park, S.Y. Han, Int. J. Precis. Eng. Manuf. 14 (2013) 115
121. [55] M.B. Yaylaoglu, C. Yildiz, F. Korkusuz, V. Hasirci, Biomaterials 20 (1999) 1513
1520. [56] M. Mihai, M.-T. Ton-That, Polym. Eng. Sci. 54 (2014) 446
458. [57] D. Zhang, X. Wu, J. Chen, K. Lin, Bioact. Mater 3 (2018) 129
138. [58] P. Fratzl, Nat. Mater. 7 (2008) 610
612. [59] R. Florencio-Silva, G.R. da, S. Sasso, E. Sasso-Cerri, M.J. Simo˜es, P.S. Cerri, Biomed Res. Int. 2015 (2015) 1
17. [60] M.M. Owen, J Cell Sci Suppl. 1988, 10, 63
76. [61] S.P. Bruder, A.I. Caplan, Connect. Tissue Res. 20 (1989) 65
71. [62] P.J. Simmons, B. Torok-Storb, Am. Soc. Heamtol. 78 (1991) 2848
2853. [63] R. Unger, A. Sartoris, K. Peters, A. Motta, C. Miliaresi, M. Kunkel, et al., Biomaterials 28 (2007) 3965
3976.

Handbook of Chitin and Chitosan

544

16. Chitin and chitosan composites for bone tissue regeneration

[64] L.M. Calvi, G.B. Adams, K.W. Weibrecht, J.M. Weber, D.P. Olson, M.C. Knight, et al., Nature 425 (2003) 841
846. [65] S. Nomura, T. Takano-Yamamoto, Matrix Biol. 19 (2000) 91
96. [66] D.L. Krebs, Stem Cells 19 (2001) 378
387. [67] B.R. Macdonald, M. Gowen, Baillieres. Clin. Rheumatol. 7 (1993) 421
443. [68] E. Kon, M. Marcacci, R. Cancedda, A. Lavroukov, M. Mastrogiacomo, R. Quarto, et al., N. Engl. J. Med. 344 (2002) 385
386. [69] S. Weiner, H.D. Wagner, Annu. Rev. Mater. Sci. 28 (2002) 271
298. [70] R. Bartl and B. Frisch, Biology of Bone, in Osteoporosis, Berlin, Heidelberg, Springer Berlin Heidelberg, 2009, pp. 7
28. [71] A. Lavroukov, S. Kutepov, M. Mastrogiacomo, R. Cancedda, M. Marcacci, R. Quarto, et al., Tissue Eng. 13 (2007) 947
955. [72] M.I. Santos, R.E. Unger, R.A. Sousa, R.L. Reis, C.J. Kirkpatrick, Biomaterials 30 (2009) 4407
4415. [73] S.E. Haynesworth, J. Goshima, V.M. Goldberg, A.I. Caplan, Bone 13 (1992) 81
88. [74] H. Nakahara, V.M. Goldberg, A.I. Caplan, J. Orthop. Res. 9 (1991) 465
476. [75] H.M. Kronenberg, Nature 423 (2003) 332
336. [76] P.G. Robey, J.D. Termine, Calcif. Tissue Int. 37 (1985) 453
460. [77] J.N. Beresford, Clin. Orthop. Relat. Res. (1989) 270
280. [78] M.J. Olszta, X. Cheng, S.S. Jee, R. Kumar, Y.Y. Kim, M.J. Kaufman, et al., Mater. Sci. Eng. R. Reports 58 (2007) 77
116. [79] E. Beniash, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 3 (2011) 47
69. [80] R.B. Martin, Mater. Sci. Forum 293 (1998) 5
16. [81] A.J. Friedenstein, U.F. Deriglasova, N.N. Kulagina, A.F. Panasuk, S.F. Rudakowa, E.A. Luria´, et al., Exp. Hematol. 2 (1974) 83
92. [82] D.T. Reilly, A.H. Burstein, V.H. Frankel, J. Biomech. 7 (1974) 271
275. [83] K. Choi, J.L. Kuhn, M.J. Ciarelli, S.A. Goldstein, J. Biomech 23 (1990) 1103
1113. [84] J.Y. Rho, T.Y. Tsui, G.M. Pharr, Biomaterials 18 (1997) 1325
1330. [85] F. Guilak, D.L. Butler, S.A. Goldstein, F.P.T. Baaijens, J. Biomech 47 (2014) 1933
1940. [86] M. Mullender, A.J. El Haj, Y. Yang, M.A. van Duin, E.H. Burger, J. Klein-Nulend, Med. Biol. Eng. Comput. 42 (2004) 14
21. [87] R.B. Martin, J. Biomech 24 (1991) 79
88. [88] J. Klein-Nulend, R.G. Bacabac, M.G. Mullender, Pathol. Biol. 53 (2005) 576
580. [89] V. dos Santos, R.N. Brandalise, M. Savaris, Engineering of Biomaterials, Springer International Publishing, Cham, 2017, pp. 5
15. [90] M.M. Stevens, Mater. Today 11 (2008) 18
25. [91] S. Bose, M. Roy, A. Bandyopadhyay, Trends Biotechnol. 30 (2012) 546
554. [92] W. Elshahawy, Biocompatibility, in C. Sikalidis (ed.), Advances in ceramics - electric and magnetic ceramics, bioceramics, ceramics and environment, Hrsg., 2011, pp. 359
378. [93] D.F. Williams, Biomaterials 35 (2014) 10009
10014. [94] G. Schmalz, K.M. Galler, Dent. Mater. 33 (2017) 382
393. [95] V.I. Sevastianov, Trends Biomater. Artif. Organs 15 (2001) 20
30. [96] M. Bernard, E. Jubeli, M.D. Pungente, N. Yagoubi, Biomater. Sci. 6 (2018) 2025
2053. [97] M. Lotfi, M. Nejib, M. Naceur, in: Advances in Biomaterials Science and Biomedical Applications, InTech, 2013, p. 64. [98] D.F. Williams, Biomaterials 29 (2008) 2941
2953. [99] J. Wilson-Hench, in: D.F. Williams (Ed.), Progress in biomedical engineering, vol 4. Definitions in biomaterials, Elservier, Amsterdam, 1987, p. 29. [100] P.O. Glantz, in: D.F. Williams (Ed.), Prog. Biomed. Eng. Defin. Biomater., vol. 4, Elsevier, Amsterdam, 1987, p. 24.

Handbook of Chitin and Chitosan

References

545

[101] T. Albrektsson, C. Johansson, Eur. Spine J 10 (2001) S96
S101. [102] X. Lin, S. Yang, K. Lai, H. Yang, T.J. Webster, L. Yang, Nanomed. Nanotechnol., Biol. Med. 13 (2017) 123
142. [103] A. Matsiko, J.P. Gleeson, F.J. O’Brien, Tissue Eng. Part A 21 (2014) 486
497. [104] P. Duan, Z. Pan, L. Cao, Y. He, H. Wang, Z. Qu, et al., J. Biomed. Mater. Res.—Part A 102 (2014) 180
192. [105] D. Griffon, M. Sedighi, D. Schaeffer, J. Eurell, A. Johnson, Acta Biomater. 2 (2006) 313
320. [106] Q. Zhang, H. Lu, N. Kawazoe, G. Chen, Acta Biomater. 10 (2014) 2005
2013. [107] T. Furuta, N. Shirai, M. Takashima, F. Xiao, H. Hanai, K. Nakagawa, et al., Pharmacogenetics 11 (2001) 341
348. [108] F.J. O’Brien, B.A. Harley, I.V. Yannas, L. Gibson, Biomaterials 25 (2004) 1077
1086. [109] F.J. O’Brien, B.A. Harley, M.A. Waller, I.V. Yannas, L.J. Gibson, P.J. Prendergast, Technol. Heal. Care 15 (2007) 3
17. [110] L.J. Gibson, I.V. Yannas, H.-D. Do Kim, B.A.C. Harley, D.A. Lauffenburger, M.H. Zaman, et al., Biophys. J. 95 (2008) 4013
4024. [111] S.H. Oh, T.H. Kim, G. Il Im, J.H. Lee, Am. Chem. Soc. 11 (2010) 1948
1955. [112] A. Gregor, E. Filova´, M. Nova´k, J. Kronek, H. Chlup, M. Buzgo, et al., J. Biol. Eng. 11 (2017) 1
21. [113] T. Ghassemi, A. Shahroodi, M.H. Ebrahimzadeh, A. Mousavian, J. Movaffagh, A. Moradi, Arch. Bone Jt. Surg. 6 (2018) 90
99. [114] V. Karageorgiou, D. Kaplan, Biomaterials 26 (2005) 5474
5491. [115] H.D. Kim, R.F. Valentini, J. Biomed. Mater. Res. 59 (2002) 573
584. [116] F. Shen, Y. Cui, L. Yang, K. Yao, X. Dong, W. Jia, et al., Polym. Int. 49 (2000) 1596
1599. [117] S.H. Oh, S.G. Kang, E.S. Kim, S.H. Cho, J.H. Lee, Biomaterials 24 (2003) 4011
4021. [118] H.W. Kim, J.C. Knowles, H.E. Kim, Biomaterials 25 (2004) 1279
1287. [119] R. Nazarov, H.J. Jin, D.L. Kaplan, Biomacromolecules 5 (2004) 718
726. [120] R. Zhang, P.X. Ma, J. Biomed. Mater. Res. 44 (1999) 446
455. [121] P. Li, J. Biomed. Mater. Res.—Part A 66 (2003) 79
85. [122] A.R. El-Ghannam, J. Biomed. Mater. Res.—Part A 69 (2004) 490
501. [123] G. Chen, T. Ushida, T. Tateishi, J. Biomed. Mater. Res. 57 (2001) 8
14. [124] E.A. Botchwey, S.R. Pollack, E.M. Levine, C.T. Laurencin, J. Biomed. Mater. Res. 55 (2001) 242
253. [125] J. Dong, H. Kojima, T. Uemura, M. Kikuchi, T. Tateishi, J. Tanaka, et al., Mater. Res 57 (2001) 208
216. [126] F.A. Maspero, K. Ruffieux, B. Mu¨ller, E. Wintermantel, J. Biomed. Mater. Res. 62 (2002) 89
98. [127] Y. Hu, D.W. Grainger, S.R. Winn, J.O. Hollinger, J. Biomed. Mater. Res. 59 (2002) 563
572. [128] A.S.P. Lin, T.H. Barrows, S.H. Cartmell, R.E. Guldberg, Biomaterials 24 (2003) 481
489. [129] G.R. Cartmell, S. Huynh, K. Lin, A. Nagaraja, S, J Biomed Mater Res A 69 (2004) 97
104. [130] S.-N. Park, J. Park, H.O. Kim, M.J. Song, H. Suh, Biomaterials 23 (2002) 1205
1212. [131] F. Zhao, Y. Yin, W.W. Lu, J.C. Leong, W. Zhang, J. Zhang, et al., Biomaterials 23 (2002) 3227
3234. [132] K. Zhang, Y. Fan, N. Dunne, X. Li, Regen. Biomater 5 (2018) 115
124. [133] F.S.L. Bobbert, A.A. Zadpoor, J. Mater. Chem. B 5 (2017) 6175
6192. [134] B. Feng, Z. Jinkang, W. Zhen, L. Jianxi, C. Jiang, L. Jian, et al., Biomed. Mater. 6 (2011) 015007. [135] A.A. Zadpoor, Biomater. Sci. 3 (2015) 231
245.

Handbook of Chitin and Chitosan

546

16. Chitin and chitosan composites for bone tissue regeneration

[136] M. Bohner, Y. Loosli, G. Baroud, D. Lacroix, Acta Biomater. 7 (2011) 478
484. [137] M.R. Dias, P.R. Fernandes, J.M. Guedes, S.J. Hollister, J. Biomech. 45 (2012) 938
944. [138] H. Yuan, J.D. De Bruijn, X. Zhang, C.A. Van Blitterswijk, K. De Groot, J. Biomed. Mater. Res. 58 (2001) 270
276. [139] L.B. Rocha, G. Goissis, M.A. Rossi, Biomaterials 23 (2002) 449
456. [140] W. Gong, A. Abdelouas, W. Lutze, J. Biomed. Mater. Res. 54 (2001) 320
327. [141] E. Damien, K. Hing, S. Saeed, P.A. Revell, J. Biomed. Mater. Res.—Part A 66 (2003) 241
246. [142] K.-U. Lewandrowski, D.D. Hile, B.M.J. Thompson, D.L. Wise, W.W. Tomford, D.J. Trantolo, Tissue Eng. 9 (2003) 85
93. [143] J.M. Taboas, R.D. Maddox, P.H. Krebsbach, S.J. Hollister, Biomaterials 24 (2003) 181
194. [144] S.F. Hulbert, F.A. Young, R.S. Mathews, J.J. Klawitter, C.D. Talbert, F.H. Stelling, J. Biomed. Mater. Res. 4 (1970) 433
456. [145] C.M. Cabello, W.B. Bair, S.D. Lamore, S. Ley, S. Alexandra, S. Azimian, G.T. Wondrak, J. Biomed. Mater. Res. A, 46 (2010) 220
231. [146] C.M. Murphy, F.J. O’Brien, Cell Adhes. Migr. 4 (2010) 377
381. [147] M. Rouahi, O. Gallet, E. Champion, J. Dentzer, P. Hardouin, K. Anselme, J. Biomed. Mater. Res. Part A 78A (2006) 222
235. [148] N. Sachot, M.A. Mateos-Timoneda, J.A. Planell, A.H. Velders, M. Lewandowska, E. Engel, et al., Nanoscale 7 (2015) 15349
15361. [149] T.T. Yu, F.Z. Cui, Q.Y. Meng, J. Wang, D.C. Wu, J. Zhang, et al., Sci. Eng 3 (2017) 1119
1128. [150] R. Bosco, J. Van Den Beucken, S. Leeuwenburgh, J. Jansen, Coatings 2 (2012) 95
119. [151] E.A. Nauman, K.E. Fong, T.M. Keaveny, Ann. Biomed. Eng. 27 (1999) 517
524. [152] R.A. Gittens, T. McLachlan, R. Olivares-Navarrete, Y. Cai, S. Berner, R. Tannenbaum, et al., Biomaterials 32 (2011) 3395
3403. [153] D.D. Deligianni, N.D. Katsala, P.G. Koutsoukos, Y.F. Missirlis, Biomaterials 22 (2001) 87
96. [154] D.D. Deligianni, N. Katsala, S. Ladas, D. Sotiropoulou, J. Amedee, Y.F. Missirlis, Biomaterials 22 (2001) 1241
1251. [155] F. Guilak, D.M. Cohen, B.T. Estes, J.M. Gimble, W. Liedtke, C.S. Chen, Cell Stem Cell 5 (2009) 17
26. [156] X. Li, H. Liu, X. Niu, Y. Fan, Q. Feng, F.Z. Cui, et al., J. Biomed. Mater. Res.—Part B Appl. Biomater. 97 B (2011) 10
19. [157] A. Itl, H.O. Ylnen, J. Yrjans, T. Heino, T. Hentunen, M. Hupa, et al., J. Biomed. Mater. Res. 62 (2002) 404
411. [158] O. Andrukhov, R. Huber, B. Shi, S. Berner, X. Rausch-Fan, A. Moritz, et al., Dent. Mater. 32 (2016) 1374
1384. [159] T.P. Kunzler, T. Drobek, M. Schuler, N.D. Spencer, Biomaterials 28 (2007) 2175
2182. [160] A.B. Faia-Torres, S. Guimond-Lischer, M. Rottmar, M. Charnley, T. Goren, K. Maniura-Weber, et al., Biomaterials 35 (2014) 9023
9032. [161] X. Rausch-fan, Z. Qu, M. Wieland, M. Matejka, A. Schedle, Dent. Mater. 24 (2008) 102
110. [162] R. Olivares-Navarrete, P. Raz, G. Zhao, J. Chen, M. Wieland, D.L. Cochran, et al., Proc. Natl. Acad. Sci. 105 (2008) 15767
15772. [163] J. Knychala, N. Bouropoulos, C.J. Catt, O.L. Katsamenis, C.P. Please, B.G. Sengers, Ann. Biomed. Eng. 41 (2013) 917
930. [164] S. Van Bael, Y.C. Chai, S. Truscello, M. Moesen, G. Kerckhofs, H. Van Oosterwyck, et al., J. Schrooten, Acta Biomater. 8 (2012) 2824
2834.

Handbook of Chitin and Chitosan

References

547

[165] P.O. Bagnaninchi, M. Tabrizian, C. Holmes, J. Tissue Eng. Regen. Med. 9 (2013) 641
645. [166] H.Y. Mi, X. Jing, M.R. Salick, T.M. Cordie, X.F. Peng, L.S. Turng, J. Mater. Sci. 49 (2014) 2324
2337. [167] C. Correia, S. Bhumiratana, L.P. Yan, A.L. Oliveira, J.M. Gimble, D. Rockwood, et al., Acta Biomater. 8 (2012) 2483
2492. [168] S. Amin Yavari, S.M. Ahmadi, R. Wauthle, B. Pouran, J. Schrooten, H. Weinans, et al., J. Mech. Behav. Biomed. Mater. 43 (2015) 91
100. [169] A. Abarrategi, C. Moreno-Vicente, F.J. Martı´nez-Va´zquez, A. Civantos, V. Ramos, J.V. Sanz-Casado, et al., PLoS One 7 (2012) 1
10. [170] J.Y. Park, D.H. Lee, E.J. Lee, S.-H. Lee, Lab Chip 9 (2009) 2043. [171] S. Scaglione, P. Giannoni, P. Bianchini, M. Sandri, R. Marotta, G. Firpo, et al., Sci. Rep. 2 (2012) 1
6. [172] J. Zhao, W. Han, H. Chen, M. Tu, S. Huan, G. Miao, et al., J. Mater. Sci. Mater. Med 23 (2012) 517
525. [173] X. Liu, M.N. Rahaman, Q. Fu, Acta Biomater. 9 (2013) 4889
4898. [174] D.W. Hutmacher, M. Sittinger, M.V. Risbud, Trends Biotechnol. 22 (2004) 354
362. [175] C.J. Little, N.K. Bawolin, X. Chen, Tissue Eng. Part B Rev 17 (2011) 213
227. [176] N.K. Bawolin, W.J. Zhang, X.B. Chen, J. Biomimetics, Biomater. Tissue Eng 6 (2010) 19
33. [177] Y. Wan, H. Wu, X. Cao, S. Dalai, Polym. Degrad. Stab. 93 (2008) 1736
1741. [178] Y. Xu, D. Xia, J. Han, S. Yuan, H. Lin, C. Zhao, Carbohydr. Polym. 177 (2017) 210
216. [179] O.J. Fergal, Mater. Today 14 (2011) 88
95. [180] Y. Huang, S. Onyeri, M. Siewe, A. Moshfeghian, S.V. Madihally, Biomaterials 26 (2005) 7616
7627. [181] H. Wu, Y. Wan, S. Dalai, R. Zhang, J. Biomed. Mater. Res.—Part A 92 (2010) 238
245. [182] A. Georgopoulou, F. Papadogiannis, A. Batsali, J. Marakis, K. Alpantaki, A.G. Eliopoulos, et al., J. Mater. Sci. Mater. Med. 29 (2018) 59. [183] U. Adhikari, N.P. Rijal, S. Khanal, D. Pai, J. Sankar, N. Bhattarai, Bioact. Mater 1 (2016) 132
139. [184] K.M. Sajesh, R. Jayakumar, S.V. Nair, K.P. Chennazhi, Int. J. Biol. Macromol. 62 (2013) 465
471. [185] Y.J. Seol, J.Y. Lee, Y.J. Park, Y.M. Lee, Y. Ku, I.C. Rhyu, et al., Biotechnol. Lett. 26 (2004) 1037
1041. [186] Y. Zhang, M. Ni, M. Zhang, B. Ratner, Tissue Eng. 9 (2003) 337
345. [187] P.J. VandeVord, H.W.T. Matthew, S.P. DeSilva, L. Mayton, B. Wu, P.H. Wooley, J. Biomed. Mater. Res. 59 (2002) 585
590. [188] P.R. Klokkevold, L. Vandemark, E.B. Kenney, G.W. Bernard, J. Periodontol. 67 (1996) 1170
1175. [189] S.-Y. Shin, H.-N. Park, K.-H. Kim, M.-H. Lee, Y.S. Choi, Y.-J. Park, et al., J. Periodontol. 76 (2005) 1778
1784. [190] R.L. Reis, M. Bhattacharya, N.M. Neves, B. Delorme, P. Charbord, V.M. Correlo, et al., Biomacromolecules 10 (2009) 2067
2073. [191] R.A.A. Muzzarelli, C. Zucchini, P. Ilari, A. Pugnaloni, M. Mattioli Belmonte, G. Biagini, et al., Biomaterials 14 (1993) 925
929. [192] R.A.A. Muzzarelli, G. Biagini, M. Bellardini, L. Simonelli, C. Castaldini, G. Fratto, Biomaterials 14 (1993) 39
43. [193] T. Barroso, R. Viveiros, T. Casimiro, A. Aguiar-Ricardo, J. Supercrit, Fluids 94 (2014) 102
112. [194] S.V. Madihally, H.W.T. Matthew, Biomaterials 20 (1999) 1133
1142.

Handbook of Chitin and Chitosan

548

16. Chitin and chitosan composites for bone tissue regeneration

[195] N.Y. Yuan, Y.A. Lin, M.H. Ho, D.M. Wang, J.Y. Lai, H.J. Hsieh, Carbohydr. Polym. 78 (2009) 349
356. [196] M.-H. Ho, P.-Y. Kuo, H.-J. Hsieh, T.-Y. Hsien, L.-T. Hou, J.-Y. Lai, et al., Biomaterials 25 (2004) 129
138. [197] K. Tuzlakoglu, C.M. Alves, J.F. Mano, R.L. Reis, Macromol. Biosci. 4 (2004) 811
819. [198] C. Hsieh, S. Tsai, M. Ho, D. Wang, C. Liu, C. Hsieh, et al., Carbohydr. Polym. 67 (2007) 124
132. [199] C. Heinemann, S. Heinemann, A. Bernhardt, H. Worch, T. Hanke, Biomacromolecules 9 (2008) 2913
2920. [200] W. Abdelfattah, T. Jiang, G. Elbassyouni, C. Laurencin, Acta Biomater. 3 (2007) 503
514. [201] S. Dhivya, S. Saravanan, T.P. Sastry, N. Selvamurugan, J. Nanobiotechnol. 13 (2015) 1
13. [202] Y. Cao, L. Zhu, Y. Liu, J. Wang, Y. Xu, H. Lv, et al., J. Mater. Sci. Mater. Med. 23 (2012) 1941
1949. [203] Z. Sha, X. Li, Q. Feng, X. Niu, Y. Fan, J. Wang, et al., Artif. Organs 35 (2011) E119
E128. [204] K.R. Mohamed, H.H. Beherei, Z.M. El-Rashidy, J. Adv. Res. 5 (2014) 201
208. [205] B. De la Riva, C. E´vora, A. Herna´ndez, M.K. Pec, A. Delgado, E. Sa´nchez, et al., Eur. J. Pharm. Biopharm. 73 (2009) 50
58. [206] P. Gentile, V.K. Nandagiri, J. Daly, V. Chiono, C. Mattu, C. Tonda-Turo, et al., Mater. Sci. Eng. C 59 (2016) 249
257. [207] S. Zhang, M.P. Prabhakaran, X. Qin, S. Ramakrishna, J. Mech. Behav. Biomed. Mater. 51 (2015) 88
98. [208] J. Xie, C. Peng, Q. Zhao, X. Wang, H. Yuan, L. Yang, et al., Acta Biomater. 29 (2016) 365
379. [209] W. Jiang, L. Li, D. Zhang, S. Huang, Z. Jing, Y. Wu, et al., Acta Biomater. 25 (2015) 240
252. [210] M. Pourhaghgouy, A. Zamanian, M. Shahrezaee, M.P. Masouleh, Mater. Sci. Eng. C 58 (2016) 180
186. [211] I.R. Serra, R. Fradique, M.C.S. Vallejo, T.R. Correia, S.P. Miguel, I.J. Correia, Mater. Sci. Eng. C 55 (2015) 592
604. [212] L.S. Moreira Teixeira, C.A. Van Blitterswijk, R.L. Reis, N.M. Alves, L. Moroni, M. Karperien, et al., Biomaterials 32 (2010) 1068
1079. [213] Z. Shao, H. Huang, L. Dai, X. Zhang, Q. Meng, J. Zhang, et al., Sci. Rep. 5 (2015) 1
14. [214] Z. Dong, B. Liu, J. Deng, G. Mo, R. She, W. Huang, J. Mater. Sci. Mater. Med. 24 (2013) 2037
2046. ˘ [215] T.T. Demirta¸s, G. Irmak, M. Gu¨mu¨s¸ derelioglu, Biofabrication 9 (2017) 035003. [216] J. Hou, J. Wang, L. Cao, X. Qian, W. Xing, J. Lu, et al., Biomed. Mater. 7 (2012) 035002. [217] W. Gordon-Evans, G.R. Ragetly, Y.S. Chung, L.P. Fredericks, D.J. Griffon, H.-B. Lee, Acta Biomater. 6 (2009) 1430
1436. [218] S.W. Whu, K.C. Hung, K.H. Hsieh, C.H. Chen, C.L. Tsai, S.H. Hsu, Mater. Sci. Eng. C 33 (2013) 2855
2863. [219] C.C. Yu, J.J. Chang, Y.H. Lee, Y.C. Lin, M.H. Wu, M.C. Yang, et al., Mater. Lett. 93 (2013) 133
136. [220] K. Nazemi, P. Azadpour, F. Moztarzadeh, A.M. Urbanska, M. Mozafari, Mater. Lett. 138 (2015) 16
20. [221] M. Kucharska, B. Butruk, K. Walenko, T. Brynk, T. Ciach, Mater. Lett. 85 (2012) 124
127. [222] Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 55 (2001) 304
312.

Handbook of Chitin and Chitosan

References

549

[223] Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 62 (2002) 378
386. [224] D. Li, Q. Lian, Z. Jin, J. Wang, A. Li, Z. Wang, J. Bioact. Compat. Polym. 24 (2009) 113
124. [225] Y. Zhang, M. Zhang, J. Biomed. Mater. Res. 61 (2002) 1
8. [226] Q. Hu, B. Li, M. Wang, J. Shen, Biomaterials 25 (2004) 779
785. [227] J.Y. Lee, S.H. Nam, S.Y. Im, Y.J. Park, Y.M. Lee, Y.J. Seol, et al., J. Control. Release 78 (2002) 187
197. [228] L. Leroux, Z. Hatim, M. Fre`che, J. Lacout, Bone 25 (1999) 31S
34S. [229] H.H.K. Xu, J.B. Quinn, S. Takagi, L.C. Chow, Biomaterials 25 (2004) 1029
1037. [230] W. Pon-On, N. Charoenphandhu, J. Teerapornpuntakit, J. Thongbunchoo, N. Krishnamra, I.M. Tang, Mater. Sci. Eng. C 38 (2014) 63
72. [231] P.A.L. Lima, C.X. Resende, G.D. De Almeida Soares, K. Anselme, L.E. Almeida, Mater. Sci. Eng. C 33 (2013) 3389
3395. [232] P. Ghosh, A.P. Rameshbabu, D. Das, N.K. Francis, H.S. Pawar, B. Subramanian, et al., Colloid. Surf. B 125 (2015) 160
169. [233] J.A. Sowjanya, J. Singh, T. Mohita, S. Sarvanan, A. Moorthi, N. Srinivasan, et al., Colloids Surf. B Biointerfaces 109 (2013) 294
300. [234] S.A. Martel-Estrada, B. Rodrı´guez-Espinoza, E. Santos-Rodrı´guez, F. Jime´nez-Vega, P.E. Garcı´a-Casillas, C.A. Martı´nez-Pe´rez, et al., J. Alloys Compd. 643 (2015) S119
S123. [235] A.R.C. Duarte, J.F. Mano, R.L. Reis, J. Supercrit, Fluids 54 (2010) 282
289. [236] F. Heidari, M.E. Bahrololoom, D. Vashaee, L. Tayebi, Ceram. Int. 41 (2015) 3094
3100. [237] S.B. Kim, Y.J. Kim, T.L. Yoon, S.A. Park, I.H. Cho, E.J. Kim, et al., Biomaterials 25 (2004) 5715
5723. [238] L. Zhao, E.F. Burguera, H.H.K. Xu, N. Amin, H. Ryou, D.D. Arola, Biomaterials 31 (2010) 840
847. [239] T.H. Ang, F.S.A. Sultana, D.W. Hutmacher, Y.S. Wong, J.Y.H. Fuh, X.M. Mo, et al., Mater. Sci. Eng. C 20 (2002) 35
42. [240] Z. Ge, S. Baguenard, L.Y. Lim, A. Wee, E. Khor, Biomaterials 25 (2004) 1049
1058. [241] T. Kawakami, M. Antoh, H. Hasegawa, T. Yamagishi, M. Ito, S. Eda, Biomaterials 13 (1992) 759
763. [242] X. Wang, J. Ma, Y. Wang, B. He, Biomaterials 23 (2002) 4167
4176. [243] Y.-M. Lee, Y.-J. Park, S.-J. Lee, Y. Ku, S.-B. Han, P.R. Klokkevold, et al., J. Periodontol. 71 (2000) 418
424. [244] B.C. Cho, J.W. Park, B.S. Baik, I.C. Kwon, I.S. Kim, J. Craniofac. Surg. 13 (2002) 783
793. [245] I.-S. Kim, J.W. Park, I.C. Kwon, B.S. Baik, B.C. Cho, Plast. Reconstr. Surg. 109 (2002) 1966
1977. [246] J. Wang, J. De Boer, K. De Groot, J. Dent. Res. 83 (2004) 296
301. [247] J.D. Bumgardner, R. Wiser, P.D. Gerard, P. Bergin, B. Chestnutt, M. Marini, et al., J. Biomater. Sci. Polym. Ed. 14 (2003) 423
438. [248] J.D. Bumgardner, R. Wiser, S.H. Elder, R. Jouett, Y. Yang, J.L. Ong, J. Biomater. Sci. Polym. Ed. 14 (2003) 1401
1409. [249] Y. Zhang, M. Zhang, J. Mater. Sci. Mater. Med. 15 (2004) 255
260. [250] M. Maruyama, M. Ito, J. Biomed. Mater. Res. 32 (1996) 527
532. [251] B.M. Chesnutt, Y. Yuan, K. Buddington, W.O. Haggard, J.D. Bumgardner, Tissue Eng. Part A 15 (2009) 2571
2579. [252] H.-H. Jin, D.-H. Kim, T.-W. Kim, K.-K. Shin, J.S. Jung, H.-C. Park, et al., Int. J. Biol. Macromol. 51 (2012) 1079
1085. [253] K. Dahlan, S.U. Dewi, A. Nurlaila, D. Soejoko, Int. J. Basic Appl. Sci. 12 (2012) 50
57.

Handbook of Chitin and Chitosan

550 [254] [255] [256] [257] [258] [259] [260] [261] [262] [263] [264] [265] [266] [267] [268] [269] [270] [271] [272] [273] [274] [275] [276] [277] [278] [279] [280] [281] [282] [283]

16. Chitin and chitosan composites for bone tissue regeneration

J.G. Saltarrelli Jr., D.P. Mukherjee, J. Biomed. Sci. Eng. 02 (2009) 24
29. G.J. Lai, K.T. Shalumon, S.H. Chen, J.P. Chen, Carbohydr. Polym. 111 (2014) 288
297. J. Zhang, G. Liu, Q. Wu, J. Zuo, Y. Qin, J. Wang, J. Bionic Eng 9 (2012) 243
251. A. Teimouri, R. Ebrahimi, R. Emadi, B.H. Beni, A.N. Chermahini, Int. J. Biol. Macromol. 76 (2015) 292
302. S.B. Qasim, S. Husain, Y. Huang, M. Pogorielov, V. Deineka, M. Lyndin, et al., Polym. Degrad. Stab. 136 (2017) 31
38. A. Rogina, P. Rico, G.G. Ferrer, M. Ivankovi´c, H. Ivankovi´c, Eur. Polym. J 68 (2015) 278
287. V.M. Correlo, L.F. Boesel, E. Pinho, A.R. Costa-Pinto, M.L. Alves da Silva, M. Bhattacharya, et al., J. Biomed. Mater. Res. Part A 91A (2009) 489
504. Y. Yin, F. Ye, J. Cui, F. Zhang, X. Li, K. Yao, J. Biomed. Mater. Res.—Part A 67 (2003) 844
855. S.-J. Lee, P.R. Klokkevold, S.-B. Han, Y. Ku, C.-P. Chung, Y.-M. Lee, et al., J. Periodontol. 71 (2005) 410
417. R.A.A. Muzzarelli, M. Mattioli-Belmonte, C. Tietz, R. Biagini, G. Ferioli, M.A. Brunelli, et al., Biomaterials 15 (1994) 1075
1081. A. Olad, F. Farshi Azhar, Ceram. Int. 40 (2014) 10061
10072. X. Li, X.Y. Ma, Y.F. Feng, L. Wang, C. Wang, Compos. Sci. Technol. 117 (2015) 78
84. S. Shokri, B. Movahedi, M. Rafieinia, H. Salehi, Appl. Surf. Sci. 357 (2015) 1758
1764. E. Wers, H. Oudadesse, B. Lefeuvre, O. Merdrignac-Conanec, A. Barroug, Appl. Surf. Sci. 353 (2015) 200
208. M. Nieto-Sua´rez, M.A. Lo´pez-Quintela, M. Lazzari, Carbohydr. Polym. 141 (2016) 175
183. S. Puvaneswary, S. Talebian, H.B. Raghavendran, M.R. Murali, M. Mehrali, A.M. Afifi, et al., Carbohydr. Polym. 134 (2015) 799
807. ´ .J. Leite, J.F. Mano, Carbohydr. Polym. 123 (2015) 39
45. C.O. Correia, A X. Jing, H.-Y. Mi, J. Peng, X.-F. Peng, L.-S. Turng, Carbohydr. Polym. 117 (2015) 941
949. H. Park, B. Choi, J. Nguyen, J. Fan, S. Shafi, P. Klokkevold, et al., Carbohydr. Polym. 97 (2013) 587
596. W.B. Tsai, Y.R. Chen, W.T. Li, J.Y. Lai, H.L. Liu, Carbohydr. Polym. 89 (2012) 379
387. N. Bhardwaj, S.C. Kundu, Carbohydr. Polym. 85 (2011) 325
333. M. Peter, N. Ganesh, N. Selvamurugan, S.V.V. Nair, T. Furuike, H. Tamura, et al., Carbohydr. Polym. 80 (2010) 687
694. Y. Yuan, B.M. Chesnutt, G. Utturkar, W.O. Haggard, Y. Yang, J.L. Ong, et al., Carbohydr. Polym. 68 (2007) 561
567. K. Zhang, S. Yan, G. Li, L. Cui, J. Yin, Biomaterials 71 (2015) 24
34. S. Ravindran, M. Kotecha, C.-C. Huang, A. Ye, P. Pothirajan, Z. Yin, et al., Biomaterials 71 (2015) 58
70. H. Liu, T. Zhu, P. Lu, Z. Yin, C. Zhang, X. Chen, et al., Biomaterials 53 (2015) 716
730. H. Li, A.M. Koenig, P. Sloan, N.D. Leipzig, Biomaterials 35 (2014) 9049
9057. P. Yilgor, K. Tuzlakoglu, R.L. Reis, N. Hasirci, V. Hasirci, Biomaterials 30 (2009) 3551
3559. T. Jiang, W.I. Abdel-Fattah, C.T. Laurencin, Biomaterials 27 (2006) 4894
4903. B. Lowe, J. Venkatesan, S. Anil, M.S. Shim, S.-K. Kim, Int. J. Biol. Macromol. 93 (2016) 1479
1487.

Handbook of Chitin and Chitosan

References

551

[284] R. Sainitya, M. Sriram, V. Kalyanaraman, S. Dhivya, S. Saravanan, M. Vairamani, et al., Int. J. Biol. Macromol. 80 (2015) 481
488. [285] A. Shavandi, A.E.D.A. Bekhit, M.A. Ali, Z. Sun, Int. J. Biol. Macromol. 80 (2015) 445
454. [286] J.S. Lee, S.D. Baek, J. Venkatesan, I. Bhatnagar, H.K. Chang, H.T. Kim, et al., Int. J. Biol. Macromol. 67 (2014) 360
366. [287] K.C. Kavya, R. Jayakumar, S. Nair, K.P. Chennazhi, Int. J. Biol. Macromol. 59 (2013) 255
263. [288] J. Venkatesan, R. Pallela, I. Bhatnagar, S.K. Kim, Int. J. Biol. Macromol 51 (2012) 1033
1042. [289] J. Venkatesan, B.M. Ryu, P.N. Sudha, S.K. Kim, Int. J. Biol. Macromol 50 (2012) 393
402. [290] S. Pattnaik, S. Nethala, A. Tripathi, S. Saravanan, A. Moorthi, N. Selvamurugan, Int. J. Biol. Macromol. 49 (2011) 1167
1172. [291] R. Jayakumar, R. Ramachandran, P.T. Sudheesh Kumar, V.V. Divyarani, S. Srinivasan, K.P. Chennazhi, et al., Int. J. Biol. Macromol. 49 (2011) 274
280. [292] S. Saravanan, S. Nethala, S. Pattnaik, A. Tripathi, A. Moorthi, N. Selvamurugan, Int. J. Biol. Macromol. 49 (2011) 188
193. [293] R. Jayakumar, R. Ramachandran, V.V. Divyarani, K.P. Chennazhi, H. Tamura, S.V. Nair, Int. J. Biol. Macromol. 48 (2011) 336
344. [294] C. Sharma, A.K. Dinda, P.D. Potdar, C.-F. Chou, N.C. Mishra, Mater. Sci. Eng. C 64 (2016) 416
427. [295] Q. Yao, W. Li, S. Yu, L. Ma, D. Jin, A.R. Boccaccini, et al., Mater. Sci. Eng. C 56 (2015) 473
480. [296] D.-J. Kim, Y.-J. Park, M. Zhang, J.-H. Yoon, D.-G. Kim, J.-S. Han, et al., Mater. Sci. Eng. C 54 (2015) 20
25. [297] J.H. Cho, S.H. Kim, K.D. Park, M.C. Jung, W.I. Yang, S.W. Han, et al., Biomaterials 25 (2004) 5743
5751. [298] J.M. Dang, D.D.N. Sun, Y. Shin-Ya, A.N. Sieber, J.P. Kostuik, K.W. Leong, Biomaterials 27 (2006) 406
418. [299] Y. Zhang, X. Cheng, J. Wang, Y. Wang, B. Shi, C. Huang, et al., Biochem. Biophys. Res. Commun. 344 (2006) 362
369. [300] Z. Li, H.R. Ramay, K.D. Hauch, D. Xiao, M. Zhang, Biomaterials 26 (2005) 3919
3928. [301] X. Cai, H. Tong, X. Shen, W. Chen, J. Yan, J. Hu, Acta Biomater 5 (2009) 2693
2703. [302] Z. Yang, J. Liu, R. Liao, G. Yang, X. Wu, Z. Tang, et al., Compos. Sci. Technol. 132 (2016) 68
75. [303] H. Ni, L.Z. Fan, J. Power Sources 214 (2012) 195
199. [304] Y. Shi, L. Wen, F. Li, H.M. Cheng, J. Power Sources 196 (2011) 8610
8617. [305] K. Shang, W. Liao, J. Wang, Y.T. Wang, Y.Z. Wang and D.A. Schiraldi, ACS Appl. Mat. Inter. 8 (1) (2016) 643
650. [306] E. Lucarelli, D. Donati, A. Cenacchi, P.M. Fornasari, Transfus. Apher. Sci. 30 (2004) 169
174. [307] P. Arpornmaeklong, P. Pripatnanont, N. Suwatwirote, Int. J. Oral Maxillofac. Surg. 37 (2008) 357
366. [308] Y.C. Kuo, C.F. Yeh, J.T. Yang, Biomaterials 30 (2009) 6604
6613. [309] L. Kong, Y. Gao, G. Lu, Y. Gong, N. Zhao, X. Zhang, Eur. Polym. J 42 (2006) 3171
3179. [310] I. Manjubala, S. Scheler, J. Bo¨ssert, K.D. Jandt, Acta Biomater. 2 (2006) 75
84. [311] I.F. Amaral, P. Sampaio, M.A. Barbosa, J. Biomed. Mater. Res.—Part A 76 (2006) 335
346. [312] C. Shi, Z. Yuan, F. Han, C. Zhu, B. Li, Ann. Jt 1 (2016). 27
27. [313] S.K.L. Levengood, M. Zhang, J. Mater. Chem. B 2 (2014) 3161.

Handbook of Chitin and Chitosan

552

16. Chitin and chitosan composites for bone tissue regeneration

[314] C.-Y. Hsieh, S.-P. Tsai, D.-M. Wang, Y.-N. Chang, H.-J. Hsieh, Biomaterials 26 (2005) 5617
5623. [315] C. Mota, D. Puppi, D. Dinucci, M. Gazzarri, F. Chiellini, J. Bioact. Compat. Polym. 28 (2013) 320
340. [316] L. Li, B. Yuan, S. Liu, S. Yu, C. Xie, F. Liu, et al., J. Mater. Chem. 22 (2012) 8585. [317] G.C. East, Y. Qin, J. Appl. Polym. Sci. 50 (1993) 1773
1779. [318] C.K. Lee, S.J. Kim, S.I. Kim, B.-J. Yi, S.Y. Han, Smart Mater. Struct. 15 (2006) 607
611. [319] V.E. Yudin, I.P. Dobrovolskaya, I.M. Neelov, E.N. Dresvyanina, P.V. Popryadukhin, E.M. Ivan’Kova, et al., Carbohydr. Polym. 108 (2014) 176
182. [320] H. Tamura, Y. Tsuruta, K. Itoyama, W. Worakitkanchanakul, R. Rujiravanit, S. Tokura, Carbohydr. Polym. 56 (2004) 205
211. [321] J. Li, D. Liu, C. Hu, F. Sun, W. Gustave, H. Tian, et al., Carbohydr. Polym. 136 (2016) 1137
1143. [322] B. Ma, A. Qin, X. Li, C. He, Carbohydr. Polym. 97 (2013) 300
305. [323] A. Watthanaphanit, P. Supaphol, H. Tamura, S. Tokura, R. Rujiravanit, Carbohydr. Polym. 79 (2010) 738
746. [324] X. Geng, O. Kwon, J. Jang, Biomaterials 26 (2005) 5427
5432. [325] A. Neamnark, R. Rujiravanit, P. Supaphol, Carbohydr. Polym. 66 (2006) 298
305. [326] R. Grande, L.A. Pessan, A.J.F. Carvalho, Ind. Crops Prod. 72 (2015) 159
165. [327] V.M. Correlo, L.F. Boesel, M. Bhattacharya, J.F. Mano, N.M. Neves, R.L. Reis, Mater. Sci. Eng. A 403 (2005) 57
68. [328] J. Feng, D. Zhang, M. Zhu, et al., J. Mater. Chem. B 5 (2017) 5176
5188. [329] D.J. Mooney, D.F. Baldwin, N.P. Suh, J.P. Vacanti, R. Langer, Biomaterials 17 (1996) 1417
1422. [330] A.G. Mikos, A.J. Thorsen, L.A. Czerwonka, Y. Bao, R. Langer, D.N. Winslow, et al., Polymer (Guildf) 35 (1994) 1068
1077. [331] L.E. Freed, J.C. Marquis, A. Nohria, J. Emmanual, A.G. Mikos, R. Langer, J. Biomed. Mater. Res. 27 (1993) 11
23. [332] A.G. Mikos, Y. Bao, L.G. Cima, D.E. Ingber, J.P. Vacanti, R. Langer, J. Biomed. Mater. Res. 27 (1993) 183
189. [333] R.C. Thomson, M.J. Yaszemski, J.M. Powers, A.G. Mikos, Biomaterials 19 (1998) 1935
1943. [334] S.L. Ishaug-Riley, G.M. Crane, A. Gurlek, M.J. Miller, A.W. Yasko, M.J. Yaszemski, et al., J. Biomed. Mater. Res. 36 (1997) 1
8. [335] S.L. Ishaug, G.M. Crane, M.J. Miller, A.W. Yasko, M.J. Yaszemski, A.G. Mikos, J. Biomed. Mater. Res. 36 (1997) 17
28. [336] U. Sampath, Y. Ching, C. Chuah, J. Sabariah, P.-C. Lin, Materials (Basel) 9 (2016) 991. [337] Q. Liu, Q. Li, S. Xu, Q. Zheng, X. Cao, Polymers (Basel) 10 (2018) 664. [338] W.L. Ng, S. Wang, W.Y. Yeong, M.W. Naing, Trends Biotechnol. 34 (2016) 689
699. [339] W.L. Ng, W.Y. Yeong, M.W. Naing, Int. J. Bioprint. 2 (2016) 53
62. [340] F. Yan, Y. Liu, H. Chen, F. Zhang, L. Zheng, Q. Hu, AIP Adv 4 (2014) 031321. [341] C. Bergonzi, A. Di Natale, F. Zimetti, C. Marchi, A. Bianchera, F. Bernini, et al., Sci. Rep. 9 (2019) 1
11. [342] U. Jammalamadaka, K. Tappa, J. Funct. Biomater. 9 (2018) 22. [343] J. Zhang, B.J. Allardyce, R. Rajkhowa, Y. Zhao, R.J. Dilley, S.L. Redmond, et al., Sci. Eng 4 (2018) 3036
3046. [344] W. Qinghua, M. Marion, L. Sophie, T. Daniel and H. Marie-Claude, Solvent-cast 3D printing of Chitosan hydrogel scaffolds for guided cell growth, in Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress, 2016. Jean-PaulRiopelle, Montre´al, Canada.

Handbook of Chitin and Chitosan

References

553

[345] C.R. Almeida, T. Serra, M.I. Oliveira, J.A. Planell, M.A. Barbosa, M. Navarro, Acta Biomater. 10 (2014) 613
622. [346] Q. Wu, D. Therriault, M.C. Heuzey, A.C.S. Biomater, Sci. Eng 4 (2018) 2643
2652. [347] J. Wang, B. Jiang, W. Guo, Y. Zhao, J. Orthop, Surg. Res 14 (2019) 102. [348] L. Elviri, R. Foresti, C. Bergonzi, F. Zimetti, C. Marchi, A. Bianchera, et al., Biomed. Mater. 12 (2017) 045009. [349] P. Garstecki, M.J. Fuerstman, H.A. Stone, G.M. Whitesides, Lab Chip 6 (2006) 437
446. [350] J.H. Xu, S.W. Li, G.G. Chen, G.S. Luo, AIChE J. 52 (2006) 2254
2259. [351] A. Testouri, C. Honorez, A. Barillec, D. Langevin, W. Drenckhan, Macromolecules 43 (2010) 6166
6173. [352] P. Marmottant, J.P. Raven, Soft Matter 5 (2009) 3385
3388. [353] P. Garstecki, I. Gitlin, W. DiLuzio, G.M. Whitesides, E. Kumacheva, H.A. Stone, Appl. Phys. Lett. 85 (2004) 2649
2651. [354] E. Lorenceau, Y.Y.C. Sang, R. Ho¨hler, S. Cohen-Addad, Phys. Fluids 18 (2006) 097103. [355] G.F. Christopher, S.L. Anna, J. Phys. D. Appl. Phys. 40 (2007) R319
R336. [356] S. Prasadh, R.C.W. Wong, Oral Sci. Int. 15 (2018) 48
55.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

17 Drug delivery and tissue engineering applications of chitosan-based biomaterial systems Rajitha Panonnummal, Nitheesh Antony and M. Sabitha Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Amrita Health Science Campus, Kochi, India

O U T L I N E 17.1 Introduction 17.1.1 Demineralization 17.1.2 Deproteination 17.1.3 Deacetylation (formation of chitosan) 17.1.4 Properties and drug delivery systems 17.1.5 Limitations of chitosan as drug delivery system

556 557 558 558 558 561

17.2 Chitosan-based nanosytems in drug therapy of neoplastic diseases

561

17.3 Chitosan-based nanosystems in drug therapy of infectious diseases 17.3.1 Cell wall destruction and leakage by the ionic interaction 17.3.2 Inhibition of protein and mRNA synthesis

566 566 567

17.4 Blocking the supply of essential elements for microbes

567

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00017-0

555

© 2020 Elsevier Inc. All rights reserved.

556

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

17.5 Chitosan-based nanosytems in drug therapy of inflammatory diseases

570

17.6 Tissue engineering applications of chitosan-based nanosytems 17.6.1 Bone 17.6.2 Liver 17.6.3 Skin 17.6.4 Nerve

573 574 577 578 580

17.7 Conclusion

581

References

582

17.1 Introduction The development and synthesis of newer therapeutic agents are very challenging, expensive, and time consuming. Much research is focused on the development of novel drug delivery devices with an aim to overcome the biopharmaceutical problems of existing drugs with increased stability and safety profile. Drug delivery systems play a crucial role in delivering the drug in desired amounts to the specific site of action. For the past few decades polymers have been considered as one of the versatile agents used for developing suitable drug delivery systems to deliver the drugs to the target sites. The drug delivery devices are engineered according to the route of administration and target of action. Polymers of natural origin, such as cellulose derivatives, sodium alginate, starch, and chitin, as well as those of synthetic origin, such as polyethylene glycol, polyvinyl chloride, and nylon, are being used for this purpose. The novel drug delivery systems developed by using such polymers include the micro- and nanosized drug carriers such as microparticles, nanoparticles, liposomes, niosomes, hydrogels, and patches. The major factors of importance in the case of a polymeric drug delivery system are the drug-holding capacity, stability, drug release mechanisms, and safety. Cellulose is the most abundant polymer from a natural origin which is an accepted carrier in the development of drug delivery systems [1
4]. Chitosan is the second most naturally occurring linear nitrogenous polysaccharide obtained by the partial deacetylation of chitin, a polymer obtained from the internal scaffold and exoskeleton of invertebrates and some vertebrates [5]. Chitosan is much preferred as a natural polymer due to its structure, biocompatibility, tenability, and lesser side effects. The molecular weight and properties of this

Handbook of Chitin and Chitosan

557

17.1 Introduction

Sources Demineralization Remove minerals

Acid treatment method

Deproteination Chemical, enzymatic and fermentation method

Remove proteins

Decolouration Pure chitin is obtained

Remove residual pigments

Deacetylation Enzymatic or chemical treatment method

FIGURE 17.1

Formation of chitosan

Representation of preparation of chitosan from its sources.

polymer differ according to the source. The sources include arthropods, marine sponges, protists, foraminiferan mollusks, diatoms, hydroids, coelenterates, brachiopods, polychetes, pogonophorans, and crustacean shells. Among these, crustacean shells are the major source and commercial products are isolated from crabs and shrimps [6,7]. Chitosan is derived from chitin which is the most abundant amino polysaccharide polymer and is found along with proteins, minerals (calcium carbonate), and residual pigments. To obtain chitin in the pure form, the collected sources are treated with acids for demineralization followed by treatment with alkali to remove proteins (Fig. 17.1). Demineralization and deproteination can be achieved by enzymatic, chemical, and fermentation methods too [8,9].

17.1.1 Demineralization Acids such as hydrochloric acid (HCl), nitric acid (HNO3), sulfuric acid(H2SO4), acetic acid (CH3COOH), and formic acid (HCOOH) are used for demineralization of chitin. Dilute HCl is preferred (up to 10%) and treatment conditions, such as duration of reaction, temperature, and solute
solvent ratio, are varied according to the source. After the treatment the formed salts are separated using deionized water [10,11].

Handbook of Chitin and Chitosan

558

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

17.1.2 Deproteination Deproteination is carried out chemically by using alkalis such as sodium hydroxide (NaOH), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), potassium hydroxide (KOH), potassium carbonate (K2CO3), calcium hydroxide [Ca(OH)2], sodium sulfate (Na2SO3), sodium bisulfate (NaHSO3), calcium bisulfate (CaHSO3), sodium phosphate (Na3PO4), and sodium sulfide (Na2S). NaOH is the most preferred one at a concentration of 0.125
5 M and temperature of 160 C. The reaction time varies from minutes to a few days. The enzymatic method of separation involves the use of proteolytic enzymes such as pepsin, papain, pancreatin, devolvase, and trypsin. In another method, selected strains of microorganisms are used for fermentation (lactic acid and nonlactic acid fermentation) to remove proteins. Decoloration is needed if residual pigments are present so as to obtain chitin in a pure colorless form [11
13].

17.1.3 Deacetylation (formation of chitosan) The deacetylation of chitin using enzymatic or chemical treatment leads to the formation of chitosan. Different methods are followed but a standard preparation method has not been established. The N-deacetylation of chitin using alkalis leads to the formation of chitosan with varying percentages of acetylation according to the experimental conditions. The method involved is classified as either homogeneous or heterogeneous deacetylation. In the heterogeneous method, 85%
99% deacetylated chitosan is obtained by treating chitin with a higher concentration of NaOH for a few hours at a temperature above 60 C. In the homogeneous method, 48%
55% deacetylated chitosan is obtained by treating alkylated chitin at 25 C for 3 h [14]. The degree of deacetylation will influence the molecular weight, distribution of acetyl groups, solubility, viscosity, biocompatibility, and biodegradability, as well as its pharmacological activity. Chitosan is composed of β-(1-4)-linked 2-amino-2-deoxy-β-Dglucopyranose (GlcN) and 2-acetamido-2-deoxy-β-D-glucopyranose (GlcNAc) residues. The presence of reactive primary and secondary hydroxyl groups and primary amino group make it suitable for the preparation of derivatives. Chitosan is insoluble in water and soluble in dilute acids [15].

17.1.4 Properties and drug delivery systems Chitosan is a safe, biodegradable, nontoxic, biocompatible polymer with wide applications in the cosmetic, food, agriculture, and pharmaceutical industries. Chitosan is reported to have antimicrobial, antioxidant, antiinflammatory, analgesic, hemostatic, angiogenic, adsorption

Handbook of Chitin and Chitosan

559

17.1 Introduction

Nanoparticles and microparticles Membrane and sponges

Microspheres and beads Drug delivery systems

Nanogels

Scaffolds

Films

FIGURE 17.2

Different drug delivery systems developed using chitosan.

enhancing, and mucoadhesive properties. Based on the physiochemical and biological properties of the polymer it is engineered into a variety of micro- and nanodrug delivery systems (Fig. 17.2) such as fibers, scaffolds, films, sponges, hydrogels, beads, and particles [15
17]. 17.1.4.1 Nanoparticles Chitosan nanoparticles were first synthesized in 1994 by Ohya and coworkers by an emulsification and cross-linking method for the intravenous delivery of 5-fluorouracil. Researchers developed various techniques like emulsion-droplet coalescence, emulsion solvent diffusion, reverse micellar, ionic gelation, polyelectrolyte complexation, spray drying, and desolvation methods for the effective loading of active moieties in chitosan nanoparticles for various drug delivery applications. Chitosan nanoparticles are reported to have controlled drug release properties, improved cell attachment and rate of permeation which offers improved drug absorption and bioavailability in addition to the general advantages of other polymeric nanoparticles [18]. 17.1.4.2 Hydrogels Chitosan-based hydrogels are a promising drug delivery system for the localized delivery of drugs, proteins, and genes with an aim to attain increased drug concentration at the application site at a controlled rate. Hydrogels can be prepared by chemical or physical cross-linking method. The chemical cross-linking is possible through hydrogen bonding or through the interaction of hydrophobic and electrostatic forces achieved with the use of cross-linkers like gluturaldehyde or by photopolymerization using UV light, by using ionic

Handbook of Chitin and Chitosan

560

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

cross-linkers like tripolyphosphate or by using polyelecrolytes like alginate. The hydrogels prepared by physical cross-linking are more biocompatible but are sensitive to changes in environmental conditions, like pH and temperature, and are less stable mechanically [18
20]. 17.1.4.3 Microsphere and beads Chitosan is a versatile carrier for the delivery of drugs with solubility and lipophilicity problems, with harmful side effects, and for drugs with faster degradation profiles. Chitosan microspheres can be prepared by cross-linking chitosan using a specific amount of multivalent anions. According to the method of preparation, acidic, basic, or neutral phases are used. A spray drying method is also suitable for the preparation of drug-loaded chitosan microsphere and beads. The process uses the principle of atomization of the acidic solution of chitosan containing drug in hot air. However the polyelectrolyte nature of chitosan causes coacervation of polymer which makes the spray drying method of preparation difficult. Other methods used for the preparation of chitosan microspheres are precipitation, complex coacervatrion, modified emulsification, and ionic gelation. Among these, complex coacervatrion and ionic gelation methods are also suitable for chitosan bead preparation [18,19,21]. 17.1.4.4 Scaffolds Scaffolds are porous polymeric systems that can deliver drugs and at the same time provide mechanical support. Chitosan scaffolds are widely used for tissue engineering applications and organ substitution because of structural similarities to cellulose. Scaffolds are prepared by freeze-drying chitosan solution and the required pore size is achieved by the addition of polyvinyl alcohol or polycaprolactone. Newer methods used for the preparation of scaffold include electrospinning and supercritical fluid technology. The pore size, adsorption, and mechanical strength of scaffolds depend upon the degree of deacetylation and molecular weight of chitosan. Usually chitosan with a molecular weight of 105
106 Dalton and a deacetylation of 5%
20% is used for scaffold preparation [22,23]. 17.1.4.5 Films Films are membrane structures that release the drug at a controlled rate. Chitosan films are permeable to specific gases and are reported to have improved mechanical properties. The modifications are possible to overcome the moisture absorbing nature of films. Hydrophobicity is increased by adding fatty acids or lipids. Chemical stability and mechanical properties are improved by combining chitosan with starch, gelatin, and other hydrocolloids. The films are prepared by dissolving

Handbook of Chitin and Chitosan

17.2 Chitosan-based nanosytems in drug therapy of neoplastic diseases

561

chitosan in dilute acids, and then they are transferred into a petri plate for drying, in order to obtain films with uniform thickness and then dried under constant temperature. Films are commonly used for the local drug delivery applications [22,24].

17.1.5 Limitations of chitosan as drug delivery system The internal (intermolecular interactions) and external parameters (degree of acetylation, molecular weight, and purity) are needed to be considered while selecting chitosan for the development of specific drug delivery systems. It is reported that the chemical structure of chitosan is not correlated well with the effective biological responses. In addition the biological safety and activity of chitosan is reported to depend upon its biological origin and the host to which it is intended to be applied. The effect of other characteristics such as size, crystallinity, and morphology on degradation profile and biocompatibility of chitosan are not yet well established. There are insufficient reports regarding the exact molecular mechanism behind the biological activity of chitosan [25].

17.2 Chitosan-based nanosytems in drug therapy of neoplastic diseases The diagnosis and treatment of neoplastic diseases remain challenging and demand newer strategies for early diagnosis and effective treatment. Solid tumors acquire a lot of characters and properties which make them fit enough to survive even in the worst atmosphere created by the treatment strategies and thus become difficult to eradicate. Advanced solid tumors in comparison with normal cells are reported to be characterized by a permissive microenvironment, deregulated metabolic properties, altered cellular and extracellular pH, enhanced blood supply, deformed cellular matrix etc. In addition, the overexpression of the efflux pumps like P glycoprotein (Pgp) offers multidrug resistance to tumors and remains as one of the biggest challenges in the treatment of neoplastic disorders. Upregulated transcriptional processes involved in controlling DNA repair, drug detoxification, and cell survival pathways make cancers difficult to treat. Toxicity to normal cells by the offtarget effect of anticancer drugs leads to patient noncompliance to drug therapy [1]. There is a demand for newer pharmaceutical and biomedical technologies to develop drug delivery systems that can overcome the demerits of conventional therapeutic strategies of neoplastic diseases to an extent. Development of targeted nanosystems is expected to improve the

Handbook of Chitin and Chitosan

562

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

effectiveness of therapy by avoiding the off-target effects and thus reduce the toxicity. It is possible to increase the circulation time of drug molecules by encapsulating them into proper nanosystems and then by functionalizing them with agents like polyethylene glycol (PEG) (for stearic protection and hydrophilicity) that give protection from reticuloendothelial system uptake and thus make them stable [1,26]. Using such types of systems, it is possible to target the drug therapy to tumor sites by conjugating ligands which are agents that can bind specifically to tumor cell markers, like Prostate Specific Antigen in the case of prostate cancer. Use of selective and specific polymers that can withstand the unfavorable environment with the capability to release the loaded moieties at the tumor environment is expected to be useful and promising. Among various polymeric systems, carbohydrate polymers seem to have many advantages due to their biocompatible and biodegradable nature. Being a carbohydrate-based polymer, chitosan has a lot of unique properties that make it the favorite choice of researchers. The presence of amino and hydroxyl groups make it suitable for functionalization with conjugating agents like folic acid, antibodies, radioactive isotopes, quantum dots, etc. (Fig. 17.3). Its cationic nature helps to target it to negatively charged cell surface receptors expressed at tumor sites and tumor stem cells. Its pH-dependent swelling property ensures the drug release at acidic tumor environment, as drug release is a function of the swelling property of the polymer. Its rich functionalities facilitate

Pegylation for stearic protection

Functionalization by quantum dots, radio isotopes, etc.

Chitosan polymer

Drug molecules Active targeting by antibody conjugation

Coating with thermo sensitive polymers

Genetic materials Coating with pH sensitive polymers

Active targeting by FA conjugation

Tumor cell sensitizers

FIGURE 17.3 Schematic representation of modification of chitosan polymer for effective drug-loading and active targeting in the treatment of cancer.

Handbook of Chitin and Chitosan

563

17.2 Chitosan-based nanosytems in drug therapy of neoplastic diseases

active targeting of loaded moieties to tumor sites through functionalization with specific tumor markers. The versatility of chitosan allows for the development of a lot of functionalized nanodrug delivery systems (Table 17.1), such as micelles, nanoparticles, hydrogels, and nanoplexus, against different types of neoplastic diseases [1,26,36,37]. Jama et al. developed chitosan
poly vinyl alcohol (PVA) hydrogel loaded with the anticancer drug doxazocin. The prepared system exhibited significant toxicity toward the human cervical cancer HeLa cell line. Significant inhibition in angiogenesis is reported in vivo and the system is expected to be useful to overcome the tumor metastasis and invasion by blocking the angiogenesis. Here, it is the compatibility of the chitosan with PVA that helps to form an ideal system suitable for TABLE 17.1 List of various chitosan-based systems with benefits achieved in antineoplastic drug delivery. Sl. No:

Delivery systems

Active moieties

1

Hydrogel

2

Benefits achieved

References

Doxazocin

• Significant inhibition in angiogenesis and toxicity toward cancer HeLa cell line

[27]

Hydrogel

5-FU

• Good cellular internalization with apoptosis of cancer cell lines • Simultaneous monitoring with improved therapeutic efficacy

[28]

3

Hydrogel

Doxorubicin

• Simultaneous monitoring with improved therapeutic efficacy

[29]

4

Nanoparticles

Legustrazine

• Targeting efficacy to folic acid receptor overexpressed tumors

[30]

5

Nanoparticles

Ursolic acid

• Targeting efficacy to folic acid receptor overexpressed tumors • Good internalization by cancer cells and reduction in tumor volume

[31]

6

Nanoparticles

Paclitaxel

• Good cellular internalization with improved toxicity to tumor cells

[32]

7

Nanoparticles

Trastuzumab

• Improved pharmacokinetics with increased drug bioavailability and good cellular internalization

[33]

8

Nanoplexus

mir 34

• Greater transfection efficacy with reduced tumor burden

[34]

9

Micelle

Gambogic acid

• Improved pharmacokinetics with good cellular internalization

[35]

Handbook of Chitin and Chitosan

564

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

the delivery of the selected anticancer drug doxazocin [27]. Li Sun et al. developed chitosan hydrogel loaded with anticancer drug 5-FU, functionalized with fluorescent carbon dots for simultaneous monitoring and therapy. A549 cancer cells exhibited good cellular internalization with significant apoptosis when treated with the developed system. Here the advantage attained is the cell apoptosis (indication of therapeutic effectiveness) along with simultaneous monitoring of the cellular internalization of the particles [28]. Similarly radiolabeled doxorubicin loaded chitosan hydrogel is prepared and reported to exhibit improved radiolabeling stability and slow drug release profile. Here also, monitoring and therapy is possible by the use of a single system. The simultaneous conjugation of radio isotopes to outer shells (for imaging) with entrapment of therapeutic moieties to the polymeric core is achieved because of the rich functionalities of chitosan [29]. Legustrazine is a vasodilator, which is reported to enhance the sensitivity of tumor cells toward the drug therapy. In order to target its delivery to folic acid receptor (FR) overexpressed tumor cells, chitosan nanoparticles loaded with legustrazine, conjugated with folic acid was synthesized by Lichun Cheng et al. Folic acid conjugation is expected to allow the system to target the FR overexpressed tumors. Chitosan makes the system biocompatible and ensures the proper release of loaded moiety at the acidic tumor environment. The system is limited by use only in FR overexpressed tumors [30]. Chitosan nanoparticles loaded with the antitumor drug ursolic acid, conjugated with folic acid for the targeted delivery to FR positive breast cancer is developed by Hua Jin et al. The system is reported to be free of the off-target effects and exhibited good internalization by breast cancer MCF-7 cell lines. The particle entry into the cell through endocytic pathway due to folic acid conjugation, followed by destruction of the lysosomal membrane and relocalization into mitochondria is well demonstrated. Mitochondrial localization and subsequent production of reactive oxygen species caused lysis of tumor cells. In vivo studies done on MCF-7 xenograft model revealed a significant antitumor effect with much reduction in tumor weight on nanoparticles treatment in comparison with free drug ursolic acid. The system is found to be promising as it can be used for other types of tumors in which overexpressed FR are confirmed [31]. In another study chitosan nanoparticles loaded with anticancer drug paclitaxel was prepared by solvent evaporation-cross-linking method. The particles exhibited a sustained drug release profile, higher cytotoxicity, and greater internalization by human ovarian cancer A2780 cell line. Even though there is lack of in vivo efficacy studies, good internalization and toxicity toward tumor cells are demonstrated in vitro; may be considered as an indirect indication of the improved therapeutic benefits of the developed system in comparison with conventional dosage

Handbook of Chitin and Chitosan

17.2 Chitosan-based nanosytems in drug therapy of neoplastic diseases

565

forms [32]. Trimethyl chitosan (TMC) is a soluble cationic chitosan derivative, which retains the biocompatibility and biodegradability of chitosan. Nanoparticles using TMC loaded with anticancer drug paclitaxel is developed by Rong et al. for the treatment of gastro-enteric tumors. The system exhibited sustained drug release profile, good cellular internalization with potent antiproliferative effect against NCI-N87 and SGC-7901 cell lines. In vivo efficacy studies done on NCI-N87 and SGC-7901 xenograft model revealed superior efficacy with safety in comparison with free drug paclitaxel. Greater efficacy toward such a highly proliferating type of tumor seems to be very useful [38]. Chitosan nanoparticles loaded with trastuzumab (monoclonal antibody against Epidermal Growth Factor); decorated with D-α-tocopheryl polyethylene glycol 1000 succinate monoester (TPGS) is prepared for its targeted delivery in Epidermal Growth Factor Receptor (EGFR) overexpressed breast cancer. The developed particle exhibited enhanced cellular uptake and cytotoxicity with improved pharmacokinetic parameters like drug absorption and bioavailability in comparison with nontargeted nanoparticles and control drug solution. The system seems to be useful for the treatment of EGFR overexpressed tumors such as breast cancer [33]. In a study methotrexate loaded chitosan nanoparticles is prepared by ionic gelation technique by Mehlia et al. The particles are further labeled by radioactive technitium 99. The formulation exhibited good entrapment efficiency and greater cellular uptake as demonstrated using breast cancer (MCF-7) and epidermal keratinocyte (HaCaT) cell lines. High cellular localization by cancerous and proliferating immortal cell lines suggests it to be useful for the treatment of malignancies [39]. Chitosan
PLGA nanoplexus loaded with tumorsuppressing mRNA, mir-34, was prepared by Donato et al. Being a cationic polymer, chitosan can easily combine with negatively charged genetic material to form nanoplexus. Encapsulation of mir-34 into the nanoplexus protects it from degradation by RNAse and confirms higher transfection efficiency. In vivo efficacy studies done on the NOD-SCID xenograft model of multiple myeloma demonstrated improved survival of animals with reduced tumor burden [34]. Chitosan lecithin nanoparticles loaded with epicatechin and conjugated by TPGS was prepared by a self-assembly method. Improved cytotoxicity is observed toward breast cancer cell lines like MCF-7, MDA-MB 231, MDA-MB-436, and SG-Br3. TPGS improves solubilization of drug in nanoparticle systems and offers a considerable degree of stability. The system showed higher selectivity toward cancer cells with a fourfold reduction in IC-50 value to induce cytotoxicity in comparison with control drug solution [40]. A hybrid micelle composed of carboxy methylauryl chitosan conjugated with folic acid and superparamagnetic iron oxide was prepared. The system exhibited improved MRI contrast, allowed better tumor

Handbook of Chitin and Chitosan

566

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

diagnosis, and actively targeted the therapy to FR overexpressed tumor cells [41]. Polymeric micelle composed of chitosan, l-arginine, and noctyl was synthesized and loaded with anticancer drug cambogic acid (GA) with an aim to alter the pharmacokinetic profile of the drug. The developed system exhibited good cellular uptake when tested against hepatic cancer (Hepg2) cell line. Lack of rapid clearance of drug with improved half life is observed when tested in vivo [35].

17.3 Chitosan-based nanosystems in drug therapy of infectious diseases Infectious diseases are major causes of death worldwide and are triggered by the invasion of pathogenic microbes into the body [42]. Diseases are transmitted directly or indirectly with the involvement of organisms such as bacteria, fungus, virus, and prions, and are classified as contagious and noncontagious. The imbalance between the body’s defense mechanism and microbial resistance increases the severity of disease. So the repeated assessment of emergence and remission of infectious diseases is necessary to control the severity of disease and effective treatment strategies are needed for the eradication of the disease [43]. Treatment of infectious diseases remains a challenge, due to the ability of microbes to adapt to changes in factors like population, medicines, environment, lack of proper technologies for exact identification of microbes, the microbial resistance offered by the presence of efflux pumps, and drug degrading enzymes and adverse reactions caused by available drugs [44,45]. Chitin and chitosan are reported to be active against Gram-positive and Gram-negative bacteria, algae, yeast, and fungi. There are a lot of scientific reports which support the use of these materials for the fabrication of drug delivery systems for the treatment of infectious diseases. Initially, chitosan was reported to be both a bacteriocidal and bacteriostatic agent but later, based on investigations, it became evident that bacteriostatic action is more prominent than bactericidal activity [46
50]. The mechanism of antibacterial activity of chitosan is not exactly proven but it is explained by three proposed models (Fig. 17.4).

17.3.1 Cell wall destruction and leakage by the ionic interaction The positively charged chitin or chitosan can interact with negatively charged microbial cell membrane leading to cell destruction [51
53]. It is the amine group of chitosan that reacts with the negatively charged

Handbook of Chitin and Chitosan

17.4 Blocking the supply of essential elements for microbes

567

Cell wall destruction

Inhibit mRNA and protein synthesis Ribosome Blocks the entry of essential components

FIGURE 17.4

Possible mechanisms of antimicrobial activity of chitosan.

bacterial cell membrane through electrostatic interaction which leads to the hydrolysis of the peptidoglycan layer followed by the leakage of intracellular components such as potassium, proteins, glucose, etc. out of the membrane. Under this situation the osmotic pressure balance of the bacterial cell gets altered leading to cell destruction [46
49,53,54]. Raafat et al. reported that E coli when treated with chitosan particles shows the presence of oligomeric units of chitosan in its plasma membrane which has been demonstrated by confocal microscopy [51].

17.3.2 Inhibition of protein and mRNA synthesis Another reported mechanism is that chitosan can enter into the cells through the multilayered cell membrane and then into the nuclei of bacteria resulting in the inhibition of protein synthesis leading to antimicrobial activity [49,54,55].

17.4 Blocking the supply of essential elements for microbes The blocking of essential elements supply to microbes is also reported to be a reason responsible for cell death caused by chitosan. Chitosan is a strong chelating agent; it can chelate the metal ions and essential elements. Thus it is able to block the supply of these things to microbes and prevent microbial growth. Downregulated growth of spore component due to the presence of reactive amine group of chitosan is also reported. This mechanism is more effective at high pH [56,57]. The antimicrobial activity of chitosan is influenced by lot of factors (Table 17.2) like degree of deacetylation, pH, water solubility, and

Handbook of Chitin and Chitosan

568

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

TABLE 17.2

Factors influencing the antimicrobial activity of chitosan.

Factors

Effect on antimicrobial activity

Increases in degree of polymerization

Increases

Increases in degree of deacetylation

Increases

Increases in molecular weight s

Increases

Increases in pH of medium

Decreases

Type of bacterial medium and bacterial strain

Varied

Increases in ionic strength

Increases

Presence of chitosan-degrading enzyme

Decreases

Presence of metal ions and chelating agents

Decreases

Increases in aqueous solubility

Increases

molecular weight. The water-soluble chitosan derivatives are suitable for microbial eradication and thus researchers focus on developing water-soluble derivatives like thiolated, alkylated, and quaternized forms of chitosan with maximum biocompatibility and antimicrobial activity [58]. There is an influence of the molecular weight of chitosan on antimicrobial activity since low-molecular-weight oligosaccharides and D-glucose amines show lesser or no toxicity against microbes. A molecular weight of more than 10 kDa is considered to inhibit bacterial growth. The increase in protonated amine group by deacetylation will also increase the antimicrobial activity. At lower pH and increased degree of polymerization an increase in activity is observed. With an increase in ionic strength of chitosan, increased solubility and potentiation of activity is observed. The presence of metal ions and chelating agents will reduce the antimicrobial activity. The solvents (organic or inorganic) used to dissolve chitosan will not have a significant effect. Chitosan-degrading enzymes are reported to have less effect on the antimicrobial activity of chitosan [59,60]. From the reported studies it is clear that chitosan is compatible for oral, ocular, nasal, and topical route of administration for the treatment of infectious diseases (Fig. 17.5). Ying-Chien Chung et al. prepared watersoluble chitosan derivatives by Maillard reaction and evaluated the antimicrobial activity against Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Shigella dysenteriae, and Salmonella typhimurium. It is found that the antibacterial activity of the water-soluble derivatives is higher than acid-soluble chitosan, with better activity in deionized water than saline water [61]. Chitosan liposome in combination with gold is prepared for vancomycin delivery in methicillin-resistant

Handbook of Chitin and Chitosan

17.4 Blocking the supply of essential elements for microbes

569

Ocular (film) Nasal(nano particles)

Oral (nanoparticles)

Chistosan-drug delivery systems used for infectious disease Buccal (nanoparticle, microsphere)

Topical (sponges, scaffolds, films, gels) Colon and vaginal (nano particle microsphere)

FIGURE 17.5 Chitosan-based drug delivery systems according to route of administration in infectious diseases.

S. aureus infections. The system exhibits improved stability due to the presence of chitosan in combination with gold and the antimicrobial potential of vancomycin is found to be enhanced [62]. The thiolated carboxymethyl chitosan-g-cyclo dextrin nanoparticle is prepared by ionic gelation method using thioglycolic acid as thiolating agent and tripolyphosphate as gelling agent for the delivery of albendazole in worm infection. Improved mucoadhesviness and controlled drug release are observed [63]. The chitosan nanoparticle loaded with cephazolin has been developed for the prevention of multidrug resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and extended Spectrum Beta Lactamasepositive E. coli. The prepared particles exhibited higher entrapment of drug cephazolin with maintained activity against the selected microbes [64]. Biocompatibility of chitosan nanoparticles leads to the formulation of rifampicin inhalation powder for the treatment of tuberculosis by combining ionic gelation with a spray drying technique with the aim to deliver the drug in a sustained manner [65,66]. Chitosan can act as a carrier and enhance the penetration of smaller polar molecules which are otherwise difficult to pass through the skin [67]. The topical application of silver containing nanocrystalline chitosan dressing is found to promote the wound-healing activity of the antibacterial agent silver sulfadiazine when compared with topically applied silver sulfadiazine alone [68]. Chitosan is also found to be useful for antimicrobial delivery in dental infections and is used for tetracycline delivery for periodontal diseases [69]. Another study shows the superior antibacterial efficacy of tetracycline-loaded chitosan sponge against S. aureus and E. coli infections when compared with conventional formulation [70]. The mucoadhesive

Handbook of Chitin and Chitosan

570

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

nature of chitosan
sodium alginate helps to deliver the antibiotic (Gatifloxacin) through an ocular route and the increased residence time of a drug suitable for ophthalmic use is reported [71]. Chitosan is also reported to have antifungal activity. Chitosan exhibits antifungal activity against different species of fungi due to its distinct mechanism of action intrinsic to it. The antifungal activity is explained by the microscopic evaluation of fungus treated with chitosan. It is demonstrated that chitosan can directly interact with fungus, penetrate through the hyphae, alter the enzyme activity, and thus inhibit cell wall morphogenesis [72,73]. In addition the inhibition in biofilm formation of fungus by chitosan is also reported. Saharan et al. developed stable, porous copper nanoparticles using chitosan with the ionic gelation technique, which showed potential to inhibit mycelia growth and spore germination when tested against Alternaria solani and Fusarium oxysporum [74].

17.5 Chitosan-based nanosytems in drug therapy of inflammatory diseases Inflammation is the biological response of the body toward external stimuli of chemical or physical means and or pathogens. Inflammation can be classified as acute and chronic according to the duration of response. In an acute condition, the inflammatory response lasts for a short duration and the activated leukocytes are responsible for the repair of injured tissue. In chronic inflammation several chemical mediators along with various transcription factors associated with the expression of cyclooxygenase-2, adhesion molecules, proinflammatory cytokines, inducible nitric oxide synthase, matrix metalloproteinase, and nuclear factor
kappa B are found to be involved [75
79]. Oxidative stress in chronic inflammation is responsible for cell dysfunction and is induced by macrophages, leukocytes, etc. The current treatment approach for inflammatory diseases is based on COX inhibition by NSAIDs and the use of biologics [80]. The challenges that need to be overcome for drug delivery in inflammatory diseases are the rapid clearance of drug due to increased blood perfusion at the site of inflammation, the need for administration of multiple drugs to target different phases of inflammation, and the possibility of adverse reactions due to multiple drug treatments. Stratum corneum remains the major physiological barrier for the topical delivery of antiinflammatory drugs. The topical delivery is further affected by the pH of skin, skin conditions such as hydration and aberrations, thickness of stratum corneum, and the presence of permeation enhancers in the delivery systems [81]. Studies related to the use of chitosan and its

Handbook of Chitin and Chitosan

17.5 Chitosan-based nanosytems in drug therapy of inflammatory diseases

571

derivatives for modulating inflammatory conditions are reported. Chitosan is reported to exhibit antiinflammatory activity through a variety of mechanisms such as inhibition of proinflammatory cytokines, promotion of tissue granulation by fibroblast proliferation, production of type III collagen, inhibition of nitric oxide synthase activity, and by increasing the expression of antiinflammatory cytokines. But some studies revealed the proinflammatory activity of chitosan through the activation of inflammasomes (multiprotein oligomers responsible for the activation of inflammatory responses) such as NLRP3. The antiinflammatory or proinflammatory activity of chitosan depends upon the specific characters such as degree of acetylation, molecular weight, size, polydispersity index, purity, and dose. The variation in the activity according to these factors is observed in all reported studies [82]. The primary concern is the size and a study conducted by using small-, medium-, and large-sized chitosan in murine peritoneal macrophages revealed that small- and medium-sized compounds stimulate TNF production, activate nuclear factor kappa B and spleen tyrosine kinase, but larger-sized chitosan is inert in nature [83]. The proposed intracellular pathways involved are cGAS-STING and NLRP3 and are demonstrated by in vitro cell line studies. The activation of immune cells by chitosan is dependent on its dose and the presence of a number of tandem glucosamine motifs. Chitosan can interact with macrophages at different stages. When treated at low doses with macrophages primed with phorbol ester, it induces the cGAS-STING pathway and causes interferon type 1 response through the release of antiinflammatory mediators like IL10 and chemokines. But at a high dose, it activates inflammasome and modulates the release of inflammatory mediators from macrophages [82,84]. The inhibition of myeloperoxidase activity by chitosan using human myeloid cells and inhibition of DNA and protein oxidation in mouse macrophages was demonstrated by Ngo et al. [85]. The partial inhibition of IL-8 and TNF-α by water-soluble chitosan is suitable for allergic inflammatory responses [86]. The matrix metalloproteinase (MMP 2) expression and activity is suppressed by chitosan oligosaccharide and is confirmed by studies done using human dermal fibroblast [87]. It is reported that the prolonged inflammatory phase of wound healing is reduced by chitosan through the inhibition of the release of inflammatory cytokines [88]. The high-molecular-weight chitosan undergoes enzymatic degradation to form low-molecular-weight chitosan oligosaccharides and exhibist antiinflammatory activity in allergic asthma. The in vitro and in vivo results of a study showed that the low-molecular-weight chitosan, when combined with glucosamine, inhibits cytokine synthesis and antigenactivated degranulation of basophils in a rat model of basophilic leukemia. The ovalbumin-mediated lung inflammation seen in asthma is also

Handbook of Chitin and Chitosan

572

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

cured by chitosan in a mice model. Significant inhibition in expression of interleukins mRNA and proteins is observed in bronchoalveolar lavage fluid and lung tissue after treatment with chitosan [89]. Hirva et al. developed zaltoprofen (an NSAID)-loaded chitosan nanoparticle and evaluated the antiinflammatory activity in rat paw edema model. The results revealed the sustained delivery of drug with improved bioavailability and reduced toxic effects when compared with conventional drug-loaded gel [90]. Hydrocortisone-loaded chitosan nanoparticles are prepared by ionic gelation method for percutaneous delivery in the treatment of atopic dermatitis. Here a controlled drug release is attained; which improves the efficacy and reduces the toxic effects of selected drugs when compared with marketed formulations [91]. Galactosylated chitosan nanoparticles loaded with sylimarin are synthesized using glutaraldehyde as a cross-linker by the condensation method for the targeted delivery of sylimarin toward liver. The biodistribution studies revealed the organ-specific delivery of the drug with improved liver protective activity when compared with marketed product [92]. Chitosan is reported to help in avoiding multiple administration, chemical instability, and skin irritation of tretinoin (vitamin A supplement, used for skin inflammatory disease like acne, psoriasis, and aging) through the formulation of its solid lipid nanoparticles [93]. Chitosan nanoparticles loaded with Ketorolac tromethamine were developed for ocular delivery to treat ocular inflammatory diseases. The system is found to be promising with an initial burst release followed by a sustained release profile and the mucoadhesiveness of chitosan helps to retain sufficient drug concentration at the site of action for a prolonged period [94]. Skin permeable bilayered chitosan nanoparticles are developed for the topical delivery of spantide II and ketoprofen (antiinflammatory). The bilayered nanoparticles are treated with oleic acid for surface modification and hydroxyl propyl methyl cellulose and carbopol are used to adjust the rheological properties of nanogel. The nanogel is evaluated for the effectiveness in allergic contact dermatitis and plague psoriatic model. Results indicate a significant increase in the antiinflammatory activity of the selected drugs against both the models of inflammation with improved skin contact time and surface hydration [95]. The delivery of hydrophilic drugs to brain is still a barrier. Brain targeting of methotrexate (hydrophilic drug) is successfully achieved using intravenous delivery of surface-modified chitosan nanogel formulation. The neuropharmacokinetic studies showed evidence of an increase in plasma and brain concentration of methotrexate on treatment with drug-loaded chitosan nanogel in comparison with administered free drug. Results prove that chitosan-based nanogel is a promising tool to target the brain due to its enhanced permeability [96].

Handbook of Chitin and Chitosan

17.6 Tissue engineering applications of chitosan-based nanosytems

573

17.6 Tissue engineering applications of chitosan-based nanosytems Tissue engineering refers to the development of biological substitutes to replace the failing part of the human body and makes use of the combination of engineering technology with molecular biology. It aims to develop artificial systems that can replace the damaged living part of human body and has great therapeutic potential in various conditions like severe trauma, accidents, major surgeries, and congenital abnormalities where critical damage to a vital system might have happened [97,98]. In such situations, autograft is considered as the gold standard treatment option for the correction of damaged tissue, but is limited by the need for an expensive surgical procedure, blood transfusion (in case of severe trauma), and it may cause significant donor morbidity. The method is also limited in terms of quantity of tissue supply [99,100]. The second option is allograft; here a tissue collected from a human cadaver is transferred to a living person. Although it has the advantage of being devoid of donor morbidity, there is a chance of transmission of diseases from the cadaver to the receiver and the occurrence of serious immunological reactions [100]. The third option is xenograft, in which tissues isolated from nonhuman sources, such as bovine, are transferred to a human receiver. The method is limited by a higher risk of viral disease transmission and immune-rejection reactions [100]. Tissue engineering helps to replace these types of conventional treatment methods with artificial scaffolds or materials loaded with cells and growth regulators that are capable of regenerating the damaged tissue [97,100]. The construction of artificial systems is possible through the combined use of suitable polymers, living cells, and signal molecules (Fig. 17.6). The incorporated cells proliferate, differentiate, and construct proper extracellular matrix that migrate suitably to regenerate the damaged tissue. All these processes are under the regulation of loaded signal molecules [97
105]. The major aspect is the polymeric support that provides adequate space for the cells to grow, proliferate, migrate, and differentiate to regenerate the damaged tissue. The polymeric supports not only act as an inert support to cells, but also control the release of loaded signal molecules and growth factors; which actually regulate the entire process of cell differentiation and regeneration [97,99,106]. A lot of natural, semisynthetic, and synthetic polymers are widely used to develop such types of tissue engineering systems. Among these the natural polymers have got considerable importance due to the expectation of being biochemically similar to living tissues. Being a natural polymer with carbohydrate origin, chitosan has got wide accessibility to tissue engineering applications. Its biocompatibility confirms the absence of immunological reactions and its slow biodegradability

Handbook of Chitin and Chitosan

574

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

Chitosan based matrix support systems like scaffolds, hydrogels, sponges, films, etc. Versatality

Bio compatibility

Tissue engineering

Controlled swellability

Antimicrobial activity

Wound healing activity Adhesiveness

Cells like stem cells, mesenchyma cells, hepatocytes, etc.

Bio degradability

Mechanical stability

Porosity

Signal molecules, growth promoters, regulators, nutrients, gases, etc.

FIGURE 17.6 Showing the construction of chitosan-based tissue engineering systems with various advantages.

matches with the time needed for tissue regeneration. It is having intrinsic antibacterial activity and is a potent wound-healing agent. The most important property that makes chitosan popular for tissue engineering applications is its versatility to form different structures like microspheres, hydrogels, scaffolds, sponges, etc by simple methods; with sufficient porosity and mechanical strength. Porosity is an important factor that regulates the flow of cells, signal molecules, nutrients, growth factors, genetic materials, etc in the system as well as in its surrounding environment, which is essential to regenerate the damaged tissue with its proper extracellular matrix [99
101,107]. Nowadays chitosan-based artificial systems are gaining rapid attraction for use in tissue engineering applications for different vital systems such as skin, bone, liver, nerve, and cartilage. Advantages of various chitosan-based nanosystems in tissue engineering applications are shown in Table 17.3.

17.6.1 Bone Fracture management remains a challenging task to the medical practitioners, particularly, if there occurs a significant loss of bone tissue. It may affect the quality of life of patients by limiting the ability to

Handbook of Chitin and Chitosan

17.6 Tissue engineering applications of chitosan-based nanosytems

TABLE 17.3 applications. Sl. No.

575

Advantages of various chitosan-based systems in tissue engineering

Tissue

Benefits achieved with the use of chitosan-based systems

1

Bone

• Improved osteogenic differentiation and osteocalcein expression • Increased porosity with improved flow properties of essential materials • Improved ostoinductive properties and osteoblastic differentiation • Improved bone matrix formation, cell adhesion, attachment, and migration • Overall improvement in bone tissue regeneration

2

Liver

• Increased hepatocytes infiltration, attachment, growth, and proliferation • Facilitated growth of hepatocytes with metabolic activities • Improved mechanical strength, stability, and biocompatibility • Increased porosity with improved flow properties of essential materials

3

Skin

• Reduction in pain sensation at wound site • Activation of platelets, induce blood coagulation • Increased secretion of growth factors, blood vessel formation, accelerated fibroblast proliferation • Fasten collagen deposition and hastens wound healing • Improved reepitheliazation and neovascularization

4

Nerve

• Higher affinity toward the system with enhanced growth • Supported the attachment, growth, proliferation, and migration of Schwann cells • Increased bridging of nerve defects, myelin sheath thickness, and action potential

perform routine activities and basic tasks such as standing, running, and walking [99]. Even though bone tissue has a remarkable capacity to regenerate, it may fail in some cases, due to significant loss of bone tissue or due to unfavorable conditions created by deformalities, severe trauma, tumors, etc. In these situations, bone tissue substitutes have significant therapeutic potential. The technique uses the implantation of artificial substitutes loaded with either primary osteoblasts or adult stem cells with osteogenic capacity to regenerate the bone tissue in the defective area. The artificial substitutes are constructed by using polymers with the desired porosity, mechanical stability, and nontoxicity, and the success of the functional ability of the developed system is determined by the polymers used [99,100,107]. Among various polymers, chitosan has got wide acceptability due to its various advantages. Its degree of deacetylation can affect the cell adhesiveness and low cell adhesion is observed with less deacetylated chitosan. It is noted that the cell adhesion and growth can be controlled by controlling the degree of deacetylation [107,108]. Similarly the molecular weight of chitosan can

Handbook of Chitin and Chitosan

576

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

be varied by varying the degree of deacetylation. Thus the higher the molecular weight, the greater the viscosity it provides and it can control the flow of materials, enabling it to control the cell interactions and tissue regeneration [100,108]. Porosity provided by chitosan is considered to be proper for cell growth, adhesion, proliferation, migration, and transport of essential elements within the system. Chitosan is reported to have good water absorption capacity, removes fluids from body parts, and helps the proper distribution of growth factors and regulators in between the cells and extracellular matrix. Even though chitosan when used alone is reported as brittle, mechanical strength can be improved by cross-linking it with agents like formaldehyde and epoxides. The presence of rich functionalities in chitosan allows easy crosslinking and modification to achieve the desired level of mechanical strength [100,108]. Its greater swelling ability at acidic pH also seems to be useful, as greater swelling provides sufficient mobility to cells and regulators, so as to perform their role in regenerating the damaged tissue. Also it can be combined with calcium phosphate to enhance its similarity with bone tissue components and increases its affinity for bone cells [97]. Christan et al. developed chitosan scaffolds loaded with bone marrow stem cells and surface-modified with collagen type I for bone tissue regeneration application. The system provided a favorable environment for the proliferation of cells and showed osteogenic differentiation with confirmed activity of alkaline phosphatase and osteocalcin expression [109]. Chitosan scaffolds doped with TiO2 synthesized by the freezedrying method exhibited greater porosity and low density with proper mechanical strength. Improved proliferation of fibroblast is demonstrated and seems to be promising for tissue engineering application [110]. It is the versatility of chitosan that helps to construct such a scaffold in combination with TiO2. Chitosan-based bioactive glass is prepared by the lyophilization technique for bone tissue regeneration applications. The system implanted under muscles and condyles of femoral vein of rats showed the presence of osteogenic cells along with osteoinductive property after 28 days. It indicated the usability of the system for bone tissue regenerative applications [111]. Zinc-doped hydrogel using chitosan in combination with hydroxyapatite (HA) and β-glycerophosphate has been prepared by the sol
gel method. The system is found to be biocompatible and has enhanced osteoblastic activity with accelerated bone formation, when tested using an in vivo rat model of bone defect [112]. A hybrid of chitosan hydrogel and polycaprolactone scaffolds loaded with rabbit bone marrow mesenchymal stem cells and bone morphogenic protein has been prepared. The system exhibited greater cell proliferation with osteogenesis and bone matrix formation after 14 days when tested using a nude mice model. It

Handbook of Chitin and Chitosan

17.6 Tissue engineering applications of chitosan-based nanosytems

577

suggested the feasibility to deliver the cells along with growth factors for bone tissue regenerative applications using a single system [113].

17.6.2 Liver Liver transplantation is the only treatment option available for patients having end-stage hepatic failure. The major problem here is the need for an expensive surgical procedure and the shortage of availability of an allograft donor with an exactly matching liver tissue. The insufficient availability of a donor greatly increases the need for tissue engineering techniques to develop artificial systems like bioactive liver to replace the failing tissue. The technique uses the cultured hepatocytes anchored in a suitable matrix support loaded with suitable signal molecules, growth factors, etc. to regenerate the damaged liver tissue. The matrix system has got considerable importance as it determines the viability and differentiation of loaded cells. Chitosan is found to be exceptionally suitable for tissue engineering application of liver because of its structural similarity with glucosamino glycan, the major component of the extracellular matrix of liver [114]. It is reported that chitosan scaffold has enough porosity for the cells to attach while allowing the movement of nutrients and gases properly for the cells to grow [114]. Forrest et al. revealed that chitosan in combination with alginate can act as a suitable matrix support for hepatocytes to attach and grow with improved viability [115]. A matrix system of chitosan along with collagen was prepared by the EDC cross-linking technique and exhibited good mechanical strength, stability, and biocompatibility suggestive of usability in liver tissue engineering application [116]. Chitosan scaffolds modified with heparin galactosylated hyaluronic acid (GHA) have been synthesized for liver tissue engineering applications. Here the use of GHA helps in increasing the porosity of the scaffold and increases the affinity of hepatocytes toward the scaffold. Heparin addition prolongs the activities of growth factors and provides suitable cultural environment for the cells to grow. The system induces the formation of cellular aggregates with metabolic activities specific to liver and indicates the functional use of the system in case of liver failures [117]. Chiitosan gelatine hybrid scaffold with 3D architecture and intrinsic flow channels mimicking liver microstructures has been prepared with sufficient porosity. Well-structured scaffold is found to facilitate the growth of hepatocytes with functional metabolic activities such as albumin and urea synthesis suggesting the possibility of use in liver diseases [118]. In another study, nanofibers of chitosan in combination with polycaprolactone (PCL) were synthesized by an electrospinning technique. Here the use of chitosan in combination with

Handbook of Chitin and Chitosan

578

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

PCL improves the mechanical property of chitosan, the spinnability of PCL, and provides sufficient porosity to allow the movement of nutrients, growth factors, gases, and regulators throughout the system. Also it provides a stable natural microenvironment within the system to allow the hepatocytes to filter, attach, grow, and proliferate as demonstrated using mouse liver cells [119]. Agarose chitosan composite scaffold is prepared for a tissue engineering application. The system exhibited optimum porosity, with enough void spaces with interconnected macropores mimicking biological liver architecture for the hepatic cells to grow. The improved growth of hepatocytes with metabolic activities like urea and albumin synthesis is observed [120].

17.6.3 Skin Correction of skin defects is considered to be difficult due to the complicated biological and physiological nature of the skin. Skin is the largest organ with three layers of the epidermis, dermis, and a deep layer of subcutaneous tissue. Skin is also equipped with hair follicles, sweat glands, sebaceous glands, and immunological cells. The immediate use of wound-dressing material is a cornerstone in the management of skin defects [97,121]. Superficial wounds will heal slowly with time, while some wounds will heal by reepithelization upon proper dressing. But deep wounds across the entire skin thickness fail to reepithelize and heal; that necessitates the need for skin grafts. Auto, allo, and xeno grafts are available, but are limited by the reasons explained previously. So, artificial skin graft plays an important role in such cases. Chitosanbased systems seem to be promising for the development of skin substitutes due to their biocompatibility and biodegradability. Besides this, the excellent wound-healing capacity, hemostatic activity, and tissue regenerating ability of chitosan make it a valuable choice for the development of such systems [121]. In addition it can be grafted with other polymers like PVA, PEG, etc. to modify its mechanical strength, porosity, and swelling behavior in order to construct a system with desired properties [97,121]. Reports indicate that chitosan can provide beneficial effects in multiple phases of the wound-healing process. Chitosan is a natural agent which can block the sensitive nerve endings and thus is reported to be capable of reducing pain sensations at wound sites. Further it can activate platelets, induce blood coagulation, and limit bleeding at wound areas. The next phase of wound healing is the inflammatory phase characterized by the involvement of mediators such as cytokines, chemokines, and growth factors along with inflammatory and immune cells. Chitosan is reported to regulate and create a suitable microenvironment for these cells to act so that the effective

Handbook of Chitin and Chitosan

17.6 Tissue engineering applications of chitosan-based nanosytems

579

healing process is possible. It is reported that chitosan can promote the infiltration of polymorphs, nuclear cells, neutrophils, etc. to the wound site and facilitate the release of mediators like prostaglandins and interleukins from these cells, a step involved in granulation tissue formation in the rapid wound-healing process. This process can further be aided by the inclusion of basic skin fibroblast growth factors along with chitosan. The next step of wound healing is the proliferative phase characterized by new blood vessel formation, reepithelization, and extracellular matrix construction. Chitosan is reported to induce the secretion of growth factors from platelets, promote growth factors involved in blood vessel formation, accelerate fibroblast proliferation, and fasten the collagen deposition, thus hastening the proliferative phase for effective wound healing [121
127]. Biocompatible chitosan sponges have been prepared for a woundhealing application which exhibited properties ideal for cell adhesion, proliferation, and migration with effective wound-healing activity when tested in vivo [122]. In another study PLGA mesh is loaded into scaffold made up of chitosan in combination with collagen and it is reported that the system improves the infiltration of cells with effective neovascularization and growth of blood vessels on subcutaneous implantation in rats. Here, the use of a combination of polymers provides sufficient mechanical strength to the system for its intended application of wound healing [123]. Chitosan scaffold constructed by combining its nanofibers made of electrospinning technique is found to facilitate the attachment, growth, and proliferation of keratinocytes, endothelial cells, and fibroblasts. The system exhibited good in vivo wound-healing activity with granulation tissue formation, reepithelization, and neovascularization indicating its effectiveness in the wound-healing process of skin defects [124]. Chitosan thin film loaded with silver nanoparticles has been prepared for wound-healing application in infectious wounds. The natural wound-healing and antimicrobial activity of chitosan along with the antimicrobial activity provided by loaded silver nanoparticles make the system efficient in the treatment of infectious wounds. The system exhibited good wound-healing activity as revealed by the development of well-organized layers of epidermis and dermis, neovascularization, and extracellular matrix formation after 12 days treatment when tested on surgical wound created in BALB/C mice [125]. Wounds due to third degree burns are difficult to heal as there is damage to underlying structures like muscle, nerves, bones, etc. Chitosan collagen scaffolds using lauric acid as plasticizer have been synthesized for application to burn wounds. The system is found to exhibit standard porosity, biodegradability, tensile strength, and mechanical properties suitable for application in wounds due to deep burns [126].

