Biological Macromolecules: Bioactivity and Biomedical Applications 9780323857598, 0323857590

Table of contents :
Front Cover
Biological Macromolecules
Copyright Page
Contents
List of contributors
Preface
I. Background
1 Biological macromolecules: sources, properties, and functions
1.1 Introduction
1.2 Carbohydrates
1.2.1 Monosaccharides
1.2.2 Oligosaccharides
1.2.3 Polysaccharides
1.3 Lipids
1.3.1 Simple lipids
1.3.2 Compound or conjugate lipids
1.3.3 Derived lipids
1.4 Proteins
1.4.1 Simple proteins
1.4.2 Conjugated proteins
1.4.3 Derived proteins
1.5 Nucleic acids
1.5.1 Nucleotides
1.5.2 Nucleosides
1.5.3 DNA
1.5.4 RNA
1.6 Conclusion
References
2 Structure–activity relationship of biological macromolecules
2.1 Introduction
2.2 Enzymes as bioactive proteins
2.2.1 l-amino acid oxidases
2.2.2 Lysostaphin
2.2.3 Metallo-β-lactamase-like lactonase
2.3 Chitosan as a bioactive polysaccharide
2.3.1 Relationship of chitosan physicochemical property and its bioactivity
2.3.2 Bioactivity of chitosan with modified functional group
2.4 Conclusion
References
3 The importance of biological macromolecules in biomedicine
3.1 Introduction
3.2 Biological macromolecules in biomedicine and therapies
3.3 Carbohydrates
3.3.1 Therapeutics based on carbohydrates
3.4 Peptides
3.4.1 Therapeutics based on peptides
3.5 Proteins
3.5.1 Therapeutics based on proteins (proteins and monoclonal antibodies)
3.5.1.1 Protein-based therapeutics
3.5.1.2 Monoclonal antibodies
3.5.1.3 First generation monoclonal antibodies
3.5.1.4 Second generation monoclonal antibodies
3.5.1.5 Third generation monoclonal antibodies
3.5.1.6 Fourth generation monoclonal antibodies
3.6 Lipids
3.6.1 Drug delivery-based on lipids
3.7 Nucleic acids and oligonucleotides
3.7.1 Therapeutics based on oligonucleotides
3.7.1.1 Antisense oligonucleotides
3.7.1.2 Small interfering RNA
3.7.1.3 Aptamer
3.8 Synthesis of macromolecules
3.9 Biomedicine
3.10 Conclusions
References
4 Modification techniques for carbohydrate macromolecules
4.1 Introduction
4.2 Cellulose
4.3 Hemicelluloses
4.4 Lignin
4.5 Chitin and chitosan
4.6 Modification of carbohydrate biological macromolecules
References
II. Bioactivity
5 Biological macromolecules as nutraceuticals
5.1 History of the applications of nutraceutical compounds in health care
5.2 Alkaloids
5.2.1 Caffeine
5.2.2 Capsaicin
5.2.3 Theobromine
5.3 Phenolic compounds
5.3.1 Curcumin
5.3.2 Resveratrol
5.3.3 Quercetin
5.3.4 Anthocyanins
5.3.5 Luteolin
5.3.6 Naringenin
5.3.7 Catechins
5.4 Terpenes
5.4.1 Lycopene
5.4.2 β-Carotene
5.4.3 Lutein
5.4.4 Zeaxanthin
5.5 Future views
5.6 Proteins and peptides with biological activity of medical interest
5.7 Nucleic acids and their nutraceutical properties used in biomedicine
5.7.1 Nucleic acids overview
5.7.1.1 DNA/RNA nanostructures
5.7.1.1.1 Biosensing
5.7.1.1.2 Drug delivery
5.7.1.1.3 Immunomodulatory
5.7.1.2 DNA/RNA functional sequence
5.7.1.2.1 Non-mRNA sequences
5.7.1.2.2 Gene therapy
5.7.1.2.3 CRISPR
5.7.2 Perspectives
5.8 Introduction of lipids
5.8.1 Polyunsaturated fatty acids
5.8.2 The role of Omega-3 PUFAs in some disorders
5.9 The potential use of bioactive lipids in cancer stem cells and coronavirus disease (COVID-19)
5.9.1 Bioactive lipids in cancer
5.9.2 Bioactive lipids in COVID-19
5.10 Carbohydrates as nutraceuticals
5.10.1 Brief overview of carbohydrates
5.10.2 Role of polysaccharides in extracellular membrane
5.10.3 Immunostimmulatory effect of carbohydrates
5.10.4 Carbohydrates from plants with nutraceutical activity
5.10.5 Cellulose and hemicellulose
5.10.6 Animal derived carbohydrates with nutraceutical activity
5.10.7 Heparin
5.10.8 Hyaluronic acid
5.10.9 Chitosan and chitin
5.10.10 Carbohydrates with nutraceutical activity from microorganisms
5.10.11 Alginate
5.10.12 Dextran
5.10.13 Bacillus striatum polysaccharide
5.11 Credit
References
6 Biological macromolecules as antioxidants
6.1 Introduction
6.2 Types and sources of biological macromolecules
6.2.1 Polysaccharides
6.2.1.1 Plant-derived polysaccharides
6.2.1.2 Animal-derived polysaccharides
6.2.1.3 Marine-derived polysaccharides
6.2.1.4 Bacterial and fungal polysaccharides
6.2.2 Proteins
6.2.2.1 Plant-derived proteins
6.2.2.2 Animal-derived proteins
6.2.2.3 Algal-derived proteins
6.2.3 Other antioxidative macromolecules
6.2.3.1 Nonextractable polyphenols
6.3 Macromolecules as antioxidants
6.3.1 Polysaccharides as antioxidants
6.3.2 Proteins as antioxidants
6.3.3 Nonextractable polyphenols as antioxidants
6.4 Applications
6.4.1 Food-based applications
6.4.1.1 Functional foods
6.4.1.2 Food packaging
6.4.1.3 Food additives
6.4.2 Other applications
6.5 Limitations of biological macromolecules
6.6 Future trends
References
7 Biological macromolecules as antimicrobial agents
7.1 Introduction
7.2 Classification of biological macromolecule
7.2.1 Carbohydrate
7.2.1.1 Monosaccharide
7.2.1.2 Disaccharide
7.2.1.3 Oligosaccharide
7.2.1.4 Polysaccharide
7.2.2 Protein
7.2.3 Lipid
7.2.3.1 Triglyceride
7.2.3.2 Phospholipid
7.2.3.3 Sterol
7.2.4 Nucleic acid
7.3 Antimicrobial activity of biological macromolecules
7.3.1 Polysaccharides
7.3.1.1 Chitosan
7.3.1.2 Cellulose
7.3.1.3 Alginate
7.3.1.4 Heparin
7.3.1.5 Chondroitin
7.3.1.6 Hyaluronic acid
7.3.2 Proteins
7.3.2.1 Collagen
7.3.2.2 Gelatin
7.3.2.3 Keratin
7.3.2.4 Soy protein
7.3.2.5 Fibrin
7.3.2.6 Lactoferrin
7.3.3 Fatty acids
7.3.3.1 Fatty acid methyl ester
7.3.3.2 Oleic acid
7.3.3.3 Linoleic acid
7.3.3.4 Arachidonic acid
7.4 Antimicrobial activity of macromolecule composites
7.4.1 Chitosan-alginate
7.4.2 Gelatin-chitosan
7.4.3 Keratin-chitosan
7.4.4 Collagen-alginate
7.4.5 Chitosan-cellulose
7.4.6 Lactoferrin-Oleic Acid
7.5 Nanotechnology based antimicrobial macromolecule
7.5.1 Chitosan based nanocomposite
7.5.2 Alginate-based nanocomposite
7.5.3 Cellulose based nanocomposite
7.5.4 Gelatin based nanocomposite
7.5.5 Collagen based nanocomposite
7.5.6 Keratin-based nanoparticle
7.5.7 Oleic acid based nanoparticle
7.6 Applications
7.6.1 Food packaging
7.6.2 Drug delivery
7.6.3 Wound dressing
7.7 Conclusion
References
8 Biological macromolecules from algae and their antimicrobial applications
8.1 Introduction
8.2 Bioactive macromolecules
8.2.1 Terpenoids
8.2.2 Steroids
8.2.3 Phenolics
8.2.4 Alkaloids
8.2.5 Polysaccharides
8.2.6 Peptides
8.2.7 Polyketide
8.2.8 Polyunsaturated fatty acids
8.3 Conclusion
References
9 Biological macromolecules acting on central nervous system
9.1 Introduction
9.1.1 Proteins
9.1.1.1 Amyloid-beta and tau protein
9.1.2 Cell cycle proteins
9.1.3 Homer/vesl proteins
9.1.4 Central fatty hypothesis
9.1.5 Carbohydrates
9.1.6 Role of carbohydrates on nervous system
9.1.6.1 In retino-tectal system
9.1.7 In sensory organs
9.1.8 Glycans
9.1.9 Role of glycan in neural development
9.1.10 Lipids
9.1.10.1 Phospholipids
9.1.11 Role of cPLA2 in cerebral ischemia
9.1.12 In the case of neurodegenerative diseases
9.1.13 Lipid peroxidation
9.2 Conclusion
References
10 Biological macromolecules as antidiabetic agents
10.1 Introduction
10.2 Types of biological macromolecules
10.3 Biological macromolecules
10.3.1 Carbohydrates
10.3.2 Lipids
10.3.3 Proteins
10.3.4 Nucleic acids
10.4 Advantages, limitations, and future perspectives
10.5 Conclusion
References
11 Biological macromolecules as anticancer agents
11.1 Introduction
11.2 Biological macromolecules for cancer therapy
11.2.1 Carbohydrates
11.2.1.1 Cervical cancer
11.2.1.2 Colon cancer
11.2.1.3 Breast cancer
11.2.1.4 Lung cancer
11.2.1.5 Pancreatic cancer
11.2.1.6 Others
11.2.2 Proteins and nucleic acid
11.2.2.1 Colon cancer
11.2.2.2 Colorectal and breast cancer
11.2.2.3 Hepatic cancer
11.2.2.4 Non-small cell lung cancer
11.2.3 Lipids
11.2.3.1 Colon cancer
11.2.3.2 Breast cancer
11.2.3.3 Brain cancer
11.2.3.4 Liver cancer
11.2.3.5 Ovarian cancer
11.2.3.6 Skin cancer
11.2.3.7 Others
11.3 Conclusion
References
12 Biological macromolecules as immunomodulators
12.1 Introduction
12.2 Immunomodulation
12.3 Immunomodulation, biomolecules, and applications
12.4 Polysaccharides
12.4.1 Immunomodulatory polysaccharides
12.4.2 Gut microbiota modulation
12.5 Lipids
12.5.1 Immunomodulatory effect of lipids
12.6 Proteins
12.6.1 Known immunomodulatory proteins
Acknowledgments
References
13 Biological macromolecules acting on gastrointestinal systems
13.1 Introduction
13.2 Role of carbohydrates in gastrointestinal system
13.3 Role of proteins in gastrointestinal system
13.4 Role of fatty acids in gastrointestinal system
13.5 Role of nucleic acids in gastrointestinal system
13.6 Conclusion
References
14 Synthetic macromolecules with biological activity
14.1 Introduction
14.2 Synthetic macromolecules with antimicrobial activity
14.2.1 History of antimicrobial agents and antimicrobial polymers
14.2.2 Classification of antimicrobial polymers
14.2.3 Preparation routes for antimicrobial polymers
14.2.4 Factors affecting the antimicrobial activity
14.2.4.1 Hydrophilic/hydrophobic balance
14.2.4.2 Molecular weight
14.2.4.3 Counter ion effect
14.2.4.4 Charge density
14.2.4.5 Polymeric architecture
14.2.4.6 Alkyl spacer
14.2.5 Synthetic macromolecules with antibacterial activity
14.2.6 Synthetic macromolecules with antiviral activity
14.2.7 Synthetic macromolecules with antifungal activity
14.2.8 Synthetic macromolecules with antiparasitic activity
14.3 Synthetic macromolecules with antioxidant activity
14.4 Polymer sequestrants
14.5 Conclusions
References
Further reading
III. Functional Applications
15 Biological macromolecules in drug delivery
15.1 Introduction
15.2 Drug delivery using various biological macromolecules
15.2.1 Drug delivery using carbohydrates
15.2.2 Drug delivery using proteins and peptides
15.2.3 Drug delivery using nucleic acids
15.2.4 Drug delivery using lipids
15.3 Conclusion
References
16 Biological macromolecules in tissue engineering
16.1 Introduction
16.2 Bone tissue engineering
16.3 Biological macromolecules in bone tissue engineering
16.3.1 Alginate
16.3.2 Chitosan
16.3.3 Carrageenan
16.3.4 Fucoidan
16.3.5 Ulvan
16.3.6 Gelatin
16.4 Conclusion
Acknowledgment
References
17 Biological macromolecules for drug delivery in tissue engineering
17.1 Introduction
17.2 Drug-loaded electrospun fibers used in tissue engineering applications and drug delivery
17.2.1 Drug-loaded polysaccharides-based electrospun fibers
17.2.2 Drug-loaded protein-based electrospun fibers
17.3 Drug-loaded injectable hydrogels used in tissue engineering applications and drug delivery
17.4 Conclusions
References
18 Biological macromolecules for growth factor delivery
18.1 Introduction
18.2 Delivery systems for growth factors
18.3 Materials for delivery systems of growth factors
18.4 Biological macromolecules for delivery systems of growth factors
18.4.1 Protein-based materials for growth factor delivery
18.4.2 Polysaccharide-based materials for growth factor delivery
18.4.3 Polysaccharide combinations for growth factor delivery
18.4.4 Composites materials for growth factor delivery
18.4.5 Protein-based composite for growth factor delivery
18.4.6 Protein-polysaccharide composites for growth factor delivery
18.4.7 Polysaccharide-polysaccharide composites for growth factor delivery
References
19 Biological macromolecules for growth factor delivery in bone regeneration
19.1 Introduction
19.2 Bone regeneration
19.3 Growth factors in tissue and bone regeneration
19.4 Biomacromolecules as carriers of growth factors
19.5 Hydrogels and sponges
19.6 Scaffolds and fibers
19.7 Nanoparticles and nanoassemblies
19.8 Concluding remarks
References
20 Biological macromolecules for nutrients delivery
20.1 Introduction
20.2 Nutrients
20.2.1 Water-soluble nutrients
20.2.1.1 Ascorbic acid
20.2.1.2 Riboflavin
20.2.2 Oil-soluble nutrients
20.2.2.1 Curcumin
20.2.2.2 β-Carotene
20.2.2.3 Lutein
20.3 Biological macromolecules used for nutrients delivery
20.3.1 Polysaccharides
20.3.1.1 Pectin
20.3.1.2 Alginate
20.3.1.3 Carrageenan
20.3.1.4 Starch
20.3.2 Proteins
20.3.2.1 Whey protein
20.3.2.2 Casein
20.3.3 Glycoproteins and proteoglycans
20.3.4 Others (lignin as example)
20.4 Molecular interactions that maintain the stability of biopolymer-based delivery systems
20.4.1 Electrostatic interactions
20.4.2 Hydrogen bonding
20.4.3 Hydrophobic interactions
20.4.4 Covalent interactions
20.5 Retention and release mechanisms
20.6 Nutrient delivery systems based on biological macromolecules
20.6.1 Composition and structure
20.6.2 Fabrication
20.6.2.1 Fabrication of hydrogel
20.6.2.2 Fabrication of microcapsules
20.6.2.3 Fabrication of nanoparticles
20.6.3 Properties
20.6.4 Applications
20.6.4.1 Flavors
20.6.4.2 Enzymes
20.6.4.3 Nutraceuticals
20.6.4.4 Omega-3 fatty acids
20.6.4.5 Vitamins
20.6.4.6 Minerals
20.6.4.7 Antimicrobials
20.7 Future trends
20.7.1 Co-encapsulation of multiple nutrients
20.7.2 Targeted and controlled release of bioactive molecules
20.7.3 In vivo testing
References
21 Biological macromolecules for nucleic acid delivery
21.1 Introduction
21.2 Nucleic acids structure and functions
21.3 Biological macromolecules for nucleic acid delivery
21.3.1 Lipid-based drug delivery systems
21.3.2 Protein-based drug delivery systems
21.3.3 Carbohydrate-based drug delivery systems
21.3.3.1 Chitosan
21.3.3.2 Pullulan
21.4 Conclusions
References
22 Biological macromolecules in cell encapsulation
22.1 Introduction
22.2 Biopolymers used for cell encapsulation in TE
22.2.1 Agarose
22.2.2 Alginate
22.2.3 Chitin and chitosan
22.2.4 Collagen
22.2.5 Gelatin
22.2.6 Fibrin
22.2.7 Glycosaminoglycans
22.2.8 Silk (fibroion and spidroin)
22.2.9 Other natural polymers in TE
22.2.9.1 Cellulose
22.2.9.2 Starch (starch, amylose, amylopectin)
22.2.9.3 Carrageenans
22.2.9.4 Dextrans
22.2.9.5 Gellan
22.2.9.6 Pullulans
22.2.9.7 Elastin
22.2.9.8 Laminin
22.2.9.9 Fibronectin
22.3 Advantages, drawbacks, applications, forms and manufacturing methods
22.4 Conclusions
Acknowledgment
References
23 Biological macromolecules for enzyme immobilization
23.1 Introduction
23.2 Biological macromolecules for enzyme immobilization
23.2.1 Chitin and chitosan
23.2.2 Agarose
23.2.3 Alginate
23.2.4 Cellulose and its derivatives
23.2.5 Gelatin for enzyme immobilization
23.2.6 Dextran for enzyme immobilization
23.2.6.1 Carrageenan for enzyme immobilization
23.2.7 Pectin for enzyme immobilization
23.2.8 Xanthan for enzyme immobilization
23.3 Conclusions and future outlook
Acknowledgment
Conflicts of interest
References
Further reading
24 Carbohydrates mimetics: enzyme inhibitors and target molecules in several diseases
24.1 Introduction
24.1.1 Biomass and biobased materials
24.1.2 Carbohydrates
24.1.3 Biological and medicinal interest of carbohydrates
24.1.4 Glycosidases
24.2 Glycomimetics
24.2.1 Iminosugars
24.2.2 Carbasugars
24.2.3 Thiosugars
24.3 Hybrid carbohydrates
24.4 Macromolecules
24.4.1 Multivalents
24.4.2 Polysaccharides
24.5 Conclusions
References
IV. Others
25 Current challenging issues of biological macromolecules in biomedicine
25.1 Introduction
25.2 Biological macromolecules
25.3 Macromolecules in biomedical applications
25.4 Macromolecules in targeted drug delivery
25.5 Biomaterials as targeted drug delivery
25.5.1 Hydrogels for drug delivery
25.5.1.1 Injectable hydrogels as drug delivery systems
25.5.1.2 Oral hydrogels as drug delivery systems
25.5.1.2.1 Hydrogel-based microparticles as drug delivery systems
25.5.1.2.2 Hydrogel-based nanoparticles as drug delivery systems
25.5.2 Gene delivery
25.6 Macromolecules on tissue engineering
25.6.1 Wound management
25.6.2 Development of skin substitutes
25.7 Conclusion
References
26 Future perspectives of biological macromolecules in biomedicine
26.1 Bio-nanotechnology
26.1.1 Delivery systems
26.1.1.1 Nonviral vectors
26.2 Mitochondrial gene therapy
26.2.1 Mitochondrion
26.2.2 Mitochondrial mutations
26.2.3 Targeting Mitochondria
26.2.3.1 Gene and protein expression
26.3 Crosstalk between chronobiology and cancer
26.3.1 Circadian clock and cancer development
26.3.2 Chronobiology and cancer treatment
26.3.2.1 Cancer chronodrug/gene delivery
26.4 Concluding remarks
References
Index
Back Cover

Citation preview

BIOLOGICAL MACROMOLECULES

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BIOLOGICAL MACROMOLECULES BIOACTIVITY AND BIOMEDICAL APPLICATIONS

Edited by

Amit Kumar Nayak Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, India

Amal Kumar Dhara Department of Pharmacy, Contai Polytechnic, Contai, India

Dilipkumar Pal Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2022 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: 978-0-323-85759-8 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre G. Wolff Acquisitions Editor: Michelle Fisher Editorial Project Manager: Barbara Makinster Production Project Manager: Swapna Srinivasan Cover Designer: Matthew Limbert Typeset by MPS Limited, Chennai, India

Contents 2.2.2 Lysostaphin 28 2.2.3 Metallo-β-lactamase-like lactonase 32 2.3 Chitosan as a bioactive polysaccharide 36 2.3.1 Relationship of chitosan physicochemical property and its bioactivity 37 2.3.2 Bioactivity of chitosan with modified functional group 41 2.4 Conclusion 43 References 43

List of contributors xiii Preface xix

I Background 1. Biological macromolecules: sources, properties, and functions

3. The importance of biological macromolecules in biomedicine

AMAL KUMAR DHARA AND AMIT KUMAR NAYAK

AHMED OLATUNDE, OMAR BAHATTAB, ABDUR RAUF, NAVEED MUHAMMAD, YAHYA S. AL-AWTHAN, TABUSSAM TUFAIL, MUHAMMAD IMRAN AND MOHAMMAD S. MUBARAK

1.1 Introduction 3 1.2 Carbohydrates 4 1.2.1 Monosaccharides 5 1.2.2 Oligosaccharides 5 1.2.3 Polysaccharides 5 1.3 Lipids 9 1.3.1 Simple lipids 10 1.3.2 Compound or conjugate lipids 10 1.3.3 Derived lipids 10 1.4 Proteins 11 1.4.1 Simple proteins 12 1.4.2 Conjugated proteins 13 1.4.3 Derived proteins 13 1.5 Nucleic acids 14 1.5.1 Nucleotides 15 1.5.2 Nucleosides 15 1.5.3 DNA 15 1.5.4 RNA 16 1.6 Conclusion 18 References 18