Handbook of Chitin and Chitosan

580

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

17.6.4 Nerve Injuries that happen to nerve tissue are very complicated as the mature neurons are unable to undergo cell division or replication. Therefore once the nerves get damaged, that may result in the abnormal functioning of a body part. Research is now concentrating on developing artificial endonervous tubes in order to regenerate the nerves. A lot of materials have been used for the development of such tubes. These tubes should have a sufficient internal surface area for the nerve and Schwann cells to cohere and regenerate. They should have optimum mechanical, cellular, and structural properties so as to avoid collapsing under conditions like muscle contractions, body weight changes, etc. [128
134]. Chitosan has been extensively studied for nerve tissue engineering applications due to its biocompatibility, biodegradability, antibacterial and antitumor activities. Moreover, the cationic nature of chitosan facilitates the attachment of negatively charged cells including nerve cells toward it. It is further reported that nerve tissue can grow well on chitosan support and chitosan has an inherent ability to repair the damaged nerves of the peripheral nervous system. In addition chitosan is reported to support the attachment, growth, proliferation, and migration of Schwann cells. Nerve conduits of chitosan are reported to be effective in bridging the peripheral nerves to correct major nerve defects. It is possible to modify the swelling behavior of chitosan by combining with HA and improved affinity to nerve cells is reported in combination with gelatin. Its amino group is reported to support the extension of neurons and facilitate their attachment and growth [128,129]. In a study, a chitosan conduit for nerve tissue regeneration application has been prepared by a phase separation technique. By optimizing the concentration of chitosan, it is possible to develop a biodegradable system with the desired level of mechanical strength and swelling behavior. It is also observed that neuroblastoma cells of mice exhibited higher affinity toward the system with enhanced growth [130]. In another study chitosan nerve conduits have been prepared by a lyophilization technique and then the tubes were seeded with mononuclear cells derived from bone marrow. In vivo efficacy of the system is evaluated in nerve defects created in goat and the results demonstrated the bridging of nerve defects by myelinated neurons having the appropriate diameter with optimum density, myelin sheath thickness, and action potential. The system is seemed to be promising for use in correcting large peripheral nerve defects [131]. Chitosan in combination with synthetic polymers like polyhydroxy butyrate is used to prepare nanofibers by electrospinning techniques. A system with desired length, hydrophillicity, thickness, mechanical, and textile strength suitable for nerve

Handbook of Chitin and Chitosan

17.7 Conclusion

581

tissue engineering applications has been constructed by optimizing the ratio of polymers [132]. Chitosan in combination with polylactic coglycolic acid (PLGA) and HA has been used to construct nerve conduits by a solvent casting
particulate leaching technique for tissue engineering application and exhibited the desired level of porosity and tensile strength in vitro. In vivo tests using a sciatic nerve defect model created in Sprague
Dawley rats revealed the deposition of nerve cells at the application site with desired density and cellular functions sufficient to correct the created nerve defect [133]. A hydrogel using a water-soluble derivative of chitosan, carboxymethyl chitosan in association with amino ethyl methacrylate has been prepared by photocross-linking technique. The system exhibited optimum rheological properties which are reported to support neuronal cell growth and promote the extension of dorsal root ganglionic cells and cortical neurons when tested in vitro, thus indicating its suitability for in vivo tissue engineering applications in nerve defects [134].

17.7 Conclusion Chitosan, a polymer from the chitin family, has diverse pharmaceutical and biomedical applications and recent pharmaceutical research has focused on the use of chitosan-based systems for drug delivery applications for various diseases. The availability of rich functionalities, biodegradation, mucoadhesiveness, pH-dependent swelling, and drug release properties of chitosan make it an ideal candidate for drug delivery applications. The presence of amino and hydroxyl groups make chitosan suitable for functionalization with conjugating agents like folic acid, antibodies, radioactive isotopes, and quantum dots. Its cationic nature helps it to target negatively charged cell surface receptors expressed at tumor sites and tumor stem cells, making it suitable for anticancer drug delivery. Positively charged chitosan can interact with negatively charged microbial cells, leading to inhibition of their growth and hence antimicrobial activity. Chitosan is reported to possess antiinflammatory properties and promote tissue granulation. The antiinflammatory and proinflammatory activity of chitosan depends upon the specific characteristics, such as degree of acetylation, molecular weight, size, polydispersity index, purity, and dose. Tissue engineering is an important therapeutic strategy for present and future medicine. The porous structure, gel-forming properties, ease of chemical modification, biocompatibility, biodegradability, antibacterial, and wound-healing activity of chitosan make it a promising supporting material for tissue engineering applications for different conditions of skin, bone, nerve, and liver.

Handbook of Chitin and Chitosan

582

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

References [1] Z. Antosova, M. Mackova, V. Kral, T. Macek, Therapeutic application of peptides andproteins: parenteral forever? Trends Biotechnol. 27 (2009) 628
635. [2] A. Grenha, D. Carrio´n-Recio, D. Teijeiro-Osorio, B. Seijo, C. Remun˜a´n-Lo´pez, Nanoandmicro-particulate carriers for pulmonary drug delivery, in: M.N.V. Ravi Kumar (Ed.), Handbook of Particulate Drug Delivery, 2, American Scientific Publishers, Valencia, CA, USA, 2008, pp. 165
192. [3] J.A. DiMasi, R.W. Hansen, H.G. Grabowski, The price of innovation: new estimates of drug development costs, J. Health Econ. 22 (2003) 151
185. [4] R. Langer, Drug delivery and targeting, Nature 392 (1998) 5
10. [5] B.S. Andreas, D. Sarah, Chitosan-based drug delivery systems, Eur. J. Pharm. Biopharm. 81 (2012) 463
469. [6] K. Eugene, Y.L. Lee, Implantable applications of chitin and chitosan, Biomaterials 24 (2003). 2339
234. [7] H. Ehrlich, E. Steck, Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part II: biomimetic potential and applications, Int. J. Biol. Macromol. 47 (2010) 141
145. [8] Y. Islem, R. Marguerite, Chitin and chitosan preparation from marine sources: structure, properties and applications, Mar. Drugs 13 (2015) 1133
1174. [9] W. Arbia, L. Arbia, L. Adour, A. Amrane, Chitin extraction from crustacean shells using biological methods—a review, Food Technol. Biotechnol. 51 (2013) 12
25. [10] P. Sneha, J. Aiswarya, S.S. Changam, M.C. Sanjay, Extraction and purification of chitosan from chitin isolated from sea prawn (fennerpenaeusoindicus), Asian J. Pharm. Clin. Res. 7 (4) (2014) 201
204. [11] E.L. Johnson, Q.P. Peniston, Utilization of shellfish waste for chitin and chitosan production, Chemistry & Biochemistry of Marine Food Products, 19, AVI Publishing Co, Westport, CT, 1982. [12] F. Shahidi, J. Synowiecki, Isolation and characterization of nutrients and value-added products from snow crab (Chifroecelesopilio) and shrimp (Panda- 111sb orealis) processing discards, J. Agri. Food Chem. 39 (1991) 1527
1532. [13] M.B. Rao, A.M. Tanksale, M.S. Ghatge, V.V. Deshpande, Molecular and biotechnological aspects of microbial proteases, Microbiol. Mol. Biol. Rev. 62 (1998) 597
635. [14] K. Kurita, T. Sannan, Y. Iwakura, Studies on chitin, 4: evidence for formation of block and random copolymers of N-acetyl-D-glucosamine and D-glucosamine by heteroand homogeneous hydrolyses, Macromol. Chem. 178 (1977) 3197
3202. [15] S. Chinmayee, G. Pankaj, K.M. Tarun, Chitosan: a promising biopolymer in drug delivery applications, J. Mol. Genet. Med. 006 (2015) 1
10. S4. [16] O. Felt, P. Buri, R. Gurny, Chitosan: a unique polysaccharide for drug delivery, Drug. Dev. Ind. Pharm. 24 (1998) 979
993. [17] A. Anitha, S. Sowmya, P.T. Sudheesh, Chitin and chitosan in selected biomedical applications, Prog. Polym. Sci. 39 (2014) 1644
1667. [18] G. Ana, Chitosan nanoparticles: a survey of preparation methods, J. Drug. Target. 20 (4) (2012) 291
300. [19] P. Giunchedi, I. Genta, B. Conti, R.A.A. Muzzarelli, Spray dried microspheres based on methylpyrrolidione chitosan: in vitro and ex vivo studies, Biomaterials 19 (1998) 157
161. [20] J. Berger, M. Reist, J.M. Mayer, O. Felt, R. Gurny, Structure and interactions in covalently and ionicallycross-linked chitosan hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. 57 (2004) 35
52. [21] K.S. Sudheesh, K.M. Ajay, A.A. Omotayo, B.M. Bhekie, Chitosan-based nanomaterials: a state-of-the-art review, Int. J. Biol. Macromol. 59 (2013) 46
58.

Handbook of Chitin and Chitosan

References

583

[22] M. Maria, M. Iwona, B. Jacek, T. Michal, Chitosan scaffolds, films and microgranules for medical application-preparation and drug release studies, Polimery 57 (2012) 714
721. [23] N. Nitar, F. Tetsuya, T. Hiroshi, The mechanical and biological properties of chitosan scaffolds for tissue regeneration templates are significantly enhanced by chitosan from Gongronellabutleri, Materials 2 (2009) 374
398. [24] S. Bhuvaneshwari, D. Sruthi, S. Velmurugan, N. Kalyani, J. Sugunabai, Development and characterisation of chitosan film, Int. J. Eng. Res. Appl. 1 (2011) 292
299. [25] B. Barbara, D.A. Ilenia, The good, the bad and the ugly of chitosans, Mar. Drugs 14 (2016) 99. [26] M.H. Fathi, S. Majidi, P.S. Zangabad, J. Barar, H.E. Niya, Y. Omidi, Chitosan-based multifunctional nanomedicines and theranostics for targeted therapy of cancer, Med. Res. Rev. 38 (2018) 2110
2136. [27] A. Jamal, L. Shahzadi, S. Ahtzaz, S. Zahid, A.A. Chaudhry, I.U. Rehman, et al., Identification of anti-cancer potential of doxazocin: loading into chitosan based biodegradable hydrogels for on-site delivery to treat cervical cancer, Mater. Sci. Eng. C: Mater. Biol. Appl. 82 (2018) 102
109. [28] L. Sun, Y. Chen, Y. Zhou, D.D. Guo, Y. Fan, F. Guo, et al., Preparation of 5-fluorouracil-loaded chitosan nanoparticles and study of the sustained release in vitro and in vivo, Asian J. Pharm. Sci. 12 (2017) 418
423. [29] A.K. Verma, A. Leekha, V. Kumar, I. Moin, S. Kumar, Biodistribution and in-vivo efficacy of doxorubicin loaded chitosan nanoparticles in ehrlich ascites carcinoma (EAC) bearing balb/c mice, J. Nanomed. Nanotechnol. 9 (2018) 1
6. [30] L. Cheng, M.A. Hui, M. Shao, Q. Fan, L.V. Huiyi, J. Peng, et al., Synthesis of folatechitosan nanoparticles loaded with ligustrazine to target folate receptor positive cancer cells, Mol. Med. Rep. 16 (2017) 1101
1108. [31] H. Jin, J. Pi, F. Yang, J. Jiang, X. Wang, H. Bai, et al., Folate chitosan nanoparticles loaded with ursolic acid confer anti-breast cancer activities in vitro and in vivo, Sci. Rep. 6 (2016) 1
6. [32] F. Li, J. Li, X. Wen, S. Zhou, X. Tong, P. Su, et al., Anti-tumor activity of paclitaxelloaded chitosan nanoparticles: an in vitro study, Mater. Sci. Eng. 29 (2009) 2392
2397. [33] A.K. Mehata, S. Bhartic, P. Singh, M.K. Viswanadha, L. Kumari, P. Agrawald, et al., Trastuzumab decorated TPGS-g-chitosan nanoparticles for targeted breastcancer therapy, Colloids Surf. B, Biointerfaces 173 (2019) 366
377. [34] D. Cosco, F. Cilurzo, J. Maiuolo, C. Federico, M. Teresa, D. Martino, et al., Delivery of miR-34a by chitosan/ PLGA nanoplexes for the anticancer treatment of multiple myeloma, Sci. Rep. 5 (2015) 1
5. [35] F. Yu, C. He, A.Y. Waddad, L.L. Munyendo, L.V. Huixia, J. Zhou, N-octyl-Narginine-chitosan (OACS) micelles for gambogic acid oral delivery: preparation, characterization and its study on in situ intestinal perfusion, Drug. Dev. Ind. Pharm. 40 (2014) 774
782. [36] P. Rajitha, Divya Gopinath, Raja Biswas, M. Sabitha, R. Jayakumar, Chitosan nanoparticles in drug therapy of infectious and inflammatory diseases, Expert Opin. Drug. Deliv. 13 (2016) 1177
1194. [37] M. Fathi, P.S. Zangabad, S. Majidi, J. Barar, H.E. Niya, Y. Omidi, Stimuli-responsive chitosan-based nanocarriers for cancer therapy, Bioimpacts 7 (2017) 269
277. [38] F. Song, X.J. Li, X.L. Cheng, A.R. Fu, Y.H. Wang, Y.J. Feng, et al., Paclitaxel-loaded trimethyl chitosan-based polymeric nanoparticle for the effective treatment of gastro enteric tumors, Oncol. Rep. 32 (2014) 1481
1488. [39] M. Ekinci, D.I. Ozdemir, E. Gundogdu, M. Asikoglu, Methotrexate loaded chitosan nanoparticles: preparation, radiolabeling and in vitro evaluation for breast cancer diagnosis, J. Drug. Deliv. Sci. Technol. 30 (2015) 107
113.

Handbook of Chitin and Chitosan

584

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

[40] A.G. Perez-Ruiz, A. Ganem, I.M. Corichi, JoseRuben, G.S. Anchez, Lecithin
chitosan
TPGS nanoparticles as nanocarriers of epicatechin enhanced its anticancer activity in breast cancer cells, RSC Adv. 8 (2018) 1
19. [41] H.P. Chen, M.H. Chen, F.I. Tung, T.Y. Liu, A novel micelle-forming material used for preparing a theranostic vehicle exhibiting enhanced in vivo therapeutic efficacy, J. Med. Chem. 58 (2015) 3704
3719. [42] Monarul, Antibacterial activity of crab-chitosan against Staphylococcus aureus and Escherichia coli, J. Adv. Sci. Res. 2 (2011) 63
66. [43] S.F. Anthony, Infectious disease: considerations for the 21st century, Clin. Infect. Dis. 32 (2001) 675
685. [44] L. Zhang, D. Pornpattananangkul, M. Huang, Development of nanoparticles for antimicrobial drug delivery, Curr. Med. Chem. 17 (2010) 585
594. [45] H.K. Chia, Y.H. Chien, Y.H. Tin, Assessment of chitosan-affected metabolic response by peroxisome proliferator-activated receptor bioluminescent imaging-guided transcriptomic analysis, PLoS ONE 4 (2012) 4
15. [46] C.S. Chen, W.Y. Liau, G.J. Tsai, Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation, J. Food Prod. 61 (1998) 1124
1128. [47] A.M. Papineau, D.G. Hoover, D. Knorr, D.F. Farkas, Antimicrobial effect of water soluble chitosan’s with high hydrostatic pressure, Food Biotechnol. 5 (1991) 45
57. [48] F. Shahidi, J. Arachchi, Y.J. Jeon, Food application of chitin and chitosan, Trends Food Sci. Technol. 10 (1999) 37
51. [49] N.R. Sudarshan, D.G. Hoover, D. Knorr, Antibacterial action of chitosan, Food Biotechnol. 6 (1992) 257
272. [50] V. Coma, A. Martial-Gros, S. Garreau, A. Copinet, F. Salin, A. Deschamps, Edible antimicrobial films based on chitosan matrix, J. Food Sci. 67 (2002) 1162
1169. [51] D. Raafat, V. Bargen, K.A. Haas, H.G. Sahl, Insights into the mode of action of chitosan as an antibacterial compound, Appl. Environ. Microbiol. 74 (2008) 3764
3773. [52] G.J. Tsai, W.H. Su, Antibacterial activity of shrimp chitosan against Escherichia coli, J. Food Prod. 62 (1999) 239
243. [53] F. Devlieghere, A. Vermeulen, J. Debevere, Chitosan: antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables, Food Microbiol. 21 (2004) 703
714. [54] S.W. Fang, C.F. Li, D.Y.C. Shih, Natural antimicrobial compounds to preserve quality and assure safety of fresh horticultural produce, J. Food Prod. 57 (1994) 136
140. [55] I. Sebti, A. Martial-Gros, A. Carnet-Pantiez, S. Grelier, V. Coma, Chitosan polymer as bioactive coating and film against Aspergillusniger contamination, J. Food Sci. 70 (2005) 100
104. [56] R.G. Cuero, G. Osuji, A. Washington, N-Carboxy methyl chitosan inhibition of aflatoxin production: role of zinc, Biotechnol. Lett. 13 (1991) 441
444. [57] S. Roller, N. Covill, The antifungal properties of chitosan in laboratory media and apple juice, Int. J. Food Microbiol. 47 (1999) 67
77. [58] D. Raafat, H.G. Sahl, Chitosan and its antimicrobial potential: a critical literature survey, Microb. Biotechnol. 2 (2009) 186
201. [59] P. Sony, N.P. Seema, In vitro antibacterial potential of chitosan and its derivatives on pathogenic enterobacteriaceae, Natl J. Physiol. Pharm. Pharmacol. 5 (2) (2015) 119
124. [60] B.K. Park, M.M. Kim, Applications of chitin and its derivatives in biological medicine, Int. J. Mol. Sci. 11 (2010) 152
164. [61] Y.C. Chung, J.Y. Yeh, C.F. Tsa, Antibacterial characteristics and activity of watersoluble chitosan derivatives prepared by the maillard reaction, Molecules 16 (2011) 8504
8514.

Handbook of Chitin and Chitosan

References

585

[62] P. Dissaya, L. Zhang, S. Olson, Bacterial toxin-triggered drug release from gold nanoparticle-stabilized liposomes for the treatment of bacterial infection, J. Am. Chem. Soc. 1339 (2011) 4132
4139. [63] G. Alamdarnejad, A. Sharif, S. Taranejoo, Synthesis and characterization of thiolated carboxymethyl chitosan-graft-cyclodextrin nanoparticles as a drug delivery vehicle for albendazole, J. Mater. Sci. Mater. Med. 24 (2013) 1939
1949. [64] J. Bushra, H. Huma, A. Shahid, Cefazolin loaded chitosan nanoparticles to cure multi drug resistant Gram-negative pathogens, Carbohydr. Polym. 78 (2015) 1
33. [65] G. Tarun, R. Goutam, K.G. Amit, Inhalable chitosan nanoparticles as anti-tubercular drug carriers for an effective treatment of tuberculosis, Artif. Cell Nanomed. Biotechnol. 14 (2015) 1
5. [66] K.P. Bhavin, H.P. Rajesh, S.A. Pooja, Development of oral sustained release rifampicin loaded chitosan nanoparticles by design of experiment, J. Drug. Deliv. Sci. Technol. 1 (2013) 1
10. [67] A.D. Sezer, E. Cevher, Topical drug delivery using chitosan nanoand microparticles, Expert. Opin. Drug. Deliv. 9 (9) (2012) 1129
1146. [68] S.Y. Lu, W.J. Gao, H.Y. Gu, Construction, application and biosafety of silver nanocrystalline chitosan wound dressing, Burns 34 (2008) 623
628. [69] S. Govender, D. Lutchman, V. Pillay, Enhancing drug incorporation into tetracyclineloaded chitosan microspheres for periodontal therapy, J. Microencapsul. 23 (2006) 750
761. [70] E.C. Shen, C. Wang, E. Fu, Tetracycline release from tripolyphosphate-chitosan cross-linked sponge: a preliminary in vitro study, J. Periodontal. Res. 43 (2008) 642
648. [71] A.E. Ghaouth, J. Arul, J. Grenier, A. Asselin, Antifungal activity of chitosan on two postharvest pathogens of strawberry fruits, Phytopathology 82 (1992) 398
402. [72] M. Eweis, S.S. Elkholy, M.Z. Elsabee, Antifungal efficacy of chitosan and its thiourea derivatives upon the growth of some sugar- beet pathogens, Int. J. Biol. Macromol. 38 (2006) 1
8. [73] K. Roy, H.Q. Mao, S.K. Huang, K.W. Leong, Oral gene delivery with chitosan- DNA nano particles generates immulogic protection in a murine model of peanut allergy, Nat. Med. 5 (1999) 387
391. [74] V. Saharan, G. Sharma, M. Yadav, M.K. Choudhary, Synthesis and in vitro antifungal efficacy of Cu- chitosan nanoparticles against pathogenic fungi of tomato, Int. J. Biol. Macromol. 75 (2015) 346
353. [75] D. Drayton, S. Liao, R. Mounzer, N. Ruddle, Lymphoid organ development: from ontogeny to neogenesis, Nat. Immunol. 7 (2006) 344
353. [76] J. Hu, P. Van den Steen, Q. Sang, G. Opdenakker, Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases, Nat. Rev. Drug. Discov. 6 (2007) 480
498. [77] E. Jimi, V. Ghosh, Role of nuclear factor-kB in the immune system and bone, Immunol. Rev. 208 (2005) 80
87. [78] F. Epstein, P. Barnes, M. Karin, Nuclear factor-kB--a pivotal transcription factor in chronic inflammatory diseases, N. Engl. J. Med. 336 (1997) 1066
1071. [79] S. Albert, J. Baldwin, Perspective series-NF-kB in defence and disease-The transcription factor NF-kB and human disease, J. Clin. Invest. 107 (2001) 3
6. [80] M. Macarthur, G. Hold, E. El-Omar, Inflammation and cancer II. Role of chronic inflammation and cytokine gene polymorphisms in the pathogenesis of gastrointestinal malignancy, Am. J. Physiol.-Gastrointestest. Liver Physiol. 286 (2004) 515
520. [81] S. Jessy, L. Maria, Nanocarriers for targeting in inflammation, Asian J. Pharm. Clin. Res. 6 (2013) 3
12.

Handbook of Chitin and Chitosan

586

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

[82] C.L. Bueter, C.K. Lee, J.P. Wang, G.R. Ostroff, C.A. Specht, S.M. Levitz, Spectrum and mechanisms of inflammasome activation by chitosan, J. Immunol. 192 (12) (2014) 5943
5951. [83] C. Da Silva, C. Chalouni, A. Williams, D. Hartl, C. Lee, J. Elias, Chitin is a sizedependent regulator of macrophage TNF and IL-10 productio, J. Immunol. 182 (2009) 3573
3582. [84] D. Fong, P.G. Gelinas, A.P. Cheng, Lysosomal rupture induced by structurally distinct chitosans either promotes a type 1 IFN response or activates the inflammasome in macrophages, Biomaterials 129 (2017) 127
138. [85] D. Ngo, S. Lee, M. Kim, S. Kim, Production of chitin oligosaccharides with different molecular weights and their antioxidant effect in RAW 264.7 cells, J. Funct. Foods 1 (2009) 188
198. [86] M. Kim, H. You, N. Kim, B. Shim, H. Kim, Inhibitory effect of water soluble chitosan on TNF α and IL 8 secretion from HMC 1, Cells 26 (2004) 401
409. [87] M. Kim, S. Kim, Chitooligosaccharides inhibit activation and expression of matrix metalloproteinase-2 in human dermal fibroblasts, FEBS Lett. 580 (2006) 2661
2666. [88] S. Tanodekaew, M. Prasitsilp, S. Swasdison, B. Thavornyutikarn, T. Pothsree, et al., Preparation of acrylic grafted chitin for wound dressing application, Biomaterials 25 (2004) 1453
1460. [89] M.J. Chung, J.K. Park, Y.I. Park, Anti-inflammatory effects of low molecular weight chitosan oilgosachhrides in igE antigen complex srtimulated RBL-2H3 cells and asthma model in mice, Int. Immunopharmcol. 12 (2) (2012) 453
459. [90] A.S. Hirva, P.P. Rakesh, Statistical modeling of zaltoprofen loaded biopolymeric nanoparticles: characterization and anti-inflammatory activity of nanoparticles loaded gel, Int. J. Pharm. Invest. 5 (2015) 120
127. [91] K. Haliza, H. Zahid, C.L. Tay, Chitosan nanoparticles as a percutaneous drug delivery system for hydrocortisone, J. Nanomater. 1 (2012) 1
11. [92] V. Venugopal, J. Kumar, S. Muralidharan, Targeted delivery of silymarin to liver cells by galactosylated nanoparticles: in-vitro and in-vivo evaluation studies, Am. J. Publichealth Sci. 2 (2014) 1
8. [93] M.R. Daniela, D.M. Priscyla, Z.J. Giselle, Chitosan-solid lipid nanoparticles ascarriers for topical delivery of tretinoin, Colloids Surf. B: Biointerfaces 93 (2012) 36
40. [94] D. Mehmet, U. Nagihan, Y. Fatma, Ketorolac tromethamine loaded chitosan nanoparticles as a nanotherapeutic system for ocular diseases, J. Biol. Chem. 41 (2013) 81
86. [95] S. Punit, D. Pinaki, P. Apurva, S. Mandip, Skin permeating nanogel for the cutaneous co-delivery of two anti-inflammatory drugs, Biomaterials 33 (5) (2012) 1607
1617. [96] A. Amir, R.R. Mohammad, H. Mehrdad, Neuropharmacokinetic evaluation of methotrexate-loaded chitosan nanogels, Int. J. Biol. Macromol. 79 (2015) 326
335. [97] I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo, I.Y. Park, B.C. Kim, et al., Chitosan and its derivatives for tissue engineering applications, Biotechnol. Adv. 26 (1) (2008) 1
21. [98] S. Ahmed, A. Annu, J. Ali, et al., A review on chitosan centred scaffolds and their applications in tissue engineering, Int. J. Biol. Macromol. 116 (2018) 849
862. [99] G.M. Marcondes, F.S. No´brega1, L. Correˆa, V.E. Arana- Chavez, A.M.G. Plepis, V.C. A. Martins, et al., Investigation of biological interactions of chitosan-collagenhydroxyapatite composite with bone tissue in sheep, Arq. Bras. Med. Vet. Zootec. 68 (6) (2016) 1531
1538. [100] A.R. Costa-Pinto, R.L. Reis, N.M. Neves, Scaffolds based bone tissue engineering: the role of chitosan, Tissue Eng. Part B: Rev. 17 (5) (2011) 331
347. [101] J. Zahra, Chitosan: a brief review on structure and tissue engineering application, J. Appl. Tissue Eng. 1 (1) (2014) 3
7.

Handbook of Chitin and Chitosan

References

587

[102] D. Archana, L. Upadhyay, R. Tewari, J. Dutta, Chitosan-pectin-alginate as a novel scaffold for tissue engineering applications, Indian. J. Biotechnol. 12 (4) (2013) 475
482. [103] S. Pokhrel, P.N. Yadav, R. Adhikari, Applications of chitin and chitosan in industry and medical science: a review, Nepal. J. Sci. Technol. 16 (1) (2015) 99
104. [104] A.R.C. Duarte, J.F. Mano, R.L. Reis, Preparation of chitosan scaffolds loaded with dexamethasone for tissue engineering applications using supercritical fluid technology, Eur. Polym. J. 45 (1) (2009) 141
148. [105] M. Dash, S.K. Samal, T.E.L. Douglas, D. Schaubroeck, S.C. Leeuwenburgh, P. Van Der Voort, et al., Enzymatically biomineralized chitosan scaffolds for tissueengineering applications, J. Tissue Eng. Regene. Med. 11 (5) (2017) 1500
1513. [106] C.A. IBbekwe, G.M. Oyatogun, T.A. Esan, K.M. Oluwasegun, Synthesis and characterization of chitosan/gum arabic nanoparticles for bone regeneration, Am. J. Mater. Sci. Eng. 5 (1) (2017) 28
36. [107] F. Ding, H. Deng, Y. Du, X. Shi, Q. Wang, Emerging chitin and chitosan nanofibrous materials for biomedical applications, Nanoscale 6 (16) (2014) 9477
9493. [108] M. Rodrı´guez-Va´zquez, B. Vega-Ruiz, R. Ramos-Zu´n˜iga, D.A. Saldan˜a-Koppel, L.F. Quin˜ones-Olvera, Chitosan and its potential use as a scaffold fortissue engineering in regenerative medicine, BioMed. Res. Int. 2015 (821279) (2015) 1
15. [109] C. Heinemann, S. Heinemann, A. Lode, A. Bernhardt, H. Worch, T. Hanke, In vitro evaluation of textile chitosan scaffolds for tissue engineering using human bone marrow stromal cells, Biomacromolecules 10 (5) (2009) 1305
1310. [110] K. Pavan, Nano-TiO2 doped chitosan scaffold for the bone tissue engineering applications, Int. J. Biomater. 2018 (6576157) (2018) 1
10. [111] S. Jebahi, H. Oudadesse, F.Z. Faouzi, J. Elleuch, T. Rebai, H. Keskes, et al., Osteoinduction and antiosteoporotic performance of hybrid biomaterial chitosanbioactive glass graft: effects on bone remodeling, J. Mater. Sci. Eng. 2 (3) (2013) 1
8. [112] S. Dhivya, S. Saravanan, T.P. Sastry, N. Selvamurugan, Nanohydroxyapatite reinforced chitosancomposite hydrogel for bone tissue repair in vitro and in vivo, J. Nanobiotechnol. 13 (40) (2015) 2
13. [113] D. Liang, W. Shao-Jie, Z. Xin-Rong, Z. Yu-Fang, Y. Jia-Kuo, 3D- printed poly (ε-caprolactone) scaffold integrated with cell-laden chitosan hydrogels for bone tissue engineering, Sci. Rep. 7 (13412) (2017) 1
9. [114] E. Zakhem, K.N. Bitar, Development of chitosan scaffolds with enhanced mechanical properties for intestinal tissue engineering applications, J. Funct. Biomater. 6 (4) (2015) 999
1011. [115] F.M. Kievit, S.J. Florczyk, M.C. Leung, O. Veiseh, J.O. Park, M.L. Disis, et al., Chitosan-alginate 3D scaffolds as a mimic of the gliomatumor microenvironment, Biomaterials 31 (22) (2010) 5903
5910. [116] W. Li, Y. Long, Y. Li, K. Long, S. Liu, Z. Wang, et al., Fabrication and characterization of chitosan
collagen cross-linked membranes for corneal tissue engineering, J. Biomater. Sci. Polym. Ed. 25 (17) (2014) 1962
1972. [117] J. Fan, J. Yang, Preparation and characterization of a chitosan/galactosylated hyaluronic acid/heparin scaffold for hepatic tissue engineering, J. Biomater. Sci. Polym. Ed. 28 (6) (2017) 569
581. [118] H. Jiankang, L. Dichen, L. Yaxiong, Y. Bo, Z. Hanxiang, L. Qin, et al., Preparation of chitosan
gelatin hybrid scaffolds with well-organized microstructures for hepatic tissue engineering, Acta Biomater. 5 (1) (2009) 453
461. [119] D. Semnani, E. Naghashzargar, M. Hadjianfar, F.D. Manshadi, S. Mohammadi, S. Karbasi, et al., Evaluation of PCL/chitosan electrospunnanofibers for liver tissue engineering, Int. J. Polym. Mater. Polym. Biomater. 66 (3) (2017) 149
157.

Handbook of Chitin and Chitosan

588

17. Drug delivery and tissue engineering applications of chitosan-based biomaterial systems

[120] A. Tripathi, J. SavioMelo, Preparation of a sponge-like biocomposite agarose
chitosan scaffold with primary hepatocytes for establishing an vitro 3D liver tissue model, RSC Adv. 5 (39) (2015) 30701
30710. [121] Sumrita Bhat, Anuj Tripathi, Ashok Kumar, Supermacroprous chitosan
agarose
gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering, J. R. Soc. Interface 8 (57) (2011) 540
554. [122] A.R. Pandey, U.S. Singh, M. Momin, C. Bhavsar, Chitosan: application in tissue engineering and skin grafting, J. Polym. Res. 24 (8) (2017) 1
22. [123] S.P. Noel, H.S. Courtney, J.D. Bumgardner, W.O. Haggard, Chitosan sponges to locally deliver amikacin and vancomycin: a pilot in vitro evaluation, Clin. Orthop. Relat. Res. 468 (8) (2010) 2074
2080. [124] A.S. Avalshahr, S. Nokhasteh, A.M. Molavi, M.K. Ghayeni, M.M. Shahri, Synthesis and characterization of collagen/PLGA biodegradable skin scaffold fibers, Regen. Biomater. 4 (5) (2017) 309
314. [125] V.T. Tchemtchoua, G. Atanasova, A. Aqil, P. File´e, N. Garbacki, O. Vanhooteghem, et al., Development of a chitosan nanofibrillar scaffold for skin repair and regeneration, Biomacromolecules 12 (9) (2011) 3194
3204. [126] M.L. Ojeda-Martinez, I. Yan˜ez, C. Velasquez-Ordonez, M.M. Martinez-Palomar, Skin wound healing withchitosan thin films containing supported silver nanospheres, J. Bioact. Compat. Polym. 30 (6) (2015) 1
16. [127] P. Widiyanti, E.D. Setyadi, D.I. Rudyardj, Collagen-chitosan scaffold-lauric acid plasticizer for skin tissue engineering on burn cases, AIP Conf. Proc. 1817 (020013) (2017) 1
8. [128] H. Liu, C. Wang, C. Li, Y. Qin, Z. Wang, F. Yang, et al., A functional chitosan-based hydrogel as a wounddressing and drug delivery system in the treatmentof wound healing, RSC Adv. 8 (2018) 7533
7549. [129] J. Ali, A.K. Dolatabadi, S.E. Barough, A. Ai, N. Lotfibakhshaiesh, A. NorouziJavidan, et al., Polymeric scaffolds in neural tissue engineering: a review, Arch. Neurosci. 1 (1) (2013) 15
20. [130] A.O. Qiang, W. Aijun, C. Wenling, Z. Chang, G. Yandao, Z. Nanming, et al., Fabrication and characterization of chitosan nerve conduits with microtubular architectures, Tsinghua Sci. Technol. 10 (4) (2005) 435
438. [131] A. Muheremu, L. Chen, X. Wang, Y. Wei, K. Gong, Q. Ao, Chitosan nerve conduits seeded with autologous bone marrow mononuclear cells for 30 mm goat peroneal nerve defect, Sci. Rep. 7 (44002) (2017) 1
12. [132] A. Karima, S. Karbasi, S. Razavi, E.N. Zargar, Poly(hydroxybutyrate)/ chitosan aligned electrospun scaffold as a novelsubstrate for nerve tissue engineering, Adv. Biomed. Res. 27 (2018) 1
7. [133] X. Shen, J. Ruan, Z. Zhou, Z. Zeng, L. Xie, Evaluation of PLGA/chitosan/HA conduits fornerve tissue reconstruction, J. Wuhan. Univ. Technol.-Mater. Sci. Ed. 24 (4) (2009) 566
570. [134] C.M. Valmikinathan, V.J. Mukhatyar, R.V. Bellamkonda, Photo cross-linkable chitosan hydrogels for neural tissue engineering, Soft Matter 8 (2012) 1964
1976.

Handbook of Chitin and Chitosan

C H A P T E R

18 Future aspects of biomedical applications of chitin and chitosan in diseases associated with oxidative stress Makoto Anraku1,2, Shinsuke Ifuku3, Daisuke Iohara1,2, Fumitoshi Hirayama1,2, Masaki Otagiri1,2 and Janusz M. Gebicki4 1

Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan, DDS Research Institute, Sojo University, Kumamoto, Japan, 3Graduate School of Engineering, Tottori University, Tottori, Japan, 4Department of Biological Sciences, Macquarie University, Sydney, Australia 2

O U T L I N E 18.1 Introduction

590

18.2 Chitosan and its derivatives as antioxidants with protective effects for chronic renal failure

591

18.3 SDACNFs as a material for pharmaceutical formulation in diseases associated with oxidative stress

597

18.4 Therapeutic effects of PD/NFs-CDs gel in model mice

601

18.5 Conclusions

604

Abbreviations

605

References

605

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00018-2

589

© 2020 Elsevier Inc. All rights reserved.

590

18. Future aspects of biomedical applications of chitin and chitosan

18.1 Introduction Living organisms are constantly exposed to reactive oxygen species (ROS) formed as by-products of normal respiration, metabolism, and autoxidation of xenobiotics, or as the result of stress that accompanies a range of diseases [1,2]. Formation of a certain level of ROS is essential for the maintenance of cell homeostasis and living organisms achieve this by a system of antioxidant defenses, allowing them to maintain a balance between oxidative challenge and protection. Under conditions of oxidative stress, these defenses can be overwhelmed by excessive production of ROS, leading to the activation of specific signaling pathways and general damage, which can result in disease and/or death [3
5]. Healthy organisms have the ability to resist or repair most of this damage, but the strong correlation between oxidative stress and cancer, diabetes, atherosclerosis, arthritis, pulmonary disorders, chronic inflammation, and other diseases indicates that their defenses are not always equal to the challenge [6]. In general, the most potentially lethal types of ROS are free radicals, which can generate a chain of damage in cells and tissues [7]. An organism can protect itself by inhibiting this chain by a system of endogenous or exogenous antioxidant compounds that scavenge or repair the free radicals. Under normal conditions endogenous antioxidants, such as glutathione, vitamins C and E, and urate, are adequate, but under conditions of oxidative stress they are not sufficient, as shown by the development of various forms of damage. In addition, some antioxidant vitamins are unstable and can have prooxidant effects [8]. The option of enhancing their effectiveness by increasing the concentrations is not available, because their levels in vivo are tightly regulated and oral administration is not effective. It is therefore clear that under oxidative stress additional biocompatible antioxidants are required to maintain the body’s defenses at full capacity [9]. A wide range of chemical compounds are known and used as antioxidants, particularly in the food industry. They include butylated hydroxyanisole, butylated hydroxytoluene, and tertiary butylhydroxyquinone, which are authorized for use by several national authorities as food additives [10,11]. However, they are not acceptable to a growing range of consumers because they are synthetic compounds, raising concerns about the safety related to their absorption, accumulation in body organs and tissues, and possible metabolic effects [12]. There is therefore a growing view that many of these objections can be overcome by replacing the synthetic antioxidants with effective natural compounds for general use in the food and medicinal fields.

Handbook of Chitin and Chitosan

18.2 Chitosan and its derivatives as antioxidants

591

One such class of compounds are natural biopolymers, for example, chitin and chitosan, recycled resources derived from marine organisms such as crab or shrimp shell, which are finding application in many fields. Chitosan, the linear polymer of β-(1
4)-linked D-glucosamine units, has been recommended as a suitable functional material for providing protection from a variety of challenges because of its biocompatibility, biodegradability, nontoxicity, adsorption properties, and free radical scavenging activities [13
15]. In recent worldwide studies, chitosans were tested as a dietary supplement for possible inhibition of the absorption of certain lipids and bile acids. In a previous study, we showed that chitosan has a high antioxidant activity as well as antilipidemic effects in model rats with a metabolic syndrome [15]. However, although several studies have reported the antioxidant activities of chitosan in some diseases, including the metabolic syndrome, the relationships between their antioxidant activities and progression of diseases linked to oxidative stress, such as renal failure and ulcerous colitis, have not been extensively reported. In this chapter, we review the in vivo antioxidant and free radical scavenging activities of various chitosans and their derivatives established from animal models and clinical trials [16
20]. The results strongly suggest that chitosan and its derivatives are likely to find new applications as natural biological defenses against the consequences of diseases and other deleterious conditions, especially those associated with oxidative stress.

18.2 Chitosan and its derivatives as antioxidants with protective effects for chronic renal failure CRF is a worldwide health problem which frequently requires renal replacement therapy because it can lead to the development of cardiovascular and end-stage kidney diseases [21]. Although the pathogenesis and pathophysiology of this syndrome have not been fully elucidated, progression of CRF is accompanied by the accumulation of uremic substances with deleterious effects. For example, protein-bound uremic solutes, such as indoxyl sulfate (IS), normally excreted into the urine via the kidneys, accumulate in patients with CRF [22,23]. It is well-known that uremic toxins damage renal tubular, endothelial, and other cells by increasing the level of cellular oxidative stress [24,25]. Treatment with an oral carbonaceous adsorbent, AST-120 (Kremezin), is commonly applied in predialysis in cases of patients with uremic-stage renal failure. The drug acts by absorbing the biologically active uremic toxins, such as IS, from the circulation, thus delaying progression of CRF and

Handbook of Chitin and Chitosan

592

18. Future aspects of biomedical applications of chitin and chitosan

the need to start dialysis treatments [26,27]. The antioxidative potential of AST-120 in the systemic circulation of 5/6 nephrectomized rats was also reported as an effective treatment for reducing the oxidative stress associated with CRF [28]. However, AST-120 is typically used for a very short period of time in cases of predialysis and uremic-stage renal failure patients and there are frequent problems with noncompliance due to the fact that the procedure involves the use of activated charcoal which many patients find very difficult to take [29]. A related frequent well-known complication in patients with renal failure is hypercholesterolemia. Since antilipid pharmaceuticals used to reduce the levels of serum cholesterol, such as statins, can have significant side effects, aggressive treatment of hypercholesterolemia patients with nephrotic syndrome has not yet been attempted. Our demonstration of the ability of ingested chitosan to reduce serum cholesterol levels suggest that it might be very useful in the treatment of hypercholesterolemia in patients with renal failure and even in normal subjects [30, 31]. Jing et al. demonstrated for the first time that chitosan treatment lowered the levels of creatinine and urea in humans [32]. However, although numerous studies have reported the antioxidant effects of chitosan in several diseases, including metabolic syndrome and acute renal failure, the antioxidant influence of chitosan on the progression of CRF has not been extensively examined. In one example, Yoon et al. [33] estimated the beneficial effects of low molecular weight chitosans on glycerol-induced acute renal failure or paraquat-induced nephrotoxicity in vitro and in vivo [33,34]. We investigated the effects of a commercial supplement comprised of chitosan on the renal function and indices of oxidative stress in CRF using 5/6 nephrectomized rats. Ingestion of chitosan over a 4-week period resulted in a significant decrease in total body weight and in the levels of glucose, serum creatinine, and IS levels compared with the control group. It also resulted in a lowered ratio of oxidized to native albumin and an increase in biological antioxidant potential as measured by the reduction in copper ions [35]. Interestingly, the ratio of oxidized albumin correlated with serum IS levels in in vivo studies. Our results suggested that the ingestion of chitosan may lead to a significant reduction in the levels of prooxidants, such as uremic toxins in the gastrointestinal tract, thereby inhibiting subsequent development of oxidative stress in the systemic circulation (Fig. 18.1) [20]. A possible mechanism of the antioxidant activity of chitosans might be related to the amount and reactivity of the hydroxyl group at C6 and of the amino group at C2 of the chitosan molecule [13]. In fact, when these functional groups were substituted reducing their numbers in the polymer chains, the antioxidant activity was also reduced [36]. In general, chitosans show considerable N2
O6 and O3
O5 hydrogen bonding, giving compact

Handbook of Chitin and Chitosan

593

18.2 Chitosan and its derivatives as antioxidants

Intestine Protein

Blood Production of IS

Tryptophan

IS IS IS ROS

Indole Liver Chitosan Excretion into feces

Oxidative stress Cardiovascular disease

Stimulation

Inhibition

FIGURE 18.1 Proposed mechanism for the renal protective and antioxidant effects of chitosan. Source: Reproduced with permission from M. Anraku, Elucidation of the mechanism responsible for the oxidation of serum albumin and its application in treating oxidative stress-related diseases, Yakugaku Zasshi, 134(9), (2014) 973
9. Copyright 2014, The Pharmaceutical Society of Japan.

structures to chitosans with high molecular weight. This makes the intramolecular hydrogen bonds stronger, decreasing the reactivity of the hydroxyl and amino groups, which would be expected to also reduce the antioxidant activity. On the other hand, LMC chitosan has a less compact structure, making the overall effect of intramolecular hydrogen bonding weaker than that of HMC chitosan (Fig. 18.2). We should therefore in future be selective in the use of various molecular weight chitosans, depending on the circumstances of each treated disease. We also examined the effects of chitosan administration on oxidative stress and related factors in hemodialysis patients. Ingestion of chitosan over a 12-week period resulted in a significant decrease in serum IS and phosphate levels, lowered ratio of oxidized albumin, and a decrease in the level of advanced oxidized protein products [19,37]. The lowered oxidized albumin ratio correlated with the lowering of the serum IS in in vivo studies, consistent with the finding in vitro that chitosan binds 38.5% of IS and 17.8% of phosphate, compared with nontreated chitosan control [19]. It appears that, particularly in the case of hemodialysis patients with hyperphosphatemia, long-term ingestion of chitosan has the potential to improve the outcomes of normal treatment without inducing any side effects [19]. In fact, Lim et al. reported that oxidized albumin ratio is a positive predictor of mortality in hemodialysis patients, especially if they had preexisting cardiovascular disease [38].

Handbook of Chitin and Chitosan

594

18. Future aspects of biomedical applications of chitin and chitosan

Intramolecular hydrogen bonds Low

4 H

Molecular weight

CH2OH

6 O

H

High

4 H

1

5

ROS

O

O OH

CH2OH H

1

5

O

O OH

H

H H

H 3

2

H

High

6

3 NH3+

Antioxidant activity

H

2

NH3+

Low

FIGURE 18.2

Possible mechanism of antioxidant activity of chitosan. Source: From M. Anraku, J.M. Gebicki, D. Iohara, H. Tomida, K. Uekama, T. Maruyama, et al., Antioxidant activities of chitosans and its derivatives in in vitro and in vivo studies, Carbohydr. Polym. 199 (2018) 141
149.

It appears that increased oxidative stress resulting from biological changes in serum oxidized albumin ratio levels could contribute to accelerated atherosclerosis and the development of cardiovascular disease in hemodialysis patients [38]. On the other hand, because chitosan has adsorption activity as a polycationic biopolymer, patients taking certain types of drugs must be careful to avoid interactions between these medications and supplements such as chitosan. However, as of this writing, there appear to be few interactions between drugs and chitosan supplements, although this may require further critical evaluation. In recent years, surface-deacetylated chitin nanofibers (SDACNFs) have attracted considerable interest because their surface as well as macroscopic properties can be altered by chemical modifications. This usually affects the properties of surface amino groups, or creates electrostatic interactions between the cationic amino groups and second components with an anionic charge. Not surprisingly, the modified fibers had new physicochemical and biological functions [39-41]. Fig. 18.3A and B shows SEM images of the oven-dried chitin gel. The isolated chitin consists of highly uniform nanofibers with a width of 10 2 20 nm, suggesting that the fibrillation process was facilitated in acidic solution, as expected. It should be emphasized that the width of the nanofibers extracted in this study corresponded to the width of the chitin nanofibers observed from crab shell surface after removal of matrix without grinder treatment. Furthermore, because broken

Handbook of Chitin and Chitosan

18.2 Chitosan and its derivatives as antioxidants

(A)

1.5kV

595

(B)

X30,000

200nm WD 7.9 mm

X50,000

100nm

WD 6.1 mm

FIGURE 18.3 FE-SEM micrographs of chitin nanofibers from crab shell after one pass through the grinder with acetic acid (pH 3). The length of the scale bar is (A) 200, and (B) 100 nm, respectively [40].

fibers were not observed even over a wide observation area, the aspect ratios of the nanofibers are very high. These results indicate that chitin nanofibers were successfully isolated from crab shells without altering their natural shape. Thus the never-dried process that has previously been used to extract the cellulose nanofibers from wood cell walls was shown to be applicable for the preparation of chitin nanofibers from crustacean shells by mechanical grinding under an acidic condition (Fig. 18.3). The surface-modified fibers had the ability to function as multifunctional nanomaterials, since they shared the properties of both chitin and of the second component, giving them the potential for use in a wide variety of fields such as biotechnology, food and cosmetic industries, and agriculture [42,43]. One potential new property of the SDACNFs derivatives, antioxidant activity, has received little attention until recently, when their ability to scavenge free radicals such as N-centered radicals derived from 1,10 -diphenyl-2-picrylhydrazyl and 2,20 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and to function as general antioxidants was studied in vitro. The major findings were that the modified fibers had significant radical scavenging properties, and that this depended on the degree of deacetylation [44]. This observation added yet another potentially important function of the SDACNFs derivatives to the emerging array of current chitosan-based pharmaceutical antioxidants. High dispersability allows SDACNFs to be used as an oral adsorbent at a substantially lower dose than required for deacetylated chitin (DAC) or AST-120.

Handbook of Chitin and Chitosan

596

18. Future aspects of biomedical applications of chitin and chitosan

Tests carried out on the protective effects of the SDACNFs on oxidative stress and CRF in 5/6 nephrectomized rats showed that the treatment enhanced the antioxidant potential of the animals and inhibited renal failure progression more effectively than DAC or AST-120 [17]. In general, IS are a major dysfunctional kidney-specific risk factor and the most widely studied uremic toxin. Schulman et al. have shown that it causes an increase in formation of free radicals and induces the production of inflammatory cytokines in the kidneys and in blood circulation [45]. Clearly, lowering of IS levels would have desirable effects on renal functions. Experimental studies showed in vitro that the binding of indole to the SDACNFs as well as AST-120 was much higher than that for the equivalent DAC treatment (Fig. 18.4). These results suggest that indole levels in the gastrointestinal tract of the CRF rats would be expected to be reduced by a relatively small dose of SDACNFs. Azuma et al. proposed that oral administration of SDACNFs increases the plasma levels of ATP and 5-HT via the activation of gut microbiota [46,47], so that the antiobesity effects of orally administered SDACNFs might be due to changes in the population of the intestinal flora. Since intestinal flora is responsible for metabolizing amino acids to precursors of uremic toxins (e.g., tryptophan to indole), pre- and probiotics are generally thought to be potent agents for reducing the concentrations of uremic toxins in serum or in fecal and urinary excretions in rats.

#

Adsorption rate of indole (%)

100

#

#

#

# 80 #

60

40

20

0 0.25

0.5

1

Concentration (mg/mL)

FIGURE 18.4 Binding capacity on indole for SDACNFs, DAC, and AST-120. Source: From M. Anraku, R. Tabuchi, S. Ifuku, T. Nagae, D. Iohara, H. Tomida, et al., An oral absorbent, surface-deacetylated chitin nano-fiber ameliorates renal injury and oxidative stress in 5/6 nephrectomized rats, Carbohydr. Polym. 161 (2017) 21
25.

Handbook of Chitin and Chitosan

18.3 SDACNFs as a material for pharmaceutical formulation

597

Results are shown for SDACNFs (’), DAC (&), and AST-120 (’). Results are expressed as the mean 6 SEM. #P , .05, compared with CRF rats. [17]. Future studies are likely to confirm the suggestion that, as a probiotic, SDACNFs can exhibit renal protective effects in CRF rats and other species by modulating the gut environment and regulating systemic inflammation [48]. Early findings supporting this possibility include the results of analysis of the fecal organic levels, indicating that oral administration of SDACNFs stimulated and activated the functions of microbiota and the detection of the ability of orally administrated SDACNFs to affect the metabolism of acyl-carnitines and fatty acids. The results showed that the SDACNFs increased plasma levels of ATP and 5-HT via the activation of gut microbiota. In addition, SDACNFs have antiinflammatory actions derived from their ability to suppress the activation of NF-κB and monocyte chemotactic protein-1 and to inhibit fibrosis in an acute ulcerative colitis (UC) mouse model. Taken together, these observations are consistent with the proposition that SDACNFs are potentially a novel group of medicinal compounds or functional food additives in treatment of patients with inflammatory bowel disease [49,50]. Another potential application of the SDACNFs lies in the manufacture of cosmetics or textiles; when the nanofibers were applied to the skin of rats, the epithelial granular layer could be improved and its granular density increased, resulting in a lower production of TGF-β [50]. Although the bioavailability of SDACNFs after oral administration to animals has not been extensively investigated, in rats chitosan was reported to be eliminated from the body within 4 h [51]. In addition, the demonstrated biodegradability of chitosan and SDACNFs also increases their acceptance and potential in a wide variety of applications. In prophylactic or therapeutic applications, SDACNFS could be coadministrated with AST-120, representing a new strategy for antioxidative treatment of several diseases including renal failure, since the antioxidative effect of SDACNFs is unique and differs from that of typical conventional antioxidants such as vitamins and N-acetyl cysteine (Fig. 18.5).

18.3 SDACNFs as a material for pharmaceutical formulation in diseases associated with oxidative stress As mentioned above, the preparation of SDACNFs with diameters of approximately 10
20 nm has recently been reported [41]. A major interest in SDACNFs fibers derives from the ability to alter their surface and macroscopic properties by chemical modification of the surface amino

Handbook of Chitin and Chitosan

598

18. Future aspects of biomedical applications of chitin and chitosan

Mild Mild

Progressive course of renal failure

Massive

Increased oxidative stress

Massive

Dietary restriction (protein, etc.) Medicinal therapy

Dosing period of Kremezin

Hemodialysis/peritoneal dialysis Kidney transplantation

Renal impaiment Nephritis Proteinuria

Uremia

Exacerbation Chronic anemia Cardiovascular disease

Prevention and treatment with functional foods Dosing period of SDACNFs

FIGURE 18.5 Strategy to control renal failure progression. Source: From M. Anraku, J.M. Gebicki, D. Iohara, H. Tomida, K. Uekama, T. Maruyama, et al., Antioxidant activities of chitosans and its derivatives in in vitro and in vivo studies, Carbohydr. Polym. 199 (2018) 141
149.

groups or by manipulating the electrostatic interactions between the cationic amino groups on the surface and second components with an anionic charge. Both these modifications have direct effects on many physicochemical and biological functions of the fibers. The modified fibers have the potential to act as a multifunctional nanomaterial, since they share the properties of both chitin and the second component. As a result, they have the potential for use in various fields, such as biotechnology, food, cosmetic industries, and agriculture. In this section, we give some examples of attempts to confer the shared properties of a cyclodextrin (CD) and SDACNFs by utilizing electrostatic interactions between amino groups on the surface of the fiber and a negatively charged CD derivative, sulfobutyl ether β-CD (SBE-β-CD). The SBE-β-CD has been approved as a safe additive and is employed as a solubilizer and stabilizer in several commercially available pharmaceutical products. We prepared new very stiff elastic gels by utilizing the ionic interaction between the surface amino acid groups of the SDACNFs and the anionic SBE-β-CD (Fig. 18.6). Actually, when the SDACNFs/SBE-β-CD gel was prepared in the presence of NaCl (10% w/v), the gel strength was significantly decreased, i.e., a large decrease in viscosity was observed. The intact chitin-NF (ICNF) consisted of nonfibrous chitin and nonDAC-NF. The preparation had less than 1% of free amino groups (i.e., more than 99% of the amino groups were acetylated) and was in a suspension, with the supernatant clearly

Handbook of Chitin and Chitosan

18.3 SDACNFs as a material for pharmaceutical formulation

(A)

(F)

(B)

(G)

(C)

(H)

(D)

(I)

599

(E) (J)

FIGURE 18.6 SDACNFs or nonfiber of chitin /CDs hydrogels [52]. (A) Non-fiber form of chitin alone, (B) Intact chitin-NF alone; (C) SDACNFsalone; (D) with α-CDs; (E) With β-CDs; (F) with γ-CD; (G) with 2-hydroxypropyl-β-CD; (H) with 2,6-di-O-dimethyl-β-CD; (I) with SBE-β-CyD; (J) free-standing SDACNFs/SBE-β-CD hydrogel.

nonviscous because the chitin-NF was only sparingly soluble in water or in 1% acetic acid solution. The ICNF and SDACNFs formed a weak, quite fluid gel (Fig. 18.6B and C). Addition of neutral CDs, such as natural α-, β-, and γ-CDs, 2-hydroxypropyl-β-CD or 2, 6-di-O-methyl-β-CD, had no effect on the gel consistency of the SDACNFs, which remained in a fluid state (Fig. 18.6D
H). In sharp contrast, addition of SBE-β-CD with anionic charges converted the suspension from a fluid to a very stiff gel which could be easily retrieved by forceps (Fig. 18.6I, J). The nonfiber

Handbook of Chitin and Chitosan

600

18. Future aspects of biomedical applications of chitin and chitosan

Shear viscosity (mPa▪ s)

form of chitin and ICNF did not form a stiff gel in the presence of either SBE-β-CD or natural CDs. The pH of the SDACNFs /SBE-β-CD gel was about 3.5, nearly the same as the value of approximately 3.0 for the other gels (Fig. 18.6A
H), indicating that the pH of the solution had no effect on the formation of the stiff gel. These results show that formation of the stiff gels depends on the presence of deacetylated nanofibers and on the electrostatic interaction of the surface groups of SDACNFs with the SBEβ-CD. Tests showed that in the absence of SBE-β-CD, the SDACNFs had a constant viscosity of around 108 mPa at lower shear stresses (10
25 Pa) which decreased at higher shear stresses ( . 25 Pa), when the fibers became less viscous. In contrast, in the presence of 1.0 or 10% w/v SBE-β-CD the viscosity of the SDACNFs was higher by about one order of magnitude (109 mPa s) and was maintained up to a shear stress of about 650 Pa. This was 26-fold higher than the 25 Pa measured in the absence of SBE-β-CD, indicating that the flow properties of the SDACNFs/SBE-β-CD gel were markedly restricted, probably by crosslinking of the fibers through electrostatic interactions between charges of the component molecules. On the other hand, a large viscosity increase was not observed in the case of ICNF/SBE-β-CD gel and the increase in yield was only fourfold, because most of the amino groups were acetylated and not able to participate in charge interactions (Fig. 18.7). The anionic SBE-β-CD markedly reinforced the physical characteristics of the SDACNFs gel through electrostatic interactions between the opposite charges of the SDACNFs and SBE-β-CD which served to anchor the SDACNFs into a three-dimensional network [52]. In a new development, we found that incorporation of some drugs into the gel was increased due to the presence and solubilizing ability of SBE-β-CD [53,54]. This suggests that the SDACNFs/SBE-β-CD elastic gel would be useful for

108 106

X 26 X4

104 102 100 10

100 Shear stress (Pa)

1000

FIGURE 18.7 Dependence of shear modulus ( 6 SD) for SDACNFs/SBE-β-CD hydrogels at a SBE-β-CD concentration of 10% (open squares), 1% (open triangles), and 0% (open circles) and intact chitin-NF/SBE-β-CD hydrogels at a SBE-β-CD concentration of 10% (full squares), 1% (full triangles), and 0% (full circles) [52].

Handbook of Chitin and Chitosan

18.4 Therapeutic effects of PD/NFs-CDs gel in model mice

601

preparing homogeneous high-content gels of poorly water-soluble drugs, a subject that is currently under investigation in our laboratory.

18.4 Therapeutic effects of PD/NFs-CDs gel in model mice Inflammatory bowel disease (IBD) is classified into Crohn’s disease and UC. In particular, UC is a chronic and refractory disease with oxidative stress, and the number of the patients is large in various developed countries [55]. In clinical situations, biologic or immunosuppressive agents, such as corticosteroids, 5-aminosalicylic acid, and antitumor necrosis factor-α, are routinely used for the treatment of such diseases. Prednisolone (PD) is frequently applied in moderate and severe UC. Use of conventional antiinflammatory drugs, including PD, frequently results in the development of toxic side effects as the result of long-term administration [56]. Recent studies indicate that long-term treatment with PD can lead to osteoporosis, diabetes, and increased susceptibility to infections [57,58]. Since these toxic side effects are attributed to systemic absorption and a lower selectivity for the diseased area, it has been suggested that a drug that specifically targets the diseased site would elevate its efficacy and reduce the severity of toxic side effects. A number of methods for delivering drugs to the area diseased in UC have been developed, including pH-sensitive polymers, controlled release through the use of a matrix system, prodrugs, and timed-release systems [59-61]. However, the potential of UC therapy using SDACNFs/SBE-β-CD gel (NFs-CDs elastic gels) for the controlled release of drugs with proven efficacy has not been evaluated. In this section we present the results of studies of the incorporation of PD, a poorly soluble UC drug, in NFs-CDs elastic gels and its estimated release under a variety of conditions. We also evaluated the therapeutic effect of a NFs-CDs elastic gel with incorporated PD on model mice with dextran sulfate sodium (DSS)-induced colitis. As mentioned above, we reported that the viscosity of the NFs-CDs gel was higher by about one order of magnitude compared to that in the absence of SBE-β-CD, demonstrating that the flow properties of the NFs-CDs gel were significantly limited, probably by the fibers being held through electrostatic interactions between cationic and anionic charges of the SBE-β-CD [52]. It seems likely that the soluble SBE-β-CD and SDACNFs formed an interpolymer complex on the surface or inside the gel, from which the drug was slowly released as the erosion of the gel proceeded. In fact, the pharmacokinetic parameters, as determined from PD plasma time
concentration data, also indicated that the ratio of the area under the blood concentration
time curve (AUC) for the PD/NFs-CDs gel to the AUC of the PD only or to the PD/SDACNFs

Handbook of Chitin and Chitosan

602

18. Future aspects of biomedical applications of chitin and chitosan

mixture was accompanied by an increase in maximum blood concentration (Cmax) value. Based on these results, we concluded that the increase in the relatively high oral bioavailability of the PD/NFs-CDs gel is due to the higher dissolution rate and the extended-release effect, resulting from the high adhesion of SDACNFs and interpolymer complexation between the SDACNFs and SBE-β-CD. Since CDs were previously reported to function as enhancers of absorption, this may be another possible explanation for the improvement in the oral bioavailability of PD in SBE-β-CD. Furthermore, SBE-β-CD is available in various dosage forms and is listed in the Food and Drug Administration’s compilation of Inactive Pharmaceutical Ingredients. A consensus appears to be building among regulators that SBE-β-CD is an excipient and not part of an actual drug formulation, although various opinions concerning this continue to exist and interpretations related to this point can be divisive and product-specific [62]. The behavior of the NFs-CDs gel in the gastrointestinal tract was examined using an MRI technique. MRI images clearly showed that GdDTPA incorporated in NFs-CDs gel was retained in the stomach for periods in excess of 60 min after the initial ingestion, while the Gd-DTPA solution was transported from the stomach to the intestine within 60 min (Fig. 18.8). These findings suggest that a gel prepared by simple mixing of SDACNFs and SBE-β-CD has the potential for use in the controlled release of PD, and possibly other drugs as well. We therefore examined the therapeutic effects of PD/NFs-CDs gel on DSS-induced colitis in Gd-DTPA

Gd-DTPA/NFs-CDs gel

FIGURE 18.8 MRI images of mice 60 min after oral administration of either an aqueous solution of Gd-DTPA or the NFs/CDs gel containing Gd-DTPA [18]. Circles indicate upper abdominal area including the stomach. Position of Gd-DTPA is shown white.

Handbook of Chitin and Chitosan

18.4 Therapeutic effects of PD/NFs-CDs gel in model mice

603

model mice. The results of histological examination of colon tissues from each group of treated mice supported the protective or therapeutic effect of the PD/NFs-CDs gel on colitis. The PD/NFs-CDs treated population showed much less tissue damage and the histological appearance was similar to that of control mice even at 8 days after the induction of colitis, whereas the other treatment groups (PD suspension, PD/SBEβ-CD solution, and PD/SDACNFs mixture) showed tissue damage, including necrosis and ulcers, and leukocyte infiltration typical of mice with DSS-induced colitis. The DSS treatment also led to anemia because of bleeding or hemorrhaging in the colon. The PD/NFs-CDs gel reversed the reduction in RBC count and Hb levels while other treatments (PD suspension, PD/SBE-β-CD solution, and PD/SDACNFs mixture) were shown to be ineffective at 8 days after the induction of colitis. These results indicate that the PD/NFs-CDs gel has the ability to exert effective protection against DSS-induced colitis. An additional result of DSS-induced intestinal inflammation was mucosal infiltration of inflammatory cells, especially neutrophils [63]. We therefore assayed for colonic myeloperoxidase (MPO) activity, to evaluate the extent of neutrophil infiltration in colonic tissues of the colitis mice (Fig. 18.9). The PD/NFs-CDs gel treatment clearly

1.2

2.5

2.0

2.0 1.5 1.0

#

#

0.5

Blood free SH contents

(B) × 102

3.0

1.6 1.2 #

0.8

#

0.4

N or m al

eP D os

/S PD 3% BE s u D S os PD -β- spe S eP CD n s / SD io D /S A sol n BE CN ut -β F ion -C m ix D tu /S re D A CN F hy dr og N el or m al D

PD

/S PD 3% BE s u D D PD -β-C spen SS /S BE /S D sio D -β A solu n -C CN t D F ion /S m D ix A tu CN re F hy dr og el

0

0

PD

MPO activity

(A) × 10–5

FIGURE 18.9 Effects of PD samples on clinical disease activity in DSS-induced colitis, assessed by MPO activity (A) and thiol content (B) [18]. xP , .05 for control (3%DSS-treated mice).

Handbook of Chitin and Chitosan

604

18. Future aspects of biomedical applications of chitin and chitosan

suppressed MPO activity in the colitis mice, while the induction of MPO was much higher in mice treated with the other treatment groups (listed in Fig. 18.9) than in untreated controls. Actually, tetramethylbenzidine was used as a substrate in the MPO activity assay to initiate the reaction. In the assay, 10 μL of the standard and sample were added to appropriately labeled tubes. TMB was added at a volume of 100 μL to initiate the reaction, and 100 μL of 0.1 M H2SO4 was added after 10 min of initiation to terminate the MPO reaction. Changes in absorbance were measured using a spectrophotometer at 450 nm. Moreover, since MPO is the main source of ROS in inflammatory diseases, we estimated the extent of protein oxidation in the DSS-treated mice by assaying the blood disulfide levels. The PD/NFs-CDs gel treatment clearly suppressed the level of this oxidative stress marker, while a decrease in thiol levels was observed for the colitis mice exposed to alternative treatments. Importantly, no severe side effects such as abnormal behavior or dizziness with lethargy were observed during the experimental period, indicating that this system is a promising candidate for safe use in IBD treatments. It was previously reported [64] that PD alone is distributed and absorbed mainly in the stomach and small intestine within a period of several hours. Thus, the high retention and efficient controlled release of the PD conferred by the PD/NFs-CDs gels might lead to reduced toxicity of PD. In fact, only the NFs-CDs gel inhibited the enlarged spleen commonly produced by PD administration. The widely used colitis models induced in mice by DSS in the drinking water or in a pathogenic mechanism are similar to IBD in humans, characterized by disruption of the epithelial barrier leading to induction of an exaggerated mucosal inflammatory response [65]. Taken together, these findings indicate that the PD/NFs-CDs gel has the potential for use in the therapeutic treatment of colitis in humans and would constitute a novel material for use in the treatment of colitis.

18.5 Conclusions In this review we present the status of current research on the use of natural chitosan and its derivatives in in vivo studies. Chitosan and its derivatives, especially the SDACNFs, have many well-documented biological activities with significant health-related benefits, including the prevention of renal failure, wound healing, reduction of gastric ulcers, antiinflammatory effects, inhibition of antigen toxicity, and anticancer activities. Further, SDACNFs might be very useful as multifunctional materials for pharmaceutical applications in diseases associated with oxidative stress. In future, the functional properties and biological

Handbook of Chitin and Chitosan

References

605

activities of SDACNFs could be further enhanced by appropriate modifications, leading to further novel applications.

Abbreviations AST-120 CRF DAC 5-HT IBD ICNF IS MPO ROS SBE-β-CD SDCHNFs UC

kremezin chronic renal failure deacetylated chitosan 5- hydroxytryptamine inflammatory bowel disease intact chitin-NF indoxy sulfate myeloperoxidase reactive oxygen species sulfobutyl ether β-cyclodextrin deacetylated chitin nanofibers ulcerative colitis

References [1] E. Bourdon, D. Blache, The importance of proteins in defense against oxidation, Antioxid. Redox Signal. 3 (2) (2001) 293
311. [2] B. Halliwell, Antioxidants in human health and disease, Annu. Rev. Nutr. 16 (1996) 33
50. [3] J.M. Gebicki, Oxidative stress, free radicals and protein peroxides, Arch. Biochem. Biophys. 595 (2016) 33
39. [4] J.M. Gebicki, Protein hydroperoxides as new reactive oxygen species, Redox Rep. 3 (2) (1997) 99
110. [5] M.P. Murphy, A. Holmgren, N.G. Larsson, B. Halliwell, C.J. Chang, B. Kalyanaraman, et al., Unraveling the biological roles of reactive oxygen species, Cell Metab. 13 (4) (2011) 361
366. [6] B. Halliwell, J.M. Gutteridge, The definition and measurement of antioxidants in biological systems, Free Radic. Biol. Med. 18 (1) (1995) 125
126. [7] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, fourth ed., Oxford University Press, Oxford, 2007. [8] C. Sa´nchez-Moreno, A. Jime´nez-Escrig, A. Martı´n, Stroke: roles of B vitamins, homocysteine and antioxidants, Nutr. Res. Rev. 22 (1) (2009) 49
67. [9] B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci. USA 90 (17) (1993) 7915
7922. [10] S. Koudelka, P. Turanek Knotigova, J. Masek, L. Prochazka, R. Lukac, A.D. Miller, et al., Liposomal delivery systems for anti-cancer analogues of vitamin E, J. Control. Release 207 (2015) 59
69. [11] A.M. Pisoschi, A. Pop, The role of antioxidants in the chemistry of oxidative stress: a review, Eur. J. Med. Chem. 97 (2015) 55
74. [12] P. Kulawik, F. Ozogul, R. Glew, Y. Ozogul, Significance of antioxidants for seafood safety and human health, J. Agric. Food Chem. 61 (3) (2013) 475
491. [13] P.-J. Park, S. Koppula, S.-K. Kim, Antioxidative activity of chitosan, chitooligosaccharides and their derivatives, in: S-K. Kim (Ed.),Chitin, Chitosan, Oligosaccharides and Their Derivatives: Biological Activities and Applications CRC press, Taylor & Francis group. (2011) pp. 241
250.

Handbook of Chitin and Chitosan

606

18. Future aspects of biomedical applications of chitin and chitosan

[14] H. Tomida, T. Fujii, N. Furutani, A. Michihara, T. Yasufuku, K. Akasaki, et al., Antioxidant properties of some different molecular weight chitosans, Carbohydr. Res. 344 (13) (2009) 1690
1696. [15] M. Anraku, A. Michihara, T. Yasufuku, K. Akasaki, D. Tsuchiya, H. Nishio, et al., The antioxidative and antilipidemic effects of different molecular weight chitosans in metabolic syndrome model rats, Biol. Pharm. Bull. 33 (12) (2010) 1994
1998. [16] M. Anraku, J.M. Gebicki, D. Iohara, H. Tomida, K. Uekama, T. Maruyama, et al., Antioxidant activities of chitosans and its derivatives in in vitro and in vivo studies, Carbohydr. Polym. 199 (2018) 141
149. [17] M. Anraku, R. Tabuchi, S. Ifuku, T. Nagae, D. Iohara, H. Tomida, et al., An oral absorbent, surface-deacetylated chitin nano-fiber ameliorates renal injury and oxidative stress in 5/6 nephrectomized rats, Carbohydr. Polym. 161 (2017) 21
25. [18] R. Tabuchi, M. Anraku, D. Iohara, T. Ishiguro, S. Ifuku, T. Nagae, et al., Surfacedeacetylated chitin nanofibers reinforced with a sulfobutyl ether β-cyclodextrin gel loaded with prednisolone as potential therapy for inflammatory bowel disease, Carbohydr. Polym. 174 (2017) 1087
1094. [19] M. Anraku, M. Tanaka, A. Hiraga, K. Nagumo, T. Imafuku, Y. Maezaki, et al., Effects of chitosan on oxidative stress and related factors in hemodialysis patients, Carbohydr. Polym. 112 (2014) 152
157. [20] M. Anraku, H. Tomida, A. Michihara, D. Tsuchiya, D. Iohara, Y. Maezaki, et al., Antioxidant and renoprotective activity of chitosan in nephrectomized rats, Carbohydr. Polym. 89 (1) (2012) 302
304. [21] A.S. Go, G.M. Chertow, D. Fan, C.E. McCulloch, C.Y. Hsu, Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization, N Engl. J. Med. 351 (13) (2004) 1296
1305. [22] F.C. Barreto, D.V. Barreto, M.E.F. Canziani, Uremia retention molecules and clinical outcomes, Contrib. Nephrol. 191 (2017) 18
31. [23] T. Niwa, The role of carbon adsorbent in the conservative management of chronic kidney disease, Panminerva Med. 59 (2) (2017) 139
148. [24] H. Shimizu, Y. Hirose, S. Goto, F. Nishijima, H. Zrelli, N. Zghonda, et al., Indoxyl sulfate enhances angiotensin II signaling through upregulation of epidermal growth factor receptor expression in vascular smooth muscle cells, Life Sci. 91 (5
6) (2012) 172
177. [25] H. Watanabe, Molecular mechanisms for uremic toxin-induced oxidative tissue damage via a cardiovascular-renal connection, Yakugaku Zasshi 133 (8) (2013) 889
895. [26] T. Niwa, Role of indoxyl sulfate in the progression of chronic kidney disease and cardiovascular disease: experimental and clinical effects of oral sorbent AST-120, Ther. Apher. Dial. 15 (2) (2011) 120
124. [27] A. Owada, M. Nakao, J. Koike, K. Ujiie, K. Tomita, T. Shiigai, Effects of oral adsorbent AST-120 on the progression of chronic renal failure: a randomized controlled study, Kidney Int. Suppl. 63 (1997) S188
S190. [28] K. Shimoishi, M. Anraku, K. Kitamura, Y. Tasaki, K. Taguchi, M. Hashimoto, et al., An oral adsorbent, AST-120 protects against the progression of oxidative stress by reducing the accumulation of indoxyl sulfate in the systemic circulation in renal failure, Pharm. Res. 24 (7) (2007) 1283
1289. [29] T. Niwa, Targeting protein-bound uremic toxins in chronic kidney disease, Expert. Opin. Ther. Targets 17 (11) (2013) 1287
1301. [30] M. Anraku, T. Fujii, N. Furutani, D. Kadowaki, T. Maruyama, M. Otagiri, et al., Antioxidant effects of a dietary supplement: reduction of indices of oxidative stress in normal subjects by water-soluble chitosan, Food Chem Toxicol. 47(1), 2009, 104
109.

Handbook of Chitin and Chitosan

References

607

[31] M. Anraku, T. Fujii, Y. Kondo, E. Kojima, T. Hata, N. Tabuchi, et al., Antioxidant and renoprotective activity of chitosan in nephrectomized rats, Carbohydr. Polym. 89 (1), 2012, 302
304. [32] S.B. Jing, L. Li, D. Ji, Y. Takiguchi, T. Yamaguchi, Effect of chitosan on renal function in patients with chronic renal failure, J. Pharm. Pharmacol. 49 (7) (1997) 721
723. [33] S.P. Yoon, M.S. Han, J.W. Kim, I.Y. Chang, H.L. Kim, J.H. Chung, et al., Protective effects of chitosan oligosaccharide on paraquat-induced nephrotoxicity in rats, Food Chem. Toxicol. 49 (8) (2011) 1828
1833. [34] H.J. Yoon, M.E. Moon, H.S. Park, H.W. Kim, S.Y. Im, J.H. Lee, et al., Effects of chitosan oligosaccharide (COS) on the glycerol-induced acute renal failure in vitro and in vivo, Food Chem. Toxicol. 46 (2) (2008) 710
716. [35] E. Straface, P. Matarrese, L. Gambardella, R. Vona, A. Sgadari, M.C. Silveri, et al., Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: a pilot study, FEBS Lett. 579 (13) (2005) 2759
2766. [36] M. Anraku, M. Kabashima, H. Namura, T. Maruyama, M. Otagiri, J.M. Gebicki, et al., Antioxidant protection of human serum albumin by chitosan, Int, J. Biol. Macromol. 43 (2) (2008) 159
164. [37] M. Anraku, V.T. Chuang, T. Maruyama, M. Otagiri, Redox properties of serum albumin, Biochim. Biophys. Acta 1830 (12) (2013) 5465
5472. [38] P.S. Lim, Y. Jeng, M.Y. Wu, M.A. Pai, T.K. Wu, C.S. Liu, et al., Serum oxidized albumin and cardiovascular mortality in normoalbuminemic hemodialysis patients: a cohort study, PLoS One 8 (7) (2013) e70822. [39] S. Ifuku, M. Nogi, K. Abe, M. Yoshioka, M. Morimoto, H. Saimoto, et al., Preparation of chitin nanofibers with a uniform width as alpha-chitin from crab shells, Biomacromolecules 10 (6) (2009) 1584
1588. [40] S. Ifuku, H. Saimoto, Chitin nanofibers: preparations, modifications, and applications, Nanoscale 4 (11) (2012) 3308
3318. [41] S. Ifuku, Chitin and chitosan nanofibers: preparation and chemical modifications, Molecules 19 (11) (2014) 18367
18380. [42] K. Azuma, T. Osaki, S. Ifuku, H. Saimoto, M. Morimoto, O. Takashima, et al., Antiinflammatory effects of cellulose nanofiber made from pear in inflammatory bowel disease model, Bioact. Carbohydr. Diet. Fibre 3 (1) (2014) 1
10. [43] R. Koizumi, K. Azuma, H. Izawa, M. Morimoto, K. Ochi, T. Tsuka, et al., Oral administration of surface-deacetylated chitin nanofibers and chitosan inhibit 5-Fluorouracilinduced intestinal mucositis in mice, Int. J. Mol. Sci. 18 (2) (2017). [44] M. Anraku, R. Tabuchi, S. Ifuku, T. Ishiguro, D. Iohara, F. Hirayama, Surface-deacetylated chitin nano-fiber/hyaluronic acid composites as potential antioxidative compounds for use in extended-release matrix tablets, Int. J. Mol. Sci. 16 (10) (2015) 24707
24717. [45] G. Schulman, R. Vanholder, T. Niwa, AST-120 for the management of progression of chronic kidney disease, Int. J. Nephrol. Renovasc. Dis. 7 (2014) 49
56. [46] K. Azuma, M. Nishihara, H. Shimizu, Y. Itoh, O. Takashima, T. Osaki, et al., Biological adhesive based on carboxymethyl chitin derivatives and chitin nanofibers, Biomaterials 42 (2015) 20
29. [47] K. Azuma, S. Ifuku, T. Osaki, Y. Okamoto, S. Minami, Preparation and biomedical applications of chitin and chitosan nanofibers, J. Biomed. Nanotechnol. 10 (10) (2014) 2891
2920. [48] K. Azuma, R. Izumi, M. Kawata, T. Nagae, T. Osaki, Y. Murahata, et al., Effects of oral administration of chitin nanofiber on plasma metabolites and gut microorganisms, Int. J. Mol. Sci. 16 (9) (2015) 21931
21949. [49] K. Azuma, T. Osaki, T. Wakuda, S. Ifuku, H. Saimoto, T. Tsuka, et al., Beneficial and preventive effect of chitin nanofibrils in a dextran sulfate sodium-induced acute ulcerative colitis model, Carbohydr. Polym. 87 (2) (2012) 1399
1403.

Handbook of Chitin and Chitosan

608

18. Future aspects of biomedical applications of chitin and chitosan

[50] I. Ito, T. Osaki, S. Ifuku, H. Saimoto, Y. Takamori, S. Kurozumi, et al., Evaluation of the effects of chitin nanofibrils on skin function using skin models, Carbohydr. Polym. 101 (2014) 464
470. [51] S.Y. Chae, M.K. Jang, J.W. Nah, Influence of molecular weight on oral absorption of water soluble chitosans, J. Control. Release 102 (2) (2005) 383
394. [52] M. Anraku, D. Iohara, A. Hiraga, K. Uekama, S. Ifuku, J. Pipkin, F. Hirayama, Formation of elastic gels from deacetylated chitin nanofibers reinforced with sulfobutyl ether β-cyclodextrin, Chem. Lett. 44 (3) (2015) 285
287. [53] R. Tabuchi, K. Azuma, R. Izumi, T. Tanou, Y. Okamoto, T. Nagae, et al., Biomaterials based on freeze dried surface-deacetylated chitin nanofibers reinforced with sulfobutyl ether β-cyclodextrin gel in wound dressing applications, Int. J. Pharm. 511 (2) (2016) 1080
1087. [54] M. Anraku, A. Hiraga, D. Iohara, D. Pipkin James, K. Uekama, F. Hirayama, Slowrelease of famotidine from tablets consisting of chitosan/sulfobutyl ether β-cyclodextrin composites, Int. J. Pharm. 487 (1
2) (2015) 142
147. [55] K. Asakura, Y. Nishiwaki, N. Inoue, T. Hibi, M. Watanabe, T. Takebayashi, Prevalence of ulcerative colitis and Crohn’s disease in Japan, J. Gastroenterol. 44 (7) (2009) 659
665. [56] P. Rutgeerts, F. Baert, Immunosuppressive drugs in the treatment of Crohn’s disease, Eur. J. Surg. 164 (12) (1998) 911
915. [57] S. Ardizzone, G. Bianchi Porro, A practical guide to the management of distal ulcerative colitis, Drugs 55 (4) (1998) 519
542. [58] B.L. Love, A.D. Miller, Extended-release mesalamine granules for ulcerative colitis, Ann. Pharmacother. 46 (11) (2012) 1529
1536. [59] H. Ali, B. Weigmann, M.F. Neurath, E.M. Collnot, M. Windbergs, C.M. Lehr, Budesonide loaded nanoparticles with pH-sensitive coating for improved mucosal targeting in mouse models of inflammatory bowel diseases, J. Control. Release 183 (2014) 167
177. [60] E.R. Bendas, J.M. Christensen, J.W. Ayres, Development and in vitro evaluation of mesalamine delayed release pellets and tableted reservoir-type pellets, Drug Dev. Ind. Pharm. 36 (4) (2010) 393
404. [61] H. Yano, F. Hirayama, M. Kamada, H. Arima, K. Uekama, Colon-specific delivery of prednisolone-appended alpha-cyclodextrin conjugate: alleviation of systemic side effect after oral administration, J. Control. Release 79 (1
3) (2002) 103
112. [62] M.E. Brewster, T. Loftsson, Cyclodextrins as pharmaceutical solubilizers, Adv. Drug Deliv. Rev. 59 (7) (2007) 645
666. [63] I. Monteleone, P. Vavassori, L. Biancone, G. Monteleone, F. Pallone, Immunoregulation in the gut: success and failures in human disease, Gut 50 (Suppl. 3) (2002). III60-4. [64] T. Oosegi, H. Onishi, Y. Machida, Novel preparation of enteric-coated chitosan-prednisolone conjugate microspheres and in vitro evaluation of their potential as a colonic delivery system, Eur. J. Pharm. Biopharm. 68 (2) (2008) 260
266. [65] S. Wirtz, C. Neufert, B. Weigmann, M.F. Neurath, Chemically induced mouse models of intestinal inflammation, Nat. Protoc. 2 (3) (2007) 541
546.

Handbook of Chitin and Chitosan

C H A P T E R

19 Immunomodulatory activities of chitin and chitosan microparticles Mostafa Haji Molla Hoseini1, Sahar Sadeghi2, Mahdieh Azizi1 and Ramin Pouriran3 1

Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 2Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 3School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

O U T L I N E 19.1 Chitinous microparticles and immune responses 19.1.1 Immunoregulation 19.1.2 Difference in the immunopotentiating effects of chitin and chitosan 19.1.3 Chitinases and chitolectins 19.1.4 Chitinous microparticles and trained immunity 19.1.5 Antimicrobial properties of chitin and chitosan microparticles

610 610

19.2 Chitin 19.2.1 19.2.2 19.2.3

614 614 615 617

and chitosan microparticles as adjuvant Adjuvanticity of chitin microparticles Adjuvanticity of chitosan microparticles Controversy about chitinous microparticles adjuvanticity

19.3 Anticancer effect of chitin and chitosan microparticles 19.3.1 Chitin and chitosan as anticancer agents 19.3.2 Chitin and chitosan microsphere as carriers of anticancer drug

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00019-4

609

611 611 612 613

619 620 622

© 2020 Elsevier Inc. All rights reserved.

610

19. Immunomodulatory activities of chitin and chitosan microparticles

19.4 Allergy and chitinous microparticles 623 19.4.1 The reasons for the controversy about chitin microparticles in allergic disorders 625 19.4.2 Chitinase activity in allergic disease 626 19.5 Inflammation and chitin, chitosan microparticles

627

19.6 Conclusion

629

References

631

19.1 Chitinous microparticles and immune responses 19.1.1 Immunoregulation Many studies have focused on the immune-potentiating effects of chitinous microparticles (CMPs) since 1982 [1]. The vertebrate immune system detects and degrades CMPs as a pathogen-associated molecular pattern (PAMPs). It has been shown that chitin is recognized by TLR-2, dectin-1, mannose receptor, FIBCD1, TLR-9, ficolin, and NOD2 [2]. Therefore several immune cell subsets could recognize the chitinous stimulant to redirect immune response. In order to study the immunestimulating properties of CMPs, they were applied to different murine models, and various immunomodulatory capacities were identified ranging from antiinflammatory [3] to proinflammatory [4]. In our research, we found that subcutaneous injection of chitin microparticles in a murine model of leishmaniasis induced IL-10 and IFN-γ production, which results in marked protection against leishmaniasis [5]. Nagatani et al. demonstrated that chitin microparticles upregulate IL-10 and IFN-γ production in a murine colitis model as well [6]. Recently, we reported that the addition of chitin microparticles under TH1/TH17 skewing conditions induced the production of IL-10 [7]. Generally, a literature review indicates that chitin microparticle stimulation may lead to deviation of the immune response. Yet contrasting reports exist on the immunomodulatory effect of chitin particles [8]. Shibata et al. believed that chitin microparticles are IFN-γ inducers (TH1 adjuvants) and could redirect TH2 allergic responses [3]. However, Da Silva et al. introduced chitin as a TLR-2 agonist to serve as an effective multifaceted adjuvant for TH2/ TH17 responses [9]. They demonstrated that chitin’s activity is sizedependent. They believe that the recognition of chitin together with the release of proinflammatory cytokines induces the secretion of chitinases. Therefore chitin digests and leads to the generation of small size chitin

Handbook of Chitin and Chitosan

19.1 Chitinous microparticles and immune responses

611

particles that induce IL-10 secretion. The IL-10, as an antiinflammatory cytokine, dampens the TH2/TH17 response by downregulating proinflammatory cytokine production. Da Silva et al. do not believe in the redirection of TH2 responses via IFN-γ production by chitin microparticles [10]. Therefore the exact regulatory mechanisms of the chitin treatment among animal models remain unknown.

19.1.2 Difference in the immunopotentiating effects of chitin and chitosan The immunopotentiating effects of chitin and chitosan in living models (cell culture and murine models) vary in terms of quantity and quality. The protective effects of chitin and chitosan microparticles were assessed in comparison with each other against Leishmania infection in BALB/c mice; the findings indicated that chitin seemingly outperforms chitosan in terms of the effects against leishmaniasis [11]. Da Silva et al. [9], Shibata et al. [12], and Strong et al. [13] reported immune responses to chitin particles in mice. Yet, Bueter et al. [14] argued that chitosan, not chitin, provokes an immune response. Bueter et al. discussed that, compared with chitosan, chitin does not exert an immune stimulatory effect by stimulating the murine macrophages to release the inflammasomeassociated cytokine. Nevertheless, it has been shown that Fc-receptor dependent phagocytosis of IgG-opsonized chitin is an IL-1Ra-inducing mechanism by chitin. IL-1Ra production induced by chitin relied on Syk kinase and phosphatidylinositol 3-kinase activation. Yet, costimulation of chitin with the pattern recognition receptor ligands like lipopolysaccharide influences the induction of proinflammatory cytokines synergistically [15]. Thus according to the microenvironments, chitin and chitosan are capable of having both pro- and antiinflammatory features. These two types of immune responses have been confirmed using both human and mouse in vitro and in vivo models.

19.1.3 Chitinases and chitolectins The glycoside hydrolase family, which includes true chitinase (Acidic mammalian chitinase and chitotriosidase) and chitinase-like proteins (CLPs) (chitolectins) with a lack of chitinase activity, is produced as part of the immune responses to chitin-containing pathogens [16]. To start hydrolysis, all chitinases require the presence of the carbonyl oxygen located on the acetyl group, and thus chitinase activity depends on the presence of chitin rather than chitosan [17]. It is tempting to speculate that true chitinases induce chitin fragmentation and produce small chitin fragments; then, the small fragment based on its acetylation status

Handbook of Chitin and Chitosan

612

19. Immunomodulatory activities of chitin and chitosan microparticles

regulates the expression and function of chitinase-like cytokines for managing the local inflammatory responses. It is also possible that commensal fungi in mucosal surfaces release chitinous particles and this release can potentially enhance attenuating the inflammatory-mediated diseases and subsequent immune homeostasis [18]. Chitolectins are capable of regulating local inflammatory cell functions like cytokine. YKL-40, and SI-CLP, as chitinase-like cytokines take part chiefly in TH2-associated immune responses [16]. It is believed that one of the key characteristics in patients with chronic inflammatory airway diseases is the overproduction of mucus. Mucin5AC production is boosted by YKL-40 human bronchial epithelial cells [19]; it is also involved significantly in the pathogenesis of airway allergic diseases [20]. SI-CLP is overexpressed by IL-4. Unbridle SI-CLP expression has been reported in patients with sarcoidosis undergoing corticoid therapy [21]. Some studies believed that chitin recognition and chitinase production are linked. It has been shown that the serum chitinase activity was increased in rats following the injection of yeast cell wall derived products containing chitin intraperitoneally [22]. The survival of mice infected with Candida albicans is prolonged through recombinant chitinase [23]. Consequently, it could be said that immune responses could be modulated through artificial induction of chitinase in chitin-treated mice.

19.1.4 Chitinous microparticles and trained immunity In general, researchers believed that immunological memory is built only by adaptive immunity. Recent findings have shown that innate immune cells are capable of mounting some kind of immunological memory that makes them more resistant to secondary infections; this is called “trained immunity” or “innate immune memory” [24]. It relies on the epigenetic remodeling and histone modifications with chromatin reconfiguration and is most likely related to DNA methylation or modulation of microRNA and/or long noncoding RNA expression in innate immune cells (monocytes/macrophages, NK cells) after they have been stimulated with numerous infectious or noninfectious agents. These changes boost responsiveness to secondary stimulation by both PAMPs and a number of danger-associated molecular patterns, boost production of inflammatory cytokines, and improve the capability for phagocytosis and killing pathogens [25]. Bacillus Calmette-Gue´rin and β-glucan (a component of the fungal cell wall) are two of the antigens that have been studied widely and the trained immune cells induced by these antigens could be effective against reinfections. Many other antigens have also been reported to induce trained innate immunity, such as Saccharomyces cerevisiae-derived chitin and LPS [26]. Like β-glucan, chitin

Handbook of Chitin and Chitosan

19.1 Chitinous microparticles and immune responses

613

is a main element of the fungal cell wall and a few studies reported that training is also induced by chitin. Rizzetto et al. [27] used various strains of S. cerevisiae, as well as S. cerevisiae-derived chitin to induce innate immunity training in vivo and in vitro. According to their findings, mice were made more resistant to subsequent infection with C. albicans by chitin through a fine modulation of inflammatory/antiinflammatory cytokines. Moreover, this study reported monocyte training is inhibited by S. cerevisiae or chitin following the inhibition of histone methyltransferases, so histone methylation plays a role in the training of monocytes.

19.1.5 Antimicrobial properties of chitin and chitosan microparticles Antimicrobial agents have the ability to prevent or limit the microbial growth. The use of antimicrobial agents is common in various industrial, medical, and agricultural fields. So far, numerous studies have been carried out to show and confirm the antimicrobial properties of chitin, chitosan, and their derivatives by various researchers [28]. The major differences between chitin and chitosan are in solubility and charge. Chitin has less antimicrobial properties than chitosan due to its lower positive charge. Chitosan has antimicrobial behavior because of the positive charges of its amino groups, which can interact with the negative charges of the cell membrane of microbes, changing the permeability of their membranes, so through leakage of intracellular components, chitosan could kill the microorganism. In addition to charge, characteristics such as molecular weight (MW), degree of deacetylation (DD), concentration, and environmental pH affect antimicrobial activity [29]. The mechanism of antimicrobial activity of chitosan in the form of a microparticle is different to the soluble form. In solid forms, only surfaces of the molecule that are in contact with the pathogen can have antimicrobial properties. In the soluble state, since the molecule is extended, entire molecule surfaces have the potential to interact with organisms. However, in the soluble form environmental factors, such as pH, affect the antimicrobial properties, but in the solid forms antimicrobial properties are less dependent on the environmental factors [30]. Furthermore, in the numerous studies since 1982, scientists have examined the immunopotentiating and antimicrobial effects of chitin and chitosan against various pathogens, for example Staphylococcus aureus, C. albicans, Pseudomonas aeruginosa, and Listeria monocytogenes [30]. CMPs by stimulating the innate immune cells, form protective responses against pathogenic challenges. Intraperitoneal or intravenously administrations

Handbook of Chitin and Chitosan

614

19. Immunomodulatory activities of chitin and chitosan microparticles

of chitin or chitosan in mice have elicited active oxygen production and myeloperoxidase activity of phagocyte cells.

19.2 Chitin and chitosan microparticles as adjuvant A vaccine which could stimulate both cellular and humoral immunity and make an immune memory would be quite desirable. Vaccine adjuvants should: 1. Well accompany antigen (or carry it) without changing the antigen structure and by maintaining the antigen properties. 2. Increase the antigen presentation and absorption to immune system 3. Do the depot and gradual release of the antigen. 4. Make an appropriate pattern of immune response and act as a beneficial immune response-modulator. 5. Have some features to allow their use in routes that are less invasive in individuals (e.g., oral or intranasal administration). 6. Easy and cheap for preparation. In the regard of vaccination, local and mucosal responses along with systemic responses are valuable. An adjuvant that can produce an appropriate stimulatory pattern without causing side effects and allergies would be valuable. Chitosan and chitin have a mucoadhesive property, which can provide satisfactory mucosal immunity. They possess features, such as intrinsic immune stimulation and bioadhesion; and can be easily applied in various shapes and sizes (including nanoparticles and microparticles).

19.2.1 Adjuvanticity of chitin microparticles In the 1980s, chitin and chitosan’s adjuvant properties were described for the first time by Suzuki et al. [31]. Over the past four decades, this feature has been reexamined and verified several times. Hasegawa and his colleagues [32] examined the mucosal adjuvant effects of chitin microparticles (1
20 μm) followed by coadministration with influenza H1N1 vaccine. The results indicated strong adjuvant properties of the chitin microparticles. Induction of mucosal and blood antibodies following complete protection against viral challenge showed immunizing in conjunction with antibody. Also they showed a well-cross-immunity when they challenged mice with different influenza strains (H3N2, B). Antibody class switching to IgG2a in this study confirmed a TH1 immune response profile which was induced by vaccine plus chitin microparticles. Asahi et al. [33] reported that chitin microparticles, prepared by

Handbook of Chitin and Chitosan

19.2 Chitin and chitosan microparticles as adjuvant

615

sonication, coadministered intranasally with avian influenza vaccine in mice, can induce a protective immunity against the infection. They showed that both chitin microparticles and dsRNA have synergistic effects with each other in antibody and cytokine responses leading to the enhancement of mucosal adjuvant effects. Chitin microparticles can have prophylactic effects and tackle influenza infection by intranasal administration. Ichinohe and colleagues [34], before viral challenge (with H1N1 and H5N1), treated mice intranasally by chitin microparticles for 2 or 3 days. They observed that the number of natural killer cells in the local lymphoid tissue increased. Additionally, cytokine responses are relevant to eliminate the pathogenesis of influenza virus infection. Chitin microparticles are candidates as a prophylactic drug against influenza virus infection. Intranasal administration of chitin microparticles prior to challenge by H3N2 virus can induce the production of cytokines, such as IFN-γ, and they can increase the influx of neutrophils, DCs, and NK cells in the airways. This accumulation of innate cells caused the reduction in viral load and morbidity due to infection with a lethal dose of virus [35]. The TH1 adjuvant pattern in chitin microparticles (1
10 μm) was also attained in intraperitoneal application along with the mycobacterial MPB59 antigen. On the one hand, chitin microparticles were introduced as an immune-adjuvant; On the other hand, they were recognized as allergic suppressors. This suppression was shown by no observation of IgE, IgG1, and interleukins 4, 5, and 10 [36]. Chitin microparticles as an antiallergic agent have been presented by Strong et al. [37]. They used chitin microparticles intranasally in mice model and showed the effect of CMPs on cytokine IL-4 level reduction and IL-12, IFN-γ, and TNF-α cytokines promotion. The nasal usage of chitin microparticles with the DNA vaccine could be effective against HIV [13].

19.2.2 Adjuvanticity of chitosan microparticles In several studies, chitosan microparticles have been studied both as carriers and adjuvants. Chitosan microparticles can act as antigen carriers with a high capacity for accurate loading and release at desired places, for example, to intestinal Peyer’s patches (in oral usage) or NALT (in nasal usage). Also at the same time, they have an adjuvant property to stimulate the immune system by antigens. In Lubben et al.’s study [38], it is noted that only microparticles below 10 μm of chitosan are capable of being absorbed by M cells in Peyer’s patches. The capacity and efficiency of antigen loading are high for chitosan microparticles; therefore they carry high levels of antigens and present them into immune cells. Tetanus [39] and diphtheria [40] toxoids transported orally by chitosan microparticles resulted in IgA and IgG production; consequently, this led

Handbook of Chitin and Chitosan

616

19. Immunomodulatory activities of chitin and chitosan microparticles

to both systemic and local protection against Clostridium tetani and Corynebacterium diphtheria, respectively. Coating of chitosan microparticles by an acid-resistant polymer, such as sodium alginate, causes its further hydrophilicity and solubility in the acidic environment of the stomach. Onuigbo et al. [41] used alginate-coated chitosan microparticles (0.55
10 μm), to orally deliver fowl typhoid vaccine into birds. This vaccine is sensitive to the acidic condition of gastrointestinal tract and is destroyed in such conditions. However, its entrapment in mucoadhesive alginate-coated chitosan microparticles maintains the structural integrity of the vaccine and provides stronger humoral and cellular immune responses than chitosan microparticles without the coating polymer. Microparticles made by mannosylated chitosan have been shown to stimulate the immune response stronger than chitosan in the intranasal delivery of Bordetella bronchiseptica antigens, due to the interaction of mannose groups with mannose receptors on macrophages [42]. Ghendon et al. [43] showed that 0.5% solution of chitosan admixed with inactivated influenza vaccines used parentally, not only increases the immunogenicity of them but also can induces the production of antibodies against different virus. Additionally, it has the ability of long-lasting depot in antigen in injection site and presenting much more peptides and antigenic regions of antigen in injection site. In another study Ghendon et al. [44] showed the ability of chitosan to induce cellular immune response by increasing the expansion and cytotoxicity of cells in the spleen. Also they showed that chitosan has no potential toward IgE antibody induction which induces allergenic reactions and no antibodies could be induced against chitosan itself. Thus chitosan as an adjuvant has two crucial properties: inability to induce negative immunological reactions and inability to induce allergic reactions. Sui and his colleagues investigated chitosan solution as a proper mucosal adjuvant for M1 [45] and M2 [46] protein (highly conserved matrix proteins in all influenza A strains)-based vaccine. The attained results demonstrated that IgG and IgA titers were higher when they used proteins with chitosan solution than using proteins solo. Also they showed that lower doses of antigen were required when chitosan was used. Thereby, it was achieved that chitosan could be a potent mucosal adjuvant which can be used nasally as an enhancer in the design of vaccines. Chitosan efficiency as a proper adjuvant for parental administration of influenza vaccines also was studied and it was compared with aluminum hydroxide; the results showed an equal effect on protection against lethal dose of virus for both used adjuvants [47]. Wang et al. [48] investigated the chitosan for its ability to improve the immunogenicity of live attenuated influenza vaccine; chitosan can provide a desirable protection against influenza viruses by increasing antibody titers and T cell responses. Conclusively their data showed that chitosan can be a proper mucosal

Handbook of Chitin and Chitosan

19.2 Chitin and chitosan microparticles as adjuvant

617

adjuvant with the ability to enhance both humoral and cellular immune responses; additionally, it can be an effective protection against various types of influenza virus. Due to its cationic property, chitosan with positive charge has an electrostatic interaction with the negative charge of mucosal surfaces. This mucoadhesive polymer, with the ability to open epithelial cells tight junctions, increases the mucus absorption of antigens. Opening the tight junctions is related to the presence of amine groups in the chitosan structure. Chitosan is attached to the β-integrins at the epithelial cell surface, causing them to cluster; an intracellular signaling cascade is evoked which leads to the entry of cladin-4 proteins into the cells. Therefore tight junctions open which allows the antigen to penetrate easily [49]. Furthermore, chitosan, especially high-molecular-weight chitosan, reduces the rapid removal of antigens from the nasal cavity; therefore it would keep antigen in the mucosa for a longer period.

19.2.3 Controversy about chitinous microparticles adjuvanticity The effect of the size of CMPs on the activation and stimulation of macrophages has been studied. Nishyama et al. [50] pointed out that following the phagocytosis of microparticles of chitin by macrophage and entering it into the cell, the mitogen-activated protein kinases (MAPK) signaling pathway is activated and induces TNF-α production. They used chitosan microparticles and chitin with the size of 1
10 μm, in addition to microparticles of chitin with a size greater than 50 μm. Particles above 50 μm, as well as chitosan microparticles in this study, could not activate the MAPK pathway. Da Silva and his colleagues [10] mentioned that the production of IL-10 by chitin microparticles is related to their size. They declared that chitin microparticles with a size of 40
70 μm induce TNF-α (and not IL-10) production from a totally dependent TLR-2 pathway. On the other hand, microparticles below 40 μm (often 2
10 μm), in addition to TNF-α, stimulated the production of antiinflammatory cytokine, interleukin-10, by macrophages. The dectin-1 receptor and the Syk signaling pathway played a major role in producing IL-10 by microparticles below 40 μm. Moreover, the particle size has an effect on the type of receptors involved and the cellular signaling pathways. The large sizes (70
100 μm) or very small ones (,2 μm) cannot interact with cellular receptors in macrophages; consequently, they do not provoke any responses. The researchers have described that IL-10 production by microparticles ,40 μm suppresses inflammation and TH2 responses, thus these particles can be considered as an innate and even acquired immune regulator (modulator).

Handbook of Chitin and Chitosan

618

19. Immunomodulatory activities of chitin and chitosan microparticles

Chitin with larger sizes than macrophages (nonphagocytosis-able) activate M2 macrophages and cause the allergic response. However, microparticles between 1
10 μm activate M1 macrophages. This activation property of M1 for chitin is related to the presence of acetyl groups in the molecule, and depends on the size of the microparticles of chitin. The TLR1 and TLR6 receptors that act in conjunction with TLR2 are capable of detecting PAMPs containing acetyl [9]. In addition to the size, other properties of chitin and chitosan affect their adjuvant activity. Characteristics, such as MW, the DD, purity of the molecule, viscosity, and the presence of impurities are effective in adjuvanicity. It could not be assumed which factor has the main effect on the adjuvant property. The higher MW of chitosan can lead to more stability of the antigen
adjuvant complex at the injection site. In this way, depot and slow release of antigen increase the presentation of APCs and produce more potent responses. Chitosan has amino groups that are protonated and carry positive charges. This causes chitosan to be better dissolved in acidic PH, and can also bond to negative molecules, such as proteins. A unique capability of chitosan is the ability for cross-presentation of antigens by MHC-I molecules in APCs, which leads to the stimulation of cellular immune responses. As antigens along with chitosan are absorbed by APCs, amino groups in the chitosan chains are protonated within the lysosome. In fact, this causes water and ions inflow into the lysosome, thus lysosome disrupt, and antigen as an endogenous antigen can be presented by MHC-I and triggers CD8 1 T cell responses. The disrupted lysosome releases cathepsin B into the cytoplasm, ultimately activating inflammasomes by activating NLRP3, and thus resulting in an inflammatory response [49]. One of the properties of chitosan that affects the bioactivity is the DD. In addition, two other factors—MW and the deacetylation pattern (which refers to the distribution of remaining acetyl groups on polymer chain)—also affect the bioactivity [51]. A lower DD results in more immune responses due to faster degradation in the body. In the medical field, chitosan with 35%
70% DD is more operational than chitosan with higher DD (70%
95%), because it has more solubility, and also more biodegradability. When the distribution pattern of acetyl groups is uniform and homogeneous on the chitosan polymer chain, the solubility of the molecule is higher than when it has a heterogeneous pattern [52]. Recently, a form of chitosan called viscosan is produced, which has approximately 50% DD and a homogeneous distribution of acetyl groups. A hydrogel compound can be made with 99% water and 1% viscosan. It can be used to make particles with a controllable size and features, such as quick biodegradability and large surface area, for medical use. In heterogeneous polymers, due to the accumulation of positive charges in one part of the molecule, when interacting with the cells, a

Handbook of Chitin and Chitosan

19.3 Anticancer effect of chitin and chitosan microparticles

619

stronger connection between the positive and negative charges was obtained; while in polymers with a homogeneous distribution pattern because of steric hindrance which acetyl groups exert, interactions are weaker. Studies have shown potent immunological responses when using chitosan with a heterogeneous pattern [53]. The pathways for cellular signaling in the chitosan-induced immune response are known as cGAS-STING and NLRP3 pathways. The researchers mentioned that, besides MW and DD, the chitosan dosage also had an effect on the immune response profile, and by setting the exact dose the development of the desired modulatory response could be possible. They explained that high-dose chitosan, in an encounter with macrophages, stimulates a condition similar to intracellular pathogens, such as mycobacterium tuberculosis. Meanwhile, by the activation of inflammasomes and stimulation of the release of PGE2, it reduced the production of IL-10 and type 1 IFNs. However, at low doses without activating inflammasomes it triggers the responses of type 1 IFNs [54]. Therefore the variations in the results of cytokine responses following stimulation by CMPs are the following: the different methods of preparation, different sources of molecule, shape, size, purity, MW, DD, and dose. In general, chitin, chitosan, and their derivatives in soluble and particle forms as adjuvants have the following characteristics: • Enabling the delivery of antigens through mucosal tissues. • Increasing antigen presentation to APCs in mucosal lymphoid tissues due to their positive charges and mucoadhesive properties. • Ability to open tight junctions in mucosal epithelium cells. • Ability to deliver large molecules, such as plasmid DNA. • Having immunomodulatory properties by inducing regulatory cytokines secretion.

19.3 Anticancer effect of chitin and chitosan microparticles Treatment of tumors by the assistance of chemotherapy, immunotherapy, and gene therapy all require effective drug delivery systems. Among the numerous carriers, as well as the antitumor agents studied by the researchers, chitin, chitosan, and their derivatives have been investigated extensively. Studies on the antitumor properties of chitin and chitosan are categorized in the following: 1. Those which have introduced chitin and chitosan themselves as anticancer agent. 2. Those which use chitin and chitosan in various forms as carriers of antitumor drugs.

Handbook of Chitin and Chitosan

620

19. Immunomodulatory activities of chitin and chitosan microparticles

19.3.1 Chitin and chitosan as anticancer agents There have been many studies regarding the antitumor effects of chitin and chitosan and the mechanisms of these effects. Inhibition of tumor cell growth, induction of proliferation of T cells, induction of apoptosis in cancer cells, antimetastatic activity, and antioxidant effects have been precisely investigated. Some of the intrinsic characteristics of CMPs are effective in antitumor activity. Chitosan and derivatives with antioxidant properties could prevent cancer [55]. Chitin and chitosan have a mucoadhesive property in the surface of epithelial cells and mucous membranes, due to their positive charges. By attaching chitin and chitosan to the membrane of the mammalian cells, the ionic environment and the surface electric charges are affected and altered. Based on the electrostatic interactions, tumor cell’s membrane permeability changes occur and cell viability could reduce; consequently, necrosis takes place [56]. The hexamer and heptamer oligosaccharides of chitosan have a significant antitumor effect [57]. Chitosan oligosaccharides (COS) are obtained from the hydrolysis of chitosan and since they have higher solubility and lower viscosity than chitosan, they have beenwell studied as an antitumor agent. COS usually consist of 2
10 monomers of glucosamine and by adding hydrophilic groups, can increase the solubility or by adding hydrophobic factors, the particles rapidly join together to make micro- and nanoparticle structures [58]. COS can inhibit angiogenesis by a mechanism dependent on the DD and polymerization [59]. Furthermore, amino-COS derivatives, such as amino ethyl derivatives, would have a greater inhibitory effect on the proliferation of cancerous cells [60]. Primarily, in 1986 Suzuki et al. [61] defined antitumor properties for COS against sarcoma-180 solid tumors in Balb/c mice and MM-46 solid tumors in C3H/HC mice. In 1988 inhibition of the growth of the Meth-A solid tumor in Balb/c mice was reported by Tokoro et al. [62] with the aid of chitosan oligomers. This group has pointed to an increase in IL-1 and IL-2, followed by activation, maturation, proliferation, and the migration of cytotoxic T-lymphocytes into the tumor site. COS, when used in the culture medium of murine peritoneal macrophages with IFN-γ, stimulate nitric oxide synthesis in macrophages and exert antitumor effects on Meth-A fibrosarcoma cells [63]. The cytotoxicity effect, in the Carreno et al. [64] study was also reported following the use of chitosan microspheres in the mouse melanoma cell line (B16F10). Therefore having an immune-stimulating property would inhibit and control the tumor growth by applying immunological effects of COS. COS by the ability to stimulate the production of IL-1β and TNF-α are capable of antitumor activity [65]. Lowmolecular-weight COS by increasing the activity of NK cells showed

Handbook of Chitin and Chitosan

19.3 Anticancer effect of chitin and chitosan microparticles

621

their anticancer effects in mice with Sarcoma 180-bearing mice [66]. Also, COS exert their antitumor effects by increasing the differentiation and stimulation of the proliferation of cytotoxic T-lymphocytes in acquired immunity [67]. Inhibition of tumor cell proliferation and induction of apoptosis in cancerous cells is an indispensable issue in the cancer therapy. Hasegawa et al. [68] showed that chitosan inhibits proliferation and induces apoptosis in bladder tumor cells; moreover, it can be used to treat bladder cancer. It was explained that chitosan in soluble form induces apoptosis by interacting with the cell membrane; however, nanoparticles and microparticles have the ability to enter the cell, where they induce apoptosis by binding to specific targets. Chitosan particles with a higher charge density could have a greater toxic effect on the gastric carcinoma cell line (MGC803) [69]. The negative charge on the surface of the tumor cells is much higher than normal cells which cause the invasion and metastasis of the cells. Chitosan through positive charges binds to cancer cells (and not to normal cells) and applies the cytotoxicity effects by membrane disruption and induction of apoptosis, as well as necrosis in cancerous cells [56]. Reduction of DNA replication and inhibition of cell proliferation in hepatocellular tumor cells and lung cancer metastasis suppression, has been reported in Shen et al. study [70]. Cell cycle genes, including PCNA, CyclinA, and CDK-2, showed downregulation after treatment by chitosan oligomers; nevertheless, P21 was overexpressed. Induction of apoptosis from the mitochondrial pathway and through upregulation of proapoptosis genes, for example, Bax, and an increase in caspase 9 activity was shown by Gibot and his colleagues [71]. Angiogenesis is a necessary process for tumor growth. One of the major molecules which is required for angiogenesis of tumors (as a proangiogenesis molecule) is matrix metalloproteinase-9 (MMP-9). Shen et al. [70] and Quan et al. [72] mention the reduction in the production and secretion of MMP-9 and also tumor metastasis. CHI3L1, which is a member of the family of CLPs, is a chitin-binding glycoprotein that is secreted from various immune cells. This molecule stimulates the production of proangiogenesis molecules. The usage of microparticles of chitin inhibits CHI3L1 and reduces the expression of proangiogenesis molecules (like MMP-9, CCL2 and CXCL2). Chitin stimulates the secretion of IL-12 by acting on macrophages, and in the presence of this cytokine, IFN-γ production increases by T-lymphocytes. In the tumor, chitin, by interacting with and inhibiting CHI3L1, changes the immune response against cancer from TH2 (tumorigenic) to TH1 (antitumorogenic) type; thereby limiting its metastasis and tumor growth [73]. Overall, chitin, chitosan, and their derivatives induce the following antitumor properties: antiangiogenesis activity, reduction of the production and secretion of MMP-9, inhibition of proliferation and growth of

Handbook of Chitin and Chitosan

622

19. Immunomodulatory activities of chitin and chitosan microparticles

cancer cells, reduction of tumor metastasis, increase in the proliferation of innate immune cells, and stimulation of their migration to the tumor site.

19.3.2 Chitin and chitosan microsphere as carriers of anticancer drug As of now, there are many antitumor drugs available, but appropriate and effective drug delivery systems are not effective enough. Intravenous injection of chemotherapeutics drugs causes systematic distribution, along with the heterogeneity of the vessels in the tumor, to form barriers preventing the release of sufficient amounts of drugs within the tumor. Solid tumor mass has a high interstitial pressure, acidic pH, hypoxic conditions, and does not contain lymphatic drainage. According to the aforementioned information, the drug would not be able to pass to the center of the tumor mass. To prevent the systemic distribution of chemotherapeutic medication a productive carrier system is needed. The carrier ought to be biodegradable, being adjustable to acidic conditions of the tumor and if used orally would be resistant to gastric acid and digestive system conditions. As mentioned, the above conditions are essential for a drug carriage system development. The high loading capacity and pH-sensitive drug release at the site of the tumor are the benefits of chitosan-based carrier systems [74]. Chitosan microspheres allow the release of the drug in a controlled and stable manner. These microspheres also reduce drug toxicity and facilitate the transfer of hydrophilic or hydrophobic drugs. The chemotherapeutic agent, PTX (paclitaxel), is insoluble, but when it is carried by chitosan microspheres, the dissolution increases. Compared to the other carriers, such as liposomes [75], chitosan microspheres show a high efficiency in drug entrapping depending on pH conditions (pH 3
5) and the MW [76]. The presence of functional groups in chitosan has made it possible to make chemical changes on it and to acquire unique and suitable features [77]. The concentration of polymer is effective in the drug release profile from chitosan microparticles. The concentration of chitosan was effective in controlling the size and morphology of microspheres. At high concentration, the thickness of the microparticles would be higher and the number of pores would be less; thus the release pathway of the drug would be prolonged and the release would be slower. Also, the higher polymer concentrations cause an increase in the viscosity of the aqueous phase, which leads to the particles’ stability and prevent the outflow of the drug, thus increasing the efficiency of drug entrapping. The increased amount of crosslinking agents to chitosan which cause microparticles to have higher density also has a quite significant effect on drug entrapment [78].

Handbook of Chitin and Chitosan

19.4 Allergy and chitinous microparticles

623

One feature of the drug delivery system is the optimal drug volume released to the desired location. Biodegradability is a factor that affects the rate and amount of drug release. Chitin and 31% acetylated chitin, are well degraded enzymatically by lysozyme, so they both have high biodegradability. Also, the incorporation of Pluronic F-108 (which is a three-part copolymer) to chitin, gives chitin an amphipathic property. When Nsereko et al. [79] used ,80 μm chitin/pluronic microparticles in their study (by an average of approximately 40% chitin acetylation), their enzymatic biodegradability and drug release were significantly higher than the chitin microparticles solo. Chitosan, like chitin, is well degraded in the living environment and in the presence of lysozyme and chitinase enzymes. Chitosan is sensitive to pH and dissolves in the acidic environments and is insoluble in water and alkali. A chitosanbased delivery system designed by Tian and his colleagues, which carried an anticancer prodrug (LZC-2b), was sensitive to pH changes [74]. The drug was released at low pH (pH 5 5) with high efficacy (90.3%). Generally, these microparticles can reduce the side effects of anticancer drugs by transferring and delivering drugs to the exact location of the tumor, and the limitations that a drug can have in the body, such as insolubility, would be eliminated by encapsulating the drugs in these particles.

19.4 Allergy and chitinous microparticles Allergic diseases rank among the main public health concerns in the industrialized world; this is probably due to the effects of lifestyle in the developed world leading to more exposure to outdoor and indoor allergens [3]. A specific immune reaction to an antigen starts the allergic reaction leading to the induction of TH2 (CD4 1 ) lymphocytes differentiation and activation. Then, TH2 cells trigger synthesizing allergenspecific IgE from plasma cell by the production of IL-4, IL-5, and IL-13. Consequently, immediate-type hypersensitivity is achieved and eosinophil and M2 macrophages are recruited in allergic inflammatory sites leading to unbridled TH2 immune response upregulation [80]. Hence, boosting TH1 and Treg immune responses could be helpful for protecting against allergic diseases. Some studies reported that chitin microparticles possess TH1 adjuvant feature and is capable of preventing allergic disorders. According to Shibata et al., administration of phagocytosable chitin particles orally to a mice model of ragweed allergy downregulated allergen-induced IgE levels and lung eosinophilia. TH2 responses were considerably inhibited by chitin particles via the reduction of IL-4, IL-5, and IL-10 levels and increased IFN-γ in spleen cells isolated from the ragweed-immunized mice [3]. Moreover, Strong et al. [37] focused on the efficiency of the intranasal application of chitin microparticles

Handbook of Chitin and Chitosan

624

19. Immunomodulatory activities of chitin and chitosan microparticles

,10 μm for treating the symptoms of allergic asthma; they confirmed the efficiency in mouse models of house dust mite (HDM) and Aspergillus fumigatus. They also reported that intranasal delivery of chitin microparticles could decrease serum IgE, peripheral blood eosinophilia, and airway hyperresponsiveness efficiently. Besides, applying chitin microparticles in both allergy models increased IL-12, IFN-γ, and TNF-α and reduced IL-4 during allergen challenge. Administration of chitin microparticles intranasally in the neonatal mice, prevented and treated histopathologic changes and their administration prior to sensitization and during allergen challenge, improving histopathologic variations in the goblet cell numbers, basement membrane, epithelial and subepithelial smooth muscle layer thicknesses of asthmatic airways in mice [81]. Also chitin and chitosan helped mice fight peanut-induced anaphylaxis reactions. Likewise, the levels of IL-5, IL-13, and IL-10 were considerably suppressed in the treated mice [82]. In addition, Sigsgaard et al. [83] reported the antiallergy effects of chitin microparticles and argued that in comparison with the placebo instillation of saline in healthy volunteers, the TH2 cytokines (IL-4 and IL-6) was downregulated with the instillation of chitin (,20 μm). However, other studies argued that the major allergens are mites and cockroaches, and chitin is their abundant component capable of activating immune cells, thus helping the development of allergic disorders such as asthma [84]. Consistent with this issue, the ability of chitin for inducing recruitment of eosinophils and basophils, and the development of TH2-cell response and allergic disorders, such as asthma, was approved when administered to the lungs in mice by Reses et al. [85]. Da saliva et al. [9] demonstrated that intraperitoneal administration of chitin particles facilitated OVA-induced airway inflammation with development of type 2-cytokine-mediated eosinophilia and IL-17mediated neutrophilia, and these effects were mediated by pathways involving TLR-2, MyD88, and IL-17A. Also, according to O’Dea et al. [86] lung transcription of the TH2-associated chemokines CCL11 (eotaxin) and CCL22 (macrophage-derived chemokine) was increased following the multiple aspirations of the high-chitin expressing isolate of A. fumigatus along with reduced IFN-γ and also induced airway eosinophilia in the lungs of recipient mice. Another study reported that the accumulation of eosinophils and alternatively activated macrophages in mice requires the activation of resident innate lymphoid type 2 cells (ILC2s) to express IL-5 and IL-13 by the inhaled chitin [87]. Moreover, another study reported that chitin increased OVA-induced airway inflammation in HDM [84]. Kelin et al. argued that intratracheal administration of chitin in mice generated C3a, thus chitin induces allergic inflammation based on C3 and C3aR signaling in the lung by boosting

Handbook of Chitin and Chitosan

19.4 Allergy and chitinous microparticles

625

TH2 responses [88]. Furthermore, a case study reported that body moisturizing cream with chitin or chitosan can cause atopic dermatitis in some people [89].

19.4.1 The reasons for the controversy about chitin microparticles in allergic disorders The role of chitin in the induction of the innate and adaptive immune responses is complex and very controversial. Possible reasons for disparate findings regarding this polysaccharide include the size of the chitin particles, the use of different sources (e.g., shrimp, crab, fungal), concentration, and anatomical site of encounter [90,91]. One study showed that intraperitoneal administration of large chitin beads ( . 50 μm), not chitin microparticles (,10 μm), induced innate eosinophilia and increased the expression of arginase, manose receptor, and Dectin-1 in macrophages, thus promoting TH2 immune responses while chitin microparticles ,10 μm induced TH1 immune responses. Consequently, this study argued that the effects of chitin in vivo are highly dependent on particle size [92]. In another study, Kim et al. [93] found that chitin enhanced the IL-33 production, but enzymatically cleaved chitin is taken up by phagocytosis, which then causes the activation of caspase-1 and caspase-7. The activation of these caspases inactivates IL-33, thereby resolving type 2 immune responses. Although they also reported that intranasal administration of chitin itself could elicit pulmonary inflammation. On the other hand, there is evidence that chitin polarizes the immune responses in different directions depending on the route of administration. Arae et al. [94] showed that any size of chitin particles has similar potency as an allergen. Besides, they showed that the mechanisms for the effect of chitin via the intranasal route are not similar to those via the intraperitoneal route. In the intranasal route, IL-4/ IL-13-STAT6 mediates the effects of chitin independent from IL-17A, whereas the mediation of the intraperitoneal route was dependent on IL-17A. In this context, another study reported that in response to inhaled chitin
MP, CCL2 is produced by airway epithelial cells and the polarization, recruitment, and activation of eosinophils need CCR2 signaling. Hence, chitin could be possibly be a type 2-cytokineassociated regulator [95]. Another possible explanation for the varied immunological response in these studies could be the LPS contamination of the used chitin
MP material because it is difficult to purify chitin extensively. Overall, these observations suggest that chitin has the capacity to stimulate an immune response in multiple directions depending on experimental conditions [14,91].

Handbook of Chitin and Chitosan

626

19. Immunomodulatory activities of chitin and chitosan microparticles

19.4.2 Chitinase activity in allergic disease Given the data on the function of chitinase for the degradation of chitin, recently researchers have focused on the association between the exposure to chitin with allergic mediators and the expression of chitinases. Chitinases consist of a group of related enzymes capable of degrading chitin; they are maintained within mammalian species including humans. Mammalian chitinase and CLPs are significantly involved in host defense against chitin-containing pathogens. Reses et al. [85] reported that allergen-induced TH2-type airway inflammation enhanced by a commercially available chitin was inhibited by chitin digestion with acid mammalian chitinase (AMCase). Another study showed that chitinase may inhibit the TH2-cell response to inhaled allergens, enhanced by HDM-derived chitin [84]. Moreover, Kim et al. found that AMCase plays a crucial role in the control of type 2 immune responses to chitin or HDM [93]. Unlike the reported findings, one study demonstrated that chitin stimulates AMCase and eotaxin expression by primary human sinonasal epithelial cells derived from sinusitis patients and induces eosinophilic inflammation within the sinonasal tract [96]. Interestingly, expression of some of the chitinases or CLPs is upregulated by TH2 cytokines in certain inflammatory and allergic conditions [91,97,98]. Several studies reported that the increase in AMCase expression and chitinase activity in TH2 inflammation may indicate that AMCase is acting to exacerbate allergic reactions in animal models [99
101]. Shen et al. demonstrated that AMCase was increased in the lung tissues of mice primed and sensitized with OVA or Der-P2 [4]. A landmark study by Zhu et al. [102] showed that AMCase is produced in epithelial cells and macrophages through a TH2-specific (IL-13
mediated) pathway in an aeroallergen asthma model. Also, the administration of anti-AMCase sera in the aeroallergen challenge model inhibited AMCase activity and ameliorated TH2 inflammation and airway hyperresponsiveness by inhibiting chemokine induction, so these results suggested that AMCase may be an important mediator in TH2-dominated disorders such as asthma. In support of this contention, subsequent studies also demonstrated that AMCase is an effective mediator in development human asthma [103], ocular allergies [104], and allergic airway inflammation [16]. Some experiments also demonstrated that the level of expression of the CLPs YKL-40 (chitinase 3-like 1) in serum and tissue is increased during TH2-type inflammation [93,105,106]. Overall, it seems that in nonchitin-dependent allergic models, such as the ovalbumin-induced allergic inflammation model and IL-13
overexpressing transgenic mice, the inhibition of chitinase activity ameliorated type 2 inflammation and airway hyperreactiveness. In contrast, in adaptive TH2 responses induced by chitin, AMCase inhibits chitin-induced inflammation [98];

Handbook of Chitin and Chitosan

19.5 Inflammation and chitin, chitosan microparticles

627

however, this topic has not been fully verified and also further studies are needed to demonstrate whether chitinase and CLP are the cause or effect? Overall, chitinases are involved in the regulation of the immune response, but it is not clear whether chitinases and chitolectines exacerbate or restrict the inflammatory process.

19.5 Inflammation and chitin, chitosan microparticles The usual response to pathogens, injuries, toxic molecules, or physical damage like burns or cuts is inflammation involving the innate and adaptive immune systems. The initial phase of inflammation is called the acute inflammatory response, which could grow into a chronic inflammatory condition and then probably lead to autoimmune or autoinflammatory disorders, neurodegenerative disease, or cancer; thus dealing with the inflammatory response is of tremendous importance. Antiinflammatory agents are mediators that reduce the production or activities of proinflammatory cytokines and block immune cell trafficking into tissues, hence they may develop to treat inflammation. Nishimura et al. [107
109] described that some chitin/chitosan derivatives can activate macrophages function; these particles also induce the production of cytokines such as IL-1 and colony-stimulating factors in vivo and in vitro, so they discussed that chitin and chitin derivatives may enhance immunologic adjuvant effects via a proinflammatory response. In another study, Suzuki et al. treated mice intraperitoneal with purified chitin microparticles three times on days 2, 4, and 6; they observed that chitin microparticles increased the number of mouse peritoneal exudate cells (PEC) and these induced PEC can cause the generation of reactive oxygen intermediates and candidacidal activities [110]. Researcher demonstrated that the intravenous administration of phagocytosable chitin particles (1
10 μm) prime alveolar macrophages to give a large oxidative burst via enhancement of superoxide anion release [12,111]. In another study, stimulation of RAW 264.7 cells, a murine macrophage-like cell line, with chitin led to the activation of MAPK associated with TNF cytokine production and cyclooxygenase-2 expression with the increased PGE2 release and these factors induce inflammation [50]. In Alvarez’s study [8], it was also noted that crab chitin particles can cause a proinflammatory immune response through secretion of IL-6 and IL-1β in human peripheral blood mononuclear cells (PBMCs), so it seems that chitin can cause inflammatory responses via activated M1 macrophages. In another study, PBMCs were isolated from obese individuals stimulated with chitin microparticles (1
10 μm). The data showed that chitin microparticles induce IL-6 production, a proinflammatory activator, in obese individuals and this result is

Handbook of Chitin and Chitosan

628

19. Immunomodulatory activities of chitin and chitosan microparticles

correlated with BMI, elevated plasma IL-6 levels, and insulin resistance [111]. Also, Koller et al. showed that chitin activated innate immune cells and explained that proinflammatory effects of chitin are mediated through upregulating the production of CXCL8 (IL-8), IL-6, and thymic stromal lymphopoietin and by increasing TLR4 surface expression on primary and immortalized keratinocytes [112]. However, some of the studies reported that chitin induces inflammatory responses by alternative activation of macrophages in vivo and in vitro. Chitin led to the expression of IL-5 and IL-13 from ILC2s that are necessary for the accumulation of alternatively activated macrophages [87]. Hu et al. demonstrated that chitin-induced M2 macrophages’ polarization through transcription factor RBP-J in vitro and in vivo [113]. In another study, it was found that M2 macrophages’ polarization was induced in response to chitin administration and helminth infection [114]. Da Silva et al. found that chitin is a size-dependent PAMP that induces acute inflammation via MyD88-, TLR-2-, and IL-17-dependent pathways [115]. In another study, this group demonstrated that big chitin particles (70
100 μm) and supersmall chitin particles (,2 μm) are inert but intermediate-sized chitin (40
70 μm) stimulates TNF-α production and induces proinflammatory responses through TLR2-, dectin-1-, and NF-dependent pathways. Also, small chitin particles (,40 μm) inhibit tissue inflammation via induction of IL-10 production in a dectin-1- and Syk-dependent pathway [10]. According to another study, IL-10 is highly secreted and inflammatory responses are decreased at low chitin concentrations (1
10 μg/mL). However, high-chitin concentrations (250
1000 μg/mL) strongly induce TNF secretion via dectin-1 and TLR2 and intermediate concentrations of chitin (50
100 μg/mL) induce equal amounts of both cytokines. Gow et al. believed that ultrapure fungal chitin (1
10 μm) induced an antiinflammatory response (IL-10 secretion) through mannose receptors and signaling that activated NOD2, and TLR-9 decreased LPS-induced inflammation in vivo [18]. Nagatani et al. showed that chitin microparticles exert antiinflammatory effects in the acute and chronic types of intestinal inflammatory conditions. In this study, administering CMPs orally significantly inhibited acute and chronic colitis in chitintreated mice in comparison with PBS-treated mice [6]. According to our findings, the secretion of IL-10 was induced in a murine model of leishmaniasis following treatment with an intermediate concentration of CMPs (,40 μm), thus chitin regulates the immune response and helps mice fight inflammation from leishmaniasis [5,11]. Chitosan has a stimulatory effect in vivo; it has been reported that it can act as a proinflammatory mediator. However, other results argued that chitosan does not stimulate an inflammatory response. For example, Shibata et al. reported that chitosan particles (1
10 μm) are unable

Handbook of Chitin and Chitosan

19.6 Conclusion

629

to prime alveolar macrophages to release superoxide anion; also the production of IFN-γ is not induced by these particles [116]. Also, several studies have shown that chitosan particles (1
10 μm) cannot induce either MAPK activation or cytokine production in RAW 264.7 cells, so M1 macrophages activation does not happen in response to chitosan [50,92,117]. Moreover, Huang et al. declared that neither large chitin beads (40
100 μm) and chitosan microparticles (1
10 μm), nor soluble chitin induce cytokine production by PBMCs isolated from nonobese PBMCs ex vivo [111]. Contrary to the above findings, in others studies it was noted that chitosan induces candidacidal activities through activated PEC when given intraperitoneally [31,110]. In addition, Bueter et al. reported that particles of purified chitosan (,20 μm) activate the NLRP3 (NLR family, pyrin domain-containing 3) inflammasome-dependent IL-1β release, and an inflammatory response in bone marrow
derived macrophages. They also demonstrated that cytochalasin D inhibits the IL-1β stimulatory activity of chitosan, so phagocytosis is essential for inflammasome activation. In this study, chitin microparticles could not activate the NLRP3 that it seems acetylation of the chitosan to chitin lead to loss of this activity [14]. This group, in another study, declared that highly purified chitosan, not chitin, could induce IL-1β and IL-18 release via NLRP3 inflammasome activation in multiple murine and human cell types. They also characterized the mechanisms responsible for inflammasome activation by chitosan. The results showed that K1 efflux, mitochondrial ROS, and lysosomal destabilization each contributed to chitosan activation of the NLRP3 [118]. Furthermore, Huang et al. reported that intratracheal administration of chitosan microparticles results in proinflammation in rat lung tissues. Increased bronchoalveolar lavage fluid protein, lactate dehydrogenease activity, increased lung tissue myeloperoxidase activity, and leukocyte migration are involved significantly in this process [119]. Chitolectin (YKL-40) cause angiogenesis and suppress inflammasome activation. In another study, it was reported that chitosan and chitosan derivatives decrease YKL-40 secretion in primary macrophages and also induce inflammation via inflammasome dysregulation [120]. Overall, these studies revealed that CMPs are capable of stimulating the production of pro- and antiinflammatory cytokines differently according to size, concentration, and acetylation degree.

19.6 Conclusion There is no consensus regarding the immunogenic properties of chitin and chitosan. Data on CMPs is misleading. Variation of biological

Handbook of Chitin and Chitosan

630

19. Immunomodulatory activities of chitin and chitosan microparticles

sources, deacetylation degree, solubility and biodegradability, purity, dose, size, shape, route of administration, and cellular model used (e.g., primary or cell line, murine, plant or human origin) are capably of inducing inconsistent immune responses to CMPs. It is believed that immune recognition is probably dependent on the shape of the chitin particles [8]. The exact effect of the shape of the chitin particles on their capability for inducing an immune response requires further studies. The magnitude of acetylase and deacetylase activity for chitosan and/or chitin in the body also might be a factor in the regulation of inflammation. The presence of proinflammatory costimulation agents (e.g., endotoxin content) can lead to those inconsistent immune responses. It is not easy to purify chitin since it is insoluble and it is usually found crosslinked to other cell wall components that could not be eliminated easily. Variable immune response to CMPs probably could be explained by the difficulty in purifying chitin and the effect of the chemical and physical treatments during purification on the structure of the chitin. Inconsistent immune responses could be induced by concurrent stimulations. The S. aureus immune evasion protein, “staphylococcal superantigen-like protein 3,” is capable of blocking chitin-binding and TLR2 activation, thus coinfections of S. aureus and pathogenic fungi such as C. albicans are frequently observed [121]. Multiple attributes have been shown from the in vivo setting that cannot be replicated in vitro. For example, CMPs are capable of possessing both pro- and antiinflammatory features according to the presence of antichitin antibody [15]. So the interaction of CMPs with serum proteins like complement and antibody can influence the recognition and introduction to cells, which influence the host response greatly. Cytokine profiles resulting from chitin stimulation in vitro could not be used as a basis for judging the effect of in vivo chitin immunomodulation. It should be noted that different procedures were used to perform the experiments in different studies, thus making comparison problematic, and further studies should be done by applying standardized procedures to settle the current debate. Weber et al. were able to detect six-subunit-long chitin chains as the smallest immunologically active motif using defined chitin (N-acetyl-glucosamine) oligomers [2]. Contradictions in the immune response are due to the snapshot views compared with the whole immune response during the time course of the CMPs digestion in the body. Chitinous macromolecules appear to be the key factor to stimulate the secretion of chitinases and chitosanase, and these enzymes break down nonphagocytable macromolecules into phagocytable microparticles, then into receptor-stimulating oligomeric agents, and ultimately full degradation into glucosamine resulting in the immunometabolism effect. Handbook of Chitin and Chitosan

References

631

References [1] K. Nishimura, S. Nishimura, N. Nishi, I. Saiki, S. Tokura, I. Azuma, Immunological activity of chitin and its derivatives, Vaccine 2 (1984) 93
99. Available from: https://doi. org/10.1016/S0264-410X(98)90039-1. [2] K. Fuchs, Y. Cardona Gloria, O. Wolz, F. Herster, L. Sharma, C.A. Dillen, et al., The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size, EMBO Rep. 19 (2018) e46065. Available from: https://doi.org/ 10.15252/embr.201846065. [3] Y. Shibata, L.A. Foster, J.F. Bradfield, Q.N. Myrvik, Oral administration of chitin downregulates serum ige levels and lung eosinophilia in the allergic mouse, J. Immunol. 164 (2000) 1314
1321. Available from: https://doi.org/10.4049/jimmunol.164.3.1314. [4] C.R. Shen, H.H. Juang, H.S. Chen, C.J. Yang, C.J. Wu, M.H. Lee, et al., The correlation between chitin and acidic mammalian chitinase in animal models of allergic asthma, Int. J. Mol. Sci. 16 (2015) 27371
27377. Available from: https://doi.org/10.3390/ ijms161126033. [5] S. Ghotloo, M.H.M. Hoseini, M.H. Alimohammadian, V. Khaze, A. Memarnejadian, A. Rostami, Immunomodulatory effects of chitin microparticles on Leishmania majorinfected BALB/c mice, Parasitol. Int. 64 (2015) 219
221. Available from: https://doi. org/10.1016/j.parint.2014.12.007. [6] K. Nagatani, S. Wang, V. Llado, C.W. Lau, Z. Li, A. Mizoguchi, et al., Chitin microparticles for the control of intestinal inflammation, Inflamm. Bowel Dis. 18 (2012) 1698
1710. Available from: https://doi.org/10.1002/ibd.22874. [7] S. Mami, F. Yeganeh, E. Farahani, A. Anissian, M. Haji Molla Hoseini, Chitin micro particles regulate splenocytes immune response in experimental autoimmune encephalomyelitis, Iran J. Allergy Asthma Immunol. 18 (2019) 190
199. [8] F.J. Alvarez, The effect of chitin size, shape, source and purification method on immune recognition, Molecules 19 (2014) 4433
4451. Available from: https://doi. org/10.3390/molecules19044433. [9] C.A. Da Silva, P. Pochard, C.G. Lee, J.A. Elias, Chitin particles are multifaceted immune adjuvants, Am. J. Respir. Crit. Care Med. 182 (2010) 1482
1491. Available from: https://doi.org/10.1164/rccm.200912-1877OC. [10] C.A. Da Silva, C. Chalouni, A. Williams, D. Hartl, C.G. Lee, J.A. Elias, Chitin is a size-dependent regulator of macrophage TNF and IL-10 production, J. Immunol. 182 (2009) 3573
3582. Available from: https://doi.org/10.4049/jimmunol.0802113. [11] M. Haji Molla Hoseini, M. Moradi, M.H. Alimohammadian, V. Khaze Shahgoli, H. Darabi, A. Rostami, Immunotherapeutic effects of chitin in comparison with chitosan against Leishmania major infection, Parasitol. Int. 65 (2016) 99
104. Available from: https://doi.org/10.1016/j.parint.2015.10.007. [12] Y. Shibata, L.A.N.N. Foster, W.J. Metzger, Q.N. Myrvik, M. Enterprises, N. Carolina, Alveolar macrophage priming by intravenous administration of chitin particles, polymers of N -Acetyl-D
glucosamine, in mice, Infect. Immun. 65 (1997) 1734
1741. [13] P. Strong, K. Okuda, Chitin micro-particles (CMP): a useful adjuvant for inducing viral specific immunity when delivered intranasally with an HIV-DNA Vaccine, Viral Immunol. 16 (2003) 541
547. Available from: https://doi.org/10.1089/ 088282403771926355. [14] C.L. Bueter, C.K. Lee, V.A.K. Rathinam, G.J. Healy, C.H. Taron, C.A. Specht, et al., Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis, J. Biol. Chem. 286 (2011) 35447
35455. Available from: https://doi. org/10.1074/jbc.M111.274936. [15] K. Becker, V. Aimanianda, X. Wang, M. Gresnigt, A. Ammerdorffer, C. Jacobs, et al., Aspergillus cell wall chitin induces anti- and proinflammatory cytokines in human

Handbook of Chitin and Chitosan

632

[16]

[17]

[18]

[19]

[20]

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

19. Immunomodulatory activities of chitin and chitosan microparticles

PBMCs via the Fc-γ receptor/Syk/PI3K pathway, Am. Soc. Microbiol. 7 (2016) 1
11. Available from: https://doi.org/10.1128/mBio.01823-15.Editor. C.G. Lee, C.A. Da Silva, C.S. Dela Cruz, F. Ahangari, B. Ma, J. Kang, et al., Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodeling, and injury, Annu. Rev. Physiol. 73 (2011) 1
28. Available from: https://doi.org/10.1146/ annurev-physiol-012110-142250.Role. M. Krolicka, S.W.A. Hinz, M.J. Koetsier, R. Joosten, G. Eggink, L.A.M. Van Den Broek, et al., Chitinase Chi1 from Myceliophthora thermophila C1, a thermostable enzyme for chitin and chitosan depolymerization, J. Agric. Food Chem. 66 (2018) 1658
1669. Available from: https://doi.org/10.1021/acs.jafc.7b04032. J. Wagener, R.K.S. Malireddi, M.D. Lenardon, M. Ko¨berle, S. Vautier, D.M. MacCallum, et al., Fungal chitin dampens inflammation through IL-10 induction mediated by NOD2 and TLR9 activation, PLoS Pathog. 10 (2014). Available from: https://doi.org/10.1371/journal.ppat.1004050. C. Liu, Q. Li, X. Zhou, V.P. Kolosov, J.M. Perelman, The chitinase-like protein YKL40 increases mucin5AC production in human bronchial epithelial cells, Exp. Cell Res. 319 (2013) 2866
2873. Available from: https://doi.org/10.1016/j.yexcr.2013.08.009. M.H. Sohn, J.H. Lee, K.W. Kim, S.W. Kim, S.H. Lee, K.E. Kim, et al., Genetic variation in the promoter region of chitinase 3-like 1 is associated with atopy, Am. J. Respir. Crit. Care Med. 179 (2009) 449
456. Available from: https://doi.org/10.1164/ rccm.200809-1422OC. J. Kzhyshkowska, A. Gratchev, S. Goerdt, Human chitinases and chitinase-like proteins as indicators for inflammation and cancer, Biomark. Insights 2 (2007) 128
146. Available from: https://doi.org/10.1177/117727190700200023. K. Vega, M. Kalkum, Chitin, chitinase responses, and invasive fungal infections, Int. J. Microbiol. (2012) 10. Available from: https://doi.org/10.1155/2012/920459. N. Gao, F.-S.X. Yu, Chitinase 3-like 1 promotes Candida albicans killing and preserves corneal structure and function by controlling host antifungal responses, Infect. Immun. 83 (2015) 4154
4164. Available from: https://doi.org/10.1128/iai.00980-15. M.G. Netea, J. Quintin, J.W.M. Van Der Meer, Trained immunity: a memory for innate host defense, Cell Host Microbe. 9 (2011) 355
361. Available from: https://doi.org/ 10.1016/j.chom.2011.04.006. C.D.C.C. van der Heijden, M.P. Noz, L.A.B. Joosten, M.G. Netea, N.P. Riksen, S.T. Keating, Epigenetics and trained immunity, Antioxid. Redox Signal. 29 (2018) 1023
1040. Available from: https://doi.org/10.1089/ars.2017.7310. ´ P. Rusek, M. Wala, M. Druszczynska, M. Fol, Infectious agents as stimuli of trained innate immunity, Int. J. Mol. Sci. 19 (2018). Available from: https://doi.org/10.3390/ ijms19020456. L. Rizzetto, D.C. Ifrim, S. Moretti, N. Tocci, S.C. Cheng, J. Quintin, et al., Fungal chitin induces trained immunity in human monocytes during cross-talk of the host with Saccharomyces cerevisiae, J. Biol. Chem. 291 (2016) 7961
7972. Available from: https://doi. org/10.1074/jbc.M115.699645. F.I. Khan, S. Rahman, A. Queen, S. Ahamad, S. Ali, J. Kim, et al., Implications of molecular diversity of chitin and its derivatives, Appl. Microbiol. Biotechnol. 101 (2017) 3513
3536. Available from: https://doi.org/10.1007/s00253-017-8229-1. X.F. Liu, Y.L. Guan, D.Z. Yang, Z. Li, K.D. Yao, Antibacterial action of chitosan and carboxymethlyated chitosan, J. Appl. Polym. Sci. 79 (2000) 1324
1335. Available from: http://doi.org/10.1002/1097-4628(20010214)79:7 , 1324::AID-APP210 . 3.0.CO;2-L. S.M. Okawa, Y. Kobayashi, S.M. Suzuki, Comparative study of protective effects of chitin, chitosan, and N-acetyl chitohexaose against pseudomonas aeruginosa and listeria monocytogenes infections in mice, Biol. Pharm. Bull. 26 (2003) 902
904. Available from: https://doi.org/10.1248/bpb.26.902.

Handbook of Chitin and Chitosan

References

633

[31] K. Suzuki, Y. Okawa, K. Hashimoto, S. Suzuki, M. Suzuki, Protecting effect of chitin and chitosan on experimentally induced Murine candidiasis, Microbiol. Immunol. 28 (1984) 903
912. Available from: https://doi.org/10.1111/j.1348-0421.1984.tb00746.x. [32] H. Hasegawa, T. Ichinohe, P. Strong, I. Watanabe, S. Ito, S.I. Tamura, et al., Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant, J. Med. Virol. 75 (2005) 130
136. Available from: https://doi.org/10.1002/jmv.20247. [33] Y. Asahi-Ozaki, S. Itamura, T. Ichinohe, P. Strong, S. Ichi Tamura, H. Takahashi, et al., Intranasal administration of adjuvant-combined recombinant influenza virus HA vaccine protects mice from the lethal H5N1 virus infection, Microbes Infect. 8 (2006) 2706
2714. Available from: https://doi.org/10.1016/j.micinf.2006.07.018. [34] T. Ichinohe, N. Nagata, P. Strong, S. Tamura, H. Takahashi, A. Ninomiya, et al., Prophylactic effects of chitin microparticles on highly pathogenic H5N1 influenza, Virus 819 (2007) 811
819. Available from: https://doi.org/10.1002/jmv. [35] B.J.G. Baaten, B. Clarke, P. Strong, S. Hou, Nasal mucosal administration of chitin microparticles boosts innate immunity against influenza A virus in the local pulmonary tissue, Vaccine 28 (2010) 4130
4137. Available from: https://doi.org/10.1016/j. vaccine.2010.04.026. [36] Y. Shibata, I. Honda, J.P. Justice, M.R.V.A.N. Scott, R.M. Nakamura, Q.N. Myrvik, Th1 adjuvant N-acetyl-D-glucosamine polymer up-regulates Th1 immunity but down-regulates Th2 immunity against a mycobacterial protein (MPB-59) in interleukin-10-knockout and wild-type mice, Infect. Immun. 69 (2001) 6123
6130. Available from: https://doi.org/10.1128/IAI.69.10.6123. [37] P. Strong, H. Clark, K. Reid, Intranasal application of chitin microparticles downregulates symptoms of allergic hypersensitivity to dermatophagoides pteronyssinus and Aspergillus fumigatus in murine models of allergy, Clin. Exp. Allergy 32 (2002) 1794
1800. Available from: https://doi.org/10.1046/j.1365-2222.2002.01551.x. [38] I.M. van der Lubben, J.C. Verhoef, A.C. van Aelst, G. Borchard, H.E. Junginger, Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine\rPeyer’s patches, Biomaterials 22 (2001) 687
694. [39] V.J. Ahire, K.K. Sawant, J.B. Doshi, S.D. Ravetkar, Chitosan microparticles as oral delivery system for tetanus toxoid, Drug Dev. Ind. Pharm. 33 (2007) 1112
1124. Available from: https://doi.org/10.1080/03639040701377847. [40] I.M. Van Der Lubben, G. Kersten, M.M. Fretz, C. Beuvery, J.C. Verhoef, H.E. Junginger, Chitosan microparticles for mucosal vaccination against diphtheria: Oral and nasal efficacy studies in mice, Vaccine 21 (2003) 1400
1408. Available from: https://doi.org/10.1016/S0264-410X(02)00686-2. [41] E. Onuigbo, J. Iseghohimhen, K. Chah, M. Gyang, A. Attama, Chitosan/alginate microparticles for the oral delivery of fowl typhoid vaccine: innate and acquired immunity, Vaccine 36 (2018) 4973
4978. Available from: https://doi.org/10.1016/j. vaccine.2018.05.087. [42] H.L. Jiang, M.L. Kang, J.S. Quan, S.G. Kang, T. Akaike, H.S. Yoo, et al., The potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization, Biomaterials 29 (2008) 1931
1939. Available from: https://doi.org/10.1016/j.biomaterials.2007.12.025. [43] Y. Ghendon, S. Markushin, G. Krivtsov, I. Akopova, Chitosan as an adjuvant for parenterally administered inactivated influenza vaccines, Arch. Virol. 153 (2008) 831
837. Available from: https://doi.org/10.1007/s00705-008-0047-4. [44] Y. Ghendon, S. Markushin, Y. Vasiliev, I. Akopova, I. Koptiaeva, G. Krivtsov, et al., Evaluation of properties of chitosan as an adjuvant for inactivated influenza vaccines administered parenterally, Int. J. Clin. Exp. Pathol. 10 (2009) 2599
2608. Available from: https://doi.org/10.1002/jmv.

Handbook of Chitin and Chitosan

634

19. Immunomodulatory activities of chitin and chitosan microparticles

[45] Z. Sui, Q. Chen, F. Fang, M. Zheng, Z. Chen, Cross-protection against influenza virus infection by intranasal administration of M1-based vaccine with chitosan as an adjuvant, Vaccine 28 (2010) 7690
7698. Available from: https://doi.org/10.1016/j. vaccine.2010.09.019. [46] Z. Sui, Q. Chen, R. Wu, H. Zhang, M. Zheng, H. Wang, et al., Cross-protection against influenza virus infection by intranasal administration of M2-based vaccine with chitosan as an adjuvant, Arch. Virol. 155 (2010) 535
544. Available from: https://doi.org/10.1007/s00705-010-0621-4. [47] H. Chang, X. Li, Y. Teng, Y. Liang, B. Peng, F. Fang, et al., Comparison of adjuvant efficacy of chitosan and aluminum hydroxide for intraperitoneally administered inactivated influenza H5N1 vaccine, DNA Cell Biol 29 (2010) 563
568. Available from: https://doi.org/10.1089/dna.2009.0977. [48] X. Wang, W. Zhang, F. Liu, M. Zheng, D. Zheng, T. Zhang, et al., Intranasal immunization with live attenuated influenza vaccine plus chitosan as an adjuvant protects mice against homologous and heterologous virus challenge, Arch. Virol. 157 (2012) 1451
1461. Available from: https://doi.org/10.1007/s00705-012-1318-7. [49] Y. Xia, Q. Fan, D. Hao, J. Wu, G. Ma, Z. Su, Chitosan-based mucosal adjuvants: sunrise on the ocean, Vaccine 33 (2015) 5997
6010. Available from: https://doi.org/ 10.1016/j.vaccine.2015.07.101. [50] A. Nishiyama, S. Tsuji, M. Yamashita, R. Ann, Q.N. Myrvik, Y. Shibata, Phagocytosis of N-acetyl-D-glucosamine particles, a Th1 adjuvant, by RAW 264. 7 cells results in MAPK activation and TNF-, but not IL-10, production, Cell Immunol. 239 (2006) 103
112. Available from: https://doi.org/10.1016/j.cellimm.2006.04.003. [51] H.B.T. Moran, J.L. Turley, M. Andersson, E.C. Lavelle, Immunomodulatory properties of chitosan polymers, Biomaterials 184 (2018) 1
9. Available from: https://doi. org/10.1016/j.biomaterials.2018.08.054. [52] I. Aranaz, M. Mengı´bar, R. Harris, I. Pan˜os, B. Miralles, N. Acosta, et al., Functional characterization of chitin and chitosan, Curr. Chem. Biol. 3 (2009) 203
230. [53] T. Neimert-Andersson, A.C. Ha¨llgren, M. Andersson, J. Langeba¨ck, L. Zettergren, J. Nilsen-Nygaard, et al., Improved immune responses in mice using the novel chitosan adjuvant ViscoGel, with a Haemophilus influenzae type b glycoconjugate vaccine, Vaccine 29 (2011) 8965
8973. Available from: https://doi.org/10.1016/j.vaccine.2011.09.041. [54] D. Fong, C.D. Hoemann, Chitosan immunomodulatory properties: perspectives on the impact of structural properties and dosage, Future Sci. OA 4 (2018). Available from: https://doi.org/10.4155/fsoa-2017-0064. FSO225. [55] D.N. Ngo, S.H. Lee, M.M. Kim, S.K. Kim, Production of chitin oligosaccharides with different molecular weights and their antioxidant effect in RAW 264.7 cells, J. Funct. Foods. 1 (2009) 188
198. Available from: https://doi.org/10.1016/j.jff.2009.01.008. [56] R. Huang, E. Mendis, N. Rajapakse, S.K. Kim, Strong electronic charge as an important factor for anticancer activity of chitooligosaccharides (COS), Life Sci. 78 (2006) 2399
2408. Available from: https://doi.org/10.1016/j.lfs.2005.09.039. [57] C. Xiong, H. Wu, P. Wei, M. Pan, Y. Tuo, I. Kusakabe, et al., Potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro, Carbohydr. Res. 344 (2009) 1975
1983. Available from: https://doi.org/10.1016/j.carres.2009.06.036. [58] P. Zou, X. Yang, J. Wang, Y. Li, H. Yu, Y. Zhang, et al., Advances in characterisation and biological activities of chitosan and chitosan oligosaccharides, Food Chem. 190 (2016) 1174
1181. Available from: https://doi.org/10.1016/j.foodchem.2015.06.076. [59] H. Wu, B.B. Aam, W. Wang, A.L. Norberg, M. Sørlie, V.G.H. Eijsink, et al., Inhibition of angiogenesis by chitooligosaccharides with specific degrees of acetylation and polymerization, Carbohydr. Polym. 89 (2012) 511
518. Available from: https://doi. org/10.1016/j.carbpol.2012.03.037.

Handbook of Chitin and Chitosan

References

635

[60] D. Ngo, T. Vo, D. Ngo, K. Kang, Biological effects of chitosan and its derivatives, Food Hydrocoll 51 (2015) 200
216. Available from: https://doi.org/10.1016/j. foodhyd.2015.05.023. [61] K.O. Suzuki, T. Mikami, Y. Okawa, A. Tokoro, M. Suzuki, Antitumor effect of hexaN-acetylchitohexaose and chitohexaose, Carbohydr. Res. 151 (1986) 403
408. [62] A. Tokoro, N. Tatewaki, K. Suzuki, T. Mikami, S. Suzuki, M. Suzuki, Growthinhibitory effect of hexa-N-acetylchitohexaose and chitohexaose against Meth-A solid tumor, Chem. Pharm. Bull. (Tokyo) 36 (1988) 784
790. [63] W.G. Seo, H.O. Pae, N.Y. Kim, G.S. Oh, I.S. Park, Y.H. Kim, et al., Synergistic cooperation between water-soluble chitosan oligomers and interferon-g for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages, Cancer Lett. 159 (2000) 189
195. Available from: https://doi.org/10.1002/ 0470011815.b2a03094. [64] B. Carren˜o-Go´mez, R. Duncan, Evaluation of the biological properties of soluble chitosan and chitosan microspheres, Int. J. Pharm. 148 (1997) 231
240. Available from: https://doi.org/10.1016/S0378-5173(96)04847-8. [65] J. Feng, L. Zhao, Q. Yu, Receptor-mediated stimulatory effect of oligochitosan in macrophages, Biochem. Biophys. Res. Commun. 317 (2004) 414
420. Available from: https://doi.org/10.1016/j.bbrc.2004.03.048. [66] Y. Maeda, Y. Kimura, Antitumor effects of various low-molecular-weight chitosans are due to increased natural killer activity of intestinal intraepithelial lymphocytes in sarcoma 180-bearing mice, J. Nutr. 134 (2004) 945
950. Available from: https://doi. org/10.1093/jn/134.4.945. [67] K. Azuma, T. Osaki, S. Minami, Y. Okamoto, Anticancer and anti-inflammatory properties of chitin and chitosan oligosaccharides, J. Funct. Biomater. 6 (2015) 33
49. Available from: https://doi.org/10.3390/jfb6010033. [68] M. Hasegawa, K. Yagi, S. Iwakawa, M. Hirai, Chitosan induces apoptosis via caspase-3 activation in bladder tumor cells, Jpn. J. Cancer Res 92 (2001) 459
466. [69] L. Qi, Z. Xu, Y. Li, X. Jiang, X. Han, In vitro effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 cells, World J. Gastroenterol. 11 (2005) 5136
5141. [70] K. Te Shen, M.H. Chen, H.Y. Chan, J.H. Jeng, Y.J. Wang, Inhibitory effects of chitooligosaccharides on tumor growth and metastasis, Food Chem. Toxicol. 47 (2009) 1864
1871. Available from: https://doi.org/10.1016/j.fct.2009.04.044. [71] L. Gibot, S. Chabaud, S. Bouhout, S. Bolduc, F.A. Auger, V.J. Moulin, Anticancer properties of chitosan on human melanoma are cell line dependent, Int. J. Biol. Macromol. 72 (2015) 370
379. Available from: https://doi.org/10.1016/j.ijbiomac.2014.08.033. [72] H. Quan, F. Zhu, X. Han, Z. Xu, Y. Zhao, Z. Miao, Mechanism of anti-angiogenic activities of chitooligosaccharides may be through inhibiting heparanase activity, Med. Hypotheses 73 (2009) 205
206. Available from: https://doi.org/10.1016/j. mehy.2009.02.018. [73] S. Libreros, R. Garcia-areas, Y. Shibata, R. Carrio, M. Torroella-kouri, Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model, Int. J. Cancer. 000 (2011). Available from: https://doi.org/10.1002/ijc.26379. [74] X. Tian, J. Zhang, F. Zhang, M. Zhao, Z. Chen, K. Zhou, et al., Preparation of anticancer micro-medicine based on quinoline and chitosan with pH responsive release performance, Colloids Surf. B Biointerfaces 165 (2018) 278
285. Available from: https://doi. org/10.1016/j.colsurfb.2018.02.052. [75] T. Yang, F. De Cui, M.K. Choi, J.W. Cho, S.J. Chung, C.K. Shim, et al., Enhanced solubility and stability of PEGylated liposomal paclitaxel: in vitro and in vivo evaluation, Int. J. Pharm. 338 (2007) 317
326. Available from: https://doi.org/10.1016/j.ijpharm.2007.02.011.

Handbook of Chitin and Chitosan

636

19. Immunomodulatory activities of chitin and chitosan microparticles

[76] B.Y. Al-najjar, S.A. Hussain, Chitosan microspheres for the delivery of chemotherapeutic agents: paclitaxel as a model, Asian J. Pharm. Clin. Res. 10 (2017) 15. Available from: https://doi.org/10.22159/ajpcr.2017.v10i8.18765. [77] M. Prabaharan, Chitosan-based nanoparticles for tumor-targeted drug delivery, Int. J. Biol. Macromol. 72 (2015) 1313
1322. Available from: https://doi.org/10.1016/j. ijbiomac.2014.10.052. [78] K. Patel, M. Patel, Preparation and evaluation of chitosan microspheres containing nicorandil, Int. J. Pharm. Investig. 4 (2014) 32. Available from: https://doi.org/ 10.4103/2230-973X.127738. [79] S. Nsereko, M. Amiji, Localized delivery of paclitaxel in solid tumors from biodegradable chitin microparticle formulations, Biomaterials 23 (2002) 2723
2731. Available from: https://doi.org/10.1016/S0142-9612(02)00005-4. [80] T.S. Vo, D.H. Ngo, K.H. Kang, W.K. Jung, S.K. Kim, The beneficial properties of marine polysaccharides in alleviation of allergic responses, Mol. Nutr. Food Res. 59 (2015) 129
138. Available from: https://doi.org/10.1002/mnfr.201400412. [81] C. Ozdemir, D. Yazi, M. Aydogan, T. Akkoc, N.N. Bahceciler, P. Strong, et al., Treatment with chitin microparticles is protective against lung histopathology in a murine asthma model, Clin. Exp. Allergy 36 (2006) 960
968. Available from: https://doi.org/10.1111/j.1365-2222.2006.02515.x. [82] M.J. Bae, H.S. Shin, E.K. Kim, J. Kim, D.H. Shon, Oral administration of chitin and chitosan prevents peanut-induced anaphylaxis in a murine food allergy model, Int. J. Biol. Macromol. 61 (2013) 164
168. Available from: https://doi.org/10.1016/j. ijbiomac.2013.06.017. [83] T. Sigsgaard, P.S. Thorne, V. Schlu¨nssen, J. Bønløkke, I.S. Riddervold, K.A. Hoppe, et al., The change in nasal inflammatory markers after intranasal challenges with particulate chitin and lipopolysaccharide: a randomized, double-blind, placebocontrolled, crossover study with a positive control, Int. Forum Allergy Rhinol. 5 (2015) 716
723. Available from: https://doi.org/10.1002/alr.21534. [84] J.P. Choi, S.M. Lee, H.I. Choi, M.H. Kim, S.G. Jeon, M.H. Jang, et al., House dust mite-derived chitin enhances Th2 cell response to inhaled allergens, mainly via a TNF-α-dependent pathway, Allergy, Asthma Immunol. Res. 8 (2016) 362
374. Available from: https://doi.org/10.4168/aair.2016.8.4.362. [85] T.A. Reese, H. Liang, A.M. Tager, A.D. Luster, N. Van, D. Voehringer, et al., Chitin induces tissue accumulation of innate immune cells associated with allergy tiffany, Nature. 447 (2007) 92
96. Available from: https://doi.org/10.1038/nature05746.Chitin. [86] E.M. O’Dea, N. Amarsaikhan, H. Li, J. Downey, E. Steele, S.J. Van Dyken, et al., Eosinophils are recruited in response to chitin exposure and enhance th2-mediated immune pathology in Aspergillus fumigatus infection, Infect. Immun. 82 (2014) 3199
3205. Available from: https://doi.org/10.1128/iai.01990-14. [87] S.J. Van Dyken, A. Mohapatra, J.C. Nussbaum, A.B. Molofsky, E.E. Thornton, S.F. Ziegler, et al., Chitin activates parallel immune modules that direct distinct inflammatory responses via innate lymphoid type 2 and gd T cells, Immunity 40 (2014) 414
424. Available from: https://doi.org/10.1016/j.immuni.2014.02.003. [88] R.M. Roy, H.C. Paes, S.G. Nanjappa, R. Sorkness, D. Gasper, A. Sterkel, et al., Chitindependent allergic sensitization to Aspergillus fumigatus but, MBio 4 (2013) 1
10. Available from: https://doi.org/10.1128/mBio.00162-13.Editor. [89] D.L.M.B. Cleenewerk, P. Martin, Allergic contact dermatitis due to a moisturizing body cream with chitin, Contact Dermatitis 31 (1994) 196. Available from: https://doi. org/10.1016/j.pedex. papers2://publication/. [90] C.J. Wagner, S. Huber, S. Wirth, D. Voehringer, Chitin induces upregulation of B7H1 on macrophages and inhibits T-cell proliferation, Eur. J. Immunol. 40 (2010) 2882
2890. Available from: https://doi.org/10.1002/eji.201040422.

Handbook of Chitin and Chitosan

References

637

[91] B.C. Brinchmann, M. Bayat, T. Brøgger, D.V. Muttuvelu, A. Tjønneland, T. Sigsgaard, A possible role of chitin in the pathogenesis of asthma and allergy, Ann. Agric. Environ. Med. 18 (2011) 7
12. [92] M. Kogiso, A. Nishiyama, T. Shinohara, M. Nakamura, E. Mizoguchi, Y. Misawa, et al., Chitin particles induce size-dependent but carbohydrate-independent innate eosinophilia, J. Leukoc. Biol. 90 (2011) 167
176. Available from: https://doi.org/ 10.1189/jlb.1110624. [93] L.K. Kim, R. Morita, Y. Kobayashi, S.C. Eisenbarth, C.G. Lee, J. Elias, et al., AMCase is a crucial regulator of type 2 immune responses to inhaled house dust mites, Proc. Natl. Acad. Sci. 112 (2015) E2891
E2899. Available from: https://doi.org/10.1073/ pnas.1507393112. [94] K. Arae, H. Morita, H. Unno, K. Motomura, S. Toyama, N. Okada, et al., Chitin promotes antigen-specific Th2 cell-mediated murine asthma through induction of IL33-mediated IL-1β production by DCs, Sci. Rep. 8 (2018) 1
15. Available from: https://doi.org/10.1038/s41598-018-30259-2. [95] R.M. Roy, M. Wuthrich, B.S. Klein, Chitin elicits CCL2 from airway epithelial cells and induces CCR2-dependent innate allergic inflammation in the lung, J. Immunol. 189 (2012) 2545
2552. Available from: https://doi.org/10.4049/jimmunol.1200689. [96] A. Lalaker, L. Nkrumah, W.K. Lee, M. Ramanathan, A.P. Lane, Chitin stimulates expression of acidic mammalian chitinase and eotaxin-3 by human sinonasal epithelial cells in vitro, Am. J. Rhinol. Allergy 23 (2009) 8
14. Available from: https://doi. org/10.2500/ajra.2009.23.3256. [97] T.E. Sutherland, R.M. Maizels, J.E. Allen, Chitinases and chitinase-like proteins: Potential therapeutic targets for the treatment of T-helper type 2 allergies, Clin. Exp. Allergy 39 (2009) 943
955. Available from: https://doi.org/10.1111/j.13652222.2009.03243.x. [98] C.G. Lee, Chitin, chitinases and chitinase-like proteins in allergic inflammation and tissue remodeling, Yonsei Med. J. 50 (2009) 22
30. Available from: https://doi.org/ 10.3349/ymj.2009.50.1.22. [99] D.L. Goldman, X. Li, K. Tsirilakis, C. Andrade, A. Casadevall, A.G. Vicencio, Increased chitinase expression and fungal-specific antibodies in the bronchoalveolar lavage fluid of asthmatic children, Clin. Exp. Allergy 42 (2012) 523
530. Available from: https://doi.org/10.1111/j.1365-2222.2011.03886.x. [100] D. Hartl, C.H. He, B. Koller, C.A. Da Silva, R. Homer, C.G. Lee, et al., Acidic mammalian chitinase is secreted via an ADAM17/epidermal growth factor receptordependent pathway and stimulates chemokine production by pulmonary epithelial cells, J. Biol. Chem. 283 (2008) 33472
33482. Available from: https://doi.org/ 10.1074/jbc.M805574200. [101] M. Ramanathan, W.-K. Lee, A.P. Lane, Increased expression of acidic mammalian chitinase in chronic rhinosinusitis with nasal polyps, Am. J. Rhinol. 20 (2006) 330
335. Available from: https://doi.org/10.2500/ajr.2006.20.2869. [102] Z. Zhu, T. Zheng, R.J. Homer, Y.K. Kim, N.Y. Chen, L. Cohn, et al., Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation, Science 304 (2004) 1678
1682. Available from: https://doi.org/10.1126/ science.1095336. [103] S. Bierbaum, R. Nickel, A. Koch, S. Lau, K.A. Deichmann, U. Wahn, et al., Polymorphisms and haplotypes of acid mammalian chitinase are associated with bronchial asthma, Am. J. Respir. Crit. Care Med. 172 (2005) 1505
1509. Available from: https://doi.org/10.1164/rccm.200506-890OC. [104] M. Musumeci, M. Bellin, A. Maltese, P. Aragona, C. Bucolo, S. Musumeci, Chitinase levels in the tears of subjects with ocular allergies, Cornea 27 (2008) 168
173. Available from: https://doi.org/10.1097/ICO.0b013e318159a950.

Handbook of Chitin and Chitosan

638

19. Immunomodulatory activities of chitin and chitosan microparticles

[105] G.L. Chupp, C.G. Lee, N. Jarjour, Y.M. Shim, C.T. Holm, S. He, et al., Protein in the lung and circulation of patients with severe asthma, N. Engl. J. Med. 357 (2007) 2016
2027. Available from: https://doi.org/10.1056/nejmoa073600. [106] J. Salomon, Ł. Matusiak, D. Nowicka-Suszko, J.C. Szepietowski, Chitinase-3-like protein 1 (YKL-40) reflects the severity of symptoms in atopic dermatitis, J. Immunol. Res. 2017 (2017) 1
5. Available from: https://doi.org/10.1155/2017/5746031. [107] K. Nishimura, C. Ishihara, S. Ukei, S. Tokura, I. Azuma, Stimulation of cytokine production in mice using deacetylated chitin, Vaccine 4 (1986) 151
156. Available from: https://doi.org/10.1016/0264-410X(86)90002-2. [108] K. Nishimura, S.-I. Nishimura, H. Seo, N. Nishi, S. Tokura, I. Azuma, Macrophage activation with multi-porous beads prepared from partially deacetylated chitin, J. Biomed. Mater. Res. 20 (1986) 1359
1372. Available from: https://doi.org/10.1002/ jbm.820200910. [109] K. Nishimura, S. Nishimura, H. Seo, N. Nishi, Effect of multiporous microspheres derived from chitin and partially deacetylated chitin on the activation of mouse peritoneal macrophages, Vaccine 5 (1987) 136
140. [110] K. Suzuki, Y. Okawa, S. Suzuki, M. Suzuki, Candidacidal effect of peritoneal exudate cells in mice administered with chitin or chitosan: the role of serine protease on the mechanism of oxygen-independent: candidacidal effect, Microb. Immunol. 31 (1987) 375
379. [111] C.J. Huang, K.N. Beasley, E.O. Acevedo, R.L. Franco, T.L. Jones, D.C. Mari, et al., Chitin enhances obese inflammation ex vivo, Hum. Immunol. 75 (2014) 41
46. Available from: https://doi.org/10.1016/j.humimm.2013.09.005. [112] R. Rupec, H.C. Korting, T. Ruzicka, B. Koller, A.S. Mu, Chitin modulates innate immune responses of keratinocytes, PLoS One 6 (2011) 1
7. Available from: https://doi.org/10.1371/journal.pone.0016594. [113] J. Foldi, Y. Shang, B. Zhao, L.B. Ivashkiv, X. Hu, RBP-J is required for M2 macrophage polarization in response to chitin and mediates expression of a subset of M2 genes, Protein Cell. 7 (2016) 201
209. Available from: https://doi.org/10.1007/ s13238-016-0248-7. [114] T. Satoh, O. Takeuchi, A. Vandenbon, K. Yasuda, Y. Tanaka, Y. Kumagai, et al., The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection, Nat. Immunol. 11 (2010) 936
944. Available from: https://doi. org/10.1038/ni.1920. [115] C.A. Da Silva, D. Hartl, W. Liu, C.G. Lee, J.A. Elias, TLR-2 and IL-17A in chitininduced macrophage activation and acute inflammation, J. Immunol. 181 (2014) 4279
4286. Available from: https://doi.org/10.4049/jimmunol.181.6.4279. [116] Y. Shibata, W.J. Metzger, Q.N. Myrvik, Chitin particle-induced cell-mediated immunity is inhibited by soluble mannan: mannose receptor-mediated phagocytosis initiates IL-12 production, J. Immunol. 159 (1997). 2462 LP
2467. http://www. jimmunol.org/content/159/5/2462.abstract. [117] A. Nishiyama, T. Shinohara, T. Pantuso, S. Tsuji, M. Yamashita, S. Shinohara, et al., Depletion of cellular cholesterol enhances macrophage MAPK activation by chitin microparticles but not by heat-killed Mycobacterium bovis BCG, Am. J. Physiol. Physiol. 295 (2008) C341
C349. Available from: https://doi.org/10.1152/ajpcell.00446.2007. [118] C.L. Bueter, C.K. Lee, J.P. Wang, G.R. Ostroff, C.A. Specht, S.M. Levitz, Spectrum and mechanisms of inflammasome activation by chitosan, J. Immunol. 192 (2014) 5943
5951. Available from: https://doi.org/10.4049/jimmunol.1301695. [119] Y.C. Huang, A. Vieira, K.L. Huang, M.K. Yeh, C.H. Chiang, Pulmonary inflammation caused by chitosan microparticles, J. Biomed. Mater. Res. Part A 75 (2005) 283
287. Available from: https://doi.org/10.1002/jbm.a.30421.

Handbook of Chitin and Chitosan

References

639

[120] S. Gudmundsdottir, R. Lieder, O.E. Sigurjonsson, P.H. Petersen, Chitosan leads to downregulation of YKL-40 and inflammasome activation in human macrophages, J. Biomed. Mater. Res. Part A 103 (2015) 2778
2785. Available from: https://doi.org/ 10.1002/jbm.a.35417. [121] E.F. Kong, C. Tsui, S. Kucharı´kova´, D. Andes, P. Van Dijck, et al., Commensal protection of Staphylococcus aureus against antimicrobials by Candida albicans biofilm matrix, Am. Soc. Microbiol 7 (2016) 1
12. Available from: https://doi.org/10.1128/ mBio.01365-16.Editor.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

20 Chitosan/chitin-based composites for food packaging applications Muhammad Zubair, Muhammad Arshad, Rehan Ali Pradhan and Aman Ullah Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

O U T L I N E 20.1 Introduction

642

20.2 Various chitosan/chitin-based composites for food packaging applications 20.2.1 Chitosan/gelatin proteins-derived composite 20.2.2 Chitin/gelatin-based composites 20.2.3 Chitin/chitosan-derived composites with other proteins

643 644 648 651

20.3 Chitin/chitosan and carbohydrates-derived composites 20.3.1 Chitin/chitosan composites with cellulose 20.3.2 Chitin/chitosan composites with starch 20.3.3 Chitin/chitosan composites with other polysaccharides

652 652 654 656

20.4 Chitin/chitosan composites with synthetic polymer

657

20.5 Inorganic materials-derived composites

659

20.6 Conclusion and future trends

663

References

664

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00020-0

641

© 2020 Elsevier Inc. All rights reserved.

642

20. Chitosan/chitin-based composites for food packaging applications

20.1 Introduction Worldwide, food packaging is one of the most essential components of the food supply network that provides protection and safety to the food [1,2]. Packaging does not just act as a buffer against the number of contaminants such as dirt, chemicals, and microorganisms, but also helps to preserve food quality during transportation and storage [3,4]. Most of the packaging materials are being developed from modern petrobased polymers that control the exposure of food to light, moisture, oxygen, and microbial spoilage [5,6]. These materials have numerous benefits such as light weight, economical, recyclable, and resilient to chemical or physical damage [7,8]. Although petroleum-derived polymeric materials can offer many essential requirements for the packaged food, yet a number of serious efforts are continue to maintain packaging performance while reducing the environmental pollution [9]. The ever growing demand of eco-friendly materials to address the innumerable issues, particularly related to the environment, sustainability, biodegradability, and biocompatibility [10
13], have compelled the scientific community to explore new horizons of research mainly directed toward naturally derived food packaging materials. Biopolymers-derived materials have laid the foundation to reduce the use of nonrenewable resources and are one of the significant elements of a circular economy [14]. Moreover, they are available in many different forms in nature and can be derived from renewable and sustainable bioresources [15,16]. The novel biobased materials have high performance, light weight, and most importantly green in nature, making them viable alternatives to nonbiodegradable food packaging materials [17
19]. Generally, biopolymers are categorized on the basis of their origin into three classes. The first class is called polysaccharides and includes starch, celluloses, gums, chitin, chitosan, and pectins. Proteins are the second class of biopolymers and include caseins, collagen, myofibrillar, whey proteins isolates, soy proteins isolates, zein, sunflower, and gelatin. While the third category is lipids such as fatty acids and waxes [20
22]. Among biopolymers, polysaccharide-based bionanocomposites have become increasingly significant materials over the last two decades. Polysaccharides provide a green and environment-friendly substitute to petroleum-based synthetic polymers for various applications particularly for food packaging applications [23
25]. Polysaccharides are encompassed of many saccharide units which are joined together via glycosidic linkages [26] and have several exceptional attributes that make them an attractive choice compared with other classes of biopolymers. Most of the polysaccharides are abundantly available and can be obtained economically from microorganisms, animals, and plants [27].

Handbook of Chitin and Chitosan

20.2 Various chitosan/chitin-based composites for food packaging applications

643

Chitin, poly (b-(1
4)-N-acetyl-D-glucosamine) is the second most naturally available polysaccharide after cellulose. It was identified almost 125 years ago [28] and has been used in many applications. Chitin is present in nature abundantly, including in the exoskeletons of crustaceans, insects, mollusks, and fungi. Presently, crustacean exoskeleton is the main industrial biomass source for the production of chitin with an estimated amount of 1560 MT annually [29
31]. Chitin’s derivative, that is, chitosan, is one of the most active biomolecules and is being used in various food applications which have progressed rapidly in recent years [28]. Chitosan is a linear amino polysaccharide with D-glucosamine and N-acetyl-D-glucosamine units which can be synthesized by deacetylating the chitin [32]. Chitosan has exceptional properties such as antioxidant, antibacterial, biocompatible, lipid-lowering, biodegradable, film-forming, and gelling [33]. The unique structures and exceptional properties of both chitin and chitosan deliver many advantages for their use in the advance of materials or composites for multiple applications particularly in the field of food packaging. The chapter emphasizes the latest trends in the development of chitin and chitosan-derived materials including films, membranes, coatings, and as a nanofiller in novel applications related to food packaging. The different composites of chitosan and chitin with other biopolymers, synthetic polymers, and inorganic particles are discussed in detail along with their physicochemical properties, such as mechanical, thermal, antibacterial, antioxidant, and barrier properties. At the end, this chapter provides future perspectives for chitosan and chitin-derived materials in the modern food packaging technology.

20.2 Various chitosan/chitin-based composites for food packaging applications Chitin, chitosan, and their derivatives have gained remarkable attention for food packaging applications due to their notable properties such as their biodegradability, biocompatibility, and their biofunctional and nontoxic nature. Owing to their efficient oxygen barrier property, film formability, antibacterial, physical, and mechanical properties, these materials have been utilized for food packaging applications to preserve food [34
38]. Chitin, chitosan, and their derivatives have been used with proteins, carbohydrates, synthetic polymers, and inorganic nanoparticles to develop composites for food packaging applications (shown in Fig. 20.1). In this section, various composites of chitin and chitosan have been discussed in detail with other biopolymers and nanofillers and synthetic polymers.

Handbook of Chitin and Chitosan

644

20. Chitosan/chitin-based composites for food packaging applications

Chitin/chitosanbased composites

Proteins Carbohydrates

Gelatin Quinoa Casein Collagen Kidney bean

Cellulose Starch Carragenenan Xylan Pectin Alginate

Synthetic polymer

Poly(vinyl alocohol) Poly(lactic acid) Low-density polyethylene

Inorganic materials

Silver Zinc oxide Graphene oxide Titanium dioxide Silica

FIGURE 20.1 Chitin/chitosan-based composites for food packaging applications.

20.2.1 Chitosan/gelatin proteins-derived composite Proteins are one of the biopolymers that have been used extensively to advance chitin and chitosan composites. Among proteins, gelatin is the most commonly used protein to make composites for food packaging with chitin and chitosan. Halim et al. prepared biopolymer films, that is, chitosan, gelatin, and methylcellulose with tannic acid (TA) using a casting method for the preservation of cherry tomatoes and grapes. The obtained films were characterized, and their physicomechanical properties were studied and compared. The incorporation of 15% w/w tannic acid improved the transparency of the films and the highest increase in transparency value (0.572 to 4.73 A/mm) was observed in methylcellulose-tannic acid-derived film. The antimicrobial study indicated that tannic acid also increased the antibacterial activities of the films against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Mechanical strength of gelatin and methylcellulose films were improved with the introduction of tannic acid, while it was reduced in the case of chitosan, which is ascribed to the poor network bonding between TA and chitosan. The preservation ability of the films was assessed in a 14-day period and it was observed that films reduced the loss in weight and browning index of the fruits in comparison with control films (commercial cling films). A substantial decrease in the weight loss was seen in cherry tomatoes, that is, 1.3% to 19.6%, 22.1% to 15.5%, and 26.2% to 20.5% for chitosan gelatin- and methylcellulose-derived films with TA, respectively. The tannic acid addition also enhanced the heat stability of the derived films compared to control films. This finding validated with FTIR data where it showed a strong interaction between TA and polymer to form a chemical bond [39].

Handbook of Chitin and Chitosan

20.2 Various chitosan/chitin-based composites for food packaging applications

645

Kumar et al. reported the hybrid nanocomposites of chitosan with gelatin and silver nanoparticles for food packaging. They used polyethylene glycol as a plasticizer for the nanocomposite preparation using a solution casting method. The study indicated that the incorporation of silver nanoparticles decreased the mechanical strength from 28.87 6 0.49 to 21.19 6 1.01 MPa, while they increased the elongation at break from 17.99% 6 0.68% to 27.23% 6 0.76%. Furthermore, nanocomposites showed a reduction in the transmission of light in the visible region [40]. In another study, gelatin (G) from eggshell membrane was used with chitosan (Ch) to prepare composite edible films. The physicomechanical and structural properties of the films were measured using FTIR, universal traction-testing machine, and SEM. The results showed that chitosan containing films (75 G:25Ch, 50 G:50Ch) improved the elongation at break greatly but caused no substantial change in tensile strength compared with gelatin-derived films. However, water solubility and water vapor permeability (WVP) of the film containing 50 G:50Ch were reduced greatly in comparison with chitosan alone or gelatin, as well as with other composite films. FTIR analysis of films demonstrated that both gelatin and chitosan polymers are totally miscible, whereas SEM images of the films revealed a homogenous film formation and a compact structure of the films, particularly blends (75:25, 50:50) of gelatin and chitosan. The study suggested that these films have good potential to be used in packaging to advance food quality [41]. Active food packaging material from chitosan/gelatin films have also been reported using essential oils, such as eugenol and ginger, as antioxidants agents by Bonilla et al. They prepared the films using glycerol (as a plasticizer) and Tween 80 (as a surfactant) by the casting method. The films were analyzed for the antioxidant, mechanical, optical, microstructural, and barrier properties to check their suitability for food packaging. FTIR investigation revealed that eugenol or ginger essential oils caused the presence of new bands while SEM and AFM studies presented an increase in roughness values that was ascribed to the poor distribution of ginger and eugenol oils in the polymer matrix. Whereas the addition of oils caused a substantial improvement in the elasticity of all films, no major effect was noticed in the WVP. Gelatin
chitosan blends with eugenol showed the maximum antioxidant activity compared with other films. Furthermore, films with oils were yellow in color and exhibited better protection against the UV
Vis light, thus providing an advantage for avoiding food deterioration by oxidizing reactions [42]. Garlic essential oil was also used to synthesize the gelatin/chitosanderived films using the casting method [43]. The study reported films based on gelatin/chitosan (G-Ch) with active compounds nanoemulsion in water. The garlic essential oil, α-tocopherol, and cinnamaldehyde were used as active compounds. The physical and antioxidant properties of the

Handbook of Chitin and Chitosan

646

20. Chitosan/chitin-based composites for food packaging applications

films containing gelatin and chitosan with oils were compared with the pure gelatin (G) and gelatin/sodium caseinate (G-Cs)-derived films. The angle contact measurements (as shown in Fig. 20.2) indicated that gelatin/ chitosan films with oils had the minimum swelling and solubility and maximum hydrophobicity, whereas films derived from G-Cs had the maximum antioxidant activity. They assumed that the greater contact angle of film based on the G-Ch along with oils was ascribed to chitosan instead of the oils (garlic essential oil, α-tocopherol, and cinnamaldehyde), and it showed a lower water affinity compared with alone gelatin and sodium caseinate films. Bonilla et al. conducted a study to investigate the effect of boldo-do-chile (B), guarana (G), cinnamon (C), and rosemary (R) ethanolic extracts on the antimicrobial, mechanical, antioxidant, barrier, and optical properties of the films with varying concentrations of gelatin and chitosan [44]. The study outcomes showed that films’ elasticity was increased and WVP was reduced by increasing the chitosan concentration. However, there was not any significant reduction observed with the extracts addition. The films

(A)

(B)

 = 45º

 = 59º

(C)

 = 36º

(D)

 = 79º

FIGURE 20.2 Images for contact angles of water droplet on the films (A) control, (B) gelatin with active compounds, (C) gelatin/sodium caseinate with active compounds, and (D) gelatin/chitosan with active compounds. Source: Reproduced with permission from Elsevier L.J.P. Co´rdoba, P.J. Sobral, Physical and antioxidant properties of films based on gelatin, gelatin
chitosan or gelatin
sodium caseinate blends loaded with nanoemulsified active compounds, J. Food Eng. 213 (2017) 47
53.

Handbook of Chitin and Chitosan

20.2 Various chitosan/chitin-based composites for food packaging applications

S. aureus

647

E. coli

(A)

(B)

C

B C R

G

B

R

G

FIGURE 20.3 Inhibition of Staphylococcus aureus and Escherichia coli A and B: gelatin: chitosan 50:50 films with extracts against S. aureus and E. coli, respectively. Source: Reproduced with permission from Elsevier J. Bonilla, P.J. Sobral, Investigation of the physicochemical, antimicrobial and antioxidant properties of gelatin
chitosan edible film mixed with plant ethanolic extracts, Food Biosci. 16 (2016) 17
25.

also displayed excellent antioxidants behavior in TEAC (Trolox-equivalentantioxidant-capacity) test and better growth inhibition against E. coli and S. aureus, as shown in Fig. 20.3. Ahmed et al. synthesized the biodegradable packaging films based on chitosan/gelatin by the solution casting method, using polyethylene glycol as a plasticizer to make films with more flexibility and transparency. Chitosan and gelatin blend were cross-linked using boric acid to advance their light transparency, water barrier, total solubility, and mechanical strength. The characterization of the films showed that there was good compatibility among the films’ components and the resulting films were transparent and homogenous. The chitosan/gelatin-based films also exhibited a good barrier effect against UV light. Furthermore, the addition of cross-linker caused an increase in the tensile strength and stiffness as high as 4%. However, when cross-linker contents were increased to 5%, the film became brittle and strength reduced, which was attributed to a reduction in the film’s flexibility [45]. In some cases, chitosan nanoparticles were used to improve the biopolymer-derived films. Hosseini et al. reported two studies on the use of chitosan nanoparticles to develop fish gelatin-based bionanocomposites. In the first study [46], they prepared chitosan nanoparticle using sodium tripolyphosphate (TPP) and later used them as a filler to make gelatin-based bionanocomposite films. SEM images of films showed that chitosan nanoparticles were very well dispersed at low concentration in gelatin polymer matrix. However, at higher concentration (8%, w/w) the aggregation of nanoparticles occurred in the composites. FTIR data demonstrated that hydrogen bonding interactions were present between chitosan nanoparticles and fish gelatin. The study also indicated that chitosan nanoparticles’

Handbook of Chitin and Chitosan

648

20. Chitosan/chitin-based composites for food packaging applications

incorporation into the fish gelatin resulted in a significant improvement (7.44 6 0.17 to 11.28 6 1.02 MPa) in the mechanical strength while reducing the elongation at break. Furthermore, chitosan nanoparticles also contributed to the substantial decline (50% at 6% (w/w) filler) in the WVP of the films. The light barrier properties of the film were measured and indicated that films were clear and showed a good protection against the UV light. In the second study [47], they also used Origanum vulgare L. essential oil (OEO) along with chitosan nanoparticles to improve the physicochemical properties of the films. The results indicated that the addition of OEO induced an increase in crystallinity of the films. However, its incorporation had no effect on the thermal stability of the films as demonstrated by TGA analysis. In terms of barrier and mechanical properties, the presence of OEO led to less strong (decreased from 10.57 6 0.19 to 3.28 6 0.43 MPa) and more flexible (44.71% 6 11.80% to 151.82% 6 4.78%) films, with a reduction in WVP. The obtained films also displayed excellent antimicrobial activity against food pathogens, viz. E. coli, Listeria monocytogenes, Salmonella enteritidis, and S. aureus. Gelatin
chitosan films were studied by Go´mez-Estaca et al. using essential oils of clove, fennel, lavender, herb-of-the-cross, pine, rosemary, cypress, and thyme, and their antimicrobial performance was assessed on 18 genera of bacteria (including food pathogens and spoilage bacteria). The results indicated that clove oil displayed the maximum inhibition among all oil followed by rosemary and lavender. The oils were also tested for their use as food preservatives while clove and thyme were the most active in all used oils. Furthermore, films having clove inhibited all microorganisms regardless of the film polymer matrix and microorganism type [48].

20.2.2 Chitin/gelatin-based composites Chitin nanofiber (ChNF)/gelatin-derived nanocomposites were prepared using the immersion method [49]. Different concentrations of chitin nanofibers were used, that is, 73% 6 2%, 62.5% 6 2.5%, and 50.2% 6 2.7% with gelatin content of 2, 5, and 10 wt.%, respectively. The field emissionscanning electron microscopy (FE-SEM) images showed that ChNF/gelatinderived nanocomposites had uniform dispersion of nanofiber network structures into the gelatin polymer matrix. Mechanical strength of the pure gelatin was 87.2 6 3.2 MPa which was increased to 101.0 6 5.3 MPa with the addition of 50.2% chitin contents while the Young’s modulus was increased to almost double that of the pure gelatin. The nanocomposites mechanical strength was improved and ascribed to the nanoreinforcement effect from chitin nanofibers and hydrogen bonding between chitin and gelatin molecules. In addition to that, transmittance of the nanocomposites was

Handbook of Chitin and Chitosan

649

20.2 Various chitosan/chitin-based composites for food packaging applications

determined using UV
Vis near-infrared spectrometer VU 600 nm and it was shown to improve up to 88.7% from 65% irrespective of the nanofiber contents. Gelatin-based bionanocomposites were synthesized using surfacemodified (deacetylated) chitin nanofiber by the immersion method [50]. The results showed that the reinforcing effect of surface-deacetylated chitin nanofiber led to an improvement in the mechanical properties (as shown in Fig. 20.4) of the gelatin-derived nanocomposites with a transparency value about 89%, stress increased from 89.2 6 2.83 to 126.3 6 3.07 MPa, while Young’s modulus improved from 2598 6 214 to 6356 6 261 MPa. The field emission SEM investigation revealed that there was a dense network structure because of modified chitin high aspect ratio and small diameter. A good interfacial interaction existed between the modified chitin and gelatin which became the reason for improvement in the mechanical strength of the nanocomposite. Additionally, FTIR data showed the presence of hydrogen bonding between the nanoparticle and polymer matrix. Sahraee et al. reported two studies on the gelatin
chitin-derived bionanocomposites. In the first study [51], they used corn oil (0.10, 0.20, and 0.30 g/g of dry gelatin) along with nanochitin (0.05 g/g of dry gelatin) to prepare emulsion gelatin films. The films’ physicochemical properties were analyzed and it was shown that films comprising nanochitin showed better mechanical properties compared with pure gelatin film and reduced the films’ ability to absorb the water. However, the addition of oil gave better barrier properties than films with just nanoparticles. The films’ thermal properties were improved by adding nanochitin which was further enhanced by the incorporation of the oil. Nanochitin-based films showed good antimicrobial activity

120

(A)

120

S-ChNF/G-2

100 Stress (MPa)

100 Stress (MPa)

(B)

80 60

S-ChNF Gelatin S-ChNF/G-2 S-ChNF/G-5 S-ChNF/G-10

40 20

ChNF/G

80 60

Fiber content: ~ 45 wt%

40 20

0

0 0

1

2

3

6 4 5 Strain (%)

7

8

0.0

0.5

1.0

1.5 2.0 2.5 Strain (%)

3.0

3.5

FIGURE 20.4 Stress
strain curves of (A) surface-modified chitin nanofibers, gelatin, and surface-modified chitin nanofibers/gelatin nanocomposites; (B) surface-modified chitin nanofibers/gelatin and surface-modified chitin nanofibers/gelatin nanocomposite with fiber concentration of 45 wt.%. Source: Reproduced with permission from Elsevier C. Chen, Y. Wang, Y. Yang, M. Pan, T. Ye, D. Li, High strength gelatin-based nanocomposites reinforced by surface-deacetylated chitin nanofiber networks, Carbohydr. Polym. 195 (2018) 387
392.

Handbook of Chitin and Chitosan

650

20. Chitosan/chitin-based composites for food packaging applications

that could be attributed to the presence of active amino groups in the nanochitin. As a result, nanochitin can bind with anionic groups present on the cell wall surface and disturb the activity of the microbial cell wall. However, the addition of oil into the films showed no antifungal activity, which can be explained in terms of oil that can cover the active groups of nanoparticles and prevent the films’ antimicrobial activity. In the second study [52], they prepared a gelatin-based nanocomposite by varying the concentration of chitin nanoparticles (0%, 3%, 5%, and 10%) and their thermal, antimicrobial, and physical properties were studied. The study indicated that the addition of chitin nanoparticles had a significant influence on the apparent color and transparency of the films. As the concentration of the nanoparticles was increased, the films’ WVP and solubility were reduced while the surface hydrophobicity was increased. Furthermore, chitin nanoparticles content up to 5% resulted in films with improved mechanical properties. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to study the thermal stability of nanocomposite films at high temperatures, and the obtained films showed better thermal stability compared with the pure gelatin film, as shown in Fig. 20.5. In terms of microbial activity, nanocomposite films exhibited better antifungal properties against Aspergillus niger in comparison with pure gelatin-based films. Inhibition increased up to 5% content of chitin nanoparticles and there was less inhibition shown for films containing 10% chitin nanoparticles. Lastly, all composite films displayed an excellent barrier to UV light (200
280 nm). This improvement was attributed to the presence of aromatic amino acids in the peptide chain of gelatin that have the ability for UV light absorption. Reactions between the amine groups and carbonyl groups of gelatin and chitin (Schiff’s base reaction) also contributed toward better barriers against UV light. 0.2 100

0%N-Chitin 5%N-Chitin 10%N-Chitin

90

0%N-Chitin 5%N-Chitin 10%N-Chitin

0.1

80 0 DTG (mg/ºC)

Weight (%)

70 60 50 40

–0.1 –0.2 –0.3

30 20

–0.4 10 0

–0.5 100

200

300

400

500

600

100

200

Temperature (ºC)

300

400

500

600

Temperature (ºC)

FIGURE 20.5 TGA and DTG curves of gelatin nanocomposite having nanochitin (N-chitin). Source: Reproduced with permission from Elsevier S. Sahraee, J.M. Milani, B. Ghanbarzadeh, H. Hamishehkar, Physicochemical and antifungal properties of bionanocomposite film based on gelatin
chitin nanoparticles, Int. J. Biol. Macromol. 97 (2017) 373
381.