3.1 Introduction 53 3.2 Biological macromolecules in biomedicine and therapies 53 3.3 Carbohydrates 54 3.3.1 Therapeutics based on carbohydrates 55 3.4 Peptides 56 3.4.1 Therapeutics based on peptides 57 3.5 Proteins 58 3.5.1 Therapeutics based on proteins (proteins and monoclonal antibodies) 58 3.6 Lipids 60 3.6.1 Drug delivery-based on lipids 60 3.7 Nucleic acids and oligonucleotides 61 3.7.1 Therapeutics based on oligonucleotides 61 3.8 Synthesis of macromolecules 63 3.9 Biomedicine 64 3.10 Conclusions 65 References 65

2. Structure
activity relationship of biological macromolecules

4. Modification techniques for carbohydrate macromolecules

AURELIE SARAH MOK TSZE CHUNG, YONG KIAT TEO, WAI TENG CHENG AND JOASH BAN LEE TAN

AJAY VASUDEO RANE, DEEPTI YADAV AND KRISHNAN KANNY

2.1 Introduction 23 2.2 Enzymes as bioactive proteins 24 2.2.1 L-amino acid oxidases 25

4.1 Introduction

v

69

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Contents

4.2 4.3 4.4 4.5 4.6

Cellulose 69 Hemicelluloses 71 Lignin 72 Chitin and chitosan 72 Modification of carbohydrate biological macromolecules 73 References 86

II Bioactivity 5. Biological macromolecules as nutraceuticals IRERI ALEJANDRA CARBAJAL-VALENZUELA, NUVIA MARINA APOLONIO
HERNANDEZ, DIANA VANESA GUTIERREZ-CHAVEZ, ´ LEZ-ARIAS, BEATRIZ GONZA ALEJANDRA JIMENEZ-HERNANDEZ, IRINEO TORRES-PACHECO, ENRIQUE RICO-GARCI´A, ANA ANGELICA FEREGRINO-PE´REZ ´ LEZ ´ N GERARDO GUEVARA-GONZA AND RAMO

5.1 History of the applications of nutraceutical compounds in health care 97 5.2 Alkaloids 99 5.2.1 Caffeine 99 5.2.2 Capsaicin 101 5.2.3 Theobromine 101 5.3 Phenolic compounds 102 5.3.1 Curcumin 102 5.3.2 Resveratrol 103 5.3.3 Quercetin 103 5.3.4 Anthocyanins 103 5.3.5 Luteolin 104 5.3.6 Naringenin 104 5.3.7 Catechins 104 5.4 Terpenes 105 5.4.1 Lycopene 105 5.4.2 β-Carotene 105 5.4.3 Lutein 106 5.4.4 Zeaxanthin 107 5.5 Future views 107 5.6 Proteins and peptides with biological activity of medical interest 107 5.7 Nucleic acids and their nutraceutical properties used in biomedicine 112 5.7.1 Nucleic acids overview 112 5.7.2 Perspectives 119 5.8 Introduction of lipids 119

5.8.1 Polyunsaturated fatty acids 120 5.8.2 The role of Omega-3 PUFAs in some disorders 121 5.9 The potential use of bioactive lipids in cancer stem cells and coronavirus disease (COVID-19) 123 5.9.1 Bioactive lipids in cancer 123 5.9.2 Bioactive lipids in COVID-19 123 5.10 Carbohydrates as nutraceuticals 124 5.10.1 Brief overview of carbohydrates 124 5.10.2 Role of polysaccharides in extracellular membrane 124 5.10.3 Immunostimmulatory effect of carbohydrates 125 5.10.4 Carbohydrates from plants with nutraceutical activity 125 5.10.5 Cellulose and hemicellulose 125 5.10.6 Animal derived carbohydrates with nutraceutical activity 126 5.10.7 Heparin 126 5.10.8 Hyaluronic acid 127 5.10.9 Chitosan and chitin 127 5.10.10 Carbohydrates with nutraceutical activity from microorganisms 128 5.10.11 Alginate 128 5.10.12 Dextran 128 5.10.13 Bacillus striatum polysaccharide 128 5.11 Credit 129 References 129

6. Biological macromolecules as antioxidants T. MADHUJITH, N.E. WEDAMULLA AND D.A.S. GAMAGE

6.1 Introduction 139 6.2 Types and sources of biological macromolecules 141 6.2.1 Polysaccharides 141 6.2.2 Proteins 149 6.2.3 Other antioxidative macromolecules 152 6.3 Macromolecules as antioxidants 152 6.3.1 Polysaccharides as antioxidants 152 6.3.2 Proteins as antioxidants 155 6.3.3 Nonextractable polyphenols as antioxidants 156 6.4 Applications 156 6.4.1 Food-based applications 156 6.4.2 Other applications 158 6.5 Limitations of biological macromolecules 158 6.6 Future trends 159 References 159

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7. Biological macromolecules as antimicrobial agents MD. SHAHRUZZAMAN, SHAFIUL HOSSAIN, TANVIR AHMED, SUMAYA F. KABIR, MD. MINHAJUL ISLAM, ASHIQUR RAHMAN, MD. SAZEDUL ISLAM, SABRINA SULTANA AND MOHAMMED MIZANUR RAHMAN

7.1 Introduction 165 7.2 Classification of biological macromolecule 167 7.2.1 Carbohydrate 167 7.2.2 Protein 169 7.2.3 Lipid 170 7.2.4 Nucleic acid 171 7.3 Antimicrobial activity of biological macromolecules 172 7.3.1 Polysaccharides 172 7.3.2 Proteins 175 7.3.3 Fatty acids 177 7.4 Antimicrobial activity of macromolecule composites 180 7.4.1 Chitosan-alginate 180 7.4.2 Gelatin-chitosan 181 7.4.3 Keratin-chitosan 182 7.4.4 Collagen-alginate 183 7.4.5 Chitosan-cellulose 184 7.4.6 Lactoferrin-Oleic Acid 185 7.5 Nanotechnology based antimicrobial macromolecule 185 7.5.1 Chitosan based nanocomposite 185 7.5.2 Alginate-based nanocomposite 187 7.5.3 Cellulose based nanocomposite 187 7.5.4 Gelatin based nanocomposite 189 7.5.5 Collagen based nanocomposite 190 7.5.6 Keratin-based nanoparticle 190 7.5.7 Oleic acid based nanoparticle 190 7.6 Applications 190 7.6.1 Food packaging 190 7.6.2 Drug delivery 191 7.6.3 Wound dressing 192 7.7 Conclusion 193 References 193

8. Biological macromolecules from algae and their antimicrobial applications NATANAMURUGARAJ GOVINDAN, GAANTY PRAGAS MANIAM, MOHD HASBI AB. RAHIM, AHMAD ZIAD SULAIMAN AND AZILAH AJIT

8.1 Introduction 203 8.2 Bioactive macromolecules

203

8.2.1 Terpenoids 203 8.2.2 Steroids 206 8.2.3 Phenolics 207 8.2.4 Alkaloids 208 8.2.5 Polysaccharides 208 8.2.6 Peptides 209 8.2.7 Polyketide 210 8.2.8 Polyunsaturated fatty acids 8.3 Conclusion 213 References 213

210

9. Biological macromolecules acting on central nervous system DILIPKUMAR PAL AND KHUSHBOO RAJ

9.1 Introduction 219 9.1.1 Proteins 219 9.1.2 Cell cycle proteins 220 9.1.3 Homer/vesl proteins 220 9.1.4 Central fatty hypothesis 222 9.1.5 Carbohydrates 222 9.1.6 Role of carbohydrates on nervous system 223 9.1.7 In sensory organs 223 9.1.8 Glycans 223 9.1.9 Role of glycan in neural development 224 9.1.10 Lipids 224 9.1.11 Role of cPLA2 in cerebral ischemia 225 9.1.12 In the case of neurodegenerative diseases 225 9.1.13 Lipid peroxidation 225 9.2 Conclusion 226 References 226

10. Biological macromolecules as antidiabetic agents JAISON JEEVANANDAM, CALEB ACQUAH AND MICHAEL K. DANQUAH

10.1 Introduction 229 10.2 Types of biological macromolecules 230 10.3 Biological macromolecules 232 10.3.1 Carbohydrates 232 10.3.2 Lipids 233 10.3.3 Proteins 235 10.3.4 Nucleic acids 237 10.4 Advantages, limitations, and future perspectives 238

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Contents

10.5 Conclusion References 239

14. Synthetic macromolecules with biological activity

238

STEFANIA RACOVITA, MARCEL POPA, LEONARD IONUT ATANASE AND SILVIA VASILIU

11. Biological macromolecules as anticancer agents HIMJA TIWARI, HARSHAL DESHMUKH, NILESH SHIRISH WAGH AND JAYA LAKKAKULA

11.1 Introduction 243 11.2 Biological macromolecules for cancer therapy 11.2.1 Carbohydrates 244 11.2.2 Proteins and nucleic acid 258 11.2.3 Lipids 262 11.3 Conclusion 269 References 269

244

12. Biological macromolecules as immunomodulators EDUARDO COSTA, MANUELA MACHADO, MANUELA PINTADO AND SARA SILVA

12.1 Introduction 273 12.2 Immunomodulation 274 12.3 Immunomodulation, biomolecules, and applications 274 12.4 Polysaccharides 275 12.4.1 Immunomodulatory polysaccharides 275 12.4.2 Gut microbiota modulation 277 12.5 Lipids 277 12.5.1 Immunomodulatory effect of lipids 278 12.6 Proteins 280 12.6.1 Known immunomodulatory proteins 280 Acknowledgments 282 References 282

13. Biological macromolecules acting on gastrointestinal systems DILIPKUMAR PAL AND SUPRIYO SAHA

13.1 Introduction 289 13.2 Role of carbohydrates in gastrointestinal system 289 13.3 Role of proteins in gastrointestinal system 295 13.4 Role of fatty acids in gastrointestinal system 298 13.5 Role of nucleic acids in gastrointestinal system 300 13.6 Conclusion 301 References 302

14.1 Introduction 305 14.2 Synthetic macromolecules with antimicrobial activity 306 14.2.1 History of antimicrobial agents and antimicrobial polymers 307 14.2.2 Classification of antimicrobial polymers 308 14.2.3 Preparation routes for antimicrobial polymers 309 14.2.4 Factors affecting the antimicrobial activity 311 14.2.5 Synthetic macromolecules with antibacterial activity 313 14.2.6 Synthetic macromolecules with antiviral activity 317 14.2.7 Synthetic macromolecules with antifungal activity 318 14.2.8 Synthetic macromolecules with antiparasitic activity 319 14.3 Synthetic macromolecules with antioxidant activity 320 14.4 Polymer sequestrants 324 14.5 Conclusions 328 References 328 Further reading 335

III Functional applications 15. Biological macromolecules in drug delivery AMIT KUMAR NAYAK, MD SAQUIB HASNAIN, ANINDITA BEHERA, AMAL KUMAR DHARA AND DILIPKUMAR PAL

15.1 Introduction 339 15.2 Drug delivery using various biological macromolecules 340 15.2.1 Drug delivery using carbohydrates 341 15.2.2 Drug delivery using proteins and peptides 349 15.2.3 Drug delivery using nucleic acids 355

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Contents

15.2.4 Drug delivery using lipids 359 15.3 Conclusion 367 References 367

16. Biological macromolecules in tissue engineering PANDURANG APPANA DALAVI, SESHA SUBRAMANIAN MURUGAN, SUKUMARAN ANIL AND JAYACHANDRAN VENKATESAN

16.1 Introduction 381 16.2 Bone tissue engineering 381 16.3 Biological macromolecules in bone tissue engineering 382 16.3.1 Alginate 382 16.3.2 Chitosan 383 16.3.3 Carrageenan 384 16.3.4 Fucoidan 385 16.3.5 Ulvan 385 16.3.6 Gelatin 386 16.4 Conclusion 387 Acknowledgment 387 References 387

17. Biological macromolecules for drug delivery in tissue engineering MARCEL POPA AND LEONARD IONUT ATANASE

17.1 Introduction 393 17.2 Drug-loaded electrospun fibers used in tissue engineering applications and drug delivery 394 17.2.1 Drug-loaded polysaccharides-based electrospun fibers 395 17.2.2 Drug-loaded protein-based electrospun fibers 401 17.3 Drug-loaded injectable hydrogels used in tissue engineering applications and drug delivery 403 17.4 Conclusions 411 References 411

18. Biological macromolecules for growth factor delivery M.D. FIGUEROA-PIZANO

18.1 Introduction 419 18.2 Delivery systems for growth factors 421 18.3 Materials for delivery systems of growth factors 423

18.4 Biological macromolecules for delivery systems of growth factors 424 18.4.1 Protein-based materials for growth factor delivery 425 18.4.2 Polysaccharide-based materials for growth factor delivery 427 18.4.3 Polysaccharide combinations for growth factor delivery 429 18.4.4 Composites materials for growth factor delivery 430 18.4.5 Protein-based composite for growth factor delivery 430 18.4.6 Protein-polysaccharide composites for growth factor delivery 432 18.4.7 Polysaccharide-polysaccharide composites for growth factor delivery 433 References 433

19. Biological macromolecules for growth factor delivery in bone regeneration ARISTEIDIS PAPAGIANNOPOULOS AND ELENI VLASSI

19.1 Introduction 439 19.2 Bone regeneration 439 19.3 Growth factors in tissue and bone regeneration 441 19.4 Biomacromolecules as carriers of growth factors 443 19.5 Hydrogels and sponges 445 19.6 Scaffolds and fibers 446 19.7 Nanoparticles and nanoassemblies 448 19.8 Concluding remarks 449 References 449

20. Biological macromolecules for nutrients delivery LONG CHEN, ZHONGYU YANG, DAVID JULIAN MCCLEMENTS, ZHENGYU JIN AND MING MIAO

20.1 Introduction 455 20.2 Nutrients 455 20.2.1 Water-soluble nutrients 456 20.2.2 Oil-soluble nutrients 458 20.3 Biological macromolecules used for nutrients delivery 458 20.3.1 Polysaccharides 459 20.3.2 Proteins 461 20.3.3 Glycoproteins and proteoglycans 461

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Contents

20.3.4 Others (lignin as example) 462 20.4 Molecular interactions that maintain the stability of biopolymer-based delivery systems 462 20.4.1 Electrostatic interactions 463 20.4.2 Hydrogen bonding 463 20.4.3 Hydrophobic interactions 463 20.4.4 Covalent interactions 464 20.5 Retention and release mechanisms 464 20.6 Nutrient delivery systems based on biological macromolecules 465 20.6.1 Composition and structure 465 20.6.2 Fabrication 466 20.6.3 Properties 469 20.6.4 Applications 469 20.7 Future trends 471 20.7.1 Co-encapsulation of multiple nutrients 471 20.7.2 Targeted and controlled release of bioactive molecules 471 20.7.3 In vivo testing 472 References 472

21. Biological macromolecules for nucleic acid delivery AHMED S. ABO DENA AND IBRAHIM M. EL-SHERBINY

21.1 Introduction 479 21.2 Nucleic acids structure and functions 480 21.3 Biological macromolecules for nucleic acid delivery 482 21.3.1 Lipid-based drug delivery systems 482 21.3.2 Protein-based drug delivery systems 484 21.3.3 Carbohydrate-based drug delivery systems 486 21.4 Conclusions 488 References 489

22. Biological macromolecules in cell encapsulation MILAN MILIVOJEVIC, IVANA PAJIC-LIJAKOVIC AND BRANKO BUGARSKI

22.1 Introduction 491 22.2 Biopolymers used for cell encapsulation in TE 496 22.2.1 Agarose 496 22.2.2 Alginate 497 22.2.3 Chitin and chitosan 499

22.2.4 Collagen 500 22.2.5 Gelatin 503 22.2.6 Fibrin 504 22.2.7 Glycosaminoglycans 505 22.2.8 Silk (fibroion and spidroin) 506 22.2.9 Other natural polymers in TE 508 22.3 Advantages, drawbacks, applications, forms and manufacturing methods 511 22.4 Conclusions 526 Acknowledgment 527 References 527

23. Biological macromolecules for enzyme immobilization HAMZA RAFEEQ, SARMAD AHMAD QAMAR, HIRA MUNIR, MUHAMMAD BILAL AND HAFIZ M.N. IQBAL

23.1 Introduction 529 23.2 Biological macromolecules for enzyme immobilization 532 23.2.1 Chitin and chitosan 532 23.2.2 Agarose 533 23.2.3 Alginate 534 23.2.4 Cellulose and its derivatives 535 23.2.5 Gelatin for enzyme immobilization 536 23.2.6 Dextran for enzyme immobilization 537 23.2.7 Pectin for enzyme immobilization 539 23.2.8 Xanthan for enzyme immobilization 540 23.3 Conclusions and future outlook 541 Acknowledgment 541 Conflicts of interest 541 References 542 Further reading 546

24. Carbohydrates mimetics: enzyme inhibitors and target molecules in several diseases ´ NICA E. MANZANO, CUSTODIANA A. VERO COLMENAREZ LOBO AND EVANGELINA REPETTO

24.1 Introduction 547 24.1.1 Biomass and biobased materials 547 24.1.2 Carbohydrates 548 24.1.3 Biological and medicinal interest of carbohydrates 550 24.1.4 Glycosidases 551 24.2 Glycomimetics 554 24.2.1 Iminosugars 554

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Contents

24.2.2 Carbasugars 561 24.2.3 Thiosugars 564 24.3 Hybrid carbohydrates 566 24.4 Macromolecules 567 24.4.1 Multivalents 567 24.4.2 Polysaccharides 569 24.5 Conclusions 570 References 570

IV Others 25. Current challenging issues of biological macromolecules in biomedicine Y. DE ANDA-FLORES, E. CARVAJAL-MILLAN, A.C. CAMPA-MADA, K.G. MARTI´NEZ-ROBINSON, ´ N-CHU, J. LIZARDI-MENDOZA, A. RASCO ´ PEZ AND J. TANORI-CORDOVA A.L. MARTI´NEZ-LO

25.1 25.2 25.3 25.4 25.5

Introduction 581 Biological macromolecules 582 Macromolecules in biomedical applications 583 Macromolecules in targeted drug delivery 584 Biomaterials as targeted drug delivery 585 25.5.1 Hydrogels for drug delivery 585

25.5.2 Gene delivery 592 25.6 Macromolecules on tissue engineering 593 25.6.1 Wound management 597 25.6.2 Development of skin substitutes 597 25.7 Conclusion 600 References 600

26. Future perspectives of biological macromolecules in biomedicine ˆ NIA ALBUQUERQUE, ´ BEN FARIA, TA ANA R. NEVES, RU ˆ NGELA SOUSA AND DIANA COSTA TELMA QUINTELA, A

26.1 Bio-nanotechnology 607 26.1.1 Delivery systems 608 26.2 Mitochondrial gene therapy 610 26.2.1 Mitochondrion 611 26.2.2 Mitochondrial mutations 611 26.2.3 Targeting Mitochondria 612 26.3 Crosstalk between chronobiology and cancer 616 26.3.1 Circadian clock and cancer development 617 26.3.2 Chronobiology and cancer treatment 619 26.4 Concluding remarks 624 References 625

Index 633

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List of contributors

Ahmed S. Abo Dena Nanomedicine Laboratory, Center for Materials Science, Zewail City of Science and Technology, Giza, Egypt; Department of Pharmaceutical Chemistry, National Organization for Drug Control and Research (NODCAR), Giza, Egypt

Anindita Behera School of Pharmaceutical Sciences, Siksha “O” Anusandhan, Deemed to be University, Bhubaneswar, India

Caleb Acquah School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada

Branko Bugarski Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia

Tanvir Ahmed Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

A.C. Campa-Mada Biopolymers-CTAOA, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico

Muhammad Bilal School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai’an, China

Azilah Ajit Faculty of Chemical & Natural Resources Engineering, Universiti Malaysia Pahang, Kuantan, Malaysia

Ireri Alejandra Carbajal-Valenzuela Biosystems Engineering Group, School of EngineeringCampus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico

Yahya S. Al-Awthan Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Saudia Arabia; Department of Biology, Faculty of Science, Ibb University, Ibb, Yemen

E. Carvajal-Millan Biopolymers-CTAOA, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico

Taˆnia Albuquerque CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal Sukumaran Anil Department of Dentistry, Oral Health Institute, Hamad Medical Corporation, College of Dental Medicine, Qatar University, Doha, Qatar Nuvia Marina Apolonio–Hernandez Biosystems Engineering Group, School of EngineeringCampus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico Leonard Ionut Atanase Academy of Romanian Scientists, Bucuresti, Romania; Faculty of Dental Medicine, “Apollonia” University of Iasi, Iasi, Romania Omar Bahattab Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Saudia Arabia

Long Chen School of Food Science and Technology, Jiangnan University, Wuxi, P.R. China; State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, P.R. China Wai Teng Cheng School of Science, Monash University Malaysia, Bandar Sunway, Malaysia Custodiana A. Colmenarez Lobo Research Center in Carbohydrate Chemistry (CIHIDECAR), National Scientific and Technical Research Council (CONICET)-UBA, Buenos Aires, Argentina Diana Costa CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal Eduardo Costa Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina —Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal

xiii

xiv

List of contributors

Pandurang Appana Dalavi Biomaterials Research Laboratory, Yenepoya Research Centre, Yenepoya (Deemed to be University), Deralakatte, Mangalore, India

Diana Vanesa Gutierrez-Chavez Biosystems Engineering Group, School of EngineeringCampus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico

Michael K. Danquah Chemical Department, University of Chattanooga, TN, United States

Md Saquib Hasnain Department of Pharmacy, Palamau Institute of Pharmacy, Daltonganj, India

Engineering Tennessee,

Y. De Anda-Flores Biopolymers-CTAOA, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico Harshal Deshmukh Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, —Pune Expressway, Bhatan Post— Somathne, Panvel, Mumbai, India

Shafiul Hossain Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh; Department of Chemical Engineering and Polymer Science, Shahjalal University of Science and Technology, Sylhet, Bangladesh

Amal Kumar Dhara Department of Pharmacy, Contai Polytechnic, Contai, India

Muhammad Imran University Institute of Diet & Nutritional Sciences, Faculty of Allied Health Sciences, The University of Lahore, Lahore, Pakistan

Ibrahim M. El-Sherbiny Nanomedicine Laboratory, Center for Materials Science, Zewail City of Science and Technology, Giza, Egypt

Hafiz M.N. Iqbal Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico

Ru´ben Faria CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal

Jaison Jeevanandam CQM—Centro de Quı´mica da Madeira (Madeira Chemistry Center), MMRG, Universidade da Madeira (University of Madeira), Campus da Penteada (Penteada campus), Funchal, Portugal

Ana Angelica Feregrino-Pe´rez Biosystems Engineering Group, School of EngineeringCampus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico M.D. Figueroa-Pizano Biopolymers-CTAOA, Research Center for Food and Development (CIAD), Hermosillo, Sonora, Mexico

Alejandra Jimenez-Hernandez Biosystems Engineering Group, School of EngineeringCampus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico School of Food Science and Jiangnan University, Wuxi, P.R. Key Laboratory of Food Science and Jiangnan University, Wuxi, P.R.