Handbook of Chitin and Chitosan

20.2 Various chitosan/chitin-based composites for food packaging applications

651

20.2.3 Chitin/chitosan-derived composites with other proteins Chitosan/quinoa protein-derived films were developed to prolong the shelf life of fresh fruits. The films were developed using high viscosity chitosan and chitosan/quinoa nanoparticles with different concentrations, loaded with thymol, and printed by inkjet printing. The films with high mechanical strength and low capacity of water uptake were selected for printing using simulated storage conditions for fresh fruit. The results showed that thymol-printed films showed improved barrier properties in comparison with the control. Furthermore, printed films showed better activity against Listeria innocua, Enterobacter aerogenes, Pseudomonas aeruginosa, E. coli, S. aureus, and Salmonella typhimurium compared with chitosan nanoparticles and gelatin-derived films [53]. Casein protein is another protein which has also been used to blend with chitosan to develop films. Sodium caseinate is a viable choice since it forms strong intermolecular hydrogen bonds, electrostatic bonds, and hydrophobic bonds [10,54,55]. Caseinate proteins have been used with chitosan to develop biodegradable polymers for active food packaging applications as antimicrobial carriers [56]. The relative antimicrobial efficiency and their release rates (labeling of nisin fluorescently as Z) from hydroxypropyl methylcellulose, chitosan, sodium caseinate, and polylactic acid films were analyzed at 4 and 40 C in water/ethanol solution. The results indicated that with the increase of temperature, the release of nisin increased greatly from sodium caseinate and hydroxypropyl methylcellulose films because of their hydrophilic nature and glass transition temperature. Furthermore, a higher antibacterial activity was observed for hydroxypropyl methylcellulose and sodium caseinate-derived films against L. monocytogenes (CIP 82110) and S. aureus (CIP 4.83). However, these films could gradually release nisin and act as an antibacterial agents and could be used to extend packaged food shelf life. The kidney bean protein isolate was used to make chitosan composite films that were fabricated at acidic pH to make antimicrobial carriers [57]. The surface hydrophobicity, free energy, and microstructure of the films were assessed. The obtained films had less rigidity (reduced from 113.8 to 47.3 MPa) and much more flexibility (increased from 7.1% to 37.4%) with reduced elastic modulus, storage modulus, and glass transition temperature. Films were analyzed for antimicrobial activities and revealed that Bacillus subtilis, E. coli, and Salmonella were sensitive to the composite films. The results showed that kidney bean protein isolate/chitosan blend films can be a good candidate for antimicrobial food packaging. Collagen was also used to develop composite films with chitosan. The study was carried out to assess the composite films’ suitability for food packaging [58]. The films were prepared using glycerol 30% (w/w) as a plasticizer with different ratios of collagen/chitosan and collagen/soy protein isolate (10%:0%, 8%:2%, 6%:4%, 5%:5%, and 0%:10% w/w).

Handbook of Chitin and Chitosan

652

20. Chitosan/chitin-based composites for food packaging applications

The results showed that elongation at break was improved while tensile strength and modulus of elasticity were reduced for the composite films in comparison with collagen film. On the contrary, the chitosan/soy protein isolates-derived films showed lower elongation at break, whereas tensile strength and elastic values were greater than pure collagen films. All composite films showed an improved barrier effect against the UV light. FTIR data indicated the intermolecular hydrogen bonding interactions were dominant in the collagen/soy protein isolates. Thermogravimetric analysis showed that films with collagen/chitosan (8:2) and collagen/soy protein isolates (8:2) had less weight loss (%) as compare with collagen films. SEM images demonstrated that composite films of collagen/chitosan (8:2) and collagen/soy protein isolates (8:2) were somewhat rougher compared with collagen film. However, no visible cracking was observed, thus suggesting their potential use as biodegradable packaging materials.

20.3 Chitin/chitosan and carbohydrates-derived composites Chitin and Chitosan haven been utilized as the matrix material, filler or nanoreinforcer with a variety of polysaccharides such as cellulose, alginate, starch, carrageenan to obtain composite films/coatings for food packaging applications and their thermal, mechanical and barrier properties are discussed concisely.

20.3.1 Chitin/chitosan composites with cellulose Chi et al. reported a composite made with the casting/evaporation method by utilizing chitosan and carboxymethyl cellulose (CMC) as a matrix material and reinforcing it with nanocellulose ((NC), 100
250 nm length, 2
5 nm diameter) [59]. They demonstrated that the use of two oppositely charged polyelectrolyte complexes (chitosan (1) and CMC (
)) enhanced the thermomechanical and barrier performances, and achieved greater dispersion and interfacial compatibility between NC and matrix, as well as stronger interactions within matrix components. Tensile strength and Young’s modulus were increased by 40% and 52%, respectively, compared with the control film. Water vapor transmission rate was lowered by 40% to 7982 g mm/m2/d as determined using ASTM E-96 standards. The tortuous diffusive path of CNC prevents water penetration, but the hydrophilicity of CNC could lead to wettability followed by the creation of pore pathways. Therefore a greater amount of CNC is not recommended, and aggregation was also observed for nanofiller .10% (wt.% of total film). The barrier performance was still inferior than reported values for commercial

Handbook of Chitin and Chitosan

20.3 Chitin/chitosan and carbohydrates-derived composites

653

films, such as PP (197 g mm/m2/d), LDPE (512 g mm/m2/d), HDPE (118 g mm/m2/d) and PET (472 g mm/m2/d) [60]. Chitin nanofiber (CHNF) at different concentrations (1.5
6 wt.%) were added to prepare bionanocomposites comprising silver nanoparticles (1 wt.%) and chitosan (75%
85% deacetylated). Jafari et al. determined barrier and mechanical performances and obtained optimum properties of bionanocomposites with 4.55 wt.% CHNF [61]. WVP was increased with increasing CHNF up to 1.5 wt.% while mechanical strength and Youngs modulus (with 6 wt.% CHNF) were increased by 2.5 times and 3.4 times, respectively, compared with pure chitosan film. Thus the film became more rigid at the expense of 46% reduction in elasticity. Satam et al. prepared suspensions consisting of cationic chitin nanofiber and anionic cellulose nanocrystal (ChNC)/nanofiber (ChNF) and deposited it onto PLA substrate to demonstrate barrier performances [62]. The reduction in coating voids was observed due to the interaction between negatively charged sulfate ester groups and the positively charged chitin surface. Thus low oxygen permeability was observed for multilayer coated PLA in comparison with neat PLA substrate. At relative humidity of 80%, O2 permeability was reduced by 73% for three alternating coated layers of ChNF
CNC
ChNF. There were not any significant improvements in WVTR and the obtained film depicted brittleness that can be reduced through the addition of plasticizers. Ma et al. cross-linked cellulose with citric acid, followed by oxidation to form aldehyde [63]. Then modified cellulose was incorporated into the chitosan matrix, where the aldehyde group reacted with the amine group of chitosan to form Schiff base (C5N bond). The stronger interaction between filler and matrix enhanced the mechanical properties and water barrier characteristics. WVP was decreased by 38% (0.98 3 1026 g/m/h/Pa at relative humidity of 75%) with the addition of 6% modified filler. The formed composites also exhibited better UV resistance, which is an essential requirement for food packaging applications as UV light can induce lipid oxidation in food systems. Li et al. fabricated multilayer films of chitosan and NC on PET substrate and investigated the barrier properties of the composite film [64]. The oxygen permeability values for 30 bilayer coated PET was decreased by 94% compared with uncoated PET. The thickness of coated PET was measured to be 780 nm and indicated that the same level of oxygen permeability for uncoated PET could only be attained for film thickness greater than 2.7 mm. Deng et al. prepared composite films by incorporating chitosan at different Mw and concentrations into CNF film [65]. The study showed that the mechanical, physiochemical, and antibacterial properties were affected by the relative abundance of functional amino groups and crystallinity of chitosan. Furthermore, antibacterial properties were improved with 20% chitosan (wt./wt.) compared with pure CNF films. Films composed of 20% chitosan

Handbook of Chitin and Chitosan

654

20. Chitosan/chitin-based composites for food packaging applications

(wt./wt.) with a molecular weight of 287 kDa presented the least water absorption. The water resistant and antibacterial results were promising, however, further research is needed to improve the sealable characteristics of chitosan
CNF films for their effective utilization as a food packaging material. Li et al. developed carboxymethyl cellulose (CMC)-based biopolymer films by utilizing chitin and deacetylated chitin nanofiber (0, 1, 5, and 10 wt.%, based on CMC weight) as reinforcing and antimicrobial agents [66]. With 10 wt.% chitin nanofiber, tensile strength was of the films was improved marginally to 49.9 MPa from 43.6 MPa (neat CMC film). However, deacetylated chitin exhibited remarkable improvement in tensile strength with an increase of nearly 30% over neat CMC film and a 20% increase compared with the chitin-based counterpart. This was attributed to the improvement in dispersion of deacetylated chitin due to the electrostatic attraction of the amine group with the carbonyl group of CMCs. The interaction is illustrated in Fig. 20.6.

20.3.2 Chitin/chitosan composites with starch The morphological influence of nanochitin on the antimicrobial, thermal, and barrier characteristics of starch-based films were evaluated and compared by Salaberria et al. for potential application in food packaging and functional coating [67]. Nanocrystals exhibited a rod-like structure with a low aspect ratio while nanofibers showcased a web-like structure with a high aspect ratio. The kind of morphological structure had little effect on the oxygen permeability, however nanofibers with high aspect ratio exhibited superior mechanical characteristics Antimicrobial properties were also dependent on the type of morphology, with chitin nanofiber showing 96% inhibition for fungal growth compared with the 89% inhibition attained in chitin nanocrystal/starch-based film. Qin et al. examined the effects of chitin nanowhiskers at different concentrations (0%
2% wt./wt.) [68]. Tensile strength was improved to 125% by incorporating 1% chitin nanowhiskers to a starch-based film, in addition to the enhancement in the WVP values being enhanced (from 5.32 3 10212 g/m/s/Pa to 2.22 3 10212 g/m/s/Pa) with 2% nanowhisker. Chitosan was cross-linked with tripolyphosphate and mixed with glycerol-plasticized starch-based matrix. The influence of filler content was studied by Chang et al. [69]. WVP was decreased to an optimal value of 3.15 3 10210 g/m/s/Pa with 6% chitosan nanoparticle (wt./wt.) addition. At a higher level, poor dispersibility of nanofiller led to an increase in permeability. Thermal stability of composite was also improved with chitosan which was attributed to the enhanced interfacial interaction between nanofiller and starch moieties. Improvement in intermolecular

Handbook of Chitin and Chitosan

20.3 Chitin/chitosan and carbohydrates-derived composites

(A)

(B)

ChNF/CMC

655

dChNF/CMC

ChNF aggregates

OH

OH

O

ChNF: m=0.20; n=0.80

O O

O m NH3+

HO

Electrostatic attraction

HO

COO–

dChNF: m=0.31; n=0.69

n

NH O CH3

H2C HO

O

Hydrogen bond O

O

CMC

O

O n

HO

OH

HO

O CH2 –OOC

FIGURE 20.6 Interaction between chitin nanofiber and deacetylated chitin nanofiber with CMC. Source: Reproduced with permission from ACS publisher.

interaction also led to an increase in Tg as determined with DMTA. Furthermore, the addition of nanofiller also made the film stiffer. Yoksan et al. loaded silver nanoparticles (20
25 nm) to chitosan solution and prepared glycerol-plasticized starch-based films by the solution casting method [70]. The water vapor transmission rate tended to be enhanced by increasing the silver nanoparticle content. This was due to the greater availability of hydrophilic sites as well as the obstruction of intermolecular and intramolecular bonding caused by silver nanoparticles. The increase in wettability was observed and determined by contact angle measurement. However, the oxygen barrier property improved slightly, and the tensile strength increased with the addition of 0.29% Ag nanoparticle (wt./wt.) content.

Handbook of Chitin and Chitosan

656

20. Chitosan/chitin-based composites for food packaging applications

20.3.3 Chitin/chitosan composites with other polysaccharides Nanocomposites films were produced by Lorevice et al. by the addition of chitosan nanoparticles to low- and high-methyl pectin films [71]. High availability of hydroxyl groups led to greater hydrogen bonding in lowmethyl pectin films, giving rise to more compact and dense structure. The mechanical property was exceptionally improved with the addition of chitosan fillers from 30.81 to 46.95 MPa for high-methyl pectin films and from 26.07 to 58.51 MPa for low-methyl pectin films. The interaction of hydroxyl groups with water molecules was greater for low-methyl pectin films, which led to its inferior barrier performances compared to high-methyl pectin films. Wei et al. modified okra fiber via layer-by-layer coating of chitosan and pectin [72]. The positively charged chitosan coating presented smoother surfaces but had comparable hydration capacity (7.06 g/g) and water suspendability (42.06%) relative to their intact counterparts (6.94 g/g and 43.67%). The addition of another layer of negatively charged pectin considerably increased hydrodynamic radius (from 27.83 mm to 38.78 mm), hydroscopic capacity (7.56 g/g), and water suspendability (65.36%). Thus the addition of both chitosan and pectin provided the overall coating efficiency. Salama et al. demonstrated the formation of edible films based on sodium alginate (SA) and chitosan biguanidine hydrochloride (CBH) [73]. The effects on antibacterial and barrier characteristics were studied with different weight fractions of SA:CBH (100:0, 90:10, 70:30, 50:50). The presence of both ionic interactions as well as hydrogen bonding between alginate and chitosan resulted in greater cross-linking density and thereby an increase in water resistance. As expected, the increase in CBH content resulted in enhanced antibacterial performance. Films from polyelectrolyte complexes of chitosan with xylan and their mechanical attributes, water and oxygen permeability were reported by Schnell et al. [74]. Increasing the proportion of xylan resulted in tremendously lower oxygen permeability. At 50% relative humidity, film made from xylan/chitosan (70:30 wt./wt. or higher proportion) showed comparable result as those reported for synthetic polymers such as ethylene vinyl alcohol (EVOH) (values below 0.5 cm3 μ/mm2/d/kPa) [75]. However, films were highly sensitive to high humidity conditions and permeability values were increased by three orders of magnitude. The linear low molecular weight of xylan polymers resulted in crystalline structure and improvement in mechanical properties, as compared to high-molecular-weight chitosan polymers. The film containing xylan/ chitosan (85/15 wt./wt.) exhibited no inhibition zone of microorganisms. The poor antimicrobial property was attributed to lack of free cationic amino groups.

Handbook of Chitin and Chitosan

657

20.4 Chitin/chitosan composites with synthetic polymer

Shankar et al. used the solution casting technique to prepare chitin nanofibril-reinforced carrageenan nanocomposite films and demonstrated improvement in mechanical and barrier properties by the addition of nanofibrils up to 5 wt.% [76]. The tensile strength was increased to 44.7 MPa by nearly 50% compared with neat carrageenan films and the minimum value of WVP of 1.54 3 1029 g.m/m2Pa was achieved at this concentration. A higher percentage of chitin nanofibrils resulted in aggregation and thus exhibited poor barrier and mechanical attributes.

20.4 Chitin/chitosan composites with synthetic polymer Preparation of chitosan-based films blended with synthetic polymers with improved properties have been reported. A water-soluble synthetic polymer, poly(vinyl alcohol) or PVOH is nontoxic and gave good mechanical properties. The formation of strong intermolecular hydrogen bonding between PVOH and chitosan assists/supports the preparation of films [77]. The proposed mechanism for the formation of hydrogen bonding of chitosan and PVOH is shown in Fig. 20.7, as confirmed by using FTIR (Perkin-Elmer, 2000 Model spectrometer) and reported by Ngah et al. [78]. The use of a higher concentration of PVOH with chitosan for film preparation enhances its elasticity, tensile strength, elongation, oxygen and water barrier properties, proclaiming their potential as an antimicrobial material for food packaging [77,79].

CH2 CH

n

O H Intermolecular HOH2C

HOH2C

O

O

O

O

O HO

NH2

NH2

HO

Intermolecular Intermolecular

H O CH2 CH

FIGURE 20.7

n

Proposed structure and interactions/hydrogen bonding of chitosan and PVOH. Source: Reproduced by permission from Elsevier.

Handbook of Chitin and Chitosan

658

20. Chitosan/chitin-based composites for food packaging applications

Furthermore, the incorporation or functionalization of chitosan with other bioactive materials as a reinforcement in PVOH provided films with improved mechanical properties and fire resistance, hence broadening the application of chitosan/PVOH films [80]. The addition of lactic acid into chitosan for film preparation has also been reported, where blending of chitosan with poly(lactic acid) and starch, or synthesis of lactic acid oligomer-grafted-chitosan and its use as a nanofiller into poly(lactic acid) films displayed improvement in multiple properties, such as thermal and tensile properties, confirming its use as a promising material in food packaging application [81,82]. The use of synthetic acids, such as fumaric acid and salicylic acid, with chitosan is reported to diminish chilling injury in cucumber and preserve its quality [81]. The use of chitosan with low-density polyethylene as a matrix polymer to obtain films via press molding or extrusion techniques is reported to have potential antimicrobial activity on different bacterial strains. Films obtained by blending chitosan with low-density polyethylene displayed high water permeability with slightly low mechanical strength [83], while the further addition of ethylene-acrylic acid copolymer as an adhesive was also studied and showed high percentage inhibition (antifungal activity) against A. niger, suggesting they are promising materials for food packaging application [84]. The preparation of antimicrobial nanofiber mats from polyethylene oxide and chitosan after loading of zeolitic imidazolate nanoparticles by the electrospinning technique was reported by Kohsari et al. [85]. The prepared nanofibers were found to be the most suitable material for food coating applications. The incorporation of polycaprolactone and allyl isothiocyanate in chitosan-based films has also been reported to enhance the antibacterial effect of the prepared films making them useful for food packaging [86,87]. Furthermore, the blending of various synthetic polymers, such as polyethylene terephthalate, lauric arginate ester, liquid paraffin, and poly(butylene adipate-co-terephthalate), with chitosan could allow the design of the flexibility of the final films. Layer-by-layer coating of chitosan on synthetic polymers acting as a substrate to obtain composite films with multiple functionalities and sustainability have been reported [64,88]. The use of chitosan with synthetic polymers for the preparation of films by its grafting on polypropylene and layer-by-layer assembling with azopolymer via electrostatic interaction has also been investigated and found to exhibit optical and antimicrobial activities making them promising material for food packaging applications [89,90]. Chitin nanofibers have also been applied as a reinforcement material due to their high mechanical strength and Young’s modulus ( . 150 Gpa) [91,92]. These properties allow their use as a promising material to improve the mechanical properties of synthetic polymers such as polylactide [93], acrylic resins [94], and poly(ε-caprolactone) [95]. Handbook of Chitin and Chitosan

20.5 Inorganic materials-derived composites

659

A remarkable rise in the Young’s moduli and tensile strength of (meth) acrylic resins was observed after reinforcement with chitin, which fosters the utilization of chitin nanofibers as a natural and environment-friendly material [94]. The investigation of mechanical properties of developed poly(vinyl alcohol)-α-chitin films obtained by reinforcing palm oil empty fruit bunch fiber-derived NC using the solvent casting method has been reported by Mok et al. [96]. Increased tensile strength of poly(vinyl alcohol)/chitin films from 29.06 to 39.27 MPa was observed by varying α-chitin contents from 10 to 30 wt.%. These results suggested that poly (vinyl alcohol)/chitin films can be a useful or potential candidate for food packaging applications. Recently, Zhang et al. [97] reported the preparation of liquefied chitin/ polyvinyl alcohol blend membranes using different concentrations or ratios. The liquefied chitin was obtained by a liquefaction method using a PEG 400/glycerin mixture as a solvent system. Under optimized conditions, membranes obtained by adding 25 wt.% of liquefied chitin to polyvinyl alcohols displayed significant improvement in thermal and mechanical properties. Furthermore, an increase in the water absorption and water retention capacity was observed by the blend membranes. In particular, the incorporation of liquefied chitin remarkably improved the antibacterial activity of prepared membranes. In addition to other improved properties, the high mechanical strength, thermal stability, and excellent antibacterial activity of liquefied chitin/polyvinyl alcohol blend membranes suggests their utilization as a promising material in food packaging application. Another recent investigation on the development of spray-coated multilayer cellulose nanocrystal-chitin nanofiber films and their barrier properties has been reported by Satam et al. [62]. The film preparations were carried out by spraying an alternative coating of cellulose nanocrystals and chitin nanofibers onto poly(lactic acid) to minimize the permeation capability of oxygen. The prepared multilayer cellulose nanocrystal-chitin nanofiber coatings (Fig. 20.8) displayed reduced oxygen and haze permeability compared with cellulose nanocrystals and chitin nanofibers alone, while a similar water transmission rate was observed by composites for the poly(lactic acid). The study suggested the application of prepared materials in food packaging and pharmaceuticals, where the permeability of oxygen is the main concern [62].

20.5 Inorganic materials-derived composites Inorganic chitosan complexes have shown great potential to be used in the preparation of films (Fig. 20.9) due to the high chelating capacity of chitosan. Mostly, these films have displayed great potential in food packaging application. The development of films with antimicrobial Handbook of Chitin and Chitosan

660

20. Chitosan/chitin-based composites for food packaging applications

O2

Chitin nanofibers

Polymer Cellulose nanicrystal H2O

FIGURE 20.8 Schematic drawing representing cellulose nanocrystal-chitin nanofiber films and their barrier properties. Source: Reproduced with permission from ACS publisher.

FIGURE 20.9

Formation of inorganic chitosan complexes. Source: Reproduced by permission from Elsevier H. Wang, J. Qian, F. Ding, Emerging chitosan-based films for food packaging applications, J. Agric. Food Chem. 66 (2018) 395
413.

properties by the incorporation and homogenous dispersion of silver nanoparticles into a chitosan polymer matrix for active food packaging has been reported using simple and green methodology [98,99]. Chitosan and silver nanoparticle-based films were found to have substantially improved hydrophilic property, antibacterial activity, biocompatibility, degradability, and nontoxicity, which encourage the use of these films for edible food packaging application [100,101]. Another nanofiller is zinc oxide, which was also incorporated into chitosan-derived films to enhance their mechanical properties,

Handbook of Chitin and Chitosan

20.5 Inorganic materials-derived composites

661

antimicrobial activity, and transparency [102,103]. As these composite films have the ability to retard the growth of food pathogens and enhance the shelf life of food, they can be considered to be useful in food applications [104]. To improve the barrier properties of films, such carbon dioxide, oxygen and WVP, chitosan
montmorillonite composites have been investigated extensively [79,105]. Researchers have shown that the use of montmorillonite as a reinforcement material with chitosan could enhance remarkably their flame retardant and physicomechanical properties, thus accelerating the potential of these composites in food packaging and related applications [106
108]. Enhanced mechanical and oxygen barrier properties of films by crosslinking of chitosan and graphene oxide have been observed when compared to pristine chitosan [109,110]. Incorporation of graphene oxide into chitosan or nanometric graphene stacks to chitosan already functionalized with cinnamaldehyde has also been evaluated and displayed an increase in mechanical properties of the films. Furthermore, the inhibitory potential of these films against food pathogens and bacteria make them a promising material for food packaging [111,112]. Studies have also been reported on the preparation of chitosan/nanosilica films, where silicon materials (silicon carbide and silica) were used to get hybrid coatings demonstrating enhanced barrier and chemical properties, thus reducing the food decaying process and ensuring their use for prolonged storage life [113,114]. Use of nanosilicon carbide into chitosan as a filler demonstrated remarkably high thermal stability, tensile strength, and reduction in oxygen permeability of the prepared films, indicating their use as suitable material for packaging applications [115]. Nanosized titanium dioxide
chitosan hybrid packaging has been evaluated for food packaging and has displayed the potential to maintain the quality and increased shelf life of climacteric fruit. This study concluded that on exposure of UV light, the titanium dioxide
chitosan films have shown ethylene photodegradation activity, hence retarding the ripening process and maintaining the quality of tomatoes (Fig. 20.10) [116]. Preparation of composite films from copper nanoparticles and chitosan using microwave heating by a solution casting method has been reported by Ca´rdenas et al. [117] They investigated the potential of prepared composite films in food packaging application and concluded that the addition of copper nanoparticles into chitosan matrix enhanced their barrier properties for oxygen and water penetration and also increased protection against light transmission. Moreover, they claimed the current study can lead to the use of these composite films for food packaging applications [117].

Handbook of Chitin and Chitosan

662

20. Chitosan/chitin-based composites for food packaging applications

UV lamp

e–

hv

H 2O O2

O2

O2–

Photocatalytic degradation

TiO2

h+

H2O

H 2O

C2H4

H–+

OH•

CO2+H2O

H 2O O2

C 2H

C2H4

C2H4 C2H4

4

Chitosan-TiO2 nanocomposite film

FIGURE 20.10 Schematic illustration of prepared chitosan-TiO2 nanocomposite film activation to oxidize ethylene on exposure of UV light. Source: Reproduced by permission from Elsevier.

Utilization of various inorganic materials, such as titanium dioxide, cloisite 20 A, (Mg(NO3)2.6H2O, Al(NO3)3.9H2O, CaCl2, CaCO3, nanomagnesium oxide, zinc(II), and cerium(IV), in the reinforcement of chitosan [112,118,119] has been reported and proved to enhance their thermal stability, mechanical and barrier properties [120
122]. The special structural characteristics of inorganic materials [123] have also demonstrated resistance against chemical materials (dilute HCl and NaOH) [124] indicating their potential in food packaging application [125]. Synthesis of iron/chitin nanocomposite was carried out by a costeffective method, where chitin nanoparticles were obtained from Penaeus semisulcatus shells, while iron nanoparticles were produced from leaf extract of Corchorus olitorius (aqueous) which acts as a reducing and capping agent. The study revealed that, the prepared iron/chitin nanocomposites displayed remarkably high antimicrobial activity when compared with the iron nanoparticles or chitin nanoparticles individually, suggesting that these materials can be potentially applied for antimicrobial and environmental applications [126]. As discussed above, chitin/chitosan-based nanocomposites/films/ coatings have great potential to be utilized for food packaging applications because of their excellent mechanical, thermal, and antimicrobial properties, while further investigations on optimizations are required for their industrialization in the market.

Handbook of Chitin and Chitosan

20.6 Conclusion and future trends

663

20.6 Conclusion and future trends The chitosan and chitin-derived polymers for the food packaging applications have great prospects for addressing the ecological and human health risks due to the pollution related to petroleum-based food packaging materials. The technological advancements are permitting the synthesis of chitin and chitosan-based films, membranes, coatings, and as a nanofiller with several attractive functional properties, which have the significant potential to be used in food packaging applications. Chitosan and chitin-derived composites offer certain attributes in food packaging applications, such as antimicrobial effects, biocompatibility and biodegradability, and lower ecocytotoxicity. However, serious attention is desirable for the advancement of chitin and chitosan-based materials in order to fulfill the practical requirements as well as cope with the petroleum-based food packaging materials. For instance, some viable approaches are essential to improve the properties of the chitin and chitosan-derived materials, particularly mechanical strength to meet numerous food packaging applications. Future advances in the chitin and chitosan-based material can be required in the following facets: 1. The synthesis of novel chitin and chitosan derivatives to enhance the functional properties of the materials are required to be exploited to meet special requirements. 2. The production of chitin and chitosan-based materials at an industrial scale. To achieve this, current industrial processes could be adjusted or coupled with advanced technology. Similarly, new approaches or technologies are still required to be optimized for the mass production of the chitin and chitosan-based composites to attain diverse food packaging applications. 3. There is not enough data available related to the toxicity and transfer of any harmful component from the chitin and chitosan-based materials to the packaged food. The detailed studies related to transfer mechanism are required to understand the contact phenomenon with the food. 4. More research efforts are critical in order to measure and understand the degradation rate and mechanism, and most importantly the ecological impact of the chitosan and chitin-derived materials in the real world. The chitin and chitosan-based material face multifaceted challenges in order to achieve the advancement of their use in competition with the petroleum-based materials. Nevertheless, the materials have bright prospects in many food packaging applications.

Handbook of Chitin and Chitosan

664

20. Chitosan/chitin-based composites for food packaging applications

References [1] K. Marsh, B. Bugusu, Food packaging—roles, materials, and environmental issues, J. Food Sci. 72 (2007) R39
R55. [2] K. Biji, C. Ravishankar, C. Mohan, T.S. Gopal, Smart packaging systems for food applications: a review, J. Food Sci. Technol. 52 (2015) 6125
6135. [3] M. Zubair, A. Ullah, Recent advances in protein derived bionanocomposites for food packaging applications, Crit. Rev. Food Sci. Nutr. (2019) 1
29. [4] S.A. Ntim, G.O. Noonan, Nanotechnology in food packaging, Nanotechnologies in Food, 2017, pp. 118
142. [5] A.J. Jose, M. Alagar, Nanotechnology for smart and intelligent food packaging, Biopolym. Biomater. (2018). [6] L.F. Ballesteros, M. Michelin, A.A. Vicente, et al., Food applications of lignocellulosicbased packaging materials, Lignocellulosic Materials and Their Use in Bio-based Packaging, Springer, 2018, pp. 87
94. [7] F. Gironi, V. Piemonte, Bioplastics and petroleum-based plastics: strengths and weaknesses, Energy Sources Part A 33 (2011) 1949
1959. [8] M. Rabnawaz, I. Wyman, R. Auras, S. Cheng, A roadmap towards green packaging: the current status and future outlook for polyesters in the packaging industry, Green. Chem. 19 (2017) 4737
4753. [9] A.M. Youssef, S.M. El-Sayed, Bionanocomposites materials for food packaging applications: concepts and future outlook, Carbohydr. Polym. (2018). [10] A. Ali, S. Ahmed, Recent advances in edible polymer based hydrogels as a sustainable alternative to conventional polymers, J. Agric. Food Chem. 66 (2018) 6940
6967. [11] H. Wang, J. Qian, F. Ding, Emerging chitosan-based films for food packaging applications, J. Agric. Food Chem. 66 (2018) 395
413. [12] S. Eilert, New packaging technologies for the 21st century, Meat Sci. 71 (2005) 122
127. [13] Z. Li, M. Rabnawaz, Fabrication of food-safe water-resistant paper coatings using a melamine primer and polysiloxane outer layer, ACS Omega 3 (2018) 11909
11916. [14] C. Girometta, A.M. Picco, R.M. Baiguera, D. Dondi, S. Babbini, M. Cartabia, et al., Physico-mechanical and thermodynamic properties of mycelium-based biocomposites: a review, Sustainability 11 (2019) 281. [15] T.A. Hottle, M.M. Bilec, A.E. Landis, Sustainability assessments of bio-based polymers, Polym. Degrad. Stab. 98 (2013) 1898
1907. [16] M.G.A. Vieira, M.A. da Silva, L.O. dos Santos, M.M. Beppu, Natural-based plasticizers and biopolymer films: a review, Eur. Polym. J. 47 (2011) 254
263. [17] J.-W. Rhim, H.-M. Park, C.-S. Ha, Bio-nanocomposites for food packaging applications, Prog. Polym. Sci. 38 (2013) 1629
1652. [18] G.E. Luckachan, C. Pillai, Biodegradable polymers-a review on recent trends and emerging perspectives, J. Polym. Environ. 19 (2011) 637
676. [19] X. Tang, P. Kumar, S. Alavi, K. Sandeep, Recent advances in biopolymers and biopolymer-based nanocomposites for food packaging materials, Crit. Rev. Food Sci. Nutr. 52 (2012) 426
442. [20] J. Go´mez-Estaca, R. Gavara, R. Catala´, P. Herna´ndez-Mun˜oz, The potential of proteins for producing food packaging materials: a review, Packag. Technol. Sci. 29 (2016) 203
224. [21] T.B. Rouf, J.L. Kokini, Natural biopolymer-based nanocomposite films for packaging applications, Bionanocomposites for Packaging Applications, Springer, 2018, pp. 149
177.

Handbook of Chitin and Chitosan

References

665

¨ . Seydibeyoglu, ˘ [22] S. Muniyasamy, O B. Thulasinathan, A. Arun, Biopolymer synthesis and biodegradation, Sustainable Biotechnology-Enzymatic Resources of Renewable Energy, Springer, 2018, pp. 399
421. [23] Y. Zheng, J. Monty, R.J. Linhardt, Polysaccharide-based nanocomposites and their applications, Carbohydr. Res. 405 (2015) 23
32. [24] R. Zafar, K.M. Zia, S. Tabasum, F. Jabeen, A. Noreen, M. Zuber, Polysaccharide based bionanocomposites, properties and applications: a review, Int. J. Biol. Macromol. 92 (2016) 1012
1024. [25] R.A. Gross, B. Kalra, Biodegradable polymers for the environment, Science 297 (2002) 803
807. [26] R. Dassanayake, S. Acharya, N. Abidi, Biopolymer-based materials from polysaccharides: properties, processing, characterization and sorption applications, Advanced Sorption Process Applications, IntechOpen, 2018. [27] D. Severian, Polysaccharides: Structural Diversity and Functional Versatility, Marcel Dekker, New York, 2008. [28] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603
632. [29] V. Manigandan, R. Karthik, S. Ramachandran, S. Rajagopal, Chitosan applications in food industry, Biopolymers for Food Design, Elsevier, 2018, pp. 469
491. [30] H. Cauchie, An attempt to estimate crustacean chitin production in the hydrosphere, Adv. Chit. Sci. 2 (1997) 32
39. [31] T.J. Gutie´rrez, Chitosan applications for the food industry, chitosan: derivatives, Compos. Appl. (2017) 183
232. [32] K. Kurita, T. Kojima, Y. Nishiyama, M. Shimojoh, Synthesis and some properties of nonnatural amino polysaccharides: branched chitin and chitosan, Macromolecules 33 (2000) 4711
4716. [33] S. Ahmed, S. Ikram, Chitosan: derivatives, Composites and Applications, John Wiley & Sons, 2017. [34] E. Agullo´, M.S. Rodrı´guez, V. Ramos, L. Albertengo, Present and future role of chitin and chitosan in food, Macromol. Biosci. 3 (2003) 521
530. [35] L. Balau, G. Lisa, M.I. Popa, V. Tura, V. Melnig, Physico-chemical properties of chitosan films, Cent. Eur. J. Chem. 2 (2004) 638
647. [36] P.K. Dutta, S. Tripathi, G.K. Mehrotra, J. Dutta, Perspectives for chitosan based antimicrobial films in food applications, Food Chem. 114 (2009) 1173
1182. [37] M.A. Khokhlova, M.O. Gallyamov, A.R. Khokhlov, Chitosan nanostructures deposited from solutions in carbonic acid on a model substrate as resolved by AFM, Colloid Polym. Sci. 290 (2012) 1471
1480. [38] B.S. Munteanu, E. Paˆslaru, L.F. Zemljic, A. Sdrobi¸s, G.M. Pricope, C. Vasele, Chitosan coatings applied to polyethylene surface to obtain food-packaging materials, Cellulose Chem. Technol. 48 (2014) 565
575. [39] A. Kamari, E. Phillip, Chitosan, gelatin and methylcellulose films incorporated with tannic acid for food packaging, Int. J. Biol. Macromol. 120 (2018) 1119
1126. [40] S. Kumar, A. Shukla, P.P. Baul, A. Mitra, D. Halder, Biodegradable hybrid nanocomposites of chitosan/gelatin and silver nanoparticles for active food packaging applications, Food Packag. Shelf Life 16 (2018) 178
184. [41] R. Mohammadi, M.A. Mohammadifar, M. Rouhi, M. Kariminejad, A.M. Mortazavian, E. Sadeghi, et al., Physico-mechanical and structural properties of eggshell membrane gelatin-chitosan blend edible films, Int. J. Biol. Macromol. 107 (2018) 406
412. [42] J. Bonilla, T. Poloni, R.V. Lourenc¸o, P.J. Sobral, Antioxidant potential of eugenol and ginger essential oils with gelatin/chitosan films, Food Biosci. 23 (2018) 107
114.

Handbook of Chitin and Chitosan

666

20. Chitosan/chitin-based composites for food packaging applications

[43] L.J.P. Co´rdoba, P.J. Sobral, Physical and antioxidant properties of films based on gelatin, gelatin-chitosan or gelatin-sodium caseinate blends loaded with nanoemulsified active compounds, J. Food Eng. 213 (2017) 47
53. [44] J. Bonilla, P.J. Sobral, Investigation of the physicochemical, antimicrobial and antioxidant properties of gelatin-chitosan edible film mixed with plant ethanolic extracts, Food Biosci. 16 (2016) 17
25. [45] S. Ahmed, S. Ikram, Chitosan and gelatin based biodegradable packaging films with UV-light protection, J. Photochem. Photobiol. B: Biol. 163 (2016) 115
124. [46] S.F. Hosseini, M. Rezaei, M. Zandi, F. Farahmandghavi, Fabrication of bionanocomposite films based on fish gelatin reinforced with chitosan nanoparticles, Food Hydrocoll. 44 (2015) 172
182. [47] S.F. Hosseini, M. Rezaei, M. Zandi, F. Farahmandghavi, Development of bioactive fish gelatin/chitosan nanoparticles composite films with antimicrobial properties, Food Chem. 194 (2016) 1266
1274. [48] J. Go´mez-Estaca, A.L. De Lacey, M. Lo´pez-Caballero, M. Go´mez-Guille´n, P. Montero, Biodegradable gelatin
chitosan films incorporated with essential oils as antimicrobial agents for fish preservation, Food Microbiol. 27 (2010) 889
896. [49] C. Chen, S. Deng, Y. Yang, D. Yang, T. Ye, D. Li, Highly transparent chitin nanofiber/gelatin nanocomposite with enhanced mechanical properties, Cellulose 25 (2018) 5063
5070. [50] C. Chen, Y. Wang, Y. Yang, M. Pan, T. Ye, D. Li, High strength gelatin-based nanocomposites reinforced by surface-deacetylated chitin nanofiber networks, Carbohydr. Polym. 195 (2018) 387
392. [51] S. Sahraee, J.M. Milani, B. Ghanbarzadeh, H. Hamishehkar, Effect of corn oil on physical, thermal, and antifungal properties of gelatin-based nanocomposite films containing nano chitin, LWT-Food Sci. Technol. 76 (2017) 33
39. [52] S. Sahraee, J.M. Milani, B. Ghanbarzadeh, H. Hamishehkar, Physicochemical and antifungal properties of bio-nanocomposite film based on gelatin-chitin nanoparticles, Int. J. Biol. Macromol. 97 (2017) 373
381. [53] N. Caro, E. Medina, M. Dı´az-Dosque, L. Lo´pez, L. Abugoch, C. Tapia, Novel active packaging based on films of chitosan and chitosan/quinoa protein printed with chitosan-tripolyphosphate-thymol nanoparticles via thermal ink-jet printing, Food Hydrocoll. 52 (2016) 520
532. [54] T. Huppertz, P. Fox, A. Kelly, The caseins: structure, stability, and functionality, Proteins in Food Processing, Elsevier, 2018, pp. 49
92. [55] T. Huppertz, I. Gazi, H. Luyten, H. Nieuwenhuijse, A. Alting, E. Schokker, Hydration of casein micelles and caseinates: implications for casein micelle structure, Int. Dairy. J. 74 (2017) 1
11. [56] M. Imran, A. Klouj, A.-M. Revol-Junelles, S. Desobry, Controlled release of nisin from HPMC, sodium caseinate, poly-lactic acid and chitosan for active packaging applications, J. Food Eng. 143 (2014) 178
185. [57] W. Ma, C.-H. Tang, X.-Q. Yang, S.-W. Yin, Fabrication and characterization of kidney bean (Phaseolus vulgaris L.) protein isolate
chitosan composite films at acidic pH, Food Hydrocoll. 31 (2013) 237
247. [58] M. Ahmad, N.P. Nirmal, M. Danish, J. Chuprom, S. Jafarzedeh, Characterisation of composite films fabricated from collagen/chitosan and collagen/soy protein isolate for food packaging applications, RSC Adv. 6 (2016) 82191
82204. [59] K. Chi, J.M. Catchmark, Improved eco-friendly barrier materials based on crystalline nanocellulose/chitosan/carboxymethyl cellulose polyelectrolyte complexes, Food Hydrocoll. 80 (2018) 195
205. [60] N.M. Stark, Opportunities for cellulose nanomaterials in packaging films: a review and future trends, J. Renew. Mater. 4 (2016) 313
326.

Handbook of Chitin and Chitosan

References

667

[61] H. Jafari, M. Pirouzifard, M.A. Khaledabad, H. Almasi, Effect of chitin nanofiber on the morphological and physical properties of chitosan/silver nanoparticle bionanocomposite films, Int. J. Biol. Macromol. 92 (2016) 461
466. [62] C.C. Satam, C.W. Irvin, A.W. Lang, J.C.R. Jallorina, M.L. Shofner, J.R. Reynolds, et al., Spray-coated multilayer cellulose nanocrystal—chitin nanofiber films for barrier applications, ACS Sustain. Chem. Eng. 6 (2018) 10637
10644. [63] X. Ma, M. Lv, D.P. Anderson, P.R. Chang, Natural polysaccharide composites based on modified cellulose spheres and plasticized chitosan matrix, Food Hydrocoll. 66 (2017) 276
285. [64] F. Li, P. Biagioni, M. Finazzi, S. Tavazzi, L. Piergiovanni, Tunable green oxygen barrier through layer-by-layer self-assembly of chitosan and cellulose nanocrystals, Carbohydr. Polym. 92 (2013) 2128
2134. [65] Z. Deng, J. Jung, Y. Zhao, Development, characterization, and validation of chitosan adsorbed cellulose nanofiber (CNF) films as water resistant and antibacterial food contact packaging, LWT-Food Sci. Technol. 83 (2017) 132
140. [66] M.-C. Li, Q. Wu, K. Song, H. Cheng, S. Suzuki, T. Lei, Chitin nanofibers as reinforcing and antimicrobial agents in carboxymethyl cellulose films: influence of partial deacetylation, ACS Sustain. Chem. Eng. 4 (2016) 4385
4395. [67] A.M. Salaberria, R.H. Diaz, J. Labidi, S.C. Fernandes, Role of chitin nanocrystals and nanofibers on physical, mechanical and functional properties in thermoplastic starch films, Food Hydrocoll. 46 (2015) 93
102. [68] Y. Qin, S. Zhang, J. Yu, J. Yang, L. Xiong, Q. Sun, Effects of chitin nano-whiskers on the antibacterial and physicochemical properties of maize starch films, Carbohydr. Polym. 147 (2016) 372
378. [69] P.R. Chang, R. Jian, J. Yu, X. Ma, Fabrication and characterisation of chitosan nanoparticles/plasticised-starch composites, Food Chem. 120 (2010) 736
740. [70] R. Yoksan, S. Chirachanchai, Silver nanoparticle-loaded chitosan
starch based films: fabrication and evaluation of tensile, barrier and antimicrobial properties, Mater. Sci. Eng. C. 30 (2010) 891
897. [71] M.V. Lorevice, C.G. Otoni, M.R. de Moura, L.H.C. Mattoso, Chitosan nanoparticles on the improvement of thermal, barrier, and mechanical properties of high-and lowmethyl pectin films, Food Hydrocoll. 52 (2016) 732
740. [72] F. Wei, F. Ye, S. Li, L. Wang, J. Li, G. Zhao, Layer-by-layer coating of chitosan/pectin effectively improves the hydration capacity, water suspendability and tofu gel compatibility of okara powder, Food Hydrocoll. 77 (2018) 465
473. [73] H.E. Salama, M.S.A. Aziz, M.W. Sabaa, Novel biodegradable and antibacterial edible films based on alginate and chitosan biguanidine hydrochloride, Int. J. Biol. Macromol. 116 (2018) 443
450. [74] C.N. Schnell, M.V. Galva´n, M.S. Peresin, M.C. Inalbon, J. Vartiainen, M.A. Zanuttini, et al., Films from xylan/chitosan complexes: preparation and characterization, Cellulose 24 (2017) 4393
4403. [75] N.M. Hansen, D. Plackett, Sustainable films and coatings from hemicelluloses: a review, Biomacromolecules 9 (2008) 1493
1505. [76] S. Shankar, J.P. Reddy, J.-W. Rhim, H.-Y. Kim, Preparation, characterization, and antimicrobial activity of chitin nanofibrils reinforced carrageenan nanocomposite films, Carbohydr. Polym. 117 (2015) 468
475. [77] H. Wang, R. Zhang, H. Zhang, S. Jiang, H. Liu, M. Sun, et al., Kinetics and functional effectiveness of nisin loaded antimicrobial packaging film based on chitosan/poly (vinyl alcohol), Carbohydr. Polym. 127 (2015) 64
71. [78] W.S. Wan Ngah, A. Kamari, Y.J. Koay, Equilibrium and kinetics studies of adsorption of copper (II) on chitosan and chitosan/PVA beads, Int. J. Biol. Macromol. 34 (2004) 155
161.

Handbook of Chitin and Chitosan

668

20. Chitosan/chitin-based composites for food packaging applications

[79] A. Giannakas, M. Vlacha, C. Salmas, A. Leontiou, P. Katapodis, H. Stamatis, et al., Preparation, characterization, mechanical, barrier and antimicrobial properties of chitosan/PVOH/clay nanocomposites, Carbohydr. Polym. 140 (2016) 408
415. [80] X. Feng, X. Wang, W. Xing, B. Yu, L. Song, Y. Hu, Simultaneous reduction and surface functionalization of graphene oxide by chitosan and their synergistic reinforcing effects in PVA films, Ind. Eng. Chem. Res. 52 (2013) 12906
12914. [81] P. Bie, P. Liu, L. Yu, X. Li, L. Chen, F. Xie, The properties of antimicrobial films derived from poly(lactic acid)/starch/chitosan blended matrix, Carbohydr. Polym. 98 (2013) 959
966. [82] A.K. Pal, V. Katiyar, Nanoamphiphilic chitosan dispersed poly(lactic acid) bionanocomposite films with improved thermal, mechanical, and gas barrier properties, Biomacromolecules 17 (2016) 2603
2618. [83] H.-s Wang, D. Chen, C.-z Chuai, Mechanical and barrier properties of lldpe/chitosan blown films for packaging, Packag. Technol. Sci. 28 (2015) 915
923. [84] A.P. Martı´nez-Camacho, M.O. Cortez-Rocha, A.Z. Graciano-Verdugo, F. Rodrı´guezFe´lix, M.M. Castillo-Ortega, A. Burgos-Herna´ndez, et al., Extruded films of blended chitosan, low density polyethylene and ethylene acrylic acid, Carbohydr. Polym. 91 (2013) 666
674. [85] I. Kohsari, Z. Shariatinia, S.M. Pourmortazavi, Antibacterial electrospun chitosanpolyethylene oxide nanocomposite mats containing ZIF-8 nanoparticles, Int. J. Biol. Macromol. 91 (2016) 778
788. [86] S. Alix, A. Mahieu, C. Terrie, J. Soulestin, E. Gerault, M.G.J. Feuilloley, et al., Active pseudo-multilayered films from polycaprolactone and starch based matrix for foodpackaging applications, Eur. Polym. J. 49 (2013) 1234
1242. [87] M. Guo, T.Z. Jin, M.P. Yadav, R. Yang, Antimicrobial property and microstructure of micro-emulsion edible composite films against Listeria, Int. J. Food Microbiol. 208 (2015) 58
64. ´ lvarez, F. Go´mez-Galva´n, E. Lizundia, I. Kuritka, [88] S. del Hoyo-Gallego, L. Pe´rez-A V. Sedlarik, et al., Construction of antibacterial poly(ethylene terephthalate) films via layer by layer assembly of chitosan and hyaluronic acid, Carbohydr. Polym. 143 (2016) 35
43. [89] J.A. Cavallo, M.C. Strumia, C.G. Gomez, Preparation of a milk spoilage indicator adsorbed to a modified polypropylene film as an attempt to build a smart packaging, J. Food Eng. 136 (2014) 48
55. [90] R. Ferna´ndez, C. Ocando, S.C.M. Fernandes, A. Eceiza, A. Tercjak, Optically active multilayer films based on chitosan and an azopolymer, Biomacromolecules 15 (2014) 1399
1407. [91] S. Ifuku, H. Saimoto, Chitin nanofibers: preparations, modifications, and applications, Nanoscale 4 (2012) 3308
3318. [92] J.F.V. Vincent, U.G.K. Wegst, Design and mechanical properties of insect cuticle, Arthropod. Struct. Dev. 33 (2004) 187
199. [93] J.Y. Kim, C.S. Ha, N.J. Jo, Synthesis and properties of biodegradable chitin-graft-poly (L-lactide) copolymers, Polym. Int. 51 (2002) 1123
1128. [94] S. Ifuku, S. Morooka, A. Norio Nakagaito, M. Morimoto, H. Saimoto, Preparation and characterization of optically transparent chitin nanofiber/(meth)acrylic resin composites, Green. Chem. 13 (2011) 1708
1711. [95] R. Jayakumar, H. Tamura, Synthesis, characterization and thermal properties of chitin-g-poly(ε-caprolactone) copolymers by using chitin gel, Int. J. Biol. Macromol. 43 (2008) 32
36. [96] Y.C. Ching, F. Muhamad, N.A. Abu Osman, R. Singh, Poly(vinyl alcohol)-α-chitin composites reinforced by oil palm empty fruit bunch fiber-derived nanocellulose, Int. J. Polym. Anal. Charact. 22 (2017) 294
304.

Handbook of Chitin and Chitosan

References

669

[97] J. Zhang, W.-R. Xu, Y. Zhang, W. Li, J. Hu, F. Zheng, et al., Liquefied chitin/polyvinyl alcohol based blend membranes: preparation and characterization and antibacterial activity, Carbohydr. Polym. 180 (2018) 175
181. [98] G. Lo´pez-Carballo, L. Higueras, R. Gavara, P. Herna´ndez-Mun˜oz, Silver ions release from antibacterial chitosan films containing in situ generated silver nanoparticles, J. Agric. Food Chem. 61 (2013) 260
267. [99] A.M. Youssef, M.S. Abdel-Aziz, S.M. El-Sayed, Chitosan nanocomposite films based on Ag-NP and Au-NP biosynthesis by Bacillus Subtilis as packaging materials, Int. J. Biol. Macromol. 69 (2014) 185
191. [100] M. Kaur, A. Kalia, A. Thakur, Effect of biodegradable chitosan
rice-starch nanocomposite films on post-harvest quality of stored peach fruit, Starch - Sta¨rke 69 (2017) 1600208. [101] A.M. Youssef, H. Abou-Yousef, S.M. El-Sayed, S. Kamel, Mechanical and antibacterial properties of novel high performance chitosan/nanocomposite films, Int. J. Biol. Macromol. 76 (2015) 25
32. [102] L. Al-Naamani, S. Dobretsov, J. Dutta, Chitosan-zinc oxide nanoparticle composite coating for active food packaging applications, Innovat. Food Sci. Emerg. Technol. 38 (2016) 231
237. [103] S. Sanuja, A. Agalya, M.J. Umapathy, Synthesis and characterization of zinc oxide
neem oil
chitosan bionanocomposite for food packaging application, Int. J. Biol. Macromol. 74 (2015) 76
84. [104] A.M. Youssef, S.M. El-Sayed, H.S. El-Sayed, H.H. Salama, A. Dufresne, Enhancement of Egyptian soft white cheese shelf life using a novel chitosan/carboxymethyl cellulose/zinc oxide bionanocomposite film, Carbohydr. Polym. 151 (2016) 9
19. [105] L. Zhang, H. Wang, C. Jin, R. Zhang, L. Li, X. Li, et al., Sodium lactate loaded chitosan-polyvinyl alcohol/montmorillonite composite film towards active food packaging, Innovat. Food Sci. Emerg. Technol. 42 (2017) 101
108. [106] J.F. Rubilar, D. Candia, A. Cobos, O. Dı´az, F. Pedreschi, Effect of nanoclay and ethyl-Nα-dodecanoyl-l-arginate hydrochloride (LAE) on physico-mechanical properties of chitosan films, LWT - Food Sci. Technol. 72 (2016) 206
214. [107] M. Vlacha, A. Giannakas, P. Katapodis, H. Stamatis, A. Ladavos, N.-M. Barkoula, On the efficiency of oleic acid as plasticizer of chitosan/clay nanocomposites and its role on thermo-mechanical, barrier and antimicrobial properties
comparison with glycerol, Food Hydrocoll. 57 (2016) 10
19. [108] M. Zhou, Q. Liu, S. Wu, Z. Gou, X. Wu, D. Xu, Starch/chitosan films reinforced with polydopamine modified MMT: effects of dopamine concentration, Food Hydrocoll. 61 (2016) 678
684. [109] P. Tzeng, B. Stevens, I. Devlaming, J.C. Grunlan, Polymer
graphene oxide quadlayer thin-film assemblies with improved gas barrier, Langmuir: ACS J. Surf. Colloids 31 (2015) 5919
5927. [110] N. Yan, F. Capezzuto, M. Lavorgna, G.G. Buonocore, F. Tescione, H. Xia, et al., Borate cross-linked graphene oxide
chitosan as robust and high gas barrier films, Nanoscale 8 (2016) 10783
10791. [111] C. Demitri, V.M. De Benedictis, M. Madaghiele, C.E. Corcione, A. Maffezzoli, Nanostructured active chitosan-based films for food packaging applications: effect of graphene stacks on mechanical properties, Measurement 90 (2016) 418
423. [112] W. Xu, W. Xie, X. Huang, X. Chen, N. Huang, X. Wang, et al., The graphene oxide and chitosan biopolymer loads TiO2 for antibacterial and preservative research, Food Chem. 221 (2017) 267
277. [113] S. Shi, W. Wang, L. Liu, S. Wu, Y. Wei, W. Li, Effect of chitosan/nano-silica coating on the physicochemical characteristics of longan fruit under ambient temperature, J. Food Eng. 118 (2013) 125
131.

Handbook of Chitin and Chitosan

670

20. Chitosan/chitin-based composites for food packaging applications

[114] H. Song, W. Yuan, P. Jin, W. Wang, X. Wang, L. Yang, et al., Effects of chitosan/ nano-silica on postharvest quality and antioxidant capacity of loquat fruit during cold storage, Postharvest Biol. Technol. 119 (2016) 41
48. [115] G.C. Pradhan, S. Dash, S.K. Swain, Barrier properties of nano silicon carbide designed chitosan nanocomposites, Carbohydr. Polym. 134 (2015) 60
65. [116] P. Kaewklin, U. Siripatrawan, A. Suwanagul, Y.S. Lee, Active packaging from chitosan-titanium dioxide nanocomposite film for prolonging storage life of tomato fruit, Int. J. Biol. Macromol. 112 (2018) 523
529. [117] G. Ca´rdenas, M.F. Mele´ndrez, A.G. Cancino, Colloidal Cu nanoparticles/chitosan composite film obtained by microwave heating for food package applications, Polym. Bull. 62 (2009) 511
524. [118] G. Xiao, X. Zhang, Y. Zhao, H. Su, T. Tan, The behavior of active bactericidal and antifungal coating under visible light irradiation, Appl. Surf. Sci. 292 (2014) 756
763. [119] W. Xiao, J. Xu, X. Liu, Q. Hu, J. Huang, Antibacterial hybrid materials fabricated by nanocoating of microfibril bundles of cellulose substance with titania/chitosan/silver-nanoparticle composite films, J. Mater. Chem. B 1 (2013) 3477
3485. [120] A. Agalya, M.J. Umapathy, Studies on magnesium oxide reinforced chitosan bionanocomposite incorporated with clove oil for active food packaging application, Int. J. Polym. Mater. Polym. Biomater. 63 (2014) 733
740. [121] R.T. De Silva, M.M.M.G.P.G. Mantilaka, S.P. Ratnayake, G.A.J. Amaratunga, K.M.N. de Silva, Nano-MgO reinforced chitosan nanocomposites for high performance packaging applications with improved mechanical, thermal and barrier properties, Carbohydr. Polym. 157 (2017) 739
747. [122] M. Shahbazi, G. Rajabzadeh, S.J. Ahmadi, Characterization of nanocomposite film based on chitosan intercalated in clay platelets by electron beam irradiation, Carbohydr. Polym. 157 (2017) 226
235. [123] T. Pan, S. Xu, Y. Dou, X. Liu, Z. Li, J. Han, et al., Remarkable oxygen barrier films based on a layered double hydroxide/chitosan hierarchical structure, J. Mater. Chem. A 3 (2015) 12350
12356. [124] S.K. Swain, S. Dash, S.K. Kisku, R.K. Singh, Thermal and oxygen barrier properties of chitosan bionanocomposites by reinforcement of calcium carbonate nanopowder, J. Mater. Sci. Technol. 30 (2014) 791
795. [125] H. Wu, D. Wang, J. Shi, S. Xue, M. Gao, Effect of the complex of Zinc(II) and Cerium(IV) with chitosan on the preservation quality and degradation of organophosphorus pesticides in Chinese Jujube (Zizyphus jujuba Mill. cv. Dongzao), J. Agric. Food Chem. 58 (2010) 5757
5762. [126] E.Z. Gomaa, Iron nanoparticles α-chitin nanocomposite for enhanced antimicrobial, dyes degradation and heavy metals removal activities, J. Polym. Environ. 26 (2018) 3638
3654.

Handbook of Chitin and Chitosan

C H A P T E R

21 Modified release properties of glutathione-based chitosan films: Physical and functional characterization Yhors Ciro1, John Rojas1, Cristian J. Yarce2 and Constaı´n H. Salamanca2 1

Department of Pharmacy, School of Pharmaceutical and Food Sciences, University of Antioquia, Medellı´n, Colombia, 2Laboratorio de Disen˜o y Formulacio´n de Productos Quı´micos y Derivados, Departamento de Ciencias Farmace´uticas, Facultad de Ciencias Naturales, Universidad ICESI, Cali, Colombia

O U T L I N E 21.1 Introduction

672

21.2 Experiment 21.2.1 Materials 21.2.2 Synthesis of thiolated chitosan 21.2.3 Characterization of chitosan and thiolated materials 21.2.4 Swelling analysis 21.2.5 Evaluation of the in vitro release of cyclophosphamide

673 673 673 674 676 677

21.3 Results and discussion 21.3.1 Synthesis of thiolated polymers 21.3.2 Physicochemical characterization of polymeric materials 21.3.3 Swelling study 21.3.4 In vitro release of cyclophosphamide from polymeric films

677 677 679 684 686

21.4 Conclusions

686

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00021-2

671

© 2020 Elsevier Inc. All rights reserved.

672

21. Modified release properties of glutathione-based chitosan films

Acknowledgments

687

References

687

21.1 Introduction Chitosan (CH) is the deacetylated product of chitin, which is the second most abundant polymer in nature after cellulose. It is a biodegradable, biocompatible, mucoadhesive, and a cationic polymer formed from subunits of N-acetyl-glucosamine and N-glucosamine. The structure of CH can be modified by the inclusion of different ligands (i.e., stearic, palmitic, oleic, and polyaspartic acids, or hydroxyapatite) improving properties such as wettability, surface charge, permeability, solubility in organic solvents, encapsulation efficiency, and entrapment ability of hydrophobic compounds [1,2]. Particularly, the chemical modification of CH with thiol groups renders a material with fabulous features such as in situ gelling, enhancement of mucoadhesivity, and cell permeation, having a better efflux pumpinhibiting capability [3]. Moreover, ligands such as 6-mercaptonicotinic acid, cysteine 2-imiothiolane, N-acetyl-penicilamine, 4-mercaptobenzoic acid, N-acetyl-cysteine, glutathione, and thioglycolic acid can be used for the thiolation of CH, resulting in materials having different reactivity, physicochemical stability, and in vivo performance [4]. For instance, Millotti and collaborators obtained 6-mercaptonicotinic acid
chitosan conjugates showing a 30-fold viscosity increase at a pH of 5.5 and 37 C. In this case, carbodiimide was used as the mediator and the reaction took 7 h. Furthermore, this derivative was considered to be nontoxic and the medium pH did not affect its reactivity [5]. In contrast, the 4-mercaptobenzoic acid
chitosan conjugate showed a 60-fold higher mucoadhesivity compared to CH alone and exhibited a high reactivity at intestinal pH since its pKa is 6.8 [6]. On the other hand, a preferred chitosan thiolation can be achieved by CH reaction with thioglycolic acid at a pH of 6.0 showing a 3.1-fold and 2.3-fold increase in mucoadhesivity and swelling, respectively. This reaction was conducted using a 50 mM carbodiimide solution at a pH of 5.0. Further, the inclusion of cysteine in the CH backbone increased mucoadhesivity resulting in a controlled release of metformin. HCl (a highly soluble drug) [7]. Kafedjiiski and collaborators synthesized GSH
CH conjugates varying the reaction pH and the ratio of EDAC and NHS precursors. They found that a pH of 6.0, a 1:1 EDAC:NHS ratio at 25 C for 7 h was the optimal reaction condition to obtain the highest substitution degree [8]. However, this coupling reaction with carbodiimide takes over 12 h, even though there are reports of reaction times between 15 min to 24 h in other disciplines [9]. Moreover, the reported studies Handbook of Chitin and Chitosan

21.2 Experiment

673

have not conducted an in-depth characterization of these thiolated materials, and thus the aim of this study is to assess the effect of thiolation degree (TD) on the solid-state and cyclophospamide release from these materials produced via carbodiimide-mediated coupling.

21.2 Experiment 21.2.1 Materials CH with a degree of acetylation (DA) of 25% (lot STBF8219V), N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (carbodiimide) (lot, BCBR6841V, purity .97%), glutathione (lot SLBQ4892V, purity .98%), Ellman’s reagent (lot MKBK7085V), and N-hydroxysuccinimide (lot MKBX1364V, purity 98%) were purchased from Sigma Aldrich Co. (Missouri, United States). Sodium hydroxide (lot B1315798639), HCl (lot K45147217 349), L-cysteine (lot K4055438-946, purity $ 99%), deuterium oxide (D2O, lot S5725666, purity $ 99.9%), and acetic acid (lot K41575763) were obtained from Merck (Darmstadt, Germany). Sodium chloride (lot 13843802) was purchased from Scharlau (Barcelona, Spain).

21.2.2 Synthesis of thiolated chitosan 21.2.2.1 Microwave-assisted deacetylation of chitosan The deacetylation reaction was carried out using a focused microwave apparatus operated at a 10% power (Samsung, Model MW 630 WA, with dimensions: 289 mm 3 179 mm 3 326 mm) for 2 h, using additive cycles of 5 min. Approximately, 5 g of commercial CH was mixed with 50 mL of 10 N NaOH in a 500 mL round-bottom flask coupled with a 300-mm-long spiral reflux condenser through an aperture on top of microwave apparatus. 21.2.2.2 Thiolation of chitosan polymers Approximately, 100 mL of 1% (w/v) of deaceytaled CH (DCH) solution was prepared in 1% (v/v) acetic acid at a pH of 6.0 adjusted with 5 N NaOH. Subsequently, the solution was mixed with 5 g of glutathione, carbodiimide, and N-hydroxysuccinimide at final concentrations of 200 mM and the pH was adjusted to 6.0. This mixture was kept under constant stirring for 24, 72, and 120 h at 25 C. Thereafter, the mixture was dialyzed in membranes having a cutoff MW of 12 kDa and 5 mM HCl. The dialysis process was then repeated with 5 mM HCl/1% (w/v) NaCl, followed by treatment with 1 mM HCl in 24 h cycles. Subsequently, the thiolated materials were lyophilized at 245 C and 0.04 bar (Model, EYELA FDU-1100, Rikakikai Co. LTDA, Tokyo, Japan) and stored in a desiccator under silica gel at 25 C until further analysis. Handbook of Chitin and Chitosan

674

21. Modified release properties of glutathione-based chitosan films

21.2.2.3 Determination of the viscosity-average molecular weight The viscosity-average molecular weight of CH and DCH was determined by intrinsic viscosimetry using solutions with concentrations between 0.01 and 0.09 g/dL at 25 C. The dispersion medium employed was composed of a mixture of 0.1 M acetic acid/0.2 M sodium chloride. The Mark
Houwink
Sakurada equation was used to calculate the molecular weight as follows: ½η 5 k 3 Mav

(21.1)

where [η] and Mv are the intrinsic viscosity and viscosity-average molecular weight of the polymer, k and α are constants related to the solvent and the 3D conformation (linear or branched) of the polymer, and have values of 1.81 3 1025 dL/g and 0.93, respectively [10]. 21.2.2.4 Quantification of thiol groups The percentage of thiol moieties in the thiolated CH backbone was obtained using the Ellman’s reagent [11]. Briefly, a 2 mg/mL solution of thiolated CH was prepared in deionized water. Subsequently, a 250 μL aliquot of these solutions were mixed with 250 μL of 0.5 M phosphate buffer (PBS, pH of 8.0) and 500 μL of the Ellman’s reagent (0.3 mg/mL in 0.5 M PBS, pH of 8.0). The reaction was allowed to proceed for 2 h at 25 C. The sample was then submitted to centrifugation at 1550 rpm for 2 min. The absorbance of the sample was measured by a UV/VIS spectrophotometry (HACH DR5000, HACXH Company, Loveland, CO) at a wavelength of 412 nm. The percentage of thiol groups was obtained by interpolation from a calibration curve built using L-cysteine at 15, 30, 70, 150, and 400 μM/L concentrations.

21.2.3 Characterization of chitosan and thiolated materials 21.2.3.1 1H-Nuclear magnetic resonance spectroscopy 1

H-Nuclear magnetic resonance spectroscopy (1H-NMR) spectra of polymers dissolved in D2O acidified with acetic acid were recorded on a Bruker Ascend III HD spectrometer using a 5 mm TXI probe and operated at 600 MHz. The TD was determined by 1H-NMR as follows: TDð%Þ 5

ASH 3 100 Atotal

(21.2)

where ASH corresponds to the integral of SH signal at B1 ppm and Atotal is the complete area under the curve in the spectrum. 21.2.3.2 Fourier-transform infrared spectroscopy The infrared spectrum of the polymers were recorded between 400 and 4000 cm21 on a Nicolet 6700 spectrophotometer at a resolution of 4 cm21 and 32 scans. Approximately, 10 mg of polymer was mixed with Handbook of Chitin and Chitosan

21.2 Experiment

675

B200 mg of KBr (previously dried at 120 C for 3 h) with an agate mortar and pestle. The mixture was then compressed in a hydraulic press (060804 Compac, Indemec, Itagui, Colombia) using a 13-mm flat-faced punches and die tooling and a dwell time of 1 min. The DA was found by taking the ratio of the Fourier-transform infrared spectroscopy (FTIR) absorbance bands obtained at 1650 and 3450 cm21, according to the Baxter equation: DAð%Þ 5

A1650 3 115 A3450

(21.3)

where DA corresponds to the DA, 115 is the ratio of the molecular weights of the N-acetyl-glucosamine and N-glucosamine subunits, and A1650 and A3450 correspond to the type I amide and hydroxyl stretching bands, respectively [12]. The TD was also determined by FTIR as follows: TDð%Þ 5

A1654 3 2:5 A1564

(21.4)

where TD corresponds to the degree of thiolation, 2.5 is the ratio of the molecular weights of thiolated and nonthiolated N-glucosamine subunits, and A1654 and A1564 correspond to the absorbance height of the type I amide and amine stretching bands, respectively. 21.2.3.3 X-ray diffraction The X-ray diffraction patterns of CHs and thiolated-CHs were studied over a 5
45 degrees 2Ө range. An Empyrean diffractometer (XPERT Panalytica, Serie II, detector PIXCel3D, 2012, operated at 40 kV) equipped ˚ ; α2 5 1.5443 A ˚ ) was employed. with a monochromatic CuKα (α1 5 1.5406 A The degree of crystallinity (DC) was calculated using the Peakfit software (Sealsolve, Incc, Framingham, MA) separating the crystalline and amorphous scattering radiation using the baseline selection tool. 21.2.3.4 Differential scanning calorimetry This analysis was conducted on a differential scanning calorimetry (DSC) instrument (TA Q2000 instrument) modulated at 60.5 C every 40 s, at a heating rate of 10 C/min from 40 C to 440 C under a nitrogen atmosphere, using three sequential cycles of heating and cooling. Approximately, 10 mg of powder was put in the aluminum crucible covered with a lid, and an empty aluminum crucible having a lid was used as a reference. 21.2.3.5 Thermogravimetric analysis Thermogravimetric analysis (TGA) was conducted on a TGA instrument (HIRES TGA 2950, TA Instruments) at a heating rate of 10 C/min

Handbook of Chitin and Chitosan

676

21. Modified release properties of glutathione-based chitosan films

under a nitrogen atmosphere from 25 C to 800 C. Approximately, 20 mg of powder was put in an uncapped pan, and a crucible without a lid was used as a reference. 21.2.3.6 Specific surface area, porosity, and particle size distribution The specific surface area (SSA) was determined using the multipoint Brunauer
Emmett
Teller (BET) method on a gas sorption analyzer (ASAP 2020 plus, Micromeritrics) at a relative vapor pressure (P/Po) between 0.05
0.30, using nitrogen as the adsorbate (77K). The crosssectional area of the nitrogen molecule was assumed to be 0.162 nm2. The total (ε) of the polymers was calculated from the measurements of true density obtained from a Helium displacement micropycnometer (AccuPyc, II 1340, and Micromeritics, United States) using Eq. 21.5:   ρbulk εð%Þ 5 1 2 3 100 (21.5) ρtrue where εcorresponds to the total porosity, ρbulk is the powder bulk density that was obtained directly from the ratio of B0.7 g of powder and its volume measured in a 10 mL graduate cylinder, and ρtrue is the powder true density. The particle size distribution was obtained by microscopy analysis. Optical micropictures of powders spread on a glass slides were taken using an optical microscope (BM 180 P, Hamburg, Germany) at a 10 3 magnification coupled with a Fuji digital camera (FinePix S9000, Fujifilm, Tokio, Japan). The digital analysis of micropictures was conducted using the ImageJ software (v. 1.46r, NIH, Bethesda, MD) and the particle size was obtained directly from the lognormal plots of the resulting particle distributions obtained from the Minitab software (v. 15, Minitab, Inc, State College, PA). 21.2.3.7 Surface morphology determination The morphology characterization of the polymers was conducted by field emission scanning electron microscopy using a scanning electron microscope (SEM) (Joel 6490LV, Peabody, MA) at ambient temperature with an accelerated voltage of 20 kV. Samples were coated with Au using a sputter coater (Desk IV, Denton Vacuum, Moorestown, NJ) operated at a vacuum of 50 mm torr for 10 min.

21.2.4 Swelling analysis Approximately, 0.1 g of each polymeric material was immersed in a 10 mL of each buffer solution having a pH of 4.5 and 6.8 at 37 C. The swelling behavior was determined by measuring the change in the

Handbook of Chitin and Chitosan

21.3 Results and discussion

677

height of each polymer at various times between 0 and 4 h. The swelling ratio was calculated as follows:  
ht 2 ho S mL=g 5 (21.6) W where W, S, ht, and ho correspond to the sample weight, swelling ratio, and heights at a time “t” and initial time, respectively.

21.2.5 Evaluation of the in vitro release of cyclophosphamide 21.2.5.1 Preparation of polymeric films Polymeric materials and cyclophosphamide were dissolved in 1% v/v acetic acid. The resulting concentrations of polymer and cyclophosphamide were 1.0% and 0.5%, respectively. A 3.0 mL aliquot of this solution was poured into a cylindrical Teflon mold having a height, inner, and outer diameters of 1, 3.5, and 4.5 cm, respectively. These molds were dried at 30 C for 72 h. The film thickness was measured with a digimatic micrometer (IP65 Coolant proof, Mitutoyo) at five different locations and the average was then taken. 21.2.5.2 In vitro release of cyclophosphamide The in vitro release of cyclophosphamide was conducted on an Erweka (DT6-K, Erweka GmbH, Milford, CT) Type 2 dissolutor operated at 37 C and 50 rpm for 30 min. Acetate (pH of 4.5) and phosphate (pH of 6.8) buffers were used as release media. Aliquots of 3 mL each were taken and diluted (0.5 mL/4.0 mL) before measurement. The concentration of cyclophosphamide was determined by UV analysis (Genesys 10 S, Thermo Scientific, US) at 209 nm and 201 nm for acidic and neutral pH, respectively. A calibration curve was built for each media between 3 and 500 ppm. Intact films without cyclophosphamide were used as negative controls.

21.3 Results and discussion 21.3.1 Synthesis of thiolated polymers 21.3.1.1 Microwave-assisted deacetylation reaction The microwave-assisted alkaline treatment of commercial CH was a rapid and efficient method for the reduction of the DA of CH from 25% to ,10%. This is attributed to the direct hydroxyl ions attack of the carbonyl atom of the acetamide group of CH via a nucleophilic reaction [10]. Likewise, the microwave-assisted method rendered a

Handbook of Chitin and Chitosan

678

21. Modified release properties of glutathione-based chitosan films

larger deacetylation as compared to simple heating at 100 C for 8 h using a 10 N NaOH (in this case, only a 5% reduction on DA was obtained in a previous experiment). Additionally, the molecular weight decreased from 680 to 477 kDa, indicating that deacetylation was accompanied with a subtle depolymerization. This outcome is in agreement with previous studies [13]. As a result, the DCH sample presents more available amine groups which, in turn, are the active sites for the thiolation reaction. 21.3.1.2 Thiolation of chitosan Thiolation of CH was generated by coupling of glutathione to the CH structure due to the formation of an amide linkage between the amine groups in the C-2 of CH with the glycine carboxylic acid group in glutathione. Carbodiimide played a key role in the reaction because it activated the carboxylic group to form an O-urea derivative which consequently reacts with the amine group of CH rendering a thiolated product. Nevertheless, this intermediary is easily hydrolyzed. Thus Nhydroxysuccinimide is used instead since its ester derivative is gradually hydrolyzed increasing the degree of substitution of CH. In this scenario, the O-acylisourea derivatives are converted into a N-hydroxysuccinimide-activated carboxylic acid, which formed a zero-length branching with the amine groups of CH [8] (Fig. 21.1). The thiolated products exhibited a yellowish color and have good water solubility. The further quantification of thiol groups was conducted with the Ellman’s reagent and it is based on the reaction with
SH groups. As a result, the formation of a mixed disulfide and a 2-nitro-5-thiobenzoic OH

O Glutamate

N

Glycine

+

Cysteine

HN

CH2CH3 HCl–

C

O

(CH2)3 N+ CH3 CH3

N

SH

Glutamate

CH2CH3 HCl–

C

(CH2)3 N+ CH3

Glycine

CH3

Cysteine SH

Carbodiimide

Glutathione

O

O

O N OH N-hydroxysuccinimide OH O

HO HO O

NH

HO O

NH2 OH O OH

O HN O

C HN

Glutamate Cysteine

Glycine

O

CH2CH3

O

HCl– Glutamate (CH2)3 N+ CH3 H3C

Glycine

N O

Cysteine SH

Thiolated chitosan

SH

FIGURE 21.1

Presumptive reaction scheme for the thiolation of chitosan with

glutathione.

Handbook of Chitin and Chitosan

679

21.3 Results and discussion

TABLE 21.1

Thiolation degree of chitosan. Thiolation degree (%) Ellman’s reagent (n 5 3)

FTIR

CH
SH-24

2.0 6 0.1

3.1

4.4

CH
SH-72

4.1 6 0.1

4.4

5.7

CH
SH-120

5.0 6 0.1

5.1

7.0

Polymer

1

H-NMR

acid (TNB21) occurs. The latter has a high molar extinction coefficient (13600/(M cm) allowing for the absorbance measurement [14]. On the other hand, the quantification was conducted based on the magnitude of the conversion of CH amine groups to type I amide as identified in the infrared spectrum. Results indicate that the reaction time had a significant effect on the degree of thiolation (Table 21.1).

21.3.2 Physicochemical characterization of polymeric materials 21.3.2.1 Proton nuclear magnetic resonance spectroscopy The 1H-NMR spectra of DCH and thiolated derivatives are shown in Fig. 21.2. In the DCH spectrum the peak at 2.9
3.1 ppm represents the hydrogen proton of the glucosamine subunit. The peaks at 3.4
4.0 ppm are attributed to the H3
6 anomeric protons of the carbohydrate ring [15]. Furthermore, the glutathione spectrum exhibits signals at 1.3
1.4 ppm ascribed to the SH proton, and at 1.8
1.9 ppm showing the typical shoulder of acetic acid signal due to H12
13 protons. Further, the signals at 2.3
2.5 ppm are due to H10
11 protons, whereas the signals at 3.7
3.8 ppm represent the H14 proton. Likewise, the signals at 3.9
4.0 ppm and 4.4
4.5 ppm are due to the H2
3 and H8, respectively [15,16]. On the other hand, the spectra of thiolated derivatives display novel peaks at 1.0
1.2 ppm, which are attributed to the SH proton of glutathione [17]. Likewise, these conjugates present other characteristic signals typical of glutathione indicating a successful thiolation. In addition, some signals of other protons such as OH, NH2 y NH-CO-CH3 did not appear since D2O generates fast exchange reactions, eliminating these resonance signals [18]. 21.3.2.2 Fourier-transform infrared spectroscopy The FTIR spectra of chitosan and thiolated polymers are shown in Fig. 21.3. DCH showed characteristic peaks at 3464 cm21, 2913 cm21, and 2870 cm21 which are attributed to OH vibrations and C-H stretching, respectively. Moreover, it showed vibration bands at 1654 cm21 due to C5O stretching, and a shoulder due to the deacetylation process at 1542 cm21 attributed to the deformation of primary amine. Likewise, the peaks at 1443 cm21, space 1372 cm21, and 1316 cm21 are ascribed to CH2 Handbook of Chitin and Chitosan

680

21. Modified release properties of glutathione-based chitosan films

DCH H3-H6

OH O

HO

2

6

NH CH3 O

HO

H

H

11

12

16

HO

6

NH2

O NH H

7

–4

NH H8

H

NH2

SH 5 H

H

O

13

14

H5

O

O OH

DCH H

H

1

2 5

4

OH O

8

1

O

1

O

H12–13

NH2

HO

5 3

H2–3 Glutathione H6–7 H10–11 H14

H6

6

4

HO

H2

3

OH

H

9

10

O

15

1

2

Glutathione

H13–14 CH-SH-24 H19–20

H9–10 H15 H6 H7

H21 H H2

H17–18

3–6

H12

H13–14

CH-SH-72 H9–10

OH O

O

H19–20

NH 7

H0

H21 H

3–6

H2

H6 H 7 5.0

4.5

O

H12

O OH O

NH H 14 HS

O n H

12

H

NH

17

16

15

H

H13–14

22

H O

10

13

H

OH

20

23

H 21

O

H19–20

H21 H17–18 H3–6 H2

4.0

FIGURE 21.2

3.5 1

3.0

2.5

H12 2.0

1.5

1.0

H-NMR spectrum of deacetylated chitosan, glutathione, and thiolated

derivatives.

100 98 Transmitance (%)

NH2 H

Glutathione-chitosan conjugate

CH-SH-120 H9–10 H15

H17–18

10

1

2 5

H

H15 H6 H7

6

3

6

HO

NH2

HO O

96 94 92 90 88 86 84 500

1000

1500

2000

2500

Wavenumber

3000

(cm–1)

FIGURE 21.3 FTIR spectrum of polymers.

Handbook of Chitin and Chitosan

3500

4000

681

21.3 Results and discussion

bending and CH deformation, C-CH3 stretching, and secondary amide stretching, respectively. Further, the vibration signals at 1076 cm21, 1033 cm21, and 892 cm21 are attributed to the ring C-O-C stretching, CO stretching, and the C-O-C linkage stretching, respectively [19]. The DCH sample showed the same peaks as CH (not shown), and the peak attributed to the deformation of primary amine was reduced by 16% due to the deacetylation process and became a shoulder of the C5O band. The CH
SH-24, CH
SH-72, and CH
SH-120 samples exhibited a change of the amine signal at 3223 cm21 essentially due to the formation of an amide linkage. Thiolated samples also showed a small band at 2500 cm21 due to S
H stretching, and new peaks appeared in the region from 1200 to 1250 cm21 attributed to C
SH stretching [20]. 21.3.2.3 X-ray diffraction characterization The powder X-ray diffractogram of the DCH showed three crystalline peaks in the 2Ө scale at 10.3, 20.2, and 21.9 degrees corresponding to the (020), (110), and (120) lattices, respectively, conforming to the tendon hydrate allomorph of CH, or simply its hydrate form [21,22]. The peaks at 10.3 and 20.2 degrees are characteristic of the α-CH allomorph having ample interand intramolecular hydrogen bonds between OH and NH2 groups [23]. Likewise, the deacetylation of CH increased the DC from 49% (data not shown) to 56%, possibly due to the formation of a larger amount of inter and intramolecular hydrogen bonds leading to a major stabilization of polymer chains, which in turn became shorter compared with CH alone [13]. Furthermore, the diffractograms of the three thiolated derivatives showed broad and nondefined amorphous halos, indicating a substantial reduction of the crystallinity of the polymer upon thiolation (Fig. 21.4). 12000

Intensity (a.u.)

9000

6000

3000

0 0

5

10

15

20

25

30

2θ (degree)

FIGURE 21.4

Powder diffractograms of polymers.

Handbook of Chitin and Chitosan

35

40

45

682

21. Modified release properties of glutathione-based chitosan films

(A)

2 Weight loss first derivative

Heat flow exo-up (W)

5 0 –5 –10 –15 –20

(B)

1 0 –1 –2 –3

50 100 150 200 250 300 350 400 450

100 200 300 400 500 600 700 800

Temperature (ºC)

FIGURE 21.5

Temperature (ºC)

(A) DSC thermograms of polymers and (B) TGA first-derivative weight

loss.

21.3.2.4 Thermal and thermogravimetric characterization The thermal and thermogravimetric characterization of samples was carried out using DSC and TGA analyses, respectively (Fig. 21.5A,B). The thermogram of DCH polymer presented an endothermic peak at 157 C, which was attributed to the melting temperature. In addition, the exothermic peak was shifted to 307 C due to the loss of amine groups of the chitosan backbone and partial depolymerization of CH [24]. Conversely, the thiolated polymers showed an endothermic peak at 166.7 C, 158.4 C, and 177.2 C corresponding to the fusion temperature of CH
SH-24, CH
SH-72, and CH
SH-120, respectively. Further, the exothermic peak at 285 C disappeared in these thiolated samples due to the effective loss of free amine groups as resulted from the formation of amide bonds. The weight loss of thermograms is depicted in Fig. 21.5B. These curves show a defined pattern composed of three segments of weight loss indicated by the first derivative signal. Each segment shows a band or peak labeled as Ta, Tb, and Tc. The first small band is ascribed to the release of nonbound water from the polymer. The second segment has a pronounced peak due to the concomitant processes of decomposition of amine groups, dehydration of saccharide rings, and depolymerizationm [25], and the third band corresponds to pyrolysis (decomposition) of the polymer [26]. Further, it is worthwhile to mention that peaks signals are precisely listed in Table 21.2, confirming that the largest degradation temperature corresponded to Tb was found in the second segment of the thermogram. On the other hand, thiolated products showed the lowest Tb values due to the formation of amide bonds and massive loss of amine moieties. Further, the new bonds formed in thiolated polymers (
NH-COCH2-SH) are easily breakable upon heating as confirmed by the low Tb of B260 C [27]. Likewise, thiolated samples showed the largest Tc values, indicating that once amide bonds are broken, the resulting depolymerized products have a more stable chain conformation.

Handbook of Chitin and Chitosan

683

21.3 Results and discussion

TABLE 21.2

TG studies of chitosan and thiolated products.

Polymer

Inflexion point (first derivative)

DCH

CH
SH-24

CH
SH-72

CH
SH-120

Temperature ( C)

Weight loss (%)

Ta

67.3

11.0

Tb

298.9

49.6

Tc

600.6

38.9

Ta

63.9

13.1

Tb

259.0

50.3

Tc

800.0

10.1

Ta

47.9

19.9

Tb

260.7

45.3

Tc

690.5

38.7

Ta

48.3

14.54

Tb

230.5

38.91

Tc

800

33.31

21.3.2.5 Specific surface area, porosity, and particle size The SSA of polymers is listed in Table 21.3. Two different types of phenomena were observed in these polymers: (1) the SSA of thiolated samples increased as the GSH moiety appeared causing a steric repulsion resulting in a volume increase; (2) the SSA of CH
SH-24 and CH
SH120 were comparable and smaller than that of CH
SH-72, probably due to the formation of disulfide bonds. Likewise, CH
SH-72 was the least stable material as determined from the thermal analysis. Moreover, the total pore area of CH
SH-72 was higher than that of CH
SH-24 and CH
SH-120, but both samples had a comparable and large total porosity due to thiolation mainly taking place at the particle surface. As a result, a large formation of mesopore volume was obtained upon DCH thiolation. Since thiolation had a major positive effect on the formation of mesopores in DCH it is expected that there was a major surface area increase upon incorporation of thiol groups in the polymer. On the other hand, the mean particle size of all samples remained between 26 and 52 μm and particle size was not related to either surface area or porosity. 21.3.2.6 Surface morphology The SEM microphotographs of the polymers taken at 100 3 , 1000 3 , and 3000 3 magnifications are illustrated in Fig. 21.6. The surface of DCH particles exhibits small lumps of crystallites and showed cracks as resulted from the grinding process. Further, DCH showed a smoother surface due to the microwave alkaline treatment. On the other hand, Handbook of Chitin and Chitosan

684

21. Modified release properties of glutathione-based chitosan films

TABLE 21.3 Specific surface area, porosity, and particle size of chitosan and thiolated materials. Polymer sample Surface parameters

DCH

CH
SH-24

CH
SH-72

CH
SH-120

Mean particle size (μm)

35.4 6 3.0

51.8 6 4.4

38.8 6 2.9

26.3 6 2.2

73.2 6 0.1

86.5 6 0.1

86.2 6 0.1

86.2 6 0.1

2.1 6 1.0

5.7 6 0.1

8.9 6 0.1

5.9 6 0.5

Pore volume (micropores, mm /g)

0.0

0.2

0.2

0.0

Total pore volume (micropores
mesopores, mm3/g)

0.1

4.9

6.1

0.8

Pore area (macropores, m2/g)

0.0

0.7

0.0

0.0

0.3

2.4

6.2

1.3

Total porosity (%) 2

Specific surface area (m /g) 3

2

Total pore area (m /g)

thiolated products showed a flaky, spongy, porous, and flexible surface due to the amorphization induced by the formation of “NH-CO-CH2-SH” bonds. The SEM results corroborated the prevalence of a mesopore structure (pore size between 2.0 and 50.0 nm), which increased upon thiolation and was attributed to the increase of the specific volume of the polymer.

21.3.3 Swelling study The swelling properties of polymers are shown in Table 21.4. It is evident that at a pH of 4.5 DCH had a higher swelling degree than thiolated derivatives due to the prevalence of protonated (NH1 3 ) primary amine groups, increasing the positive charge density and possibly favoring the repulsion between these positive charges [28]. Moreover, the TD was inversely related to swelling at acidic pH due to the decrease of amine groups upon thiolation, and along with the presence of carboxyl and thiol groups modified the total ionization on the polymer backbone. On the other hand, the swelling degree was larger for thiolated materials than DCH at nearly neutral pH (6.8), due to the reduction of ionization of amine groups and hence causing a smaller positive charge density. Likewise, glutathione has four reported pK values of 2.12, 3.59, 8.75, and 9.65 attributed to COOH, COOH, NH2, and SH groups, respectively [29] and only a free carboxyl group is lost during the thiolation, which indicates a charge balance on the polymer backbone, and hence the swelling degree and ionization at both pHs was comparable. Furthermore, the swelling peak time was # 2.0 h in all cases, but at pH of 6.8 the swelling rate was larger for thiolated conjugates. Once they reached the swelling peak time all polymers started eroding steadily and this effect was pronounced at a pH of 4.5.

Handbook of Chitin and Chitosan

685

21.3 Results and discussion

FIGURE 21.6

TABLE 21.4

SEM of chitosan, deacetylated chitosan, and thiolated derivatives.

Swelling of chitosan and thiolated materials. Swelling rate (mL/g)

Swelling peak timea (h)

pH 5 4.5

pH 5 6.8

pH 5 4.5

pH 5 6.8

DCH

1.42 6 0.22

0.11 6 0.04

1.0 6 0.0

0.7 6 0.0

CH
SH-24

1.15 6 0.18

1.23 6 0.43

2.0 6 0.0

1.0 6 0.0

CH
SH-72

0.71 6 0.07

0.69 6 0.16

2.0 6 0.0

0.5 6 0.0

CH
SH-120

0.51 6 0.19

0.53 6 0.14

0.5 6 0.0

1.0 6 0.0

Polymer sample

a

Swelling peak time: maximum swelling time.

Handbook of Chitin and Chitosan

686 TABLE 21.5

21. Modified release properties of glutathione-based chitosan films

Dimensions and mass of polymeric films. Cyclophosphamide released (%) Mass (mg)

pH 5 4.5 (t 5 5 min)

pH 5 6.8 (t 5 15 min)

50.2 6 7.2

49.3 6 1.6

91.2 6 1.4

91.5 6 3.5

CH
SH-24

65.3 6 5.9

47.2 6 2.5

95.1 6 4.3

100.0 6 0.8

CH
SH-72

68.7 6 3.7

48.5 6 0.3

99.0 6 2.4

98.0 6 1.4

CH
SH-120

67.5 6 4.6

45.9 6 1.4

99.1 6 0.7

99.0 6 2.8

Polymeric films

Thickness (μm)

DCH

21.3.4 In vitro release of cyclophosphamide from polymeric films Polymeric films of were obtained via casting-evaporation and their dimensions and weights are listed in Table 21.5. The films’ thicknesses and masses ranged from 40
70 μm and 45
50 mg, respectively. Films of thiolated derivatives were thicker and more brittle than that of DCH due to the formation of a nonhomogeneous structure upon drying. The cyclophosphamide release from polymeric films is considered to be a burst effect as shown in Table 21.5. This phenomenon is explained by the combined effect of high drug solubility (40 mg/mL in water [30]) in the media, high porosity, and a high drug load in the matrix (B33%). Even though there was no statistical difference in drug release with pH, a slight oxidation of free thiol groups was expected at a pH of 6.8. Further, a major film erosion took place at acidic pH, but this effect had no influence on drug release. Similar studies conducted on chitosan films crosslinked with sodium tripolyphosphate have shown a modest anthocyanin release from 12% to 15% after 3 h at pH of 6.5 [31]. Moreover, Rodriguez and collaborators developed monolayer and bilayer films of chitosan (from a 2% w/v) loaded with dexamethasone obtaining release profiles from 0.5 to 60 days [32]. Moreover, the impregnation of films with NaOH decreased their mechanical characteristics, swelling behavior, and solubility in aqueous media due to the increment of interactions between polymer chains contributing to the formation of more compactable and less deformable films [33]. This effect could prolong the release profile of active compounds from the film.

21.4 Conclusions Thiolation of microwave-assisted deacetylated chitosan rendered products with a diverse content of free thiol bonds. These products in turn exhibited a reduced crystallinity and better thermal stability.

Handbook of Chitin and Chitosan

References

687

Additionally, the TD had a crucial effect on the released properties of cyclophosphamide due to the ionic nature of the polymers. The generated polymers can be a promising tool for the formulation of film matrices for the release of distinctive bioactive compounds.

Acknowledgments This research was funded by the Colombian Institute of Science (Colciencias) through the grant No. 727
2015. CHS and CJY are grateful to Icesi University for the internal research grant No. 041352. The authors would like to especially thank the committee for the development of research of University of Antioquia and its Sustainability Strategy Program (2018
2019) for their financial support.

References [1] O. Patil, D.N. Schulz, ACS Symposium Series Functional Polymers: An Overview, American Chemical Society, 1998pp. 1
14. [2] J. Wang, L. Wang, H. Yu, Zu Abdin, Y. Chen, Q. Chen, et al., Recent progress on synthesis, property and application of modified chitosan: an overview, Int. J. Biol. Macromol 88 (2016) 333
344. [3] F. Laffleur, A. Berknop-Schnu¨rch, Thiomers: promising platform for macromolecular drug delivery, Future Med. Chem 4 (17) (2012) 2205
2216. [4] S. Bonegel, A. Berknop-Schnu¨rch, Thiomers—from bench to market, J. Control. Release 195 (2014) 120
129. [5] G. Millotti, C. Samberger, E. Fro¨hlich, A. Bernkop-Schnu¨rch, Chitosan-graft-6mercaptonicotinic acid: synthesis, characterization, and biocompatibility, Biomacromolecules 10 (2009) 3023
3027. [6] G. Milloti, C. Samberger, E. Fro¨nlich, D. Sakloestsakun, A. Bernkop-Schnu¨rch, Chitosan-4-mercaptobenzoic acid: synthesis and characterization of a novel thiolated chitosan, J. Mater. Chem 20 (12) (2010) 2432
2440. [7] D.W. Kurniawan, A. Fudholi, R.A. Susidarti, Synthesis of thiolated chitosan as matrix for the preparation of metformin hydrochloride microparticles, Res. Pharm 2 (1) (2012) 26
35. [8] K. Kafedjiiski, F. Fo¨ger, M. Werle, A. Berknkop-Schnu¨rch, Synthesis and in vitro evaluation of a novel chitosan-glutathione conjugate, Pharm. Res 22 (9) (2005) 1480
1488. [9] H. Mojarradi, Coupling of substances containing a primary amine to hyaluronan via carbodiimide-mediated amidation (2010). [10] M.R. Kasaai, Calculation of Mark
Houwink
Sakurada (MHS) equation viscometric constants for chitosan in any solvent
temperature system using experimental reported viscometric constants data, Carbohydr. Polym 68 (2007) 477
488. [11] G. Alamdarnejad, A. Sharif, S. Taranejoo, M. Janmaleki, M.R. Kalaee, M. Dadgar, et al., Synthesis and characterization of thiolated carboxymethyl chitosan-graftcyclodextrin nanoparticles as a drug delivery vehicle for albendazole, J. Mater. Sci. Mater. Med 24 (8) (2013) 1939
1949. [12] A. Baxter, M. Dillon, K. Taylor, G. Roberts, Improved method for IR determination of the degree of N-acetylation of chitosan, Int. J. Biol. Macromol 14 (1992) 166
169. [13] Y. Yuan, B. Chesnutt, W.O. Haggard, J.D. Bumgardner, Deacetylation of chitosan: material characterization and in vitro evaluation via albumin adsorption and preosteoblastic cell cultures, Materials (Basel), 4, 2011, pp. 1399
1416.

Handbook of Chitin and Chitosan

688

21. Modified release properties of glutathione-based chitosan films

[14] S.K. Kim, Chitin and Chitosan Derivates: Advances in Drug Discovery and Developments, CRC Press, Boca Raton, 2014. [15] A.G.B. Pereria, E.C. Muniz, Y.L. Hsieh, 1H NMR and 1H-13C HSQC Surface characterization of chitosan-chitin sheath-core nanowhiskers, Carbohydr. Polym 123 (2015) 46
52. [16] L.G. Kaiser, M. Marja´nska, G.B. Matson, I. Iltis, S.D. Bush, B.J. Soher, et al., 1H MRS detection of glycine residue of reduced glutathione in vivo, J. Magn. Reson 202 (2010) 259
266. [17] J. Li, Y. Shu, T. Hao, Y. Wang, Y. Qian, C. Duan, et al., A chitosan-glutathione based injectable hydrogel for supression of oxidative stress damage un cardiomyocytes, Biomaterials 34 (36) (2013) 9071
9081. [18] M.R. Kassai, Determination of the degree of N-acetylation for chitin and chitosan by various NMR spectroscopy techniques: a review, Carbohydr. Polym 79 (2010) 801
810. [19] P. Negrea, A. Caunii, I. Sarac, M. Butnariu, The study of infrared spectrum of chitin and chitosan extract as potential sources of biomass, Dig. J. Nanomater. Biostructures 10 (4) (2015) 1129
1138. [20] R. Esquivel, J. Jua´rez, M. Almada, J. Ibarra, M.A. Valdez, Synthesis and characterization of new thiolated chitosan nanoparticles obtained by ionic gelation method, Int. J. Polym. Sci 2015 (2015) 1
18. ´ [21] J. Kumirska, M. Czerwicka, Z. Kaczynski, A. Bychowska, K. Brzozowski, J. Tho¨ming, et al., Application of spectroscopic methods for structural analysis of chitin and chitosan, Mar. Drugs 8 (2010) 1567
1636. [22] C.H. Hsu, S.K. Chen, W.Y. Chen, M.L. Tsai, R.H. Chen, Effect of the characters of chitosans used and regeneration conditions on the yield and physicochemical characteristics of regenerated products, Int. J. Mol. Sci 16 (2015) 8621
8634. [23] R. Ramya, P.N. Sudha, J. Mahalakshmi, Preparation and characterization of chitosan binary blend, Int. J. Sci. Res. Publ 2 (10) (2012) 1
9. [24] L.S. Guinesi, E.T. Gomes, The use of DSC curves to determine the acetylation degree of chitin/chitosan samples, Thermochim. Acta 444 (2006) 128
133. [25] C.G. Flores-Herna´ndez, A. Colı´n-Cruz, C. Velasco-Santos, V.M. Castan˜o, J.L. RiveraArmenta, A. Almendarez-Camarillo, et al., All green composites from fully renewable biopolymers: chitosan-starch reinforced with keratin from feathers, Polymers (Basel) 6 (2014) 686705. [26] Z. Zakaria, Z. Izzah, M. Jawaid, A. Hassan, Effect of degree of deacetylation of chitosan on thermal stability and compatibilty of chitosan-polyamide blend, BioResources 7 (4) (2012) 5568
5580. [27] A. Anitha, N. Deepa, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Development of mucoadhesive thiolated chitosan nanoparticles for biomedical applications, Carbohydr. Polym 83 (2011) 66
73. [28] W.C. Lin, D.G. Yu, M.C. Yang, pH-Sensitive polyelectrolyte complex gel microespheres composed of chitosan/sodium tripolyphosphate/dextran sulfate: swelling kinetics and drug delivery properties, Colloids Surf. B Biointerfaces 44 (2
3) (2005) 143
151. [29] Sigma-Aldrich, Product Information: L-Glutathione Reduced, 2018. [30] Sigma-Aldrich, Product Information: Cyclophosphamide Monohydrate, 2018. [31] M.J.C. Salazar, A.C. Valderrama, Release of anthocyanins from chitosan films crosslinked with sodium tripolyposphate, Rev. Soc. Quı´m. Peru 83 (1) (2017) 115
125. [32] L.B. Rodrigues, H.F. Leite, M.I. Yoshida, J.B. Saliba, A.S. Cunha, A.A.G. Faraco, In vitro release and characterization of chitosan films as dexamethasone carrier, Int. J. Pharm 368 (2009) 1
6. [33] E.A. Takara, J. Marchese, N.A. Ochoa, NaOH treatment of chitosan films: impact on macromolecular structure and film properties, Carbohydr. Polym 132 (2015) 25
30.

Handbook of Chitin and Chitosan

C H A P T E R

22 Chitosan-based materials as templates for essential oils Rut Ferna´ndez-Marı´n1, Susana C.M. Fernandes2, Colin McReynolds2, Jalel Labidi1 and ´ ngeles Andre´s Sa´nchez1 Ma A 1

Environmental and Chemical Engineering Departament, University of the Basque Country UPV/EHU, Donostia-San Sebastia´n, Spain, 2CNRS/Univ Pau & Pays Adour/ E2S UPPA, Institut des Sciences Analytiques et de Physico-Chimie pour l’Environnement et les Materiaux, Anglet, France

O U T L I N E 22.1 Introduction 22.1.1 Why use chitosan as a carrier of essential oils?

690 690

22.2 Chitosan-based essential oils coatings and films

692

22.3 Chitosan-based essential oils emulsions and (nano)gels

693

22.4 Chitosan (nano)capsules for essential oils encapsulation

696

22.5 Antioxidants activity in chitosan as templates for essential oils 697 22.5.1 Folin
Ciocalteu assay 697 22.5.2 DPPH assay (radical 2,2-diphenyl-1-picrylhydrazyl) 699 22.5.3 Trolox equivalent antioxidant capacity or 2,20 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt 699 22.5.4 Ferric ion reducing antioxidant power assay 701 22.5.5 Antioxidant properties of chitosan
essential oils coating and films 701 22.5.6 Antioxidant properties of chitosan
essential oils emulsions and (nano)gels 705

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00022-4

689

© 2020 Elsevier Inc. All rights reserved.

690

22. Chitosan-based materials as templates for essential oils

22.5.7 Antioxidant properties of chitosan
essential oils encapsulations

705

22.6 Antibacterial activity in chitosan as templates for essential oils 22.6.1 Antimicrobial activity of chitosan
essential oils coating and films 22.6.2 Antimicrobial activity of chitosan
essential oils emulsions and (nano)gels 22.6.3 Antimicrobial activity of chitosan
essential oils encapsulations

706

22.7 Conclusions and future perspectives

714

References

715

707 712 713

22.1 Introduction 22.1.1 Why use chitosan as a carrier of essential oils? Currently, there is increased demand for antioxidant and antimicrobial materials from naturally occurring agents and with a low negative impact on human health and the environment. An alternative to synthetic bioactive substances is the use of chitosan (CS)-based materials. CS (Fig. 22.1) is a cationic natural polymer obtained from the deacetylation of chitin (CH) (the second most abundant natural polymer in the world) by alkaline hydrolysis. CS shows bioactive properties, namely antimicrobial, antioxidant, or antiinflammatory effects [1
3]. Moreover, it also presents other excellent properties such as biocompatibility, biodegradability, low toxicity, solubility in weak acids, and great filmforming ability [4]. Furthermore, the antioxidant and antimicrobial properties of CS can be improved with the association of supplemental bioactive substances. As such, essential oils (EOs) are good candidates for the design of this type of material due to their intrinsic properties. Indeed, these compounds have shown their therapeutic potential as antioxidants [5,6], antifungals [7], insecticidals [8], or antimicrobials [9
11]. EOs are highly complex mixtures of low-molecular-weight compounds obtained from different parts of plants such as flowers, barks, roots, leaves, seeds and fruit peels [12,13]. They are secondary metabolites constituted of two main groups: (1) terpenes (also called isoprenes); and (2) terpenoids (also called isoprenoids). The terpenes group is made up of monoterpenes, which contain 10 carbon atoms, and sesquiterpenes, which

Handbook of Chitin and Chitosan

691

22.1 Introduction

HOH2C

O

NH2 HO O

HO NH2

O CH2OH

FIGURE 22.1

Chitosan chemical structure.

contain 15 carbon atoms and have a mono-, bi-, or tricyclic structures (Fig. 22.2). The terpenoids group is composed of oxygenated derivatives of isoprenes (Fig. 22.2) [12,14]. Most of them fall into the Generally Recognized as Safe (GRAS) category, as defined by the US Food and Drug Administration (USFDA) [15]. EOs are extracted using different techniques, which can be divided two groups: (1) conventional methods, and the generally more eco-friendly (2) new methods, as summarized in Fig. 22.3. Among the conventional OH O

OH O

Thymol (monoterpene)

Linalool (monoterpene)

Methyleugenol (terpenoid)

H

H OH

Humulene (terpenoid)

FIGURE 22.2

Beta-caryophyllene (sesquiterpene)

Chemical structure of some typical essential oils.

Handbook of Chitin and Chitosan

Aplha-santalol (sesquiterpene)

692

22. Chitosan-based materials as templates for essential oils

Conventional methods

• Hydrodistillation • Organic solvent extracrion • Steam distilation • Clevenger distilation

New methods

• Microwave-assisted extraction • Microwave hydrodiffusion • Microwave steam distillation • Solvent-free microwave extraction • Ultrasound-assisted extraction • Supercritical fluid extraction • Subcritical extraction liquid

Essential oil extraction methods

FIGURE 22.3 Essential oil extraction methods.

methods are hydrodistillation, organic-solvent-based extraction, steam distillation, and clevenger distillation [16]. In recent years, innovation in EO extraction has grown, with the use of methods such as microwaveassisted extraction, microwave hydrodiffusion, microwave steam distillation, solvent-free microwave extraction, supercritical fluid extraction, and subcritical liquid extraction [12,16]. EOs are very volatile compounds, and can easily be degraded during handling or on exposure to oxygen, heat, or ultraviolet light [17]. For this reason, they are protected through incorporation in emulsions [18,19], particles [20,21], films [22,23], or coatings [21,24] using different biopolymers such as lignocellulosic materials, soy protein, alginate, gelatine, and, the focus of this chapter, CS [25
29]. The aim of this chapter is to report the most recent and relevant advances concerning the use of CS as templates, in the form of films, coating, capsules, gels, and emulsions of EOs. For instance, CS active (1) films have been made with Cinnamon verum, Zingiber officinale, Thymus moroderi, and Thymus piperella [23,30,31]; (2) coatings with Zataria multiflora, Corum copticum, and Camelia sinensis [24,32,33]; (3) emulsions with Zea mays [18,19]; and (4) encapsulations with Coriandrum sativum L. and Summer savory [20,34]. These show the potential of CS as a versatile material for different applications, in particular in the food industry as well as fields as diverse as medicine, agriculture, and cosmetics.

22.2 Chitosan-based essential oils coatings and films Coatings and films are generally applied in liquid form before solidifying [35]. In this way, the first step of their preparation is solubilizing CS in an acidic aqueous solution (e.g., acetic acid) [24,36,37]. Then, in order to improve flexibility a plasticizer and/or surfactants are added, such as glycerol or Tween 80 and Tween 20, respectively [22,38,39]. In the final step,

Handbook of Chitin and Chitosan

22.3 Chitosan-based essential oils emulsions and (nano)gels

693

Solvent casting

Spray coating

Dipping

Extrusion

FIGURE 22.4

Different methods to prepare CS-based films or coatings.

several methods are used to apply the coating on foodstuffs, for example, solvent casting, spray coating, dipping, and extrusion (Fig. 22.4) [40]. In the last few years, CS-based films and coatings combined with natural bioactive agents such as EOs have increasingly gained the interest of researchers due to their useful properties. They are used to (1) protect food against gases, water vapor, UV-Vis radiation, and proliferation of microbial organisms; (2) improve the mechanical properties of the material; and (3) and to release bioactive agents in order to maintain the quality and the organoleptic properties of food [40,41]. CS provides antimicrobial and antioxidant properties, which are further improved by incorporating EOs into solution (Table 22.1). Adding EOs may improve other properties like water vapor permeation and elongation at break [2,10].

22.3 Chitosan-based essential oils emulsions and (nano)gels In the food industry, nanogels and emulsions are increasingly being studied as carriers of EOs to control their release and their stability in order to extend the shelf life of food [44,47].

Handbook of Chitin and Chitosan

694 TABLE 22.1 properties.

22. Chitosan-based materials as templates for essential oils

Summary of the materials of CS-based EO coatings and films and their Coating or films method

Final bioactive properties

References

Essential oil

Matrix

Zataria multiflora Boiss and Sumac extract

CS coating

Dip coating

Antioxidant and antimicrobial

Mojaddar [24]

Prunus armeniaca

CS
N-methyl-2pyrrolidone nanoparticles films

Solvent casting

Antioxidant and antimicrobial

Priyadarshi [2]

Carum copticum

CS film reinforced with cellulose nanofibers or lignocellulose nanofibers

Solvent casting

Antioxidant and antimicrobial

Jahed, Alizadeh, et al. [10]

Cinnamon verum, Cumbopogon citrates and Origanum vulgare

CS film

Solvent casting

Antifungal

Munhuweyi [42]

Myrcia ovata Cambessedes

CS coatings

Dip coating

Antimicrobial

Santos Fraza˜o et al. [43]

Citrus reticulate

N-palmitoyl CS
basedcoating containing nanoemulsion

Spray coating

Antimicrobial

Donsı`, Ferrari, et al. [44]

Citrus reticulata

N-palmitoyl CS
based coating nanoemulsion

Spray coating

Antimicrobial

Severino [45]

O. vulgare

Cassava starch
CS film

Extrusion

Antimicrobial

Pelissari [46]

Emulsions are a mixture of at least two immiscible liquids, usually oil and water. One of the liquids is dispersed (dispersed phase) in the form of small spherical droplets in the other (continuous phase or dispersing phase). There are two types of emulsions: (1) hydrophilic emulsions, in which the oil droplets are dispersed in water (oil-in-water emulsion, O/W); and (2) hydrophobic emulsions, in which there are water droplets dispersed in oil (water-in-oil emulsion, W/O). Emulsions are thermodynamically unfavorable systems, which tend to break down over time [44,48]. For this reason, stabilizing the drops of the emulsion can be performed by reducing the interfacial tension using surfactants or hydrocolloids and water-soluble proteins [49]. One of the most common ways to stabilize emulsions is the use of particles, known as Pickering emulsions. In this type of emulsion, an accumulation of

Handbook of Chitin and Chitosan

695

22.3 Chitosan-based essential oils emulsions and (nano)gels

Oil

Oil

Oil

Oil

Oil Oil Oil

Oil Water

Water

Platelets

Globules

Oil

Oil

Oil

Oil

Oil

Oil Water

Water

Surfactants

Fibrils

FIGURE 22.5 Nanoparticle stabilized and surfactant stabilized emulsions. Source: Adapted from V. Calabrese et al., Pickering emulsions stabilized by naturally derived or biodegradable particles. Curr. Opin. Green. Sustain. Chem. 12(Fig. 1) (2018) 83
90 [51]. Available from: https://doi.org/10.1016/j.cogsc.2018.07.002.

dispersed particles occurs at the water
oil interface which forms a mechanical barrier to protect the drops against coalescence [50]. As shown in Fig. 22.5, these particles can take on a number of different morphologies, shapes, and appearances, such as platelets, globules, surfactants, and fibrils. In factories producing foodstuffs, proteins, starch, flavonoids, and nanocrystals of CH or cellulose are used as stabilizing particles to make Pickering emulsions [19,52,53]. The use of CH and CS nanoparticles in Pickering emulsions has been the subject of numerous recent studies because of their biocompatibility and the absence of toxicity. However, because these polysaccharides are poorly water soluble, the addition of surfactants and other components is required for their use in emulsions, such as Tween 20 or Tween 80, gelatin, benzoic acid, cinnamic acid, caffeic acid, or glycerol among others [18,54,55].

Handbook of Chitin and Chitosan

696

22. Chitosan-based materials as templates for essential oils

Nanogels are colloidal systems with a semisolid appearance that flow when subjected to relatively weak forces. Normally, they are divided into two broad groups: (1) hydrogels, which absorb water, and (2) organogels (micelles) which are hydrophobic and have a tendency to absorb oils [47]. Structurally, nanogels are self-assembled three-dimensional networks connected by covalent bonds [1,54]. They can be made using proteins, organic or inorganic compounds, and polymers such as CS. In brief, adding emulsions of EOs into CS films can decrease solubility, increase elongation at break values, and increase antimicrobial performance [56,57]. When nanogels are encapsulated, the release of EOs is sustained over an extended time period, improving antioxidant and antimicrobial activity. Some of the oils incorporated as emulsions or nanogels are sunflower oil [49], garlic oil [57], corn oil [19], or rosemary oil [1].

22.4 Chitosan (nano)capsules for essential oils encapsulation Encapsulation is a technique widely used in food and medicine to release bioactive substances, such as terpenes, terpenoids, and phenolic compounds, mainly for their antioxidant and antimicrobial properties [58]. Encapsulations are individual particles of an active agent in a core that is surrounded by a membrane called a shell (Fig. 22.6). The size of (nano)capsulates usually ranges from 1 to 1000 μm [59]. The core can be liquid (in the case of EOs), solid, or a dispersion. The shell can be made up of polysaccharides and simple sugars (gum, cellulose, starch), lipids (oils, waxes, parafilms), proteins (soy, gelatin, casein), or biodegradable polymers (poly(vinyl-alcohol), poly(D,L-lactide), CS) [60,61]. The stability of the (nano)capsulate depends on factors like pressure, temperature, or pH and the method of encapsulation. The encapsulation methods most effective for EOs (nano)capsulates appear to be ionic gelation, spray drying, and coacervation [16,62]. Ionic gelation encapsulation makes use of the electrostatic interactions between the negative groups of the polyanion [e.g., tripolyphosphate (TPP)] and the positive charges of the primary amines of CS [17]. Spray drying is a method in which a solution is atomized in small droplets, Shell Core

FIGURE 22.6 A simple structure of capsule.

Handbook of Chitin and Chitosan

22.5 Antioxidants activity in chitosan as templates for essential oils

697

followed by a drying step in a heated gas. Coacervation is the separation of two liquid phases in a colloidal solution. These two phases are called coacervates, one polymer-rich, and another in which there is no polymer, an equilibrium solution. There are two main types of coarcervation: simple, involving a single polymer; and complex, involving two or more polymers [12]. In the last decade, many studies have used encapsulated EOs for diverse applications, as compiled in Table 22.2.

22.5 Antioxidants activity in chitosan as templates for essential oils One of the biggest problems for food preservation is the oxidation of lipids that occurs during the handling, thermal treatment, and storage of finished food products. This affects nutritional and sensory properties (changes in taste, smell, texture, and loss of vitamins), ultimately shortening shelf life and potentially causing consumer health problems [66]. CS has the potential to prevent or slow some of the oxidation. Indeed, several studies have shown CS may scavenge free radicals through interaction with residual free amino groups, leading to stable macromolecular radicals and ammonium groups [31,67]. This scavenging activity can be increased with the addition of EOs and plant extracts containing monoterpenes, terpenes, and polyphenols or other molecules with antioxidant activity [31,68]. The most commonly used assays to measure antioxidant activity are the Folin
Ciocalteau method, DPPH assay (radical 2,2-diphenyl-1picrylhydrazyl), ABTS assay, and FRAP assay (ferric ion reducing antioxidant power assay):

22.5.1 Folin
Ciocalteu assay The Folin
Ciocalteu assay is a simultaneous reaction of HAT (hydrogen atoms transfer reactions) and SET (single electron transfer reactions) and is used to determine and quantify total polyphenol content. The Folin
Ciocalteu reagent contains a mixture of sodium molybdate and sodium tungstate, which react with the phenolic compounds present in the sample under alkaline conditions. Molybdenum(VI) during the reaction is reduced by the electrons of the phenolic compounds to an oxidation state of 5. This change in the oxidation state produces a change in the color of the solution, from yellow to blue. The intensity of the blue color increases with the content of phenolic compounds. The Folin
Ciocalteau assay is measured by spectroscopy at 765 nm and the

Handbook of Chitin and Chitosan

TABLE 22.2 Summary of CS-based EOs nanocapsules and their properties. Encapsulation method

Size

Final bioactive properties

References

CS
TPP nanoparticles

Ionic gelation

129
1288 nm

Antifungal

Hasheminejad et al. [62]

Mentha spicata and green Camellia sinensis

CS

Emulsification
ionic gelation

20
256 nm

Antioxidant and antibacterial activities for food and medical

Shetta et al. [58]

Coriandrum sativum

CS

Spray drying

400 nm
7 μm

Antimicrobial and antioxidant

Duman and Kaya [60]

Mentha piperita

CS
cinnamic acid nanogel

Ionic gelation

, 100 nm

Antimicrobial

Beyki [54]

Pimenta dioica (L.) Merrill

CS/k-carrageenan

Complex coacervation

1172
1224 μm

Antimicrobial and antioxidant

Dima and Dima [16]

Origanum vulgare

CS
TPP nanoparticles

Ionic gelation

40
80 nm

bioactive food components delivery systems

Hosseini Fakhreddin [17]

Curcuma longa

CS

Spray-drying

80
120 nm

Antiinflamatory agent

Sowasod [63]

Eugenol

CS
TPP nanoparticles

Ionic gelation

80
100 nm

Antioxidant for the thermal processing in food packaging

Woranuch and Yoksan [64]

Lippia sidoides

CS/ cashew gum nanogel

Complex coacervation

335
558 nm

Larvicide

Abreu [65]

Essential oil

Template

Syzygium aromaticum

TPP, Tripolyphosphate.

699

22.5 Antioxidants activity in chitosan as templates for essential oils

results are represented by a calibration curve of gallic acid and expressed in gallic acid equivalents [38,69]. Na2 WO4 =Na2 MO4 -ðphenol2MoW11 O40 Þ42 MoðVIÞ 1 e2 -MoðVÞ

22.5.2 DPPH assay (radical 2,2-diphenyl-1-picrylhydrazyl) Among antioxidant assays, the most widely used is the DPPH assay (radical 2,2-diphenyl-1-picrylhydrazyl) because of its simplicity, speed, and low cost. The method measures the capacity of a substance to give a hydrogen to the DPPH radical. When the antioxidant substance is exposed to the DPPH in a violet methanol solution, DPPH is reduced, resulting in a loss of color (Fig. 22.7). Absorbance is measured at 517 nm, a greater loss of intensity indicates greater antioxidant activity of the substance analyzed [10,68,70].

22.5.3 Trolox equivalent antioxidant capacity or 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt The Trolox equivalent antioxidant capacity (TEAC) or ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assays belong to the SET reaction and were developed by Miller in 1993 [71] to easily determine the antioxidant capacity. In this method, the Trolox (6-hydroxy2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) reagent is used as the standard to measure the antioxidant capacity. The assay is based on the reaction of an aqueous solution of ABTS with potassium persulfate solution in the dark for 12
16 h. During that time, the radical ABTS•+ is generated RH (antioxidant)

NO2 N+ N

O2N

NO2 O2N

H N

N

R* NO2

NO2

DPPH

DPPH*

FIGURE 22.7 The mechanism between the DPPH radical and an antioxidant to generate DPPH.

Handbook of Chitin and Chitosan

700

22. Chitosan-based materials as templates for essential oils

(blue/green chromophore), which has three absorption maxima at the wavelengths of 645, 734, and 815 nm. At this stage, the antioxidant compounds to be tested are placed in contact with the radical and reduce it to ABTS (Fig. 22.8.), producing a decoloration in the solution and consequently, a decrease in the absorbance measured at a wavelength of 734 nm [23,69]. * –O

3S

SO3–

S

S N N

N

N C2H5 C2H5 ABTS*+

+antioxdant –K2SO5

–O

3S

S

S

SO3–

N N

N

N

C2H5 C2H5

ABTS2– (colorless)

FIGURE 22.8

Structure of radical ABTS•+ before and after the reaction with an

antioxidant.

Handbook of Chitin and Chitosan

701

22.5 Antioxidants activity in chitosan as templates for essential oils

N

N

N

N N

N Fe(III)

N N

N N

N

N

N

N

Antioxidant

Fe(III) N

– e– N

N N

N

N

N

N

[Fe(TPTZ)2]3+

FIGURE 22.9

N

N

[Fe(TPTZ)2]2–

Reaction mechanism of FRAP assay.

22.5.4 Ferric ion reducing antioxidant power assay The FRAP assay belongs to the SET reactions and was developed by Benzi and Strain in 1996 in order to measure the reducing power in plasma samples. This method consists of measuring the reduction of the 2,4,6-tripyridyls-triazine complex (TPTZ) to the ferrous complex by the action of an antioxidant compound in an acidic environment (Fig. 22.9). This reaction produces a variation in color which is measured at a wavelength of 593
595 nm. The results are represented by a calibration curve using Trolox reagent as standard and are expressed in Trolox equivalents (μmol Trolox/g or μmol Trolox/L) [57,69]. The Table 22.3 summarizes the antioxidant assays involving CS as templates of essential oil and their applications.

22.5.5 Antioxidant properties of chitosan
essential oils coating and films Many researchers have studied how different EOs in films or coatings influence antioxidant activity, and how this relates to the quality of the food and its preservation. Hormis et al. [76] used caraway seed oil and beeswax with CS as packaging films. Using the DPPH assay, they measured the antioxidant activity of the films after 2.5, 4, and 24 h. The results showed that the antioxidant activity of the CS (around 29% after 24 h) significantly increased when the oil was added (80% after 24 h). Jahed et al. [32] also used C. copticum seed essential oil (EO) and CS to make bionanocomposites for food preservation. To elaborate the bionanocomposite CS films, they reinforced C. copticum oil (CCO) with lignocellulose nanofibers (LCNFs) or cellulose nanofibers (CNFs). The highest value of antioxidant activity was observed in the CS films with 5% (w/w) of CCO with a value around 37% using the DPPH assay. This is due to the high

Handbook of Chitin and Chitosan

TABLE 22.3

Antioxidant assay and CS as templates of essential oils and their applications.

Essential oil or extract

Template

Antioxidant assay

Application

References

Helianthus annuus

Pickering emulsion CS
stearic acid nanogel

TBARS

Food manufacturers

Atarian [49]

Mentha piperita and Camellia sinensis

CS nanoparticles: encapsulation

DPPH

Nutraceuticals, cosmetics, and pharmaceuticals

Shetta et al. [58]

Zingiber officinale

Gelatin/CS films

ABTS

Food packaging

Bonilla [72]

Lippia origanoide

Nanogel emulsion CS
p-coumaric acid: encapsulation

DPPH

Antioxidant

Silva Damasceno [73]

Food active packaging

Kadam and Lele [74]

Lepidium sativum

CS film

ABTS DPPH FRAP Folin
Ciocalteu

Zataria multiflora

CS coating

TBARS

Food packaging (Beef steaks)

Mojaddar [24]

Brassica napus oil

Gelatin
CS-based films that incorporate nanoemulsions

DPPH ABTS FRAP

Food packaging

Pe´rez-Co´rdoba [57]

Prunus armeniaca

CS
N-methyl-2-pyrrolidone nanoparticles films

DPPH

Bread

Priyadarshi [2]

Apple peel polyphenols

CS film

DPPH

Food packaging

Riaz [75]

Beef cutlets during storage

Hadian [1]

α-Tocopherol/cinnamaldehyde α-Tocopherol/Allium sativum α-tocopherol/cinnamaldehyde A. sativum oil

ABTS Rosmarinus officinalis

CS
benzoic acid nanogel encapsulation

DPPH

Carum copticum

CS film reinforced with cellulose nanofibers or lignocellulose nanofibers

DPPH

Active packaging

Jahed and Alizadeh [10]

Coriandrum sativum L.

Microencapsulated CS

DPPH

Packaging technology in the food industry

Duman and Kaya [34]

Satureja hortensis L.

CS nanoparticles

DPPH

Packaging

Feyzioglu and Tornuk [20]

Eucalyptus globulus

CS film

DPPH

Packaging

Hafsa [38]

Folin
Ciocalteu NO H2O2 Carum carvi

CS film

DPPH

Active packaging

Hromiˇs [76]

Green Camellia sinensis

CS coating

Folin
Ciocalteu

Fresh walnut kernel

Sabaghi et al. [33]

Cinnamomum verum

CS
oleic acid film

ABTS

Strawberry

Perdones [23]

Thymus serpyllum L.

CS microbeads

Folin
Ciocalteu

Food applications

Trifkovi [77]

Pimenta dioica

Microencapsulated CS/κ-carrageenan

DPPH

Meat

Dima [14]

Zataria multiflora

CS film

DPPH

Food packaging

Moradi [68]

Active packaging

Siripatrawan and Harte [67]

Boiss and grape seed extract Green Camellia sinensis

Folin
Ciocalteu CS film

DPPH Folin
Ciocalteu

ABTS assay, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; DPPH assay, radical 2,2-diphenyl-1-picrylhydrazyl; FRAP assay, ferric ion reducing antioxidant power assay; H2O2, hydrogen peroxide radical scavenging; NO, nitric oxide radical scavenging activity.

704

22. Chitosan-based materials as templates for essential oils

content of carvacrol and γ-terpinene in the oil. The polymers used to reinforce the bionanocomposites also had an effect, those with LCNF showing greater antioxidant activity than those with CNF, reducing DPPH intensity by 27% and 23%, respectively. Priyadarshi et al. [2] prepared CS films with apricot kernel oil (AKEO) at different concentrations for active food packaging. Apricot kernels contain high percentages of oleic acid and N-methyl-2-pyrrolidone with antioxidant activities. Increasing the concentration of AKEO in the film increased antioxidant activity as evaluated by DPPH. Pure CS films showed 21.8% DPPH scavenging activity and with the AKEO ratios of 0.125:1 (with respect to CS) increased DPPH from 25.9% to 35.3%. Similar results were obtained by [68] when incorporating Z. multiflora Boiss oil in CS films, reaching around 38% DPPH scavenging activity with a ratio of 1:0.5 (CS:oil). Z. multiflora oil (ZEO) was also used together with hydroalcoholic extract of sumac (SE) in edible CS coating in modified atmosphere packaging (MAP) of beef meat for 20 days of storage [24]. This kind of substrate contains high protein and lipid contents, for this reason oxidation was measured using thiobarbituric acid reactive substances (TBARS) at 532 nm. The TBARS assay measures the concentration of the compounds that give off an unpleasant odor or taste resulting from lipid oxidation. Films with CS
SE showed a value of TBARS of 1.77 mg malonaldehyde/kg meat after 20 days of storage and it was observed that when adding the ZEO oil this value was decreased to 1.56 mg malonaldehyde/kg meat. The addition of the EO increases antioxidant activity of the film and aids in the preservation of bovine meat. Perdones et al. [23] used the ABTS (TEAC) assay to evaluate the antioxidant activity of CS films with cinnamon leaf EO and oleic acid. Their conclusions mirrored that of other studies, showing that the adding the EO decreases the TEAC values, indicating high antioxidant activity. DPPH, Folin
Ciocalteu, and FRAP assays were used [74] to evaluate antioxidant activity in CS films with Lepidium sativum seedcake phenolic extract (LSE) intended for use as active packaging. Extracts were extracted in water and in 50% and 95% ethanol. In both DPPH and FRAP assays, antioxidant activity was higher with water extracts. Furthermore, increasing the concentration of the oil also increased the antioxidant activity. These results followed polyphenol content as determined by the Folin
Ciocalteu method, with the most found in the water extract, followed by the 50% ethanol and 95% ethanol extracts. Despite using different EOs in the films and coatings, and the use of a variety of methods to evaluate antioxidant activity, the studies unanimously showed that EOs provide supplemental antioxidant properties and that increasing their concentration results in higher antioxidant activity.

Handbook of Chitin and Chitosan

22.5 Antioxidants activity in chitosan as templates for essential oils

705

22.5.6 Antioxidant properties of chitosan
essential oils emulsions and (nano)gels Antioxidant and antimicrobial activity of Rosmarinus officinalis (RO) EO encapsulated in CS
benzoic acid (CS
BA) nanogels on beef cutlets were studied [1]. DPPH radical scavenging activity assay was used to determine the antioxidant activity of free RO EO (REO), CS
BA nanogel, and CS
BA nanogel-encapsulated EOs. Results showed that at the beginning of the experiment, free RO had higher radical scavenging activity, but over time its effect stabilized. On the other hand, CS
BA nanogel demonstrated an increase in the radical scavenging activity until 100 h, and CS
BA nanogel-encapsulated EO showed the best DPPH radical scavenging ability, with an 85% decrease in intensity. Similar results were reported by Silva-Damasceno et al. [73], who studied the antioxidant activity of Lippia origanoides oil encapsulated in a nanogel made with p-coumaric acid and CS using DPPH and ABTS assays. In both assays the CS modified with p-coumaric acid had increased antioxidant activity as compared to pure CS. However, the addition of EOs further increased antioxidant activity, possibly due to compounds such as thymol and carvacrol that are H1 donors. Perez-Cordoba et al. [57] prepared gelatin
CS films loaded with oil/water emulsions, using canola oil, α-tocopherol/cinnamaldehyde, α-tocopherol/garlic oil, or α-tocopherol/cinnamaldehyde and garlic oil. The film loaded with nanoemulsion encapsulating α-tocopherol/cinnamaldehyde showed the highest antioxidant activity, as measured by DPPH and ABTS assays. Using the FRAP assay, however, the material with α-tocopherol/garlic oil showed the greatest antioxidant activity with an increase from 51% to 91% when compared with the control. This increase was probably due to the fact that during the reaction the FRAP reagent is in direct contact with the film samples.

22.5.7 Antioxidant properties of chitosan
essential oils encapsulations CS can be used, to directly encapsulate EOs and several studies have looked into the resulting antioxidant activity. The most commonly used method to study antioxidant capacity for CS-encapsulated EOs is the DPPH assay. Shetta et al. [58] studied the encapsulation of peppermint (PO) and green tea oil (GTO) in CS
-TPP with the emulsification
ionic gelation technique for food and pharmaceutical applications. DPPH assay was used to analyze the antioxidant capacity of the encapsulation. The results of the antioxidant activity were evaluated using the IC50 (concentration required to scavenge DPPH radicals by 50%) of each

Handbook of Chitin and Chitosan

706

22. Chitosan-based materials as templates for essential oils

composite. CS
TPP showed relatively high values (116.07 mg/mL) perhaps due to TPP masking CS amino groups, which are the groups that react with DPPH. The highest values of antioxidant activity were observed in the encapsulations containing GTO and PO with IC50 values attained at 0.34 and 1.61 mg/mL, respectively. Of the two oils, the one with the highest antioxidant activity was GTO, possibly due to the greater amount of phenolic compounds it contains. These results agree with those obtained by Dima et al. [14]. These authors showed how the encapsulation of pimento (Pimenta dioica L. Merr) oil with CS/κ-carrageen, using the complex coacervation method, shows greater antioxidant activity than pure CS and κ-carrageen. This is because this EO contains a high percentage of eugenol and methyl eugenol. In addition, they observed that the CS
EO encapsulation had greater antioxidant activity than κ-carrageen
EO, most probably from the synergy of CS and the EO. Moreover, Duman and Kaya [60] observed that coriander oil (Coriandrum sativum L.) encapsulated by the spray drying method with CS showed high DPPH antioxidant activity value of 60%. They also observed that the antioxidant activity of CS was dependent on factors such as molecular weight and degree of acetylation. Whatever the method of encapsulation used, all results show that by adding EOs, antioxidant activity increases. On the one hand, it is due to their capacity to donate hydrogen atoms and to capture the free radicals that increase the antioxidant activity. On the other hand, the encapsulation shell helps to protect the bioactive compounds from factors such as humidity, pH, temperature, and pressure among others, making them be slowly released and thus prolonging effects.

22.6 Antibacterial activity in chitosan as templates for essential oils One of the most important causes of food spoilage is the growth of microorganisms and pathogens [78]. These can accelerate the lipid oxidation, resulting in changes in the organoleptic properties, creating toxicity and/or potential pathogenicity in humans [79]. To combat these problems and extend food shelf life, several studies have shown the high potential of natural compounds such as CS and EOs [49,75,80]. CS is usually used as a food preservative due to its antimicrobial activity against a wide range of yeast, bacteria, and fungi, its biocompatibility, and its low toxicity [47,60,81]. The mechanism of the antimicrobial activity of CS is not yet known, however, there are several different hypotheses. One of these hypotheses is that cell permeability changes due to the interaction between the negative charges of the cell membrane and the positive charges of the CS groups. Another hypothesis is

Handbook of Chitin and Chitosan

22.6 Antibacterial activity in chitosan as templates for essential oils

707

that the activity is the result of the interaction of the microorganism’s DNA with diffuse hydrolysis products that inhibit the synthesis of proteins, RNA, and essential nutrients [82]. As for EOs, antimicrobial activity can be explained by the presence of bioactive phytochemicals such as phenols. The mechanism of action of these molecules are numerous. Some phenolic compounds can penetrate the cell membrane and disable its functional and lipophilic properties. These compounds can also alter the permeability of the cell, interfere with the cellular energy generation system (ATP), damage the cytoplasmic membranes, and induce cell death upon adhesion to specific receptors [83,84]. Microbes are extremely diverse unicellular organisms including viruses, fungi, and bacteria. There are two types of bacteria: Gram positive and Gram negative, as determined by the structure of their membranes [84]. Several researchers believe that EOs are more susceptible to inhibition of the growth of Gram-positive bacteria because their lipophilic nature interacts easily with Gram-positive cellular membranes. EOs can separate the lipids from the cell membrane of the bacteria and in doing so make the cell more permeable. According to these studies, Gramnegative bacteria are globally less susceptible to the effects of EOs because of their hydrophilic cell walls [82
84]. Table 22.4 shows a map of the different CS templates of EOs against different microorganisms.

22.6.1 Antimicrobial activity of chitosan
essential oils coating and films One of the most studied fungi in food is Aspergillus niger, which commonly occurs on vegetables. CS has been shown to have good antimicrobial activity when used in biomaterials [91,92], and as such CS
EOs coatings can be thought to show antimicrobial activity. Perdones et al. [23] studied the antifungal effect of CS films with the addition of basil or thyme oil against A. niger, Botrytis cinerea, and Rhizopus stolonifer. The experiments showed no effect on the inhibition of fungal growth. These same results were obtained by Sahraee et al. [18], who studied the antifungal effect of A. niger in gelatin-based nanocomposite films with corn oil. In these cases, the absence of observed antimicrobial activity may be due to the oil covering the active amino groups that interact with the anionic groups on the surface of the cell, impeding activity and effect on microorganism growth. However, the lack of antifungal effect is not systematic. Noshirvani et al. [30] used CS
carboxymethyl cellulose films emulsified with oleic acid and with cinnamon or ginger EO which resulted in antifungal

Handbook of Chitin and Chitosan

TABLE 22.4 Summary of the different microorganisms used to assess the antimicrobial activity of CS templates of essential oils. Essential oil Mentha piperita

Template

Microorganism

CS nanoparticle encapsulation

Camellia sinensis

Staphylococcus aureus

Application

References

Nutraceutical, cosmetic and Pharmaceutical

Shetta et al. [58]

Escherichia coli Syzygium aromaticum Prunus armeniaca

Zataria multiflora Boiss Apple peel polyphenols

Brassica napus oil α-tocopherol/ cinnamaldehyde

CS
tripolyphosphate nanoparticle encapsulation

Aspergillus niger

Agriculture and food industries

Hasheminejad et al. [62]

CS
N-methyl-2-pyrrolidone nanoparticles films

Bacillus subtilis

Bread

Priyadarshi [2]

Beef steaks preserved by modified atmosphere packaged

Mojaddar [24]

Food packaging

Riaz [75]

CS coating CS film

E. coli Enterobacteriaceae Pseudomonas spp. Bacillus cereus E. coli Salmonella typhimurium S. aureus

Gelatin
CS-based films that incorporate nanoemulsions

α-Tocopherol/Allium sativum

Pseudomonas aeruginosa

Food packaging

Pe´rez-Co´rdoba [57]

Listeria monocytogenes

α-Tocopherol/ cinnamaldehyde A. sativum oil Rosmarinus officinalis

CS
benzoic acid nanogel encapsulation

S. typhimurium

Carum copticum

CS film reinforced with cellulose nanofibers or lignocellulose nanofibers

E. coli B Botrytis cereus

Beef cutlets during storage

Hadian [1]

Active packaging

Jahed, Alizadeh et al. [10]

Cinnamon verum Cumbopogon citrates

CS film

Origanum vulgare C. verum

Botrytis sp.

Food

Munhuweyi [42]

Pilidiella granati Penicillium sp. CS
carboxymethyl cellulose films

A. niger

Food packaging

Noshirvani et al. [30]

CS
gelatin films with nanochitin

A. niger

Food packaging

Sahraee [18]

P. aeruginosa S. aureus

Mangaba fruits

Ribeiro-Santos [85]

Packaging technology in the food industry

Duman and Kaya [34]

Packaging

Feyzioglu and Tornuk [20]

Zingiber officinale Zea mays Myrcia ovata Cambessedes

CS coatings

B. cereus B. subtilis Enterococcus faecalis Serratia marcescens E. coli Salmonella enteritidis

Coriandrum sativum L.

Microencapsulated CS

Aeromonas hydrophila E. coli Klebsiella pneumonia P. aeruginosa S. typhimurium Yersinia enterocolitica B. cereus L. monocytogenes Candida albicans

Satureja hortensis L.

CS nanoparticles

E. coli S. aureus L. monocytogenes

(Continued)

TABLE 22.4 (Continued) Essential oil Eucalyptus globulus

Template

Microorganism CS film

S. aureus

Application

References

Food packaging

Hafsa [38]

Packaging minced rainbow trout

Kakaei and Shahbazi [86]

Strawberry

Sangsuwan et al. [80]

Modified atmosphere packaged pork

Paparella [87]

Cucumber fruit rot

Mohammadi et al. [88]

Food

Perdones [37]

Active packaging

Hromiˇs [76]

Green beans

Severino [89]

E. coli P. aeruginosa K. pneumonia Ziziphora clinopodioides

CS
gelatin film

Pseudomonas spp. L. monocytogenes Shewanella putrefaciens

Lavandula angustifolia

CS beads

Botrytis cinerea

red Thymus vulgaris O. vulgare

CS solution

L. monocytogenes

Z. multiflora Cinnamomum

CS coating

Phytophthora drechsleri

zeylanicum T. vulgaris

CS
oleic acid film-forming emulsion

Ocimum basilicum

A. niger B. cinerea Rhizopus stolonifer

Carum carvi

CS film

S. aureus E. coli

Carvacrol Citrus aurantium ssp. Citrus reticulate Citrus x limon L.

CS coating containing nanoemulsion

E. coli S. typhimurium

T. vulgaris

CS
benzoic acid nanogel encapsulation

Cuminum cyminum

CS
caffeic acid nanogel (encapsulation) CS
cinnamic acid nanogel

M. piperita Citrus

CS film

Limonum

Aspergillus flavus

Grape

Khalili Tahereh [47]

A. flavus

Food packaging

Zhaveh [55]

A. flavus

Food

Beyki [54]

Active food packaging

Peng and Li [22]

Food packaging

Wu [90]

E. coli S. aureus

T. vulgaris Cinnamomum zeylanicum O. vulgare

Skin gelatin
CS film

E. coli S. aureus B. subtilis S. enteritidis Shiga bacillus

T. moroderi Thymus piperella

CS edible films

Listeria innocua Serratia marcenscens A. hydrophila Achromobacter denitrificans Alcaligenes faecalis

Food

Ruiz-Navajas [31]

712

22. Chitosan-based materials as templates for essential oils

activity against A. niger. In this case, increasing the concentration of EOs resulted in decreased fungal growth. Comparing the two EOs, it was shown that cinnamon oil has higher antifungal effects. The results reflect a synergistic effect between the oils and the fatty acid, and lead to the shortening of the chains in the CS fraction. Other authors such as Priyadarshi et al. [2] studied the effect of CS
(N-methyl-2-pyrrolidone) films with AKEO (Prunus armeniaca) against Bacillus subtilis (Gram positive) and Escherichia coli (Gram negative). Decrease of bacterial growth in both bacteria could be observed on addition of the EOs. Furthermore, it was shown that N-methyl-2-pyrrolidone, which is a known antimicrobial, improved microbial inhibition when included with the oil. N-methyl-2-pyrrolidone helped to dissolve the lipids of the cell membrane, provoking its disintegration and, consequently, cell death. The antimicrobial effects of CS
EOs coatings have been the subject of several recent studies. Mojaddar [24] analyzed the antimicrobial effect of Z. multiflora Boiss oil and sumac hydroalcoholic extract in CS coatings for beef steaks stored over 20 days. The microorganisms studied were Pseudomonas spp., Lactic acid bacteria, Enterobacteriaceae, and yeasts: for all less microbial growth was observed with higher concentration of EOs and the extract. In addition, it was demonstrated that the antimicrobial effect was greater when using EOs and extracts together. This synergistic effect was also found by [86], with ethanolic red grape seed extract and Ziziphora clinopodioides oil in a CS
gelatin film inhibiting Listeria monocytogenes growth in minced trout fillet. The synergistic effect between the extracts and EOs is explained by the combination of different mechanisms such as the inhibition of protective enzymes and concurrent alteration of cell walls.

22.6.2 Antimicrobial activity of chitosan
essential oils emulsions and (nano)gels Hadian et al. [1] studied how to preserve beef cutlets against Salmonella tryphimurium during 12 days in refrigeration. (REO) was encapsulated in a CS
benzoic acid nanogel. Meat was inoculated by 109 CFU S. typhimurium/mL, then sprayed with free REO and nanoencapsulated REO. The results showed encapsulation resulted in a greater inhibitory effect as measured by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). This was explained by the greater accessible surface area of CS particles interacting with the cell wall. In addition, it was also observed that on day 1 of refrigeration, the nanoencapsulation with the highest concentration (2 mg nanoencapsulated REOs/g beef) showed a large decrease in the population of S. typhimurium

Handbook of Chitin and Chitosan

22.6 Antibacterial activity in chitosan as templates for essential oils

713

(3.3 log CFU/g) and after 12 days of refrigeration, reduction was maximum with a value of 2.5 log CFU/g. Severino et al. [89] also evaluated the inhibition of the growth of S. typhimurium and E. coli in green beans. The antimicrobial effect of modified CS coatings containing nanoemulsions of EOs (carvacrol, bergamot, lemon, and mandarin oil), gamma irradiation treatment, and MAP, were examined alone or in combination. Carvacrol showed the highest antimicrobial inhibition on both microbes, so it was used in the other experiments. In the next step, the behavior of E. coli and S. typhimurium was evaluated against gamma irradiation. In the experiments a synergic effect was observed with the coating under MAP, which increased the radiosensitivity in E. coli and S. typhimurium of 1.80-fold and 1.89-fold, respectively. The combination of CS coating with carvacrol, gamma irradiation treatment, and MAP caused the greatest decrease in the population of E. coli and S. typhimurium after 7 days of storage. One of the fungi that most proliferates in food systems is Aspergillus flavus and it is believed to be the cause of infections in corneas [47]. These authors studied the antimicrobial activity of thyme oil encapsulated in CS
benzoic acid nanogel against A. flavus on grapes during 1 month of storage. In vitro, sealed, and unsealed experiments were performed. In the sealed experiments, the MIC of the free oils was 400 and 300 mg/L for encapsulated oils. In the unsealed experiment, the MICs were much higher. The free oils did not achieve complete inhibition, not even at 1000 mg/L; for the encapsulate MIC was 500 mg/L. This explained by the volatility of the compounds present in EOs. In in vivo analysis, A. flavus by was inhibited by encapsulates at a concentration around 700 mg/L. Similar results were obtained in the work of Zhaveh et al. [55], where Cuminum cyminum oil encapsulated in CS
caffeic acid nanogel antimicrobial activity was measured against A. flavus. Free oil in unsealed flasks failed to prevent the growth of the fungus, even using a concentration of 1000 mg/L. Under the same conditions, encapsulates succeeded in inhibiting the growth of A. flavus at 950 mg/L, which was much higher than under sealed conditions (350 mg/L). Encapsulation of the oil in CS
caffeic acid nanogel is nevertheless the best option, with slower evaporation of the oil and slow and delayed release of the EO into the medium.

22.6.3 Antimicrobial activity of chitosan
essential oils encapsulations The application of crayfish CS microencapsulated with coriander (Coriandrum sativum L.) EO for its antioxidant properties and antimicrobial activity against Aeromonas hydrophila, E. coli, Klebsiella pneumoniae,

Handbook of Chitin and Chitosan

714

22. Chitosan-based materials as templates for essential oils

Pseudomonas aeruginosa, Salmonella typhimurium, Yersinia enterocolitica, Bacillus cereus, L. monocytogenes, and Candida albicans was reported [60]. Although numerous authors have demonstrated the antimicrobial activity of coriander oil [93,94], in this work the authors did not observe any antimicrobial effect. In addition, the effect against microorganisms was greater with the pure crayfish CS than with the microcapsules of CS
coriander oil. This result could be because the microcapsules are covered with an oil without antimicrobial effect and which effectively prevents CS from having an effect. In the work of Shetta et al. [58] the encapsulations of PO and GTO in CS nanoparticles were compared and the antimicrobial activity against Staphylococcus aureus (Gram positive) and E. coli (Gram negative) was studied. Antimicrobial activity was observed for both encapsulated oils, although it was lower against E. coli than against S. aureus. MBC values of pure EOs were greater than encapsulated oils against S. aureus. This could be because the walls of the Gram-positive bacteria are hydrophilic, while the molecules of the EOs are hydrophobic. Therefore by encapsulating the EOs in CS nanoparticles that contain hydrophilic compounds, they can penetrate the bacteria. In the Gram-negative bacteria the antimicrobial activity depends on the interaction of the lipophilic oil with the bacterial membrane of phospholipids generating a passive permeability of the oil. CS nanoparticle encapsulation improved the antifungal activity of clove oil against A. niger [62]. This could be because the encapsulation helps the oil compounds to not evaporate and results in a better inhibitory effect. In 2015, Zhaveh et al. [55] obtained the same conclusions. In addition, they found that the organoleptic alterations that the oils can cause are reduced by means of the encapsulation.

22.7 Conclusions and future perspectives This chapter compiles information related to the antimicrobial and antioxidant activity of CS templates, namely films, coatings, nanocapsules, nanogels, and emulsions of EOs. A special emphasis was placed on CS active films with C. verum, Z. officinale, T. moroderi, and T. piperella; CS-based coatings with Z. multiflora, C. copticum, and C. sinensis and emulsions with Zea mays. The final bioactive properties of these materials show the potential of CS
EOs materials for different applications, in particular in the food industry as well as in medicine, agriculture, and cosmetics. Future research should focus on the application of the different CS templates of EOs for different food matrices and the cytotoxicity of the EOs and final materials.

Handbook of Chitin and Chitosan

References

715

References [1] M. Hadian, et al., Encapsulation of Rosmarinus officinalis essential oils in chitosanbenzoic acid nanogel with enhanced antibacterial activity in beef cutlet against Salmonella typhimurium during refrigerated storage, LWT—Food Sci. Technol. 84 (2017) 394
401. Available from: http://dx.doi.org/10.1016/j.lwt.2017.05.075. [2] R. Priyadarshi, et al., Chitosan films incorporated with apricot (Prunus armeniaca) kernel essential oil as active food packaging material, Food Hydrocoll. 85 (July) (2018) 158
166. Available from: https://doi.org/10.1016/j.foodhyd.2018.07.003. [3] G. Yuan, X. Chen, D. Li, Chitosan films and coatings containing essential oils: the antioxidant and antimicrobial activity, and application in food systems, FRIN 89 (2016) 117
128. Available from: http://dx.doi.org/10.1016/j.foodres.2016.10.004. [4] M.Z. Elsabee, E.S. Abdou, Chitosan based edible films and coatings: a review, Mater. Sci. Eng. C. 33 (4) (2013) 1819
1841. Available from: http://dx.doi.org/10.1016/j. msec.2013.01.010. [5] M.M.A. Rashed, et al., Isolation of essential oil from Lavandula angustifolia by using ultrasonic-microwave assisted method preceded by enzymolysis treatment, and assessment of its biological activities, Ind. Crop. Prod. 100 (2017) 236
245. Available from: http://dx.doi.org/10.1016/j.indcrop.2017.02.033. [6] A.F.M.S. Ud-daula, et al., Chemical composition, antioxidant and antimicrobial activities of essential oils from leaves, aerial stems, basal stems, and rhizomes of Etlingera fimbriobracteata (K. Schum.) R. M. Sm, Ind. Crop. Prod. 84 (2016) 189
198. Available from: http://dx.doi.org/10.1016/j.indcrop.2015.12.034. [7] J. Bravo, et al., Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae, J. Invertebr. Pathol. 149 (July) (2017) 141
147. Available from: http://dx.doi.org/10.1016/j.jip.2017.08.012. [8] S.S. Cheng, et al., Chemical compositions and larvicidal activities of leaf essential oils from two eucalyptus species, Bioresour. Technol. 100 (1) (2009) 452
456. [9] I. Bajalan, et al., Antioxidant and antibacterial activities of the essential oils obtained from seven Iranian populations of Rosmarinus officinalis, Ind. Crop. Prod. 107 (February) (2017) 305
311. Available from: http://dx.doi.org/10.1016/j.indcrop.2017.05.063. [10] E. Jahed, M. Alizadeh, et al., Physicochemical properties of Carum copticum essential oil loaded chitosan films containing organic nanoreinforcements, Carbohydr. Polym. 164 (2017) 325
338. Available from: http://dx.doi.org/10.1016/j.carbpol.2017.02.022. [11] H. Xiang, et al., Chemical compositions, antioxidative, antimicrobial, anti-inflammatory and antitumor activities of Curcuma aromatica Salisb. essential oils, Ind. Crop. Prod. 108 (May) (2017) 6
16. Available from: http://dx.doi.org/10.1016/j.indcrop.2017.05.058. [12] A. El Asbahani, et al., Essential oils: from extraction to encapsulation, Int. J. Pharm. 483 (1
2) (2015) 220
243. Available from: http://dx.doi.org/10.1016/j.ijpharm.2014.12.069. [13] R. Ribeiro-santos, M. Andrade, Application of encapsulated essential oils as antimicrobial agents in food packaging, Curr. Opin Food Sci. 14 (2017) 78
84. [14] C. Dima, et al., Reprint of ‘Microencapsulation of essential oil of pimento [Pimenta dioica (L) Merr.] by chitosan/k-carrageenan complex coacervation method’, Innov. Food Sci. Emerg. 25 (C) (2014) 97
105. Available from: ,https://doi.org/10.1016/j. ifset.2014.07.008.. [15] S. Manso, et al., Diminution of aflatoxin B1 production caused by an active packaging containing cinnamon essential oil, Food Control. 45 (2014) 101
108. Available from: http://dx.doi.org/10.1016/j.foodcont.2014.04.031. [16] C. Dima, S. Dima, Science direct essential oils in foods: extraction, stabilization, and toxicity, Curr. Opin. Food Sci. 5 (2015) 29
35. Available from: http://dx.doi.org/ 10.1016/j.cofs.2015.07.003.

Handbook of Chitin and Chitosan

716

22. Chitosan-based materials as templates for essential oils

[17] S. Hosseini Fakhreddin, et al., Two-step method for encapsulation of oregano essential oil in chitosan nanoparticles: preparation, characterization and in vitro release study, Carbohydr. Polym. 95 (1) (2013) 50
56. Available from: ,https://doi.org/ 10.1016/j.carbpol.2013.02.031.. [18] Sahraee et al., Effect of corn oil on physical, thermal, and antifungal properties of gelatin-based nanocomposite films containing nano chitin, LWT—Food Sci. Technol. 76 (2017) 33
39. [19] M.V. Tzoumaki, et al., Oil-in-water emulsions stabilized by chitin nanocrystal particles, Food Hydrocoll. 25 (6) (2011) 1521
1529. Available from: ,https://doi.org/ 10.1016/j.foodhyd.2011.02.008.. [20] G.C. Feyzioglu, F. Tornuk, Development of chitosan nanoparticles loaded with summer savory (Satureja hortensis L.) essential oil for antimicrobial and antioxidant delivery applications, LWT—Food Sci. Technol. 70 (2016) 104
110. Available from: ,https://doi.org/10.1016/j.lwt.2016.02.037.. [21] A. Mohammadi, M. Hashemi, S. Masoud, Chitosan nanoparticles loaded with Cinnamomum zeylanicum essential oil enhance the shelf life of cucumber during cold storage, Postharvest Biol. Technol. 110 (2015) 203
213. Available from: ,https://doi. org/10.1016/j.postharvbio.2015.08.019.. [22] Y. Peng, Y. Li, Combined effects of two kinds of essential oils on physical, mechanical and structural properties of chitosan films, Food Hydrocoll. 36 (2014) 287
293. Available from: http://dx.doi.org/10.1016/j.foodhyd.2013.10.013. ´ . Perdones, et al., Physical, antioxidant and antimicrobial properties of chitosan e [23] A cinnamon leaf oil films as affected by oleic acid, Food Hydrocoll. 36 (2014) 256
264. Available from: http://dx.doi.org/10.1016/j.foodhyd.2013.10.003. [24] A. Mojaddar, et al., Effects of sumac extract dipping and chitosan coating enriched with Zataria multi flora Boiss oil on the shelf-life of meat in modi fied atmosphere packaging, LWT—Food Sci. Technol. 98 (August) (2018) 372
380. Available from: https://doi.org/10.1016/j.lwt.2018.08.063. [25] A. Acevedo-fani, et al., Edible films from essential-oil-loaded nanoemulsions: physicochemical characterization and antimicrobial properties, Food Hydrocoll. 47 (2015) 168
177. Available from: http://dx.doi.org/10.1016/j.foodhyd.2015.01.032. [26] M. Ahmad, et al., Physico-mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils, Food Hydrocoll. 28 (1) (2012) 189
199. Available from: http://dx.doi.org/10.1016/j. foodhyd.2011.12.003. [27] L. Atare´s, et al., Characterization of SPI-based edible films incorporated with cinnamon or ginger essential oils, J. Food Eng. 99 (3) (2010) 384
391. Available from: http://dx.doi.org/10.1016/j.jfoodeng.2010.03.004. [28] M.H. Hosseini, S.H. Razavi, M.A. Mousavi, Antimicrobial, physical and mechanical properties of chitosan-based films incorporated with thyme, clove and cinnamon essential oils, J. Food Process Pres. 33 (2009) (2008) 727
743. [29] E. Jahed, M.A. Khaledabad, M.R. Bari et al., Effect of cellulose and lignocellulose nano fibers on the properties of Origanum vulgare ssp. gracile essential oil-loaded chitosan films, React. Funct. Polym. 117 (May) (2017) 70
80. [30] N. Noshirvani, B. Ghanbarzadeh, C. Gardrat, Cinnamon and ginger essential oils to improve antifungal, physical and mechanical properties of chitosan-carboxymethyl cellulose films, Food Hydrocoll. 70 (2017) 36
45. [31] Y. Ruiz-Navajas, et al., In vitro antibacterial and antioxidant properties of chitosan edible films incorporated with Thymus moroderi or Thymus piperella essential oils, Food Control. 30 (2) (2013) 386
392. Available from: http://dx.doi.org/10.1016/j. foodcont.2012.07.052.

Handbook of Chitin and Chitosan

References

717

[32] E. Jahed, M.A. Khaledabad, H. Almasi, et al., Physicochemical properties of Carum copticum essential oil loaded chitosan films containing organic nanoreinforcements, Carbohydr. Polym. 164 (2017) 325
338. [33] M. Sabaghi, Y. Maghsoudlou, M. Khomeiri, Active edible coating from chitosan incorporating green tea extract as an antioxidant and antifungal on fresh walnut kernel, Postharvest Biol. Technol. 110 (2015) 224
228. Available from: http://dx.doi. org/10.1016/j.postharvbio.2015.08.025. [34] F. Duman, M. Kaya, Crayfish chitosan for microencapsulation of coriander (Coriandrum sativum L.) essential oil, Int. J. Biol. Macromol. 92 (2016) 125
133. [35] V. Falguera, et al., Edible films and coatings: structures, active functions and trends in their use, Trends Food Sci. Technol. 22 (6) (2011) 292
303. [36] Bonilla, J. et al., 2012. Effect of essential oils and homogenization conditions on properties of chitosan-based films, Food Hydrocoll. 26 (2012). ´ . Perdones, et al., Effect of chitosan—lemon essential oil coatings on volatile profile [37] A of strawberries during storage, Food Chem. 197 (2016) 979
986. Available from: http://dx.doi.org/10.1016/j.foodchem.2015.11.054. [38] J. Hafsa, et al., Physical, antioxidant and antimicrobial properties of chitosan films containing Eucalyptus globulus essential oil, LWT—Food Sci. Technol. 68 (2016) 356
364. [39] Sa´nchez-gonza´lez et al., Study of the release of limonene present in chitosan films enriched with bergamot oil in food simulants, J. Food Eng. 105 (2011) 138
143. [40] H. Wang, J. Qian, F. Ding, Emerging chitosan-based films for food packaging applications, J. Agric. Food Chem. 66 (2) (2018) 395
413. ´ [41] S. Galus, J. Kadzinska, Food applications of emulsion-based edible films and coatings, Trends Food Sci. Technol. 45 (2) (2015) 273
283. [42] K. Munhuweyi, et al., In vitro and in vivo antifungal activity of chitosan-essential oils against pomegranate fruit pathogens, Postharvest Biol. Technol. 129 (2017) 9
22. Available from: http://dx.doi.org/10.1016/j.postharvbio.2017.03.002. [43] G.G. Santos Fraza˜o, A.F. Blank, L.C.L. de Aquino Santana, Optimisation of edible chitosan coatings formulations incorporating Myrcia ovata Cambessedes essential oil with antimicrobial potential against foodborne bacteria and natural microflora of mangaba fruits, LWT—Food Sci. Technol. 79 (2017) 1
10. [44] F. Donsı`, G. Ferrari, Essential oil nanoemulsions as antimicrobial agents in food, J. Biotechnol. 233 (2016) 106
120. Available from: http://dx.doi.org/10.1016/j. jbiotec.2016.07.005. [45] R. Severino, et al., Antibacterial and physical effects of modified chitosan basedcoating containing nanoemulsion of mandarin essential oil and three non-thermal treatments against Listeria innocua in green beans, Int. J. Food Microbiol. 191 (2014) 82
88. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.007. [46] F.M. Pelissari, et al., Antimicrobial, mechanical, and barrier properties of cassava starch-chitosan films incorporated with oregano essential oil, J. Agric. Food Chem. 57 (16) (2009) 7499
7504. [47] Khalili Tahereh et al., Encapsulation of thyme essential oils in chitosan-benzoic acid nanogel with enhanced antimicrobial activity against Aspergillus flavus, LWT—Food Sci. Technol. 60 (2015) 502
508. [48] D.J. McClements, Principles, practicies, and techniques, CRC Press, Florida, 2005. [49] M. Atarian, et al., Formulation of pickering sunflower oil-in-water emulsion stabilized by chitosan-stearic acid nanogel and studying its oxidative stability, Carbohydr. Polym. 210 (January) (2019) 47
55. [50] E. Dickinson, Use of nanoparticles and microparticles in the formation and stabilization of food emulsions, Trends Food Sci. Technol. 24 (1) (2012) 4
12.

Handbook of Chitin and Chitosan

718

22. Chitosan-based materials as templates for essential oils

[51] V. Calabrese, et al., Pickering emulsions stabilized by naturally derived or biodegradable particles, Curr. Opin. Green. Sustain. Chem. 12 (Fig. 1) (2018) 83
90. Available from: https://doi.org/10.1016/j.cogsc.2018.07.002. [52] M. Errezma, et al., Surfactant-free emulsion pickering polymerization stabilized by aldehyde-functionalized cellulose nanocrystals, Carbohydr. Polym. 202 (August) (2018) 621
630. [53] X. Song, et al., Preparation and characterizations of pickering emulsions stabilized by hydrophobic starch particles, Food Hydrocoll. 45 (2015) 256
263. Available from: http://dx.doi.org/10.1016/j.foodhyd.2014.12.007. [54] Beyki et al., Encapsulation of Mentha piperita essential oils in chitosan—cinnamic acid nanogel with enhanced antimicrobial activity against Aspergillus flavus, Ind. Crop. Prod. 54 (2014) 310
319. [55] S. Zhaveh, et al., Encapsulation of Cuminum cyminum essential oils in chitosan-caffeic acid nanogel with enhanced antimicrobial activity against Aspergillus flavus, Ind. Crop. Prod. 69 (2015) 251
256. Available from: http://dx.doi.org/10.1016/j.indcrop.2015.02.028. [56] I. Gu¨lseren, M. Corredig, Interactions of chitin nanocrystals with β-lactoglobulin at the oil-water interface, studied by drop shape tensiometry, Colloid. Surface. B 111 (2013) 672
679. [57] L.J. Pe´rez-Co´rdoba, et al., Physico-chemical, antimicrobial and antioxidant properties of gelatin-chitosan based films loaded with nanoemulsions encapsulating active compounds, Food Hydrocoll. 79 (2018) 544
559. [58] A. Shetta, J. Kegere, W. Mamdouh, Comparative study of encapsulated peppermint and green tea essential oils in chitosan nanoparticles: encapsulation, thermal stability, in-vitro release, antioxidant and antibacterial activities, Int. J. Biol. Macromol. 126 (2019) 731
742. Available from: https://doi.org/10.1016/j.ijbiomac.2018.12.161. [59] J. Rodrı´guez, et al., Current encapsulation strategies for bioactive oils: from alimentary to pharmaceutical perspectives, Food Res. Int. 83 (2016) 41
59. Available from: http://dx.doi.org/10.1016/j.foodres.2016.01.032. [60] F. Duman, M. Kaya, Crayfish chitosan for microencapsulation of coriander (Coriandrum sativum L.) essential oil, Int. J. Biol. Macromol. 92 (2016) 125
133. Available from: http://dx.doi.org/10.1016/j.ijbiomac.2016.06.068. [61] C.E. Mora-Huertas, H. Fessi, A. Elaissari, Polymer-based nanocapsules for drug delivery, Int. J. Pharm. 385 (1
2) (2010) 113
142. [62] N. Hasheminejad, F. Khodaiyan, M. Safari, Improving the antifungal activity of clove essential oil encapsulated by chitosan nanoparticles, Food Chem. 275 (September 2018) (2019) 113
122. [63] N. Sowasod, et al., Encapsulation of curcumin loaded oil droplets with chitosan based cryogel: influence of freezing condition on nanocapsule properties, Food Sci. Technol. Res. 19 (4) (2013) 633
640. [64] S. Woranuch, R. Yoksan, Eugenol-loaded chitosan nanoparticles: I. Thermal stability improvement of eugenol through encapsulation, Carbohydr. Polym. 96 (2) (2013) 578
585. Available from: http://dx.doi.org/10.1016/j.carbpol.2012.08.117. [65] F.O.M.S. Abreu, et al., Chitosan/cashew gum nanogels for essential oil encapsulation, Carbohydr. Polym. 89 (4) (2012) 1277
1282. Available from: http://dx.doi.org/ 10.1016/j.carbpol.2012.04.048. [66] G. Yuan, X. Chen, D. Li, Chitosan films and coatings containing essential oils: the antioxidant and antimicrobial activity, and application in food systems, Food Res. Int. 89 (2016) 117
128. Available from: http://dx.doi.org/10.1016/j.foodres.2016.10.004. [67] U. Siripatrawan, B.R. Harte, Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract, Food Hydrocoll. 24 (8) (2010) 770
775. Available from: http://dx.doi.org/10.1016/j.foodhyd.2010.04.003.

Handbook of Chitin and Chitosan

References

719

[68] M. Moradi, et al., Characterization of antioxidant chitosan film incorporated with Zataria multi flora Boiss essential oil and grape seed extract, LWT—Food Sci. Technol. 46 (2012) 477
484. [69] D. Huang, O.U. Boxin, R.L. Prior, The chemistry behind antioxidant capacity assays, J. Agric. Food Chem. 53 (6) (2005) 1841
1856. [70] W. Brand-Williams, M.E. Cuvelier, C. Berset, Respostas perceptivas E, LWT—Food Sci. Technol. 28 (1) (1995) 25
30. [71] N.J. Miller, et al., A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates, Clin. Sci. 84 (4) (1993) 407
412. [72] J. Bonilla, et al., Food bioscience antioxidant potential of eugenol and ginger essential oils with gelatin/chitosan films, Food Biosci. J. 23 (August 2017) (2018) 107
114. [73] E.T. Silva Damasceno, et al., Lippia origanoides Kunth essential oil loaded in nanogel based on the chitosan and ρ-coumaric acid: encapsulation efficiency and antioxidant activity, Ind. Crop. Prod. 125 (September) (2018) 85
94. [74] D. Kadam, S.S. Lele, Cross-linking effect of polyphenolic extracts of Lepidium sativum seedcake on physicochemical properties of chitosan films, Int. J. Biol. Macromol. 114 (2018) 1240
1247. [75] A. Riaz, et al., Preparation and characterization of chitosan-based antimicrobial active food packaging film incorporated with apple peel polyphenols, Int. J. Biol. Macromol. 114 (2018) 547
555. [76] Hromiˇs et al., Optimization of chitosan biofilm properties by addition of caraway essential oil and beeswax, J. Food Eng. 158 (2015) 86
93. [77] Trifkovi et al., Chitosan microbeads for encapsulation of thyme (Thymus serpyllum L.) polyphenols, Carbohyd. Polym. 111 (2014) 901
907. [78] M. Viuda-Martos, et al., In vitro antioxidant and antibacterial activities of essentials oils obtained from Egyptian aromatic plants, Food Control. 22 (11) (2011) 1715
1722. Available from: http://dx.doi.org/10.1016/j.foodcont.2011.04.003. [79] L. Atare´s, A. Chiralt, Essential oils as additives in biodegradable films and coatings for active food packaging, Trends Food Sci. Technol. J. 48 (2016) 51
62. [80] J. Sangsuwan, T. Pongsapakworawat, P. Bangmo, Effect of chitosan beads incorporated with lavender or red thyme essential oils in inhibiting Botrytis cinerea and their application in strawberry packaging system, LWT—Food Sci. Technol. 74 (2016) 14
20. Available from: http://dx.doi.org/10.1016/j.lwt.2016.07.021. [81] J. Go´mez-Estaca, et al., Biodegradable gelatin e chitosan films incorporated with essential oils as antimicrobial agents for fish preservation, Food Microbiol. 27 (7) (2010) 889
896. Available from: http://dx.doi.org/10.1016/j.fm.2010.05.012. [82] P. Cazo´n, et al., Polysaccharide-based films and coatings for food packaging: a review, Food Hydrocoll. 68 (2017) 136
148. [83] S. Burt, Essential oils: their antibacterial properties and potential applications in foods—a review, Int. J. Food Microbiol. 94 (3) (2004) 223
253. [84] J.R. Calo, et al., Essential oils as antimicrobials in food systems—a review, Food Control. 54 (2015) 111
119. Available from: http://dx.doi.org/10.1016/j.foodcont.2014.12.040. [85] R. Ribeiro-Santos, et al., Use of essential oils in active food packaging: recent advances and future trends, Trends Food Sci. Technol. 61 (2017) 132
140. [86] S. Kakaei, Y. Shahbazi, Effect of chitosan-gelatin film incorporated with ethanolic red grape seed extract and Ziziphora clinopodioides essential oil on survival of Listeria monocytogenes and chemical, microbial and sensory properties of mince, LWT—Food Sci. Technol. 72 (2016) 432
438. Available from: http://dx.doi.org/10.1016/j.lwt.2016.05.021. [87] Paparella et al., Chitosan boosts the antimicrobial activity of Origanum vulgare essential oil in modified atmosphere packaged pork, 59 (2016) 23
31.

Handbook of Chitin and Chitosan

720

22. Chitosan-based materials as templates for essential oils

[88] A. Mohammadi, M. Hashemi, S. Masoud, Integration between chitosan and Zataria multi flora or Cinnamomum zeylanicum essential oil for controlling Phytophthora drechsleri, the causal agent of cucumber fruit rot, LWT—Food Sci. Technol. 65 (2016) 349
356. Available from: http://dx.doi.org/10.1016/j.lwt.2015.08.015. [89] R. Severino, et al., Antimicrobial effects of modified chitosan based coating containing nanoemulsion of essential oils, modified atmosphere packaging and gamma irradiation against Escherichia coli O157:H7 and Salmonella typhimurium on green beans, Food Control. 50 (2015) 215
222. [90] J. Wu, et al., Science direct properties and antimicrobial activity of silver carp (Hypophthalmichthys molitrix) skin gelatin-chitosan films incorporated with oregano essential oil for fish preservation, Food Packag. Shelf Life 2 (1) (2014) 7
16. Available from: http://dx.doi.org/10.1016/j.fpsl.2014.04.004. [91] A.M. Salaberria, et al., Preparing valuable renewable nanocomposite films based exclusively on oceanic biomass—Chitin nanofillers and chitosan, React. Funct. Polym. 89 (2015) 31
39. Available from: http://dx.doi.org/10.1016/j.reactfunctpolym.2015.03.003. [92] S. Shankar, et al., Preparation, characterization, and antimicrobial activity of chitin nanofibrils reinforced carrageenan nanocomposite films, Carbohydr. Polym. 117 (2015) 468
475. Available from: http://dx.doi.org/10.1016/j.carbpol.2014.10.010. [93] P. L.O. Cantore, et al., Antibacterial activity of Coriandrum sativum L. and Foeniculum vulgare Miller Var. vulgare (Miller) essential oils, J. Agric. Food Chem. 52 (26) (2004) 7862
7866. [94] F. Silva, et al., Coriander (Coriandrum sativum L.) essential oil: its antibacterial activity and mode of action evaluated by flow cytometry, J. Med. Microbiol. 60 (10) (2011) 1479
1486.

Handbook of Chitin and Chitosan

C H A P T E R

23 Chitosan and chitosan-based biomaterials for wound management Md. Sazedul Islam, Md. Shirajur Rahman, Tanvir Ahmed, Shanta Biswas, Papia Haque and Mohammed Mizanur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

O U T L I N E 23.1 Introduction

722

23.2 Wound healing and its different stages 23.2.1 Wound healing models 23.2.2 Wound healing phases 23.2.3 Factors for delayed wound healing

723 723 724 726

23.3 Properties of chitosan advantageous for wound management 23.3.1 Biocompatibility and biodegradability 23.3.2 Hemostatic and analgesic properties 23.3.3 Antimicrobial and antiinflammatory nature

727 727 728 730

23.4 Different forms of chitosan-based biomaterials in wound management 23.4.1 Hydrogels 23.4.2 Membranes 23.4.3 Sponges 23.4.4 Scaffolds

731 731 735 736 737

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00023-6

721

© 2020 Elsevier Inc. All rights reserved.

722

23. Chitosan and chitosan-based biomaterials for wound management

23.4.5 Gels 23.4.6 Fibers

737 738

23.5 Chitosan-based biocomposites for wound healing 23.5.1 Chitosan
starch 23.5.2 Chitosan
alginate 23.5.3 Chitosan
dextran 23.5.4 Chitosan
collagen 23.5.5 Chitosan
gelatin 23.5.6 Chitosan
polylactic acid 23.5.7 Chitosan
polyvinyl alcohol

739 739 740 741 744 745 747 748

23.6 Chitosan derivatives in wound management 23.6.1 N,O-carboxymethyl chitosan 23.6.2 N-Carboxybutyl chitosan 23.6.3 5-Methylpyrrolidinone chitosan

749 749 750 751

23.7 Future aspects of chitosan-based material for wound management

752

23.8 Conclusion

753

References

753

23.1 Introduction A wound is a breach of the epidermis of the skin that can be caused by major or minor injuries, surgery, or burns, etc., and these are very susceptible to infection and sepsis. In order to avoid such danger, proper wound management is essential, which may be achieved by using dressing materials. Dressing materials are used to cover the site of wound to protect it from contamination and infection that may impede healing. However, conventional dressing materials are usually dry, allow evaporation of moisture, and tend to become more adherent to wounds [1]. As a result they are difficult and painful to remove and these dressings do not support tissue regeneration. To overcome these problems regarding traditional dressing materials, biopolymer-based advanced wound dressing materials are investigated for their inherent properties like biocompatibility, biodegradability, hemostatic activity, ability to maintain a moist environment around the wound, and the ability to support tissue regeneration and differentiation resulting in accelerated wound healing [2]. Wound healing is a complex and dynamic regenerative process that progresses through a series of

Handbook of Chitin and Chitosan

23.2 Wound healing and its different stages

723

interreliant and corresponding stages in which a variety of cellular and matrix components act together to regenerate the integrity of damaged tissue and replacement of lost tissue. Different researches are currently being conducted to discover ways for humans to heal via regeneration and the use of a variety of dressing materials to facilitate proper wound management. Efforts are focused on the use of biologically derived materials such as chitosan, its derivatives, and biocomposites, which are capable of accelerating the healing processes at the molecular, cellular, and systemic level. Chitosan, a natural polymer, has received great attention for medical and pharmaceutical applications due to its beneficial intrinsic properties [3]. It has a high potential for wound healing application because it stimulates hemostasis, absorbs exudates, and accelerates tissue regeneration [1]. Moreover, chitosan’s monomeric unit, N-acetylglucosamine, occurs in hyaluronic acid, an extracellular macromolecule that is important in wound repair. However, pure chitosan-based dressing materials suffer from poor tensile strength and elasticity due to their brittleness. As a result, chitosan is often modified to its derivatives and biocomposites to achieve dressing materials with improved strength and elasticity, as well as vapor penetration, water uptake, oxygen penetration, and protection against microbial penetration [4]. Biocomposites obtained from chitosan and other natural and synthetic polymers showed better attachment and growth of epithelial cells than pure chitosan [5]. Chitosan and its derivatives and biocomposites have been found to be promising for wound management.

23.2 Wound healing and its different stages Wound healing naturally proceeds through some physiological stages, usually showing signs of recovery within 4 weeks. Wounds that heal by maintaining a normal rate of recovery are called acute wounds. A natural wound healing process can be disordered and delayed by various factors, such as abnormal growth of inflammatory mediators, local infection, development of hypoxia, protective bacterial layer formation, and poor nutrition. This is then called a chronic wound and it is quite often observed in many types of ulcers.

23.2.1 Wound healing models There are differences in the various wound healing models described by many scientists many times. However, they can be broadly classified into primary, secondary, and tertiary intention wound healing [6,7].

Handbook of Chitin and Chitosan

724

23. Chitosan and chitosan-based biomaterials for wound management

23.2.1.1 Superficial wound healing Ulcerations in the superficial skin cause damage to the soft tissues and they can be healed over time via an inflammatory repair process. 23.2.1.2 Primary intention wound healing Connective tissue deposition and reepithelialization, in which cells grow in from the margins of the wounds. Sometimes, primary intention healing may be delayed by keeping the wound open to promote drainage and reduce bacterial burden. A small surgical incision to close the edges neatly may be required for this case. This type of wound usually heals within 4
14 days. 23.2.1.3 Secondary intention wound healing It involves the loss of some degree of tissues and may cause the loss of partial or full thickness of skin. Partial thickness wounds pass through the epidermis layer of the skin, whereas full-thickness wounds extend up to the epidermis and dermis of the skin and even subcutaneous tissues and muscles. In this situation, wounds first fill with granulation tissues, a scar forms, and then reepithelialization starts from the edge of the wound. It takes a longer time to heal than the primary intention healing. 23.2.1.4 Tertiary intention wound healing If a wound is severe and it releases exudate, it is then delayed for closure targeting to clear off all inflammatory exudates and to observe the underlying structures. This causes more scarring and takes a longer time to heal.

23.2.2 Wound healing phases There are four different but overlapping phases in wound healing process. Phase Phase Phase Phase

1: 2: 3: 4:

Hemostasis phase Defensive/inflammatory phase Proliferative phase Remodeling/maturation phase

23.2.2.1 Phase 1: hemostasis phase Hemostasis is the first phase of wound healing and starts immediately after injury. Any injury to the skin which extends to the dermis results in bleeding and immediate vasoconstriction. On exposure to air, blood initiates clotting and the process accelerates by platelet aggregation and thromboplastin formation within 5
10 min. A fibrin mesh is

Handbook of Chitin and Chitosan

23.2 Wound healing and its different stages

725

finally formed which covers the wound area and after a few hours it dries out to become a scab. At this stage, wounds usually produce a large amount of serous fluid, which helps to cleanse the wound of surface contaminants [8]. 23.2.2.2 Phase 2: defensive/inflammatory phase The second phase of wound healing is vasodilation. The vascular response in the hemostasis process stimulates the release of inflammatory mediators, such as prostaglandins and histamine from mast cells. The mediators cause swelling of the injured area due to the accumulation of serous fluid and increase capillary permeability for blood flow, oxygen, and nutrients. Some symptoms of localized heat, erythema, discomfort, and functional disturbance are observed in this phase of wound healing. The active vasodilation process takes about 10
30 min. Phagocytosis is another important part in this stage. The exudate production is natural at this time and exudate contains various proteins, growth factors, nutrients, and enzymes, which help the healing. Neutrophils (white blood cells) are the first phagocytic cells that prevent the wound area from microbial action by releasing free O2 radicals and lysosomal enzymes, such as proteases, collagenases, and elastases. After 2
3 days, polymorphonuclear leukocytes and mononuclear leukocytes, which mature to macrophages and later to lymphocytes, further continue the antimicrobial action and clear old neutrophils debris from the wound area. If the cellular debris accumulates on the wound surface, a creamy yellow deposition is observed, which is called slough. In the worst cases, a fibrous tissue generation may cover the wound base underneath the slough. Then new tissue formation in the wound area will be hampered and it may cause chronic wounds. Macrophages are then necessary to stimulate the next healing stage, the proliferative phase. 23.2.2.3 Phase 3: proliferative phase After 1 week of wounding, macrophages produce transforming growth factor (TGF) and tumor necrosing factor (TNF), which facilitate the formation of extracellular matrix (ECM) and collagen for the formation and proliferation of granulation tissues [9,10]. The healthy new granulation tissues are pinky red in color and if it darkens in color that may signal that the wound is ischemic or infected. The tissues later form a scaffold-like porous and channel structure to produce connective tissues for new blood vessels from endothelial cells of damaged vessels. This is called angiogenesis. New blood vessels supply more blood to the tissues for its healthy growth. Thus produced fibroblasts start to contract the wound from the edge. During this time, new collagen fibers are

Handbook of Chitin and Chitosan

726

23. Chitosan and chitosan-based biomaterials for wound management

continuously produced, deposited, and cross-linked to get back the mechanical strength of the skin. Finally, reepithelialization occurs when cells migrate in the moist wound surface toward the cut of the wound until they form a continuous layer. Necrotic tissues and scab formation can hinder the cell proliferation process in this stage by forming a mechanical barrier underneath cells in the wound. New epithelial cells are translucent whitish-pink in color and restore the epidermis. 23.2.2.4 Phase 4: remodeling/maturation phase After approximately 3 weeks, the remodeling stage of the wound starts and continues for months or even 2
3 years [11]. At this stage degeneration and synthesis of collagen goes on to form scar tissues and mature collagen fibers are oriented properly to increase the tensile strength of the wound. However, the number of fibroblasts decreases at this stage. The scar tissue can return a maximum 80% of tensile strength of the previous skin. In mature scar tissues blood supply decreases, this is why the swelling of wound area reduces and the wound area looks smoother and flatter. The formation of keloid and hypertrophic (raised) scars are abnormalities associated with this stage of healing. Hypertrophic scarring occurs directly after initial repair and it develop in areas of thick corium and is confined to the incision line, while keloid scarring may occur sometime after healing and continue to grow and spread, invading the surrounding healthy tissue. Keloid is mostly observed in African and Asian populations. It forms a well-defined edge in wounds and it forms due to over proliferation of collagen fibers in the subcutaneous tissue.

23.2.3 Factors for delayed wound healing There are basically three types of factors that can impede wound healing [12]. They are intrinsic factors, extrinsic factors, and iatrogenic factors. Intrinsic factors are pathological factors such as age, chronic disease and immunosuppression, perfusion and oxygenation, neurologically impaired skin, etc. Extrinsic factors are environment-related factors. They can be medications, nutrition, irradiation/chemo, stress, bioburden, etc. Iatrogenic factors are improper management-related problems. These include local stress or tears on wound, inappropriate dressing, misuse of antiseptics, etc. However, a healthy nutritious diet, proper intake of vitamin A, vitamin C, zinc, and iron, adequate exercise, control of diabetes, etc. can help the wound healing process. Some complications may arise in wound healing and may delay the healing or require special treatment. Seroma: When the wound is filled with serous fluid, lymph, or blood and causes severe fluctuation, swelling, redness, tenderness, etc., it is

Handbook of Chitin and Chitosan

23.3 Properties of chitosan advantageous for wound management

727

called seroma. It requires spontaneous absorption of fluid, sterile puncture and compression, and opening up of a suction drain by surgical exploration Hematoma: If bleeding continues from the wound with short drainage time and with anticoagulant, then the risk of infection increases in wounds and it causes local swelling, fluctuation, pain, redness, etc. Its treatment is the same as that applied for seroma. Wound disruption: Partial or complete disruption can be happened in wounds in some cases due to surgical error, increased intraabdominal pressure, hypoproteinemia, etc. Its treatment is provided by using U-shaped sutures. Mixed wound infection: This may cause serious problems due to necrotic tissues and putrid and anaerobic infection, which ultimately cause gangrene. As for its treatment, aggressive surgical debridement with effective antibiotic medications is necessary. Atrophic scar: Atrophic scar is a condition of skin when normal tissue does not regenerate from the wound. It can be of many types, such as hypertrophic scar, keloid formation, necrosis, inflammatory infiltration, abscesses, insufficient collagen production, staphylococcus infection, acne, pox, etc. Excision is the ultimate treatment for such scars.

23.3 Properties of chitosan advantageous for wound management 23.3.1 Biocompatibility and biodegradability For many reasons, natural material is preferred over synthetic product for biomedical application. Natural products are more biocompatible than the synthetic ones. Chitosan is metabolized by certain human enzymes, such as lysozyme, which makes it biodegradable. [13]. Importantly, a similar kind of structure and composition are observed in chitosan compared to glycosaminoglycans (GAGs) and thus exhibits a minimal immune response when implanted in the human body. Therefore chitosan is considered as a nontoxic, biodegradable, and biocompatible macromolecule that has gained interest for the biomedical field of applications [14]. There are many evidences which proved that chitosan is more biocompatible than chitin in terms of in vitro application. Moreover, in chitosan, the number of positive charges is greater than in chitin, so the interaction between cells and chitosan increases, which tends to improve the overall biocompatibility [15]. Considering the biodegradation phenomena, chitosan releases amino sugars which can be incorporated into GAGs and glycoproteins metabolic pathways or otherwise excreted [16]. The biodegradation of chitosan also

Handbook of Chitin and Chitosan

728

23. Chitosan and chitosan-based biomaterials for wound management

leads to the formation of oligosaccharides of variable length, which are nontoxic in nature. These oligosaccharides can be introduced in metabolic pathways or can be further excreted from the system. The rate of degradation of chitosan molecule is mainly controlled not only by its degree of deacetylation but also by the distribution of N-acetyl D-glucosamine residues and the molecular weight [17].

23.3.2 Hemostatic and analgesic properties Although the accurate mechanism of the hemostatic action of chitosan remains undiscovered, a scientist suggested three possible ways to control the bleeding by analyzing data. The mechanisms are: (1) sorption of plasma, (2) erythrocytes coagulation, and (3) platelet adhesion, aggregation, and activation (Fig. 23.1) [18]. The first one is plasma sorption which is a key factor in chitosan application as a hemostatic, capable of absorbing from 50% to 300% liquid thanits primary weight. This absorption leads to the concentration of erythrocytes and platelets in the injured site. The antimicrobial property, sorption rate etc. also depends on molecular weight and degree of deacetylation of the chitosan molecule. Moreover, lyophilization or freeze-gelation increases sorption. However, sorption is not the principal factor that can stop bleeding from the injured site. The second mechanism explained is the erythrocytes coagulation which is directly associated with hemostatic properties. In presence of the chitosan molecule, cross-linking of the erythrocytes increases which results in the elevated agglutination of erythrocytes. They were Chitosan

Plasma absorption

hemoconcentration

Erythrocyte coagulation

Platelet adhesion and aggregation Blood clotting Blood clotting

FIGURE 23.1 Mechanisms of hemostatic action of chitosan.

Handbook of Chitin and Chitosan

23.3 Properties of chitosan advantageous for wound management

729

compiled together by chitosan polymer chains and polymerized to generate a lattice that captured cells, fabricating an artificial clot. Contact of chitosan with blood leads to the alteration of the erythrocytes’ morphology while losing the typical biconcave morphology and they are observed to have an unusual affinity towards one another. Lima et al. showed that chitosan solution causes hemolytic activity in human erythrocytes at low pH, whereas pH neutralization of these solutions induced a higher hemagglutination index [19]. Chitosan can directly attract any plasma protein firstly or is capable of inducing erythrocytes adhesion without introducing any dimensional network structure. When chitosan is in solution, it can absorb fibrinogen and other plasma protein, thus boosting erythrocytes adhesion and coagulation. Fan et al. suggested that the main mechanism of action of chitosan in hemagglutination is that at its low
molecular weight, chitosan can directly bind with erythrocytes’ walls due to its cationic nature [20]. But chitosan’s effect on erythrocytes may only explain part of its hemostatic function because of arresting the loss of the formulated clot. The third mechanism illustrated in the literature is related to platelet adhesion, aggregation, and activation. It was stated that chitosan films can incite platelet adhesion, aggregation, and the activation of intrinsic blood coagulation. Shen et al. prove that the concentration of the platelets in the plasma is connected to the aggregation [21]. While comparing between chitin and chitosan in inducing the hemostatic property, some relevant data concluded that chitosan acted more efficiently than chitin for platelets aggregation. Scanning electron microscope analysis showed that platelets were more strongly attached on the surface of chitin and chitosan particles with an elongated process. It was found that platelets were attached and bound to each other by developing an aggregated mass in irregular patterns. Also, chitosan can bring forth intracellular signal reactions, activating glycoprotein IIb/IIIa and discharging thromboxane A2/ADP as well as elevating platelet spreading and thereby restoring the stability of adhesion [18]. The increased level of integrin α2β3 was found to be expressed by platelets that adhered to chitosan [22]. To summarize this hemostatic effect by the chitosan molecule it can be concluded that the hemostatic mechanism of chitosan-based materials is very promising and the primary plasma sorption leads to blood cells concentration at the injured site. Moreover, coagulation of erythrocytes and platelets adhesion and aggregation cause fast clot formation without systemic hemostasis activation. Another significant property of chitosan is the analgesic effect. The polycationic nature of chitosan is mainly responsible for such kind of effect. Indeed, the amino groups of the D-glucosamine residues can protonate in the presence of proton ions that are released in the inflammatory area, resulting in an analgesic effect [23].

Handbook of Chitin and Chitosan

730

23. Chitosan and chitosan-based biomaterials for wound management

23.3.3 Antimicrobial and antiinflammatory nature Chitosan possesses a broader spectrum of activity, satisfactory antimicrobial activity and a higher killing rate with lower toxicity toward mammalian cells [24]. However, the actual mechanism of bacterial inhibition is not fully discovered by the researchers. The most probable hypothesis is a change in cell permeability. This change happens due to interaction between positively charged polysaccharide (for chitosan pH , 6) and the negatively charged membrane of anionic components such as N-acetylmuramic acid, sialic acid, and neuraminic acid, present on the cell surface. Low molecular weight chitosan shows the maximum probability to penetrate into the cell wall of bacteria and attach with DNA and thus inhibit the synthesis of mRNA and DNA transcription [25]. Whereas high molecular weight chitosan is found to alter the cell permeability while interacting with the cell wall surface [26] or form an impermeable layer around the cell. This is how the chitosan molecule blocks the transport of essential solutes into the cell [27]. Chung et al. reported that the hydrophilicity and negative charge on the cell wall of the bacteria and the pattern of chitosan adsorption dominates the overall antimicrobial property [28]. The hydrophilicity and cell wall negativity were higher in gram-negative bacteria compared to gram-positive. Moreover, the charge distribution is different in two types of bacteria and some of the researchers suggested that chitosan is more effective for gram-negative bacteria than gram-positive bacteria [29]. In addition, Zheng et al. stated the effect of the molecular weight of chitosan on gram-negative and gram-positive bacteria. For gram-positive Staphylococcus aureus, the antimicrobial activity increases with the increase inmolecular weight of chitosan. On the other hand, for gram-negative E. coli, the antibacterial activity increased with a decrease in molecular weight [30]. Chitosan possesses some immunological functions including inhibition of proinflammatory cytokines, promotion of tissue granulation through fibroblast recruitment [31], and production of type III collagen [32]. For three decades the immune-modulating properties (antiinflammatory effect) of chitosan have been studied with equal importance, whereas the intracellular chitosan response pathways have only been recently started to elaborate. Currently, the best-described intracellular signaling pathways involve two methods, one is cGAS-STING, and the second one is NLRP3 [33]. Again, these two pathways were observed to be deployed following macrophage exposure to chitosans with 80% to 98% DD and 3 to 400 kDa of MW. The cGAS-STING pathway triggers a type 1 IFN response and specific downstream expression of the chemokine CXCL10/IP-10. Moreover, type 1 IFN responses induce the release

Handbook of Chitin and Chitosan

23.4 Different forms of chitosan-based biomaterials in wound management

731

of a well-known and therapeutically relevant antiinflammatory factor, IL-1ra, signaling through STAT1/STAT2 activation. By contrast, activation of NLRP3 leads to inflammation activation and release of proinflammatory factors IL-1β and PGE2. These two types of cytokine responses have been reported in both primary and cell line derived human and mouse macrophage models. Moreover in vivo analysis of these materials in living animal provides great confidence to the researchers. It is noted that these responses to chitosan are reproducible and conserved across different species when functioning as antiinflammatory agent.

23.4 Different forms of chitosan-based biomaterials in wound management 23.4.1 Hydrogels Chitosan has always been a popular choice by the researchers as a wound-healing promoting agent. As a cationic polysaccharide, chitosan is nontoxic, biodegradable, and biocompatible, widely used in several biomedical fields including drug delivery, wound dressing, and tissue engineering. Chitosan has one primary amino and two free hydroxyl groups for each glucose unit. The cationic amino groups react with a number of multivalent anions to form hydrogels. Various forms of chitosan are available which could be applied in fabricating different forms of hydrogels, beads, and composites with or without modification. It is found that modifications provide additional scope to improve the property of chitosan for the desired field of application. Based on the size and type, modified chitosan hydrogels have been proven to be a potential carrier for the delivery of different drug molecules in wound management [34]. The main biochemical aspects of chitosan in wound healing are fibroblast activation, cytokine production, giant cell migration, and stimulation of type IV collagen synthesis [35]. One of the most common uses of the chitosan-based hydrogel is in the field of biomedical application. Chitosan can be used alone or sometimes it is combined with other suitable materials for better performance and also helps to mitigate the drawbacks while using the chitosan alone. Mohsen et al. developed a cross-linked polyvinyl alcohol (PVA)/chitosan blend hydrogel for the release of an antibiotic (sparfloxacin). This method is described as an eco-friendly procedure where no chemical cross-linking agents have been introduced, which made the hydrogels nontoxic and safe to use. Properties like swelling percent of the freeze-thawed hydrogels are dependent on the various factors like pH, chitosan percentage, time, and number of freezing cycles. The antimicrobial activity

Handbook of Chitin and Chitosan

732

23. Chitosan and chitosan-based biomaterials for wound management

also showed a positive effect on both gram-positive and gram-negative bacteria. It is already mentioned that the fabricated hydrogel is applied for sparfloxacin delivery, which actually gets activated in the acidic medium and the drug delivery is dependent on both pH and temperature [36]. The hydrogel can be fabricated in the form of films which were used as a potential wound dressing material, as reported by Zhang et al. For the hydrogel film formation PVA was modified by introducing carboxyl group which was then cross-linked with chitosan. In this case, as a chemical cross-linking agent, succinate acid was preferred to obtain carboxyl-modified poly(vinyl alcohol) (PVA
COOH). The cross-linking was developed by the formation of amide bonds between PVA
COOH and chitosan (Fig. 23.2). This cross-linked hydrogel is capable of maintaining the appropriate moisture over the wound bed. Moreover, the high swelling ratio and biocompatibility (showing negative cytotoxicity and hemolytic potential result) makes it very suitable for further applications in drug delivery. In a continuation, gentamicin sulfate was incorporated into this hydrogel film, which showed a sustained drug release profile and could effectively suppress bacterial proliferation and protect the wound from infection. In terms of in vitro cell viability properties, mouse fibroblast cell line (NIH3T3) and rabbit blood were used

NH2

PVA–COOH

Chitosan

Cross-linking

H N

C O

FIGURE 23.2 The formation of cross-linked PVA
COOH/CS hydrogels resulted from the amide linkage between the chitosan and PVA
COOH. Source: Reprinted with permission from D. Zhang, et al., Carboxyl-modified poly (vinyl alcohol)-cross-linked chitosan hydrogel films for potential wound dressing. Carbohydr. Polym. 125 (2015) 189
199. Copyright r 2015 Elsevier Ltd.

Handbook of Chitin and Chitosan

23.4 Different forms of chitosan-based biomaterials in wound management

733

to determine the effects of cross-linked hydrogel films on the epidermis and hemolytic potential, respectively [37]. The above studies showed that chitosan can be a promising agent for wound management when combining with other polymeric materials with or without using chemical cross-linkers. However, it is also reported that chitosan when used alone can initiate many problems, for example, easy deformation through external stress and lower absorption profile in the physiological fluid. These obstacles really hamper the use of chitosan in the biomedical sector, especially for wound dressing purposes [38]. Besides freeze-thawing and chemical cross-linking methods, hydrogels can be fabricated by using chitosan, PVA, and gelatin by introducing gamma irradiation into the system. This technique was described by Fan et al., where the researchers found the best results when applying 40 kGy dose gamma irradiation in all five different compositions (20
60 kGy doses of radiation were analyzed) [35]. These three component hydrogels were characterized by their physical properties and blood clotting activity. The freeze-dried hydrogels showed a uniform porous structure (Fig. 23.3). As a part of the physical and mechanical characterization, structure, tensile strength, blood clotting activity, coagulation effect, hemostatic effect, water evaporation, pH effect, swelling nature, etc. were evaluated. The highest tensile strength was found at 2.2 MPa. All hydrogels have been shown to have an optimal coagulation effect as well as the hemostatic effect of hydrogel was also satisfactory. So, this kind of material is very feasible for wound dressing purposes. As it closes on the skin surface, it should not be easily torn when stretched out. Chitosan is considered as one of the best hemostatic materials found, whereas gelatin and PVA are well known for their mechanical support. In this case, these three components were mixed in order to optimize the mechanical and hemostatic properties. Chitosan has been demonstrated to be a natural polysaccharide with a nontoxic effect both in animal and

FIGURE 23.3 SEM images of chitosan/gelatin/PVA hydrogel. Source: Reprinted with permission from L. Fan, et al., Preparation and characterization of chitosan/gelatin/PVA hydrogel for wound dressings, Carbohydr. Polym. 146 (2016) 427
434. Copyright r 2016 Elsevier Ltd.