D.A.S. Gamage Department of Chemical and Process Engineering, Faculty of Engineering, University of Peradeniya, Peradeniya, Sri Lanka

Zhengyu Jin Technology, China; State Technology, China

Beatriz Gonza´lez-Arias Biosystems Engineering Group, School of Engineering-Campus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico

Sumaya F. Kabir Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

Natanamurugaraj Govindan Algae Culture Collection Center & Laboratory, Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Kuantan, Malaysia; Centre for Research in Advanced Tropical Bioscience, Universiti Malaysia Pahang, Kuantan, Malaysia

Krishnan Kanny Composite Research Group, Department of Mechanical Engineering, Durban University of Technology, Durban, South Africa

Ramo´n Gerardo Guevara-Gonza´lez Biosystems Engineering Group, School of EngineeringCampus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico

Jaya Lakkakula Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, —Pune Expressway, Bhatan Post—Somathne, Panvel, Mumbai, India J.

Lizardi-Mendoza Biopolymers-CTAOA, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico

xv

List of contributors

Manuela Machado Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal T. Madhujith Department of Food Science and Technology, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka Gaanty Pragas Maniam Algae Culture Collection Center & Laboratory, Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Kuantan, Malaysia; Centre for Research in Advanced Tropical Bioscience, Universiti Malaysia Pahang, Kuantan, Malaysia Vero´nica E. Manzano Faculty of Exact and Natural Sciences, Department of Organic Chemistry, University of Buenos Aires, Buenos Aires, Argentina; Research Center in Carbohydrate Chemistry (CIHIDECAR), National Scientific and Technical Research Council (CONICET)-UBA, Buenos Aires, Argentina A.L. Martı´nez-Lo´pez NANO-VAC Research Group, Department of Chemistry and Pharmaceutical Technology, University of Navarra, Pamplona, Spain K.G. Martı´nez-Robinson Biopolymers-CTAOA, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico David Julian McClements Department of Food Science, University of Massachusetts, Amherst, MA, United States Ming Miao Technology, China; State Technology, China

School of Food Science and Jiangnan University, Wuxi, P.R. Key Laboratory of Food Science and Jiangnan University, Wuxi, P.R.

Milan Milivojevic Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia

Mohammad S. Mubarak Department of Chemistry, The University of Jordan, Amman, Jordan Naveed Muhammad Department of Pharmacy, Abdul Wali Khan University, Khyber Pakhtunkhwa, Pakistan Hira Munir Department of Biochemistry and Biotechnology, University of Gujrat, Gujrat, Pakistan Sesha Subramanian Murugan Biomaterials Research Laboratory, Yenepoya Research Centre, Yenepoya (Deemed to be University), Deralakatte, Mangalore, India Amit Kumar Nayak Department Pharmaceutics, Seemanta Institute Pharmaceutical Sciences, Jharpokharia, India

of of

Ana R. Neves CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal Ahmed Olatunde Department of Biochemistry, Abubakar Tafawa Balewa University, Bauchi, Nigeria Ivana Pajic-Lijakovic Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia Dilipkumar Pal Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, India Aristeidis Papagiannopoulos Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Manuela Pintado Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal

Md. Minhajul Islam Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

Marcel Popa “Apollonia” University of Iasi, Faculty of Dental Medicine, Iasi, Romania; Academy of Romanian Scientists, Bucuresti, Romania; Faculty of Chemical Engineering and Environmental Protection, Department of Natural and Synthetic Polymers, “Gheorghe Asachi” Technical University of Iasi, Iasi, Romania

Aurelie Sarah Mok Tsze Chung School of Science, Monash University Malaysia, Bandar Sunway, Malaysia

Sarmad Ahmad Qamar Biochemistry, University Faisalabad, Pakistan

Department of of Agriculture,

xvi

List of contributors

Telma Quintela CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal

Supriyo Saha School of Pharmaceutical Sciences and Technology, Sardar Bhagwan Singh University, Dehradun, India

Stefania Racovita “Petru Poni” Institute Macromolecular Chemistry, Iasi, Romania

Md. Sazedul Islam Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

of

Hamza Rafeeq Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan Mohd Hasbi Ab. Rahim Algae Culture Collection Center & Laboratory, Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Kuantan, Malaysia; Centre for Research in Advanced Tropical Bioscience, Universiti Malaysia Pahang, Kuantan, Malaysia Ashiqur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh; National Institute of Textile Engineering and Research (NITER), Dhaka, Bangladesh Mohammed Mizanur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Khushboo Raj School of Pharmacy, Arka Jain university, Tata, Jamshedpur, India

Md. Shahruzzaman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Sara Silva Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina —Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal ˆ ngela A Sousa CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal Ahmad Ziad Sulaiman Faculty of Bio-Engineering & Technology, University Malaysia Kelantan Kampus Jeli, Kelantan, Malaysia Sabrina Sultana Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh; Department of Arts and Sciences, Ahsanullah University of Science and Technology, Dhaka, Bangladesh

Ajay Vasudeo Rane Composite Research Group, Department of Mechanical Engineering, Durban University of Technology, Durban, South Africa

Joash Ban Lee Tan School of Science, Monash University Malaysia, Bandar Sunway, Malaysia

A. Rasco´n-Chu Biotechnology-CTAOV, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico

J. Tanori-Cordova Department of Polymers and Materials Research, University of Sonora, Hermosillo, Mexico

Abdur Rauf Department of Chemistry, University of Swabi Anbar, Khyber Pakhtunkhwa, Pakistan Evangelina Repetto Faculty of Exact and Natural Sciences, Department of Organic Chemistry, University of Buenos Aires, Buenos Aires, Argentina; Research Center in Carbohydrate Chemistry (CIHIDECAR), National Scientific and Technical Research Council (CONICET)-UBA, Buenos Aires, Argentina Enrique Rico-Garcı´a Biosystems Engineering Group, School of Engineering-Campus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico

Yong Kiat Teo School of Science, Monash University Malaysia, Bandar Sunway, Malaysia Himja Tiwari Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, —Pune Expressway, Bhatan Post—Somathne, Panvel, Mumbai, India Irineo torres-Pacheco Biosystems Engineering Group, School of Engineering-Campus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico Tabussam Tufail University Institute of Diet & Nutritional Sciences, Faculty of Allied Health Sciences, The University of Lahore, Lahore, Pakistan

List of contributors

Silvia Vasiliu “Petru Poni” Institute Macromolecular Chemistry, Iasi, Romania

of

Jayachandran Venkatesan Biomaterials Research Laboratory, Yenepoya Research Centre, Yenepoya (Deemed to be University), Deralakatte, Mangalore, India Eleni Vlassi Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Nilesh Shirish Wagh Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, —Pune Expressway, Bhatan Post— Somathne, Panvel, Mumbai, India

xvii

N.E. Wedamulla Department of Export Agriculture, Faculty of Animal Science and Export Agriculture, Uva Wellassa University, Badulla, Sri Lanka Deepti Yadav Department of Biotechnology and Food Science, Durban University of Technology, Durban, South Africa Zhongyu Yang School of Food Science and Technology, Jiangnan University, Wuxi, P.R. China

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Preface

The scope of this book, entitled Biological Macromolecules: Bioactivity and Biomedical Applications, is the coverage and review of recent trends and applications of biological macromolecules, such as carbohydrates, lipids, proteins, peptides, and nucleic acids in biomedicines, drug delivery, growth factors delivery, nutrients and nucleic acids delivery, cell encapsulation, enzyme mobilization, and tissue engineering. The mysteries of life lie in biological macromolecules. A large volume of biological macromolecules is obtained from different biological origins such as plants, algae, fungi, animals, and microbial sources. Biological macromolecules exhibit some significant and favorable advantages over synthetic macromolecules, such as sustainable and economic production, biocompatibility, biodegradability, and improved bioavailability. In recent years, a plethora of biological macromolecules (carbohydrates, lipids, proteins, peptides, and nucleic acids) has been used in the biomedical and healthcare fields. They showed varieties of bioactivities such as antioxidant, anticancer, antidiabetic, antimicrobial, immunomodulatory activities on the central nervous system, and gastrointestinal activity. The other biomedical applications include drug delivery, growth factors delivery, nutrients and nucleic acids delivery, cell encapsulation, enzyme mobilization, and tissue engineering. The structure
property relationship is also an important aspect for a thorough understanding of the bioactivity of biological macromolecules.

This book, containing 4 sections and 26 chapters, provides a systematic insight into the inclusive discussions on bioactivity and biomedical applications of different biological macromolecules. We are glad to see that many authors across the globe accepted our invitation and contributed valued chapters for this book, covering a wide spectrum of fields. A concise account of the contents of each chapter has been described to provide a glimpse of the book to the potential readers of various fields. The topics in the book (in order of preference) include the following: Biological Macromolecules: Sources, Properties, and functions (Chapter 1)—this chapter describes sources physicochemical properties, bioactivity and biomedical applications of different biological macromolecules concisely; Structure
Activity Relationship of Biological Macromolecules (Chapter 2)—this chapter aims to provide an overview of the structural features influencing the bioactivities of biological macromolecules, namely L-amino acid oxidases, lysostaphin, and metallo-β-lactamase-such as lactonase and chitosan; The Importance of Biological Macromolecules in Biomedicine (Chapter 3)—this chapter highlights the therapeutic aspects of macromolecules and the medicinal use of biological macromolecules against various diseases and ailments; Modification Techniques for Carbohydrate Macromolecules (Chapter 4)—this chapter characteristically abridges the significant developments of the last five to ten years and discusses critically in the area of modification of carbohydrates macromolecules;

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Preface

Biological Macromolecules as Nutraceuticals (Chapter 5)—this chapter aims to demonstrate some recent knowledge regarding the nutraceutical and biological activities of the macromolecules of biological origin, as well as some frontier applications of these in healthcare; Biological Macromolecules as Antioxidants (Chapter 6)—this chapter highlights the potential applications of biological macromolecules as antioxidants to scavenge reactive oxygen species and control oxidative stress, which leads to various pathogenesis; Biological Macromolecules as Antimicrobial Agents (Chapter 7)—the chapter describes the antimicrobial activity of biological macromolecules (chitosan, cellulose, alginate, gelatin, collagen, and keratin) and also, comprehensively elucidates their applications in addressing challenges associated with drug delivery, wound dressing, food packaging, and so on Biological Macromolecules From Algae and Their Antimicrobial Applications (Chapter 8)
this chapter provides an overview of bioactive macromolecules and their antimicrobial activities with particular reference to algal sources; Biological Macromolecules Acting on Central Nervous System (Chapter 9)—in this chapter, the role of biological macromolecules on central nervous system and their critical role in downregulation after the various neurological disorders have been discussed; Biological Macromolecules as Antidiabetic Agents (Chapter 10)—this chapter is an overview of different types of biological macromolecules and their applications as potential antidiabetic agents and also, highlights the advantages, limitations and future perspectives of biological macromolecules as antidiabetic agents; Biological Macromolecules as Anticancer Agents (Chapter 11)—this chapter presents the extraction of macromolecules such as carbohydrate, proteins, lipids, and nucleic acid (miRNAs) from different biological sources, such as plants, animal, algae and fungi. The various mechanisms by which the macromolecules exhibit their anticancer activity have been

discussed briefly along with several assays done to evaluate cytotoxicity of the macromolecules against various cancers such as lung cancer, breast cancer, cervical cancer, and colon cancer. Biological Macromolecules as Immunomodulators (Chapter 12)—this topic focuses on the potential modulations of immune response of biomacromolecules (three major classes of compounds: lipids, proteins and polysaccharides); Biological Macromolecules Acting on Gastrointestinal Systems (Chapter 13)—this chapter describes the role of biological macromolecules for the management of gastrointestinal system and related disorders; Synthetic Macromolecules With Biological Activity (Chapter 14)—this chapter describes some classes of synthetic macromolecules with biological activity that have a great importance on the human comfort and health, including antimicrobial polymers, antioxidant polymers, and polymeric sequestrants; Biological Macromolecules in Drug Delivery (Chapter 15)— this chapter focuses on the advancements in the uses of various biological macromolecules in drug delivery applications; Biological macromolecules in tissue engineering (Chapter 16)—this chapter provides an overview on the important role of natural-derived biomaterials (alginate, chitosan, carrageenan, fucoidan, ulvan, collagen, and gelatin) combining with ceramic biomaterials for bone tissue construction; Biological Macromolecules for Drug Delivery in Tissue Engineering (Chapter 17)—This chapter is focused on the preparation and physicochemical characterization of engineered biomaterials, based on biological macromolecules (polysaccharides and proteins), as scaffolds which are capable of supporting physiological activities of cells, but also can act as drug delivery systems for tissue engineering and wound healing; Biological Macromolecules for Growth Factor Delivery (Chapter 18)—this chapter discusses the fabrication of synthetic and natural macromolecules, sometimes combined with other mineral or metallic compounds for growth factor delivery; Biological

Preface

Macromolecules for Growth Factor Delivery in Bone Regeneration (Chapter 19)—this chapter describes the process of bone tissue regeneration in healing injuries and arthritic conditions, introduces the main ideas through the scope of allogenous and autogenous transplantation and demonstrates the role of growth factors in these processes; Biological Macromolecules for Nutrients Delivery (Chapter 20)—This chapter focuses on the types of nutrients that need to be delivered, the biological macromolecules that can be used to construct edible delivery systems, the most common delivery systems currently used for this purpose, and some of the major challenges that must be addressed in the future; Biological Macromolecules for Nucleic Acid Delivery (Chapter 21)—this chapter describes the nonviral nucleic acid delivery systems made up of biological macromolecules, such as peptides, lipids, and carbohydrates and also gives an introduction on the history and structure of nucleic acids; Biological Macromolecules in Cell Encapsulation (Chapter 22)—this chapter aims to review the most examined, most promising and recently proposed biopolymers that are used in tissue engineering scaffolds, and to highlight their main properties, drawbacks, fields of applications and fabrication technologies in order to provide readers with important guidelines for selecting appropriate scaffold biomaterials; Biological Macromolecules for Enzyme Immobilization (Chapter 23)—this chapter provides a broad overview of properties and the applications of various naturally occurring biopolymers, that is, chitosan, chitin, agarose, alginates, cellulose, gelatin, dextran, carrageenan, pectin and xanthan gum for their applications in enzyme immobilization with recent literature studies indicating biopolymer-based support material development and their utilization to make biocatalysts with desired stability and catalytic functionalities; Carbohydrates for Enzyme Inhibition and Their Use as Target Molecules for the Interference of Diseases (Chapter 24)—this chapter describes the study of a widespread group of enzymes and the

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inhibition of these enzymes constitutes an interesting and novel strategy to approach new therapies against numerous diseases; Current Challenging Issues of Biological Macromolecules in Biomedicine (Chapter 25)—this chapter provides information on recent innovations in various biomaterials, engineered from macromolecules ranging from drug delivery, cancer therapies, tissue engineering, bioprinting and wound healing; Future Perspectives of Biological Macromolecules in Biomedicine (Chapter 26)— this chapter discusses the impact of the combination of nanotechnology and chronobiology in personalized cancer treatment. We sincerely acknowledge the valuable contribution of the distinguished authors and convey our sincere thanks. This book could not have been published without the cooperation of Barbara Makinster, Editorial Project Manager. We wish to express our cordial gratitude to Elsevier Inc., Michelle Fisher (Acquisition Editor), and other editorial staff for their invaluable supports in organizing the intelligent editing of the book. We also gratefully acknowledge all the permissions we received for reproducing the copyright materials from different sources. Finally, we cannot overlook the sacrifices and supports from our family members during the preparation of the current book. All our friends, colleagues, and students who have helped in the process of editing this book deserve our great appreciation. Contributing authors, the publishers, and we, the editors, will be extremely happy if our endeavor fulfills the needs of the academicians, researchers, students, pharmaceutical experts, biomedicine experts, and formulators. Amit Kumar Nayak1, Amal Kumar Dhara2 and Dilipkumar Pal3 1 Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, India 2 Department of Pharmacy, Contai Polytechnic, Govt. of West Bengal, Contai, India 3Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, India

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P A R T I

Background

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C H A P T E R

1 Biological macromolecules: sources, properties, and functions Amal Kumar Dhara1 and Amit Kumar Nayak2 1

Department of Pharmacy, Contai Polytechnic, Contai, India 2Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, India

1.1 Introduction

(RNA) are responsible for carrying genetic blueprint and information for protein synthesis (Minchin & Lodge, 2019; Schwartz, Schwartz, Mieszerski, McNally, & Kobilinsky, 1991). Biological macromolecules are abundantly available in nature and also possess properties like biocompatibility, environmental friendly, biodegradability, etc., because of their natural sources (Chandika et al., 2020; Teramoto, 2020). Various species of algae have been mentioned to be used as bioactive compounds and are also employed as antibacterial agents (Shannon & Abu-Ghannam, 2016). Various disorders related to central nervous system, such as Alzheimer’s disease, Parkinson’s disease, convulsive disorders, etc., are being treated with the biological macromolecules (Acosta & Cramer, 2020; Soderquist & Mahoney, 2010; Zhang et al., 2018). All the biological macromolecules, viz., carbohydrates, lipids, proteins and nucleic acids, have shown their significant role in the management of cancer therapy and have been advocated to be used against various cancers like lung cancer, colon cancer, breast cancer, cervical cancer, etc.

The mystery of life is in biological macromolecules. There are four important classes of biological macromolecules, viz., carbohydrates, lipids, proteins, and nucleic acids (Luo, Zhang, Wu, Liang, & Li, 2020; Zhang, Sun, & Jiang, 2018). Carbohydrates, proteins, and nucleic acids naturally exist as long chain polymers, while lipids are smaller and in true sense, these are all considered as biopolymers (Albertsson, 2019; Teramoto, 2020; Zhang et al., 2018). Carbohydrates are the storage form of energy and meet the demand as and when required (Slavin & Carlson, 2014). Lipids are also storage form of energy and are the important structural components of the cell membrane (van Meer, Voelker, & Feigenson, 2008; Zheng, Fleith, Giuffrida, O’Neill, & Schneider, 2019). Proteins serve several functions including structural support, catalyzing important metabolic reactions, signals receiving and transmission, etc. (Watford & Wu, 2018; Zaretsky & Wreschner, 2008). Nucleic acids, that is, deoxyribonucleic acid (DNA) and ribonucleic acid

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00005-1

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© 2022 Elsevier Inc. All rights reserved.