Handbook of Chitin and Chitosan

734

23. Chitosan and chitosan-based biomaterials for wound management

human models [39]. Conventional wound dressing has recorded with various clinical limitations. It adheres easily to the wound and makes it difficult to change medicine, the little effect in the face of large diffuse hemorrhage, hardly plays a role for infection and festering wound. In this case, the fabricated hydrogel adhered to the surface of the wound to block broken blood vessels while stimulating the platelet release coagulation factor, which can promote and accelerate the blood coagulation [35]. Since 19th century, ionic Ag and Ag-based compounds have been widely accepted as bacterial growth inhibitors due to their notable biocidal properties against various types of bacteria, including E. coli and S. aureus [40]. A hydrogel nanocomposite was fabricated using a synthetic polymer PVA and a natural polymer, where Ag1 functionalized nanoparticles were incorporated in the nanocomposite. The hydrogel was observed to resist bacterial activity and under some defined conditions it was capable of growing healthy cells [41]. The addition of Ag1 functionalized nanoparticles to the hydrogel nanocomposite matrix has been found to improve the structural rigidity without compromising the rate of fluid uptake (i.e., swellability). Moreover, it provides a site for controlled release (i.e., no static ion leaching) of Ag1 required for bacterial disinfection. The concentration of the nanoparticles must be greater than 5 mM to achieve optimal control of biofilm formation. The fabricated hydrogel nanocomposites showed an effective bacterial inhibition when exposed to cultures of E. coli, S. aureus, and methicillin-resistant Staphylococcus aureus (MRSA). Human dermal fibroblast test analyses revealed that when PVA concentration rose in the system, there was no considerable cell growth. On the contrary, when the ratio of biomimetic chitosan and PVA is optimized to decrease the overall amount of synthetic polymer (PVA), healthy cell growth was observed. This result directly speaks to a key point that reveals the importance of the correct balance or the removal of synthetic polymer from the system. The use of chitosan alone has been shown to limit the mechanical strength of the hydrogel during swelling [42]. It was also reported that polymer blending can be utilized to improve the overall physicochemical properties of the final product [43]. In support of this statement, Yang et al reported that chitosan-based hydrogels cross-linked with PVA showed increased mechanical strength compared to pristine chitosan [44]. However, there is a difficulty with Ag1, i.e., it is difficult to uniformly distribute it throughout the hydrogel matrix. As a part of the solution to this problem, Ag-coated semiconductor particles offer an alternative incorporation approach. When solid support, such as titanium dioxide (TiO2), functionalized with Ag is placed in a suspension, the Ag1 released from the surface is capable of killing bacteria [45]. This allows for the gradual release of Ag1, which has been shown to increase the longevity of the substrate. The application of this theory to

Handbook of Chitin and Chitosan

23.4 Different forms of chitosan-based biomaterials in wound management

735

hydrogel technology can allow for the development of burn wound management materials with increased rigidity and sustainability [41].

23.4.2 Membranes It is a well-established fact that chitosan is polycationic in nature. Chitosan solution when combined with a polyanionic aqueous solution, such as gelatin, sodium alginate, poly acrylic acid, etc., gives polyelectrolyte complexes (PECs) which have application as coating membranes and controlled release delivery systems. However, inadequate information confined its application to being a wound dressing. Feng et al. developed a membrane made of chitosan and alginate with silver sulfadiazine as a model drug incorporated in different concentrations and as a result, different membrane compositions were obtained. This membrane is considered to be a potential membrane for the controlled release of the respective drug [46]. The group of alginate and chitosan is also capable of forming a bilayer composite membrane reported by Han et al. In this case, as a drug ciprofloxacin hydrochloride is used to check the sustained release behavior of the drug from the prepared membrane. The membrane was studied based on in vitro antimicrobial activity, as well as drug permeation study, morphology, cytotoxicity, primary skin irritation, and in vivo pharmacodynamics were also investigated. The obtained results showed that the membrane could inhibit the growth of microorganisms for longer than 7 days and therefore is suitable for wound dressing [47]. In another study conducted by Gu et al. chitosan wascombined with silk fibroin (CS/SF) to blend in a membranes cross-linked with alginate dialdehyde (ADA) used as wound dressings. The prepared membrane could meet the stability, water absorption, and water vapor permeability requirements which are needed for wound dressing. Moreover, this membrane is satisfactorily biocompatible and found to promote cell attachment and proliferation. Therefore this material is considered as a promising candidate for wound dressing [48]. Morgado et al. reported that for membrane formation purposes, PVA is used with chitosan and in this case, an asymmetrical membrane is fabricated for wound dressing purposes. The asymmetrical membrane is an ideal wound dressing material due to its skin regeneration ability. However, this membrane is capable of uptaking water and therefore acts like a hydrogel which ultimately provides the moist environment needed for wound healing. The pharmacokinetic release profile of a small drug (ibuprofen) from the swollen membrane in physiological conditions was studied. In vitro studies revealed that the dressings had excellent biocompatibility and biodegradation properties that were adequate for skin wound healing [49].

Handbook of Chitin and Chitosan

736

23. Chitosan and chitosan-based biomaterials for wound management

23.4.3 Sponges A sponge can be fabricated from chitosan when interacting with gelatin. In a study conducted by Han et al. a gelatin
chitosan sponge scaffold was fabricated as a skin tissue engineering material for wound dressing application. The scaffold possessed a porous structure with a high water capacity, high water retention, satisfactory degradability, and biocompatibility that was suitable for cell adhesion and proliferation. Moreover an in vivo study also suggested that this sponge could offer effective support and attachment to cells for skin wound healing. The unique design (Fig. 23.4) of the sponge (3 M tape) prevents the bacterial invasion, controls water vapor permeation, and retains a favorable moist environment at the wound interface. In this study, a model drug ciprofloxacin hydrochloride (CIP) was selected and the antibacterial property of sponge scaffold was preliminary studied in vitro to evaluate the potential for wound healing [50]. In another study conducted by Ding et al., a spongy bilayer dressing material is fabricated by using chitosan as the major component. In the upper layer of the chemically cross-linked bilayer composite, crosslinked chitosan
Ag nanoparticles with genipin was used which effectively suppressed bacterial infection of the wound. In the lower layer, a hybrid was fabricated by cross-linking with chitosan, genipin, and partially oxidized Bletilla striata polysaccharide, which showed potent cell proliferation activities. The bilayer material showed better antibacterial and faster tissue rebuilding effects than the conventional gauze, and therefore could Prevention of bacterial invasion

Controlled water eveporation

3M Tape

Sponge

Drainage of wound exudates

FIGURE 23.4 Design of the gelatin
chitosan sponge’s scaffold. Source: Reprinted with permission from F. Han, et al., Preparation, characteristics and assessment of a novel gelatin
chitosan sponge scaffold as skin tissue engineering material, Int. J. Pharm. 476 (1
2) (2014) 124
133. Copyright r 2014 Elsevier B.V.

Handbook of Chitin and Chitosan

23.4 Different forms of chitosan-based biomaterials in wound management

737

potentially serve as an ideal wound dressing for wound protection and healing [51]. Other than using the traditional source of chitosan Sathiyaseelan et al. reported the use of fungal chitosan to fabricate a composite sponge along with Aloe vera extract and the plant Cuscuta reflexa mediated biosynthesized silver nanoparticles [52]. The sponge was fabricated by the freeze-drying method and showed potential antibacterial property in the wound dressing application. The function of Aloe vera extract in this sponge is to decrease the toxic effect of Ag nanoparticles while the antibacterial property remains unaffected. In a different study, chitosan and silver sulfadiazine composite sponges were prepared by incorporating silver sulfadiazine particles into the chitosan matrix in order to develop a spongy biocomposite for wound healing. The spongy material showed its antibacterial property against E. coli, C. albicans, S. aureus, and B. subtilis, and the results from the cell viability test indicates that this material has no significant cytotoxicity. Moreover, this fabricated sponge is biocompatible in nature and therefore can be suggested as a promising antibacterial wound dressing material [53].

23.4.4 Scaffolds For preparing scaffold material chitosan can be used alone and it can be combined with several natural and synthetic polymers. Moreover, oil immobilized, extract immobilized, and drug-loaded chitosan-based scaffold was reported by several researchers. In modern times, diabetic wounds are a common complication in patients with diabetes and often lead to amputation. It takes a long time to heal the wound because of many factors, especially the lack of tissue regeneration which leads to impaired wound healing in the diabetic patient. Focusing on this rising issue, Karri et al. described a procedure to develop a nanohybrid material by loading curcumin with chitosan nanoparticles and then incorporating it into collagen
alginate scaffolds. This scaffold helps in reducing the persistent inflammation and boosting the healing as well as tissue regeneration in the wounds of a diabetic patient. In this study, alginate was blended with collagen and then cross-linked to improve its physical stability and also to provide a moist wound environment. The investigation of the scaffold showed positive results regarding biocompatibility, antiinflammation, cell adhesion, and proliferation, which are crucial for tissue engineering in impaired wounds of diabetic patients [54].

23.4.5 Gels A burn is a type of injury to the skin caused by electricity, chemicals, heat, or radiation. Second-degree burns involve the superficial

Handbook of Chitin and Chitosan

738

23. Chitosan and chitosan-based biomaterials for wound management

(papillary) dermis and may also involve the deep (reticular) dermis layer, leading to the generation of blisters, severe pain, and redness. Degim et al. aimed to develop gels to treat this kind of second-degree burn by using chitosan. A gel material is formed by using chitosancontaining liposomes loaded with epidermal growth factor (EGF) and to evaluate their effects on the healing of second-degree burn wound. The effectiveness of this gel is examined by applying the gel on the back of rats where second-degree uniform deep burn wounds were created. The animals were divided into groups and treated with three different formulations, namely EGF
liposome, chitosan gel with EGF, and EGF
liposome
chitosan gel. The experiment was conducted for 14 days and the healing was evaluated immunohistochemically and histochemically. The results obtained showed an enhanced efficacy on burned skin with the developed liposome formulations containing EGF in chitosan gel [55]. In another study, a sterile and biocompatible gel was fabricated from modified chitosan for wound healing application. The chitosan powder was treated in an autoclave, which modified the molecular weight of the chitosan. The thermally modified chitosan exhibited some effects, for example, increased the proliferation and migration of human foreskin fetal fibroblasts at 24 hours and accelerated wound healing (measured as an area of the lesion) at 3 and 10 days in an in vivo model of pressure ulcers. The developed gel accelerated the healing process in vivo, by decreasing the inflammatory markers and raising the reepithelialization parameters. Above all, this novel chitosan gel has considerable potential in wound healing and other therapeutic applications [56].

23.4.6 Fibers Biocompatible chitosan/sericin composite nanofibers were fabricated by an electrospinning technique. The prepared fibers have good morphology with a diameter between 240 nm and 380 nm. Moreover, nanofibers were observed to have satisfactory bactericidal activity against both Gram-positive and Gram-negative bacteria and they could activate cell proliferation, and therefore are promising for wound dressing applications. The cell viability revealed the nontoxicities and good biocompatibilities of composite nanofibers toward cells [57]. In a different study conducted by Naseri et al. a randomly oriented fiber mat was developed from chitosan, polyethylene oxide, and chitin nanocrystals. Electrospinning is the technique applied for fabricating chitosan/polyethylene oxide fiber mats which are further reinforced with chitin nanocrystals and finally cross-linked via genipin for wound dressing purposes. The prepared nanofibrous mats showed suitable cell

Handbook of Chitin and Chitosan

23.5 Chitosan-based biocomposites for wound healing

739

attachment, proliferation, and potential wound dressing material. The mat is porous in nature with a high surface area and the addition of chitin enhanced the mechanical properties and improved the moisture stability of the mats and facilitated water-mediated cross-linking processes. The mats were also compatible toward adipose-derived stem cells after 7 days and therefore proved to be very beneficial for wound dressing applications [58].

23.5 Chitosan-based biocomposites for wound healing In order to improve some properties of chitosan-based wound dressing materials like tensile strength, elasticity etc., chitosan is often mixed with other biopolymers to produce biocomposites which are then used to prepare wound dressing materials. Polysaccharides like starch, dextran, alginate, hyaluronan etc., protein-origin polymers like gelatin, silk fibroin, collagen, fibrin etc., synthetic polymers like polyvinyl pyrrolidone, polylactic acid (PLA), polyvinyl alcohol (PVA), polyethylene glycol diacrylate, etc. have been combined with chitosan to fabricate biocomposites with properties suitable for wound dressing materials. Many of these biocomposites have been used as a carrier of drugs like ciprofloxacin and other nanoparticles like silver, zinc oxide, titanium oxide which promote the wound healing process.

23.5.1 Chitosan
starch Starch, a biopolymer composed of anhydroglucose units—amylase and amylopectin—has been utilized for various applications including wound dressing, tissue engineering scaffolds, drug delivery systems, bone replacement implants, and substrate for cell seeding, because of its biodegradability, availability, biocompatibility, and economic feasibility [59
61]. Wittaya-areekul et al. developed a composite film from chitosan, cornstarch, and dextran for wound healing applications [4]. In vitro results showed that the incorporation of cornstarch and dextran into chitosan has improved composites’ mechanical properties like oxygen penetration, water uptake, and vapor penetration. Moreover, composite demonstrated better resistance against microbial infiltration, which makes this composite a potential candidate for wound dressing. In another study, Arockianathan et al. prepared temporary biological wound dressings by a solvent casting method from chitosan (Ch) and sago starch (SG) impregnated with silver nanoparticles (AgNP) (Ch-SGAgNP) and additionally with antibiotic gentamicin (G) (Ch-SG-AgNP-G).

Handbook of Chitin and Chitosan

740

23. Chitosan and chitosan-based biomaterials for wound management

Their gross observation of histopathological examinations, biochemical studies, macroscopic observations, and planimetric studies on rats revealed that Ch-SG-AgNP-G healed wounds significantly in the shortest time (16 days), while the untreated control wounds took the longest time (24 days) [62]. Baghaie et al. incorporated zinc oxide (nZnO) nanoparticles in the composite of chitosan (Ch), PVA, and starch (St) by the freeze
thaw technique [63]. Histopathology and in vivo wound healing examinations recognized the Ch-St-PVA-nZnO composite membrane was a suitable wound dressing material during the early stage of wound healing as the composite showed a large spectrum of antibacterial activity. Moreover, the Ch-StPVA-nZnO hydrogel membrane displayed a tremendously low level of toxicity toward L929 fibroblast cells.

23.5.2 Chitosan
alginate Alginate is well known for wound management from the commercial point of view as well as ease of preparation in the desired form. Alginate-based dressings materials are mainly used for bleeding wounds as calcium alginate has been proven to be a natural hemostat in several investigations [64]. The gel-forming ability of alginate facilitates the removal of the alginate dressing materials without any suffering while changing bandages. It helps by providing a soggy environment that aids swift reepithelialization and granulation. In a controlled medical experiment it was found that only 10 days was adequate for wound healing for a significant number of patients, compared with patients of a paraffin gauze group. It was evident that in healing split skin graft donor sites of calcium alginate dressings offer a major improvement [65]. During the management of split-thickness skin graft donor sites, the collective utilization of calcium and sodium alginate reduced soreness and the problem of seroma formation. Moreover, a significant amount of leakage could be seen consistently when bioocclusive dressing materials were used solely [66]. Chen et al. fabricated a composite gel dressing of tetracycline hydrochloride (TH)
loaded gelatin microspheres (GMs) followed by their incorporation into oxidized alginate
carboximethyl chitosan (OAlg
CMCS) [67]. In vitro drug release showed that when the composite gel dressing was compared with pure hydrogel, the TH-loaded microsphere incorporated in OAlg
CMCS hydrogel dressing provided sustainable drug release. Furthermore, as this composite dressing material showed powerful bacterial growth inhibition effects against E. coli and S. aureus, its use against bacterial infection was a promising approach.

Handbook of Chitin and Chitosan

23.5 Chitosan-based biocomposites for wound healing

741

Meng et al. produced PEC membranes from chitosan and alginate via a solvent casting method and they incorporated silver sulfadiazine as a model drug to study various properties like drug release, water uptake, water vapor transmission, and physical properties [46]. These requisite properties and the ease of formability and biodegradability helped show this PEC to be a suitable material for wound dressing. Murakami et al. had an approach to prepare a hydrogel sheet by blending alginate, chitin/chitosan, and fucoidan (ACF) to create a moist environment for rapid wound healing [68]. Additionally they observed that ACF successfully interacted and protected the wound of a rat offering a decent moist healing atmosphere with wound exudates. During the research study, full-thickness skin scars were made on the back of the rats and mitomycin C solution (1 mg/mL in saline) was spread over the wound for 10 min in order to prepare healing-impaired wounds. After systematically rinsing out the mitomycin C, the ACF was employed easily all over the wounds with proper adherence, and after 7 days of application the ACF was peeled off from the wound. Encouraging consequences on wound enclosure, advancement in wound contraction, and reepithelialization for ACF treatment were monitored and compared with those treated with calcium alginate fiber (Kaltostat; Convatec Ltd., Tokyo, Japan) and those left untreated. Wang et al. prepared chitosan
alginate polyelectrolyte complex (PEC) membranes as potential wound dressing materials [69]. The membranes were evaluated as nontoxic toward mouse and human fibroblast cells. Where the control wound still showed the mark of an active inflammatory phase under scab on Day 21, the PEC enclosed the wound 14 days postoperatively and wound healing was comparable to Opsite (Smith & Nephew)-treated wound healing. Moreover, these wound dressings were found to be easy to formulate, batch-to-batch reproducible, easy to handle, cost effective, biodegradable, and stable upon storage. Some chitosan/alginate-based biomaterials investigated for wound healing applications are given in Table 23.1.

23.5.3 Chitosan
dextran Dextran is a group of polymers which is generally generated by specific bacteria. Therapeutically dextran is mainly used as expander and anticoagulant [78]. It has also found utilization in biological experimentation and in industry for various purposes. Many in vivo and in vitro experiments have verified its biocompatibility. For example, promotion of angiogenesis and skin regeneration were observed in mice’s burn wounds by a group of researchers when dextran hydrogels were used

Handbook of Chitin and Chitosan

742

23. Chitosan and chitosan-based biomaterials for wound management

TABLE 23.1 Some chitosan/alginate-based biomaterials investigated for wound healing applications. Sl. no

The composition of wound dressing material

Form

Special features

References

1.

Chitosan
alginate

Nanomembrane

Biocompatible and promotes bone marrow stem cell adhesion, proliferation, and differentiation

[70]

2.

Fibrin
chitosan
sodium alginate

Composite sheet

Good mechanical properties

[71]

3.

Chitosan
alginate

Sponges

Capable of loading and releasing ciprofloxacin

[72]

4.

Chitosan
alginate

Membrane

High water drainage ability, appropriate tensile strength, high stability in water, capable of preventing the permeation of bacteria

[73]

5.

Chitosan
alginate polyelectrolyte complex

Membrane

pH- and ionic strengthdependent water uptake, capable of loading and releasing silver sulfadiazine

[46]

6.

Chitosan
sodium alginate

Film

Improves epithelialization, blood vessels formation, collagenization, promotes rapid replacement of type III for type I collagen, favors the better arrangement of the newly formed collagen fibers in combination with laser therapy.

[74]

(Continued)

Handbook of Chitin and Chitosan

743

23.5 Chitosan-based biocomposites for wound healing

TABLE 23.1

(Continued)

Sl. no

The composition of wound dressing material

7.

Form

Special features

References

Heparin
functionalized chitosan
alginate

Scaffold

Capable of loading and controllable and long-term release of basic fibroblast growth factor

[75]

8.

Chitosan
alginate

Bilayer composite membrane

Capable of loading and sustained releasing ciprofloxacin, inhibits the growth of microorganisms for longer than 7 days

[47]

9.

Chitosan
alginate

Membrane

Modulates the inflammatory phase, stimulates fibroplasia and collagenesis

[76]

10.

Smad3 antisense oligonucleotidesimpregnated chitosan/ alginate

Polyelectrolyte complex

Reduces production of TGF-β1 via suppression of smad3 at cellular level

[77]

without any additives [79]. It is essential to insert intermolecular junctions via the use of cross-linkers as it is inherently water soluble and it is necessary to adapt the stability, biodegradability, and mechanical properties in wet condition [80]. Hoque et al. prepared bioadhesive injectable hydrogels from the derivatives of chitosan and dextran for the purpose of the wound healing process by sealing the wound leakage, ceasing the unwanted bleeding, and binding the tissues together [81]. Additionally these sealant hydrogels have been shown to be very effective against both Gram-negative and Gram-positive bacteria including drug resistant bacteria, for example, lactam-resistant Klebsiela pneumonia, vancomycin-resistant Enterococcus faecium (VRE), and MRSA. For the study of the rate of wound healing, a spherical wound (B18 mm diameter) on the dorsum of the Wistar rats was made by skin incision and removal. The adhesive sealant demonstrated outstanding wound healing performance

Handbook of Chitin and Chitosan

744

23. Chitosan and chitosan-based biomaterials for wound management

within a very short period of time (20 days). Moreover, it was observed that the mortality rate of the rats had been decreased five times due to the use of the hydrogels. Zhao et al. developed degradable hydrogels of an antibacterial nature from quartanized chitosan (QCS)
grafted polyaniline with oxidized dextran [82]. Hydrogels’ comprehensive antibacterial properties were evaluated using E. coli and S. aureus. It was found that the incorporation of polyaniline enhanced the antibacterial activity of QCS by a synergetic effect and significantly decreased the cytotoxicity of QCS. The conductivity of the hydrogel significantly invigorated proliferation of C2C12 myoblast cells, with an increase of polyaniline in the QCS. These inclusive studies as well as in vivo tests on Sprague Dawley rats helped the researchers to move toward an agreement that these electroactive antibacterial conductive degradable injectable hydrogels could be considered as a new class of bioactive scaffolds for tissue regeneration and wound restoration. To improve the wound healing process Ribeiro et al. evaluated the usability of a wound dressing made of dextran hydrogel impregnated with chitosan microparticles containing epidermal and vascular endothelial growth factors [83]. The applicability of hydrogel for the treatment of skin burns was evaluated through in vivo experiment on Wistar rats which suggested that the wound dressings promoted faster healing without any signs of local or systemic inflammatory response. This hydrogel wound material was recommended as a future wound dressing for the controlled delivery of bioactive regenerative medicine, as the hydrogel and its degradation by-products were biocompatible and can contribute to the restoration of skin architecture. Aziz et al. developed a chitosan
dextran-based (CD) hydrogel for utilization in the endoscopic sinus surgery [84]. The in vitro test of this hydrogel was carried out on a range of pathogenic microorganisms, namely, S. aureus, Streptococcus pyogenes, E. coli, and Clostridium perfringens. In consideration of pathological results the researchers hinted that the CD hydrogel could be mostly functional for postsurgical aid, particularly where there is a possibility of Gram-positive infections.

23.5.4 Chitosan
collagen Collagen is the chief structural protein in the extracellular space in a variety of connective tissues in the animal body. As a key constituent of connective tissue, it is the most abundant protein in mammals. Due to their verified biocompatibility and capability of alleviating the surface injury, collagen containing substances have been exploited in pharmaceuticals and dentistry. Collagen membranes are absorbable and

Handbook of Chitin and Chitosan

23.5 Chitosan-based biocomposites for wound healing

745

prevent epithelial downgrowth of the root surface of injury during the initial stage of wound healing [85]. Similarly, chitosan has the ability to improve the healing process by stimulating the migration of inflammatory cells and fibroblasts to the wound site as well as the collagen deposition [86]. Moreover, collagen-based biomaterial can be prepared easily by mixing collagen solution with chitosan. Chitosan is a polycationic molecule which can create ionic bonds with many amine groups and carboxylic groups of collagen. These bonds are sufficient enough to stabilize the biomaterial structure and have a high mechanical strength [87]. Electrospun nanofibrous membranes were prepared from chitosan, type I collagen, chitosan, and polyethylene oxide by Chen et al. to investigate their potential use as wound dressing material [88]. In vitro analysis showed that the prepared nanofibrous membranes were biocompatible as they were nontoxic toward growth of 3T3 fibroblasts. From in vivo analysis, it was observed that the membranes were better than gauze and commercial collagen sponge wound dressing in terms of wound healing rate. Such electrospun matrix from chitosan and collagen can be used as a wound dressing material for skin regeneration. Biochemical and biophysical features of chitosan-cross-linked collagen sponge were examined in research as a wound dressing material to enhance diabetic wound healing [89]. Recombinant human acidic fibroblast growth factor was loaded in the sponge which had a direct effect on accelerating diabetic wound healing. The prepared sponge showed several properties that are advantageous for wound dressing, including a uniform and porous ultrastructure with pores at small intervals, less water absorbance, high resistance to collagenase digestion, and slow release of growth factor from the matrix. An in vivo experiment was carried out in a type 1 diabetic rat model (Fig. 23.5) in which hyperglycemia was induced and the growth factor
loaded sponges showed the most efficient therapeutic effect like the shortest healing time, quickest tissue collagen generation, the earliest and highest TGF-β1 expression, and dermal cell proliferation. These results suggested that growth factor
loaded chitosan-cross-linked collagen sponge could be an ideal wound dressing to improve the recovery of healing-impaired wounds such as diabetic skin wound.

23.5.5 Chitosan
gelatin Gelatin is a derivative of collagen and is colorless, translucent, brittle (when dehydrated significantly), and a flavorless food ingredient. It is also referred to as hydrolyzed collagen, collagen hydrolysate, gelatine hydrolysate, hydrolyzed gelatin, and collagen peptides. As a proteinaceous hydrocolloid biopolymer gelatin displays outstanding properties

Handbook of Chitin and Chitosan

746

23. Chitosan and chitosan-based biomaterials for wound management

Control

Diabetes

D/CCCS/FGF(H)

d7

d 14

d 28

FIGURE 23.5 Representative images of wound healing status in control, diabetes, and diabetes treated with growth factor
loaded chitosan-cross-linked collagen sponge (D/ CCCS/FGF (H)) after 7 (d 7), 14 (d 14), and 28 (d 28) days. Source: Reprinted with permission from W. Wang, et al., Acceleration of diabetic wound healing with chitosan-cross-linked collagen sponge containing recombinant human acidic fibroblast growth factor in healing-impaired STZ diabetic rats. Life Sci. 82(3
4) (2008) 190
204. Copyright r 2007 Elsevier Inc.

of biocompatibility, biodegradability, expandability to soak up a large amount of water, and ease for chemical adaptation [90]. In many biomedical applications it was observed that chitosan and gelatin in combination showed better performance than when they used separately. In most of the cases the casting method was preferred for the preparation of composites from these ingredients [91]. Due to their film-forming nature, chitosan and gelatin were considered as the main components in various studies [92]. Patel et al. fabricated lupeol-entrapped chitosan
gelatin (LCGH) films by a solution casting technique using glyceraldehyde as a crosslinker and glycerol as plasticizer [93]. The presence of plasticizers had great influence on several properties, namely, strength, thickness, swelling capacity, and controlled release of lupeol. The prominence of antioxidant properties of lupeol was assured by the antioxidant assay, while the antibacterial activity of lupeol in LCGH film was found to be retained as assessed by the disk diffusion method. Moreover, viability and nontoxicity showed this hydrogel system to be a potential film dressing for drug delivery in wound management. Nguyen et al. prepared composite sponges of chitosan and gelatin in various ratios incorporating curcumin [94]. When comparing the in vivo

Handbook of Chitin and Chitosan

23.5 Chitosan-based biocomposites for wound healing

747

test in rabbits of the composite sponge with or without curcumin, it was found that curcumin-containing sponges displayed superior wound enclosure than those without curcumin and untreated wounds. These composite sponges were established to increase the configuration of collagen and wound closure, and therefore enhanced the wound-healing activity. Cai et al. studied the influence of Fe3O4 nanoparticles as supplements on the antibacterial and mechanical properties of chitosan
gelatin composite nanofibers membranes as wound dressing [95]. Due to the incorporation of Fe3O4 nanoparticles in the composite, thermal stability and mechanical robustness improved significantly and the swelling capability of the tailored composite was not compromised. This tailored membrane was recommended to have a potential applications as a wound dressing because of the optimum antibacterial ability and desired mechanical properties. Wang et al. developed a new dressing (HS) from chitosan, honey, and gelatin in the form of a composite hydrogel sheet, which was proven to destroy S. aureus and E. coli completely [96]. The burn of New Zealand rabbit was considered as a model for carrying out in vivo test of with HS, MEBO ointment (Shantou MEBO Pharmaceuticals Co., Ltd., Guangdong, China), and sterile gauze, respectively. The experiment showed that HS had a significant effect on wound contraction with the shortest treatment duration of 12 days compared with MEBO ointment and no treatment (Fig. 23.3). As the HS exhibited significantly improved in vivo and in vitro test results, it could be used potentially for the treatment of wounds.

23.5.6 Chitosan
polylactic acid As biodegradable and biocompatible materials chitosan and polylactic acid (PLA) have acquired extensive consideration. Nevertheless, chitosan as a scaffold material degrades little by little, dissolves only in dilute acid medium, and has a compromising response with regard to tissue regeneration. In addition, chitosan had poor mechanical properties in several cases. The application of chitosan in tissue engineering is restricted by these limiting properties to some extent, whereas PLA is considered one of the most smart biopolymers due to its biocompatibility, renewability, biodegradability, and physical properties [97]. PLA under physiological conditions degrades into naturally occurring lactic acid which is considered the best advantage [98]. PLA acid may have great potential in wound management, because of their complimentary characteristics. In combination, they may form complexes—chitosan may neutralize the acidic degradation products generated by PLA—which may improve the desired mechanical properties. Furthermore, a PLA can be processed by many recognized methods which makes it suitable for several applications.

Handbook of Chitin and Chitosan

748

23. Chitosan and chitosan-based biomaterials for wound management

Zhang et al. prepared biodegradable and biocompatible composite poly(chitosan-g-lactic acid) (PCLA) by grafting lactic acid onto the amino groups on chitosan without any catalyst [99]. To evaluate the composite’s biocompatibility histological examination and bone marrow mesenchymal stem cell (BMSC) culture experiments were performed. Mass loss in vitro, and degradation in vivo as a function of feed ratio of lactic acid/chitosan indicated the biodegradability of the composite. According to these results this group of researchers predicted PCLA was a promising material for tissue engineering. Guo et al. introduced poly(L-lactic acid) (PLLA) into chitosan microspheres to prepare a composite (CSMs) to control the acid microclimate of PLLA degradation [100]. In vivo degradation behavior of the prepared composite on rat’s subcutaneous was found to be consistent with in vitro degradation results in some degree. The comprehensive results indicated that CSMs composites have a tunable nature for degradation and could be an auspicious contender for biomedical application.

23.5.7 Chitosan
polyvinyl alcohol High antimicrobial activity, cytocompatibility, nontoxicity, and nonallergenic properties are the most anticipated characteristics for an ideal wound dressing. The fabrication of composites based on natural and synthetic biopolymers for wound dressing application via an electrospinning technique has drawn the attention of the research community in recent years. The preparation of well-fabricated dressing incorporating biomaterials has augmented the healing process many fold. Integration of chitosan in polyvinyl alcohol (PVA) following the electrospinning route was a prominent example of such a wound healing product. Electrospun composites can imitate the structure of extracellular matrix for improved cell attachment and proliferation. Undoubtedly, they can be an appropriate preference for wound-healing application because of their interconnected network, flexibility in surface functionality, high ratio of surface area to volume, and high oxygen permeability [101]. As a synthetic water-soluble polymer PVA has widespread use in biomedical applications like tissue engineering and wound healing. PVA has biodegradability, biocompatibility, nontoxicity, and electrospinnability which allowed PVA to be used to prepare mat-like electrospun composite for wound healing [102]. Huge quantities of studies have been performed regarding the formation of the composite of chitosan
PVA by many researchers. Kang et al. prepared nanofibrous wound dressings from heat-treated PVA coated with chitosan that were tested on open wound healing in a mouse. Their mechanical and histological studies showed that in the

Handbook of Chitin and Chitosan

23.6 Chitosan derivatives in wound management

749

early stage of wound healing chitosan
PVA nanofibrous matrix worked more effectively than heat-treated PVA nanofibrous matrix solely [103]. Majd et al. prepared PVA/chitosan nanofiber wound dressings and extended their studies to diabetic as well as nondiabetic wound healing [104]. It was observed that this wound dressing was noncytotoxic and had excellent odor-absorbing capability. Their microscopic and macroscopic effect on rat experimental external ulcers was promising as the length of epidermis and dermis areas were reduced notably in diabetic rats. Silver has been introduced in numerous products in numerous conformations mostly for burn dressing to defend against the contamination of microbes, as it has no viable effect on mammalian cells [105]. Abdelgawad et al. fabricated nonwoven PVA
chitosan (PVA/CS) blends and PVA
chitosan
Ag nanoparticle (PVA/CS-Ag-NP) blends by the electrospinning method and their viable cell-counting studies showed that electrospun composite mat of PVA/CS-Ag-NPs blends had better bactericidal activity toward the Gram-negative bacteria E. coli than PVA/CS blends [106].

23.6 Chitosan derivatives in wound management Various derivatives of chitosan have been produced for wound management, particularly to enhance wound healing. For example, oligochitosan (OC) and N,O-carboxymethyl chitosan (NO-CMC) derivatives have been fabricated into films for wound dressing. The use of N-carboxybutyl chitosan improves cutaneous tissue regeneration with good histoarchitecture and vascularization at the wound site and promotes tissue regeneration. Additionally, 5-methylpyrrolidinone chitosan (MPC) is compatible with other polymer solutions like gelatin, PVA, polyvinyl pyrrolidone, and hyaluronic acid, which are beneficial for the treatment of wounded meniscal tissues, decubitus ulcers, depression of capsule formation around prostheses, scar formation, and retraction during wound healing. To improve the mucoadhesive properties of chitosan various derivatives such as trimethylated chitosan, mono-Ncarboxymethyl chitosan, N-sulfochitosan, and chitosan
EDTA conjugates were developed. Different derivatives of chitosan and their application in wound management are given below.

23.6.1 N,O-carboxymethyl chitosan CMC is a chitosan derivative with the enhanced properties like water solubility over a wide pH range and good biocompatibility and these

Handbook of Chitin and Chitosan

750

23. Chitosan and chitosan-based biomaterials for wound management

enhanced properties are attributed to the carboxymethyl groups on the copolymer backbone [107]. Other properties like gel-forming capacity and ability to interact with different drugs and promotion of skin fibroblast proliferation make it attractive for wound healing [108]. NO-CMC can be prepared by following one single-step reaction directly from chitosan. By treating chitosan with sodium hydroxide in the presence of isopropanol and water followed by adding chloroacetic acid to the reaction mixture NO-CMC can be synthesized [109]. A research by Liu et al. has shown that the highest antibacterial activity against E. coli was found in N,O-CMC followed by chitosan and then O-CMC [110]. Extensive research has been conducted to study the applications of NO-CMC in wound healing and management. Chen et al. investigated in vitro effects of NO-CMC on the growth and collagen secretion of normal skin fibroblasts and keloid fibroblasts [111]. Their study showed that the use of NO-CMC promoted the proliferation of the normal skin fibroblast significantly but inhibited the proliferation of keloid fibroblast. An in vivo study in a rabbit abdominal surgery model by Zhou et al. reported that NO-CMC gel and solution can reduce adhesion formation and reformation [112]. Their investigation also suggested that NO-CMC may work as the foremost biophysical barrier as the fibroblasts were unable to adhere to NO-CMC solution-coated surfaces. To enhance the wound healing properties of NO-CMC by stimulating the recruitment of fibroblasts Chen et al. incorporated NO-CMC into the backbone of a collagen (COL) matrix without or with chondroitin sulfate or an acellular dermal matrix (ADM) [113]. Enhanced wound healing characteristics have been found in their study which open the door for NO-CMC/COL matrixes to become potential wound dressings for medical applications.

23.6.2 N-Carboxybutyl chitosan N-Carboxybutyl chitosan (N-CBC) is another chitosan derivative that enhances its properties. It is an amphoteric polymer that is soluble under acidic, neutral, and basic conditions [114]. Its properties, like the capacity to form good film polymer, excellent moisturizing agent, and good bacteriostatic capacity [115], make it capable for various biomedical applications, such as tissue expanders [116], wound management, and formation of organized cutaneous tissues [117]. N-CBC can be synthesized directly from chitosan in the presence of levulinic acid. The reaction can form N-CBC or 5-MPC depending on the chemical conditions, so it is required to control the reaction medium efficiently [115]. N-CBC is more versatile than any other polysaccharide presently used in wound management and has special characteristics for the

Handbook of Chitin and Chitosan

23.6 Chitosan derivatives in wound management

751

production of wound dressings. For this regard extensive research is being conducted to study the effects of N-CBC for wound management. Muzzarelli et al. reported the antimicrobial properties of N-CBC and tested it in different physical forms considering its use in wound management [118]. Their study with the help of electron microscopy showed that microbial cells exposed to N-CBC experienced noticeable morphological modifications, which can be regarded as an important superfluous trait of N-CBC-based wound dressings, because they could be a factor in the inhibition of secondary infections, resulting in partial scar formation. Biagini et al. [117] treated donor sites of plastic surgery patients with soft pads of N-CBC to promote ordered tissue regeneration. The study showed better histoarchitectural order, better vascularization, absence of inflammatory cells, and fewer aspects of malpighian layer proliferation due to the use of N-CBC pads, and therefore leading to the formation of regularly arranged skin tissue and reducing the abnormal healing process. Biagini et al. reported the effects of N-CBC coated expanders on surgical injuries in rabbit dorsal skin and capsular tissue formation [116]. Their study showed that the use of N-CBC helps and potentiates the correct proliferation and organization of the tissue. Muzzarelli et al. used N-CBC to assist the spontaneous tissue repair of the meniscus on rabbit [119]. Results from their experiments showed that this extremely difficult task of spontaneously repairing meniscal lesions is favored due to the use of N-CBC.

23.6.3 5-Methylpyrrolidinone chitosan 5-MPC is a derivative of chitosan that is water-soluble in a wide range of pH. The nitrogen atom is simultaneously part of the methylpyrrolidinone moiety and of the glucosamine repeating unit. Their water solubility and antimicrobial activity against a broad variety of bacteria and fungi make them beneficial to accelerate wound and ulcer healing and several other biomedical applications. MPC can be synthesized by following the same procedure of N-CBC preparation using levulinic acid. Proper reaction conditions, mainly high chitosan and borohydride concentrations, final pH higher than 5, and slow borohydride delivery rate, can favor the formation of MPC in levulinic acid [120]. Berscht et al. described MPC as one of the most biocompatible chitosans among several tested derivatives [121]. They prepared soft and flexible fleeces of MPC by freeze-drying and used them as a carrier material for basic fibroblast growth factor (bFGF) to investigate the treatment of wound-healing deficiencies. Results from their study showed that a sustained release of biologically active bFGF from the fleeces which

Handbook of Chitin and Chitosan

752

23. Chitosan and chitosan-based biomaterials for wound management

could have overproportioned effects on wound management and could be very useful for the therapy of prolonged wounds. Muzzarelli et al. used MPC as a medication for surgical wounds from wisdom tooth avulsions [122]. Osteoconduction was promoted due to the use of MPC and desirable mechanical and physiological characteristics to the healed wound site were observed. Most importantly, no adverse effects were reported by the patients during 1 year of observation. Favorable properties of MPC, like its cationic nature and ability for chelation, made it beneficial for promoting new bone tissue formation. The ultrastructural investigation from their study demonstrated that bone osteoid was followed by mineralization of the tissue. Although chitosan has shown its effectiveness as a biomaterial for wound healing and management, the modification of chitosan into its derivatives can make it more efficient and enhance the properties in these regards. Several other derivatives can be synthesized, like N,N,Ntrimethyl derivatives and carboxymethyl-trimethyl derivatives, which can be used to accelerate the wound-healing process by using them in different formulations, such as nanoparticles, gels, dressings, scaffolds, films, fibers, or sponges.

23.7 Future aspects of chitosan-based material for wound management After cutaneous injury, a number of events occur which ultimately result in tissue repair. A plethora of cells, enzymes, cytokines, hormones, and ions are involved in this process. Chitosan, its derivatives, and biocomposites are very promising for entrapping these species and delivering them to the site of wound. The localized delivery of woundhealing promoters and growth factors will certainly open a new era of wound management and chitosan-based materials can play a big role in it. Wound healing in the field of tissue engineering possesses some limitations, like chronic inflammation on application, mismatch of donor tissue, and deficiency of donor tissue, and the elimination of these restrictions is the impetus to looking forward for creating the biological alternatives to replenish the injured tissue. Heretofore, for the management of full-thickness injuries many skin substitutes had been utilized. Substitutes originated from skin are susceptible to attack from dormant viruses. Whereas synthetic skin graft can mend the far-reaching scarring, they fail to rejuvenate structures, for example, hair follicles, glands, nerves, etc. Therefore, there is an unquestionable need to develop novel and practical skin management materials in the near future. In view of future perspectives, it is anticipated that chitosan-based composite materials will achieve significant consideration. More specifically this

Handbook of Chitin and Chitosan

References

753

attention might be given on composites where inorganic/organic nanoparticles would be incorporated. In recent decades, chitosan-containing material has been in the driving seat of various fields, like agriculture, biomedicine, material science, etc. In the upcoming days, chitosan-based composites will be used in the form of advanced composite or fiber, for instance, to accelerate tissue repair and wound healing processes as well as to advance the existing tissue engineering expertise. Various biopolymers, including gelatin, alginate, collagen, carrageenan, and hyaluronic acid, in combination with various nanomaterials may find application in the field of wound management.

23.8 Conclusion Wound healing seems to be a very complex process which involves a number of cascade events, integrated responses by growth factors and different types of cells to achieve rapid restoration of skin architecture and prevent scar formation. Chitosan and its derivatives and biocomposites with natural or synthetic polymers have been found as potential biomaterials to be used in wound healing due to their intrinsic properties, such as their analgesic, antimicrobial, and antiinflammatory nature, biocompatibility, and biodegradability. Moreover, chitosan-based materials can be processed into different forms, like scaffolds, membranes, films, and hydrogels, with improved properties and are very promising for wound management. It is possible to entrap wound healing promoters and growth factors into chitosan-based biomaterials and deliver them to the site of wound which will open up new possibilities in wound healing and management.

References [1] C. Radhakumary, M. Antonty, K. Sreenivasan, Drug loaded thermoresponsive and cytocompatible chitosan based hydrogel as a potential wound dressing, Carbohydr. Polym. 83 (2) (2011) 705
713. [2] R. Jayakumar, et al., Biomaterials based on chitin and chitosan in wound dressing applications, Biotechnol. Adv. 29 (3) (2011) 322
337. [3] M.S. Islam, et al., Core
shell drug carrier from folate conjugated chitosan obtained from prawn shell for targeted doxorubicin delivery, J. Mater. Sci. Mater. Med. 28 (4) (2017) 55. [4] S. Wittaya-areekul, C. Prahsarn, Development and in vitro evaluation of chitosan
polysaccharides composite wound dressings, Int. J. Pharm. 313 (1-2) (2006) 123
128. [5] M. Risbud, A. Hardikar, R. Bhonde, Growth modulation of fibroblasts by chitosanpolyvinyl pyrrolidone hydrogel: implications for wound management? J. Biosci. 25 (1) (2000) 25
30.

Handbook of Chitin and Chitosan

754

23. Chitosan and chitosan-based biomaterials for wound management

[6] I.K. Cohen, B.A. Mast, Models of wound healing, J. Trauma. 30 (12 Suppl.) (1990) S149
S155. [7] W.K. Stadelmann, A.G. Digenis, G.R. Tobin, Physiology and healing dynamics of chronic cutaneous wounds, Am. J. Surg. 176 (2) (1998) 26S
38S. [8] M. Flanagan, The physiology of wound healing, J. Wound Care 9 (6) (2000) 299
300. [9] D.L. Steed, The role of growth factors in wound healing, Surg. Clin. North. Am. 77 (3) (1997) 575
586. [10] R.F. Diegelmann, I.K. Cohen, A.M. Kaplan, The role of macrophages in wound repair: a review, Plast. Reconstr. Surg. 68 (1) (1981) 107
113. [11] R. Clarke, Overview and general considerations of wound repair, Mol. Cell. Biol. Wound Repair. (1988) 18
22. [12] G.C. Gurtner, et al., Wound repair and regeneration, Nature 453 (7193) (2008) 314. [13] L.C. Keong, A.S. Halim, In vitro models in biocompatibility assessment for biomedical-grade chitosan derivatives in wound management, Int. J. Mol. Sci. 10 (3) (2009) 1300
1313. [14] T.C. Tseng, et al., An injectable, self-healing hydrogel to repair the central nervous system, Adv. Mater. 27 (23) (2015) 3518
3524. [15] C. Chatelet, O. Damour, A. Domard, Influence of the degree of acetylation on some biological properties of chitosan films, Biomaterials 22 (3) (2001) 261
268. [16] S. Pangburn, P. Trescony, J. Heller, Lysozyme degradation of partially deacetylated chitin, its films and hydrogels, Biomaterials 3 (2) (1982) 105
108. [17] S.-I. Aiba, Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans, Int. J. Biol. Macromol. 14 (4) (1992) 225
228. [18] P.V. Maksym, S. Vitalii, Chitosan as a hemostatic agent: current state, Eur. J. Med. Ser. B 2 (1) (2015) 24
33. [19] J.M.D. Lima, et al., Evaluation of hemagglutination activity of chitosan nanoparticles using human erythrocytes, Bio Med. Res. Int. 2015 (2015). [20] W. Fan, et al., Erythrocytes load of low molecular weight chitosan nanoparticles as a potential vascular drug delivery system, Colloids Surf. B: Biointerfaces 95 (2012) 258
265. [21] E.C. Shen, et al., Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation, Clin. Oral. Implant. Res. 17 (5) (2006) 572
578. [22] M.S. Lord, et al., The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins, Biomaterials 32 (28) (2011) 6655
6662. [23] Y. Okamoto, et al., Analgesic effects of chitin and chitosan, Carbohydr. Polym. 49 (3) (2002) 249
252. [24] J. Synowiecki, N.A. Al-Khateeb, Production, properties, and some new applications of chitin and its derivatives, Crit. Rev. Food Sci. Nutr. 43 (2), 2003, 145
171. [25] N. Sudarshan, D. Hoover, D. Knorr, Antibacterial action of chitosan, Food Biotechnol. 6 (3) (1992) 257
272. [26] J. Leuba, P. Stossel, Chitosan and other polyamines: antifungal activity and interaction with biological membranes, Chitin in Nature and Technology, Springer, 1986, pp. 215
222. [27] P. Eaton, et al., Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus, Ultramicroscopy 108 (10) (2008) 1128
1134. [28] Y.-C. Chung, et al., Relationship between antibacterial activity of chitosan and surface characteristics of cell wall, Acta Pharmacol. Sin. 25 (7) (2004) 932
936. [29] Y.-J. Jeon, P.-J. Park, S.-K. Kim, Antimicrobial effect of chitooligosaccharides produced by bioreactor, Carbohydr. Polym. 44 (1) (2001) 71
76.

Handbook of Chitin and Chitosan

References

755

[30] L.-Y. Zheng, J.-F. Zhu, Study on antimicrobial activity of chitosan with different molecular weights, Carbohydr. Polym. 54 (4) (2003) 527
530. [31] H. Ueno, T. Mori, T. Fujinaga, Topical formulations and wound healing applications of chitosan, Adv. Drug. Deliv. Rev. 52 (2) (2001) 105
115. [32] H. Ueno, et al., Accelerating effects of chitosan for healing at early phase of experimental open wound in dogs, Biomaterials 20 (15) (1999) 1407
1414. [33] D. Fong, C.D. Hoemann, Chitosan immunomodulatory properties: perspectives on the impact of structural properties and dosage, Future Sci. 4 (1), 2017. [34] N. Bhattarai, J. Gunn, M. Zhang, Chitosan-based hydrogels for controlled, localized drug delivery, Adv. Drug. Deliv. Rev. 62 (1) (2010) 83
99. [35] L. Fan, et al., Preparation and characterization of chitosan/gelatin/PVA hydrogel for wound dressings, Carbohydr. Polym. 146 (2016) 427
434. [36] A. Abdel-Mohsen, et al., Eco-synthesis of PVA/chitosan hydrogels for biomedical application, J. Polym. Environ. 19 (4) (2011) 1005
1012. [37] D. Zhang, et al., Carboxyl-modified poly (vinyl alcohol)-cross-linked chitosan hydrogel films for potential wound dressing, Carbohydr. Polym. 125 (2015) 189
199. [38] I. Sweeney, M. Miraftab, G. Collyer, Absorbent alginate fibres modified with hydrolysed chitosan for wound care dressings
II. Pilot scale development, Carbohydr. Polym. 102 (2014) 920
927. [39] Y. Yang, et al., Advances in self-assembled chitosan nanomaterials for drug delivery, Biotechnol. Adv. 32 (7) (2014) 1301
1316. [40] R. Warriner, R. Burrell, Infection and the chronic wound: a focus on silver, Adv. Skin Wound Care 18 (8) (2005) 2
12. [41] S. Rinehart, et al., Synthesis and characterization of a chitosan/PVA antimicrobial hydrogel nanocomposite for responsive wound management materials, J. Microb. Biochem. Technol. 8 (2016) 65
70. [42] W.E. Hennink, C.F. van Nostrum, Novel cross-linking methods to design hydrogels, Adv. Drug Deliv. Rev. 64 (2012) 223
236. [43] J. Stephens-Altus, et al., Development of bioactive photocross-linkable fibrous hydrogels, J. Biomed. Mater. Res. Part. A 98 (2) (2011) 167
176. [44] X. Yang, et al., Effects of PVA, agar contents, and irradiation doses on properties of PVA/ws-chitosan/glycerol hydrogels made by γ-irradiation followed by freezethawing, Radiat. Phys. Chem. 77 (8) (2008) 954
960. [45] J. Keleher, et al., Photo-catalytic preparation of silver-coated TiO2 particles for antibacterial applications, World J. Microbiol. Biotechnol. 18 (2) (2002) 133
139. [46] X. Meng, et al., Chitosan and alginate polyelectrolyte complex membranes and their properties for wound dressing application, J. Mater. Sci. Mater. Med. 21 (5) (2010) 1751
1759. [47] F. Han, et al., Alginate/chitosan based bi-layer composite membrane as potential sustained-release wound dressing containing ciprofloxacin hydrochloride, Appl. Surf. Sci. 311 (2014) 626
634. [48] Z. Gu, et al., Preparation of chitosan/silk fibroin blending membrane fixed with alginate dialdehyde for wound dressing, Int. J. Biol. Macromol. 58 (2013) 121
126. [49] P.I. Morgado, et al., Poly (vinyl alcohol)/chitosan asymmetrical membranes: Highly controlled morphology toward the ideal wound dressing, J. Membr. Sci. 469 (2014) 262
271. [50] F. Han, et al., Preparation, characteristics and assessment of a novel gelatin
chitosan sponge scaffold as skin tissue engineering material, Int. J. Pharm. 476 (1-2) (2014) 124
133. [51] L. Ding, et al., Spongy bilayer dressing composed of chitosan
Ag nanoparticles and chitosan
Bletilla striata polysaccharide for wound healing applications, Carbohydr. Polym. 157 (2017) 1538
1547.

Handbook of Chitin and Chitosan

756

23. Chitosan and chitosan-based biomaterials for wound management

[52] A. Sathiyaseelan, et al., Fungal chitosan based nanocomposites sponges—an alternative medicine for wound dressing, Int. J. Biol. Macromol. 104 (2017) 1905
1915. [53] W. Shao, et al., Construction of silver sulfadiazine loaded chitosan composite sponges as potential wound dressings, Carbohydr. Polym. 157 (2017) 1963
1970. [54] V.V.S.R. Karri, et al., Curcumin loaded chitosan nanoparticles impregnated into collagen-alginate scaffolds for diabetic wound healing, Int. J. Biol. Macromol. 93 (2016) 1519
1529. ˘ [55] Z. Degim, et al., Evaluation of chitosan gel containing liposome-loaded epidermal growth factor on burn wound healing, Int. Wound J. 8 (4) (2011) 343
354. [56] L. Mayol, et al., Design and characterization of a chitosan physical gel promoting wound healing in mice, J. Mater. Sci. Mater. Med. 25 (6) (2014) 1483
1493. [57] R. Zhao, et al., Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings, Int. J. Biol. Macromol. 68 (2014) 92
97. [58] N. Naseri, et al., Electrospun chitosan-based nanocomposite mats reinforced with chitin nanocrystals for wound dressing, Carbohydr. Polym. 109 (2014) 7
15. [59] A. Hassan, et al., Development of anti-bacterial PVA/starch based hydrogel membrane for wound dressing, J. Polym. Environ. 26 (1) (2018) 235
243. [60] J. Chen, et al., Toxicology of starch-based DDSs, Drug Delivery Applications of Starch Biopolymer Derivatives, Springer, 2019, pp. 133
137. [61] F. Torres, et al., Biodegradability and mechanical properties of starch films from Andean crops, Int. J. Biol. Macromol. 48 (4) (2011) 603
606. [62] P.M. Arockianathan, et al., Preparation, characterization and evaluation of biocomposite films containing chitosan and sago starch impregnated with silver nanoparticles, Int. J. Biol. Macromol. 50 (4) (2012) 939
946. [63] S. Baghaie, et al., Wound healing properties of PVA/starch/chitosan hydrogel membranes with nano Zinc oxide as antibacterial wound dressing material, J. Biomater. Sci. Polym. Ed. 28 (18) (2017) 2220
2241. [64] S. Sharma, A. Dua, A. Malik, Third generation materials for wound dressings, Int. J. Pharm. Sci. Res. 5 (2014) 2113
2124. [65] J. O’Donoghue, et al., Calcium alginate dressings promote healing of split skin graft donor sites, Acta Chir. Plast. 39 (2) (1997) 53
55. [66] J.J. Disa, et al., Evaluation of a combined calcium sodium alginate and bio-occlusive membrane dressing in the management of split-thickness skin graft donor sites, Ann. Plas. Surg. 46 (4) (2001) 405
408. [67] H. Chen, et al., Covalently antibacterial alginate-chitosan hydrogel dressing integrated gelatin microspheres containing tetracycline hydrochloride for wound healing, Mater. Sci. Eng. C 70 (2017) 287
295. [68] K. Murakami, et al., Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-impaired wound dressings, Biomaterials 31 (1) (2010) 83
90. [69] L. Wang, et al., Chitosan-alginate PEC membrane as a wound dressing: Assessment of incisional wound healing, J. Biomed. Mater. Rese. 63 (5) (2002) 610
618. [70] Y. Kong, et al., Fast and safe fabrication of a free-standing chitosan/alginate nanomembrane to promote stem cell delivery and wound healing, Int. J. Nanomed. 11 (2016) 2543. [71] M.P. Devi, et al., A novel wound dressing material—fibrin
chitosan
sodium alginate composite sheet, Bull. Mater. Sci. 35 (7) (2012) 1157
1163. ¨ ztu¨rk, et al., Preparation and characterization of ciprofloxacin-loaded alginate/ [72] E. O chitosan sponge as a wound dressing material, J. Appl. Polym. Sci. 101 (3) (2006) 1602
1609. [73] A.P. Rodrigues, et al., The influence of preparation conditions on the characteristics of chitosan-alginate dressings for skin lesions, J. Appl. Polym. Sci. 109 (4) (2008) 2703
2710.

Handbook of Chitin and Chitosan

References

757

[74] M. Dantas, et al., Improvement of dermal burn healing by combining sodium alginate/chitosan-based films and low level laser therapy, J. Photochem. Photobiol. B: Biol. 105 (1) (2011) 51
59. [75] Y.-C. Ho, et al., Heparin-functionalized chitosan
alginate scaffolds for controlled release of growth factor, Int. J. Pharm. 376 (1-2) (2009) 69
75. [76] G.F. Caetano, et al., Chitosan-alginate membranes accelerate wound healing, J. Biomed. Mater. Res. Part. B: Appl. Biomater. 103 (5) (2015) 1013
1022. [77] H.-J. Hong, et al., Accelerated wound healing by smad3 antisense oligonucleotidesimpregnated chitosan/alginate polyelectrolyte complex, Biomaterials 29 (36) (2008) 4831
4837. [78] M. Moscovici, Present and future medical applications of microbial exopolysaccharides, Front. Microbiol. 6 (2015) 1012. [79] G. Sun, et al., Dextran hydrogel scaffolds enhance angiogenic responses and promote complete skin regeneration during burn wound healing, Proc. Natl. Acad. Sci. 108 (52) (2011) 20976
20981. [80] R.I. Malini, et al., Crosslinking dextran electrospun nanofibers via borate chemistry: Proof of concept for wound patches, Eur. Polym. J. 110 (2019) 276
282. [81] J. Hoque, et al., Biocompatible injectable hydrogel with potent wound healing and antibacterial properties, Mol. Pharmaceutics 14 (4) (2017) 1218
1230. [82] X. Zhao, et al., Antibacterial and conductive injectable hydrogels based on quaternized chitosan-graft-polyaniline/oxidized dextran for tissue engineering, Acta Biomater. 26 (2015) 236
248. [83] M. Ribeiro, et al., Dextran-based hydrogel containing chitosan microparticles loaded with growth factors to be used in wound healing, Mater. Sci. Eng. C. 33 (5) (2013) 2958
2966. [84] M.A. Aziz, et al., Antimicrobial properties of a chitosan dextran-based hydrogel for surgical use, Antimicrob. Agents Chemother. 56 (1) (2012) 280
287. [85] P. Bunyaratavej, H.L. Wang, Collagen membranes: a review, J. Periodontol. 72 (2) (2001) 215
229. [86] S. Ahmed, S. Ikram, Chitosan based scaffolds and their applications in wound healing, Achiev. Life Sci. 10 (1) (2016) 27
37. [87] O. Damour, et al., A dermal substrate made of collagen-GA-chitosan for deep burn coverage: first clinical uses, Clin. Mater. 15 (4) (1994) 273
276. [88] J.-P. Chen, G.-Y. Chang, J.-K. Chen, Electrospun collagen/chitosan nanofibrous membrane as wound dressing, Colloids Surf. A: Physicochem. Eng. Asp. 313 (2008) 183
188. [89] W. Wang, et al., Acceleration of diabetic wound healing with chitosan-cross-linked collagen sponge containing recombinant human acidic fibroblast growth factor in healing-impaired STZ diabetic rats, Life Sci. 82 (3-4) (2008) 190
204. [90] L.-H. Peng, et al., Topical astragaloside IV-releasing hydrogel improves healing of skin wounds in vivo, Biol. Pharm. Bull. 35 (6) (2012) 881
888. [91] E. Shahram, et al., Evaluation of chitosan
gelatin films for use as postoperative adhesion barrier in rat cecum model, Int. J. Surg. 11 (10) (2013) 1097
1102. [92] Z. Nasreen, M.A. Khan, A. Mustafa, Improved biodegradable radiation cured polymeric film prepared from chitosan-gelatin blend, J. Appl. Chem. 2016 (2016). [93] S. Patel, et al., Preparation and optimization of chitosan-gelatin films for sustained delivery of lupeol for wound healing, Int. J. Biol. Macromol. 107 (2018) 1888
1897. [94] V.C. Nguyen, V.B. Nguyen, M.-F. Hsieh, Curcumin-loaded chitosan/gelatin composite sponge for wound healing application, Int. J. Polym. Sci. 2013 (2013). [95] N. Cai, et al., Tailoring mechanical and antibacterial properties of chitosan/gelatin nanofiber membranes with Fe3O4 nanoparticles for potential wound dressing application, Appl. Surf. Sci. 369 (2016) 492
500.

Handbook of Chitin and Chitosan

758

23. Chitosan and chitosan-based biomaterials for wound management

[96] T. Wang, et al., Hydrogel sheets of chitosan, honey and gelatin as burn wound dressings, Carbohydr. Polym. 88 (1) (2012) 75
83. [97] R. Scaffaro, et al., Polysaccharide nanocrystals as fillers for PLA based nanocomposites, Cellulose 24 (2) (2017) 447
478. [98] E. Llorens, L.J. del Valle, J. Puiggalı´, Electrospun scaffolds of polylactide with a different enantiomeric content and loaded with anti-inflammatory and antibacterial drugs, Macromol. Res. 23 (7) (2015) 636
648. [99] Z. Zhang, H. Cui, Biodegradability and biocompatibility study of poly (chitosan-glactic acid) scaffolds, Molecules 17 (3) (2012) 3243
3258. [100] Z. Guo, et al., Degradation properties of chitosan microspheres/poly (L-lactic acid) composite in vitro and in vivo, Carbohydr. Polym. 193 (2018) 1
8. [101] A. Rahmani Del Bakhshayesh, et al., Recent advances on biomedical applications of scaffolds in wound healing and dermal tissue engineering, Artif. Cell. Nanomed. Biotechnol. 46 (4) (2018) 691
705. [102] R.P. Gonc¸alves, et al., Effect of chitosan on the properties of electrospun fibers from mixed poly (vinyl alcohol)/chitosan solutions, Mater. Res. 20 (4) (2017) 984
993. [103] Y.O. Kang, et al., Chitosan-coated poly (vinyl alcohol) nanofibers for wound dressings, J. Biomed. Mater. Res. Part B: Appl. Biomater. 92 (2) (2010) 568
576. [104] S.A. Majd, et al., Application of Chitosan/PVA Nano fiber as a potential wound dressing for streptozotocin-induced diabetic rats, Int. J. Biol. Macromol. 92 (2016) 1162
1168. [105] D.W. Brett, A discussion of silver as an antimicrobial agent: alleviating the confusion, Ostomy Wound Manag. 52 (1) (2006) 34
41. [106] A.M. Abdelgawad, S.M. Hudson, O.J. Rojas, Antimicrobial wound dressing nanofiber mats from multicomponent (chitosan/silver-NPs/polyvinyl alcohol) systems, Carbohydr. Polym. 100 (2014) 166
178. [107] H.T. Pang, et al., Preparation and rheological properties of deoxycholate-chitosan and carboxymethyl-chitosan in aqueous systems, Carbohydr. Polym. 69 (3) (2007) 419
425. [108] L.Y. Chung, et al., Biocompatibility of potential wound management products: fungal mycelia as a source of chitin/chitosan and their effect on the proliferation of human F1000 fibroblasts in culture, J. Biomed. Mater. Res. 28 (4) (1994) 463
469. [109] V. Patrulea, et al., Chitosan as a starting material for wound healing applications, Eur. J. Pharm. Biopharm. 97 (2015) 417
426. [110] X. Fei Liu, et al., Antibacterial action of chitosan and carboxymethylated chitosan, J. Appl. Polym. Sci. 79 (7) (2001) 1324
1335. [111] X.-G. Chen, et al., The effect of carboxymethyl-chitosan on proliferation and collagen secretion of normal and keloid skin fibroblasts, Biomaterials 23 (23) (2002) 4609
4614. [112] J. Zhou, C. Elson, T.D. Lee, Reduction in postoperative adhesion formation and reformation after an abdominal operation with the use of N, O-carboxymethyl chitosan, Surgery 135 (3) (2004) 307
312. [113] R.-N. Chen, et al., Development of N, O-(carboxymethyl) chitosan/collagen matrixes as a wound dressing, Biomacromolecules 7 (4) (2006) 1058
1064. [114] M. Rinaudo, et al., NMR investigation of chitosan derivatives formed by the reaction of chitosan with levulinic acid, Carbohydr. Polym. 46 (4) (2001) 339
348. [115] R. Muzzarelli, et al., Characteristic properties of N-carboxybutyl chitosan, Carbohydr. Polym. 11 (4) (1989) 307
320. [116] G. Biagini, et al., Morphological study of the capsular organization around tissue expanders coated with N-carboxybutyl chitosan, Biomaterials 12 (3) (1991) 287
291. [117] G. Biagini, et al., Wound management with N-carboxybutyl chitosan, Biomaterials 12 (3) (1991) 281
286.

Handbook of Chitin and Chitosan

References

759

[118] R. Muzzarelli, et al., Antimicrobial properties of N-carboxybutyl chitosan, Antimicrob. Agents Chemother. 34 (10) (1990) 2019
2023. [119] R. Muzzarelli, et al., Role of N-carboxybutyl chitosan in the repair of the meniscus, J. Bioact. Compat. Polym. 7 (2) (1992) 130
148. [120] R.A. Muzzarelli, P. Ilari, M. Tomasetti, Preparation and characteristic properties of 5-methyl pyrrolidinone chitosan, Carbohydr. Polym. 20 (2) (1993) 99
105. [121] P.C. Berscht, et al., Incorporation of basic fibroblast growth factor into methylpyrrolidinone chitosan fleeces and determination of the in vitro release characteristics, Biomaterials 15 (8) (1994) 593
600. [122] R. Muzzarelli, et al., Osteoconduction exerted by methylpyrrolidinone chitosan used in dental surgery, Biomaterials 14 (1) (1993) 39
43.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

24 Chitin and chitosan as promising immunostimulant for aquaculture Dibyendu Kamilya and Md. Idrish Raja Khan Department of Aquatic Health and Environment, College of Fisheries, Central Agricultural University, Lembucherra, Tripura, India

O U T L I N E 24.1 Introduction

762

24.2 Immunostimulatory effect of chitin on finfish

763

24.3 Immunostimulatory effect of chitin on shellfish

764

24.4 Immunostimulatory effect of chitosan on finfish

765

24.5 Immunostimulatory effect of chitosan on shellfish

766

24.6 Effect of chitin and chitosan on disease resistance

766

24.7 Mechanism of action of chitin and chitosan as an immunostimulant

767

24.8 Conclusions and future perspectives

768

References

769

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00024-8

761

© 2020 Elsevier Inc. All rights reserved.

762

24. Chitin and chitosan as promising immunostimulant for aquaculture

24.1 Introduction Aquaculture, the farming of commercially important aquatic organisms, is one of the most dynamic and fastest growing food production sectors in the world. It is an important economic activity in many countries, and the contribution of aquaculture in providing the nutritional as well as the livelihood security is well recognized. The current global aquaculture practices are dominated by high stocking density, artificial feeding, and other intensive approaches. Intensification tends to adversely affect the physiological environment for the aquatic organisms and increases their susceptibility to infection by pathogenic microorganisms [1]. In fact, infectious diseases have become the major impediment for successful and profitable aquaculture [2]. Vaccines, antibiotics, and several other chemotherapeutics have been used as disease control and prevention strategies in aquaculture for the last few decades. Though vaccination has been suggested as a useful prophylactic method for fish disease control, it is not effective against intracellular pathogens and also when a wide number of pathogens are present in the fish farming system [1,3]. Application of antibiotics and other chemotherapeutics can lead to the development of antibiotic-resistant bacterial strains, environmental hazards, and food safety problems [4
6]. The frequent rise in the restrictions on the use of antibiotics and other chemotherapeutics has prompted the scientific community to look for safer and environment-friendly alternatives for the health management of the aquatic organisms. Use of immunostimulants is such an ecofriendly alternative measure to immunologically control diseases of aquatic organisms. Immunostimulants increase the immunocompetency and resistance to infectious diseases by stimulating the innate defense mechanisms of fish and shellfish [1]. Use of immunostimulants has now widely been adopted by the farming community to strengthen the immunity of fish and shellfish. A large number of compounds with highly diverse chemical structures, including microbial derivatives, animal and plant extracts, nutritional factors, synthetic chemicals, polysaccharides, and other substances, have been investigated as potential immunostimulants in fish and shellfish farming [1,7,8]. Polysaccharides are an important class of biomacromolecules abundantly present in plants, animals, and microbes and have been considered as broad-spectrum immunostimulants since the 1960s [8]. Chitin is a linear polymer of β-1,4-linked N-acetyl-d-glucosamine [9]. It is the second most abundant polysaccharide in nature and found in the exoskeleton of insects and crustaceans and also in the fungal and yeast cell wall [10]. Chitosan is a nontoxic, biodegradable, and biocompatible polycationic linear polysaccharide derived by partial deacetylation of chitin [11,12]. Both chitin and chitosan are widely used for pharmaceutical

Handbook of Chitin and Chitosan

24.2 Immunostimulatory effect of chitin on finfish

763

and biomedical applications [13]. In the recent past, chitin and chitosan have built up a lot of curiosity and interest among scientists and aquapreneurs as dietary additives in aquaculture, which is supported by a number of studies deciphering the beneficial effects (on growth, feed utilization, antimicrobial activity, modulation of intestinal microbiota, and immunity) of these polysaccharides on fish and shellfish [14,15].

24.2 Immunostimulatory effect of chitin on finfish The fundamental defense mechanisms in finfish are primarily governed by the innate immune system and the effects of any biological response modifier (e.g., immunostimulant) can be determined by measuring the responses of the components of innate immunity, especially phagocytes [16]. The effects of chitin on the immune system have been studied in various finfish species from a diverse geographical origin, primarily through dietary administration followed by measuring different immunological responses. These studies were aimed to elucidate the potential of chitin as an immunostimulatory additive for fish feed. The immunostimulatory potential of any substance can be primarily judged by studying in vitro responses of leucocytes when incubated with the immunostimulant. However, only a few studies have demonstrated the direct interaction of chitin with the leucocytes through in vitro measurement of immune responses. Leucocytes incubated in vitro with chitin remained viable up to 72 h as indicated by polyclonal activation of leucocytes [17
19]. Various innate immune responses, including superoxide anion production, myeloperoxidase activity, and nitric oxide production, were significantly enhanced in a dose-dependent manner following in vitro addition of chitin with headkidney leucocytes of catla (Catla catla) [19]. In another in vitro study, gilthead seabream (Sparus aurata) leucocytes phagocytosed only the chitin particles of ,10 μm but not the .10 μm ones [18]. These in vitro studies indicate that dose and particle sizes of chitin are important attributes to be considered while using it as a dietary supplement. In an early study on rainbow trout (Oncorhynchus mykiss), Sakai et al. [20] reported stimulated macrophage activities (phagocytosis and respiratory burst) in the fish injected with chitin. Intraperitoneal injection of chitin (particles of , 10 μm) into gilthead seabream showed significant enhancement in respiratory burst and phagocytic activity which peaked at 3 and 5 days postinjection, respectively, while natural cytotoxic activity peaked at 3 days postinjection and remained significantly elevated until 10 days postinjection [21]. Considering the facts that injection is not practical for the mass application of immunostimulants and that the oral administration is a nonstressful delivery method for mass

Handbook of Chitin and Chitosan

764

24. Chitin and chitosan as promising immunostimulant for aquaculture

application with minimum effort and cost, chitin administration through feed was also conducted in gilthead seabream [21]. Gilthead seabream was fed chitin-supplemented diets (25, 50, or 100 mg/kg), and lysozyme, natural hemolytic complement, respiratory burst, phagocytic, and cytotoxic activity were measured after 2, 4, and 6 weeks of feeding. Beside lysozyme activity, all other responses were significantly elevated by the dietary intake of chitin, each increasing at a different time. Juveniles of rohu (Labeo rohita) fed with 25 and 50 mg/kg chitin did not show any significant alteration in the respiratory burst activity after 60 days of feeding [22]. The authors speculated that the reasons could be the improper duration of feeding and low chitin dose to stimulate the immune system of rohu. In common carp (Cyprinus carpio), dietary administration of chitin (1%) for 90 days significantly elevated the lysozyme and superoxide anion production [23]. Harikrishnan et al. [24] determined the effects of chitin-enriched diets on the immune response of kelp grouper (Epinephelus bruneus) against Vibrio alginolyticus infection at weeks 1, 2, and 4. The lysozyme activity was significantly increased from weeks 1 to 4 in fish fed with 2% chitin diet, while phagocytic and complement activity remained unaltered. Dietary chitin (0.5%, 1%, and 2%) significantly stimulated superoxide anion production and lysozyme activity of catla in a dose-dependent manner. All the measured responses were higher after 1 week of feeding and declined thereafter [19]. In a more recent study, dietary chitin added with feed at 1%
2% significantly enhanced the myeloperoxidase and alkaline phosphate activity of rohu after 2
3 weeks of administration. However, no significant alteration in superoxide anion production was observed in rohu administered with chitin supplemented diets [25]. The combined effects of immunostimulant with other biological response modifiers (e.g., probiotic) have also been investigated in certain instances to achieve synergistic effects. For example, a diet comprising the probiotic Bacillus subtilis (109 cells/g) and chitin (2%) significantly enhanced some systemic and mucosal immune responses of catla [26]. Taken together, these studies indicate that chitin generally activates the innate immunity of finfish.

24.3 Immunostimulatory effect of chitin on shellfish The effects of chitin on the immune system of shellfish are relatively less studied compared to finfish. It is presumed that the shellfish defense system is completely dependent upon innate immunity. The activities of the innate immune factors of shellfish treated with immunostimulants can be detected by hemocyte-mediated responses, including phagocytosis, respiratory burst, and prophenoloxidase activity. For

Handbook of Chitin and Chitosan

24.4 Immunostimulatory effect of chitosan on finfish

765

example, white shrimp (Litopenaeus vannamei) injected with chitin (4 and 6 μg/g) showed significantly enhanced total hemocyte count, respiratory burst, phenoloxidase, and phagocytic activity, which were observed 1
6 days postinjection [27]. Adult shore crab (Carcinus maenas) showed significantly more hyaline hemocytes after 6 weeks of feeding with 10% chitin supplemented diet. However, the phagocytic activity of the hemocytes was unaffected by either 5% or 10% chitin supplementation [28]. In another study, Zhu et al. [29] reported significantly higher levels of total hemocyte count, prophenoloxidase, and superoxide dismutase activity in crayfish (Procambarus clarkia) fed with chitin at 5
15 mg/g for 4 weeks. Chitin supplemented diet (0.75%) also significantly increased the prophenoloxidase and respiratory burst activity of hemocytes of the freshwater prawn, Macrobrachium rosenbergii [30].

24.4 Immunostimulatory effect of chitosan on finfish Chitosan, prepared from alkaline deacetylation of chitin, has also attracted considerable attention in the aquaculture sector owing to its safety, biodegradability, biocompatibility, and nontoxicity [31]. Besides promoting the growth of fish, inhibiting aquatic pathogens, and purifying water in aquaculture, this biopolymer material has been found to stimulate and improve the immune status of different fish species primarily through activation of the innate immune components. For example, chitosan (1%) supplemented feed significantly enhanced the superoxide anion production and lysozyme activity of common carp under field conditions [23]. Significant enhancement of phagocytic, complement, and lysozyme activity was also observed in kelp grouper fed with chitosan (1% and 2%) for 1
4 weeks [24]. The innate immunological responses (phagocytic activity, superoxide anion production, lysozyme, and alternative complement activity) of Nile tilapia (Oreochromis niloticus) were significantly increased after 3 weeks of feeding with 1% chitosan [32]. However, Cha et al. [33] did not find any significant increase in the reactive oxygen species production and serum lysozyme activity of olive flounder (Paralichthys olivaceus) fed with chitosan (1%)coated diet. The combined effects of chitosan with probiotic were investigated in cobia (Rachycentron canadum) where the combination diet containing 1.0 g/kg B. subtilis and 6.0 g/kg chitosan significantly enhanced several innate immune parameters [34]. In another combination study, Victor et al. [35] reported significantly enhanced immunological parameters in loach (Paramisgurnus dabryanus) fed with selenium
chitosan combination diet. Dietary chitosan has also been found to enhance the immunocompetency through activation of the innate immune responses in several other fish species including Asian seabass (Lates calcarifer)

Handbook of Chitin and Chitosan

766

24. Chitin and chitosan as promising immunostimulant for aquaculture

[36], mrigal (Cirrhina mrigala) [37], gibel carp (Carassius auratus gibelio) [38], grey mullet (Mugil cephalus) [39], and juvenile loach (Misgurnus anguillicaudatus) [40]. Besides chitosan supplementation in fish feed, chitosan nanoparticles have also been explored for their potential immunostimulatory and other beneficial effects. For instance, dietary chitosan nanoparticles were found to improve certain indicators of innate immunity in Nile tilapia [41].

24.5 Immunostimulatory effect of chitosan on shellfish Like chitin, the effects of chitosan on the immune system of shellfish are not well studied compared to finfish. In one study, white shrimp injected with chitosan (2 and 4 μg/g) showed significantly enhanced total hemocyte count and respiratory burst activity after 2 days, phagocytic activity after 1 day, and prophenoloxidase activity for 6 days [27]. In another study, chitosan supplemented diet (0.05%
4.0%) showed immunostimulatory potential in Penaeus monodon as measured by total hemocyte count and hemolymph clotting time [42]. To the author’s knowledge, there is no other direct study reporting the effects of chitosan on immune parameters of shellfish, especially shrimps and prawns.

24.6 Effect of chitin and chitosan on disease resistance Immunostimulants increase resistances of fish and shellfish to infectious diseases and reduce the risk of disease outbreaks by stimulating the innate immunity [1]. As shellfish are solely dependent on innate immunity for their resistance to infections, immunostimulants may be particularly useful to render them more resistant to diseases [7]. Although some variation exists depending on the fish species, dose, duration, and other conditions of chitin and chitosan application, published literature indicate activation of the innate immunity of fish and shellfish by dietary administration of chitin and chitosan, in general. It is, therefore, expected that chitin and chitosan should also enhance the disease resistance ability of fish and shellfish. In fact, the majority of the researchers who investigated the immunostimulatory potential of chitin and chitosan in different fish and shellfish species, also observed their disease protecting ability against diverse pathogens. Administration of chitin increased resistance to a variety of pathogens responsible for disease outbreaks in aquaculture. Injection administration of chitin increased the resistance of rainbow trout to Vibrio anguillarum infection [20]. In contrast, injection of chitin failed to protect yellowtail (Seriola quinqueradiata) against Pasteurella piscicida infection

Handbook of Chitin and Chitosan

24.7 Mechanism of action of chitin and chitosan as an immunostimulant

767

[43]. Dietary chitin administration protected white shrimp to V. alginolyticus, common carp to Aeromonas hydrophila, shore crab and kelp grouper to V. alginolyticus, kelp grouper to a protozoan parasite (Philasterides dicentrarchi), mrigal to the oomycete Aphanomyces invadans, catla to Edwardsiella tarda, and rohu to gill monogenean infection [23
28,37]. However, chitin supplementation did not show any significant effect on the protection of rohu against A. hydrophila infection which might be due to insufficient dose and duration of chitin administration [22]. Similarly, protection was also not achieved in crayfish to white spot syndrome virus challenge [29]. Dietary administration of chitosan protected rainbow trout to A. salmonicida, white shrimp to V. alginolyticus, cobia to Vibrio harveyi, koi (Cyprinus carpio koi) to A. veronii, Asian seabass to V. anguillarum, kelp grouper to P. dicentrarchi, mrigal to Aphanomyces invadans, and Nile tilapia to A. hydrophila infection [27,32,34,36,37,44
46]. The findings collectively suggest the disease protecting abilities of chitin and chitosan at various application regimes.

24.7 Mechanism of action of chitin and chitosan as an immunostimulant As chitin or chitosan is not the natural constituent of fish cells, they are the potential target for recognition by the innate immune system of fish following exposure. The immunostimulatory potential of chitin and chitosan has been well demonstrated in fish showing activation of phagocytes as well as direct phagocytosis of chitin and chitosan particles. This indicates the role of specific receptors on the immune cells for recognition and subsequent activation. Although such kind of receptors has not been conclusively identified in fish, a chitin-binding interlectin-like protein has been identified in rainbow trout [47]. The protein (rainbow trout plasma interlectin) showed homology to human and murine interlectins and was detected in the gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder, and within the leucocytes of the rainbow trout. Although the protein has been detected in the sites of immune defense, its definitive role in the activation of phagocytes is yet to be demonstrated. Another type of high-affinity receptor for chitin and chitin fragments was identified in the vertebrate model [48]. The receptor, highly expressed in the gastrointestinal tract, is a novel 55-kDa type II homotetrameric transmembrane protein encoded by the FIBCD1 gene. The receptor protein is considered to be conserved among major vertebrate species, involved in endocytosis, and has high binding affinities toward acetylated components in a calcium-dependent manner. The authors

Handbook of Chitin and Chitosan

768

24. Chitin and chitosan as promising immunostimulant for aquaculture

speculated that the FIBCD1 might play an important role in controlling the exposure of intestine to chitin and chitin fragments, which was of great relevance for the modulation of the immune responses. Indirect evidence also suggests the involvement of certain other molecules including NKR-P1 (an activating receptor on rat natural killer cells), RegIIIγ (a secreted C-type lectin), galectin-3 (a lectin with affinity for β-galactosides), Toll-like receptor (TLR) 2, dectin-1, and mannose receptor in sensing chitin or chitin fragments in the mammalian system [49,50]. However, the presence of a specific chitin binding receptor in fish is yet to be demonstrated to elucidate the mechanism of chitin sensing by the fish immune system. It may be possible that intact and insoluble chitin may not be readily recognized by receptors on the immune cells, but soluble by-products of chitin digestion within the intestinal milieu can be recognized [50]. The mechanisms of action of chitosan in modulating the immune responses are not fully understood as well. It is speculated that the abundance of amino or imino residues in chitosan which can be recognized by the mannose/fucose receptors present on the leucocytes may be responsible for immune recognition and subsequent immune responses [33,51]. Therefore several aspects of the interactions between chitin and chitosan with the host’s immune system are not thoroughly understood yet and require more exploration.

24.8 Conclusions and future perspectives A great deal of research has been done on the stimulatory effects of chitin and chitosan on the immune system of fish and shellfish, which clearly indicate that they can be used as immunostimulatory additives in aquafeed. The followup impact of activation of the immune system effectively translates into enhanced protection from infectious diseases. The research observations also indicate that the immunostimulatory effects of chitin and chitosan are influenced by the species being tested, chitin and chitosan dose, duration of application, and size of chitin particle. Therefore optimization of conditions for chitin and chitosan application is very important not only to achieve maximum benefits but to prevent the possible negative effects, such as immune suppression and growth retardation. There is a need to further understand the mechanisms through which chitin and chitosan induce immune responses in fish and shellfish. The possible effects of interaction between the chitin/ chitosan and their receptors on the immune modulation, thus require more exploratory investigation. Moreover, future research should also be directed toward exploring synergistic effects when chitin and chitosan are used in conjunction with other biological response modifiers.

Handbook of Chitin and Chitosan

References

769

References [1] M. Sakai, Current research status of fish immunostimulants, Aquaculture 172 (1
2) (1999) 63
92. [2] M.G. Bondad-Reantaso, R.P. Subasinghe, J.R. Arthur, K. Ogawa, S. Chinabut, R. Adlard, et al., Disease and health management in Asian aquaculture, Vet. Parasitol. 132 (3
4) (2005) 249
272. [3] Y.B. Wang, J.R. Li, J. Lin, Probiotics in aquaculture: challenges and outlook, Aquaculture 281 (1
4) (2008) 1
4. [4] T. Aoki, Present and future problems concerning the development of resistance in aquaculture, Chemotherapy in Aquaculture: From Theory to Reality (1992) 254
262. [5] P.H. Serrano, Responsible use of antimicrobials in aquaculture, FAO Fisheries Technical Paper No. 469, FAO, Rome, 2005. [6] B. Austin, D.A. Austin, Bacterial Fish Pathogens: Diseases of Farmed and Wild Fish, fourth ed., Springer-Praxis, Chichester, UK, 2007. [7] J. Raa, The use of immunostimulatory substances in fish and shellfish farming, Rev. Fish. Sci. 4 (3) (1996) 229
288. [8] M. Wang, G. Liu, M. Lu, X. Ke, Z. Liu, F. Gao, et al., Effect of Bacillus cereus as a water or feed additive on the gut microbiota and immunological parameters of Nile tilapia, Aquacult. Res. 48 (6) (2017) 3163
3173. [9] D. Carlstro¨m, The crystal structure of α-chitin (poly-N-acetyl-D-glucosamine), J. Biophys. Biochem. Cytol 3 (5) (1957) 669
683. [10] R. Cheung, T. Ng, J.H. Wong, W.Y. Chan, Chitosan: an update on potential biomedical and pharmaceutical applications, Mar. Drugs 13 (8) (2015) 5156
5186. [11] T. Chandy, C.P. Sharma, Chitosan-as a biomaterial, Biomater. Artif. Cells Artif. Organs 18 (1) (1990) 1
24. [12] M.N.V.R. Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A.J. Domb, Chitosan chemistry and pharmaceutical perspectives, Chem. Rev. 104 (12) (2004) 6017
6084. [13] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. Structure, properties and applications, Mar. Drugs 13 (3) (2015) 1133
1174. [14] E. Ringo, Z. Zhou, R.E. Olsen, S.K. Song, Use of chitin and krill in aquaculture
the effect on gut microbiota and the immune system: a review, Aquacult. Nutr 18 (2) (2012) 117
131. [15] H.M. Abdel-Ghany, M.E.-S. Salem, Effects of dietary chitosan supplementation on farmed fish; a review, Rev. Aquacult (2019). Available from: https://doi.org/ 10.1111/raq.12326. [16] B. Magnado´ttir, Innate immunity of fish (overview), Fish Shellfish Immunol 20 (2) (2006) 137
151. [17] P. Pelegrin, V. Mulero, M.A. Esteban, P. Sepulcre, J. Meseguer, The in vitro addition of chitin particles to gilthead seabream leucocytes enhances macrophage-activating factor (MAF) production, in: Proceedings of Aqua 2000: Responsible Aquaculture in the New Millenium, Nice, France, 2000. ´ . Esteban, J. Meseguer, In vitro effect of chitin particles on the innate [18] A. Cuesta, M.A cellular immune system of gilthead seabream (Sparus aurata L.), Fish Shellfish Immunol 15 (1) (2003) 1
11. [19] T. Sangma, D. Kamilya, In vitro and dietary effects of chitin on cellular and humoral immune parameters of catla, Catla catla (Hamilton), J. World Aquacult. Soc. 46 (6) (2015) 617
623. [20] M. Sakai, H. Kamiya, S. Ishii, S. Atsuta, M. Kobayashi, The immunostimulating effects on chitin in rainbow trout, Oncorhynchus mykiss, in: Diseases in Asian aquaculture I (ed. by M. Shariff, R. P. Subasighe, J. R. Arthur), Fish Health Section, Asian Fish. Soc., Manila, 1992, pp. 413
417.

Handbook of Chitin and Chitosan

770

24. Chitin and chitosan as promising immunostimulant for aquaculture

[21] M.A. Esteban, V. Mulero, A. Cuesta, J. Ortuno, J. Meseguer, Effects of injecting chitin particles on the innate immune response of gilthead sea bream (Sparus aurata L.), Fish Shellfish Immunol 10 (6) (2000) 543
554. [22] D. Choudhury, A.K. Pal, N.P. Sahu, S. Kumar, S.S. Das, S.C. Mukherjee, Dietary yeast RNA supplementation reduces mortality by Aeromonas hydrophila in rohu (Labeo rohita L.) juveniles, Fish Shellfish Immunol 19 (3) (2005) 281
291. [23] A. Gopalakannan, V. Arul, Immunomodulatory effects of dietary intake of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds, Aquaculture 255 (1
4) (2006) 179
187. [24] R. Harikrishnan, J.S. Kim, C. Balasundaram, M.S. Heo, Immunomodulatory effects of chitin and chitosan enriched diets in Epinephelus bruneus against Vibrio alginolyticus infection, Aquaculture 326
329 (2012) 46
52. [25] R. Kumar, N. Kaur, D. Kamilya, Chitin modulates immunity and resistance of Labeo rohita (Hamilton, 1822) against gill monogeneans, Aquaculture 498 (2019) 522
527. [26] T. Sangma, D. Kamilya, Dietary Bacillus subtilis FPTB13 and chitin, single or combined, modulate systemic and cutaneous mucosal immunity and resistance of catla, Catla catla (Hamilton) against edwardsiellosis, Comp. Immunol. Microbiol. Infect. Dis. 43 (2015) 8
15. [27] S.H. Wang, J.C. Chen, The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei, Fish Shellfish Immunol 19 (3) (2005) 191
204. [28] A. Powell, A.F. Rowley, The effect of dietary chitin supplementation on the survival and immune reactivity of the shore crab, Carcinus maenas, Comp. Biochem. Physiol. A Mol. Integr. Physiol. 147 (1) (2007) 122
128. [29] F. Zhu, H. Quan, H. Du, Z. Xu, Y. Li, The effect of dietary chitosan and chitin supplementation on the survival and immune reactivity of crayfish, Procambarus clarkia, J. World Aquacult. Soc 41 (2010) 284
290. [30] B.N. Kumar, H.S. Murthy, P. Patil, P.L. Doddamani, R. Patil, Enhanced immune response and resistance to white tail disease in chitin-diet fed freshwater prawn, Macrobrachium rosenbergii, Aquacult. Rep. 2 (2015) 34
38. [31] W. Wang, J. Sun, C. Liu, Z. Xue, Application of immunostimulants in aquaculture: current knowledge and future perspectives, Aquacult. Res. 48 (1) (2017) 1
23. [32] N.M. Abu-Elala, S.H. Mohamed, M.M. Zaki, A.E. Eissa, Assessment of the immunemodulatory and antimicrobial effects of dietary chitosan on Nile tilapia (Oreochromis niloticus) with special emphasis to its bio-remediating impacts, Fish Shellfish Immunol 46 (2) (2015) 678
685. [33] S.H. Cha, J.S. Lee, C.B. Song, K.J. Lee, Y.J. Jeon, Effects of chitosan-coated diet on improving water quality and innate immunity in the olive flounder, Paralichthys olivaceus, Aquaculture 278 (1
4) (2008) 110
118. [34] X. Geng, X.H. Dong, B.P. Tan, Q.H. Yang, S.Y. Chi, H.Y. Liu, et al., Effects of dietary chitosan and Bacillus subtilis on the growth performance, non-specific immunity and disease resistance of cobia, Rachycentron canadum, Fish Shellfish Immunol 31 (3) (2011) 400
406. [35] H. Victor, B. Zhao, Y. Mu, X. Dai, Z. Wen, Y. Gao, et al., Effects of Se-chitosan on the growth performance and intestinal health of the loach Paramisgurnus dabryanus (Sauvage), Aquaculture 498 (2019) 263
270. [36] R. Ranjan, K.P. Prasad, T. Vani, R. Kumar, Effect of dietary chitosan on haematology, innate immunity and disease resistance of Asian seabass Lates calcarifer (Bloch), Aquacult. Res. 45 (6) (2014) 983
993. [37] L.S.S. Mari, C. Jagruthi, S.M. Anbazahan, G. Yogeshwari, R. Thirumurugan, J. Arockiaraj, et al., Protective effect of chitin and chitosan enriched diets on immunity

Handbook of Chitin and Chitosan

References

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49] [50] [51]

771

and disease resistance in Cirrhina mrigala against Aphanomyces invadans, Fish Shellfish Immunol 39 (2) (2014) 378
385. Y. Chen, X. Zhu, Y. Yang, D. Han, J. Jin, S. Xie, Effect of dietary chitosan on growth performance, haematology, immune response, intestine morphology, intestine microbiota and disease resistance in gibel carp (Carassius auratus gibelio), Aquacult. Nutr. 20 (5) (2014) 532
546. P. Akbary, A. Younesi, Effect of dietary supplementation of Chitosan on growth, hematology and innate immunity of grey Mullet (Mugil cephalus), Vet. Res. Biol. Prod. 30 (3) (2017) 194
203. J. Yan, C. Guo, M.A.O. Dawood, J. Gao, Effects of dietary chitosan on growth, lipid metabolism, immune response and antioxidant-related gene expression in Misgurnus anguillicaudatus, Benef. Microbes 8 (3) (2017) 439
449. F.S.A. El-Naby, M.A.E. Naiel, A.A. Al-Sagheer, S.S. Negm, Dietary chitosan nanoparticles enhance the growth, production performance, and immunity in Oreochromis niloticus, Aquaculture 501 (2019) 82
89. J. Niu, H.Z. Lin, S.G. Jiang, X. Chen, K.C. Wu, Y.J. Liu, et al., Comparison of effect of chitin, chitosan, chitosan oligosaccharide and N-acetyl-D-glucosamine on growth performance, antioxidant defenses and oxidative stress status of Penaeus monodon, Aquaculture 372 (2013) 1
8. H. Kawakami, N. Shinohara, M. Sakai, The non-specific immunostimulation and adjuvant effects of Vibrio anguillarum bacterin, M-glucan, chitin and Freund’s complete adjuvant against Pasteurella piscicida infection in yellowtail, Fish Pathol. 33 (4) (1998) 287
292. A.K. Siwicki, D.P. Anderson, G.L. Rumsey, Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis, Vet. Immunol. Immunopathol. 41 (1
2) (1994) 125
139. S. Lin, Y. Pan, L. Luo, L. Luo, Effects of dietary β-1, 3-glucan, chitosan or raffinose on the growth, innate immunity and resistance of koi (Cyprinus carpio koi), Fish Shellfish Immunol 31 (6) (2011) 788
794. R. Harikrishnan, J.S. Kim, C. Balasundaram, M.S. Heo, Dietary supplementation with chitin and chitosan on haematology and innate immune response in Epinephelus bruneus against Philasterides dicentrarchi, Exp. Parasitol. 131 (1) (2012) 116
124. S. Russell, K.M. Young, M. Smith, M.A. Hayes, J.S. Lumsden, Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin, Fish Shellfish Immunol. 25 (1
2) (2008) 91
105. A. Schlosser, T. Thomsen, J.B. Moeller, O. Nielsen, I. Tornøe, J. Mollenhauer, et al., Characterization of FIBCD1 as an acetyl group-binding receptor that binds chitin, J. Immunol. 183 (6) (2009) 3800
3809. C.L. Bueter, C.A. Specht, S.M. Levitz, Innate sensing of chitin and chitosan, PLoS Pathog. 9 (1) (2013) e1003080. D.E.A. Komi, L. Sharma, C.S.D. Cruz, Chitin and its effects on inflammatory and immune responses, Clin. Rev. Allerg. Immunol. 54 (2) (2018) 213
223. S. Tokura, H. Tamura, I. Azuma, Immunological aspects of chitin and chitin derivatives administered to animals, EXS 87 (1999) 279
292.

Handbook of Chitin and Chitosan

This page intentionally left blank

C H A P T E R

25 Chitosan-based materials for water and wastewater treatment Muhammad Zubair, Muhammad Arshad and Aman Ullah Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

O U T L I N E 25.1 Introduction

774

25.2 Various forms of chitosan-based sorbents for water/wastewater remediation 775 25.2.1 Raw or unmodified chitosan 775 25.2.2 Nanochitosan 777 25.2.3 Chitosan/biopolymer blend 778 25.2.4 Chitosan/nanoparticles composites 780 25.2.5 Chitosan hydrogels 781 25.2.6 Chitosan beads 782 25.3 Removal of different pollutants using chitosan-based material 25.3.1 Metal ions removal 25.3.2 Organic pollutants 25.3.3 Desalination 25.3.4 Antibacterial effect 25.3.5 Other pollutants

783 783 787 789 791 792

25.4 Mechanism of adsorption 25.4.1 Factors affecting adsorption performance

792 798

25.5 Summary and future perspective

800

References

801

Handbook of Chitin and Chitosan DOI: https://doi.org/10.1016/B978-0-12-817966-6.00025-X

773

© 2020 Elsevier Inc. All rights reserved.

774

25. Chitosan-based materials for water and wastewater treatment

25.1 Introduction Water is the most precious natural resource which is vital for all living organisms on the planet Earth. It is the most important commodity for modern societies because they use it for domestic purposes, in agricultural and industrial sectors, and in recreational activities [1,2]. Water preservation in terms of its safety in quantity and quality is essential for the development of a sustainable society [3,4]. Currently, scarcity of clean freshwater is one of the most challenging global issues that is predicted to get worse in the future due to rapid industrialization and greater energy needs for the ever increasing population of the world [5
7]. Around the globe, 1.2 billion people do not have access to clean drinking water, while 2.6 billion have poor or no sanitation. Countless people become sick due to contaminated water and millions die every year, including 3900 children a day [8]. Numerous methods have been established to resolve the issues related to water quality including reverse osmosis [9], nanofiltration [10], and disinfection using different chemicals [11]. There are a variety of adsorbents or chemicals that are being used in these technologies to remove particles, for example, heavy metals, microorganisms, and organic dyes, to make water drinkable. However, these treatment methods have several disadvantages as they are chemically and energetically very expensive and chemicals transfer into the water during purification that are detrimental to the environment. Such factors make their use difficult in the developing countries [12]. In addition, the adsorbing materials are occasionally replaced which limits the continuous operation and increases the overall capital cost [12
14]. In recent years, the development of biobased adsorbents, especially those comprising renewable and sustainable natural biopolymers, has increased greatly. Polysaccharides such as chitosan, modified chitosan, and its composites [15,16], are one of the promising choices that have atracted special attention as they are abundantly available in nature, renewable, sustainable, and have low cost and better performance, compared to other material used in the treatment of contaminated water [17,18]. There is a plethora of research data available that deals with the performance, modification, and characterization of chitosan as an adsorbent material for toxins, such as microorganisms, heavy metals, precious metals, fluoride, radionulcides, dyes, and phenols, from drinking or groundwater and industrial wastewater [19
21]. The chemical structure of chitosan is very unique and provides plenty of opportunities for its modification through physical or chemical approaches to broaden the applications for the removal of various contaminants from the water. Chitosan has both cationic and anionic moieties that make it suitable for the removal of pollutants present either in the form of

Handbook of Chitin and Chitosan

25.2 Various forms of chitosan-based sorbents for water/wastewater remediation

775

anions (reactive or direct dyes and acids) or cations (metals) [17]. So there are numerous reasons which enable materials made with chitosan to stop or at least minimize damage to water and the biosphere. Thus this chapter places an emphasis on recent advances in chitosan-derived materials for water and wastewater treatment applications. Herein, we discuss various forms of chitosan that have been used so far for water remediation. This chapter provides detailed recent valuable information about using chitosan-derived material to eliminate pollutants from polluted water. The chapter also explores the possibilities of adsorption mechanisms for chitosan and its derivatives during the removal process of pollutants from water. The factors affecting the adsorption process are also highlighted. The chapter ends with the current challenges and our viewpoint for chitosan-based material to be utilized to their full potential.

25.2 Various forms of chitosan-based sorbents for water/wastewater remediation The modification of chitosan is crucial to improve its adsorption capacity and broaden the applications for the removal of multiple contaminants in a single treatment and at low cost. The modification of chitosan changes the physicochemical properties of the chitosan and in turn its adsorption capacity is achieved to meet the specific application requirements. Thus, this section discusses both modified and unmodified forms (as shown in Fig. 25.1) of chitosan that have been studied so far for water and wastewater treatment.

25.2.1 Raw or unmodified chitosan The chitosan without any modification (in powder or flakes form) has been used for the removal of various contaminants from water. Chitosan is mostly derived from chitin in a solid form with high crystallinity called chitosan flakes. Various studies have been carried out on chitosan flakes/powder for dye removal and as an antimicrobial agent for aqueous solutions [22
24]. Chitosan in solution form was studied by Al-Manhel et al. [23] to disinfect the drinking water. The results demonstrated that most bacteria were removed from the water using chitosan solution. Chitosan for Cu(II) adsorption has been examined and compared with the cross-linked chitosan. They used glutaraldehyde (GLA), epichlorohydrin (ECH), and ethylene glycol diglycidyl ether (EGDE) for chitosan crosslinking [24]. The adsorption capacity (80.71 mg/g) of unmodified chitosan

Handbook of Chitin and Chitosan

776

25. Chitosan-based materials for water and wastewater treatment

Chitosan

Raw chitosan

Modified chitosan

Flakes

Nano chitosan

Powder

Chitosan/biopolymer blend Chitosan/ nanoparticle composites Chitosan hydrogels

Chitosan beads

FIGURE 25.1 Different forms of chitosan and its derivatives.

was better than the cross-linked chitosan materials. The GLA
chitosan, ECH
chitosan, and chitosan
EGDE showed 59.67 mg/g, 62.47 mg/g, and 45.94 mg/g adsorption capacities, respectively. Most importantly, Cu(II) could be recovered from the all adsorbents rapidly using an EDTA solution in water. Furthermore, authors claimed that adsorbents have the ability for regeneration and can be reused. Fluoride was removed using natural chitosan by electrodialysis from the underground brackish water reported by Sahli et al. The results showed that chitosan adsorption coupled with electrodialysis is a viable option for the removal of high contents of fluoride from brackish water [25]. Mostly, unmodified chitosan such as powder is used for dye removal from industrial wastewater. Dotto et al. used shrimp wastes for the elimination of dyes used in the food industry, that is, acid blue 9 and food yellow 3, using aqueous solution via a batch method [26]. The optimal conditions for maximum adsorption were determined by varying pH, time of contact, and rate of stirring rate and they found that adsorption performance was improved as time and pH were decreased. Furthermore, acid blue 9 dye adsorption was optimized using a 1-h contact with stirring of 150 rpm at pH 3 while in the case of yellow 3 a low stirring rate (50 rpm) was better while keeping pH and contact the same as for the acid blue dye. The adsorbent showed an adsorption capacity of 210 and 295 mg/g for acid blue 9 and food yellow 3, respectively.

Handbook of Chitin and Chitosan

25.2 Various forms of chitosan-based sorbents for water/wastewater remediation

777

The adsorption of Remazol black 13 dye was investigated using chitosan flakes from an aqueous solution. The adsorbent kinetic, isothermal, and thermodynamic studies were carried out to understand the mechanism behind the adsorption process [27]. The maximum adsorption capacity for the Remazol black 13 was found to be between 91.47 and 130.0 mg/g. The study showed that the presence of amino groups on chitosan offered sites for dye adsorption. The reactive dye adsorption kinetics were well fitted with pseudo-first as well as -second order, which revealed the greater effect of intraparticle diffusion during the adsorption, whereas the enthalpy value (0.212 kJ/mol) indicated the endothermic nature of the adsorption process. A study was reported to investigate the adsorption/desorption of a mixture of reactive dyes onto chitin and chitosan flakes or beads. Both chitosan flakes and beads-based adsorbents had adsorption capacities higher than the chitin adsorbent. Whereas the desorption efficiency of chitin was better than the other chitosan adsorbents [28]. Nevertheless, unmodified chitosan could remove heavy metals and dyes, and have an antimicrobial effect. However, the modification of chitosan has more profound effects for the removal of contaminants compare with unmodified chitosan.

25.2.2 Nanochitosan Nanochitosan is an environment-friendly materials with exceptional physicochemical properties which make it suitable for use for improving the strength and washability of textile supplies and offering them antibacterial properties [29]. Currently, the use of nanosize adsorbents has been increasing tremendously compare with the traditional microsize supports owing to their better performance. The nanosize materials are small in size (nanosize in at least in one dimension), have a large surface area, quantum size effect [30], and good diffusivity that leads to better adsorption performance [22]. Nanochitosan can be synthesized using various methods such as emulsion droplet coalescence methods, cross-linking, either covalent or ionic, and coagulation or precipitation [31]. Chitosan nanoparticles are formed because of the interaction present between the macromolecules of opposite charges. Commonly, nanochitosan is prepared by the incorporation of tripolyphosphate into chitosan solution and forms gel through ionic interactions [32]. Nanochitosan is being used to remove heavy metals, organic dyes, and as an antibacterial agent. Recently, nanochitosan/sodium alginate/ microcrystalline cellulose beads were reported for lead removal from aqueous solution while using tripolyphosphate as an anionic crosslinking agent [33]. The study revealed that the optimum pH for lead adsorption was 6 at 50 C with 62.5 mg/L initial metal concentration.

Handbook of Chitin and Chitosan

778

25. Chitosan-based materials for water and wastewater treatment

The adsorption could be explained very well with Freundlich isotherm and pseudo
second order kinetics. Similarly, in another study, tripolyphosphate was used as a cross-linking agent to convert chitosan into nanochitosan and used it as a flocculant for harvesting microalga Nannochloropsis sp. The flocculants (chitosan and nanochitosan) performance were evaluated on the basis of biomass recovery. The results showed that the use of flocculant was reduced (40%) while biomass recovery was increased (9%) when nanochitosan was used in place of chitosan. Furthermore, the water from this process was recycled which improved the microalgae growth around 7% [34]. Sometimes, the modification of nanochitosan is required to increase further its adsorption capacity or multiple metal removal using a single adsorbent. Nanochitosan has also been used for the removal of multiple heavy metals (Cu21, Cd21Hg21 and Pb21) in a single treatment, as reported by Mahmoud et al. First, they prepared nanochitosan using chitosan and tripolyphosphate ionic gelation followed by modification with acetophenone through condensation reaction [35]. Acetophenonemodified nanochitosan showed better extraction for all the metals in comparison to only nanochitosan-based absorbent. The results indicated that the metal sorption capacity at pH 7.0 in case of acetophenonemodified nanochitosan was higher (1298
1608 μmol/g) compared with nanochitosan-based adsorbent (810
1236 μmol/g). This was further verified by using this adsorbent for metal ions removal (divalent) from seawater, tap water, and industrial wastewater, with adsorption capacity between 91.3
100.

25.2.3 Chitosan/biopolymer blend There are a number of biopolymers that have been used with chitosan to develop blended adsorbents for the removal of contaminants from drinking water and wastewater. Cellulose, gelatin, and alginate are the most studied biopolymers to blend with chitosan-based adsorbent for decontamination purposes [36
39]. Luo et al. prepared an effective and recyclable magnetic chitosan/cellulose microspheres adsorbent using the sol
gel method to remove heavy metal ions from aqueous solution. The performance of the adsorbent was tested for the removal of Pb21, Cd21, or Cu21 and it was found out that selectivity order in a multimetal ion system was Pb21 . Cd21 . Cu21 [40]. A study was also reported on the removal of heavy metal ions from aqueous solution using chitosan/cellulose-based hydrogel by the batch adsorption method. They prepared hydrogels by long irradiation of concentrated carboxymethylated cellulose solution (CM-cellulose) and chitosan. The results showed that the incorporation of chitosan improved the degree of cross-linking as well as the adsorbing capacity of the blended hydrogels.

Handbook of Chitin and Chitosan

25.2 Various forms of chitosan-based sorbents for water/wastewater remediation

779

Experimental data related to Cu21 were examined for kinetic and isothermal studies. The adsorption of copper was found to be in accordance with a Langmuir isotherm. The mechanism for Cu(II) adsorption was proposed to be a complexation of the Cu(II) with the hydrogel carboxyl and amino groups. The researchers concluded that the cellulose/chitosan blend hydrogels are promising for the removal of heavy metal from water [41]. Nair et al. prepared chitosan
alkali lignin composites for the adsorption of Cr(VI) and anthraquinonic and Remazol Brilliant Blue R (RBBR) dyes from wastewater [42]. They optimized the chitosan concentration, initial pH, and effective dose of adsorbent to remove maximum contaminants from the water. The studies showed that the composite showed maximum removal of both dyes and Cr(VI) with chitosan
alkali lignin (50:50) compared with the other prepared composites. The adsorption mechanism involved an electrostatic interaction between adsorbent and the anionic group (SO32) of dye and HCrO42 of chromium(VI). Additionally, a chemical interaction between amino or hydroxyl groups of the adsorbent and the carbonyl moiety of the dye was also proposed. Similarly, Albadarin et al. explored the adsorption of methylene blue using activated lignin
chitosan extrudates. The study showed that the electrostatic attraction was developed between a quaternary nitrogen on the methylene blue and an oxygen anion on the adsorbent [39]. Alginate is another biopolymer that has been used to develop chitosan-based adsorbent for the removal of water contaminants. Removal of p-chlorophenol was reported using chitosan and sodium alginate from synthetic aqueous solutions. The obtained beads were tested for adsorption efficiency under batch equilibrium and dynamic column flow. The study found that the maximum monolayer adsorption capacity of beads was 127 mg/g and suggested chitosan
calcium alginate blend was very efficient for the removal of p-chlorophenol from an aqueous medium [43]. In the second study by the same group, three adsorbents, that is, pure calcium alginate, chitosan-coated calcium alginate, and chitosan-coated silica, were prepared and investigated as adsorbents for the removal of Ni(II) using the same experimental conditions. The results were very interesting and adsorption capacity for Ni(II) ions of both chitosan-coated calcium alginate and chitosan-coated silica were lower than the calcium alginate [38]. Another biopolymer, gelatin, has been used with chitosan (ternary nanoparticles) and nonliving biomass (Pseudomonas sp.) to remove Cd(II) from contaminated wastewaters. The results showed that the adsorbent can be used as a potential adsorbent for cadmium removal [44]. Wang et al. prepared chitosan-based semiinterpenetrating polymer network (IPN) hydrogels for Cu21 adsorption. The results showed that hydrogel with gelatin contents (2 wt.%) had the maximum adsorption

Handbook of Chitin and Chitosan

780

25. Chitosan-based materials for water and wastewater treatment

capacity (261.08 mg/g), with recovery around 95%. Moreover, the study suggested ion exchange and complexation modes of interactions during the adsorption of Cu21 ion [45]. In another study, chitosan/gelatinbased hydrogels were used for dye removal. Hydrogels were modified by introducing ion exchanger [zirconium(IV) selenophosphate] in microwave conditions. The obtained polymer network was found to be very selective for the removal of cationic dyes [37]. Chitosan-based biopolymer composite was also prepared from D. novaehollandiae feathers as a novel material for the adsorption of Pb (II) in the concentration range between 20
100 mg/L. The study proposed that the adsorption of Pb(II) is probably because of either chemisorption, metal complexation (between amine or hydroxyl group of the adsorbent and metal), or ion exchange [46].

25.2.4 Chitosan/nanoparticles composites Chitosan has been blended with various nanoparticles, such as bentonite, ZnO, and MMT [47
49], for developing composites for water treatment. Chitosan/zinc oxide-derived bionanocomposites were prepared and tested for the removal of direct blue 78 and acid black 26 dyes from textile manufacturing wastewater. The study data showed that the zinc oxide nanoparticles were immobilized onto the chitosan surface. The adsorption of acid black 26 and direct blue 78 were fitted with Langmuir and Tempkin isotherms, respectively, while kinetics for both were correlated with pseudo-second order [50]. Motshekga et al. reported an interesting study related to water disinfection. They prepared chitosan-derived nanocomposite with silver and zinc oxide nanoparticles using bentonite as a support and tested for bacterial inactivation in water. Nanocomposites for antibacterial activity were examined in water using Gram-negative and Gram-positive bacteria and compared with antibacterial activity of GLA crosslinked chitosan. The authors indicated bacterial inactivation was improved by the incorporation of nanoparticles (silver and zinc oxide
bentonite support) and showed maximum removal efficiency of at least 78% [48]. Nanosized γ-Fe2O3 was used to develop chitosan/kaolin composites by a microemulsion process and material was examined using wideangle XRD, SEM, and TEM. The analysis determined the pores and pleats existence on the composites surface that provided more opportunities for dye adsorption. Methyl orange (MO) dye was used to test the adsorption capacity of the composites. The results showed that 71% of MO was adsorbed at pH 6.0 by the adsorbent dose of 1.0 g/L. The authors recommended these composites for the removal of anionic dyes from industrial wastewater [51].

Handbook of Chitin and Chitosan

25.2 Various forms of chitosan-based sorbents for water/wastewater remediation

781

A chitosan-based composite was synthesized using Al2O3/magnetic iron oxide nanoparticles for the adsorption of MO from aqueous solution. The properties of the composite were examined by EDAX (Energy Dispersive spectrometer), BET (Brunauer
Emmett
Teller) specific surface area, XRD, TGA, SEM, and IR. The removal of dye with the nanocomposite adsorbent was explored by a batch adsorption method by varying adsorbent dose, pH, and initial dye concentration. The results demonstrated that 0.4 g/L was an optimum adsorbent amount in the pH range between 4
10. Furthermore, the adsorption isotherm was well fitted with the Langmuir model and exhibited the highest (417 mg/g) adsorption capacity at 25 C. The experimental data showed that diffusion and intraparticle diffusion were involved in the adsorption process [49].

25.2.5 Chitosan hydrogels Presently, hydrogels have become of special interest due to their promising properties, as they are very soft, smart, and have good capacity to store water [52,53]. The ability of the hydrogels to absorb water is due to the presence of hydrophilic functional groups in the polymer backbone while they are resistant to dissolution as cross-links exist between the network chains [54]. Among the hydrogels, pH-sensitive hydrogels are very good contenders as they have functional groups which are ionic in nature and are suitable for the separation and removal of metal ions or ionic dyes from wastewater. A chitosanderived hydrogel has been prepared by Milosavljevic et al. using ionic cross-linking of chitosan with itaconic acid followed by free radical polymerization and cross-linking by the addition of methacrylic acid. The resulting hydrogel was characterized by SEM/EDX, FTIR, TGA, and AFM analyses. The maximum adsorption (285.7 mg/g) of hydrogel was found at pH 5.5 and the adsorption/desorption process suggested that the hydrogel has the ability to be used as material to adsorb Cd21 in aqueous solution [55]. Vieira et al. reported chitosan-based hydrogel and chitosan/montmorillonite (acid activated) composite hydrogels, prepared by the copolymerization of radical chitosan, acrylic acid (AA), and N,N0 -methylenebisacrylamide. The composite hydrogels were tested for the adsorption of Pb21 and Ni21 in aqueous solutions. The authors mentioned that the adsorption mechanism was highly dependent on the aqueous solution pH and transfer between non-Fickian and Fickian transports. The adsorption capacities of the chitosan-based hydrogel were 41.06
30.20 mg/g for Pb21 and 42.38
36.45 mg/g for Ni21 at a pH range of 5.5
3.5, while adsorption capacities of the chitosan/acid-activated montmorillonite composite hydrogel ranged from 35.22
26.11 mg/g for Pb21

Handbook of Chitin and Chitosan

782

25. Chitosan-based materials for water and wastewater treatment

and 37.16
42.04 mg/g for Ni21 in the pH range of 5.5
3.5 [56]. In another study, the same group of researchers reported the same hydrogel for the removal and adsorption of chromium(VI) in an aqueous solutions. The results indicated that maximum adsorption capacities of the chitosan-based hydrogel for Cr(VI) were 73.14 by nonlinear Langmuir model and 93.03 mg/g using Sips isotherm models [57].

25.2.6 Chitosan beads The presence of hydroxyl and amino groups provide chitosan with a great opportunity to harvest the chitosan into beads with excellent properties, such as acid stability, better strength, variable pore size, and high surface area. The chitosan beads have also been cross-linked using different agents to obtain better surface functionality and textural properties that improve interfacial interactions and give better adsorption performance [58]. Sutirman et al. developed poly(methacrylamide) graft chitosan beads and investigated them for the removal of Cu(II) and Cd(II) from single metal ion solution. The chitosan bead modification showed a substantial improvement in terms of acid resistance. The study concluded that the maximum adsorption for Cu(II) and Cd(II) were 140.9 mg/g at pH 4 and 178.6 mg/g at and pH 5, respectively. The Langmuir equation and pseudo-second order were best fitted with the adsorption data and suggested chemisorption or electron transfer were involved during the adsorption of the metal ions onto the beads [59]. Chitosan beads have also been synthesized and reported for the removal of dyes. Recently, magnetic chitosan nanocomposite beads have been cross-linked using H2SO4 and used for methylene blue adsorption. The study showed that the adsorption capacity of the absorbent was increased by increasing the solution pH and the maximum adsorption capacity of the beads was 20.408 mg/g for methylene blue [60]. Protonated cum carboxylated cross-linked chitosan beads were synthesized to offer more NH31 and COOH groups to the beads for the removal of fluoride. The modified chitosan beads exhibited maximum adsorption of 1800 mg /F/kg while chitosan beads had only 52 mg/F/kg. Most importantly, the study indicated that the adsorption process was pH independent and affected to some extent by the existence of other anions [61]. The same group reported enriched fluoride adsorption in batch mode by chitosan beads supported mixed metal oxides of lanthanum(III) and zirconium(IV). However, in this study, they mentioned that the adsorption process is dependent on pH and maximum adsorption was obtained at pH 7. The kinetic study indicated that the experimental data was agreed with the Langmuir isotherm. Furthermore, at room temperature 50 min was an optimum equilibrium time while using 100 mg/L adsorbent

Handbook of Chitin and Chitosan

783

25.3 Removal of different pollutants using chitosan-based material

dose. Thermodynamic data showed that the process of fluoride sorption was spontaneous and endothermic [62]. Recently, chitosan beads have also been reported to remove the U(VI) from an aqueous solution. Magnetic amidoxime-functionalized chitosan beads were synthesized by Zhuang et al. and tested for U(VI) adsorption. The maximum adsorption capacity of the beads for U(VI) was found to be 117.65 mg/g at pH 6 which was ascribed to the coordination ability of amidoxime groups [63].

25.3 Removal of different pollutants using chitosan-based material Chitosan and its derivatives have been widely studied for the removal of a number of pollutants (as shown in Fig. 25.2) from water and wastewater. Dyes and heavy metals are the most common pollutants that are removed and in some cases recovered by using chitosan and modified chitosan. They have also been used for desalination purposes and as an antimicrobial agent.

25.3.1 Metal ions removal Metals ions water pollution is very common and usually generated by industrial activities. They can be toxic and affect living organisms. Biosorption using chitosan is an effective way for their adsorption from water and wastewater. Chitosan and its derivatives have been used to adsorb heavy metals (Cu, Pb, Ag, Cr, Ni, As, etc.), precious metals (Pd, Au, Pt, etc.), and radionuclides. Li et al. reported the modified chitosan material for the removal of Cr(VI). First, they prepared magnetic β-cyclodextrin
chitosan (CC) which has more adsorption capacity and better separation properties. Further, graphene oxide (GO) was introduced to chemically bind with the CC to enhance its adsorption performance for metal removal. The results showed that magnetic β-cyclodextrin
chitosan/graphene oxide Types of pollutants Metal ion removal

Organic pollutants

Desalination

Antibacterial

Other pollutants

Heavy metals

Organic dyes

NaCl removal

Oil/organic separation

Other metals

Other organic pollutants

Other salts

Antifungal

FIGURE 25.2

Types of pollutants removed using chitosan and its derivatives.

Handbook of Chitin and Chitosan

784

25. Chitosan-based materials for water and wastewater treatment

(CCGO) adsorption capacity for Cr(VI) had better removal (67.66 mg/g) than other sorbents such as ethylenediamine-functionalized Fe3O4 (61.35 mg/g) and poly(4-vinylpyridine) modified activated carbon (53.7 mg/g). This was ascribed to the large surface area due to a higher number of hydroxyl and amino groups and Fe3O4 magnetic behavior. The study found CCGO adsorption equilibrium paralleled to the Langmuir isotherm model and removal efficiency was better at low pH. They concluded that CCGO adsorption performance was highly dependent on the CCGO specific surface area and charge concentration [64]. In another study, GO was used to functionalize chitosan for arsenic (III and V) removal in aqueous solutions. The adsorption of arsenic was elucidated using Freundlich and Langmuir models while the kinetics were discussed using a pseudo
second order model. The adsorption capacity was found to be in agreement with a Langmuir isotherm model, that is, 64.2 mg/g for As(III) and 71.9 mg/g for As(V), whereas the optimum pH for removal of the As(V) was found to be 4.3
6.5 [65]. Montmorillonite/chitosan composite was prepared and its adsorption in terms of kinetic, thermodynamic, and equilibrium for Pb12, Cu12, and Cd12 were tested and compared. The composite was characterized using XRD and FTIR. Results indicated MMT
chitosan composite adsorption toward the metals in the order of Cd21 , Cu21 , Pb21 which was attributed to the electronegative behavior of metal ions, that is, the higher the electronegativity the greater is the adsorption capacity. The adsorption capacities in a binary system were better in a single system and the adsorption of each cation could increase due to the presence of other cations in the binary system. The adsorption kinetics of the metals was well fitted with the pseudo
secondorder equation. The adsorption process was exothermic on the composite surface at a temperature between 25 C and 50 C [66]. Lie et al. reported carboxylate-rich magnetic chitosan flocculants (chitosan surface graft copolymerization on magnetite particles) to remove Ni(II) from synthetic wastewater. They also studied the role of different ionic groups (amino, hydroxyl, and carboxyl) for metal removal. The successful grafting, magnetic behavior, and core
shell structure were studied using SEM, XPS, FITR TEM, XRD, and TGA. The kinetic data determined Ni(II) flocculation by surface graft magnetic chitosan and reached equilibrium (uptake rate 98.3%) within ,60 min (pH 4.0
8.0) for its removal. Carboxylate-rich magnetic chitosan flocculants showed better performance in terms of flocculation when carboxyl groups were introduced by the itaconic acid as compared to chitosan magnetic flocculant. The authors claimed that recycling of flocculants is easy, and separation is cheap and rapid with excellent adsorption capacity [67]. Recently, three different aerogels (wastepaper, chitosan, and wastepaper/chitosan aerogels) were prepared for Cu(II) adsorption. The results

Handbook of Chitin and Chitosan

25.3 Removal of different pollutants using chitosan-based material

785

indicated that wastepaper/chitosan had better Cu(II) adsorption capacity (156.3 mg/g) compare with chitosan aerogel (35.08 mg/g) and chitosan/ cellulose microspheres (88.2 mg/g). The improved mechanical strength and acid resistance of the wastepaper/chitosan aerogel was ascribed to strong hydrogen bonding between the paper cellulose and chitosan. The metal adsorption phenomenon was in accordance with the Langmuir model and kinetic results were well fitted with the pseudo-second order. Furthermore, the adsorption process showed the formation of a coordination complex between amino groups of chitosan and Cu21 [68]. Kong et al. synthesized a chitosan solution in acetic acid for Cu(II) and Ni(II) homogeneous adsorption in a single and binary system. First, chitosan and metal ions solution were mixed for the rapid chelation between metals and chitosan. Then the formation of chitosan
metal ion nanoparticles occurred due to the addition of sodium phytate and finally separation using centrifugation. The adsorption performance of chitosan solution for metal ions was tested by varying adsorption conditions. The data showed that the chitosan adsorption process was in agreement with the Langmuir model while its kinetics followed pseudo-second order. The chitosan solution adsorption capacity was found using the Langmuir isotherm and it was 384 mg/g for Ni(II) and 450 mg/g for Cu(II) at pH 5.2 and 30 C. The study claimed that adsorption capacities were many times higher than for the raw chitosan resin reported in the literature [69]. Novel chitosan-based adsorbents were prepared from chitosan crosslinking using three amino acids (glutamic acid, aspartic acid, and alanine) ionic liquids for lead(II) removal. The sorbents’ performances were studied using EDS, FTIR, XRD, and SEM. The results showed that chitosan
glutamic acid-derived adsorbent had the best Pb21 adsorption capacity (91%) at pH 4 and 30 C. The best behavior of glutamic acid
chitosan absorbent toward lead(II) removal was attributed to its more active sites (two carboxylic groups), and they had the strongest steric and electronic effects compared with other two amino acids crosslinked chitosan adsorbents. The adsorption kinetics behavior was in accordance with pseudo-second order while the Freundlich isotherm model was found to be best to describe the adsorption processes [70]. Recently, low-concentration adsorption of As(V) and Hg(II) from drinking water was reported by using a novel adsorbent, that is, biomimetic SiO2
chitosan composite. In this study, chitosan nanoparticles were coated on silica that offered more functional moieties for the adsorption of As(V) and Hg(II). The SiO2 formed hollow leaf-like structures which avoided agglomeration and chitosan loss, thus promoting an effective interaction between the chitosan functional groups and heavy metal ions. The results showed that SiO2/chitosan composite had fast adsorption process (over 60% within 2 min) with a capacity of 204.1 mg/g for Hg(II) and 198.6 mg/g for As(V) [71].

Handbook of Chitin and Chitosan

786

25. Chitosan-based materials for water and wastewater treatment

Chen et al. reported the functionalization of magnetic chitosan using ethylenediaminetetraacetic acid (EDTA) for the removal of Cu(II)), Pb(II), and methyl blue (MB) simultaneously from complex wastewater. The data indicated that EDTA (chelating groups) is primarily involved in heavy metals binding, whereas anionic dyes adsorbed to chitosan (protonated amino groups) via electrostatic interaction. Thus the adsorption of both metals and dyes occurred in a single treatment. The study found that adsorption was in accordance with Langmuir isotherm and pseudo
second order kinetics. The adsorption capacity of the chitosan (EDTA functionalized) composite was 225.0 and 220.0 mg/g for Cu(II) and Pb(II), respectively. The adsorption capacity of Cu(II) and Pb(II) was improved with increasing concentration of methylene blue, which was the most interesting finding of the study. These results established the synergetic adsorption performance of modified chitosan composite for cationic heavy metals and anionic organic dye. This study offers some basic understanding of the adsorption of multiple pollutants in a complex wastewater system [72]. Recently, a green approach has been reported to prepare novel spherical hydrogel capsules by partial ionic cross-linking of carboxylated chitosan (CCS). 2-Acrylamido-2-methyl-propanesulfonic acid (AMPS) and AA were used as monomers for the synthesis of P(AMPS-co-AA) hydrogel particles with excellent reusability for the Ni(II), Cd(II), Cu(II), and Ag(I) adsorption. The study indicated maximum adsorption capacities of 171.6, 220.5, 366.0, and 668.4 mg/g for Ni21, Cd21 Cu21, and Ag1, respectively [73]. Chitosan/GO
derived composites have also been reported for the removal of the precious metals of Au(III) and Pd(II). In this study, chitosan/graphene oxide (CSGO) composites were prepared with GO (5, 10, and 15 wt.%) to remove Au31 and Pd21 from an aqueous solution. The performances of the composites were studied and compared using surface area analyzer, FTIR, and XRD. The optimum pH for Pd(II) adsorption was 3.0
4.0 and it was 3.0
5.0 for Au(III) adsorption. The study showed that the adsorption capacity of Au(III) and Pd(II) was first improved by an increase in pH and later reduced at higher pH 5
6. That was ascribed to the formation of hydrolyzed chlorogold complex and Pd(OH)2 which could be precipitated and make the Au(III) and Pd(II) ions adsorption inaccessible. Both Langmuir and Freundlich isotherms were applied to the experimental data and Langmuir was found to best fit on the adsorption of Au(III) and Pd(II). Chitosan with 5 wt.% graphene oxide (CSGO5) was the best composite for the adsorption of Au(III) when compared with the other adsorbents. The maximum adsorption capacity for Au(III) was 1076.649 mg/g, while 216.920 mg/g was found for Pd(II) [74]. The removal and recovery of uranium (U) from seawater has also been reported using modified chitosan/bentonite composite recently. A

Handbook of Chitin and Chitosan

25.3 Removal of different pollutants using chitosan-based material

787

novel poly(amidoxime)-grafted chitosan/bentonite composite [P(AO)-g CTS/BT] was prepared from ethylene glycol dimethacrylate (crosslinker) and potassium peroxy disulfate (radical initiator) using in situ intercalative polymerization of acrylonitrile and 3-hexenedinitrile over the chitosan/bentonite composite. The material characterization was done with SEM, EDS, XRD, BET, FTIR, and XPS. The study ascribed the improved adsorption efficiency of uranium(VI) from seawater to the formation of amidoxime groups from two monomer nitrile groups. The U(VI) maximum adsorption was found to be 49.09 mg/g at 8 pH. The adsorption kinetics data of U(VI) over adsorbent were pseudo-second order and showed an ion exchange mechanism followed by complexation. Whereas the isotherm data agreed with the Langmuir model confirming the formation of U(VI) monolayer on the P(AO)-g-CTS/BT. Furthermore, an increase in temperature caused an increase in adsorption that showed the endothermic nature of the adsorption process [75].

25.3.2 Organic pollutants Thousands of organic pollutants are discharged into the environment from chemical, textile, fertilizers, and pharmaceutical industries. Organic dyes, phenols, hydrocarbons, pesticides, plasticizers, detergents, and grease are just a few examples. Among organic pollutants, chitosan and its derivatives are generally used for the removal of dyes from water and wastewater. Methylene blue is one of the most studied organic dyes using chitosan or chitosan derivatives from industrial wastewater. Fan et al. reported a novel magnetic chitosan/graphene oxide (MCGO)-derived adsorbent for the removal of MB. The adsorbent (MCGO) was characterized using XRD, SEM, and FTIR to elucidate the structural and morphological changes as well as magnetic property. MB adsorption was analyzed by changing pH, temperature, adsorption time, and initial concentration of MB. The results indicated an optimum pH of 5.3 for adsorption kinetics and isotherm. The data was best fitted to pseudo
second order and Langmuir isotherms. Adsorption of methylene blue with MCGO (95.16 mg/g) was higher than magnetic chitosan (60.4 mg/ g). Thermodynamic values showed that the process was exothermic and spontaneous. Furthermore, the MB was adsorbed onto the adsorbent surface through an ion exchange mechanism [76]. In another study, Tanhaei et al. synthesized novel xanthate-modified magnetic chitosan and used it for the adsorption of mixture of cationic dyes, that is, methylene blue and safranin O. The study demonstrated that chitosan modification with xanthate greatly improved its adsorption for methylene blue and safranin O. The optimum pH for the adsorption process was found to be 4
11. Similar to the study reported

Handbook of Chitin and Chitosan

788

25. Chitosan-based materials for water and wastewater treatment

by Fan et al., kinetic data were in accordance with the pseudo
second order model while film diffusion and intraparticle diffusion were the controlling factors. The equilibrium data were found to be fitted by the Langmuir and Sips isotherm model. The maximum adsorption capacity was found to be at 35 C, that is, 197.8 mg/g for methylene blue and 169.8 mg/g for safranin O. The thermodynamic investigations determined the adsorption process was irreversible, endothermic, and spontaneous [77]. Magnetic chitosan/GO composite has also been used to adsorb a mixture of cationic dye, that is, methyl violet, and anionic dyes, that is, alizarin yellow R(AY), from industrial wastewater. The compositional and surface properties of the composite were investigated by using FTIR, EDX, SEM, XRD, and TGA. The removal performance of the magnetic chitosan/GO composite was determined at a pH between 4 and 12, duration of 0
80 min, 2
10 mg of composite dose, 2
30 μg m/L of initial dye concentration, and at a temperature of 303, 313, and 323 K. The adsorption kinetics for these two dyes also followed the pseudo
second order equation. The equilibrium for interaction between dye and adsorbent was in accordance with Langmuir and Freundlich isotherms [78]. Chitosan-based nanocomposite films were used for the adsorption of acid black 1 (AB 1) from aqueous solution. The biobased films were synthesized using chitosan, poly(vinyl alcohol) (PVA), and ZnO nanoparticles by a solution casting method. The photoluminescent properties of chitosan/poly(vinyl alcohol)/ZnO films were compared with the chitosan/poly(vinyl alcohol)-based films. The fluorescence and UV
vis absorption spectra were taken with a fluorescence spectrometer and spectrophotometer, respectively. The study suggested that the incorporation of ZnO enhanced the red shift and intensities of the chitosan/PVA composite, which is ascribed to the cationic coordination formation in the chitosan/PVA/ZnO composite, where nitrogen or oxygen in the chitosan provide complexation sites. Moreover, the study showed that ZnO’s presence in the CS/PVA/ ZnO film improved the adsorption performance as well as the stability of the films. The most prominent part of the study that makes it different from other studies, is the investigation of toxicity using MMT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The study concluded that the viability of chitosan/PVA/ZnO films was better than pure chitosan and PVA films [79]. Xu et al. synthesized chitosan-derived adsorbent by surface grafting of poly(2-(dimethylamino) ethyl methacrylate) on magnetic chitosan microspheres for anionic dyes adsorption from water. The adsorbent showed improved adsorption performance for acid green 25 and reactive blue 19 as compared to magnetic chitosan microspheres. The study

Handbook of Chitin and Chitosan

25.3 Removal of different pollutants using chitosan-based material

789

specified efficient adsorption of dyes was carried out because of two types of interactions: electrostatic interactions which were developed between cationic tertiary amine functional groups and dye molecules, and hydrogen bonding established between the dye secondary amine groups and ester carbonyl group of the grafted polymer. Most importantly, even after five adsorption
desorption cycles regenerated adsorbent can be used for adsorption with high (about 90%) recovery efficiency [80]. Chitosan biocomposite adsorbents were developed to separate the mixture of cation dyes (safranine T and brilliant cresyl) from water. N-vinylpyrrolidone and AA (molar ratio 1.3:1) showed a high adsorption capacity of 362 and 398 mg/g at a pH 7 for safranine T and brilliant cresyl, respectively. The experimental equilibrium adsorption data for a single dye system was very well fitted to the Fritz
Schu¨lnde model, whereas the combined Langmuir
Freundlich model was well correlated for the binary dye mixture [81]. Nitrophenol is another organic pollutant which has been eliminated from the real water using chitosan-based nanocomposites. Khan et al. prepared chitosan/GO nancomposite fiber templated with copper nanoparticles to detect and reduce 4-nitrophenol. The Cu-containing nanocomposites showed a constant reduction rate of 1.310 6 0.093/min, high sensitivity (1.729 6 0.027 μA/mM/cm2) and good detection limit of 3.5 mM [82].

25.3.3 Desalination Chitosan-derived materials have also been used for water desalination. Xiaowei Qian et al. prepared chitosan/GO chitosan-derived matrix membrane for desalination. They investigated the morphological changes, wettability, and desalination capacity of the membrane by changing the GO content. The thermodynamic and kinetic behavior of water and salt permeation through the membrane was also examined. The improvement in the permeate flux (30.0 kg/m2 h) and salt rejection (99.99%) at 81 C was observed for NaCl (5 wt.% aqueous) solution with 1 wt.% GO concentration. The results indicated that the stability of the chitosan material was improved due to good compatibility between the GO and chitosan polymer. The phenomena of NaCl/H2O solubility and diffusion through the membrane demonstrated that there was a tradeoff effect on the salt/water selectivity and permeability as GO concentration was increased [83]. Deng et al. also used GO to modify chitosan for water desalination. But they also used titania oxide for the reduction of GO to enhance the salt rejection. The study mentioned that hybrid membranes displayed about 30% higher rejection rates than the only GO-containing membrane [84].

Handbook of Chitin and Chitosan

790

25. Chitosan-based materials for water and wastewater treatment

Chitosan has also been blended with different biopolymeric materials from biomass such as hemicellulose to develop chitosan-based material for salt removal from solutions of known concentration. For this purpose, first hemicellulose was grafted with penetic acid (diethylene triamine pentaacetic acid, DTPA) followed by cross-linking with chitosan. The adsorbent was studied to determine the concentration of hemicellulose
DTPA, time and temperature of reaction with chitosan for the adsorption of NaCl. The results showed a maximum salt uptake of B0.30 g/g. Moreover, the salt adsorption equilibrium followed a second-order kinetic model [85]. A chitosan-derived nanofiltration membrane was developed using piperazine for the desalination of brackish and seawater. They incorporated piperazine to increase the permeate flux of the membrane. The membrane was analyzed using different compositional and surface characterization techniques. The study demonstrated that piperazine with 25% (w/w) increased the permeate flux (60.6 L/m2/h) almost two times without any substantial decrease in rejection of Na2SO4 which was observed to be 89.1% [86]. There are many studies that report that chitosan membranes with suitable support have promising performance for pollutant removal or separation. Recently, catechin-modified chitosan loose nanofiltration membrane has been synthesized for dye desalination [87]. Chitosan conjugate was assembled on the surface of polyacrylonitrile (HPAN) ultrafiltration (UF) membrane to study its effect on the dye desalination. The results showed that a higher NaCl elimination (82%) was observed along with 10% rate loss in dye removal during the dye desalination process. In addition, the membrane showed an excellent dye antifouling ability with 87.8% flux recovery ratio. In another study, polypropylene fiber supported chitosan nanofiltration membrane was synthesized for desalination applications [88]. The results showed that the rejection rate of the membrane was higher at acidic pH while low in the basic pH region. Similarly, Kumar et al. [89] also prepared an UF membrane containing polysulfone and chitosan using a diffusion-induced phase-separation method. They concluded that flux was high at low pH because of the protonation of chitosan amine (-NH2) groups. Chitosan-based materials have also been studied in the forward osmosis (FO) desalination process. Shakeri et al. [90] synthesized chitosan-derived membrane by polymerizing chitosan with trimesoyl chloride (TMC) on the surface of sulfonated polyethersulfone
polyethersulfone (SPES
PES) as a support layer. They compared the performance of modified membrane with the commercially available thin-film composite. They concluded that the modified membrane has higher hydrophilicity, salt rejection, and water permeation.

Handbook of Chitin and Chitosan

25.3 Removal of different pollutants using chitosan-based material

791

25.3.4 Antibacterial effect In the last decade, chitosan and its derivatives have received a lot of attention because of their excellent antimicrobial activity [91,92]. Membrane distillation is one of the promising technologies for the industrial desalination. But membranes are very prone to fouling or membrane pore wetting because of the presence of a variety of complex constituents (hydrophobic or amphiphilic) in wastewaters. Wang et al. investigated the effect of the surface wetting properties and fouling behaviors on membrane distillation [93]. They studied the hydrophobic, omniphobic, and composite membranes wetting and fouling resistance with a hydrophobic substrate and a superhydrophilic top surface. Membranes with a comparatively higher concentration of crude oil (with and without Triton X-100 as a surfactants) with hypersaline feed solutions were tested. The data showed that the membranes containing superhydrophilic groups at the top surface were more resistant to oilfouling without Triton X-100, whereas in the presence of Triton X-100 they were exposed to pore wetting. Furthermore, omniphobic membranes were easily fouled by oil-in-water emulsion without surfactant but maintained stable performance of membrane distillation with a surfactant. FO is one of the viable options and has potential application in the desalination of seawater. Since external hydraulic pressure is not required during FO, it is considered to have a low fouling propensity. Salehi et al. reported a very efficient FO membrane using chitosan/GO layer by layer on a porous support (sulfonated polyethersulfone into polyethersulfone matrix) layer [94]. The results showed that chitosan/GO-coated membrane had better fouling resistance and water flux (2
4 orders of magnitude) than the thin-film composite membrane. In another study, magnetic chitosan was used along with GO to develop chitosan/GO nanocomposite for antibacterial effect. The composite antibacterial performance against E. coli was found be better due to the cell membrane damage [95]. A study was reported for the surface modification of commercially available brackish water thin-film composite (polyamide) membrane using GO-functionalized chitosan [96]. The modified composite membranes exhibited a higher performance as an antifouling membrane compared with the unmodified polyamide membrane, as well as for salt rejection and water flux rate. Two studies were reported in 2015 for the use of ZnO nanoparticles with chitosan to improve the antibacterial property. In the first study, Motshekga et al. prepared chitosan-based nanocomposites using bentonite-supported silver and zinc oxide nanoparticles to disinfect water [48]. In the study reported by Kamal et al. pure chitosan/zinc oxide composite coatings were applied on a microfibrillar cellulose mat

Handbook of Chitin and Chitosan

792

25. Chitosan-based materials for water and wastewater treatment

to prepare chitosan-coated microfibrillar cellulose and zinc oxide
chitosan-coated microfibrillar cellulose [97]. In both studies, they used E. coli to test the antibacterial activity of the membranes and the results showed excellent performance of the nanocomposites against E. coli.

25.3.5 Other pollutants Chitosan-based materials have also been used for the removal of other pollutants, such as oil or organic separation, algae removal, and CO2 fixation. A bioinspired chitosan-derived mesh was developed to improve oil/water fouling repellent property in a wide range of pH and hypersaline environments. The study indicated that the separation efficiency of oil/water mixtures was .99% and the process was merely dependent on gravity. The authors claimed that the separation process is useful in oil spill cleanup and industrial oil wastewater treatment [98]. In another study [99], chitosan-oxidized cellulose aerogels have been developed which can potentially be used for the separation of oil/ organic in oils and organic solvents spill cleanup or water/oil separation field. The kinetic analysis of the process determined that the pseudo
second order model is the most suitable for this kind of aerogel. The aerogel showed excellent absorption performance and a better absorption rate for a variety of organic solvents and oils. There is also a study presented in the literature which focused on the carbon dioxide adsorption or chemical fixation using mesoporous zeolite/chitosan composite. The composite exhibited improved adsorption of CO2 compared with pure zeolite or chitosan. The composites also displayed excellent catalytic activity for the CO2 chemical fixation into cyclic carbonates [100]. Chitosan has also been used as a flocculant for the rapid harvesting of freshwater microalgae Chlorella vulgaris. The various concentrations of chitosan were used for the recovery of C. vulgaris and it was found that chitosan exhibited the highest efficiency of 92% 6 0.4% in a span of 3 min. The harvesting efficiency was highly pH dependent and showed a highest value of 99% 6 0.5% at pH 6.0. The results recommended that chitosan can be used as a potential flocculant for microalgae harvesting because of high efficacy, low dose requirements, and short settling time [101].

25.4 Mechanism of adsorption The mechanism of adsorption is of immense importance because its better insights can advance the performance of adsorbents for the removal of pollutants. It is very difficult to understand the adsorption mechanism of modified chitosan as an adsorbent especially for the

Handbook of Chitin and Chitosan

25.4 Mechanism of adsorption

793

heavy metals removal [102]. There are only a few studies available on the interaction mechanism of chitosan with the metal ions. It is generally believed that the amine group is the most reactive site for the metal ions while the hydroxyl group at the C-position contributes toward sorption. These two functional groups play a critical role to develop different kinds of interactions with the heavy metals. In addition to that type of interaction, the nature of metal ions, system pH, and solution matrix are also determined. The presence of two lone pairs of electrons on nitrogen acts as a binding site for metal cations at a neutral pH or weakly acidic region [103,104]. While a highly acidic environment causes the protonation of amine groups that leads to cationic behavior of the chitosan polymer. Thus the chitosan-based material has the potential to interact with the metal anions too [105,106]. Generally, heavy metal adsorption through a modified chitosan surface can be considered through electrostatic or chemical interactions (chelation or complexation), ion exchange, and nonpolar interaction (van der Waals forces) [103]. The interaction mechanism of raw chitosan or modified chitosan with the metals is influenced by the presence of multiple functional groups which are characterized using various surface analyses such as X-ray photoelectron spectroscopy (XPS), X-ray diffractions (XRD), scanning electron microcopy (SEM), Fourier-transform infrared (FTIR) spectroscopy, and electron dispersive X-ray spectroscopy (EDX). XPS and FTIR were used to investigate the adsorption mechanism of Cr(VI) by γ-Fe2O3-chitosan beads. The results indicated that both hydroxyl and amino groups of the modified chitosan may participate during the metal removal. They reported three adsorption phenomena (as shown in Fig. 25.3) during the removal process, that is, chelation, electrostatic interaction, and complexation. Cr(VI) was first sorbed over the bead via electrostatic attraction and was reduced to Cr(III) by the chitosan 2 OH group. While two forces come into play for Cr(III) immobilization onto the beads’ surface, that is, precipitation of Cr(III) in the hydroxide form and chelate formation with free amino groups. These phenomena also describe the Cr(VI) higher uptake capacity of the adsorbent [107]. Kuang et al. used FTIR technique to characterize the triethylene
tetramine grafted magnetic chitosan before and after Pb(II) adsorption. The data showed that complexation was involved in the adsorption process of Pb(II). Due to the grafting of chitosan, many amino, imino, and hydroxy groups were present and a lone pair on nitrogen and oxygen atoms can be donated to the Pb21 and form a chitosan
metal complex, as shown in Fig. 25.4 [108]. Jiang et al. have reported spherical polystyrene
chitosan (PC-CS) thin films for Cu(II) removal. They characterized the adsorption mechanism of films taking XPS spectra of N 1 s before and after Cu(II)

Handbook of Chitin and Chitosan

794

25. Chitosan-based materials for water and wastewater treatment

Adsorbent[107]. (1) NH2 Cr2O72–

+

H+

Cr2O72–

NH3+

(2)

OH

OH

e– (3) NH2 Cr(OH)2+

H+

NH3+

HO

donor

Cr3+ pH>6

NH2

(4) Cr(OH)3

O

O

FIGURE 25.3 Interaction mechanism of Cr(VI) with γ-Fe2O3 2 chitosan beads. Source: Reproduced with permission from American Chemical Society (ACS) Y.-J. Jiang, X.-Y. Yu, T. Luo, Y. Jia, J.-H. Liu, X.-J. Huang, γ-Fe2O3 nanoparticles encapsulated millimeter-sized magnetic chitosan beads for removal of Cr (VI) from water: thermodynamics, kinetics, regeneration, and uptake mechanisms, J. Chem. Eng. Data 58 (2013) 3142
3149.

adsorption. The results showed Cu(II) coordinates with the N atoms of polystyrene
chitosan with 10% GLA (PC-CS-10) as a cross-linking agent [109]. Fig. 25.5 shows the two peaks of N 1 s before the Cu(II) adsorption; the first peak has a binding energy of 400.5 eV, which can be ascribed to N atoms of NH2 and NH groups of CS, while the second peak can be observed before the Cu(II) adsorption at 401.9 eV related to N atoms oxidation with positive charges of NH31 groups of CS. The same two peaks were shifted to 399.8 and 402.2 eV, respectively, that is due to the formation of R-NH2-Cu21 or R-NH-Cu. This variation in binding energies of peaks governs the complexes formation between PS
CS surface functional groups and the Cu(II) in which the N atom’s lone pair were shared with the Cu(II) ions. There are few cases where heavy metal ions are in the form of oxyanions in solutions and can bind to the modified chitosan surface with protonated amine, xanthate, hydroxyl, and carboxyl functional groups via electrostatic force [102]. Kwok et al. reported a study for the removal of arsenic by using nanochitosan and chitosan [110]. They proposed that an electrostatic force of attraction may exist between arsenic oxyanions and chitosan-protonated amine groups as an adsorption mechanism for the arsenic removal. On the other hand, metals present in a solution as a neutral species could be adsorbed to chitosan’s surface through the van der Waals force of attraction. In some cases electrostatic attraction, along with reduction and complexation mechanisms, can also take part in the adsorption of oxyanions such as chromium (VI). Chauhan et al. reported the removal of Cr(VI)

Handbook of Chitin and Chitosan

795

25.4 Mechanism of adsorption H OCH2COOFe3O4 H OCH2COO– OCH2COO– OCH2COOFe3O4 H O O O O H H H H O O O O O HO H HO H HO HO NH NH NH NH H H H H H H H H HO HO HO HO HN HN HN HN H

HN

HN

HN

HN

NH2

NH2

NH2

NH2

NH

NH

NH

NH

n

Pb2+ H OCH2COOFe3O4 OCH2COO– OCH2COO– OCH2COOFe3O4 H O O O O H H H H O O O O O HO HO H HO HO NH NH NH NH H n H H H H H H H HO HO HO HO HN HN HN HN Pb2+ Pb2+ Pb2+ NH NH NH H

Pb2+

NH

H N

H 2N

NH2

HN

N H

HN

NH2 H N 2 H OCH2COOFe3O4 OCH2COOH H OCH COOH H OCH2COOFe3O4 2 O O O H O H H H O O O O O H HO H HO H HO HO NH2 NH NH NH H + H H H H H H H HO HO HO HO H

HCI

HN +

NH2+

NH2 + H2N

NH3+

NH2+

NH + H2N

HN NH3+

HN

HN

H2N

n

HN NH3+

NH3+

Pb21 adsorption and desorption mechanism on the Fe3O4-TETA-CMCS. Source: Reproduced with permission from Elsevier S.-P. Kuang, Z.-Z. Wang, J. Liu, Z.-C. Wu, Preparation of triethylene-tetramine grafted magnetic chitosan for adsorption of Pb (II) ion from aqueous solutions, J. Hazard. Mater. 260 (2013) 210
219.

FIGURE 25.4

using xanthated chitosan and proposed adsorption mechanisms, that is, electrostatic force of attraction existed between protonated xanthated chitosan and negatively charged chromate ions. Furthermore, THE process is followed by reduction of Cr(VI) to Cr(III) due to xanthate presence and complexation of Cr(III) with unreacted xanthate [111]. Li et al. speculated the four-step adsorption mechanism for the removal of Cr(VI) using magnetic cyclodextrin
chitosan/graphene

Handbook of Chitin and Chitosan

796

25. Chitosan-based materials for water and wastewater treatment

402.2 eV

400.5 eV

399.8 eV

401.9 eV Virgin

404

403

401

402

400

Adsorbed Cu

398

399

404 403 402 401 400 399 398 397

Binding energy (eV)

Binding energy (eV)

FIGURE 25.5 XPS N 1 s spectra before (left) and after (right) adsorption of Cu(II). Source: Reproduced with permission from Elsevier W. Jiang, X. Chen, B. Pan, Q. Zhang, L. Teng, Y. Chen, et al., Spherical polystyrene-supported chitosan thin film of fast kinetics and high capacity for copper removal, J. Hazard. Mater. 276 (2014) 295
301.

oxide (CCGO), as shown in Fig. 25.6. The first step is the binding of Cr(VI) to CCGO through electrostatic interaction which was created between the negatively charged Cr(VI) and the CCGO protonated amine groups. The second step is the reduction of chromium(IV) to chromium (III) which was ascribed to the presence of π electrons on the carbocyclic group belonging to the six-membered ring in CCGO. The third step is release of Cr(III) ions into the solution due to the repulsive forces between the protonated amine groups and the cationic Cr(III), or the binding because of the attractive forces between the Cr(III) cation and negatively charged COO2 groups of CCGO. While in the last step, H+ Cr(VI)

H+

Cr(VI)

1

2 +

Cr(VI)

Cr(III)

Cr(III)

H+

e–

Cr(VI)

3

+

HCrO4–

e– Electron donor

Cr3+

– –COO– +

Cyclodextrin

Cr(III)

Cr(III)

–

–

4

Protonated amine groups CCGO

FIGURE 25.6 Proposed mechanism for the removal of chromium(VI) using CCGO. Source: Reproduced with permission from Elsevier L. Li, L. Fan, M. Sun, H. Qiu, X. Li, H. Duan, et al., Adsorbent for chromium removal based on graphene oxide functionalized with magnetic cyclodextrin
chitosan, Colloid. Surface B 107 (2013) 76
83.

Handbook of Chitin and Chitosan

797

25.4 Mechanism of adsorption

O 1s

(A)

Relative intensity (CPS)

Fe 2p Cr 2p

C 1s

Cr-loaded MCh-Fe N 1s

Cl 2p MCH-Fe

800

600

400

200

0

Binding energy (eV)

FIGURE 25.7 XPS spectra of MCh-Fe before and after Cr(VI) adsorption. Source: Reproduced with permission from American Chemical Society (ACS) Z. Yu, X. Zhang, Y. Huang, Magnetic chitosan
iron (III) hydrogel as a fast and reusable adsorbent for chromium (VI) removal, Ind. Eng. Chem. Res. 52 (2013) 11956
11966.

cyclodextrin can allow them to bind Cr(VI) and Cr(III) into their cavities to form stable host
guest inclusion complexes [64]. Later, a similar mechanism was reported in two other studies using chitosan (CS)-derived functional gel (FG), multiwalled carbon nanotube
poly(AA)
poly(4-amino diphenyl amine) [112] and chitosan crosslinked with diethylenetriaminepentaacetic acid for Cr61 removal [113]. There are a few studies reported which proposed an ion exchange mechanism for metal removal. Yu et al. studied the adsorption mechanism of Cr(VI) on magnetic chitosan
iron(III) hydrogel (MCh-Fe). They investigated the mechanism by XPS as shown in Fig. 25.7. A peak related to C1 2p with binding energy of 200.5 eV disappeared in MChFe after the loading of chromium and suggested an ion exchange interaction between the Cl2 and Cr(VI). This mechanism was corroborated with the TG results (shown in Fig. 25.8) as the thermal stability of MChFe was improved with the Cr(VI) loading and proposed the formation of a complex with more thermal stability [114]. A study reported by Kyzas et al. proposed the adsorption mechanism based on chelation for Pb(II) and Cd(II) adsorption onto the poly(itaconic acid)-grafted chitosan with GLA or ECH as a cross-linking agents. They suggested two main adsorption interactions. The first is chelation between nitrogen atoms of chitosan amino groups (left unreacted after cross-linking) and metal ions. The second is electrostatic attraction between chitosan

Handbook of Chitin and Chitosan

798

25. Chitosan-based materials for water and wastewater treatment

(C)

90

MCH-Fe Cr(VI) loaded MCh-Fe

Weight (%)

75 60 45 30 15 0

150

300

450

600

750

Temperature (ºC)

FIGURE 25.8 TG curves of MCh-Fe before and after Cr(VI) loading. Source: Reproduced with permission from American Chemical Society (ACS) Z. Yu, X. Zhang, Y. Huang, Magnetic chitosan
iron (III) hydrogel as a fast and reusable adsorbent for chromium (VI) removal, Ind. Eng. Chem. Res. 52 (2013) 11956
11966.

carboxylate ions obtained after grafting and positive metal ions [115]. The chelation mechanism could be explained by the presence of hydroxyl and amino groups in the chitosan because both have a lone pair of electrons that could add to a proton or cation by coordinated covalent bond. Recently, intermolecular hydrogen and electrostatic interactions have been proposed for GO-functionalized chitosan after As(V) adsorption. The presence of multiple oxygenated functional moieties on the surface of GO-functionalized chitosan had a critical role on As(V)/As(III) adsorption. They conceptualized several interactions (as shown in Fig. 25.9) such as cation
pi interaction, electrostatic interaction, interand intermolecular hydrogen bonding, and anion
pi interaction. FTIR and XRD analyses were performed to confirm the adsorption mechanisms, that is, electrostatic interaction, intermolecular hydrogen bonding, and surface complex for the adsorption of As(V) and As(III). Further, the claim was validated using EDS and XPS analysis [65].

25.4.1 Factors affecting adsorption performance There are many factors which play a critical role in the performance (capacity and rate) of chitosan as an adsorbent [106]. However, the physical nature of the material, source and composition, physical properties, and process application are the most influential factors which affect the adsorption process.

Handbook of Chitin and Chitosan

799

25.4 Mechanism of adsorption

n

Electrostatic interaction NH2

– H2AsO4 +

n

O

+

OH OH

OH

O

Peptide bond O C

OH2 Cl

NH

Cl

C

O

NH2 O

OH

OH2

Cl

Electrostatic interaction

n

OH

+

Peptide bond

OH

+

O

OH2

O

OH

Intermolecular H-bonding

O

C C

+

NH O

O

Cl

H2AsO4

O

O C

O

O

n

+

–

OH2

Intermolecular H-bonding

2–

H2AsO4

Electrostatic interaction

Arsenic adsorption

FIGURE 25.9 Interaction between chitosan and functionalized GO, after adsorption of As(V). Source: Reproduced with permission from Elsevier A.S.K. Kumar, S.-J. Jiang, Chitosanfunctionalized graphene oxide: a novel adsorbent an efficient adsorption of arsenic from aqueous solution, J. Environ. Chem. Eng. 4 (2016) 1698
1713.

Chitosan’s physical nature has the most profound effect on its adsorption capacity. Chitosan has been physically modified into various forms for water treatment, such as hydrogels and beads [116
119], magnetic beads [120,121], swollen beads [59,122], nanoparticles [123,124], hollow fibers [125
127], powder [128], solid fibers [129,130], flakes [131,132], membrane [133,134], and honeycomb or sponge-like structures [135,136]. The second most important factor is the source and composition of the chitosan used for water decontamination. Chitosan has been isolated from several natural materials; lobsters, crab shells, and prawns are few examples of sources of chitosan that have been used for water purification [137
139]. There are studies reported on the comparison of sorption capacities of shrimp, lobster, and crab-derived chitosan materials for Cu12 and reactive red dye 222. The results showed that shrimp-derived chitosan flakes had maximum Cu12 sorption capacity of 123 mg/g while crab beads showed maximum sorption capacity of 1106 mg/g for reactive red dye 222 [140,141]. The adsorption ability of chitosan also depends on the binding ability of metal ion with its amino group, degree of deacetylation, and molar density of the amino group on its surface. Wu et al. studied chitosan
metal ion adsorption with varying degrees of deacetylation. The

Handbook of Chitin and Chitosan

800

25. Chitosan-based materials for water and wastewater treatment

results indicated that chitosan flakes exhibited 293 (76.3%), 398 (77.9%), and 494 (80.9%) mg/g sorption capacities for reactive red dye to crab, lobster, and shrimp sources, respectively. While in case of beads the converse was true (higher DD: low sorption capacity) [142]. Physical properties particularly surface area, pore volume, density, pore size distribution, and elemental composition also have an effect on the adsorption capacity of the chitosan-derived materials [143,144]. Surface area is a very important factor for adsorption since a large surface means a greater number of moieties are available for adsorption [106]. Li et al. reported that high surface area is an advantage for Cr(VI) removal using chitosan-derived adsorbent. They also claimed that adsorbent had better removal for Cr(VI) at low pH. The study explained that there were more hydroxyl and amino groups on the modified chitosan as compared with raw chitosan. The better performance of modified chitosan for Cr(VI) adsorption was ascribed to their surface charge concentration and specific surface area [64]. Process application factors such as solution temperature, pH, presence of anion or cation, degree of agitation, and nature of contact methods are the most significant factors for application processes of chitosan. These factors have the foremost effect on the rate-controlling steps. For example, an increase in temperature during the process of activated adsorption causes an increase in the adsorption rate but lowers the adsorption capacity [145]. This is attributed to the greater effect of temperature on the desorption rate constant. Solution pH also has a critical role in metal ion adsorption, for instance, if pH is too low, that is, around 2.5, it increases the adsorption between the protons and metal ions. Also, the system pH with the zeta potential and pKa of the chitosan amine group is also important since a high pH causes metallic precipitation [106].

25.5 Summary and future perspective The utilization of chitosan-based materials for water and wastewater decontamination from dyes, desalination, heavy metals, and microorganism offers exceptional adsorption capacities with other characteristics, such as chitosan’s low cost, harmless nature, and biocompatibility. All these properties related to chitosan-based materials provide them with a promising future compared with the existing adsorbing materials. However, chitosan’s swelling is recognized as the foremost issue which prevents its use at a larger scale. There are very few industries that utilize industrial grade chitosan-derived material for water and wastewater detoxification from dyes and heavy metals. Therefore the potential of modified chitosan materials is still in its infancy and needs

Handbook of Chitin and Chitosan

References

801

to be explored further for larger-scale applications. Although extensive research is going on to utilize chitosan biosorbents for water and wastewater remediation, there are still several research questions that need to be addressed to take full advantage of chitosan to control water damage. Some of them are highlighted below: 1. Selection of a suitable chitosan-modified adsorbent based on adsorbent
adsorbate interactions to achieve the maximum adsorption. 2. Insights of the mechanism during the adsorption process by which binding sites can be increased for the pollutant and in turn the absorption capacity of the adsorbent can be maximized. 3. Comprehensive regeneration studies to recover the metals as well as adsorbent which can cut the overall cost. 4. Understanding of the effect of multiple cocontaminants on each other during the adsorption process. 5. Efficient removal of multiple contaminants simultaneously at the larger scale. 6. Scale up of lab-based batch studies to broaden the applications of chitosan-based adsorbent at industrial level. Aside from these shortcomings, the chitosan-derived materials have tremendous scope for water remediation in the coming years. The growing concerns regarding water pollution has led to the public and governments becoming quite serious at dealing with this burning issue, and concurrently the responsibilities of the researchers have also increased enormously. In the coming years, the demand for biobased materials with better performance and cheap architectures will undoubtedly be at its highest. The development of novel materials with low cost with ever greater performance for water treatment has opened up new horizons to scientists and provided them with collaborative opportunities to solve the problem of water with better approaches. The expansion of chitosan-based materials at the larger scale can contribute as a sustainable and renewable material for water remediation in the future.

References [1] J. Alcamo, T. Henrichs, T. Ro¨sch, World Water in 2025—Global modeling and scenario analysis for the World Commission on Water for the 21st Century, Kassel World Water Series 2, Center for Environmental Systems Research, University of Kassel, Germany, 7411 (2000) 5. [2] M. Venohr, S.D. Langhans, O. Peters, F. Ho¨lker, R. Arlinghaus, L. Mitchell, et al., The underestimated dynamics and impacts of water-based recreational activities on freshwater ecosystems, Environ. Rev. 26 (2018) 199
213. [3] Y. Han, Z. Xu, C. Gao, Ultrathin graphene nanofiltration membrane for water purification, Adv. Funct. Mater. 23 (2013) 3693
3700.

Handbook of Chitin and Chitosan

802

25. Chitosan-based materials for water and wastewater treatment

[4] D.E. McNabb, Water’s role in a sustainable future, Global Pathways to Water Sustainability, Springer, 2019, pp. 17
30. [5] A.Y. Hoekstra, The water footprint: the relation between human consumption and water use, The Water We Eat, Springer, 2015, pp. 35
48. [6] D.B. Parker, M.S. Brown, Water consumption for livestock and poultry production, in: B.A. Stewart, T. Howell, Encyclopedia of Water Science (Print), CRC press, 2003. [7] M.A. Shannon, P.W. Bohn, M. Elimelech, J.G. Georgiadis, B.J. Marin˜as, A.M. Mayes, Science and technology for water purification in the coming decades, Nature 452 (2008) 301
310. [8] M.A. Montgomery, M. Elimelech, Water and Sanitation in Developing Countries: Including Health in the Equation, ACS Publications, 2007. [9] K. Ikehata, Y. Zhao, H.V. Kulkarni, Y. Li, S.A. Snyder, K.P. Ishida, et al., Water recovery from advanced water purification facility reverse osmosis concentrate by photobiological treatment followed by secondary reverse osmosis, Environ. Sci. Technol. 52 (2018) 8588
8595. [10] L. Dong, W. Fan, X. Tong, H. Zhang, M. Chen, Y. Zhao, A CO 2-responsive graphene oxide/polymer composite nanofiltration membrane for water purification, J. Mater. Chem. A 6 (2018) 6785
6791. [11] S. Szklarek, I. Wagner, T. Jurczak, M. Zalewski, Sequential sedimentation-biofiltration system for the purification of a small urban river (the Sokolowka, Lodz) supplied by stormwater, J. Environ. Manag. 205 (2018) 201
208. [12] M.S. Mauter, I. Zucker, F. Perreault, J.R. Werber, J.-H. Kim, M. Elimelech, The role of nanotechnology in tackling global water challenges, Nat. Sustain. 1 (2018) 166. [13] J. Liu, X. Ge, X. Ye, G. Wang, H. Zhang, H. Zhou, et al., 3D graphene/δ-MnO 2 aerogels for highly efficient and reversible removal of heavy metal ions, J. Mater. Chem. A 4 (2016) 1970
1979. [14] M.A. Shannon, P.W. Bohn, M. Elimelech, J.G. Georgiadis, B.J. Marinas, A.M. Mayes, Science and technology for water purification in the coming decades, Nanoscience and Technology: A Collection of Reviews from Nature Journals, World Scientific, 2010, pp. 337
346. [15] A. Bhatnagar, M. Sillanpa¨a¨, Applications of chitin-and chitosan-derivatives for the detoxification of water and wastewater—a short review, Adv. Colloid Interface Sci. 152 (2009) 26
38. [16] L. Pontoni, M. Fabbricino, Use of chitosan and chitosan-derivatives to remove arsenic from aqueous solutions—a mini review, Carbohydr. Res. 356 (2012) 86
92. [17] G. Crini, P.-M. Badot, Application of chitosan, a natural aminopolysaccharide, for dye removal from aqueous solutions by adsorption processes using batch studies: a review of recent literature, Prog. Polym. Sci. 33 (2008) 399
447. [18] W.W. Ngah, L. Teong, M. Hanafiah, Adsorption of dyes and heavy metal ions by chitosan composites: a review, Carbohydr. Polym. 83 (2011) 1446
1456. [19] J. Wang, S. Zhuang, Removal of various pollutants from water and wastewater by modified chitosan adsorbents, Crit. Rev. Environ. Sci. Technol. 47 (2017) 2331
2386. [20] M. Julinova´, R. Slavı´k, Removal of phthalates from aqueous solution by different adsorbents: a short review, J. Environ. Manag. 94 (2012) 13
24. [21] I. Munnawar, S.S. Iqbal, M.N. Anwar, M. Batool, S. Tariq, N. Faitma, et al., Synergistic effect of chitosan-zinc oxide hybrid nanoparticles on antibiofouling and water disinfection of mixed matrix polyethersulfone nanocomposite membranes, Carbohydr. Polym. 175 (2017) 661
670. [22] M. Vakili, M. Rafatullah, B. Salamatinia, A.Z. Abdullah, M.H. Ibrahim, K.B. Tan, et al., Application of chitosan and its derivatives as adsorbents for dye removal from water and wastewater: a review, Carbohydr. Polym. 113 (2014) 115
130.

Handbook of Chitin and Chitosan

References

803

[23] A.J. Al-Manhel, A.R.S. Al-Hilphy, A.K. Niamah, Extraction of chitosan, characterisation and its use for water purification, J. Saudi Soc. Agric. Sci. 17 (2018) 186
190. [24] W.W. Ngah, C. Endud, R. Mayanar, Removal of copper (II) ions from aqueous solution onto chitosan and cross-linked chitosan beads, React. Funct. Polym. 50 (2002) 181
190. [25] M.M. Sahli, S. Annouar, M. Tahaikt, M. Mountadar, A. Soufiane, A. Elmidaoui, Fluoride removal for underground brackish water by adsorption on the natural chitosan and by electrodialysis, Desalination 212 (2007) 37
45. [26] G. Dotto, L. Pinto, Adsorption of food dyes acid blue 9 and food yellow 3 onto chitosan: stirring rate effect in kinetics and mechanism, J. Hazard. Mater. 187 (2011) 164
170. [27] G. Annadurai, L.Y. Ling, J.-F. Lee, Adsorption of reactive dye from an aqueous solution by chitosan: isotherm, kinetic and thermodynamic analysis, J. Hazard. Mater. 152 (2008) 337
346. [28] U. Filipkowska, Adsorption and desorption of reactive dyes onto chitin and chitosan flakes and beads, Adsorp. Sci. Technol. 24 (2006) 781
795. [29] D.-R. Ding, Y. Shen, Antibacterial finishing with chitosan derivatives and their nano particles, Dyeing Finish. 14 (2005) 002. [30] M. Zubair, A. Ullah, Recent advances in protein derived bionanocomposites for food packaging applications, Crit. Rev. Food Sci. Nutr. (2019) 1
29. [31] K.-S. Huang, Y.-R. Sheu, I.-C. Chao, Preparation and properties of nanochitosan, Polym. Technol. Eng. 48 (2009) 1239
1243. [32] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M. Alonso, Novel hydrophilic chitosanpolyethylene oxide nanoparticles as protein carriers, J. Appl. Polym. Sci. 63 (1997) 125
132. [33] K. Vijayalakshmi, B.M. Devi, S. Latha, T. Gomathi, P. Sudha, J. Venkatesan, et al., Batch adsorption and desorption studies on the removal of lead (II) from aqueous solution using nanochitosan/sodium alginate/microcrystalline cellulose beads, Int. J. Biol. Macromol. 104 (2017) 1483
1494. [34] M.S. Farid, A. Shariati, A. Badakhshan, B. Anvaripour, Using nano-chitosan for harvesting microalga Nannochloropsis sp, Bioresour. Technol. 131 (2013) 555
559. [35] M.E. Mahmoud, M.T.A. Kana, A.A. Hendy, Synthesis and implementation of nanochitosan and its acetophenone derivative for enhanced removal of metals, Int. J. Biol. Macromol. 81 (2015) 672
680. [36] S. Olivera, H.B. Muralidhara, K. Venkatesh, V.K. Guna, K. Gopalakrishna, Y. Kumar, Potential applications of cellulose and chitosan nanoparticles/composites in wastewater treatment: a review, Carbohydr. Polym. 153 (2016) 600
618. [37] K. Kaur, R. Jindal, Comparative study on the behaviour of chitosan-gelatin based hydrogel and nanocomposite ion exchanger synthesized under microwave conditions towards photocatalytic removal of cationic dyes, Carbohydr. Polym. 207 (2019) 398
410. [38] Y. Vijaya, S.R. Popuri, V.M. Boddu, A. Krishnaiah, Modified chitosan and calcium alginate biopolymer sorbents for removal of nickel (II) through adsorption, Carbohydr. Polym. 72 (2008) 261
271. [39] A.B. Albadarin, M.N. Collins, M. Naushad, S. Shirazian, G. Walker, C. Mangwandi, Activated lignin-chitosan extruded blends for efficient adsorption of methylene blue, Chem. Eng. J. 307 (2017) 264
272. [40] X. Luo, J. Zeng, S. Liu, L. Zhang, An effective and recyclable adsorbent for the removal of heavy metal ions from aqueous system: magnetic chitosan/cellulose microspheres, Bioresour. Technol. 194 (2015) 403
406. [41] L. Zhao, H. Mitomo, Adsorption of heavy metal ions from aqueous solution onto chitosan entrapped CM-cellulose hydrogels synthesized by irradiation, J. Appl. Polym. Sci. 110 (2008) 1388
1395.

Handbook of Chitin and Chitosan

804

25. Chitosan-based materials for water and wastewater treatment

[42] V. Nair, A. Panigrahy, R. Vinu, Development of novel chitosan
lignin composites for adsorption of dyes and metal ions from wastewater, Chem. Eng. J. 254 (2014) 491
502. [43] N. Siva Kumar, A. Subba Reddy, V.M. Boddu, A. Krishnaiah, Development of chitosan-alginate based biosorbent for the removal of p-chlorophenol from aqueous medium, Toxicol. Environ. Chem. 91 (2009) 1035
1054. [44] M.A. Shaker, Thermodynamics and kinetics of bivalent cadmium biosorption onto nanoparticles of chitosan-based biopolymers, J. Taiwan. Inst. Chem. Eng. 47 (2015) 79
90. [45] W.-B. Wang, D.-J. Huang, Y.-R. Kang, A.-Q. Wang, One-step in situ fabrication of a granular semi-IPN hydrogel based on chitosan and gelatin for fast and efficient adsorption of Cu2 1 ion, Colloid. Surface B 106 (2013) 51
59. [46] R.K. Anantha, S. Kota, Removal of lead by adsorption with the renewable biopolymer composite of feather (Dromaius novaehollandiae) and chitosan (Agaricus bisporus), Environ. Technol. & Innov. 6 (2016) 11
26. [47] M.P.M. da Costa, I.L. de Mello Ferreira, M.T. de Macedo Cruz, New polyelectrolyte complex from pectin/chitosan and montmorillonite clay, Carbohydr. Polym. 146 (2016) 123
130. [48] S.C. Motshekga, S.S. Ray, M.S. Onyango, M.N. Momba, Preparation and antibacterial activity of chitosan-based nanocomposites containing bentonite-supported silver and zinc oxide nanoparticles for water disinfection, Appl. Clay Sci. 114 (2015) 330
339. [49] B. Tanhaei, A. Ayati, M. Lahtinen, M. Sillanpa¨a¨, Preparation and characterization of a novel chitosan/Al2O3/magnetite nanoparticles composite adsorbent for kinetic, thermodynamic and isotherm studies of methyl orange adsorption, Chem. Eng. J. 259 (2015) 1
10. [50] R. Salehi, M. Arami, N.M. Mahmoodi, H. Bahrami, S. Khorramfar, Novel biocompatible composite (chitosan
zinc oxide nanoparticle): preparation, characterization and dye adsorption properties, Colloid. Surfaace B 80 (2010) 86
93. [51] H.-Y. Zhu, R. Jiang, L. Xiao, Adsorption of an anionic azo dye by chitosan/kaolin/ γ-Fe2O3 composites, Appl. Clay Sci. 48 (2010) 522
526. [52] M. Shibayama, T. Tanaka, Volume phase transition and related phenomena of polymer gels, Responsive Gels: Volume Transitions I, Springer, 1993, pp. 1
62. [53] T. Tanaka, Gels, Sci. Am. 244 (1981) 124-S-117. [54] O. Okay, General properties of hydrogels, Hydrogel Sensors and Actuators, Springer, 2009, pp. 1
14. ˇ [55] N.B. Milosavljevi´c, M.Ð. Risti´c, A.A. Peri´c-Gruji´c, J.M. Filipovi´c, S.B. Strbac, Z.L. Rakoˇcevi´c, et al., Hydrogel based on chitosan, itaconic acid and methacrylic acid as adsorbent of Cd2 1 ions from aqueous solution, Chem. Eng. J. 165 (2010) 554
562. [56] R.M. Vieira, P.B. Vilela, V.A. Becegato, A.T. Paulino, Chitosan-based hydrogel and chitosan/acid-activated montmorillonite composite hydrogel for the adsorption and removal of Pb 1 2 and Ni 1 2 ions accommodated in aqueous solutions, J. Environ. Chem. Eng. 6 (2018) 2713
2723. [57] P.B. Vilela, A. Dalalibera, E.C. Duminelli, V.A. Becegato, A.T. Paulino, Adsorption and removal of chromium (VI) contained in aqueous solutions using a chitosanbased hydrogel, Environ. Sci. Pollut. Res. (2018) 1
9. [58] M.H. Mahaninia, L.D. Wilson, Phosphate uptake studies of cross-linked chitosan bead materials, J. Colloid Interface Sci. 485 (2017) 201
212. [59] Z.A. Sutirman, M.M. Sanagi, K.J.A. Karim, W.A.W. Ibrahim, B.H. Jume, Equilibrium, kinetic and mechanism studies of Cu (II) and Cd (II) ions adsorption by modified chitosan beads, Int. J. Biol. Macromol. 116 (2018) 255
263. [60] I. Mustafa, Methylene blue removal from water using H2SO4 crosslinked magnetic chitosan nanocomposite beads, Microchem. J. 144 (2019) 397
402.

Handbook of Chitin and Chitosan

References

805

[61] N. Viswanathan, C.S. Sundaram, S. Meenakshi, Development of multifunctional chitosan beads for fluoride removal, J. Hazard. Mater. 167 (2009) 325
331. [62] S.M. Prabhu, S. Meenakshi, Enriched fluoride sorption using chitosan supported mixed metal oxides beads: synthesis, characterization and mechanism, J. Water Process. Eng. 2 (2014) 96
104. [63] S. Zhuang, R. Cheng, M. Kang, J. Wang, Kinetic and equilibrium of U (VI) adsorption onto magnetic amidoxime-functionalized chitosan beads, J. Clean. Prod. 188 (2018) 655
661. [64] L. Li, L. Fan, M. Sun, H. Qiu, X. Li, H. Duan, et al., Adsorbent for chromium removal based on graphene oxide functionalized with magnetic cyclodextrin
chitosan, Colloid. Surface B 107 (2013) 76
83. [65] A.S.K. Kumar, S.-J. Jiang, Chitosan-functionalized graphene oxide: a novel adsorbent an efficient adsorption of arsenic from aqueous solution, J. Environ. Chem. Eng. 4 (2016) 1698
1713. [66] C. Hu, P. Zhu, M. Cai, H. Hu, Q. Fu, Comparative adsorption of Pb (II), Cu (II) and Cd (II) on chitosan saturated montmorillonite: kinetic, thermodynamic and equilibrium studies, Appl. Clay Sci. 143 (2017) 320
326. [67] B. Liu, X. Chen, H. Zheng, Y. Wang, Y. Sun, C. Zhao, et al., Rapid and efficient removal of heavy metal and cationic dye by carboxylate-rich magnetic chitosan flocculants: role of ionic groups, Carbohydr. Polym. 181 (2018) 327
336. [68] Z. Li, L. Shao, Z. Ruan, W. Hu, L. Lu, Y. Chen, Converting untreated waste office paper and chitosan into aerogel adsorbent for the removal of heavy metal ions, Carbohydr. Polym. 193 (2018) 221
227. [69] A. Kong, Y. Ji, H. Ma, Y. Song, B. He, J. Li, A novel route for the removal of Cu (II) and Ni (II) ions via homogeneous adsorption by chitosan solution, J. Clean. Prod. 192 (2018) 801
808. [70] H. Ren, B. Li, M. Neckenig, D. Wu, Y. Li, Y. Ma, et al., Efficient lead ion removal from water by a novel chitosan gel-based sorbent modified with glutamic acid ionic liquid, Carbohydr. Polym. (2018). [71] J. Liu, Y. Chen, T. Han, M. Cheng, W. Zhang, J. Long, et al., A biomimetic SiO2@ chitosan composite as highly-efficient adsorbent for removing heavy metal ions in drinking water, Chemosphere 214 (2019) 738
742. [72] B. Chen, H. Zhao, S. Chen, F. Long, B. Huang, B. Yang, et al., A magnetically recyclable chitosan composite adsorbent functionalized with EDTA for simultaneous capture of anionic dye and heavy metals in complex wastewater, Chem. Eng. J. 356 (2019) 69
80. [73] X. Huang, D. Zhou, Y. Wang, C. He, W. Zhao, S. Sun, et al., A green approach towards functional hydrogel particles from synthetic polymers via spherical capsule mini-reactors, Chem. Eng. J. 359 (2019) 1360
1371. [74] L. Liu, C. Li, C. Bao, Q. Jia, P. Xiao, X. Liu, et al., Preparation and characterization of chitosan/graphene oxide composites for the adsorption of Au (III) and Pd (II), Talanta 93 (2012) 350
357. [75] T. Anirudhan, G. Lekshmi, F. Shainy, Synthesis and characterization of amidoxime modified chitosan/bentonite composite for the adsorptive removal and recovery of uranium from seawater, J. Colloid Interface Sci. 534 (2019) 248
261. [76] L. Fan, C. Luo, X. Li, F. Lu, H. Qiu, M. Sun, Fabrication of novel magnetic chitosan grafted with graphene oxide to enhance adsorption properties for methyl blue, J. Hazard. Mater. 215 (2012) 272
279. [77] B. Tanhaei, A. Ayati, M. Sillanpa¨a¨, Magnetic xanthate modified chitosan as an emerging adsorbent for cationic azo dyes removal: kinetic, thermodynamic and isothermal studies, Int. J. Biol. Macromol. 121 (2019) 1126
1134.

Handbook of Chitin and Chitosan

806

25. Chitosan-based materials for water and wastewater treatment

[78] K. Gul, S. Sohni, M. Waqar, F. Ahmad, N.N. Norulaini, M.O. AK, Functionalization of magnetic chitosan with graphene oxide for removal of cationic and anionic dyes from aqueous solution, Carbohydr. Polym. 152 (2016) 520
531. [79] S. Kumar, B. Krishnakumar, A.J. Sobral, J. Koh, Bio-based (chitosan/PVA/ZnO) nanocomposites film: thermally stable and photoluminescence material for removal of organic dye, Carbohydr. Polym. 205 (2019) 559
564. [80] B. Xu, H. Zheng, H. Zhou, Y. Wang, K. Luo, C. Zhao, et al., Adsorptive removal of anionic dyes by chitosan-based magnetic microspheres with pH-responsive properties, J. Mol. Liq. 256 (2018) 424
432. [81] S. Choudhury, S.K. Ray, Efficient removal of cationic dye mixtures from water using a bio-composite adsorbent optimized with response surface methodology, Carbohydr. Polym. 200 (2018) 305
320. [82] S.B. Khan, F. Ali, K. Akhtar, Chitosan nanocomposite fibers supported copper nanoparticles based perceptive sensor and active catalyst for nitrophenol in real water, Carbohydr. Polym. (2018). [83] X. Qian, N. Li, Q. Wang, S. Ji, Chitosan/graphene oxide mixed matrix membrane with enhanced water permeability for high-salinity water desalination by pervaporation, Desalination 438 (2018) 83
96. [84] H. Deng, P. Sun, Y. Zhang, H. Zhu, Reverse osmosis desalination of chitosan crosslinked graphene oxide/titania hybrid lamellar membranes, Nanotechnology 27 (2016) 274002. [85] A. Ayoub, R.A. Venditti, J.J. Pawlak, A. Salam, M.A. Hubbe, Novel hemicellulose
chitosan biosorbent for water desalination and heavy metal removal, ACS Sustain. Chem. Eng. 1 (2013) 1102
1109. [86] Y.-J. Tang, L.-J. Wang, Z.-L. Xu, H.-Z. Zhang, Novel chitosan-piperazine composite nanofiltration membranes for the desalination of brackish water and seawater, J. Polym. Res. 25 (2018) 118. [87] S. Liu, Z. Wang, P. Song, Free radical graft copolymerization strategy to prepare catechin-modified chitosan loose nanofiltration (NF) membrane for dye desalination, ACS Sustain. Chem. Eng. 6 (2018) 4253
4263. [88] M. Padaki, A.M. Isloor, J. Fernandes, K.N. Prabhu, New polypropylene supported chitosan NF-membrane for desalination application, Desalination 280 (2011) 419
423. [89] R. Kumar, A.M. Isloor, A. Ismail, S.A. Rashid, T. Matsuura, Polysulfone
Chitosan blend ultrafiltration membranes: preparation, characterization, permeation and antifouling properties, RSC Adv. 3 (2013) 7855
7861. [90] A. Shakeri, H. Salehi, M. Rastgar, Chitosan-based thin active layer membrane for forward osmosis desalination, Carbohydr. Polym. 174 (2017) 658
668. [91] M. Benhabiles, R. Salah, H. Lounici, N. Drouiche, M. Goosen, N. Mameri, Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste, Food Hydrocoll. 29 (2012) 48
56. [92] A. Khanafari, R. Marandi, S. Sanatei, Recovery of chitin and chitosan from shrimp waste by chemical and microbial methods, Iran. J. Environ. Health Sci. Eng. 5 (2008) 19
24. [93] Z. Wang, S. Lin, Membrane fouling and wetting in membrane distillation and their mitigation by novel membranes with special wettability, Water Res. 112 (2017) 38
47. [94] H. Salehi, M. Rastgar, A. Shakeri, Anti-fouling and high water permeable forward osmosis membrane fabricated via layer by layer assembly of chitosan/graphene oxide, Appl. Surf. Sci. 413 (2017) 99
108. [95] Y. Jiang, J.-L. Gong, G.-M. Zeng, X.-M. Ou, Y.-N. Chang, C.-H. Deng, et al., Magnetic chitosan
graphene oxide composite for anti-microbial and dye removal applications, Int. J. Biol. Macromol. 82 (2016) 702
710.

Handbook of Chitin and Chitosan

References

807

[96] H.M. Hegab, Y. Wimalasiri, M. Ginic-Markovic, L. Zou, Improving the fouling resistance of brackish water membranes via surface modification with graphene oxide functionalized chitosan, Desalination 365 (2015) 99
107. [97] T. Kamal, M. Ul-Islam, S.B. Khan, A.M. Asiri, Adsorption and photocatalyst assisted dye removal and bactericidal performance of ZnO/chitosan coating layer, Int. J. Biol. Macromol. 81 (2015) 584
590. [98] S. Zhang, F. Lu, L. Tao, N. Liu, C. Gao, L. Feng, et al., Bio-inspired anti-oil-fouling chitosan-coated mesh for oil/water separation suitable for broad pH range and hyper-saline environments, ACS Appl. Mater. Interfaces 5 (2013) 11971
11976. [99] Z. Li, L. Shao, W. Hu, T. Zheng, L. Lu, Y. Cao, et al., Excellent reusable chitosan/ cellulose aerogel as an oil and organic solvent absorbent, Carbohydr. Polym. 191 (2018) 183
190. [100] S. Kumar, K. Prasad, J.M. Gil, A.J. Sobral, J. Koh, Mesoporous zeolite-chitosan composite for enhanced capture and catalytic activity in chemical fixation of CO2, Carbohydr. Polym. 198 (2018) 401
406. [101] N. Rashid, S.U. Rehman, J.-I. Han, Rapid harvesting of freshwater microalgae using chitosan, Process. Biochem. 48 (2013) 1107
1110. [102] L. Zhang, Y. Zeng, Z. Cheng, Removal of heavy metal ions using chitosan and modified chitosan: a review, J. Mol. Liq. 214 (2016) 175
191. [103] E. Guibal, Interactions of metal ions with chitosan-based sorbents: a review, Sep. Purif. Technol. 38 (2004) 43
74. [104] J. Randall, V. Randall, G. McDonald, R. Young, M. Masri, Removal of trace quantities of nickel from solution, J. Appl. Polym. Sci. 23 (1979) 727
732. [105] S. Qian, G. Huang, J. Jiang, F. He, Y. Wang, Studies of adsorption behavior of crosslinked chitosan for Cr (VI), Se (VI), J. Appl. Polym. Sci. 77 (2000) 3216
3219. [106] C. Gerente, V. Lee, P.L. Cloirec, G. McKay, Application of chitosan for the removal of metals from wastewaters by adsorption—mechanisms and models review, Crit. Rev. Environ. Sci. Technol. 37 (2007) 41
127. [107] Y.-J. Jiang, X.-Y. Yu, T. Luo, Y. Jia, J.-H. Liu, X.-J. Huang, γ-Fe2O3 nanoparticles encapsulated millimeter-sized magnetic chitosan beads for removal of Cr (VI) from water: thermodynamics, kinetics, regeneration, and uptake mechanisms, J. Chem. Eng. Data 58 (2013) 3142
3149. [108] S.-P. Kuang, Z.-Z. Wang, J. Liu, Z.-C. Wu, Preparation of triethylene-tetramine grafted magnetic chitosan for adsorption of Pb (II) ion from aqueous solutions, J. Hazard. Mater. 260 (2013) 210
219. [109] W. Jiang, X. Chen, B. Pan, Q. Zhang, L. Teng, Y. Chen, et al., Spherical polystyrenesupported chitosan thin film of fast kinetics and high capacity for copper removal, J. Hazard. Mater. 276 (2014) 295
301. [110] K.C. Kwok, L.F. Koong, G. Chen, G. McKay, Mechanism of arsenic removal using chitosan and nanochitosan, J. Colloid Interface Sci. 416 (2014) 1
10. [111] D. Chauhan, N. Sankararamakrishnan, Modeling and evaluation on removal of hexavalent chromium from aqueous systems using fixed bed column, J. Hazard. Mater. 185 (2011) 55
62. [112] M.K. Kim, K.S. Sundaram, G.A. Iyengar, K.-P. Lee, A novel chitosan functional gel included with multiwall carbon nanotube and substituted polyaniline as adsorbent for efficient removal of chromium ion, Chem. Eng. J. 267 (2015) 51
64. [113] R. Bhatt, B. Sreedhar, P. Padmaja, Adsorption of chromium from aqueous solutions using crosslinked chitosan
diethylenetriaminepentaacetic acid, Int. J. Biol. Macromol. 74 (2015) 458
466. [114] Z. Yu, X. Zhang, Y. Huang, Magnetic chitosan
iron (III) hydrogel as a fast and reusable adsorbent for chromium (VI) removal, Ind. Eng. Chem. Res. 52 (2013) 11956
11966.

Handbook of Chitin and Chitosan

808

25. Chitosan-based materials for water and wastewater treatment

[115] G.Z. Kyzas, P.I. Siafaka, D.A. Lambropoulou, N.K. Lazaridis, D.N. Bikiaris, Poly (itaconic acid)-grafted chitosan adsorbents with different cross-linking for Pb (II) and Cd (II) uptake, Langmuir 30 (2014) 120
131. [116] T. Jiao, H. Zhao, J. Zhou, Q. Zhang, X. Luo, J. Hu, et al., Self-assembly reduced graphene oxide nanosheet hydrogel fabrication by anchorage of chitosan/silver and its potential efficient application toward dye degradation for wastewater treatments, ACS Sustain. Chem. Eng. 3 (2015) 3130
3139. [117] B.C. Melo, F.A. Paulino, V.A. Cardoso, A.G. Pereira, A.R. Fajardo, F.H. Rodrigues, Cellulose nanowhiskers improve the methylene blue adsorption capacity of chitosan-g-poly (acrylic acid) hydrogel, Carbohydr. Polym. 181 (2018) 358
367. [118] W. Wang, Y. Zhao, H. Bai, T. Zhang, V. Ibarra-Galvan, S. Song, Methylene blue removal from water using the hydrogel beads of poly (vinyl alcohol)-sodium alginate-chitosan-montmorillonite, Carbohydr. Polym. 198 (2018) 518
528. [119] T. Gotoh, K. Matsushima, K.-I. Kikuchi, Preparation of alginate
chitosan hybrid gel beads and adsorption of divalent metal ions, Chemosphere 55 (2004) 135
140. [120] S. Rezgui, A. Amrane, F. Fourcade, A. Assadi, L. Monser, N. Adhoum, ElectroFenton catalyzed with magnetic chitosan beads for the removal of chlordimeform insecticide, Appl. Catal. B: Environ. 226 (2018) 346
359. [121] P. Liang, R. An, R. Li, D. Wang, Comparison of La3 1 and mixed rare earths-loaded magnetic chitosan beads for fluoride adsorption, Int. J. Biol. Macromol. 111 (2018) 255
263. [122] F.-C. Wu, R.-L. Tseng, R.-S. Juang, Enhanced abilities of highly swollen chitosan beads for color removal and tyrosinase immobilization, J. Hazard. Mater. 81 (2001) 167
177. [123] M. Omidinasab, N. Rahbar, M. Ahmadi, B. Kakavandi, F. Ghanbari, G.Z. Kyzas, et al., Removal of vanadium and palladium ions by adsorption onto magnetic chitosan nanoparticles, Environ. Sci. Pollut. Res. 25 (2018) 34262
34276. [124] K.C. Petkar, S. Chavhan, N. Kunda, I. Saleem, S. Somavarapu, K.M. Taylor, et al., Development of novel octanoyl chitosan nanoparticles for improved rifampicin pulmonary delivery: optimization by factorial design, AAPS PharmSciTech (2018) 1
15. [125] M.S. Dorraji, A. Amani-Ghadim, Y. Hanifehpour, S.W. Joo, A. Figoli, M. Carraro, et al., Performance of chitosan based nanocomposite hollow fibers in the removal of selenium (IV) from water, Chem. Eng. Res. Des. 117 (2017) 309
317. [126] N. Reddy, Y. Yang, Hollow chitosan fibers, Innovative Biofibers from Renewable Resources, Springer, 2015, pp. 123
126. [127] T. Vincent, E. Guibal, Cr (VI) extraction using Aliquat 336 in a hollow fiber module made of chitosan, Ind. Eng. Chem. Res. 40 (2001) 1406
1411. [128] Y. Andres, L. Giraud, C. Gerente, P. Le Cloirec, Antibacterial effects of chitosan powder: mechanisms of action, Environ. Technol. 28 (2007) 1357
1363. [129] A. Cooper, R. Oldinski, H. Ma, J.D. Bryers, M. Zhang, Chitosan-based nanofibrous membranes for antibacterial filter applications, Carbohydr. Polym. 92 (2013) 254
259. [130] M.L. Mathew, A. Gopalakrishnan, C.T. Aravindakumar, U.K. Aravind, Low
cost multilayered green fiber for the treatment of textile industry waste water, J. Hazard. Mater. 365 (2019) 297
305. [131] A. Ahmad, S. Sumathi, B. Hameed, Adsorption of residue oil from palm oil mill effluent using powder and flake chitosan: equilibrium and kinetic studies, Water Res. 39 (2005) 2483
2494. [132] S. Jagtap, M.K. Yenkie, S. Das, S. Rayalu, Synthesis and characterization of lanthanum impregnated chitosan flakes for fluoride removal in water, Desalination 273 (2011) 267
275.

Handbook of Chitin and Chitosan

References

809

[133] M. Beppu, R. Vieira, C. Aimoli, C. Santana, Crosslinking of chitosan membranes using glutaraldehyde: effect on ion permeability and water absorption, J. Membr. Sci. 301 (2007) 126
130. [134] A. Tabriz, M.A.U.R. Alvi, M.B.K. Niazi, M. Batool, M.F. Bhatti, A.L. Khan, et al., Quaternized trimethyl functionalized chitosan based antifungal membranes for drinking water treatment, Carbohydr. Polym. 207 (2019) 17
25. [135] C. Qi, L. Zhao, Y. Lin, D. Wu, Graphene oxide/chitosan sponge as a novel filtering material for the removal of dye from water, J. Colloid Interface Sci. 517 (2018) 18
27. [136] M. Amaike, Y. Senoo, H. Yamamoto, Sphere, honeycomb, regularly spaced droplet and fiber structures of polyion complexes of chitosan and gellan, Macromol. Rapid Commun. 19 (1998) 287
289. [137] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. Structure, properties applications, Mar. Drugs 13 (2015) 1133
1174. [138] J.R. Evans, W.G. Davids, J.D. MacRae, A. Amirbahman, Kinetics of cadmium uptake by chitosan-based crab shells, Water Res. 36 (2002) 3219
3226. [139] D.S. Kim, The removal by crab shell of mixed heavy metal ions in aqueous solution, Bioresour. Technol. 87 (2003) 355
357. [140] F.-C. Wu, R.-L. Tseng, R.-S. Juang, Comparative adsorption of metal and dye on flake-and bead-types of chitosans prepared from fishery wastes, J. Hazard. Mater. 73 (2000) 63
75. [141] F.-C. Wu, R.-L. Tseng, R.-S. Juang, Kinetic modeling of liquid-phase adsorption of reactive dyes and metal ions on chitosan, Water Res. 35 (2001) 613
618. [142] L.-Q. Wu, A.P. Gadre, H. Yi, M.J. Kastantin, G.W. Rubloff, W.E. Bentley, et al., Voltage-dependent assembly of the polysaccharide chitosan onto an electrode surface, Langmuir 18 (2002) 8620
8625. [143] L.-L. Min, L.-M. Yang, R.-X. Wu, L.-B. Zhong, Z.-H. Yuan, Y.-M. Zheng, Enhanced adsorption of arsenite from aqueous solution by an iron-doped electrospun chitosan nanofiber mat: preparation, characterization and performance, J. Colloid Interface Sci. 535 (2019) 255
264. [144] J. Ma, Y. Lei, M.A. Khan, F. Wang, Y. Chu, W. Lei, et al., Adsorption properties, kinetics & thermodynamics of tetracycline on carboxymethyl-chitosan reformed montmorillonite, Int. J. Biol. Macromol. 124 (2019) 557
567. [145] M.-S. Chiou, H.-Y. Li, Equilibrium and kinetic modeling of adsorption of reactive dye on cross-linked chitosan beads, J. Hazard. Mater. 93 (2002) 233
248.