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1. Biological macromolecules: sources, properties, and functions

(Corn, Windham, & Rafat, 2020; Oana, Adriana, Mircea, Dragos, & Monica, 2018; Rodrigues Mantuano, Natoli, Zippelius, & La¨ubli, 2020; Sun, Jing, Ma, Feng, & Hu, 2020). Another important area of research is immunomodulators with respect to present SARS-CoV-2 perspective, where the biological macromolecules, mainly proteins, play significant role (Ji et al., 2020). Proteins are associated with the development process of immune system (Daly, Reynolds, Sigal, Shou, & Liberman, 1990). Lipids are also responsible to play key role as adjuvants for the development of vaccines (Martinez-Gil, Goff, & Tan, 2018; Schwendener, 2014). Day by day, the incidence of lifestyle diseases more specifically diabetes, hypertension, etc., are increasing vertically, where the roles of biological macromolecules have been studied and were found to be utilized widely as antidiabetic agents (Alam, Shafique, Amjad, & Bin Asad, 2019; Hu, Nie, & Xie, 2018; Rı´os, Francini, & Schinella, 2015; Yu, Shen, Song, & Xie, 2018). They cause increase in insulin secretion and thus, reduce the blood glucose level (Rı´os et al., 2015). Chitosan is a well-known polysaccharide, which is reported to exhibit antimicrobial and antidiabetic activities (Hasnain & Nayak, 2018; Karadeniz & Kim, 2014; Rabea, Badawy, Stevens, Smagghe, & Steurbaut, 2003). Extensive research is going on in the area of tissue engineering for the development of artificial tissue to repair and replace defective or diseased tissue or organs (Hasnain, Ahmad, Chaudhary, Hoda, & Nayak, 2019; Nayak, Ahmed, Tabish, & Hasnain, 2019; Pal, Saha, Nayak, & Hasnain, 2019). Naturally derived biological macromolecules like chitosan, alginate, carrageenan, ulvan, gelatin, etc., have been used for bone tissue regeneration (Hasnain, Nayak, Singh, & Ahmad, 2010; Maity, Hasnain, Nayak, & Aminabavi, 2021; Nayak, Ahmed, Tabish, & Hasnain, 2019). Numbers of polysaccharide have long been used in different types of drug delivery systems as biopolymeric excipients

(Hasnain et al., 2020; Kandar, Hasnain, & Nayak, 2021; Maity et al., 2021; Nayak & Hasnain, 2019a, 2019b; Nayak, Hasnain, Dhara, & Pal, 2021; Pal, Saha, Nayak et al., 2019). Polysaccharide and proteins are used extensively for the preparation of hydrogels for drug delivery, tissue regeneration, wound dressings, etc. (Del Valle, Dı´az, & Puiggalı´, 2017; Nayak & Pal, 2016b; Nayak, Hasnain, Pal, Banerjee, & Pal, 2020; Pal, Nayak, & Saha, 2019a, 2019b; Ray et al., 2020). Beside drug delivery, the biological macromolecules have continuously been used to formulate delivery carrier-systems for growth factors (Rao, Rekha, Anil, Lowe, & Venkatesan, 2019; Shariatinia, 2019). Polysaccharides are also employed for the encapsulation of various bioactive substances like vitamins and nutraceuticals (Bala, Singha, & Patra, 2019; Lauro, Amato, Sansone, Carbone, & Puglisi, 2019). The current chapter deals with a brief discussion about the sources, properties, and valuable applications of various biological macromolecules like carbohydrates, lipids, proteins and nucleic acids.

1.2 Carbohydrates The most widely found organic compounds in nature are carbohydrates. These are wellknown as very essential source of life or sustaining life itself (Slavin & Carlson, 2014). Carbohydrates are commonly found in plants, microorganisms and animal tissues (Werz & Seeberger, 2005). These are also present in blood, tissue fluids, etc. (Kilcoyne & Joshi, 2007). Carbon, hydrogen and oxygen are the three primary elements of molecular structure of carbohydrates (Luo et al., 2020; Werz & Seeberger, 2005). These are optically active polyhydroxy aldehydes or ketones. There are three major classes of carbohydrates, broadly, monosaccharides, oligosaccharides and polysaccharides (Slavin & Carlson, 2014).

I. Background

1.2 Carbohydrates

5

1.2.1 Monosaccharides

1.2.2 Oligosaccharides

These are generally called simple sugars, and the most common monosaccharide is glucose. Most of the monosaccharides comprise of the general formula CnH2nOn (Pigman & Horton, 1972). The different classes of monosaccharides include aldoses (functional group is aldehyde), and ketoses (functional group is keto) (Slavin & Carlson, 2014; Werz & Seeberger, 2005). On the basis of number of carbon in the sugar, they are also subcategorized into (Shin & Kim, 2013; Slavin & Carlson, 2014):

These yield 2
10 monosachharides on hydrolysis. On the basis of number of monosaccharide units present, these are further subclassified into (Shin & Kim, 2013; Slavin & Carlson, 2014): disaccharides (e.g., sucrose, maltose, lactose and trehalose), trisaccharides (e.g., raffinose and maltotriose), etc. These can exhibit reducing property, when these contain free aldehyde and/or ketone group, which is/ are not participated in the formation of linkage.

1. Trioses (containing three carbon atoms in the sugar), for example, glyceraldehydes and dihydroacetone, 2. Tetroses (containing four carbon atoms in the sugar), for example, erythrose and erythrulose, 3. Pentoses (containing five carbon atoms in the sugar), for example, ribose and xylulose, 4. Hexoses (containing six carbon atoms in the sugar), for example, glucose, glactose, fructose and mannose, and 5. Heptoses (containing seven carbon atoms in the sugar), for example, sedoheptulose. Physicochemical properties of monosaccharides: These are soluble in water, sweet in taste and permeable through plasma membrane. Monosaccharides react with hydrazine to form osazones (Pigman & Horton, 1972). They undergo reduction and form sugar alcohols (e.g., glucose-sorbitol; fructose-mannitol; galactose-dulcitol; glyceraldehyde-glycerol, etc.). On oxidation, these produce sugar acids like gluconic acid. Monosaccharides play varieties of important physiological functions (Pigman & Horton, 1972). These are used for energy production in living organisms. The vital components of cells are RNA and DNA, which are composed of ribose and deoxyribose and are well-recognized as the building blocks of life (Minchin & Lodge, 2019).

1.2.3 Polysaccharides These are formed by uniting monosaccharide or there derivatives. These are joined together by glycosidic linkage (Maity et al., 2021). Unlike proteins and nucleic acids, polysaccharides exist as both linear as well as branched polymers. These are colloidal in nature. Polysaccharides are grouped into two categories (Shin & Kim, 2013; Slavin & Carlson, 2014): 1. Homopolysaccharides: These yield one type of monosaccharides on hydrolysis, for example, starch, cellulose, glycogen, etc. 2. Heteropolysaccharides: These yield two or more different type of monosaccharides on hydrolysis, for example, hyaluronic acid, heparin, chondriotin sulfate, etc. Some important homopolysaccharides are described here: 1. Starches—These contain several units of glucose joined in α-1, 4-linkages and are well-known as examples of homopolysaccharides (Nayak & Pal, 2017). In plants, it is the storage form of carbohydrates. Different parts of the plants are rich in starches like tubers, roots, vegetables, cereals, etc. (Nayak & Pal, 2017; Nayak, Bera, & Hasnain, 2020). In higher

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1. Biological macromolecules: sources, properties, and functions

animals, starches are the most important source of food. These consist of two types of molecules (Fig. 1.1): (A) linear and water soluble component, e.g., amylose and (B) branched water insoluble, e.g., amylopectin. Commercially, starch is produced mostly from corn, but wheat starch, potato starch, and tapioca starch are also used (Nayak et al., 2020). 2. Cellulose—It is the chief constituent of fibrous parts of the plants and consequently, is the most abundant organic material occurring in nature (Pal et al., 2019b). It is made up of long chains of β D-glucose molecules linked by 1, 4-linkages (Fig. 1.2). It serves as bulk forming agent of the food. Undigested cellulose increases the bulk of feces and helps in the evacuation of bowels. Cellulose exhibits some of important

properties like low density, flexibility, high strength, biocompatibility, biodegradability, etc., which suggest for biomedical applications (Hasnain et al., 2020; Kandar et al., 2021). In traditional healthcare, cellulosic biomaterials play an important role and recently, some significant areas of application are being explored with the uses of cellulose like drug delivery (Hasnain et al., 2020; Kandar et al., 2021; Pal et al., 2019b), tissue engineering (Hasnain, Nayak, Singh, & Ahmad, 2010; Murizan, Mustafa, Ngadiman, Mohd Yusof, & Idris, 2020), management of wound (Alven & Aderibigbe, 2020), etc. By using quaternary ammonium salt the surface of cellulose nanocrystals (CNC) is modified, which can inhibit the growth of Staphylococcus aureus and Escherichia coli (Tavakolian, Jafari, & van de Ven, 2020).

FIGURE 1.1 Molecular structure of starch: (A) linear and water soluble component: amylose and (B) branched water insoluble: amylopectin.

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1.2 Carbohydrates

FIGURE

1.2 Molecular

structure of cellulose.

FIGURE 1.3

Molecular structure of dextran.

3. Dextran—It is a highly branched polymer of glucose, produced by yeast or bacteria (Chen, Huang, & Huang, 2020; Huang & Huang, 2018). The linear chain dextran molecular structure are formed by 1, 6-α glycosidic linkages (Fig. 1.3). Dextran occurs in honey, maple syrup, etc. It is used as plasma substitutes as it retains water in circulation for longer period, when administered intravenously (i.v.). Dextran and its derivatives are being used to formulate nanocarriers for advanced drug delivery applications (Huang & Huang, 2018).

4. Pectins—These form gel with sugar solutions. In nature, pectins are usually found in pulps and peels of citrus fruits, apple pomaces, beet roots, etc. (Nayak & Pal, 2016a). Chemically pectins are polysaccharide of galacturonic acid, galactose, and arabinose (Hasnain et al., 2020; Kandar et al., 2021). Molecular structure of pectin is presented in Fig. 1.4. Pectins have been found to possess beneficial biological activities, like antioxidant, antiinflammatory, antibacterial, immune regulation, and anticoagulation ´ activities (Rascon-Chu, Gomez-Rodriguez, Carvajal-Millan, & Campa-Mada, 2019). Biomedical applications of pectins include tissue engineering, drug delivery, wound healing and gene delivery (Hasnain et al., 2020; Kandar et al., 2021; Maity et al., 2021; ´ Nayak et al., 2019, 2021; Rascon-Chu et al., 2019). 5. Gum acacia or Gum Arabic—It is a plantderived gum containing hexoses or pentoses or both. It is extensively used in pharmaceutical, food and cosmetic industries (Nayak, Das, & Maji, 2012). It is an effective and useful excipient for the preparation of nanomaterials for drug delivery (De, Nayak, Kundu, Das, & Samanta, 2021). 6. Alginates—These consist of linear polymer of β (1-4)-linked D-mannuronic acid (M-unit) and α (1-4)-linked L-guluronic

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1. Biological macromolecules: sources, properties, and functions

FIGURE 1.4

Molecular structure of pectin.

FIGURE 1.5 Molecular structure of alginate.

acid (G-unit) (Hasnain et al., 2020; Kandar et al., 2021; Nayak et al., 2021). Molecular structure of pectin is presented in Fig. 1.5. It is processed from marine algae and giant kelp as raw materials. These are widely used as thickener, emulsifier, stabilizer, etc. (Kandar et al., 2021). Alginate based drugs are effectively used for antimicrobial as well as antiviral therapy (Szekalska, Pucilowska, ´ Szymanska, Ciosek, & Winnicka, 2016). The alginate film with EDTA exhibited stronger antimicrobial effects against Gram-negative bacteria, especially, in case of processed food packaging (Senturk Parreidt, Mu¨ller, & Schmid, 2018). Structural modifications of alginates can easily be made by using crosslinkers, improvise the mechanical strength and cell affinity and was widely

used in the biomedical applications mainly in drug delivery and tissue regeneration (Malakar, Nayak, Jana, & Pal, 2013; Malakar, Nayak, & Das, 2013). 7. Chitosan—It is a cationic natured carbohydrate polysaccharide, extracted by deacetylation of chitin (Hasnain & Nayak, 2018). It is reported to show various biological properties including antidiabetic, antioxidant, immune-enhancing, antimicrobial as well as anticancer activities (Hasnain & Nayak, 2018; Rabea et al., 2003; Rı´os et al., 2015). Chitosan is very much effective in the formulation of insulin with controlled delivery functionality at the target site (Barbosa et al., 2020). Carboxymethylhexanoyl derivative and polyethylene glycoltrimethyl complexes of chitosan have been

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1.3 Lipids

found to possess fat-lowering and fatpreventing properties. Chitosan-based collagen complex sponges showed effectiveness in the healing of diabetic wounds (Wang et al., 2008). Chitosan and its derivatives are being extremely used in many biomedical applications, such as drug delivery, tissue engineering, wound dressing, orthopedics, etc. (Hasnain et al., 2020; Hasnain, Ahmad, Chaudhary, Hoda, & Nayak, 2019; Hasnain, Nayak, Singh, & Ahmad, 2010; Kandar, Hasnain, & Nayak, 2021; Maity, Hasnain, Nayak, & Aminabavi, 2021; Nayak, Ahmed, Tabish, & Hasnain, 2019; Pal, Saha, Nayak, & Hasnain, 2019). 8. Agar—It is a natural polysaccharide obtained from seaweeds. It is a sulfuric acid ester of a complex galactose polysaccharide (Kandar et al., 2021). It is nondigestible material and is used as a bulk laxative. Recent years, a number of biocompatible agar-based composite has been formulated for their potential applications in biomedical fields including drug delivery and tissue engineering applications (Kandar et al., 2021; Nayak, Alkahtani, & Hasnain, 2021; Shah et al., 2019).

9. Glycogen—This is also known as animal starch, as it is a principal polysaccharide occurring in animal tissues, specifically in liver and muscle (Roach, Depaoli-Roach, Hurley, & Tagliabracci, 2012). Glycogen is the storage form of energy release, quickly, when needed (Kreitzman, Coxon, & Szaz, 1992). Similar to starch, this is also composed of glucose units united by 1, 4linkages and branches arising by 1, 6linkages. Sources and functions of various polysaccharides are listed in Table 1.1.

1.3 Lipids Lipids are heterogeneous group of organic compounds, related either actually or potentially, to the fatty acids (Pandey & Kohli, 2018). They are poorly soluble in water and soluble in nonpolar solvents like chloroform, benzene, petroleum ether, etc. In our body, lipids are an integral part of the cell membrane structure, metabolic fuel and storage form of energy (van Meer et al., 2008; Zheng et al., 2019). Lipids act

TABLE 1.1 Sources and functions of various polysaccharides. Polysaccharides

Source type

Functions

Cellulose

Plants

Cell structure and food additives

Starches

Plants

Storage and drug adjuvants

Pectins

Plants

Food additives

Carrageenan

Microorganism

Food additives

Alginate

Microorganism

Drug adjuvant

Hyaluronan

Animals

Animal tissue structure, therapeutic agents

Heparin

Animals

Animal tissue structure, therapeutic agents

Chondroitin sulfate

Animals

Animal tissue structure

Chitin and chitosan

Animals

Tissue scaffolds

I. Background

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1. Biological macromolecules: sources, properties, and functions

as mechanical, thermal and electrical insulators (Pandey & Kohli, 2018). Lipids are usually classified as follows:

1.3.1 Simple lipids These are esters of fatty acids with certain alcohols, generally glycerol. According to nature of alcohols, these are (Vance & Vance, 2002): 1. Fats and oils: Ester of fatty acids with glycerol. These are also known as natural fat or triglyceride or triacylglycerol. At room temperature when these are solid known as fat and when liquid these are known as oil. 2. Waxes: These are esters of fatty acid with higher molecular weight monohydric alcohol.

1.3.2 Compound or conjugate lipids These are ester of fatty acids containing groups, in addition to an alcohol and the fatty acids. These are further classified as (Vance & Vance, 2002): 1. Phospholipids: These contain fatty acids, glycerol, a phosphoric acid residue and sometimes a nitrogenous base. They are subdivided as: a. Phosphotidic acids—on hydrolysis, these produce one molecule each of glycerol and phosphoric acid with two molecules of fatty acids. b. Lecithins—contain glycerol, fatty acid, phosphoric acid and the nitrogen base choline. These are widely distributed in different animal tissues including brain, liver, blood, cardiac muscles, etc., and also found in plant seeds. Lecithins are used as emulsifying and smoothing agents in food industry (Robert, Coue¨delo, Vaysse, & Michalski, 2020). Lecithins have been mentioned to be used for the

development of nanocarriers for drug delivery (Das, Sen, Maji, Nayak, & Sen, 2017; Malakar, Sen, Nayak, & Sen, 2012). c. Cephalins—similar in structure with lecithin but the base is ethanolamine. These are found in brain, liver, cardiac muscles, erythrocytes, etc. d. Plasmalogens—found in brain, cardiac muscles, erythrocytes, etc. e. Sphingomyelins—on hydrolysis, these give a single fatty acid, a nitrogen base sphingosine, phosphoric acid and choline but no glycerol. These participate in different signaling pathways. 2. Glycolipids: These contain sphingol, a carbohydrate (galactose), and fatty acid. Large amount of glycolipids are present in the white matter of brain and in the myelin sheath of nerves (Willison, 2018). 3. Sulpholipids: Lipid material containing sulfur has long been known to be present in the different tissues like liver, kidneys, brain, etc. It is found in the white matter of the brain. 4. Lipoproteins: These are composed of lipid material bound to the protein. The lipid of lipoproteins mainly consists of cholesterol esters and phospholipids (such as stearic, palmitic, and oleic acids) (Schumaker & Adams, 1969). These are found in plasma and the four important lipoproteins are chylomicrons, preβ-lipoprotein, β-lipoprotein and α-lipoprotein. Hyperlipoproteinemia is clinically significant, nowadays, as lipoproteins are directly related with atherosclerotic cardiovascular disease (Arnao, Tuttolomondo, Daidone, & Pinto, 2019).

1.3.3 Derived lipids These are substances derived by hydrolysis of simple or compound lipids.

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1.4 Proteins

FIGURE 1.6 (A) Cyclopentano perhydro phenantherene ring system in steroids, and (B) molecular structure of cholesterol.

1. Steroids: These are abundantly found in nature. Steroids are derivative of complex ring system named as cyclopentano perhydro phenantherene (Fig. 1.6A). The important classes of steroids include sterols, bile acids, sex hormones, adrenal cortical hormones, Vitamin D, saponins and cardiac glycosides (Cole, Short, & Hooper, 2019). 2. Sterols: Cholesterol is a well-known sterol (Fig. 1.6B). It is a white and waxy substance, widely distributed in all cells of the body, particularly in nervous tissue. Cholesterol is the precursor of bile salts, adrenocorticoids, sex hormones, Vitamin D and cardiac glycosides (Schade, Shey, & Eaton, 2020). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) play important roles with respect to immune functions, neurological and cardiovascular disorders (Ochi & Tsuchiya, 2018). Basic functions of different lipids are listed in Table 1.2. Recent studies have showed the efficacy of various lipid-based drug delivery

carrier systems, such as liposomes, transferosomes, solid lipid nanoparticles, lipid nanostructures, etc.

1.4 Proteins Proteins constitute a diverse, heterogeneous class of macromolecules and these may be said as the essence of life processes (Zaretsky & Wreschner, 2008; Zhang et al., 2018). These are high molecular weight extremely complex polymers of amino acids (Watford & Wu, 2018). In addition to carbon, hydrogen, oxygen atoms, the molecular structure of proteins contains nitrogen and sometimes sulfur, phosphorus, iron, copper, manganese, iodine, zinc, and other elements. The amino acids of proteins are joined together with the help of peptide bonds (
CO
NH
). The common structure of protein is presented in Fig. 1.7. These exhibit varieties of functions in cells by acting as structural materials, carrier molecules, enzymes,

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1. Biological macromolecules: sources, properties, and functions

TABLE 1.2 Basic functions of lipids. Type of lipids

Functions

Fatty acids

Precursor of triglyceride and source of energy

Triglyceride

Storage of energy, thermal insulation and protection, binding of organs together

Cholesterol

Component of cell membranes and also the Precursor of different steroids

Phospholipids

Structural component of cell membranes and helps in digestion of fat

Bile acids

Helps in fat digestion and absorption of nutrients

Eicosanoids

Chemical messenger between cells

Dietary fat

Carry lipid soluble Vitamins (A, D, E and K)

FIGURE 1.7 Common structure of protein.

lubricants, etc. (Zaretsky & Wreschner, 2008). Proteins derived from different sources (animals and plants) have been used for the isolation of peptides, and exhibited different biological activities for humans (Daly et al., 1990; Nayak, 2010). Proteins are classified in three groups, namely simple proteins, conjugated proteins and derived proteins (Watford & Wu, 2018).

1.4.1 Simple proteins Upon hydrolysis, these types of proteins yield only amino acids or their derivatives (Murray, Harper, Granner, Mayes, & Rodwell, 2006; Watford & Wu, 2018). Simple proteins

are further classified into albumins, globulins, glutelins, prolamins, albuminoids (scleroproteins), histones and protamines. 1. Albumin: These are soluble in water, coagulated by heat, and precipitated by saturated salt solution. Examples are lactalbumin, serum albumin, egg albumin, myogen of muscle, etc. 2. Globulin: These are soluble in dilute solution of strong acids and bases and get coagulated by heat. Examples are serum globulin, ovoglobulin, myosin of muscle, etc. 3. Glutelin: These are soluble in dilute acids and alkalis and get coagulated by heat. Examples are glutenin from wheat and oryzenin from rice, etc.

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1.4 Proteins

4. Prolamines: These are soluble in 70%
80% alcohol and insoluble in water, absolute alcohol and other neutral solvents. Examples are zein (corn), hordein (barley), gliadin (wheat), etc. 5. Albuminoids (Screlroproteins): Albuminoids are insoluble proteins and form supportive tissues. These are animal proteins found in hair, nails, horns and hooves. Examples are keratin, collagen, gelatin, etc. 6. Histones: These are soluble in water, very dilute acids and salt solutions. These are not conjugated by heat and contain basic amino acids. Examples include nucleic acids. 7. Protamines: These are the simplest of proteins and are basic polypeptides, soluble in water and ammonium hydroxides. These are not conjugated by heat. Examples include salmine (salmon sperm), clupeine (herring sperm), etc.

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3. Chromoproteins: These are composed of simple proteins with chromotropic group as prosthetic group. Examples include hemoglobin (prosthetic group is heme), flavoproteins (prosthetic group is riboflavin), cytochrome (prosthetic group is heme), etc. 4. Phosphoproteins: These are composed of proteins and phosphoric acid as the prosthetic group. Caesin (milk protein) and vitelline (egg yolk protein) are the important examples of this group. 5. Lipoproteins: These proteins are the combination of proteins and lipids (fatty acid, lecithin, cephalin, etc.) as prothetic group. Lipoproteins occur in blood, cell nuclei, milk, cell membranes, egg yolk, etc. 6. Metalloproteins: These are compounds of proteins and some metals (such as iron, cobalt, zinc, manganese, copper and magnesium). Examples are ferritin, ceruloplastin, carbonic anhydrase, etc.