Handbook of Chitin and Chitosan

This page intentionally left blank

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A AA. See Acrylic acid (AA) AAO. See Anodic aluminum oxide (AAO) AATCC 100, 115
116 AATCC 147, 115 ABTS diammonium salt. See 2,20 -Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS diammonium salt) Acellular dermal matrix (ADM), 750 Acetamido-2-deoxy-D-glucopyranose-2 units, 389 2-Acetamido-2-deoxy-D-glucose, 4 Acetic acid (CH3COOH), 347, 557 Acetylated/acetylation, 46 chitin, 51, 51f chitosan, 61 reaction, 50 ACF. See Alginate, chitin/chitosan, and fucoidan (ACF) ACI. See Autologous chondrocytes implantation (ACI) Acid mammalian chitinase (AMCase), 626
627 Acrylamide, 11 2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 49
50, 786 Acrylic acid (AA), 11, 781
782 Acrylic binder, 107 Acrylic cross-binder resin, 106 Acrylonitrile (AN), 50, 163 Acute inflammatory response, 627 Acute wounds, 723 Acyl derivatives, 480 Acylation, 389 reaction, 50, 390
393, 391f Acyl-chitosan, 58
59, 58f AD. See Atopic dermatitis (AD) ADA. See Alginate dialdehyde (ADA) Adipose-derived stem cells (ADSC), 331 Adjuvanticity of chitin microparticles, 614
615

of chitosan microparticles, 615
617 ADM. See Acellular dermal matrix (ADM) ADSC. See Adipose-derived stem cells (ADSC) Adsorbent polymeric matrices, 136
137 Adsorbents, 774 Adsorption, 138 capacity, 161 factors affecting adsorption performance, 798
800 mechanism, 792
800 performance, 154 composites, nanocomposites, and fillers, 158
163 cross-linking, 156
157 derivatization, 163
166 pristine chitin, 154
156 relevant developments, 154 Adsorptive membrane, 153
154 Adsorptive processes, chitin supports for, 150
151 Adult shore crab (Carcinus maenas), 764
765 Advanced electronic devices, 77 Advanced electronics, chitin and CS composites for, 73
77 stretchable and wearable devices, 74
77 Aerogels, 784
785 Aggregation properties of CMC, 441
442 AgNPs. See Silver nanoparticles (AgNPs) Agriculture, 363
365 AIBN. See 2-Azobisizobutironitril (AIBN) AKEO. See Apricot kernel oil (AKEO) Alginate, 31
32, 244
246, 324
325, 740
741, 778
779 Alginate, chitin/chitosan, and fucoidan (ACF), 741 Alginate dialdehyde (ADA), 735 Alginate
carboxymethyl chitosan composite, 82
83 Alkaline phosphatase (ALP), 28, 263
264 Alkylated chitosans, 187
188

811

812

Index

Alkylation, 46 Alkylphenol ethoxylate (APEO), 106 Alkylsulfonated chitosan, 412 Allergy and chitinous microparticles, 623
627 chitinase activity in allergic disease, 626
627 reasons for controversy about chitin microparticles, 625 1-Allyl-3-methyl-imidazolium acetate (AMIMAc), 474 1-Allyl-3-methylimidazolium bromide (AMIMBr), 474 1-Allyl-3-methylimidazolium chloride (AMIMCl), 472
473 1-Allyl-3-methylimidazolium (AMIM), 478 ALP. See Alkaline phosphatase (ALP) α-chitin, 342
343, 354
355 Aluminum trihydroxide (ATH), 126 AMCase. See Acid mammalian chitinase (AMCase) AMIM. See 1-Allyl-3-methylimidazolium (AMIM) AMIMAc. See 1-Allyl-3-methylimidazolium acetate (AMIMAc) AMIMBr. See 1-Allyl-3-methylimidazolium bromide (AMIMBr) AMIMCl. See 1-Allyl-3-methylimidazolium chloride (AMIMCl) 2-Amino-2-deoxy-β-D-glucopyranose, 139
140, 343 Amino-COS derivatives, 620 Aminoguanidine, 189
190 Ammonium peroxy disulfate (APS), 49
50 Amphiphilic derivatives, 187 Ampicillin, 101
102 AMPS. See 2-Acrylamido-2-methyl-1propanesulfonic acid (AMPS) Amylase, 739 Amylopectin, 739 AN. See Acrylonitrile (AN) Analgesic properties, 728
729 Angiogenesis, 621, 725
726 Anodic aluminum oxide (AAO), 211 Antheraea pernyi silk fibroin, 8 Antibacterial activity, 113
121, 184
185 in chitosan as templates for essential oils, 706
714 CS
EOs coating and films, 707
712 CS
EOs emulsions and nanogels, 712
713 CS
EOs encapsulations, 713
714

microorganisms used to assess, 708t Antibacterial agents, 453
454 Antibacterial effect, 791
792 Antibiotics, 762 Anticancer effect of chitin and chitosan microparticles, 619
623 chitin and chitosan as anticancer agents, 620
622 microsphere as carriers of anticancer drug, 622
623 Antifungal phytoalexin pisatin, 183 Antifungal properties of CMC, 444
445 Antiinflammatory nature, 730
731 Antimicrobial activity, 31, 178
179 of CMC, 443
444 Antimicrobial and antiinflammatory nature, 730
731 Antioxidant activity, 188
191 in CS as templates for EOs, 697
706 DPPH assay, 699 Folin
Ciocalteu assay, 697
699 FRAP assay, 701 TEAC, 699
700 Antioxidant properties of CS
EOs coating and films, 701
704 of chitosan
EOs emulsions and nanogels, 705 of chitosan
EOs encapsulations, 705
706 of CMC, 444 APEO. See Alkylphenol ethoxylate (APEO) Apoptosis inhibitory activities of CMC, 445 Applied voltage, 245t Apricot kernel oil (AKEO), 701
704 APS. See Ammonium peroxy disulfate (APS) Aquaculture, 762 Aqueous model system, 190 Architectures, chitin and chitosan-based, 484
488 Arginase, 625 Arginine
glycine
aspartate (RGD), 220 Arthroplasty, 240 Arthropods, 345 Articular cartilage, 237 Artificial systems, 315 ASGPR. See Asialoglycoprotein receptor (ASGPR) Asia-Pacific region, 369 Asialoglycoprotein receptor (ASGPR), 216 Asian seabass (Lates calcarifer), 765
766

Index

Aspergillus flavus, 453 Aspergillus fumigatus, 623
625 Aspergillus nidulans, 350 Aspergillus niger, 409 A. niger cell-wall, 62
63 AST-120, 591
592 Asymmetric membranes as wound dressings, 326
327 Asymmetric topological chitosan (ATCS), 211 ATCS. See Asymmetric topological chitosan (ATCS) ATH. See Aluminum trihydroxide (ATH) Atom transfer radical polymerization (ATRP), 479
480 Atopic dermatitis (AD), 122 Atrophic scar, 727 ATRP. See Atom transfer radical polymerization (ATRP) Autologous chondrocytes implantation (ACI), 240
241 2,20 -Azino-bis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS diammonium salt), 699
700, 700f 2-Azobisizobutironitril (AIBN), 49
50

B Bacillus Calmette-Gue´rin, 612
613 Bacterial resistance, 418
419 BAL liver. See Bioartificial liver (BAL liver) Basic fibroblast growth factor (bFGF), 751
752 Batch adsorption processes, 146 Batch systems, 151
152 Batteries, 82
84 Beads, 9, 485
488, 560 chitosan, 782
783 BET model. See Brunauer
Emmett
Teller model (BET model) β-(1,4)-linked 2-acetamino-2-deoxy-β-Dglucopyranose, 343 β-Chitin, 91
92, 342
343, 354
355 β-cyclodextrin
chitosan (CC), 783
784 β-glucan, 612
613 β-glycerophosphate (β-GP), 414
415 β-tricalcium phosphate (β-TCP), 213
215, 519
520 bFGF. See Basic fibroblast growth factor (bFGF) BG. See Bioactive glasses (BG) BGC. See Bioactive glass ceramic (BGC)

813

BHA. See Butylated hydroxyanisole (BHA) BHT. See Butylated hydroxytoluene (BHT) Bioactive glass ceramic (BGC), 3 Bioactive glasses (BG), 262
264 Bioactive molecules, 266 Bioadhesive injectable hydrogels, 743
744 Bioartificial liver (BAL liver), 215
216 Biocompatible/biocompatibility, 2, 9, 44, 136
137, 244, 436
437, 507, 508f, 722
723, 727
728 chitosan/sericin composite nanofibers, 738
739 polymer, 47
48, 178 synthetic polymers, 244
246 Biodegradability, 2, 7
9, 44, 136
137, 244, 436
437, 507, 722
723, 727
728 Biodegradable coating material, 58
59 packaging materials, 31 polymer, 47
48, 178, 185 synthetic polymers, 244
246 transparent electrode, 74
75 Biodegradation, 205
206, 727
728 Biofilms, 185 Biofunctionality, 506, 506f Bioimaging, 459
460 Bioinspiration, 309 Bioinspired membranes, 308
310 applications, 310
316 chitosan, 316
331 preparation, 308
310 Biologic dressings, 313 Biological membrane, 308 Biologic
synthetic dressings, 313 Biomaterial immobilization, 309 Biomaterials, 237, 244 Biomedical application, chitosan for, 206 biosensing, 220
221 derivatives, 212
221 drug delivery, 217
218 gene therapy, 219
220 modification methods, 207
209 modified forms, 209
211 regenerative medicine, 219 tissue engineering, 212
217 wound healing, 218
219 Biomimetic membranes, 308
310 applications, 310
316 chitosan, 316
331 preparation, 308
310 Biomimetic sensor-based membranes, 315
316

814

Index

Biomimetics in wound closure, 313
314 Biophotonic field-effect transistors, 72 Biopolymers, 136
137, 308 biopolymers-derived materials, 642 Bioprinting, 535, 539f, 540f Bioremediation, 25
26 Bioresorbability. See Biodegradability Biosensing, 220
221 Biosensors, 460
461 Blends with natural polymers, 8 with synthetic polymers, 7
8 Blood-clotting behavior, 21 BMIM. See 1-Butyl-3-methylimidazolium (BMIM) BMIMAc. See 1-Butyl-3-methylimidazolium acetate (BMIMAc) BMIMCl. See 1-Butyl-3-methyl-imidazolium chloride (BMIMCl) BMP. See Bone morphogenetic protein (BMP) BMSC. See Bone marrow mesenchymal stem cell (BMSC) Bone, 232
233, 574
577 biology, 504
505 and bone repair, 519
528 bone-forming osteoblasts, 234
235 defects, 232
233, 235
236 heterologous grafts, 235
236 injuries, 235
236 physiology, 233
237 regeneration, 31
32 remodeling, 235 repair, 490 scaffolds for bone tissue regeneration, 236
237 structure and composition, 233
235, 234f tissue engineering, 24
25, 213
215, 420
422 chitosan electrospun materials for, 256
266, 259t tissue replacement requirements, 509
513 Bone marrow mesenchymal stem cell (BMSC), 748 Bone morphogenetic protein (BMP), 258
262 BMP-2, 258
262 Bone tissue engineering (BTE), 500
503, 502f bone tissue materials, 529t chitin and chitosan for, 513
519 for bone and bone repair, 519
528

for cartilage regeneration, 519 clinical requirements for, 506
509 dilemma of chitosan scaffold applicability, 537
539 fabrication methods, 528
537 Bordetella bronchiseptica, 615
616 Botrytis cinerea, 358
359 Bovine serum albumin (BSA), 166 Bridging mechanism, 189t Brunauer
Emmett
Teller(BET) method, 147
148, 675
676, 781 BSA. See Bovine serum albumin (BSA) BST-CarGel, 296, 297t, 298 BTCA. See Butane tetracarboxylic acid (BTCA) BTE. See Bone tissue engineering (BTE) Burn treatment/artificial skin graft, 361 Butane tetracarboxylic acid (BTCA), 120 1-Butyl-3-methyl-imidazolium chloride (BMIMCl), 474 1-Butyl-3-methylimidazolium acetate (BMIMAc), 472
473 1-Butyl-3-methylimidazolium (BMIM), 478 Butylated hydroxyanisole (BHA), 188 Butylated hydroxytoluene (BHT), 188 Butyryl-chitin, 50
51

C C. copticum oil (CCO), 701
704 CA. See Citric acid (CA) Cadmium telluride quantum dots (CdTeQD), 460
461 CAGR. See Compound annual growth rate (CAGR) Calcified final zone of cartilage tissue, 238 Calcium absorption acceleration effect in vivo, 180
181 Calcium carbonate (CaCO3), 347 Calcium hydrogen sulfite [Ca(HSO3)2], 347 Calcium hydroxide [Ca(OH)2], 347 Calcium phosphate (CaP), 453 Calorimetry, 402 Cambogic acid (GA), 564
566 Camptothecin (CPT), 422 Cancellous bone. See Porous trabecular bone Candida albicans, 358
359, 444
445, 453, 612
614 Candidiasis, 444
445 CaP. See Calcium phosphate (CaP) Carassius auratus gibelio.. See Gibel carp (Carassius auratus gibelio)

Index

Carbohydrate metabolism, 142 Carbohydrates-derived composites, 652
657 chitin/chitosan composites with cellulose, 652
654 with polysaccharides, 656
657 with starch, 654
655 Carbon black (CB), 75
77 Carbon nanotubes (CNTs), 3, 483 Carbonization
activation process, 79
80 Carboxyl group, 184
185 Carboxyl-modified poly(vinyl alcohol) (PVA
COOH), 732
733, 732f Carboxylate-rich magnetic chitosan flocculants, 784 Carboxylated cellulose nanofibrils (CCNFs), 29
30 Carboxylated chitosan (CCS), 786 Carboxymethyl cellulose (CMC), 652
653 Carboxymethyl chitin (CMCH), 46
48, 47f, 287
288 Carboxymethyl chitosan (CMC), 268, 295
296, 412, 434
437 applications, 461
462 biological properties antifungal properties, 444
445 antimicrobial activities, 443
444 antioxidant properties, 444 apoptosis inhibitory activities, 445 biomedical applications antibacterial agents, 453
454 bioimaging, 459
460 biosensors, 460
461 drug delivery, 447
449 gene therapy, 453 hydrogels for medicinal use, 454
459 targeted drug delivery, 452
453 tissue engineering and regenerative medicine, 449
451 wound healing, 445
447 CMC-based systems, 450t physicochemical properties chelating and sorption properties, 443 moisture retention and absorption properties, 442
443 solubility and aggregation properties, 441
442 preparative methods, 437
441, 438f CNBC, 440 N,N-di-CMC preparation, 439
440 N,O-CMC preparation, 439 N-CMC preparation, 439 NCEC, 440

815

NSC, 440 O-CMC synthesis, 438
439 OSC, 441 Carboxymethyl chitosan/gelatin/ nanohydroxyapatite composite, 293 1-Carboxymethyl-3-methylimidazolium hydrochloride ((IMIM-COOH)Cl), 474 Carboxymethylated cellulose solution (CMcellulose solution), 778
779 Carboxymethylated chitosan (CM chitosan), 55
56 Carboxymethylation, 46, 51
53, 438
439 Carcinus maenas.. See Adult shore crab (Carcinus maenas) Cartilage defects, 238
239 treatment, 239
241 lesions, 239 physiology, 237
241 regeneration, 519 repair, 212
213 structure and composition, 237
238 tissue, 238 tissue engineering, 314
315, 420
422 chitosan electrospun materials for, 266
269 Casein protein, 651 Castor oil, 387 Catalytic carboxylate, 143 Cationic groups, 184 CB. See Carbon black (CB) CBH. See Chitosan biguanidine hydrochloride (CBH) CC. See β-cyclodextrin
chitosan (CC) CCGO. See Cyclodextrin
chitosan/ graphene oxide (CCGO) CCL2, 625 CCL22. See Macrophage-derived chemokine (CCL22) CCMs. See Chitin/chitosan microspheres (CCMs) CCNFs. See Carboxylated cellulose nanofibrils (CCNFs) CCO. See C. copticum oil (CCO) CCS. See Carboxylated chitosan (CCS) CD. See Chitosan
dextran (CD); Cyclodextrin (CD) CDNs. See Chitosan
dextran sulfate nanoparticles (CDNs) CdTe QDs-CMC NO donors. See CdTeQD
CMC nanocomposite NO donors (CdTe QDs-CMC NO donors)

816

Index

CdTeQD. See Cadmium telluride quantum dots (CdTeQD) CdTeQD
CMC nanocomposite NO donors (CdTe QDs-CMC NO donors), 460
461 Cell permeation, 671
672 wall destruction and leakage, 566
567 Cellulose, 24, 142, 556
557, 652
654, 778 constitutes, 367 Cellulose nanofibers (CNFs), 701
704 cGAS-STING pathways, 619 CGC. See Chitin
glucan complex (CGC) CH. See Chitin (CH) CH-GP-HEC hydrogel. See Chitosan-beta glycerophosphate-hydroxyethyl cellulose hydrogel (CH-GP-HEC hydrogel) Charge patching mechanism, 189t Chelating, 191
193 properties of CMC, 443 Chemical cross-linking, 289, 298 Chemical finishing, 105 Chemical gels, 211 Chemical hydrogels, 289
294 click chemistry, 294 enzymatic cross-linking, 293
294 Michael additions, 291
293 photocross-linking hydrogels, 289
290 Schiff base cross-linking, 291, 292t Chemical injectable hydrogels, 282 Chemical modification, 479
483 Chemical properties of chitin and CS, 72
73, 73f Chemical structure and preparation of chitin and chitosan, 4
6 Chemically modified chitin, 46
53 chitin derivatives, 50
53 chitin graft copolymerization, 49
50 CM-chitin and chitin alkylated derivatives, 46
48 P-chitin, 48
49 S-chitin, 49 Chemically modified chitinous polysaccharides, 62
63 Chemically modified chitosan, 53
62 acyl-chitosan, 58
59 chitosan derivatives, 54t, 60
62 CM chitosan and chitosan alkylated derivatives, 55
56 graft copolymerization of chitosan, 59
60

P-chitosan, 56
57 quaternary chitosan derivatives, 59 S-chitosan, 57
58 Chemisorption, 138
139 ChGC. See Chitosan
glucan complex (ChGC) ChHA. See Chitin-based biocomposite (ChHA) CHI3L1, 621 Chitin (CH), 2
3, 6f, 47
48, 72, 136
137, 137t, 138f, 178, 200
201, 249, 282
283, 316
317, 317f, 342
343, 385
386, 389, 408, 435, 671
672, 690, 762
763 adsorption properties chitin chemistry, 139
144 chitin physics, 144
148 alkylated derivatives, 46
48 allomorphs, 342
343 as anticancer agents, 620
622 antimicrobial activity, 178
179 antioxidant activity, 188
191 applications of chitin and chitosan blends, 8
9 biomedical applications in ILs, 489
490 characterization of shrimp, 95
96 chemical and physical modifications, 389
397 chemical structure, 390f and preparation, 4
6, 4f chitin-based composite, 22
27 composite for advanced electronics, 73
77 conductive composites, 73
74 deacetylation reaction, 45f derivatives, 46, 47t, 50
53, 53f reactions, 46 dietary activity, 178
181 difference in immunopotentiating effects of, 611 dissolution, 474 using ILs, 474 dry weight, 93t economic potential, 367
370 effect on disease resistance, 766
767 emulsifying properties, 185
188 flocculent and chelating, 191
193 future trends, 193 graft copolymerization, 49
50 grafting, 158
159 hydrogels, 296t immunostimulatory effect

Index

of chitin on finfish, 763
764 of chitin on shellfish, 764
765 injectable polymeric gels, 283
294 IPN from, 9
14 isolation of, 94
95 mechanism of action as immunostimulant, 767
768 microparticles adjuvanticity, 614
615 antimicrobial properties, 613
614 microsphere as carriers of anticancer drug, 622
623 mixing with different types of surfactants, 396
397 NCO-functionalization reaction, 393
395 origin and sources, 343
345 parameters, 475
477 physical and chemical properties, 72
73 physics, 144
148 polymer blends from, 6
9 processability via ILs, 479
483, 487t properties, 354
359 reaction with HMDI, 395f solubilization behavior, 476t sources, 93 structure, 91
92, 474
475, 475f supports for adsorptive processes, 150
151 synthesis, 345
353 by biological method, 348
350 by chemical method, 346
348 of derivatives, 352
353 Chitin nanocrystals (CNC), 75
77 Chitin nanofiber (ChNF), 648
649, 653 papers, 72 Chitin nanofibrils (CNF), 146
147 Chitin-based biocomposite (ChHA), 490 Chitin-based gels, 14
22 applications of, 19
22 covalently cross-linked gels, 18
19 physically associated gels, 14
17 polyelectrolyte complexation, 17
18 Chitin-based nanocomposites, 27
32 nanocomposites from chitin and chitosan, 28
29 Chitin-based oleogels application, 386
387 chemical and physical modifications, 389
397 preparation method and mechanism, 387
389 rheological properties, 397
400

817

thermal behavior, 402
404 thermogravimetric properties, 400
402 Chitin-based polyurethane synthesis (TRCPU synthesis), 396, 396f Chitin-nanofibrils (CN), 191 Chitin/chitosan microspheres (CCMs), 77
78 Chitin/gelatin-based composites, 648
650 Chitinase activity in allergic disease, 626
627 Chitinase-like proteins (CLPs), 611
612, 626 Chitinases, 611
612, 626 Chitin
glucan complex (CGC), 44, 62 CGC
aldehyde, 63 Chitinous microparticles (CMPs), 610
611 allergy and, 623
627 antimicrobial properties of chitin and chitosan microparticles, 613
614 chitinases, 611
612 chitolectins, 611
612 controversy about CMPs adjuvanticity, 617
619 difference in immunopotentiating effects of chitin and chitosan, 611 immunoregulation, 610
611 and trained immunity, 612
613 Chitinous polysaccharides, 44
45, 63 Chitinous protein complexes, 74 Chitin
protein interaction, 142 Chitolectin (YKL-40), 611
612, 629 Chitooligosaccharides (COS), 180 Chitosan (CS), 2
3, 6f, 10, 12, 44, 72, 91, 136
137, 137t, 200, 212
213, 244
246, 249
255, 249f, 282
283, 308, 316
331, 317f, 319t, 343, 385
386, 388
389, 408, 413, 423
424, 434, 443
444, 461
462, 556
557, 645, 671
672, 690, 722
723, 739
740, 762
763, 797 advantageous properties for wound management antimicrobial and antiinflammatory nature, 730
731 biocompatibility and biodegradability, 727
728 hemostatic and analgesic properties, 728
729 alkylated derivatives, 55
56 as anticancer agents, 620
622 antimicrobial activity, 101
105 application, 318
323

818

Index

Chitosan (CS) (Continued) applications of chitin and chitosan blends, 8
9 biological properties, 357
359, 411
412 biomedical application, 360
362 burn treatment/artificial skin graft, 361 drug delivery, 362 ophthalmology, 362 tissue engineering, 361
362 wound dressing/wound healing, 360 biomedical applications, 318, 328t in ILs, 489
490 as carrier of essential oils, 690
692, 691f cellular interactions, 252
253 characterization, 674
676 of shrimp, 95
96 chemical and physical modifications, 389
397 acylation reactions, 390
393, 391f N-acylation, 391
392, 393f N-and O-acylation, 392
393, 394f chemical derivatization, 53 chemical structure, 200f and preparation, 4
6, 5f effect of chitin and chitosan on disease resistance, 766
767 chitosan-treated textile products, 107
121 composite for advanced electronics, 73
77 conductive composites, 73
74 CS
nanoHA electrospun scaffolds, 264
266 derivatives, 60
62, 61f in wound management, 749
752 of chitin, 500
501 difference in immunopotentiating effects of, 611 dissolution using ILs, 474 economic potential, 367
370 electrospinning, 253
255 electrospun materials for bone tissue engineering, 256
266, 259t for cartilage tissue engineering, 266
269 factors to synthesis, 99
100 films for food packaging, 322t gel for light-emitting device, 366 grafts, 60 hydrogels, 296t immunostimulatory effect

of chitosan on finfish, 765
766 of chitosan on shellfish, 766 industrial applications, 362
367 agriculture, 363
365 chitosan gel for light-emitting device, 366 cosmetics industries, 365
366 food processing, 365 paper industry, 367 photography, 366 textile industry, 366
367 water engineering, 363 injectable polymeric gels based on, 283
294 IPN from, 9
14 IPN hydrogels, 11 mechanism of action as immunostimulant, 767
768 in medical textiles, 121
124 membranes in wound dressing, 323
326 microparticles adjuvanticity, 615
617 antimicrobial properties, 613
614 microsphere as carriers of anticancer drug, 622
623 microwave-assisted deacetylation, 673 molecular weights, 98t nanocapsules for EOs encapsulation, 696
697, 696f, 698t nanosystems in infectious diseases, 566
567 in neoplastic diseases, 561
566 NCO-functionalization reaction, 393
395, 395f oleogel, 388
389 origin and sources, 343
345 parameters, 478
479 physical and chemical properties, 72
73, 96
99 physicochemical properties, 250
252, 354
357, 410
411 polymer blends from, 6
9 polymer thiolation, 673 preparation of, 557f processability via ILs, 479
483, 487t processing, 201
205, 435
436 production, 94
95 properties, 354
359 and limitations, 205
206, 436
437 reaction with HMDI, 395f scaffold applicability, 537
539 sources, 93, 408
409

Index

structure, 91
92, 409
410, 477
478, 478f synthesis, 345
353 and characterization, 93
96 of derivatives, 352
353 and synthetic matrices, 12
13 to textile products, 105
107 in textile-reinforced composites, 124
126 thiolation, 672, 677
678 as tissue supporting material, 419
424 application in bone and cartilage tissue engineering, 420
422 application in skin and liver tissue engineering, 422
424 Chitosan biguanidine hydrochloride (CBH), 656 Chitosan combined with silk fibroin (CS/SF), 735 Chitosan oligosaccharides (COS), 82
83, 620
621 Chitosan-based biocomposites for wound healing, 739
749 Chitosan-based biomaterials in wound management fibers, 738
739 gels, 737
738 hydrogels, 731
735 membranes, 735 scaffolds, 737 sponges, 736
737 Chitosan-based CNT composite fibers, 75
77 Chitosan-based composite, 22
27 applications of chitin-and chitosan-based composites, 25
27 drug delivery, 27 dye removal, 25
26 heavy metals removal, 26 tissue engineering, 27 wound healing, 26 from natural fillers, 23
25 from synthetic fillers, 22
23 Chitosan-based edible films, 185 Chitosan-based EOs coatings and films, 692
693, 694t Chitosan-based full-IPN, 10
11 Chitosan-based gels, 14
22 applications of, 19
22, 20f covalently cross-linked gels, 18
19 physically associated gels, 14
17 polyelectrolyte complexation, 17
18 Chitosan-based hydrogels, 436
437

819

Chitosan-based material for wound management, 752
753 Chitosan-based nanocomposites, 27
32 applications, 29
32 biodegradable packaging materials, 31 bone regeneration, 31
32 drug delivery, 32 dye removal, 29
30 heavy metals removal, 30 wound healing, 30
31 from chitin and chitosan, 28
29 Chitosan-based oleogels application, 386
387 preparation method and mechanism, 387
389 rheological properties, 397
400 thermal behavior, 402
404 thermogravimetric properties, 400
402 Chitosan-based polysaccharides, 45 Chitosan-based sorbents for water/ wastewater remediation, 775
783 Chitosan-based therapeutic systems, 413
419 local administration, 418
419 ocular drug delivery, 413
415 oral drug delivery, 415
417 transdermal drug delivery, 417
418 Chitosan-beta glycerophosphatehydroxyethyl cellulose hydrogel (CH-GP-HEC hydrogel), 422 Chitosan-modified poly(mPEGMA-coMAA) nanoparticles, 417 Chitosan-TGA conjugate. See Chitosan
thioglycolic acid conjugate (Chitosan-TGA conjugate) Chitosan/alginate-based biomaterials, 741, 742t Chitosan/biopolymer blend, 778
780 Chitosan/nanoparticles composites, 780
781 Chitosan
benzoic acid (CS
BA), 705 Chitosan
carbon black aerogels, 74
75 Chitosan
carbon nanotubes, 72 Chitosan
dextran (CD), 744 Chitosan
dextran sulfate nanoparticles (CDNs), 414 Chitosan
essential oils encapsulations, antioxidant properties of, 705
706 Chitosan
glucan complex (ChGC), 44, 62
63 Chitosan
thioglycolic acid conjugate (Chitosan-TGA conjugate), 361
362

820

Index

Chitosanzeolite (CS-SZ), 125 2-Chloroethylsulfonate, 63 ChNF. See Chitin nanofiber (ChNF) Cholesterol-binding capacity, 365 Cholesterol-lowering activity, 179 Cholestyramine, 178 Chondroitin, 331 Chondroitin sulfate (CS), 750 Chromatographic purification processes, 137 Chronic renal failure (CRF), 591
592 chitosan and derivatives, 591
597 Chronic wound, 723 CIP. See Ciprofloxacin hydrochloride (CIP) Ciprofloxacin hydrochloride (CIP), 736 Cirrhina mrigala.. See Mrigal (Cirrhina mrigala) Citrates, 17 Citric acid (CA), 120 Cleaving glycosidic linkages, 46 Click chemistry, 294 Clostridium tetani, 615
616 CLPs. See Chitinase-like proteins (CLPs) CM chitosan. See Carboxymethylated chitosan (CM chitosan) CM-cellulose solution. See Carboxymethylated cellulose solution (CM-cellulose solution) CMC. See Carboxymethyl cellulose (CMC); Carboxymethyl chitosan (CMC) CMCH. See Carboxymethyl chitin (CMCH) CMPs. See Chitinous microparticles (CMPs) CN. See Chitin-nanofibrils (CN) CNBC. See N-carboxybutyl chitosan (CNBC) CNC. See Chitin nanocrystals (CNC) CNF. See Chitin nanofibrils (CNF) CNFs. See Cellulose nanofibers (CNFs) CNTs. See Carbon nanotubes (CNTs) CoA. See Coenzyme A (CoA) Coacervate phase, 17
18 Coating method, 105 of shrimp chitosan, 105
107 Cobalt ions, 26 Cobalt molybdate (CoMoO4), 80
82 Coenzyme A (CoA), 456
457 Collagen (COL), 244
246, 331, 744
745, 750 Colletotrichum lindemuthianium, 350 Colloidal suspension, 3 Colony-stimulating factors, 627 Complex modulus, 399

Composites, 158
163 materials, 22 from natural fillers, 23
25 from synthetic fillers, 22
23 Compound annual growth rate (CAGR), 368
369 Concentration, 613 Conductivity, 245t Continuous-flow systems, 152
154 Conventional SPH (CSPH), 457
458 Coriander (Coriandrum sativum L.), 713
714 Cortical/compact bone, 234 Corynebacterium diphtheria, 615
616 COS. See Chitooligosaccharides (COS); Chitosan oligosaccharides (COS) Cosmetics industries, 365
366 Covalently cross-linked gels, 18
19 CPT. See Camptothecin (CPT) Crab, 343
344, 367 Crayfish (Procambarus clarkia), 764
765 Creatinase, 326 Creatininase, 326 CRF. See Chronic renal failure (CRF) CrI. See Crystalline index (CrI) Cross-linked chitosan, 82
83 Cross-linking, 156
157 Crustacean shell waste, 408
409 by-products, 178 Crustaceans, 345 Cryogels, 12 Crystalline index (CrI), 99t Crystalline polysaccharide, 44 CS. See Chitosan (CS); Chondroitin sulfate (CS) CS
BA. See Chitosan
benzoic acid (CS
BA) CS-based polymer materials antimicrobial activity, 178
179 antioxidant activity, 188
191 dietary activity, 178
181 emulsifying properties, 185
188 flocculent and chelating, 191
193 future trends, 193 CS-SZ. See Chitosanzeolite (CS-SZ) CS/SF. See Chitosan combined with silk fibroin (CS/SF) CS25TCP, 420
421 CS50HA, 420
421 CSMMA. See Methyl methacrylatemodified chitosan (CSMMA) CSPH. See Conventional SPH (CSPH) Curcumin-loaded chitosan nanoparticles, 418

Index

Cyclodextrin (CD), 598
601 Cyclodextrin
chitosan/graphene oxide (CCGO), 783
784, 795
797, 796f Cyclophosphamide, 672 from polymeric films, 685
686 in vitro release preparation of polymeric films, 676
677 in vitro release of, 677

D DA. See Degree of acetylation (DA) DAC. See Deacetylated chitin (DAC) DAHBC (thermoresponsive amphilic polysaccharide), 284
285 DC. See Degree of crystallinity (DC); Direct current (DC) DCH. See Deaceytaled CH (DCH) DD. See Degree of deacetylation (DD) Deacetylated chitin (DAC), 595
596 Deacetylation, 435
436, 558 Deaceytaled CH (DCH), 673 Decalcification process, 201
202 Decolorization, 348, 435 Dectin-1, 610
611, 625 Deep zone of cartilage tissue, 238 Defensive/inflammatory phase, 725 Degradation temperature of shrimp chitosan (DTGmin), 96 Degree of acetylation (DA), 97, 97t, 139
140, 354, 437
438, 475, 672 Degree of crystallinity (DC), 675 Degree of deacetylation (DD), 97, 97t, 200, 249, 351, 358, 408, 411, 437
438, 613, 618 Degree of substitution (DS), 182, 437
438 Degrees of hydroxypropyl molar substitution (DS), 287
288 Demineralization, 201
202, 347
348, 435, 557 Demineralized skeleton, 80
82 Deproteination, 558 Deproteinization, 201
202, 347, 435 Derivatization, 163
166 Desalination, 789
790 Dextran, 741
744 Dextran sulfate sodium (DSS), 601 D-glucosamine, 409 Dialdehydes, 18
19 Dietary activity calcium absorption acceleration effect in vivo, 180
181

821

hypocholesterolemic effect, 178
179 prebiotics ingredients, 180 Dietary compounds, 178 Diethylene triamine pentaacetic acid (DTPA), 790 Differential scanning calorimetry (DSC), 7, 144, 402
404, 650, 675, 682f Diffractograms, 680
681 Diffuse hemorrhage, 733
734 3,4-Dihydroxyphenylalanine (DOPA), 313
314 Dimethyl sulfoxide (DMSO), 258, 267
268, 483 Dimethylacetamide (DMAc), 44 Dimethylacetamide/LiCl and pyridine, 50
51 Dimethylformamide (DMF), 48, 57
58, 441 1,3-Dimethylimidazolium dimethylphosphate (MMIM (Me2PO4)), 474 1,3-Dimethylimidazolium (DMIM), 478 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 188 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 190 assay, 699, 699f radicals, 358 Direct current (DC), 11 Disease resistance, effect of chitin and chitosan on, 766
767 DLS. See Dynamic light scattering (DLS) DMAc. See Dimethylacetamide (DMAc) DMEM/PBS. See Dulbecco’s modified eagle’s medium and phosphate buffered saline (DMEM/PBS) DMF. See Dimethylformamide (DMF) DMIM. See 1,3-Dimethylimidazolium (DMIM) DMSO. See Dimethyl sulfoxide (DMSO) DOPA. See 3,4-Dihydroxyphenylalanine (DOPA) Doxorubicin (DOX), 449 DOX-loaded hydrogels, 288
289 DOX-loaded nanoparticles, 449 hydrochloride, 32 Doxorubicine hydrochloride (DOX•HCl), 284
285 DPPH. See 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Drug delivery, 27, 32, 217
218, 315, 362, 447
449, 490
491, 556 antineoplastic drug delivery, 563t blocking of essential elements, 567
570 chitosan-based nanosytems

822

Index

Drug delivery (Continued) in infectious diseases, 566
567 in inflammatory diseases, 570
572 in neoplastic diseases, 561
566 limitations of chitosan as, 560 properties and, 558
561 systems, 58, 142, 623 Drug-loaded PVA/(CS composite nanofibers, 417
418 DS. See Degree of substitution (DS); Degrees of hydroxypropyl molar substitution (DS) DSC. See Differential scanning calorimetry (DSC) DSS. See Dextran sulfate sodium (DSS) DSSC. See Dye-sensitized solar cells (DSSC) DTGmin. See Degradation temperature of shrimp chitosan (DTGmin) DTPA. See Diethylene triamine pentaacetic acid (DTPA) Dulbecco’s modified eagle’s medium and phosphate buffered saline (DMEM/ PBS), 285
286 Dye(s), 783 dyed denim fabric, 109
111 removal, 25
26, 29
30 Dye-sensitized solar cells (DSSC), 84 Dynamic light scattering (DLS), 213
215

E ECH. See Epichlorohydrin (ECH) ECM. See Extracellular matrix (ECM) EDAX. See Energy Dispersive spectrometer (EDAX) EDC. See 1-Ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) Edible electronics, 74
75 EDTA. See Ethylenediaminetetraacetic acid (EDTA) EDX spectroscopy. See Electron dispersive X-ray spectroscopy (EDX spectroscopy) EGDE. See Ethylene glycol diglycidyl ether (EGDE) EGF. See Epidermal growth factor (EGF) EGFR. See Epidermal Growth Factor Receptor (EGFR) Egg yolk, 186
187 Eggshell membrane (ESM), 331 Elastic gel, 598
601 Elasticity, 398

eLbL. See Electrostatic Layer-by-Layer (eLbL) Electrochemical processes, 13
14 Electrode material supercapacitor applications, 80
82 Electrolyte composite, 83
84 Electrolytic manganese dioxide (EMD), 82
83 Electron dispersive X-ray spectroscopy (EDX spectroscopy), 108
112, 792
793 Electrospinning, 232, 241
249, 242f, 532
533, 533f method, 3, 738
739 parameters, 242
244, 245t regulatory issues of electrospinning scaffolds, 255
256 for tissue engineering scaffolds, 244
249 Electrospun chitosan electrospun chitosan
inorganic particles composite scaffolds, 262
266 materials, 232
233 bone physiology, 233
237 for bone tissue engineering, 256
266, 259t cartilage physiology, 237
241 for cartilage tissue engineering, 266
269 chitosan, 249
255 regulatory issues of electrospinning scaffolds, 255
256 scaffolds, 257
262 Electrospun fibrillar systems, 248 Electrospun nanofibrous membranes, 745 Electrostatic action, 14 attraction, 794
795 charge, 17 interaction, 138
139, 160, 798 Electrostatic Layer-by-Layer (eLbL), 84 Electrostatic repulsions, 187
188 EMD. See Electrolytic manganese dioxide (EMD) EMIM(Me2PO4). See 1-Ethyl-3methylimidazolium dimethyl phosphate (EMIM(Me2PO4)) EMIMAc. See 1-Ethyl-3-methylimidazolium acetate (EMIMAc) EMIMCl. See 1-Ethyl-3-methylimidazolium chloride (EMIMCl) Emulsifying capacity of egg yolk, 186
187, 187t

Index

Emulsifying properties, 185
188 Emulsions antimicrobial activity of chitosan
EOs emulsions and nanogels, 712
713 antioxidant properties of chitosan
EOs emulsions and nanogels, 705 chitosan-based EOs emulsions and naogels, 693
696, 695f emulsion-stabilizing properties, 186 emulsion-templated method, 388
389 Energy Dispersive spectrometer (EDAX), 781 Energy storage devices, 77
85 batteries, 82
84 SCs, 77
82 solar cells, 84
85 Environmental pH affect antimicrobial activity, 613 Enzymatic cross-linking, 293
294 Enzymatic hydrolysis, 4 Enzyme, 143, 158
159 immobilization approach, 159 EOs. See Essential oils (EOs) Eotaxin, 624
625 Epichlorohydrin (ECH), 164
165, 774 Epidermal growth factor (EGF), 737
738 Epidermal Growth Factor Receptor (EGFR), 564
566 Escherichia coli, 3 ESM. See Eggshell membrane (ESM) Essential oils (EOs), 690 antibacterial activity in CS as templates for, 706
714 antioxidants activity in CS as templates for, 697
706 chitosan as carrier of, 690
692 chitosan-based Eos coatings and films, 692
693 emulsions and nanogels, 693
696 CS nanocapsules for EOs encapsulation, 696
697 Ethanol
water mixtures, 7 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 216, 452 1-Ethyl-3-methyl-imidazolium acetate (EMIMAc), 474 1-Ethyl-3-methylimidazolium chloride (EMIMCl), 474 1-Ethyl-3-methylimidazolium dimethyl phosphate (EMIM(Me2PO4)), 474 Ethylene glycol diglycidyl ether (EGDE), 24, 775
776

823

Ethylene vinyl alcohol (EVOH), 656 Ethylenediaminetetraacetic acid (EDTA), 348, 786 Evaporating technique, 3
4 EVOH. See Ethylene vinyl alcohol (EVOH) Expanded bed adsorption, 152 Extracellular matrix (ECM), 232
235, 237
238, 282, 314
315, 725
726 Extract of sumac (SE), 704 Extraction process of chitin and chitosan, 202, 203f Extremophile organisms, 145 Extrinsic factors, 726

F FA. See Folic acid (FA) Fabrication techniques, 258, 528
537 FA-conjugated CMCcoordinated
manganese-doped zinc sulfide quantum dot nanoparticles (FA
CMC
ZnS:Mn nanoparticles), 459
460 FA-modified CMC (FCC), 452 Farnesyl pyrophosphate, 32 Fat, 387
388 Fatty acids, 398
399 FCC. See FA-modified CMC (FCC) Fenton’s reagent, 49
50 Ferric ion reducing antioxidant power assay (FRAP assay), 701 antioxidant assay and CS as templates, 702t Ferritin, 190 FE-SEM. See field emission-scanning electron microscopy (FE-SEM) FHF. See Fulminant hepatic failure (FHF) FIBCD1, 610
611 Fibers, 738
739 bonding, 534, 536f Ficolin, 610
611 field emission-scanning electron microscopy (FE-SEM), 648
649 Fillers, 158
163 Films, 9, 485, 560
561 Finfish, 763 immunostimulatory effect of chitin on, 763
764 of chitosan on, 765
766 Finishing processes, 105 First-generation SPH. See Conventional SPH (CSPH) Fish tissue proteins, 190

824 Fixed-bed modes, 152 FL microscopy. See Fluorescence microscopy (FL microscopy) Flocculants, 777
778 Flocculation efficiency, 191
192 mechanisms of chitosan-based flocculants, 189t Flocculent, 191
193 Fluidized bed, 152 Fluorescence microscopy (FL microscopy), 449 Fluoride, 776 5-Fluorouracil (5-FU), 459
460 FO. See Forward osmosis (FO) Folic acid (FA), 452 Folic acid receptor (FR), 563
564 Folin
Ciocalteu assay, 697
699 Follicle stimulating hormone (FSH), 144 Food packaging applications, 642 carbohydrates-derived composites, 652
657 chitin/chitosan-derived composites, 651
652 chitin/gelatin-based composites, 648
650 chitosan/chitin-based composites for, 643
652 G-Ch proteins-derived composite, 644
648 inorganic materials-derived composites, 659
662 synthetic polymer, 657
659 Food processing, 365 Formic acid (HCOOH), 57
58, 557 Forward osmosis (FO), 790 Fourier-transform infrared spectroscopy (FTIR spectroscopy), 95, 107
108, 213
215, 674
675, 679
680, 680f, 792
793 FR. See Folic acid receptor (FR) FRAP assay. See Ferric ion reducing antioxidant power assay (FRAP assay) Free divalent metal ions, 143 Freeze gelation, 528, 532f Freeze-dried hydrogels, 733 Freeze-drying, 3
4, 258, 528 Freundlich isotherm, 777
778 FSH. See Follicle stimulating hormone (FSH) FT-Raman spectra, 141

Index

FTIR spectroscopy. See Fourier-transform infrared spectroscopy (FTIR spectroscopy) 5-FU. See 5-Fluorouracil (5-FU) Full-thickness defects, 239 Fulminant hepatic failure (FHF), 215
216 Fungal chitin and chitosan, 44 Fungi, 343

G GA. See Cambogic acid (GA) GAGs. See Glycosaminoglycans (GAGs) Galactosylated hyaluronic acid (GHA), 577
578 Gamma radiation, 49
50 γ-Chitin, 91
92, 342
343, 354
355 Gas sorption analyzer, 675
676 Gastric carcinoma cell line (MGC803), 620
621 Gastrointestinal tract, 767
768 GBR. See Guided bone regeneration (GBR) G-Ch. See Gelatin/chitosan (G-Ch) Gel(s), 9, 737
738 formation, 16 Gelatin (G), 32, 244
246, 645, 745
747, 778 chitin/chitosan-derived composites, 651
652 chitin/gelatin-based composites, 648
650 G-Ch proteins-derived composite, 644
648 Gelatin microspheres (GMs), 740 Gelatin/chitosan (G-Ch), 645
646 Gelation process, 15
16 Gene delivery, 60, 490
491 Gene therapy, 219
220, 453 Generally Recognized as Safe (GRAS), 690
691 Genipin, 18
19 Gentamicin (G), 101
102, 739
740 Geranyl-pyrophosphate pathways, 32 GFs. See Growth factors (GFs) GHA. See Galactosylated hyaluronic acid (GHA) GHs. See Glycoside hydrolases (GHs) Gibel carp (Carassius auratus gibelio), 765
766 GLA. See Glutaraldehyde (GLA) GlcN. See Glucosamine (GlcN) GlcNAc. See N-acetyl glucosamine (GlcNAc) Global Industry Analysts Inc., 368
369

Index

Glucosamine (GlcN), 91
92, 98
99 Glutaraldehyde (GLA), 18
19, 60, 82
83, 162, 774 cross-linker, 8 Glutathione (GSH), 448 Glutathione-based chitosan films experiment characterization of chitosan and thiolated materials, 674
676 evaluation of in vitro release of cyclophosphamide, 676
677 materials, 672 swelling analysis, 676 synthesis of thiolated chitosan, 673
674 physicochemical characterization of polymeric materials, 678
684 swelling study, 685 synthesis of thiolated polymers, 677
678 in vitro release of cyclophosphamide from polymeric films, 685
686 Glycerophosphate (GP), 212
213 Glycidyl methacrylate, 448 Glycidyl methacrylate
hydroxypropyl chitin-based hydrogel (GM-HPCH), 288 Glycine carboxylic acid group, 677
678 Glycol chitin-P4 hydrogels, 288
289 Glycosaminoglycans (GAGs), 237
238, 318, 331, 727 Glycoside hydrolases (GHs), 142 Glycyrrhizin-modified O-CMC nanoparticles, 452 GM-HPCH. See Glycidyl methacrylate
hydroxypropyl chitinbased hydrogel (GM-HPCH) GMs. See Gelatin microspheres (GMs) GO. See Graphene oxide (GO) Gold (Au), 3 GP. See Glycerophosphate (GP) Graft copolymerization, 46, 49
50, 53 of chitosan, 59
60 reactions, 46 Grafting methods, 56 Gram-negative bacteria, 363
364 Gram-positive bacteria, 363
364, 418
419 Graphene oxide (GO), 73
74, 146
147, 783
784 GRAS. See Generally Recognized as Safe (GRAS) Green tea oil (GTO), 705
706

825

Grey mullet (Mugil cephalus), 765
766 Growth factors (GFs), 312 GSH. See Glutathione (GSH) GTO. See Green tea oil (GTO) Guided bone regeneration (GBR), 315 application of chitosan-based membranes for GBR tissue engineering, 327
331 Gut species, 180

H 1

H-Nuclear magnetic resonance spectroscopy (1H-NMR spectroscopy), 674 HA. See Hyaluronic acid (HA); Hydroxyapatite (HAp) HAp. See Hydroxyapatite (HAp) HAS. See Human serum albumin (HAS) HAT. See Hydrogen atoms transfer reactions (HAT) HBC. See Hydroxybutyl chitosan (HBC) hBMSCs. See Human bone marrow mesenchymal stem cells (hBMSCs) HCl. See Hydrochloric acid (HCl) HCMs. See Hierarchically porous carbon microspheres (HCMs) HCOOH. See Formic acid (HCOOH) hDFb. See Human dermal fibroblasts (hDFb) HDM. See House dust mite (HDM) Heavy metals, 154
155, 783 removal, 26, 30 HEC. See Hydroxyethyl cellulose (HEC) Helium displacement micropycnometer, 675
676 Hematoma, 727 Hemocyte-mediated responses, 764
765 Hemoglobin, 190 Hemostatic/hemostasis activity, 722
723 phase, 724
725 properties, 728
729, 728f Hetero-COS, 189
190 Heterocyclic amines, 163
164 Heterogeneous adsorption sites, 139 Heterogeneous deacetylation, 350 Heterogeneous phase adsorption, 138, 156 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), 254, 267
268 1,6-Hexamethylenediisocyanate (HMDI), 393
394 Hexavalent chromium, 30 1,1,1,3,3,3-HFIP. See 1,1,1,3,3,3-Hexafluoro2-propanol (HFIP)

826

Index

Hierarchically porous carbon microspheres (HCMs), 77
78 High-molecular-weight chitosan (HMWC), 356, 358 Highly toxic organomercury compounds, 191 Histamine, 725 HLC. See Human-like collagen (HLC) HMDI. See 1,6-Hexamethylenediisocyanate (HMDI) hMSCs. See Human mesenchymal stem cell (hMSCs) HMWC. See High-molecular-weight chitosan (HMWC) Homogeneous deacetylation, 5 Horseradish peroxidase (HRP), 293 Hot-pressing technique, 3
4 House dust mite (HDM), 623
624 HPC. See Hydroxylpropyl cellulose (HPC) HPCHs. See Hydroxypropyl chitins (HPCHs) HRP. See Horseradish peroxidase (HRP) HTCC. See N-(2-hydroxy) propyl-3trimethylammonium chitosan chloride (HTCC) Human bone marrow mesenchymal stem cells (hBMSCs), 515
516 Human dermal fibroblasts (hDFb), 489 Human mesenchymal stem cell (hMSCs), 246
247, 421
422 Human serum albumin (HAS), 166 Human-like collagen (HLC), 283
284 Humidity, 245t Hyaluronan-like materials, 442
443 Hyaluronic acid (HA), 18, 282
283, 331 Hybrid injectable hydrogel, 21 Hydrochloric acid (HCl), 202, 347, 557 Hydrogels, 11, 149
150, 152, 154, 211, 282, 385, 485, 559
560, 696, 731
735, 733f chitosan, 781
782 for medicinal use, 454
459 precursors, 282 Hydrogen atoms transfer reactions (HAT), 697 1-Hydrogen-3-methylimidazolium (MIM), 478 Hydrolyzes β-1,4-glycosidic linkages, 143 Hydrophobic action, 14 domains, 187
188 interaction, 141 polymer organogelators, 388
389

Hydrothermal carbonization, 78
79, 84
85 doping reaction process, 79
80 Hydroxyapatite (HAp), 3, 263
264, 486, 504
505 Hydroxybutyl chitosan (HBC), 284
286 Hydroxyethyl cellulose (HEC), 212
213 Hydroxylpropyl cellulose (HPC), 8 Hydroxypropyl chitins (HPCHs), 287
288 Hydroxypropyl group, 182 Hypocholesterolemic effect, 178
179

I Iatrogenic factors, 726 IBD. See Inflammatory bowel disease (IBD) IBL. See Implantable bioartificial liver (IBL) ICNF. See Intact chitin-NF (ICNF) ICSE. See Instant catapult steam explosion (ICSE) IFN-γ production, 610
611 ILC2s. See Innate lymphoid type 2 cells (ILC2s) ILs. See Ionic liquids (ILs) Immediate-type hypersensitivity, 623
624 Immobilized lysozyme, 156 Immune-modulating properties, 730
731 Immunoregulation, 610
611 Immunostimulants, 762 mechanism of action of chitin and chitosan as, 767
768 Immunostimulatory effect of chitin on finfish, 763
764 on shellfish, 764
765 of chitosan on finfish, 765
766 on shellfish, 766 Implantable bioartificial liver (IBL), 216 In situ gelling, 671
672 In vitro release of cyclophosphamide, 676
677 from polymeric films, 685
686 In vivo calcium absorption acceleration effect, 180
181 Infectious diseases, 566
567 Inflammation and chitin, chitosan microparticles, 627
629 Inflammatory bowel disease (IBD), 601 Inflammatory diseases, 570
572 Influenza H1N1 vaccine, 614
615 Infrared spectroscopy (IR), 140 Infrared spectrum, 674
675

Index

Injectable chitosan/beta glycerophosphate hydrogels (CS/β-GP hydrogels), 283
284 Injectable hydrogels, 282, 448 potential use of, 294
295 Injectable polymeric gels based on chitosan and chitin, 283
294 chemical hydrogels, 289
294 thermosensitive physical hydrogels, 283
289 biomedical applications, 294
298 clinical trial and human applications, 295
298 Innate immune memory. See Trained immunity Innate lymphoid type 2 cells (ILC2s), 624
625 Inorganic hydrothermal condensation, 145 Inorganic materials-derived composites, 659
662 Insect cuticle, 343 Instant catapult steam explosion (ICSE), 145
146 Intact chitin-NF (ICNF), 598
601 Integrin β, 252
253 Interleukins (ILs) IL-1, 627 IL-1Ra-inducing mechanism, 611 IL-6, 445
446 IL-8, 446 IL-10, 610
611 Intermediate zone of cartilage tissue, 238 Intermolecular hydrogen, 798 Interpenetrated polymers, 158
159 Interpenetrating polymer networks (IPN), 9, 779
780 applications of chitin/chitosan-based, 13
14 from chitin/chitosan, 9
14 chitosan-based full-IPN, 10
11 cryogels on chitosan and synthetic matrices, 12
13 hydrogels based on chitosan and synthetic ionic matrices, 11 nonionic matrices, 12 semi-IPN systems, 10
11 Intestinal enzymes, 180 Intestinal Peyer’s patches, 615
616 Intraparticle diffusion, 139 Intrinsic factors, 726 Ionic liquids (ILs), 472
473 application of, 481t

827

biomedical applications of chitin and chitosan in, 489
490 bone repair, 490 dissolution of chitin and chitosan using, 474 drug and gene delivery, 490
491 1-ethyl-3-methylimidazolium acetate, 156 neuron repair, 491 processability of chitin and chitosan via, 479
483, 487t IPN. See Interpenetrating polymer networks (IPN) IR. See Infrared spectroscopy (IR) Isophorone diisocyanate, 396 Isoprenes. See Terpenes Isoprenoids. See Terpenoids Isopropyl alcohol, 63

J Juvenile loach (Misgurnus anguillicaudatus), 765
766

K Kinetic equilibrium, 146 KIO014 (chitosan-based biomaterial), 295 Knee surgical treatments, 240 KPS. See Potassium persulphate (KPS) Kremezin. See AST-120

L Lactic acid, 436 Lactobacillus helveticus, 349 Lactococcus lactis, 349
350 Lamella, 387
388 Langmuir and Freundlich models, 157 Langmuir isotherm, 782
783 model, 140
141 Langmuir
Blodgett method (LB method), 309 Lates calcarifer.. See Asian seabass (Lates calcarifer) Layer-by-layer adsorption (LbL adsorption), 309 LB method. See Langmuir
Blodgett method (LB method) LbL adsorption. See Layer-by-layer adsorption (LbL adsorption) LCDNs. See Lutein-loaded CDNs (LCDNs) LCGH. See Lupeol-entrapped chitosan
gelatin (LCGH) LCNFs. See Lignocellulose nanofibers (LCNFs)

828

Index

LCST. See Lower critical solution temperature (LCST) “Learn from nature” philosophy, 308 LEDs. See Light-emitting devices (LEDs) Legustrazine, 563
564 Lepidium sativum seedcake phenolic extract (LSE), 704 Leucocytes, 763 LiBs. See Lithium-ion batteries (LiBs) Ligands, 671
672 Light-emitting devices (LEDs), 366 Light-harvesting efficiency, 84
85 Lignin/chitin film, 158 Lignocellulose nanofibers (LCNFs), 701
704 Linear viscoelastic region (LVE), 398 (1
4)-Linked 2-acetamido-2-deoxy-β-Dglucopyranose units, 139
140 (1,4)-Linked 2-amino-deoxy-β-D-glucan, 72
73 Lipid oxidation, 190 Lipid-based oleogelators, 385 Liquid electrolytes, 84
85 Listeria monocytogenes, 613
614 Lithium-ion batteries (LiBs), 77, 82
83, 83f Liver, 577
578 tissue engineering, 215
216, 422
424 transplantation, 577 LMWC. See Low-molecular-weight chitosan (LMWC) LMWSC. See Low-molecular-weight scorpion chitosan (LMWSC) Loach (Paramisgurnus dabryanus), 765
766 Lobster, 343 Loss modulus, 397 Lower critical solution temperature (LCST), 284
285 Low-molecular-weight carboxymethylated CGC, 62
63 Low-molecular-weight chitosan (LMWC), 356, 358
359, 730 Low-molecular-weight scorpion chitosan (LMWSC), 104 LPS, 612
613 LSE. See Lepidium sativum seedcake phenolic extract (LSE) Lubricants, 384 Lubrication greases, 386
387 Lupeol-entrapped chitosan
gelatin (LCGH), 746 Lutein-loaded CDNs (LCDNs), 414 LVE. See Linear viscoelastic region (LVE)

Lysozyme, 143, 158
159, 436
437, 727 Lysyloxidase, 293
294

M MAC. See Myristic acid (MAC) Macrophage-derived chemokine (CCL22), 624
625 Magnetic amidoxime-functionalized chitosan beads, 783 Magnetic chitosan/graphene oxide (MCGO), 787 Magnetic resonance imaging (MRI), 423 Maillard reaction, 184 Malachite green dye (MG dye), 21
22 Maleilated chitosan/methacrylated silk fibroin hydrogels (MCS/MSF hydrogels), 421 MAm. See Methacrylamide (MAm) Mannose receptor, 610
611, 625 MAP. See Modified atmosphere packaging (MAP) MAPK signaling pathway. See Mitogenactivated protein kinases signaling pathway (MAPK signaling pathway) Marine gastropods, 343
345 Marine sponge skeleton, 161 Marine zooplankton, 345 Marine-based sources, 343 Mark
Houwink
Sakurada equation, 673 Matrix metalloproteinase (MMP), 570
571 MMP-1, 445
446 MMP-9, 621 Mayonnaise, 183, 186
187 MB. See Methyl blue (MB) MB dye. See Methylene blue dye (MB dye) MBA. See N,N0 -methylenebisacryl amide (MBA) MBC. See Minimum bactericidal concentration (MBC) MCC. See Microcrystalline chitin (MCC) MCGO. See Magnetic chitosan/graphene oxide (MCGO) MCS/MSF hydrogels. See Maleilated chitosan/methacrylated silk fibroin hydrogels (MCS/MSF hydrogels) MD. See Molecular dynamics (MD) MDI. See 4,40 -Methylenebis (phenyl isocyanate) (MDI) Mechanical blending method, 7 Medical textiles, 121
124 Medium-molecular-weight chitosan (MMWC), 356, 358

Index

Medium-molecular-weight commercial chitosan (MMWCC), 104 Melt spinning, 533, 534f Melt-molding, 258 Membranes, 154, 211, 735 6-Mercaptopurine (6-MP), 448 MES. See 2-Morpholinoethane sulfonic acid (MES) Mesenchymal stem cells (MSCs), 213
215, 234
235, 491 Mesoporous silica nanoparticles (MSiNps), 264 Metal binding by chitosan, 357 Metal ions, 152 removal, 783
787 Metal soaps, 384
385 Metal
chitosan nanocomposites, 3 Metal
organic framework (MOFs), 145 Metals absorption capacity, 50 Methacrylamide (MAm), 49
50 Methacrylated hexanoyl glycol chitosans, 290 Methacryloyl chloride, 49
50, 50f Methicillin-resistant Staphylococcus aureus (MRSA), 734 Methyl blue (MB), 786 Methyl methacrylate-modified chitosan (CSMMA), 220 Methyl orange dye (MO dye), 22
23, 25
26, 145, 780 5-Methyl pyrrolidinone chitosan (5-MPCs), 440, 749, 751
752 Methylene blue dye (MB dye), 25
26, 145 Methylene chloride, 267
268 4,40 -Methylenebis (phenyl isocyanate) (MDI), 75
77 Mevalonate, 32 MG dye. See Malachite green dye (MG dye) MIC. See Minimum inhibitory concentration (MIC) Michael additions, 291
293 Micro-CT. See Microcomputed tomography (Micro-CT) Microbes, 707 Microbial fermentation, 345 Microcomputed tomography (Micro-CT), 510 Microcontact printing, 209 Microcrystalline chitin (MCC), 365 Microemulsion formulations, 418 Microfractures, 240 Microparticles, 9

829

Microporous oxygen, 80
82 Microspheres, 152, 154, 560 Microwave-assisted deacetylation of chitosan, 673 Microwave-assisted deacetylation reaction, 677 Milano, 349 MIM. See 1-Hydrogen-3methylimidazolium (MIM) Mineral
protein-based matrix, 148 Minimum bactericidal concentration (MBC), 712
713 Minimum inhibitory concentration (MIC), 712
713 MIPs. See Molecularly imprinted polymers (MIPs) Misgurnus anguillicaudatus.. See Juvenile loach (Misgurnus anguillicaudatus) Mitogen-activated protein kinases signaling pathway (MAPK signaling pathway), 617 MMIM(Me2PO4). See 1,3Dimethylimidazolium dimethylphosphate (MMIM (Me2PO4)) MMP. See Matrix metalloproteinase (MMP) MMWC. See Medium-molecular-weight chitosan (MMWC) MMWCC. See Medium-molecular-weight commercial chitosan (MMWCC) MO dye. See Methyl orange dye (MO dye) Modification methods, 207
209 Modified atmosphere packaging (MAP), 704 Modified chitosan, 774 MOFs. See Metal
organic framework (MOFs) Moisture absorption properties of CMC, 442
443 retention properties of CMC, 442
443 Molecular dynamics (MD), 144 Molecular weight (MW), 249, 408, 412, 436
438, 475, 613, 618 Molecularly imprinted membranes from chitosan as biosensors, 326 Molecularly imprinted polymers (MIPs), 315
316 Molybdenum disulfide (MoS2), 483 Monocomponent systems, 207 Monodispersion foaming, 537 Monolith, 153
154 Mononuclear leukocytes, 725

830 Montmorillonite (MTM), 326 Montmorillonite/chitosan composite, 784 2-Morpholinoethane sulfonic acid (MES), 216 Mosaicoplasty, 240 Mouse melanoma cell line (B16F10), 620
621 5-MPCs. See 5-Methyl pyrrolidinone chitosan (5-MPCs) 6-MP. See 6-Mercaptopurine (6-MP) MPO. See Myeloperoxidase (MPO) MRI. See Magnetic resonance imaging (MRI) Mrigal (Cirrhina mrigala), 765
766 mRNA synthesis, 567 MRSA. See Methicillin-resistant Staphylococcus aureus (MRSA) MSCs. See Mesenchymal stem cells (MSCs) MSiNps. See Mesoporous silica nanoparticles (MSiNps) MTM. See Montmorillonite (MTM) Mucin5AC production, 612 Mucoadhesive chitosan films, 415 Mucoadhesivity enhancement, 671
672 Mucor rouxii, 350, 409 Mugil cephalus.. See Grey mullet (Mugil cephalus) Multicomponent systems, 207 Multiwalled carbon nanotubes (MWNTs), 268
269 Muramidases, 143 MW. See Molecular weight (MW) MWNTs. See Multiwalled carbon nanotubes (MWNTs) Myeloperoxidase (MPO), 603
604 Myoglobin, 190 Myristic acid (MAC), 50
51

N N-(2-hydroxy) propyl-3trimethylammonium chitosan chloride (HTCC), 120 N-(2, 6-dichlorobenzyl) chitosan, 358
359 N-acetyl glucosamine (GlcNAc), 44, 143, 409, 671
672 N-acetylation of chitosan derivatives, 288
289 N-acetylglucosamine groups, 5 N-acylation, 389, 391
392, 393f N-alkylation, 389 NALT, 615
616 N-and O-acylation, 392
393, 394f

Index

Nanocarriers, 207 Nanocellulose (NC), 652
653 Nanochitosan, 777
778 Nanocomposites, 158
163 from chitin and chitosan, 28
29 films, 31 interpenetrated polymers and chitin grafting, 158
159 Nanoemulsion formulations, 418 Nanofibers, 263
264 Nanofibrils, 148
149, 152 Nanofillers, 159
160 Nanofiltration, 774 Nanogels, 453 antimicrobial activity of CS
EOs emulsions and, 712
713 antioxidant properties of CS
EOs emulsions and, 705 chitosan-based EOs, 693
696 Nanomaterials, 210 Nano-microfibers, 485 Nano-microparticles, 485 Nanoobjects, 161 Nanoparticles, 148
149, 152, 559 of thiolated chitosan, 414 Nanosized γ-Fe2O3, 780 Nanostructured biohybrid materials, 309 Nano-TiO2
chitosan with collagen artificial skin (NTCAS), 26, 28 Nanowhiskers, 148
149, 152 Naproxen nanoparticles, 283
284 National Medical Products Administration (NMPA), 295
296 Natural biopolymer, 72 Natural fillers, composites from, 23
25 Natural polymers, 2, 244
246 blends with, 8 Natural rubber (NR), 75
77 NBAC. See p-nitrobenzoic acid (NBAC) NC. See Nanocellulose (NC) N-carboxyalkylation, 389 N-carboxybutyl chitosan (CNBC), 440, 750
751 N-carboxyethyl chitosan (NCEC), 56, 440 N-carboxymethyl chitosan (N-CMC), 439, 442
444 NCEC. See N-carboxyethyl chitosan (NCEC) N-chloroacetyl sulfonamides production, 61
62 NCO-functionalization reaction, 393
395, 395f

Index

N-deacetylation, 385
386 N-dodecylchitosan, 358
359 Neoplastic diseases, 561
566 Nerve, 580
581 tissue engineering, 216
217 Neuron repair, 491 Neutral Red (NR), 160
161 Neutrophils, 725 Newtonian behavior, 398 Next-generation wearable green optoelectronics, 74
75 N-glucosamine, 671
672 NHS. See N-hydroxysuccinimide (NHS) N-hydroxysuccinimide (NHS), 216 NHS-activated carboxylic acid, 677
678 Nile tilapia (Oreochromis niloticus), 765
766 Nitric acid (HNO3), 347, 557 Nitric oxide (NO), 460
461 Nitrogen adsorption isotherms, 162
163 nitrogen-doped activated carbon, 80
82 carbon materials, 77
78 hierarchical porous carbon materials, 79
80 porous carbons, 78
79 Nitrophenol, 789 NLR family, pyrin domain-containing 3 (NLRP3), 619, 629 N-methyl-pyrrolidinone (NMP), 59 NMP. See N-methyl-pyrrolidinone (NMP) NMPA. See National Medical Products Administration (NMPA) N,N-dicarboxymethyl chitosan (N,N-diCMC), 56, 439
440 N,N-di-CMC. See N,N-dicarboxymethyl chitosan (N,N-di-CMC) N,N-dimethylacetamide/LiCl, 148
149 N,N0 -methylenebisacryl amide (MBA), 11 NO. See Nitric oxide (NO) N,O-acyl chitosan, 58, 182 N,O-carboxymethyl chitosan (N,O-CMC), 439, 443
444, 454
455, 461
462, 749
750 N,O-CMC. See N,O-carboxymethyl chitosan (N,O-CMC) NOCMC. See Water soluble N,O-CMC (NOCMC) N,O-CM-chitosan, 55, 55f NOD2, 610
611 Nonaqueous solvent dispersion technique, 3
4

831

Nonionic monomers, 12 Nonporous, 147
148 Nonsurgical treatments, 239
240 Nontoxic compounds, 178 Nontoxicity, 9, 136
137, 436
437 N-palmitoyl chitosan hydrogel, 293 N-phthaloylation, 53 N-(p-isopropylbenzyl) chitosan, 358
359 NR. See Natural rubber (NR); Neutral Red (NR) NR/CNC-CB sensors, 75
77 NSC. See N-succinyl chitosan (NSC) NSHBC hydrogels, 285
286 N-succinyl chitosan (NSC), 440 NTCAS. See Nano-TiO2
chitosan with collagen artificial skin (NTCAS)

O O-acetylation, 16 O-acylation, 389 OAlg
CMCS. See Oxidized alginate
carboximethyl chitosan (OAlg
CMCS) O-carboxymethyl chitosan (O-CMC), 438
439, 442, 452
453, 458
459, 461
462 Ocular drug delivery, 413
415 OE-MSCs. See Olfactory ectomesenchymal stem cells (OE-MSCs) OEO. See Origanum vulgare L. essential oil (OEO) Oil, 387
388 Oil-in-water emulsion (O/W), 694 Oleogelation, 385 Oleogels, 385 effect of chemical modification on thermogravimetric characteristics, 401
402 influence of chemical modification on rheological properties, 399
400 on thermal behavior, 402
404 Olfactory ectomesenchymal stem cells (OEMSCs), 217 Oncorhynchus mykiss.. See Rainbow trout (Oncorhynchus mykiss) Ophthalmology, 362 Oral administration, 763
764 Oral drug delivery, 415
417 Oreochromis niloticus.. See Nile tilapia (Oreochromis niloticus) Organic pollutants, 787
789 Organic solar cells, 84

832

Index

Organogels, 385, 696 Origanum vulgare L. essential oil (OEO), 648 OSC. See O-succinyl chitosan (OSC) Osteoarthritis, 239 Osteoconductive matrices, 31
32 Osteoconductive properties of materials, 236, 236t Osteoconductivity, 507
508 Osteogenicity, 509 Osteoid, 234
235 Osteoinductive properties, 48
49 of materials, 236, 236t Osteoinductivity, 509 Osteointegrity, 509 O-succinyl chitosan (OSC), 441 O/W. See Oil-in-water emulsion (O/W) Oxalic acid, 348 Oxidative stress, 597
601 Oxidized alginate
carboximethyl chitosan (OAlg
CMCS), 740

P P glycoprotein (Pgp), 561 P2HEM. See Poly(2hydroxyethylmethacrylate) (P2HEM) P3HB. See Poly3-hydroxybutyrate (P3HB) PAA. See Polyacrylic acid (PAA) Paclitaxel (PTX), 55
56, 448
449, 452
453 Palladium (Pd), 3 PAMPs. See Pathogen-associated molecular pattern (PAMPs) PANI. See Polyaniline (PANI) Paper industry, 367 Paramisgurnus dabryanus.. See Loach (Paramisgurnus dabryanus) Parapenaeus longirostris, 349 Partial thickness defects, 239 Partially N-lauroyl (PNL), 182 Particle leaching, 535 Particle size, 682
683 distribution, 675
676 Pathogen-associated molecular pattern (PAMPs), 610
611 PBMCs. See Peripheral blood mononuclear cells (PBMCs) PBS. See Phosphate buffer solution (PBS); Poly(butylenes succinate) (PBS) PBSA. See Poly(butylene succinate adipate) (PBSA) PBTA. See Poly(butylene terephthalate adipate) (PBTA) PCA. See Polycaproactone (PCA)

P-chitin. See Phosphorylated chitin (Pchitin) P-chitosan. See Phosphorylated chitosan (Pchitosan) PC-CS. See Polystyrene
chitosan (PC-CS) PCL. See Polycaprolactone (PCL) PD. See Prednisolone (PD) PD/NFs-CDs gel in model mice, 601
604 PDMS. See Polydimethylsiloxane (PDMS) pDNA. See Plasmid DNA (pDNA) PEC. See Peritoneal exudate cells (PEC) PECs. See Polyelectrolyte complexes (PECs) Pectin/CH blend membranes, 331 PEDOT. See Poly(3,4ethylenedioxythiophene) (PEDOT) PEG. See Polyethylene glycol (PEG) PEG-CMCS. See PEG-grafted CMC (PEGCMCS) PEG-grafted CMC (PEG-CMCS), 453 PEI. See Polyethylenimine (PEI) Penicillium notatum, 358
359 PEO. See Polyethylene oxide (PEO) Peppermint (PO), 705
706 Peripheral blood mononuclear cells (PBMCs), 627
628 Peritoneal exudate cells (PEC), 627
628 Permeabilizing effect, 184 Perovskite solar cells, 84 Pervaporation method, 7 PG. See Pristine graphene (PG) PGA. See Polyglycolic acid (PGA) Pgp. See P glycoprotein (Pgp) Phagocytosis, 725 Pharmaceutical applications, 49 Phase separation, 258, 535
537, 540f PHB. See Polyhydroxybutyrate (PHB) PHBV. See Poly-3-hydroxybutyrate-co-3hydroxyvalerate (PHBV) Phosphate buffer solution (PBS), 213
215 Phosphates, 17 Phosphorylated chitin (P-chitin), 48
49, 48f Phosphorylated chitosan (P-chitosan), 56
57, 57f Phosphorylation, 46, 53 Phosvitin (PV), 266 Photocross-linking hydrogels, 289
290 Photoelectrical applications, 72 Photography, 366 Photoinduced methods, 49
50 Photoinitiators, 290 pH-responsive composite, 32 pH-sensitive swelling behavior, 47
48

Index

Phthalic anhydride, 441 Phthalimide chitosan, 441 Physical gels, 211 Physical injectable hydrogels, 282 Physical properties of chitin and CS, 72
73 Physically associated gels, 14
17, 17f Physicochemical characterization of polymeric materials FTIR spectroscopy, 679
680 proton nuclear magnetic resonance spectroscopy, 678
679 SSA, porosity, and particle size, 682
683 surface morphology, 683
684 thermal and thermogravimetric characterization, 681
682 X-ray diffraction characterization, 680
681 Physisorption, 138
139 Pickering emulsions, 694
695 Piezoelectric effect, 72 Pirfenidone, 418 PLA. See Polylactic acid (PLA) Plant-defense enzyme, 183 Plasma cholesterol level, 178 Plasma sorption, 728 Plasmid DNA (pDNA), 491 Platinum (Pt), 3 PLGA. See Polylactic coglycolic acid (PLGA) PLLA. See Poly(L-lactic acid) (PLLA) Pluronic F-108, 623 PMAA. See Poly(methacrylic acid) (PMAA) PMAA-g-CTS/B composite. See Poly (methacrylic acid)-grafted chitosan/ bentonite composite (PMAA-g-CTS/ B composite) PMMA. See Poly(methyl methacrylate) (PMMA) p-nitrobenzoic acid (NBAC), 50
51 p-nitrophenol, 141 PNL. See Partially N-lauroyl (PNL) PNVI. See Poly(N-vinyl imidazole) (PNVI) PO. See Peppermint (PO) Pollutants using chitosan-based material, 783
792, 783f antibacterial effect, 791
792 desalination, 789
790 metal ions removal, 783
787 organic pollutants, 787
789 other pollutants, 792 Poly (b-(1
4)-N-acetyl-D-glucosamine), 643

833

Poly-3-hydroxybutyrate-co-3hydroxyvalerate (PHBV), 262 Poly(2-hydroxyethylmethacrylate) (P2HEM), 7 Poly(3-hydroxybutyric acid) (PHB). See Polyhydroxybutyrate (PHB) Poly(3,4-ethylenedioxythiophene) (PEDOT), 74
75 Poly(butylene succinate adipate) (PBSA), 7
8 Poly(butylene terephthalate adipate) (PBTA), 7
8 Poly(butylenes succinate) (PBS), 7
8 Poly(e-caprolactone), 3 Poly(L-lactic acid) (PLLA), 520
521, 748 Poly(methacrylic acid) (PMAA), 11 Poly(methacrylic acid)-grafted chitosan/ bentonite composite (PMAA-g-CTS/ B composite), 23
24 Poly(methyl methacrylate) (PMMA), 523
524 Poly(mPEGMA-co-MAA), 417 Poly(N-acryloylglycine), 11 Poly(N-isopropyl acrylamide), 448, 456
457 Poly(N-vinyl imidazole) (PNVI), 454 Poly3-hydroxybutyrate (P3HB), 268 Polyacrylamide
chitosan, 14 Polyacrylic acid (PAA), 11, 217, 410 Polyaniline (PANI), 10
11, 60, 74, 77
78 Polycaproactone (PCA), 7 Polycaprolactone (PCL), 7
8, 212
213, 244
246, 513
514, 577
578 Polydimethylsiloxane (PDMS), 74 Polyelectrolyte, 10
11 complexation, 17
18 Polyelectrolyte complexes (PECs), 183, 325
326, 735 Polyethylene glycol (PEG), 7, 79
80, 220, 282
283, 396, 424 Polyethylene oxide (PEO), 254
255 Polyethylenimine (PEI), 220, 483 Polyglycolic acid (PGA), 244
246 Polyhedral oligomeric silsesquioxanes (POSS), 28 Polyhydroxybutyrate (PHB), 8, 268 Polylactic acid (PLA), 7, 244
246, 739, 747
748 Polylactic coglycolic acid (PLGA), 580
581 Polymer binder, 82
83 blends from chitin and chitosan, 6
9

834

Index

Polymer (Continued) applications of chitin and chitosan blends, 8
9 blends with natural polymers, 8 blends with synthetic polymers, 7
8 concentration, 245t grafting, 3
4 solution flow rate, 245t Polymer
ceramic/metallic composite materials, 244 Polymeric films preparation of, 676
677 in vitro release of cyclophosphamide from, 685
686, 686t Polymeric gel formation, 19 Polymeric materials, 2, 459
460 physicochemical characterization, 678
684 Polymer
polymer interactions, 388
389 Polymer
solvent interactions, 388
389 Polymorphisms, 44 Polymorphonuclear leukocytes, 725 Polypropylene-NIPAAm-CH membrane, 325 Polypyrrole moiety, 163
164 Polysaccharides, 19, 45, 142, 144, 187, 436
437, 642, 656
657, 695, 762
763, 774 matrices, 157 polysaccharide-based materials, 156 Polystyrene
chitosan (PC-CS), 793
794 Polyurethane (PU), 7, 75
77 PU-based electrospun fibers, 247 Polyvinyl alcohol (PVA), 3, 254
255, 324
325, 563
564, 731
732, 748
749, 788 film, 415 Polyvinyl pyrrolidone (PVP), 7, 254
255 Polyvinylidene fluoride (PVDF), 82
83 Pore shape, 511
512 Pore size and pore size distribution, 511 Porosity, 510, 675
676, 682
683 Porous trabecular bone, 504
505 Porphyrin compounds, 366 POSS. See Polyhedral oligomeric silsesquioxanes (POSS) Potassium carbonate (K2CO3), 347 Potassium hydroxide (KOH), 347 Potassium permanganate (KMnO4), 348 Potassium persulphate (KPS), 454 Prebiotics ingredients, 180 Prednisolone (PD), 601 Primary drying, 528 Primary intention wound healing model, 724

Pristine chitin, 154
156 Pristine CS electrospun scaffolds, 254
255 Pristine graphene (PG), 74
75 Proangiogenesis molecules, 621 Procambarus clarkia.. See Crayfish (Procambarus clarkia) Processing of chitosan, 201
205 Proliferative phase, 725
726 Prostaglandins, 725 Proteases, 436 Protein(s), 313
314 inhibition, 567 protein-based adhesives, 313 purification, 142 Proton nuclear magnetic resonance spectroscopy (1H-NMR spectroscopy), 678
679, 679f Pseudo-second-order model, 140
141, 792 Pseudomonas aeruginosa, 613
614 P. aeruginosa bacterium, 436 Pseudo
second order kinetics, 777
778 PTX. See Paclitaxel (PTX) PU. See Polyurethane (PU) PV. See Phosvitin (PV) PVA. See Polyvinyl alcohol (PVA) PVA/CS. See PVA
chitosan (PVA/CS) PVA/CS-Ag-NP. See PVA
chitosan
Ag nanoparticle (PVA/CS-Ag-NP) PVA
chitosan (PVA/CS), 749 PVA
chitosan
Ag nanoparticle (PVA/CSAg-NP), 749 PVA
COOH. See Carboxyl-modified poly (vinyl alcohol) (PVA
COOH) PVDF. See Polyvinylidene fluoride (PVDF) PVP. See Polyvinyl pyrrolidone (PVP) Pyrene, 187
188 Pyricularia grisea, 358
359 Pyridoxamine, 189
190 Pyrogallol, 313
314

Q Quantum-dot (QD), 84 Quartanized chitosan (QCS), 744 Quaternary ammonium salt, 182 Quaternary chitosan derivatives, 59, 59f Quaternization, 46 Quercetin, 63

R Radiation-induced cross-linked hydrogel, 457 Rainbow trout (Oncorhynchus mykiss), 763
764

Index

Raman spectroscopy, 141 Raw calico fabric, 108 Raw chitosan, 775
777 RB. See Remazol Black (RB) RBBR. See Remazol Brilliant Blue R (RBBR) Reactive oxygen species (ROS), 188, 590 Reduced GO, 73
74 Regenerative medicine, 219, 314
315, 449
451 Remazol Black (RB), 160
161 Remazol Brilliant Blue R (RBBR), 779 Remodeling/maturation phase, 726 Renewability, 2 Renewable resources, 6 REO. See Rosmarinus officinalis EO (REO) Reverse osmosis, 774 Reversible chitosan
dextran sulfate nanogel, 18 RGD. See Arginine
glycine
aspartate (RGD) Rhodotorula rubra, 358
359 RO. See Rosmarinus officinalis (RO) ROS. See Reactive oxygen species (ROS) Rosmarinus officinalis (RO), 705 Rosmarinus officinalis EO (REO), 705 Round window membrane (RWM), 218

S S-chitin. See Sulfated chitin (S-chitin) S-chitosan. See Sulfated chitosan (Schitosan) SA. See Sodium alginate (SA) Saccharomyces cerevisiae-derived chitin, 612
613 Sago starch (SG), 739
740 Salt-leaching, 258 Sarcosine oxidase, 326 SBE-β-CD. See Sulfobutyl ether β-CD (SBEβ-CD) SBF. See Simulated body fluid (SBF) SC. See Starch and chitosan (SC) Scaffolds, 209
210, 241, 485
488, 560, 737 architecture, 509 for bone tissue regeneration, 236
237 chitosan scaffold applicability, 537
539 Scanning electron microcopy (SEM), 95, 108
112, 213
215, 676, 684f, 729, 792
793 Scavenging effect, 188 SCF. See Supercritical fluid drying (SCF) Schiff base cross-linking, 291, 292t Schottky diodes, 72

835

Schwann cell (SCs), 216 SCNT. See Single-walled CNTs (SCNT) SCO2 technologies. See Supercritical CO2 technologies (SCO2 technologies) SCs. See Schwann cell (SCs); Supercapacitors (SCs) SDACNFs. See Surface-deacetylated chitin nanofibers (SDACNFs) SE. See Extract of sumac (SE) Seafood processing industries, 367 Secondary intention wound healing model, 724 Self-activation, 78
79 Self-assembly, 207 Self-assembly in biological systems, 309 SEM. See Scanning electron microcopy (SEM) Semi-interpenetrating polymer network (Semi-IPN), 12
13 hydrogel, 456
457 systems, 10
11 Semisolid systems, 385 Separative performance, 154 Sequential adsorption
desorption cycles, 22
23 Sequential IPN, 10 Seroma, 726
727 Serum cholesterol, 178 Sesquiterpenes, 690
691 SFC. See Solid fat content (SFC) SG. See Sago starch (SG) Shaping chitin materials, 148
154 batch systems, 151
152 chitin supports for adsorptive processes, 150
151 continuous-flow systems, 152
154 Shellfish, 762 immunostimulatory effect of chitin on, 764
765 immunostimulatory effect of chitosan on, 766 Shrimp, 343
344, 367 characterization, 95
96 chitosan antimicrobial analysis results, 102
105 antimicrobial assay, 101
102 coating, 105
107 isolation of chitin, 94
95 SI-CLP, 612 Silk fibroin, 331 Silver (Ag), 3

836

Index

Silver nanoparticles (AgNPs), 123
124, 462, 739
740 Silver zeolite (SZ), 125 Silylation, 52
53 reaction, 50 Simple charge neutralization, 189t Simple solution mixing
evaporation method, 80
82 Simulated body fluid (SBF), 263
264 Simultaneous IPN, 10 Single-walled CNTs (SCNT), 74
75 Skin, 578
579 grafts, 312 injuries, 311 tissue engineering, 215, 422
424 Slough, 725 Smad3 gene expression, 446 Smart materials, 211 SMS. See Surface-modifying systems (SMS) Sodium alginate (SA), 656 Sodium bromide, 51
52 Sodium carbonate (Na2CO3), 347 Sodium caseinate, 651 Sodium hydrogen carbonate (NaHCO3), 347 Sodium hydrogen sulfite (NaHSO3), 347 Sodium hydroxide (NaOH), 202, 347, 351 Sodium hypochlorite, 51
52 Sodium sulfide (Na2S), 347 Sodium sulfite (Na2SO3), 347 Sodium tetradecyl sulfate, 283
284 Solar cells, 84
85, 85f Sol
gel, 207 fabrication, 309 transition, 149
150 transition, 162
163 Solid fat content (SFC), 398 Solid tumor mass, 622 Solid-like behavior, 138 Solid
liquid separation, 191 Solubility properties of CMC, 441
442 Solvent choice, 253
254 Solvents, 435 Solvent
solvent interactions, 388
389 Sorption properties of CMC, 443 Soybean oil, 387 Specific surface area (SSA), 675
676, 682
683, 683t SPES
PES. See Sulfonated polyethersulfone
polyethersulfone (SPES
PES) SPH. See Superporous hydrogels (SPH)

Spinneret
collector distance, 245t Sponges, 485
488, 736
737, 736f Spongy bone. See Porous trabecular bone Sprague
Dawley rats, 446
447 Spray drying method, 560, 696
697 Squid, 343, 367 SSA. See Specific surface area (SSA) Staphylococcus aureus, 3, 418
419, 443
444, 613
614, 714 Starch, 654
655, 739
740 Starch and chitosan (SC), 74
75 Stirred tank, 151
152, 152f Storage modulus, 397 Strain sensitivity of NR/CNC-CB sensors, 75
77 sensors, 75
77 Stratum corneum, 418 Streptomyces, 409 Stretchable device, 74
77, 75f Sulfate, 412 Sulfated chitin (S-chitin), 49, 49f Sulfated chitosan (S-chitosan), 57
58, 57f Sulfation, 46, 53 Sulfobutyl ether β-CD (SBE-β-CD), 598
601 Sulfonamide, 61
62 Sulfonated chitosan, 61
62 modifications, 412 Sulfonated polyethersulfone
polyethersulfone (SPES
PES), 790 Sulfuric acid (H2SO4), 347, 557 Supercapacitors (SCs), 77
82, 78f Supercritical CO2 technologies (SCO2 technologies), 155 Supercritical fluid drying (SCF), 485
486 Superficial wound healing model, 724 Superficial zone of cartilage tissue, 238 Supermolecular assembly, 387
388 Superporous hydrogels (SPH), 457
458 SPH-IPNs, 459 Surface area, 800 morphology, 676, 683
684 properties, 511 tension, 245t Surface-deacetylated chitin nanofibers (SDACNFs), 594
595, 597
601 Surface-modifying systems (SMS), 366
367 Surfactant
chitosan membranes, 186 Surgical treatments, 239
240 Sustainability, 136
137, 154
155

Index

Sustainable technology, 308 Suturing, 313 Sweeping mechanism, 189t Swelling, 251 analysis, 676 study, 685, 685t Syk signaling pathway, 617 Synergic radical scavenging activity, 191 Synthetic composites from synthetic fillers, 22
23 dressings, 313 ionic matrices, 11 matrices, 12
13 nonionic matrices, 12 plastics, 367
368 Synthetic polymers, 2, 136
137, 657
659 blends with, 7
8 Synthetic tissue adhesives, 313 SZ. See Silver zeolite (SZ)

T TA. See Tannic acid (TA) TAA. See Triamcinolone acetonide (TAA) Tablets, 9 Tannic acid (TA), 644 Targeted drug delivery, 452
453 TAT. See Twin-arginine translocation (TAT) Taylor cone, 232, 242
243 TBARS. See Thiobarbituric acid reactive substances (TBARS) TBHQ. See Tertiary butylhydroquinone (TBHQ) TD. See Thiolation degree (TD) TEAC. See Trolox equivalent antioxidant capacity (TEAC) Teflon, 327 Temperature, 245t Template synthesis, 309 TEMPO. See 2,2,6,6-Tetramethylpiperidine1-oxyl radical (TEMPO) TEOS. See Tetraethoxysilane (TEOS); Tetraethylorthosilicate (TEOS) Teredinobacter turnirae, 349
350 Terpenes, 690
691 Terpenoids, 690
691 Tertiary butylhydroquinone (TBHQ), 190 Tertiary intention wound healing model, 724 Tetracycline hydrochloride (TH), 740 Tetraethoxysilane (TEOS), 158
159 Tetraethylorthosilicate (TEOS), 480
483 Tetrahydrofuran, 57
58

837

2,2,6,6-Tetramethylpiperidine-1-oxyl radical (TEMPO), 51
52 Textile industry, 366
367 Textile production, 90
91 Textile-reinforced composites, 124
126 TGA. See Thermogravimetric analysis (TGA) TGF. See Transforming growth factor (TGF) TH. See Tetracycline hydrochloride (TH) TH2 cells, 623
624 TH2-associated chemokines CCL11, 624
625 Thermal characterization, 681
682 Thermogravimetric analysis (TGA), 96, 144, 400, 650, 675 Thermogravimetric characterization, 681
682 TG studies of chitosan and thiolated products, 683t Thermosensitive HBC hydrogels, 284
285 Thermosensitive physical hydrogels, 283
289 HBC, 284
286 injectable chitosan/beta glycerophosphate hydrogels, 283
284 thermosensitive modified chitin, 286
289 thermosensitive modified chitosan, 286 Thermosensitive polymer hydrogels, 21 Thiobarbituric acid reactive substances (TBARS) Thiobarbituric acid reactive substances (TBARS), 190, 704 Thiol groups, quantification of, 673
674 Thiolated chitosan nanoparticles, 61 Thiolated chitosan synthesis microwave-assisted deacetylation of chitosan, 673 quantification of thiol groups, 673
674 thiolation of chitosan polymers, 673 viscosity-average molecular weight, 673 Thiolated materials DSC, 675 FTIR, 674
675 1 H-NMR spectroscopy, 674 SSA, porosity, and particle size distribution, 675
676 surface morphology determination, 676 TGA, 675 X-ray diffraction, 675 Thiolated polymer synthesis microwave-assisted deacetylation reaction, 677

838 Thiolated polymer synthesis (Continued) thiolation of chitosan, 677
678 Thiolation degree (TD), 672 Thiolation of chitosan, 677
678, 678f polymers, 673 thiolation degree of chitosan, 678t Thiophenol, 5
6 Three-dimension (3D) cell carriers, 240
241 cross-linked structure, 282 fibrillar macrostructures, 248
249 printing, 258, 535, 538f structure, 147
148 Time-consuming process, 151
152 TISSEEL (injectable hydrogel-based bioadhesives), 295 Tissue engineering, 27, 210, 212
217, 314
315, 361
362, 419, 449
451 applications, 72 of chitosan-based nanosytems, 573
581, 575t approaches, 312 electrospinning for tissue engineering scaffolds, 244
249 Tissue incisions, 313 Tissue regeneration, 722
723 Titanium dioxide (TiO2), 734
735 TLR. See Toll-like receptor (TLR) TMC. See Trimesoyl chloride (TMC); Trimethyl chitosan (TMC) TNF. See Tumor necrosis factor (TNF) Toll-like receptor (TLR), 767
768 TLR-2, 610
611 TLR-9, 610
611 TOPA. See 3,4,5-Trihydroxyphenylalanine (TOPA) Tosyl (CH3C6H4SO2) group, 52 Tosyl-chitin synthesis, 52, 52f Tosylation reaction, 50 TPP. See Tripolyphosphate (TPP) TPTZ. See 2,4,6-Tripyridyls-triazine complex (TPTZ) Trabecular/spongy bone, 234 Trained immunity, 612
613 Transdermal drug delivery, 417
418 Transferrin, 190 Transforming growth factor (TGF), 725
726 TGF-β1, 445
446 Transglutaminase, 293
294 Transient electronics, 74
75 Transitional zone of cartilage tissue, 238

Index

TRCPU synthesis. See Chitin-based polyurethane synthesis (TRCPU synthesis) Triamcinolone acetonide (TAA), 217 Trichophyton rubrum, 358
359 Trichophyton violaceum, 358
359 3,4,5-Trihydroxyphenylalanine (TOPA), 313
314 Trimesoyl chloride (TMC), 790 Trimethyl chitosan (TMC), 422
423, 564
566 Tripolyphosphate (TPP), 17, 647
648, 696
697 2,4,6-Tripyridyls-triazine complex (TPTZ), 701 Trisodium phosphate (Na3PO4), 347 Triton X100, 267
268 Trolox, 189
190 Trolox equivalent antioxidant capacity (TEAC), 699
700 Tryptic soy broth (TSB), 101 TSB. See Tryptic soy broth (TSB) Tumor necrosis factor (TNF), 725
726 TNF-α, 446 Tunicates, 313
314, 314f Tunicin, 313
314 Twin-arginine translocation (TAT), 220 Two-dimensional flat collector (2D flat collector), 248
249 Tyramines, 424 Tyrosinase, 293

U UA technologies. See Ultrasound-assisted technologies (UA technologies) UC. See Ulcerative colitis (UC) UDP-N-acetylglucosamine (UDP-GlcNAc), 345 UDP-N-acetylglucosamine. See Uridinediphosphate-N-acetylglucosamine (UDP-N-acetylglucosamine) UF. See Ultrafiltration (UF) Ulcerative colitis (UC), 597 Ultrafiltration (UF), 790 Ultrasound-assisted technologies (UA technologies), 155 Ultraviolet (UV), 289 Unmodified chitosan, 775
777 Uranium (U), 786
787 Urea, 384
385 Urease, 293 Uridine-diphosphate-N-acetylglucosamine (UDP-N-acetylglucosamine), 345

Index

US Food and Drug Administration (USFDA), 690
691 UV. See Ultraviolet (UV)

V Vaccines, 762 van der Waals interactions, 151 Vancomycin-resistant Enterococcus faecium (VRE), 743
744 Vegetable oil, 385 with mineral oil, 387 1-Vinylimidazole (VIM), 49
50 Viscosity, 245t Viscosity-average molecular weight, 673 VRE. See Vancomycin-resistant Enterococcus faecium (VRE)

W W/O. See Water-in-oil emulsion (W/O) Water, 774 engineering, 363 mass, 410
411 purification processes, 158 Water soluble N,O-CMC (NOCMC), 456 Water vapor permeability (WVP), 645 Water-in-oil emulsion (W/O), 694 Water-in-water emulsions (W/o/w emulsions), 185
186 Water-soluble carboxymethyl chitin, 3 Water-soluble derivatives, 178, 192 of chitosan, 434, 567
568 Water/wastewater remediation, 775
783 chitosan beads, 782
783 chitosan hydrogels, 781
782 chitosan/biopolymer blend, 778
780 chitosan/nanoparticles composites, 780
781 nanochitosan, 777
778 raw or unmodified chitosan, 775
777 WAXD. See Wide-angle X-ray diffraction (WAXD) Wearable device, 74
77, 75f Wearable electronics and energy storage devices chitin and CS composites for advanced electronics, 73
77 energy storage devices, 77
85 physical and chemical properties of chitin and CS, 72
73 Wet spinning, 146, 528
532, 533f extrusion process, 30
31 Wheat germ agglutinin (WGA), 165
166 Whey protein isolate (WPI), 186

839

White calico fabric, 108
109 Wide-angle X-ray diffraction (WAXD), 7 Woolen fabric, 111
112 Wound, 722
723 Wound dressing, 311
313, 360 applications, 8 asymmetric membranes as, 326
327 chitosan membranes in, 323
326 Wound healing, 26, 30
31, 218
219, 311, 360, 445
447, 722
723, 746f bandages, 436
437 chitosan-based biocomposites for, 739
749 material, 55
56 process, 723 and stages, 723
727 factors for delaying, 726
727 models, 723
724 phases, 724
726 Wound management chitosan derivatives in, 749
752 chitosan-based biocomposites for wound healing, 739
749 chitosan-based biomaterials in, 731
739 chitosan-based material for, 752
753 properties of chitosan advantageous for, 727
731 wound healing and stages, 723
727 WPI. See Whey protein isolate (WPI) WVP. See Water vapor permeability (WVP)

X X-ray diffraction (XRD), 213
215, 675, 792
793 characterization, 680
681 powder diffractograms of polymers, 681f measurements, 144
145 X-ray photoelectron spectroscopy (XPS), 792
793, 797f

Y Yeast, 343 YKL-40. See Chitolectin (YKL-40)

Z Z. multiflora oil (ZEO), 704 Zinc oxide (ZnO), 660
661 Zinc-doped iron oxide, 80
82 ZnO/carboxymethyl chitosan (ZnO/ CMCS), 125 Zygomycytes, 358
359