1.4.2 Conjugated proteins These contain simple protein combined with nonprotein prosthetic group (Murray et al., 2006). These include: (1) nucleoproteins (2) proteoglycans and glycoproteins (3) chromoproteins (4) phosphoproteins (5) lipoproteins and (6) metalloproteins (Murray et al., 2006; Watford & Wu, 2018). 1. Nucleoproteins: These proteins are composed of simple proteins with nucleic acids as prosthetic group. In nucleoproteins, protein moiety is usually a basic protein like protamine and histone. Examples include chromosomal proteins and some glandular proteins. 2. Proteoglycans and glycoproteins: These proteins contain carbohydrates as prosthetic group like hyaluronic acid and chondroitin sulpahte. These are mainly found in blood plasma, gastric and salivary mucine, immunoglobulins, human chorionic gonadotropins, etc.

1.4.3 Derived proteins These are formed from simple and conjugate proteins by denaturation or partial hydrolysis (Murray et al., 2006). They are of two types (1) denatured or primary derived proteins and (2) secondary derived proteins (Murray et al., 2006; Watford & Wu, 2018). 1. Denatured or primary derived proteins: These proteins may be of different types. a. Proteans—derived in the early stages of protein hydrolysis by water, dilute acids or alkalis or enzymes. Examples include fibrin from fibrinogen, myosan from myosin and edestan from edestin. b. Metaproteins—derived by further hydrolysis by stronger acids or alkalies, which are insoluble in very dilute acids and alkalis. Examples of such proteins include acid metaproteins and alkali metaproteins.

I. Background

14

1. Biological macromolecules: sources, properties, and functions

c. Coagulated proteins—derived by the action of heat, ultraviolet (UV)-rays, Xrays, very high pressure, mechanical shaking, etc. Examples are coagulated albumin, cooked meat, etc. 2. Secondary derived proteins: Progressive hydrolysis of peptide bond caused breakdown of proteins into smaller molecules, which include (Murray et al., 2006): a. Proteoses—formed by the action of pepsin and trypsin, b. Peptones—produced by further hydrolytic decomposition and c. Peptides—composed of two or more amino acids (such as dipeptides, and polypeptides). Plethora of proteins and peptides with varieties of biological activities has already been identified (Nayak, 2010). Some of the proteins and peptides exhibit bioactivities like antioxidant, antihypertensive, antibiotics, immunomodulatory, anticancer activities, etc. (Giromini, Cheli, Rebucci, & Baldi, 2019).

Bioactive proteins obtained from legumes have been found beneficial in the prevention of obesity and type-II diabetes (Moreno-Valdespino, Luna-Vital, Camacho-Ruiz, & Mojica, 2020). Anticancer activities are also demonstrated by some important biological proteins like lectins, glycoproteins, etc. (Laaf, Bojarova´, Pelantova´, Kˇren, & Elling, 2017; Rodrigues Mantuano et al., 2020; Singh, Kaur, & Kanwar, 2016). Gelatin obtained from animal collagen, either acid or alkali treated, is used in bone tissue engineering as it is biocompatible, low immunogenic and biodegradable (Hasnain et al., 2019). Pharmacological effects of bioactive proteins/peptides along with their sources are listed in Table 1.3.

1.5 Nucleic acids Nucleic acids are macromolecules, in remain linked to each bonds in-between the

nonprotein nitrogenous which the nucleotides other by phosphodiester 30 and 50 position of the

TABLE 1.3 Pharmacological effects of bioactive proteins/peptides along with their sources. Bioactive proteins/ peptides Sources

Pharmacological effects

Fibrinolytic enzymes

Eisenia fetida (earthworm)

Antitumor activity against several hepatoma cell lines, in vitro and in vivo (Chen et al., 2007)

Hirudin

Hirudo medicinalis

Anticoagulation activity through inhibition of thrombin activity (Markwardt, 2002)

Cordymin

Cordyceps militaris

Antifungal activity (Wong et al., 2011), anticancer and inhibitory effect on HIV-1 reverse transcriptase (Wong et al., 2019), antiinflammatory and antinociceptive activities (Qian, Pan, & Guo, 2012)

Lectin

Cordyceps militaris

Hemagglutinating activity and mitogenic activity (Wong, Wang, & Ng, 2009)

Ginkbilobin

Ginkgo biloba seeds

Antifungal activity (Wang & Ng, 2000)

Dioscorin

Dioscorea batatas

Trypsin inhibitory activities (Hou et al., 1999)

Trichosanthin

Trichosanthes kirilowii

Anti-HIV activity (Zhao, Ben, & Wu, 1999) and antiviral activity against hepatitis B virus (Wen et al., 2015)

I. Background

1.5 Nucleic acids

sugars (Minchin & Lodge, 2019; Nelson & Cox, 2005). A nucleotide is composed of a pentose, a phosphate and a nitrogen base. The nitrogen base may be a purine or a pyrimidine. In case of RNA and DNA, the pentose is ribose and deoxyribose, respectively (Brosius & Raabe, 2016; Schwartz et al., 1991). Adenine and guanine are the major purine bases (others are methyladenine, methylguanine, hypoxanthine, etc.) whereas cytosine, uracil and thymine are the major pyrimidine bases (others include 5methylcytosine, 5, 6-dihydrouracil, etc.) of nucleic acids (Brosius & Raabe, 2016; Schwartz et al., 1991). Various functions of nucleic acids are (Minchin & Lodge, 2019; Nelson & Cox, 2005): 1. Nucleic acids direct the metabolism process of the cell throughout the life 2. Synthesis of protein is directed by nucleic acids 3. They regulate the synthesis of enzymes 4. They play important role in the transfer of genetic information from one offspring to another 5. Nucleic acids contribute essential substances of the genes and the apparatus by which the genes act 6. Nucleic acids are intimately involved with the varieties of disease like cancers, etc. and are major areas for research

1.5.1 Nucleotides When the phosphate diester bond gets hydrolyzed, the monomeric nucleic acids are separated which consist of nitrogenous base, a sugar and a phosphate, and that unit is called nucleotide (Nelson & Cox, 2005; Zaharevitz et al., 1992). According to the presence of ribose or deoxyribose, these may be ribonucleotides or deoxyribonucleotides, respectively. Nucleotides carrying more than one phosphate group are called higher nucleotides, for example, adenosine triphosphate (ATP), adenosine

15

diphosphate (ADP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), cytidine triphosphate (CTP), cytidine diphosphate (CDP), uridine triphosphate (UTP), etc. (Murray et al., 2006).

1.5.2 Nucleosides It is a structural subunit of nucleic acids. In living cell, nucleoside is the heredity controlling component (Murray et al., 2006; Nelson & Cox, 2005). When the ester bond between the sugar and the phosphate group in a nucleotide is hydrolyzed, a fragment consists of nitrogenous base and a sugar moiety is obtained which is called as nucleoside. Sugar moiety in nucleoside is either ribose or deoxyribose, whereas nitrogenous bases consist of either a pyridine, that is, cytosine, thymine, or uracil or a purine, that is, adenine or guanine (Nelson & Cox, 2005).

1.5.3 DNA DNA is a biological macromolecule where genetic information of cell is confined, which is known as genome of the cell (Nelson & Cox, 2005; Watson & Crick, 1953). It is a polymer of deoxyribonucleotides. It occurs in chromosomes, mitochondria and chloroplasts. The chemical nature of monomeric units of DNA is deoxyadenylate, deoxyguanylate, deoxycytidylate and thymidylate (Watson & Crick, 1953). The monomeric units are held in polymeric form by 30 , 50 -phophodiester bridges constituting a single strand (Murray et al., 2006). The genetic information resides in the sequence of the monomeric unit. The polymer possesses a polarity, that is, one end has a 50 -hydroxyl or phosphate terminus while other has a 30 -phosphate or hydroxyl moiety. In DNA, the concentration of adenosine nucleotide equals to thymidine and the concentration of guanosine nucleotide equals to cytosine nucleotide

I. Background

16

1. Biological macromolecules: sources, properties, and functions

(Nelson & Cox, 2005; Watson & Crick, 1953). The secondary structure of DNA consists of a double stranded helix (Watson & Crick, 1953). The two strands of right handed DNA molecules are held by hydrogen bonds. Each strand is again compactly held by hydrophobic forces between the rings of its consecutive bases. The pairing between the purine and pyrimidine nucleotides on opposite strands is (Chang, 2017) specific and dependent upon hydrogen bonding of adenine (A) residue with thymine (T) residue and guanine (G) with cytosine (C) residue (Malhotra & Ali, 2018; Watson & Crick, 1953). Schematic representation of the structure of DNA with its nitrogenous bases is presented in Fig. 1.8 (right-hand side). Important biological roles of DNA are (Nelson & Cox, 2005; Pisetsky, 2017; Watson & Crick, 1953): 1. The function of DNA is to act as a storage house of genetic information and to control the synthesis of protein in the cell.

2. Cell replication—Hereditary characteristics are passed on to daughter cells through replication of DNA. 3. Control protein synthesis. 4. Transcription and translation.

1.5.4 RNA There are three types of RNA known to exist (Brosius & Raabe, 2016): 1. Messenger RNA (mRNA) 2. Transfer RNA (tRNA) 3. Ribosomal RNA (rRNA) These are polymer of purine and pyrimidine ribonucleotides linked together by phophodiester bonds (Nelson & Cox, 2005). Schematic representation of the structure of RNA with its nitrogenous bases is presented in Fig. 1.8 (lefthand side). Major nucleotides in RNA are adenylic, guanylic, cytidylic and uridylic acids. However, thymine is absent except in tRNA. FIGURE 1.8 Schematic representation of the structure of RNA (left-hand side) and DNA (right-hand side) with its nitrogenous bases (Malhotra & Ali, 2018). Source: With permission, Copyright r 2018 Elsevier Inc.

I. Background

1.5 Nucleic acids

RNA is distributed throughout the cell, most of which remains present in cytoplasm as soluble and rRNA, but about 10% is found in nucleus with very small quantities being also present in the mitochondria (Higgs & Lehman, 2015; Nissen et al., 2000). 1. Messenger RNA (mRNA): These are homogenous in size and stability. Amongst all RNAs, mRNAs exhibit highest molecular weight (Sergeeva, Koteliansky, & Zatsepin, 2016). An mRNA carries adenine, guanine, cytosine and uracil as the major bases along with some minor bases, such as methylpurines and methylpyrimidines (Guan & Rosenecker, 2017). mRNAs give signal for the synthesis of very important substances like the enzymes, the proteins, a variety of polypeptide hormones, etc. (Guan & Rosenecker, 2017; Sergeeva et al., 2016). 2. Transfer RNA (tRNA): This consists of approximately 75 nucleotides and generated by nuclear processing of precursor molecule (Balatti, Pekarsky, & Croce, 2017; Phizicky & Hopper, 2010). It serves as an adapter molecule for the translation of information in sequence of nucleotides of mRNA into specific amino acids. tRNA is participated in protein synthesis (Phizicky & Hopper, 2010). Beside the presence of regular bases, that is, adenine, guanine, uracil and cytosine, tRNA has been found to contain some very unusual bases like ribothymidine, dihydrouracil, inosine, dihydrouridine (DHU), pseudouridine, etc., which possess an unusual linkage in-between the sugar ribose and the base (Higgs & Lehman, 2015; Nelson & Cox, 2005). All tRNA molecules consist of an ACC sequence at the 30 termini. It is through an ester bond to the 30 hydroxyl group of the adenosyl moiety that the carboxyl groups of amino acids are attached (Phizicky & Hopper, 2010). The

17

anticodon group at the end of base paired stem recognizes the triplet nucleotide or codon of the template mRNA. The DHU loop helps to recognize the specific enzyme which activates the specific amino acids. The Thymidine-pseudouridine cytidine binds the tRNA in ribosomes for protein synthesis (Phizicky & Hopper, 2010). 3. Ribosomal RNA (rRNA): It constitutes nearly 50%
60% of the total RNA of the cell and is single stranded fibrous molecules which are highly elongated (Nelson & Cox, 2005; Urlaub, Kruft, Bischof, Mu¨ller, & Wittmann-Liebold, 1995). An mRNA carries adenine, guanine, cytosine and uracil as the major bases along with some minor bases such as methylpurines and methylpyrimidines. One or more segments of mRNA strand carry the genetic code or message, which is translated into the primary structure of a protein. Each genetic code consists of many consecutive nucleotide triplets called codons, each of which helps to incorporate specific amino acids in the peptide being synthesized (Higgs & Lehman, 2015). Nucleic Acids, that is, DNA and RNA are significantly employed as biomedicine for the management of varieties of physiological conditions (Minchin & Lodge, 2019). In a study, it was observed that chitosan nanoparticles loaded with probiotic DNA showed hypoglycemic activity (Kaur, Bhatia, Sethi, Kaur, & Vig, 2017). Antidiabetic activity has been reported by some other researchers by combining of berberine and noncoding RNA (Chang, 2017). Floxuridine, a cytotoxic nucleoside analog, is a very good anticancer drug and it can be incorporated into DNA strands by synthesis or incorporated into RNA by transcription (Ma et al., 2018). This can be used as a real nucleoside. DNases II of tumor cells hydrolyze the nucleotide strands and cytotoxic drug is released.

I. Background

18

1. Biological macromolecules: sources, properties, and functions

1.6 Conclusion Biological macromolecules are large cellular components abundantly obtained naturally and are responsible for varieties of essential functions for the growth and survival of living organisms. Biological macromolecules play an important role in the biomedical and related fields. These possess some favorable characteristics, such as good biocompatibility, excellent biodegradability, desired mechanical strength, better bioavailability, etc. These exhibit various bioactivities like anticancer, antidiabetic, antimicrobial, antioxidant, immunomodulatory, etc. Important carbohydrates like alginate, chitosan, pectin, starches, carrageenan, fucoidan, etc., are used commercially. Proteins (polymer of amino acids) and lipids are being extensively used in materials sciences as well as in biomedical field. Carbohydrates, lipids, proteins, and/or nucleic acids can modulate the pathophysiology of neurodegenerative disorders/diseases.

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I. Background

C H A P T E R

2 Structure
activity relationship of biological macromolecules Aurelie Sarah Mok Tsze Chung, Yong Kiat Teo, Wai Teng Cheng and Joash Ban Lee Tan School of Science, Monash University Malaysia, Bandar Sunway, Malaysia

2.1 Introduction

Gong, Wang, & Yang, 2010; Mitragotri, Burker, & Langert, 2014). Higher specificity may also be associated with higher potency, where the desirable therapeutic effect can be reached with a low dose of the macromolecular drug (Mitragotri et al., 2014). Moreover, advances in synthetic chemistry and molecular biology approaches have further enabled the mass production of many biomacromolecules (He et al., 2010; Tripathi & Shrivastava, 2019). Like most compounds, the functions and bioactivities of biomacromolecules are often influenced by their corresponding molecular structures. As such, structure
activities relationship (SAR) studies are one of the most actively pursued areas of molecular biology and biochemistry. With a growing arsenal of imaging and visualization technologies, molecular structures can now be elucidated more readily (Maveyraud & Mourey, 2020; Olson, 2018). Conversely, the real challenge lies in comprehensively describing the influence of molecular structure on the bioactivity, and how it pertains to the mechanisms of action for

Biological macromolecules are naturally occurring large cellular compounds that can be generally categorized into four major classes: proteins, carbohydrates, lipids, and nucleic acids. Apart from their ability to carry out a diverse range of physiological functions for the growth and survival of living organisms, many biological macromolecules also possess various bioactivities, including antimicrobial, antioxidant, antitumor, antiviral, antiinflammatory, antiproliferative, and hypoglycemic activities (Shi, 2016). Although bioactivity research has focused heavily on small molecules, there has nevertheless always been an interest in the use of macromolecules as drug candidates. These large compounds may hold certain advantages over small molecule drugs. For instance, the structural complexity of some biological macromolecules allows for relatively higher specificity, which may lower the risk of systemic adverse effects (Atyabi, Zahir, Khonsari, Shafiee, & Mottaghitalab, 2017; He, Dong,

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00002-6

23

© 2022 Elsevier Inc. All rights reserved.

24

2. Structure
activity relationship of biological macromolecules

these biomacromolecules. A better understanding of this SAR would also allow for the biological properties of newly discovered compounds to be inferred from those of similar existing compounds whose risks have already been evaluated (McKinney, Richard, Waller, Newman, & Gerberick, 2000), and betterinform future design of structural analogs or derivatives. Therefore, a better understanding of SAR may accelerate the prototyping stages and shorten the time of development process, thereby reducing costs (Guha, 2013; Weida, William, Leming, Hong, & Roger, 2003). Moreover, this knowledge may allow for the designing of synthetic derivatives with improved pharmacokinetics and pharmacodynamics (Guha, 2013). Given the extensive number of bioactive macromolecules that exist, it would be impossible to discuss all of them within a single chapter. Instead, this chapter describes several macromolecules as examples to demonstrate the relationship between structure and their various bioactivities, be it antimicrobial, antioxidant, or antitumor activities.

2.2 Enzymes as bioactive proteins The proteins have been extensively studied in recent decades for their remarkable broad scope of bioactivities and health benefits, including antioxidant, antimicrobial, antiinflammatory, antitumour, anti-HIV and wound healing activities (Jakubczyk, Karas, Rybczynska-Tkaczyk, Zielinska, & Zielinski, 2020; Łojewska et al., 2020; Moghadam, Niazi, Afsharifar, & Taghavi, 2016; Peng et al., 2021; Tao, Cai, Wang, Zuo, & He, 2021). They are the most functionally versatile and abundant biological macromolecules omnipresent in living organisms, responsible for a wide range of cellular functions. Proteins are made up of different monomers known as amino acids. Each amino acid consists of the same basic structure:

a central carbon atom bonded to a carboxyl group (2COOH), an amino group (2NH2) and a side chain which can harbor diverse functional R-groups (Littlechild, 2013). Based on the various interactions between the amino acids, the proteins may hold up to four organizational levels and acquire a three-dimensional molecular shape (Littlechild, 2013). The composition and sequence of amino acids, together with an array of R-groups and the inner dynamic folds, thus determine the overall protein structure while also contributing to their chemical properties like solubility, reactivity and stability, amongst many others (Hvidsten et al., 2009). As such, the functions and bioactive properties of each protein are greatly dependent on its three-dimensional structure. The vast diversity of different sequences and structures allows proteins to exhibit a broad range of biological functions and bioactive properties. At present, over 150,000 protein structures have been recorded in the Protein Data Bank (PDB) (Guzenko, Burley, & Duarte, 2020). Interestingly, over the course of evolution, most sequences essential for a given function are usually conserved, thus facilitating the identification of many bioactive protein families (Jensen, Ussery, & Brunak, 2003). Besides that, although proteins are considered as macromolecules, shorter peptides consisting of only a few amino acids may still exert a bioactive effect (Newstead, Varjonen, Nuttall, & Paterson, 2020). Usually, peptides with less than 50 amino acids are referred as small proteins or short peptides (Chen & Lu, 2020; Storz, Wolf, & Ramamurthi, 2014). Given the macromolecular scope of this book chapter, the focal point in this chapter will be on large bioactive proteins like the enzymes, with potential applicability in the pharmaceutical industry. Enzymes are globular multidomain proteins, consisting of one or more polypeptide chains, which act as biological catalysts in living organisms (Robinson, 2015). Due to their ubiquity in nature, their applications within the

I. Background

2.2 Enzymes as bioactive proteins

biomedical field have been actively growing over the years. While enzymes are essential for fundamental physiological functions, they also possess several bioactive therapeutic properties. Some enzymes can be used as antitumor and antimicrobial drugs, whereas others can be used to treat genetic disorders and cardiovascular diseases (Baldo, 2015; Gurung, Ray, Bose, & Rai, 2013; Sabala, Jonsson, Tarkowski, & Bochtler, 2012; Thallinger, Prasetyo, Nyanhongo, & Guebitz, 2013). Moreover, unlike conventional drugs, enzymes are highly specific to their substrate, to which they can bind and act on their targets with great affinity (Robinson, 2015). The spatial arrangement and type of amino acids at the active site can provide a conformation complementary to that of the substrate, allowing considerable specificity in their catalytic activity (Ghanem & Raushel, 2012). In certain cases, some enzymes may also contain another component, known as a cofactor, essential for their catalytic activity (Andreo-Vidal, Sanchez-Amat, & CampilloBrocal, 2018). Other than that, the rest of the enzyme structure stabilizes the active site, creating a suitable environment for the interaction of the site with the corresponding substrate (Robinson, 2015). In view of the vast array of enzymes with bioactive properties that exist, a few examples of enzymes, whose crystalline structures have been studied and deposited in the PDB have been selected for the discussion of their SAR. These include the L-amino acid oxidases (LAOs), lysostaphin and metallo-β-lactamaselike lactonase (MLL). All three enzymes are active against pathogenic bacteria, except for LAO which is also active against tumorigenic cells (Cheleuitte-Nieves et al., 2020; Costa et al., 2015; Lo´pez-Ja´come et al., 2019; Mukherjee, Saviola, Burns, & Mackessy, 2015). Each of the aforementioned enzymes act through catalyzing a distinct type of reaction and using different substrates. For these reasons, the LAO, lysostaphin and MLL are categorized as

25

oxidative, proteolytic and quorum-quenching enzymes respectively (Sabala et al., 2012; Sikdar & Elias, 2020; Ullah, 2020). The selection of these specific enzymes highlights the therapeutic potential of enzymes, while also underpinning their associated structural diversity.

2.2.1 L-amino acid oxidases The LAOs are one of the most-studied members of the flavoenzyme family. They are widely distributed in nature across diverse phyla, from fungi and bacteria to plants and animals (Sabotiˇc et al., 2020; Yang et al., 2011). In particular, LAOs from snake venoms (SV) have been extensively investigated to probe understanding on their ability to induce toxic physiological effects and to develop snakebite envenomation treatment (Hossain et al., 2014). However, it was later demonstrated that SVLAOs may additionally possess dosedependent antibacterial, antiparasitic, antifungal and antitumor properties (Costa et al., 2015; Mukherjee et al., 2015; Rey-Sua´rez et al., 2018; Soares et al., 2020; Zainal Abidin et al., 2018). These properties were also observed in bacterial and fungal LAOs (Andreo-Vidal et al., 2018; Chen, Lin, Chen, Wang, & Sheu, 2010; Yang et al., 2011). Hence, due to the high availability of LAOs in nature, their potential use for therapeutic purposes has been further spurred. These enzymes catalyze the oxidative deamination of L-amino acids with a strict stereospecificity under aerobic conditions, producing the corresponding α-ketoacids and ammonia, while also generating hydrogen peroxide (H2O2) (Wellner & Meister, 1961). They are composed of homodimers with a flavin adenine dinucleotide (FAD) as a cofactor and each protomer (50
70 kDa) contains three conserved domains: the substrate-binding, FADbinding and helical domains (Fig. 2.1) (Feliciano, Rustiguel, Soares, Sampaio, &

I. Background

26

2. Structure
activity relationship of biological macromolecules

Cristina Nonato, 2017; Pawelek et al., 2000; Sabotiˇc et al., 2020; Wiezel et al., 2019). Few exceptions have been reported where LAOs can exist as monomers or tetramers, with the latter being only biologically active in their multimeric form (Andreo-Vidal et al., 2018; Georgieva, Murakami, Perband, Arni, & Betzel, 2011; Rey-Sua´rez et al., 2018). A highly conserved motif β-α-β and a glutamic acid-rich motif are observed in the N-terminal sequence of LAOs, important for the FAD binding (Izidoro et al., 2014; Yu, Zhou, Qiao, & Qiu, 2014). Moreover, 3%
4% of the molecular mass of most LAOs consist of carbohydrates,

which play a functional role for their antibacterial and antitumor activities (Soares et al., 2020; Ullah, 2020). Although the substrate specificity of LAOs may vary, most LAOs demonstrate a high affinity for hydrophobic L-amino acids (Andreo-Vidal et al., 2018; Naumann et al., 2011; Rey-Sua´rez et al., 2018; Sabotiˇc et al., 2020; Soares et al., 2020; Wiezel et al., 2019). Nevertheless, there are exceptions where hydrophilic L-amino acids can be the best substrate for some LAOs (Ben et al., 2019; Nuutinen, Marttinen, Soliymani, Hilden, & Timonen, 2012). The variation in LAO

FIGURE 2.1 (A) Cartoon representation of the LAO dimer from the Malayan Pit viper Calloselasma rhodostoma. Opposite charges on the surface of each protomer stabilize the functional dimeric form of the protein. The substratebinding, FAD-binding and helical domains for each protomer are colored in green, red and blue respectively. The glycan moiety is located at the surface of the protein while the cofactor, FAD in buried inside each protomer. (B) Close-up view of the protomer. Residues Ile374 and Ile430 account for substrate preference of the LAO from Viper a. ammodytes to L-phenylalanine. Residue His223 plays an important role in regulating substrate specificities. Source: (A) From the RCSB PDB (rcsb. org) of PDB ID 1F8R (Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., et al. (2000). The Protein Data Bank. Nucleic Acids Research, 28(1), 235
242; Burley, S.K., Bhikadiya, B., Bi, C., Bittrich, S., Chen, L., Crichlow, G.V., et al. (2020). RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. Nucleic Acids Research, 49(1), 437
451; Pawelek, P.D., Cheah, J., Coulombe, R., Macheroux, P., Ghisla, S., Vrielink, A. (2000). The structure of Lamino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site. The EMBO Journal, 19(16), 4204
4215) (Berman et al., 2000; Burley et al., 2020).

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2.2 Enzymes as bioactive proteins

substrate specificities may be explained by the differences in amino acid composition and sequence in the loops region, which in turn influence the surface charge distribution, as well as the cavity volume and depth at the active site (Ullah, 2020). For example, the hydrophobic amino acid residues Ile374 and Ile430 at the substrate-binding site account for the preference of the LAO from Vipera ammodytes ammodytes to L-phenylalanine (Georgieva et al., 2011). Interestingly, the amino acid residue at position 223 in the active site has been reported to play an important role in regulating the substrate specificities of LAOs. It was observed that when alanine or serine occupied this position, the LAO from Daboia venoms had higher specificity towards L-arginine as the substrate-binding cavity was further expanded and the steric repulsion towards this substrate was reduced (Chen, Wang, Huang, Huang, & Tsai, 2012). Hence, position 223 could be further exploited for drug design. The ability of LAOs to inhibit bacterial and fungal pathogens has been associated with the exogenous production of H2O2 during the oxidative deamination of L-amino acids, as this effect was inhibited by the presence of catalase (Andreo-Vidal et al., 2018; Costa et al., 2015; Sabotiˇc et al., 2020). It was also suggested that the most hydrophobic sequences in the amphipathic N-terminus of LAAOs are able to interact with the cell surface of bacteria such as Staphylococcus aureus and Escherichia coli, subsequently destabilizing the cell membrane integrity, which further enhanced the antimicrobial effect (Abdelkafi-Koubaa et al., 2016; Costa et al., 2015; Yang et al., 2011). The cell membrane permeabilization may result in an accumulation of H2O2, or the production of other reactive-oxygen species (ROS) intracellularly due to an unusual metabolic cytosol environment. Consequently, cellular damages such as lipid peroxidation and DNA fragmentation may occur, leading to bacterial growth inhibition (Yang et al., 2011). An illustration of the

27

antimicrobial activity of LAOs is summarized in Fig. 2.2. Apart from their antimicrobial activity, LAOs can also exert a cytotoxic and antiproliferative effect on different tumorigenic cells such as leukemia, lung cancer, gastric cancer, prostate cancer, breast cancer and colon carcinoma cells (Costa et al., 2015; Li Lee, Chung, Yee Fung, Kanthimathi, & Hong Tan, 2014; Naumann et al., 2011; Salama et al., 2018; Zainal Abidin et al., 2018). It was reported that the glycan moiety at position 172 on the protein surface mediates the interaction of SV-LAOs with the cell surface for a targeted local release of H2O2. This further enhances oxidative stress, which consequently leads to DNA damage and cell apoptosis (BedoyaMedina et al., 2019; Bregge-Silva et al., 2012; Feliciano et al., 2017; Geyer et al., 2001; Machado et al., 2018; Naumann et al., 2011). Furthermore, the LAOs can induce proteolytic enzyme release, which is essential in programmed cell death via two pathways: via their interaction with death receptors in the plasma membrane of tumorigenic cells, such as the Fas receptor; or through the activation of the mitochondria-mediated caspase pathway upon depolarization of the mitochondrial membrane due to the accumulation of ROS (Bedoya-Medina et al., 2019; Mukherjee ˇ et al., 2015; Piˇslar, Sabotiˇc, Slenc, Brzin, & Kos, 2016; Tan, Ler, Gunaratne, Bay, & Ponnampalam, 2017; Tavares et al., 2016; Zhang & Cui, 2007). In the absence of the glycan moiety, the catalytic activity of the LAO is not affected, but the apoptotic activity is significantly reduced (Ande et al., 2006; Lu et al., 2018; Ullah, 2020). This demonstrates that the apoptotic effect is not only brought about by the production of H2O2, but also depends on the interaction of the glycan moiety, thus underpinning its importance in the structure of LAOs. In summary, based on the general structural framework of LAOs, it can be observed

I. Background

28

2. Structure
activity relationship of biological macromolecules

FIGURE 2.2 (A) Representation of the catalytic activity of LAO, using L-amino acids as a substrate to release H2O2 as one of its by-products. (B) Binding of the glycan moiety to the cell surface receptor helps to produce a localized high concentration of H2O2, which may accumulate intracellularly. (C) Interaction between the most hydrophobic sequences in the N-terminus with cell surface may destabilize the cell membrane integrity. (D) Formation of endogenous H2O2 due to unusual metabolic environment. (E) Accumulation of ROS may lead to DNA fragmentation. (F) ROS production may lead to lipid peroxidation in the membrane layer.

that the amino acid chain composition at the active site is a major determinant for substrate specificity of the enzyme. Besides that, hydrophobic residues at the N-terminal and the glycan moiety are key structural components for LAOs to exert their bioactive effect on bacterial pathogens and tumorigenic cells. The important structural features are summarized in Table 2.1. Nevertheless, although LAOs seem to hold great antibacterial and antitumor potential, the delivery system of the LAOs to the site of infection and their therapeutic dose still need to be optimized before they can be used for pharmaceutical purposes.

2.2.2 Lysostaphin Lysostaphin is a 27 kDa monomeric zinccontaining metalloenzyme of 246 amino acids produced by Staphylococcus simulans (Schindler & Schuhardt, 1964). It has been mainly studied for its ability to inhibit S. aureus, including methicillin-resistant strains and its biofilms, by cleaving the crosslinking pentaglycine bridges in the peptidoglycan layer (Askari, Ahmad, Abhishek, Waris, & Malakar, 2014; Boksha et al., 2016; Ceotto-Vigoder et al., 2016; Cheleuitte-Nieves et al., 2020; Chen et al., 2014). This enzyme consists of two distinct domains: the N-terminal peptidase domain

I. Background

2.2 Enzymes as bioactive proteins

29

TABLE 2.1 Summary of the important structural components of LAO, lysostaphin and MLL associated with their bioactivities. L-amino

oxidase

Bioactivity

Antibacterial and antitumor

Substrate

L-amino

Cofactor

FAD

Domains

Substrate-binding domain, FAD-binding domain, Helical domain

Active site

Amino acid at position 223 can alter substrate specificity

N-terminal

Most hydrophobic sequences interact with bacterial surface to disrupt membrane integrity

Glycan moiety

Position 172 mediates interaction with tumor cell surface for localized production of H2O2

acids

Lysostaphin Bioactivity

Antibacterial and antibiofilm

Substrate

Pentaglycine cross-bridges

Cofactor

Zn21

Domains

Catalytic (CAT) domain, cell wall targeting (CWT) domain

Active site

His 278, Asp 283 and His 362 residues coordinates the zinc metal ion

Flexible linker

Adjusts orientation of CAT and CWT domains for cleaving process

CWT domain

Confers target-cell specificity

Binding sites

• Located at opposite faces of CWT domain, allowing for lysostaphin clustering • β1 and β2 in first binding site strands essential for peptidoglycan binding • Lower affinity of first binding site to pentaglycine for rapid access to CAT domain.

Metallo-β-lactamase-like lactonase Bioactivity

Antivirulence and antibiofilm

Substrate

N-acyl homoserine lactones

Cofactor

Zn21

Domains

HXHXDH motif domain

Active site

• • • • •

Five His and two Asp residues coordinate the dinuclear metal cationic center Some AAs may act as hydrogen donors for substrate binding Hydrophobic channel accommodates for the amide linkage of AHLs Types of Aas influence acyl chain length preference Structural variations may influence KM values

responsible for its catalytic activity, and the Cterminal cell-wall targeting (CWT) domain for the binding to the peptidoglycan layer (Fig. 2.3) (Baba & Schneewind, 1996; Sabala

et al., 2014). The catalytic (CAT) domain at the active site consists of an antiparallel β-sheet which anchors catalytic residues, grouped around a tightly bound central Zn21 cofactor,

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FIGURE 2.3 (A) Cartoon representation of lysostaphin. The CAT domain, CWT domain and linker is colored in pink, blue and orange respectively. The cofactor, zinc ion (yellow sphere) is buried inside the catalytic domain. (B) The zinc ion is coordinated by His279, Asp283 and His362 residues. Source: (A) From the RCSB PDB (rcsb.org) of PDB ID 5NMY (Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., et al. (2000). The Protein Data Bank. Nucleic Acids Research, 28(1), 235
242; Burley, S.K., Bhikadiya, B., Bi, C., Bittrich, S., Chen, L., Crichlow, G.V., et al. (2020). RCSB Protein Data Bank: Powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. Nucleic Acids Research, 49(1), 437
451; Tossavainen, H., Raulinaitis, V., Kauppinen, L., Pentika¨inen, U., Maaheimo, H., Permi, P. (2018). Structural and functional insights into lysostaphin-substrate interaction. Frontiers in Molecular Biosciences, 5:60).

coordinated by His279, Asp283 and His362 (Chandra Ojha et al., 2018; Tossavainen et al., 2018). The CAT domain is connected to the CWT domain through a flexible linker of 14 amino acid residues, allowing the orientations of the domains to be adjusted upon cleavage of the pentaglycine bridges in peptidoglycan (Tossavainen et al., 2018). Although the CAT domain is essential for the catalytic activity of the lysostaphin, yet the CWT domain appears to also be essential for its antibacterial activity. Upon the removal of this particular domain, lysostaphin can no longer distinguish between the producer cell and the target cells, demonstrating that this particular domain confers target-cell specificity (Baba & Schneewind, 1996). The CWT domain

contains two well-defined binding sites, namely a narrow groove accommodating for interaction with pentaglycine and a more open site for the interaction with the peptide stem within the peptidoglycan (Gonzalez-Delgado et al., 2020). The two binding sites are located at opposite faces of the CWT domain, making the simultaneous biding of the enzyme to the two peptidoglycan moieties not feasible. However, this special structural organization was reported to provide a biological benefit as this mutually exclusive recognition of the pentaglycine and the peptide stem leads to the clustering (and thus higher concentration) of lysostaphin at the peptidoglycan (GonzalezDelgado et al., 2020). Hence, the CWT domain can anchor to the peptidoglycan to cleave

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2.2 Enzymes as bioactive proteins

various pentaglycine cross-bridges within a localized region, which weakens the peptidoglycan locally in a more effective way, rather than random cleavage (Sabala et al., 2014). In the first binding site, two β strands (β1 and β2) were reported to be essential for the activity of lysostaphin as the removal of these strands drastically impaired binding to lysostaphinsensitive peptidoglycan (Mitkowski et al., 2019). In general, the CWT domain has a relatively higher affinity to the peptide stem through the second binding site of the peptidoglycan as compared to the pentaglycine through the first binding site (GonzalezDelgado et al., 2020; Tossavainen et al., 2018). It was suggested that the weaker affinity to the pentaglycine led to rapid-off rates, which in turn increased the access of the catalytic domain to quickly cleave the pentaglycine crosslinks (Gonzalez-Delgado et al., 2020). The schematic illustration of the antibacterial activity is presented in Fig. 2.4. Besides that, it appears that lysostaphin has a preference towards the exclusive recognition and cleavage of crosslinks composed only of glycine residues, which is a distinct and unique

31

feature present in staphylococci peptidoglycan (Fig. 2.5) (Monteiro et al., 2019). This could explain the observation made by Bagwat et al. where lysostaphin only targeted S. aureus cells in a coculture of different gram-positive bacteria, leaving Micrococcus luteus and Bacillus cereus cells intact (Bhagwat, Collins, & Dordick, 2019). The CWT domain adopts a special conformation at the narrow groove of the substrate-binding site which is accessible to glycine residues only. Simultaneously, steric repulsions are created between the substrate-recognition residues (Tyr472 and Asn405) and the carbon β atoms in all other amino acid residues, thus allowing little tolerance for other amino acid residues (Gonzalez-Delgado et al., 2020; Mitkowski et al., 2019). This also explains why lysostaphin does not affect its producer cell since the peptidoglycan of S. simulans consists of cross-bridges made up of serine residues, which then hampers the binding of the CWT domain of lysostaphin to its peptidoglycan (Gru¨ndling & Schneewind, 2006; Mitkowski et al., 2019). Thus, these observations further underpin the selectivity of lysostaphin towards glycine residues to exert its antibacterial activity.

FIGURE 2.4 Schematic representation of antibacterial activity of lysostaphin. NAM, N-acetylglucosamine; NAG, N-acetylmuramic acid. (A) The two binding sites (1 and 2) are located at opposite faces of the CWT domain, allowing interaction of the domain only to either the peptide stem or the pentaglycine. (B) Once identification of the pentaglycine is done by the CWT, access is provided to the catalytic domain for the cleavage of the pentaglycine cross-bridge.

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activity relationship of biological macromolecules

FIGURE 2.5 (A) Lysostaphin enzyme with both domains recognizes and cleaves cross-bridges made of glycine residues only. (B) Without the CWT domain, lysostaphin can cleave cross-bridges made of amino acid residues, other than glycine. It can no longer distinguish between its producer cell, S. simulans and the target cell, S. aureus.

The points presented in this section highlight the importance of both CAT and CWT domains for the antistaphylococcal activity. The coordination between these two domains is an interesting dynamic that allows the enzyme to recognize the peptidoglycan and exclusively cleave the pentaglycine crossbridges. Besides that, it should be noted that lysostaphin works best in its monomeric form, albeit it may be rapidly cleared from the systemic circulation. Recent attempts to dimerize lysostaphin monomers have been conducted to improve its pharmacokinetics, but dimerization was found to decrease its inhibitory

activity towards S. aureus (Grishin et al., 2019). Nevertheless, all the structural features of lysostaphin influencing its interaction with the peptidoglycan layer and antibacterial activity (summarized in Table 2.1) should be holistically taken into account, for further engineering of novel lysostaphin variants with improved properties.

2.2.3 Metallo-β-lactamase-like lactonase Lactonases are a class of enzymes known to disrupt bacterial quorum sensing (QS), a communication system used in many bacterial

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2.2 Enzymes as bioactive proteins

pathogens, through the hydrolysis of the ester bond in the lactone ring of acylated homoserine lactones (AHLs) (Bergonzi, Schwab, Naik, & Elias, 2019; Cai et al., 2018). The latter is produced by many bacterial pathogens as signaling molecules in response to high cell density, which mediates the regulation of gene expression associated with their pathogenesis once a certain threshold is reached (Chu, Liu, Jiang, Zhu, & Zhuang, 2013; Liu et al., 2018). Thus, by degrading AHLs, lactonases can act as quorum quenching (QQ) enzymes to reduce the capability of bacterial pathogens in triggering virulent expression, which consequently may inhibit the production of virulence factors and biofilm formation (Fig. 2.6) (Bergonzi et al., 2018, 2019; Lo´pez-Ja´come et al., 2019; Mahan et al., 2020; Maleˇsevi´c et al., 2020). This quorum quenching approach has gained more attention as lactonases do not generally inhibit bacterial growth and as

33

such contribute less to antibacterial resistance development (Chen, Gao, Chen, Yu, & Li, 2013). Different types of lactonases have been isolated from mammals, bacteria, fungi, insects and plants (Grandcle´ment, Tannie`res, More´ra, Dessaux, & Faure, 2015; Morohoshi, Kamimura, Sato, & Iizumi, 2019; See-Too, Convey, Pearce, & Chan, 2018). The most studied lactonases belong to the MLLs superfamily of proteins. Some examples are AiiA from Bacillus thuringiensis, AiiB from Agrobacterium tumefacien, MomL from Muricauda olearia and AaL from Alicyclobacillus acidoterrestris among many others, with the former being the most characterized lactonase from this family (Bergonzi et al., 2018; Liu et al., 2007; Tang et al., 2015; Thomas, Stone, Costello, Tierney, & Fast, 2005). The MLLs are structured into two quasirepeats exhibiting a canonical αβ/βα fold

FIGURE 2.6 Schematic representation of the quorum-quenching activity of MLL. (A) In response to high cell density, AHLs are produced by the bacterial cells, triggering the expression of virulent genes for biofilm formation. (B) MLLs degrade AHLs, preventing biofilm formation.

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2. Structure
activity relationship of biological macromolecules

where the N-terminal contains a single mixed β sheet with seven strands and three α helices, along with six short helices where the Cterminal contains a single mixed β sheet with five strands and three α helices (Fig. 2.7) (Bergonzi et al., 2018; Kim et al., 2005). The protein identity between different MLLs may vary, but the HXHXDH motif required for the AHL-degrading activity at the active site is conserved among all identified MLLs (Cai et al., 2018; Morohoshi et al., 2019). This motif is associated with the formation of the dinuclear metal cation center which is essential for the enzyme activity (Kim et al., 2005). The two metal cations are generally coordinated with five histidine residues and two aspartic acid residues at the active site (Charendoff, Shah, & Briggs, 2013). The number and type of amino acids binding the dinuclear metal ion center

remain the same within different MLLs, albeit the position of these residues may vary (Bergonzi et al., 2018; Kim et al., 2005; Mascarenhas et al., 2015). If the amino acids coordinating the two metal cations at the active site are replaced by uncharged nonmetal binding residues, the catalytic activity of MLLs may decrease (Tang et al., 2015). For instance, when the histidine and aspartic acid residues were substituted with alanine, leucine or serine, either a decrease in the enzyme activity or the inactivation of the enzyme was observed (Kim et al., 2005; Tang et al., 2015). Moreover, it was reported that the formation of a hydrogen bond between the active site and lactone carboxyl of the AHL is important for substrate binding and catalysis. To illustrate, the phenol side chain of Tyr194 in AiiA or the N-H part of the side chain of His214 in MomL act as

FIGURE 2.7 (A) Ribbon representation of the AiiA monomer produced by B. thuringiensis. The α-helices, β-sheets and loops are shown in red, pink and white, respectively. An αβ/βα fold is observed. The two zinc ions cofactors are represented as yellow spheres. (B) The zinc ions are coordinated by five histidine residues and two aspartic acid residues. Source: (A) From the RCSB PDB (rcsb.org) of PDB ID 4J5F (Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., et al. (2000). The Protein Data Bank. Nucleic Acids Research, 28(1), 235
242; Burley, S.K., Bhikadiya, B., Bi, C., Bittrich, S., Chen, L., Crichlow, G.V., et al. (2020). RCSB Protein Data Bank: Powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. Nucleic Acids Research, 49(1), 437
451; Liu, C.F., Liu, D., Momb, J., Thomas, P.W., Lajoie, A., Petsko, G.A., et al. (2013). A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis. Biochemistry, 52(9), 1603
1610) (Liu et al., 2013).

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2.2 Enzymes as bioactive proteins

hydrogen bond donors. However, if replaced by alanine and glycine respectively to prevent the hydrogen bond formation, the enzyme was rendered nonfunctional (Lu, Yuan, Xue, Zhang, & Zhou, 2006; Tang et al., 2015). In terms of the substrate preference, the MLLs generally exhibit a broad substrate spectrum with respect to the acyl chain length of AHLs (long and short), although in some cases, it may have a higher affinity for a specific acyl chain length depending on the type of amino acid residues at the active site (Bergonzi et al., 2019; Cai et al., 2018; Lo´pez-Ja´come et al., 2019; Ryu et al., 2020; Tang et al., 2015). For instance, when comparing a MLL exhibiting broad catalytic spectrum with another MLL having a particular preference for C10-AHL, the residues Ile73 and Met138 were replaced by valine and leucine respectively (Kim et al., 2005). Nevertheless, the ability of MLLs to catalyze a wide range of AHLs may be beneficial for its quorum-quenching activity as bacterial pathogens usually secrete multiple AHLs with varied chain lengths during pathogenesis (Honda et al., 2019; Mahan et al., 2020). Besides that, it is to be noted that MLLs are only active against AHLs and not the homoserine lactone (HL) itself, which demonstrates that the amide linkage is essential for proper substrate binding (Kim et al., 2005). This amide linkage is hydrophobic in nature and is suggested to fit into the hydrophobic channel of the active site during catalysis (Bergonzi et al., 2018). Recently, three subsites within the active site of the GcL MLL from Parageobacillus caldoxylosilyticus were identified. The first subsite is a hydrophobic and overhanging bimetallic active site center which harbors the lactone ring of AHLs, irrespective of their chain lengths. Upon the binding of the lactone ring to this site, the active-site loop is structurally reorganized to lock the loop in a closed conformation while positioning the rings for a nucleophilic attack from the bridging water molecules. In contrast, the second subsite is a ring of hydrophobic

35

residues harboring the amide group and short acyl chains of AHLs, while the third subsite is a ring of hydrophilic residues, which opens up to the protein surface to accommodate longer acyl chains of AHLs (Bergonzi et al., 2019). Given the hydrophobic nature of these long acyl chains, the hydrophilic ring in the third subsite is quite an unusual observation. Indeed, other lactonases showing a preference for long acyl chains usually contain a long hydrophobic channel which fully accommodates the whole hydrophobic substrate (Elias et al., 2008; Hiblot, Gotthard, Chabriere, & Elias, 2012). However, in the case of GcL, the authors argue that the active site cleft is too short to fully accommodate the long acyl chain length, thus leaving the aliphatic chain largely exposed to the solvent. Moreover, they also suggested that the absence of the long hydrophobic channel in GcL could explain its broadsubstrate spectrum (Bergonzi et al., 2019). Other than that, it was reported that the KM value (Michaelis constant, an inverse measure of the affinity of the enzyme for a particular substrate) may vary significantly across MLLs sharing a broad substrate spectrum (Re´my et al., 2020). For example, the KM values with respect to the optimal substrate of some MLLs like AiiA, AiiB and AiiE, can be in the range of 0.1
6.4 mM, but this value can decrease to a range of 0.005
0.080 mM for MLLs like GcL, AaL and AidC (Bergonzi et al., 2018, 2019; Liu et al., 2007; Mascarenhas et al., 2015; Shastry, Dolan, Abdelhamid, Vittal, & Welch, 2018; Thomas et al., 2005). One study suggested that the low KM values of AaL could be attributed to the structural variations in the active site, as it contains a hydrophobic ring patch in the vicinity of the active site which could potentially lower the KM value (Bergonzi et al., 2018). Meanwhile another study suggested that the low KM values for AidC could be attributed to the N-acyl binding pocket structure at the active site. This is because unlike AiiA, AidC contains an unusual closed, well-defined

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binding pocket which could prevent short chain AHLs from readily reaching deep inside and fully occupying the binding pocket (Mascarenhas et al., 2015). However, the exact mechanisms behind the low KM value of some MLLs are still unclear and further studies are warranted for QQ optimization strategies. Indeed, the KM value of MLLs is an important factor for their anti-QS activity as the threshold concentration of AHLs for QS activation is usually in the nM range (Bergonzi et al., 2019; Honda et al., 2019; Scholz & GreenBerg, 2017). Hence, although different MLLs may catalyze the same AHLs and inhibit QS, yet a MLL with a lower KM value may be more desirable to maximize the magnitude of AHLs inhibition. In summary, it can be observed that the major structural features of MLLs responsible for their quorum quenching activity are located at the active site (Table 2.1). Although the AHL-degrading motif is conserved among all MLLs, their substrate preference and KM value may differ based on structural variations in the active site of each MLL. Through the literature, many MLLs from different sources have been isolated and their substrate spectrum have been identified. However, for some of the isolated MLLs, there is still a paucity of explanation regarding the mechanisms influencing its substrate preference and its low KM values towards specific substrates. Hence, the elucidation of these mechanisms could provide essential information moving forward, for the development of MLL variants with tailored selectivity as potential treatments against QSdependent bacterial infections.

2.3 Chitosan as a bioactive polysaccharide In contrast to the proteins, the carbohydrates have been mainly studied for their relevance in cell metabolism and pathology, as well as their application as carriers for drug delivery

(Brown & Higgins, 2010; Dai et al., 2012; Hardy, Brand-Miller, Brown, Thomas, & Copeland, 2015; Kang, Opatz, Landfester, & Wurm, 2015). Nevertheless, continuous investigations on the properties of carbohydrates have demonstrated their potential as a relatively untapped source of bioactive compounds (Hayes & Tiwari, 2015). They could potentially act as antimicrobial agents, antioxidant agents, functional foods, and biomaterials (Appelt et al., 2013; Hu et al., 2016; Kokkinidou, Peterson, Bloch, & Bronston, 2018; Oldenkamp, Vela Ramirez, & Peppas, 2019). Differing monosaccharide composition, chain length, and glycosidic linkages are some of the factors responsible for the different properties of macromolecular polymeric polysaccharides. Common polysaccharides include starch, cellulose and chitin, each having its own role in living plants and animals. Cellulose provides structural support to plant’s cell wall, while chitin forms fibrous exoskeletons of arthropods and fungal cell wall (Blackwell, 1982). Polysaccharides typically demonstrate high biocompatibility and biodegradability (Yadav, Yadav, Shah, Shah, & Dhaka, 2015), as well as low cytotoxicity (Queiroz et al., 2006; Seedevi et al., 2019). Since the biological activities of polysaccharides are strongly governed by their distinct structural features (Grube et al., 2020; Ja¨ntschi, 2019; Solanki, Mehta, & Thakore, 2014), much research has focused on understanding how their structure relates to their bioactivity. Given the myriad of potential polysaccharides, there currently exists no universal SAR consensus. Although bioactivity studies on certain polysaccharides can be relatively limited, our focus in this chapter will be on chitosan given their comparatively wellstudied bioactivities. Chitosan is a cationic macromolecule naturally found in fungi such as Agaricus bisporus and Pleurotus sajor-caju, consisting of linear chains of β-(1,4)-linked glucosamine and Nacetylglucosamine (Abasian, Shafiei Alavijeh,

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2.3 Chitosan as a bioactive polysaccharide

Satari, & Karimi, 2020). It can also be obtained via deacetylating chitin, a polysaccharide abundant in the exoskeleton of crustaceans and insects (Rinaudo, 2006). Compared to chitin, chitosan has comparatively superior antimicrobial activity and higher aqueous solubility, hence potentially giving it greater pharmaceutical value (Rinaudo, 2006). Although chitosan is generally the second-most studied biomacromolecule after cellulose, the number of bioactivity-related publications surrounding chitosan far eclipses that of cellulose within the past few years due to its comparatively superior bioactivity (Khattak et al., 2019; Kingkaew, Kirdponpattara, Sanchavanakit, Pavasant, & Phisalaphong, 2014), particularly in terms of its antimicrobial activity. Although chitosan shares a similar primary structure to cellulose with β-glycoside linkages, a major difference lies in the type of functional group at the C-2 position; where chitosan has an amino group while cellulose has a hydroxyl group. This also provides a little insight into the SAR, where the presence of certain functional groups (in this case, amino groups) could enhance the given activity. Given its biocompatibility and bioactive properties, researchers have also made synthetic derivatives, each with different properties (Chen & Park, 2003; Choi, Kim, Pak, Yoo, & Chung, 2007; Sajomsang, Ruktanonchai, Gonil, & Warin, 2010). For instance, crosslinking with active compounds, carboxy-methylation, carboxyalkylation, hydroxy-alkylation, alkylation, acylation, sulfation and thiolation have been carried out on the amino and/or hydroxyl reactive sites of chitosan, where promising enhancement of bioactivities have been reported for these modified derivatives (Fernandes, Francesko, Torrent-Burgue´s, & Tzanov, 2013; Khalil et al., 2017; Lim & Hudson, 2003; Sajomsang et al., 2010; Zhong et al., 2008). Aside from that, the physicochemical properties of chitosan, namely its molecular weight (MW) and deacetylation

37

degree (DD) are found to strongly impact its bioactivity (Kim, 2018).

2.3.1 Relationship of chitosan physicochemical property and its bioactivity Several comprehensive reviews on chitosan have acknowledged the relationship between its physicochemical properties and bioactivity (Abd El-Hack et al., 2020; Kong, Chen, Xing, & Park, 2010; Sahariah & Ma´sson, 2017). In nature, chitosan is composed of repeated monomers linked in long chains of varying MW, typically ranged from 50 to 300 kDa, depending on the source and extraction methods (Zhang et al., 2016). Further chemical modifications allow the formation of chitosan analogs with desired MW via either depolymerization, irradiation, acetylation and/or deacetylation without changing its monomer conformation (Xing et al., 2005; Xuan Du, Xuan Vuong, & Mai, 2019; Yaghobi, 2012). Such approaches have been useful in SAR studies to determine the role of MW with respect to the bioactivity of chitosan. As such, the apparent antimicrobial activity of chitosan seems to depend on its MW. In general, chitosan with low MW (#100 kDa) appears to exhibit better antimicrobial activity than those with MW greater than 100 kDa; implying an inverse relationship between chitosan MW and its antimicrobial activity (Table 2.2). As shown in a comparative study, Benhabiles et al. demonstrated that short chain chitosan or oligochitosan derived from shrimp (Palaemon longirostris) shells (H. Lucasm, 1846) exhibited a considerably stronger antimicrobial activity (two to fourfold) as compared to the extracted low MW chitosan (12 kDa) (Benhabiles et al., 2012). These findings are contradicted by a small handful of studies however, where higher MW chitosan demonstrated better antimicrobial activity, and/or lower MW failed to

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TABLE 2.2 Chitosan analogs of different MW and DD, and their antimicrobial effect against Candida spp., E. coli and S. aureus. MW (kDa)

DD (%)

Strain tested (no. of strains)

MIC (μg/mL)a

15.06

94

Candida spp. (5)

16
64

Kulikov, Lisovskaya, Zelenikhin, Bezrodnykh, and Shakirova (2014)

15.06

94

Candida spp. (1)

256

Kulikov et al. (2014)

19.99

98

Candida spp. (5)

16
32

Kulikov et al. (2014)

19.99

98

Candida spp. (1)

256

Kulikov et al. (2014)

19.99

98

Candida spp. (36)

8
256

Kulikov et al. (2014)

70

.75

Candida spp. (8)

1
4

Alburquenque et al. (2010)

70

80

Candida spp. (1)

16

Kulikov et al. (2014)

70

.75

Candida spp. (6)

16
32

Alburquenque et al. (2010)

70

80

Candida spp. (4)

64
128

Kulikov et al. (2014)

70

80

Candida spp. (1)

1024

Kulikov et al. (2014)

107

75
85

Candida spp. (1)

3000

Costa, Silva, Tavaria, and Pintado (2014)

600

75

Candida spp. (1)

128

Kulikov et al. (2014)

600

75

Candida spp. (4)

512
1024

Kulikov et al. (2014)

600

75

Candida spp. (1)

$ 2048

Kulikov et al. (2014)

28.4

84

E. coli (1)

30

Mellega˚rd, Strand, Christensen, Granum, and Hardy (2011)

42.5

52

E. coli (1)

170

Mellega˚rd et al. (2011)

43

77

E. coli (9)

30
250

Jiang et al. (2013)

67

62

E. coli (9)

60
16,000

Jiang et al. (2013)

70

75
85

E. coli (1)

370

Mohammadi, Hashemi, and Masoud Hosseini (2016)

80

-

E. coli (1)

125

Tamara, Lin, Mi, and Ho (2018)

107

75
85

E. coli (1)

1500

Madureira, Pereira, Castro, and Pintado (2015)

140

83

E. coli (3)

1750
2000

Tayel et al. (2010)

190

84.9

E. coli (3)

2000
2500

Tayel et al. (2010)

190

75
85

E. coli (1)

370

Mohammadi et al. (2016)

684

75
85

E. coli (1)

1000

Mohammadi et al. (2016)

21

95

S. aureus (2)

750
1000

Tayel et al. (2010)

27

84.9

S. aureus (2)

500
1250

Tayel et al. (2010)

43

77

S. aureus (1)

250

Jiang et al. (2013)

Reference

(Continued)

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2.3 Chitosan as a bioactive polysaccharide

39

TABLE 2.2 (Continued) MW (kDa)

DD (%)

Strain tested (no. of strains)

MIC (μg/mL)a

Reference

67

62

S. aureus (1)

60

Jiang et al. (2013)

107

75
85

S. aureus (1)

1000

Madureira et al. (2015)

140

83

S. aureus (2)

1000
1500

Tayel et al. (2010)

190

84.9

S. aureus (2)

750
1750

Tayel et al. (2010)

a

MW means molecular weight; DD means degree of deacetylation; MIC means minimum inhibitory concentration which is determined via broth microdilution method.

demonstrate better activity (Abedian et al., 2019; Park, Nah, & Park, 2011). It is possible that the differing experimental conditions (i.e., pH, bacterial concentration) and/or assessment methods (i.e., broth microdilution, agar diffusion, or disk diffusion) chosen to evaluate the antimicrobial activity may have played a role in this contradiction. For instance, Chang et al. reported that chitosan antimicrobial activity was positively associated with increasing MW in acidic conditions (pH 5 5
6); but declined with increasing MW in neutral conditions (Chang, Lin, Wu, & Tsai, 2015). Meanwhile, as illustrated in Jiang et al. and Younes et al. studies, different methods (i.e., broth microdilution and agar diffusion) chosen to assess the antimicrobial property of chitosan could result in drastic varying outcomes; for instance, broth microdilution might show inhibitory action, but agar diffusion showed otherwise (Jiang et al., 2013; Younes, Sellimi, Rinaudo, Jellouli, & Nasri, 2014). Although most studies seemed to indicate that MW did not influence specificity against gram-positive or gram-negative bacteria (Kaya, Asan-Ozusaglam, & Erdogan, 2016; Park et al., 2015), Younes et al. reported greater gram-negative bacteria susceptibility towards chitosan with MW ranged from 42 to 135 kDa than gram-positive bacteria (Younes et al., 2014). Additionally, as with all antibacterial testing, the specific strain used also plays a significant role; where there can be a 10-fold

difference in MIC between different strains of the same species, even when tested with the exact same chitosan compound (Younes et al., 2014). Another physicochemical property of chitosan found to be crucial in determining its antimicrobial activity is its DD, which can also be referred to as the molar fraction of N-glucosamine units. Chitosan with high DD would have a higher number of free amino groups in the chain. This in turn is positively associated with the cationic charge of chitosan, contributed by the protonation at its N position in solution (Li et al., 2017). As a result, DD of chitosan greatly governs its solubility where higher DD results in greater water solubility (Lv, 2016). The solubility of chitosan is generally a point of concern, as although chitosan was found to exhibit better solubility and antimicrobial activity under acidic conditions at pH 5 (Ardila, Daigle, Heuzey, & Ajji, 2017; Sudarshan, Hoover, & Knorr, 1992), this pH range unfortunately limits it applications under physiological conditions, where its solubility becomes a hurdle. Intriguingly, increase in DD improved the antimicrobial activity of both low and high MW chitosan against various bacterial and fungal strains, albeit the effect was more pronounced for low MW chitosan (Jung et al., 2010; Younes et al., 2014). Li et al. suggested that chitosan with high DD would have more free N groups, thereby

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2. Structure
activity relationship of biological macromolecules

further increasing its cationic charge once dissociated in solution. Its cationic form would then have higher binding affinity towards bacterial membranes, which typically has a net negative charge (Li, Wu, & Zhao, 2016). This provides further evidence to support that the amino group or cationic charge plays an important role in the antimicrobial activity of chitosan. It has been proposed that high MW chitosan typically demonstrates weaker antimicrobial activity due to its larger compact size and stronger intramolecular bonding which hinders it from binding and penetrating the bacterial cellular membrane (Raafat, Von Bargen, Haas, & Sahl, 2008). On the other hand, low MW chitosan shows improved cellular penetration typically via perturbation of the bacterial membrane, which in turn causes leakage of cellular components, membrane damage, and consequently cell growth inhibition (Abedian et al., 2019; Kulikov et al., 2014). Besides, low MW chitosan with high DD also exhibits DNAbinding ability which could lead to the interference of DNA transcription, inhibition of mRNA synthesis, and impaired protein production with potentially deleterious effects (He et al., 2014; Yang et al., 2017). As aforementioned, the reported antimicrobial activity of a compound is also highly dependent on the experimental conditions and type of microorganism chosen. This further highlights the need to carefully interpret these antimicrobial findings; and perhaps consider standardizing the protocols and bacterial selection to enable better evaluation of chitosan MW and DD influence on its antimicrobial activity. Aside from antimicrobial activity, the effects of chitosan MW and DD on in vitro antioxidant activity has also been well-studied. Likewise, lower MW chitosan was found to exhibit more profound in vitro antioxidant activity as compared to higher MW chitosan, whereas chitosan with higher DD also exhibited better antioxidant activity. For instance, on the basis

of noncell-based antioxidant assays, chitosan with MW of 2 kDa showed stronger scavenging effect on hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and better ferrous ion chelating activity than chitosan with MW of 300 kDa (Chang, Wu, & Tsai, 2018). Besides, Chien et al. found that the MW of chitosan was inversely related to its radical scavenging activity (Chien, Sheu, Huang, & Su, 2007). In contrast, chitosan with higher DD (90%) exhibited better DPPH, hydroxyl and superoxide radical scavenging activity than those with lower DD (50% and 75%) (Park, Je, & Kim, 2004). As suggested by Igberase et al., the numerous amino and hydroxyl groups could also act as ligands to chelate metal ions such as Fe21 (Igberase, Osifo, & Ofomaja, 2017). Besides, Tamer et al. and Xie et al. postulated that the radical scavenging activity of chitosan could be attributed to its hydroxyl groups which reacted with hydroxyl radicals via hydrogen abstraction reaction, and free amino groups which formed stable complexes with hydroxyl radicals (Tamer, Valachova´, Mohyeldin, & Soltes, 2016; Xie, Xu, & Liu, 2001). The poor antioxidant potency of large MW chitosan may be due to their compact structure, where intramolecular hydrogen bonding is more likely to occur, thereby restricting the number of free amino and hydroxyl groups available for radical scavenging (Xing et al., 2005). In contrast, small MW or high DD chitosan may have a higher number of electron-donating terminals exposed to the environment, thus providing greater radical scavenging activity. Unlike the contradicting results observed regarding the relationship between chitosan’s antimicrobial activity and MW, the tendency of small MW and high DD chitosan to exhibit better antioxidant activity than larger MW/low DD chitosan appears mostly consistent in reports. In summary, physicochemical properties of chitosan, namely MW and DD, play an important role in determining its bioactivity.

I. Background

2.3 Chitosan as a bioactive polysaccharide

2.3.2 Bioactivity of chitosan with modified functional group As aforementioned, several limiting factors such as poor solubility and large MW have restricted the pharmaceutical applications of chitosan. Therefore, synthetic approaches deliberately targeting the amino and/or hydroxyl group at the C-2, C-3 and/or C-6 positions of chitosan have been conducted, aimed at discovering a more potent and watersoluble chitosan derivative with improved functionality. Substitution has been actively conducted on these positions as they are the most reactive sites on chitosan. Thus, numerous modified chitosan derivatives with markedly improved antimicrobial property have been documented, albeit the relationship between the structures of such derivatives and their bioactivity remains unclear. Antimicrobial testing of N-vanillyl and 4hydroxylbenzyl chitosan-incorporated film against the agricultural-related pathogenic fungus, Aspergillus flavus, resulted in a .90% (w.r.t. chitosan) reduction in aflatoxin production and reduced fungal biomass (Jagadish, Divyashree, Viswanath, Srinivas, & Raj, 2012). As hydrophobicity was found to have an impact on chitosan antimicrobial activity, the introduction of hydrophobic (e.g., alkyl or aromatic) groups on the amino group of low MW chitosan has been studied. In general, the antimicrobial activity of modified chitosan with alkyl chains was found to be proportional to its carbon length. As this chain increased from one to six carbons, a gradual improvement of its antimicrobial activity against both gram-positive and gram-negative bacteria was observed. All modified chitosan showed notable inhibition on bacterial growth and biofilm formation when compared to its parental compound (Sahariah et al., 2015; Sahariah, Ma´sson, & Meyer, 2018). Besides, mono-alkyl substituted chitosan and those with di-alkyl substitution of same length were found to have a similar effect in preventing bacterial growth

41

(Sahariah et al., 2015). Hence it is suggested that, on the same N position, increasing alkyl chain length has a more prominent effect on chitosan antimicrobial activity than having a greater degree of alkyl substitution. For instance, Badawy reported that the elongation of chitosan amino group with alkyl (butyl, pentyl, hexyl, octyl and N,N,N-dimethyl pentyl) substituents could greatly enhance its inhibitory activity towards bacteria and fungi (Badawy, 2010). After successfully alkyl chain grafting a 12carbon hydrophobic tail on the amino group of chitosan, Vo and Lee (2017) reported effective bactericidal effect of the N-dodecyl modified chitosan dry-coated on a glass surface, where more than 50% of E. coli suspended on the glass surface were killed in 2 h of contact time (Vo & Lee, 2017). The enhancement brought upon by the dodecyl substituent on chitosan was further confirmed via the addition of α-cyclodextrin, which counteracted the observed improvement by sequestering the hydrophobic tail. In a similar setup, Vo and Lee (2018) demonstrated that dodecyl chitosaninfused sponge, as compared to unmodified chitosan, was more effective in trapping and killing gram-negative bacteria than grampositive bacteria within the sponge (Vo & Lee, 2018). It could be inferred from this result that the hydrophobic tail of chitosan might have higher binding affinity towards the outer membrane of gram-negative bacteria as compared to the peptidoglycan layer in gram-positive bacteria which could restrict its interaction with dodecyl chitosan. However, elongation of chitosan alkyl chain does not always improve antimicrobial activity. For instance, in a series of O-quaternary ammonium salt-chitosan derivatives with alkyl bromide chains ranging up to 18 carbons long, the octadecyl derivative displayed weaker antimicrobial activity than other derivatives with shorter carbon chains (Wang et al., 2016). This indicates that there is a cut-off/critical length of alkyl chain in affecting chitosan antimicrobial activity, which

I. Background

42

2. Structure
activity relationship of biological macromolecules

aligns with the diminishing returns in antimicrobial effect for long-chain alcohols or compounds with alkyl chains exceeding the cut-off carbon length. It is likely that the increasing hydrophobicity of the given compound eventually limits its antimicrobial activity (Altay, Yapao¨z, Keskin, Yucesan, & Eren, 2015; Kubo, Muroi, Masaki, & Kubo, 1993; Łuczak, Jungnickel, Ła˛cka, Stolte, & Hupka, 2010). N,N,N-trimethyl chitosan (TMC) is a quaternized chitosan derivative with triple methyl substitution that shows considerably higher aqueous solubility as compared to chitosan (Gec¸er, Yıldız, C ¸ alımLı, & Turan, 2010). This overcomes a major challenge in deploying chitosan as an antimicrobial agent
poor solubility. In comparison to chitosan, TMC showed drastic improvement in antimicrobial activity against S. aureus and E. coli under both acidic (pH 5.5) and neutral conditions (pH 7.2) (Xu, Xin, Li, & Huang, 2010). Notably, TMC with smaller MW was found to possess better inhibitory activity against S. aureus when compared to unmodified chitosan. Increasing MW from 2 to 20 kDa was positively associated with improved TMC antimicrobial activity, albeit further MW increment showed little or no improvement (Sahariah et al., 2019). The superior antimicrobial activity of TMC (relative to chitosan) against E. coli, and S. aureus was also corroborated by Bakshi, Selvakumar, Kadirvelu, and Kumar (2018). Another hydrophilic chitosan derivative that has been widely studied is carboxymethyl chitosan (CMC) due to its better solubility governed by its degree of carboxymethylation at the N and/or O position of chitosan (Shariatinia, 2018). O-CMC and N,O-CMC demonstrated greater bactericidal effects against S. aureus than chitosan; while N,O-CMC exhibited the most prominent bactericidal effect, suggesting the degree of CMC carboxymethylation is closely related to its antimicrobial activity (Anitha et al., 2009). Besides, CMC also showed better antifungal activity against Candida albicans than

unmodified chitosan (Kurniasih, Purwati, Cahyati, & Dewi, 2018). However, as shown in a comparative study by Bakshi et al. and Rathinam et al., CMC exhibited weaker antimicrobial activity than TMC under neutral conditions as zwitterionic CMC primarily exhibits anionic charge at neutral pH, thereby having poorer solubility than TMC (Bakshi et al., 2018; Rathinam, Solodova, Kristja´nsdo´ttir, Hja´lmarsdo´ttir, & Ma´sson, 2020). When more than one substitution is simultaneously introduced to the chitosan at the N and OH position, the presence of these substituents may influence the overall bioactivity of chitosan. For instance, TMC inhibited bacteria better than most quaternary chitosan derivatives; albeit such effect declined or diminished when its O position was substituted with a carboxymethyl group (Xu et al., 2010). Besides, TMC substituted with a quaternary ammonium moiety, such as 2-hydroxy-3trialkylammonium propyl, at the O position showed a maximum of 16-fold reduction in its MIC towards S. aureus and E. coli (Xu et al., 2011). When O-CMC was used as the synthetic precursor, introduction of additional acetyl, chloro-acetyl, dimethyl aminobenzyl, benzoyl, benzoyl thiourea, acyl thiourea, thiosemicarbazone or polyvinyl substituents at either the O or N position resulted in enhanced antimicrobial activity (Mohamed & Abd El-Ghany, 2012, 2017; Mohamed, Mohamed, & Seoudi, 2014; Qin et al., 2012; Rahmani et al., 2016; Sabaa, Mohamed, Mohamed, Khalil, & Abd El Latif, 2010). A similar finding was observed when quaternary ammonium moieties introduced to N-CMC resulted in a decrease in MIC against Bacillus subtilis, Streptococcus pneumoniae and E. coli (6.25
300 μg/mL) from their respective precursors (MIC B525 μg/mL) (Mohamed et al., 2014). Overall, it appears that under many circumstances, the presence of substituents with better aqueous solubility would have positive reinforcement on chitosan’s antimicrobial activity.

I. Background

References

2.4 Conclusion Understanding the SAR of a group of biological macromolecules can play a role in improving their bioactivity and subsequent therapeutic activity. This chapter describes that the catalytic activities and consequently the bioactivities of enzymes are influenced by the type of amino acid residues at specific positions within their structure. Such changes in their sequence could alter their chemical properties and the overall three-dimensional protein structure, which may then affect substrate interaction. As observed for LAOs, lysostaphin, and MLL, their structural components are very distinct and so are their SARs. Thus, for each enzyme, the structural basis associated with their respective bioactivities needs to be scrupulously studied in order to identify the major amino acid residues that could be substituted to improve their therapeutic potential. In contrast, for chitosan, the MW appears to be an important factor influencing its bioactivities. Further chemical modifications such as deacetylation and the addition of substituents with better aqueous solubility may improve its antimicrobial and antioxidant activities. Inherently, established SARs are the key to many aspects of drug discovery, whether it is for primary screening or for lead optimization.

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936. Xuan Du, D., Xuan Vuong, B., & Mai, H. D. (2019). Study on preparation of water-soluble chitosan with varying molecular weights and its antioxidant activity. Advances in Materials Science and Engineering. Yadav, P., Yadav, H., Shah, V. G., Shah, G., & Dhaka, G. (2015). Biomedical biopolymers, their origin and evolution in biomedical sciences: A systematic review. Journal of Clinical and Diagnostic Research, 9(9), 21
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Yaghobi, N. (2012). Controlling chitosan’s molecular weight via multistage deacetylation. Biotechnology and Bioprocess Engineering, 17(4), 812
817. Yang, C. A., Cheng, C. H., Liu, S. Y., Lo, C. T., Lee, J. W., & Peng, K. C. (2011). Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323. The FEBS Journal, 278 (18), 3381
3394. Yang, Y., Guo, M., Qian, R., Liu, C., Zong, X., Li, Y. Q., et al. (2017). Binding efficacy and kinetics of chitosan with DNA duplex: The effects of deacetylation degree and nucleotide sequences. Carbohydrate Polymers, 169, 451
457. Younes, I., Sellimi, S., Rinaudo, M., Jellouli, K., & Nasri, M. (2014). Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities. International Journal of Food Microbiology, 185, 57
63. Yu, Z., Zhou, N., Qiao, H., & Qiu, J. (2014). Identification, cloning, and expression of L-amino acid oxidase from marine Pseudoalteromonas sp. B3. The Scientific World Journal, 2014. Zainal Abidin, S. A., Rajadurai, P., Chowdhury, M. E. H., Ahmad Rusmili, M. R., Othman, I., & Naidu, R. (2018). Cytotoxic, antiproliferative and apoptosis-inducing activity of L-amino acid oxidase from malaysian Calloselasma rhodostoma on human colon cancer cells. Basic and Clinical Pharmacology and Toxicology, 123(5), 577
588. Zhang, H., Li, Y., Zhang, X., Liu, B., Zhao, H., & Chen, D. (2016). Directly determining the molecular weight of chitosan with atomic force microscopy. Frontiers in Nanoscience and Nanotechnology, 2. Zhang, L., & Cui, L. (2007). A cytotoxin isolated from Agkistrodon acutus snake venom induces apoptosis via Fas pathway in A549 cells. Toxicology In Vitro: An International Journal Published in Association with BIBRA, 21(6), 1095
1103. Zhong, Z., Xing, R., Liu, S., Wang, L., Cai, S., & Li, P. (2008). Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro. Carbohydrate Research, 343(3), 566
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C H A P T E R

3 The importance of biological macromolecules in biomedicine Ahmed Olatunde1, Omar Bahattab2, Abdur Rauf3, Naveed Muhammad4, Yahya S. Al-Awthan2,5, Tabussam Tufail6, Muhammad Imran6 and Mohammad S. Mubarak7 1

Department of Biochemistry, Abubakar Tafawa Balewa University, Bauchi, Nigeria 2Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Saudia Arabia 3Department of Chemistry, University of Swabi Anbar, Khyber Pakhtunkhwa, Pakistan 4Department of Pharmacy, Abdul Wali Khan University, Khyber Pakhtunkhwa, Pakistan 5Department of Biology, Faculty of Science, Ibb University, Ibb, Yemen 6University Institute of Diet & Nutritional Sciences, Faculty of Allied Health Sciences, The University of Lahore, Lahore, Pakistan 7Department of Chemistry, The University of Jordan, Amman, Jordan

3.1 Introduction

greater specificity compared to other interventions. In this regards, 1 billion SEK was invested into this promising field by Swedish government and some major key players (Martinez et al., 2017). Based on this, this chapter gives a detailed data on the field of biological macromolecules and their use as novel interventions for various diseases.

Recently, there is an increasing interest in the use of therapeutic biomolecules in the medical field, and they are assumed to be seen as the next generation of therapeutics and pharmaceuticals (Valeur et al., 2017). Study in the field of medicine focuses mainly on biological molecules such as carbohydrates, peptides, oligonucleotides, lipids, and proteins with their subgroup monoclonal antibodies (Martinez, Mohammadi, Holowacz, Krans, & Walle´n, 2017). Biomolecules-based therapeutics have been found to be both efficacious in the management of different kinds of diseases, and have lower adverse effects as a result of their

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00003-8

3.2 Biological macromolecules in biomedicine and therapies Macromolecules such as carbohydrates, proteins, and nucleic acids have been broadly utilized for managing diseases such as cancer,

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3. The importance of biological macromolecules in biomedicine

cerebrovascular and cardiovascular diseases, and others. They are compounds with molecular weight greater than 1500 Da, making them impermeable to cell membranes (He, Dong, Gong, Wang, & Yang, 2010). The use of biological macromolecules in medicine is gaining much attention. Based on this, in the National Program for Science and Technology Development Plan (2006
2010), protein drugs were among the “Significant Scientific Research Plans.” Furthermore, biomoleculesbased therapeutics have some important merits when compared to conventional drugs. These include high organ specificity, high efficacy, and repetitive mode of action. In addition, they can be produced in large amounts through modern molecular methods, they show high solubility, they are normally active at normal body temperature, and they are not influenced by multidrug resistant issues (Buchner, Pastan, & Brinkmann, 1992; He et al., 2010). In case of proteins, it was reported that about 100 real therapeutic proteins have been recommended for use in clinics in the United States and European Union by 2011. However, there is an interest for the first approved therapeutic DNA (Dimitrov, 2012). Based on all of the aforementioned merits, biomolecules-based therapeutics have shown a promise in the management of several diseases.

3.3 Carbohydrates In the current modern era, biologically active constitutes of natural products such as carbohydrates have attracted the attention of researchers because of their pharmacological potentials. According to earlier research reports, it was claimed that carbohydrates isolated from natural sources are not toxic. These positive reports stimulated researchers to focus on carbohydrates isolation and identification followed by testing for various biological activities. Different types of carbohydrates have

been isolated from natural sources (such as wheat bran, orange peel, fungi, and lichen, among others) and were found to be significantly effective pharmacologically (Chang, 2012). The key pharmacological active class of carbohydrates is polysaccharides. These polysaccharides are important part of various cell organelles and cell membrane, and are involved in different physiological activities. Due to the involvement of carbohydrates in these biological activities, the concept of importance of carbohydrates as medicine is developing day by day. The carbohydrates-based therapies or modified form of carbohydrates are extensively used for the treatment or management of a wide range of health issues, especially in cardiovascular, inflammation, and others. The best example of carbohydratesbased therapeutic agent is heparin (Fig. 3.1) (Kilcoyne & Joshi 2007). This complex carbohydrate is used as an anticoagulant, to prevent the coagulation of blood in clinical settings. In addition to polysaccharides, other carbohydrates such as monosaccharides are used as therapeutic agents. A good example on this is mannitol (Fig. 3.2), used as a therapeutic molecule to reduce eye pressure. Pharmacologically active polysaccharides are found widely in nature for examples, dietary fibers (indigestible polysaccharides) help to promotes adequate motility in the

FIGURE 3.1 The chemical structure of heparin.

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3.3 Carbohydrates

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functions, such as, antioxidant, antivirus, antimicrobial, anticancer, antidiabetic, antistress, and immunomodulatory actions.

FIGURE 3.2

3.3.1 Therapeutics based on carbohydrates

The chemical structure of mannitol.

gastrointestinal tract and they can be used to treat various gastrointestinal disorders (PoolZobel, 2005). More so, medicinal plants accumulating polysaccharides as active chemical constitutes are responsible for their large number of pharmacological activities. A large number of pharmacologically active saccharides, ranging from monosaccharides to polysaccharides are also found in fungi. Depending upon their presence in the fungi, they can be summarized as intracellular or extracellular polysaccharides (Zhang et al., 2019). Polysaccharides found in algae and lichens are of great importance due to their exceptional physical properties such as gelling, stabilizing, and thickening capability, and biological potentials including antiviral, immunomodulating, anticoagulant, antioxidant, antitumor, antiinflammatory, and antithrombotic activity. The potentiality of biologically active polysaccharides is strongly influenced by configuration and chemical structure. Polysaccharides present in plants, animals, microorganisms (bacteria, fungi, and yeasts), and algae are chemically and/or physically bound to other biomolecules like lignin, lipids, proteins, polynucleotides, and a few minerals. Hence, understanding the importance of bioactive polysaccharides in the area of life sciences requires the multidisciplinary cooperation of scientists from the fields of phytology, microbiology, glycol-biology, food sciences, nutrition, and glycol-medicine. Polysaccharides both in the simple and complex glycol-conjugated form are famous for various bioactive

Current pharmacological studies on medicinal plants or other natural products have revealed that the principal components of herbal medicines include polysaccharides, alkaloids, and phenolic compounds among others. Among these constitutes, polysaccharides are sometimes found as active constituents and are responsible for several pharmacological activities (Tang et al., 2003). Polysaccharides and compounds derived from them are pharmacologically more preferred to synthetic polymers because of their nontoxic nature, high bioavailability, and high solubility. Stated benefits associated with polysaccharides extracted from natural sources make them a valuable ingredient in the fields of nutraceuticals, pharmaceuticals, cosmetics, and food industries (Khan & Ahmad 2013; Klein, 2009). Currently, polysaccharides are used in disease control and to promote healthcare, while numerous novel areas have also been identified; this includes cancer diagnosis and treatment, viral and bacterial managements, and in tissue engineering (Khan & Ahmad 2013; Klein, 2009). Among the types of polysaccharides derivatives, sulfated polysaccharides are mostly found in marine algae. They have numerous pharmacological potentials such as their anticoagulant characteristic. Within this context, the carrageenan (sulfated galactans) extracted from red algae and sulfated fucoidans isolated from brown algae are recognized as excellent anticoagulants. Research findings indicated that sulfated polysaccharides present in algae have equal or even stronger activities than those associated with heparin (Fig. 3.1) (Klein,

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3. The importance of biological macromolecules in biomedicine

2009). Experimentally, fucoidan has been reported to exhibit extensive antioxidant activities followed by alginate and laminaran, therefore, protecting cells from the damaging action of reactive oxygen species (ROS). Likewise, the anticancer and antitumor characteristics of algae-based sulfated polysaccharides are due to their antioxidant and free radical scavenging properties. Similarly, scientists attributed inhibition of the human immunodeficiency virus (HIV) and herpes viruses in cells to the antiviral potentials of sulfated rhamnogalactans, carrageenans, and fucoidans. In addition, algae-based sulfated polysaccharides demonstrated immunomodulating characteristics by increasing the secreting and phagocytic activities of macrophages. In this respect, polysaccharides isolated from lichens have significant immunomodulatory, anticancer, and antiviral actions (Schepetkin & Quinn 2006). Lichenin, also known as lichenan, consists of a galactoglucomannan (water-soluble hemicellulose) structure that exhibits antithrombotic and anticoagulant properties (Ullah et al., 2019). Thus, carbohydrates in biomedicine have been evidenced as significant molecules with various pharmacological actions. Consistently, antimicrobial effect of carbohydrates has been reported, where they displayed significant effect against various bacteria and viruses. The pharmacologically active potential of fucoidans (sulfated polysaccharide) obtained from marine brown seaweeds have shown significant actions against HIV and HSV (Dinesh et al., 2016; Hayashi et al., 2008). On the other hand, research findings indicated that ginseng polysaccharides have an exciting effect on dendritic cells leading to an elevated formation of interferon-g. It has also been stated that acidic ginseng polysaccharides (GP) promote production of cytotoxic cells against tumors and helped macrophages for the production of Th1 and Th2 (helper type 1 and 2) cytokines. Acidic GP also

showed modulating action on the activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase; this was attributed to the induction of regulating cytokines (Ma, Liu, Ma, Liu, & Wang, 2014). In this regard, numerous clinical trial indicated that polysaccharides in dietary fibers act as an antiobesity and antihypercholesterolemic agents (Howarth et al., 2001). Moreover, published research revealed that β-glucan, a carbohydrate, can help to regulate glycemic responses. Such interactions are affected by numerous factors such as concentration, nature of the food, and molecular weight of β-glucan among others. Based on this discussion, the dose of β-glucan is the main factor in controlling the effect of fiber on glycemic responses. In comparison to other fibers, a small dose of β-glucan is enough to decrease the insulin and postprandial glucose responses in healthy, type 2 diabetic, and hyperlipidemic subjects. Furthermore, findings showed that ingestion of breakfast consisting of 4, 6, and 8.6 g of β-glucan significantly reduced the mean concentration of serum glucose and insulin as compared to control noninsulin-dependent diabetic mellitus subjects (Tappy, Gu¨golz, & Wu¨rsch, 1996). In addition to these therapeutic effect, the polysaccharides play an important role such as antiinflammatory, wound healing, antioxidant, and others.

3.4 Peptides Peptides are widespread in nature in the form of hormones, growth factors, and neurotransmitters among others; these compounds play significant roles in normal human physiological processes (Hruby, Li, Haskell-Luevano, & Shenderovich, 1997; Sable, Parajuli, & Jois, 2017). According to published research, around 7000 peptides or peptidomimetics have been

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3.4 Peptides

reported from different natural sources (Sable et al., 2017). With respect to drug design, peptides serve as effective pharmacophores because of their selectivity and high binding affinity to carry out signaling processes in cells (Agnes et al., 2006; Hruby et al., 1997). Moreover, peptides are the natural macromolecules found in various marine fungi, algae, vegetables, and other plants (Sable et al., 2017). In this context, naturally occurring peptides are often not directly suitable for use as convenient therapeutics because they have intrinsic deficiencies such as poor chemical and physical stability, and a short circulating plasma half-life (Hui, Farilla, Merkel, & Perfetti, 2002). These aspects should be addressed before their use as medicines. Some of these drawbacks have been successfully resolved through the “traditional design” of therapeutic peptides. In addition to traditional peptide design, a range of peptide technologies has been emerging that represents the opportunities and future directions within the peptide field. These include multifunctional and cell penetrating peptides (Zhang, Wang, Xu, Zhang, & Wang, 2016). There are several functions of peptides obtained from natural sources including antimicrobial peptides (Ko´sciuczuk et al., 2012), an abundant and diverse group of molecules that are produced by many tissues and cell types in a variety of invertebrate, plant, and animal species. Furthermore, immunomodulatory peptides derived from hydrolysates of some bean proteins act to stimulate reacROStive oxygen species, which triggers nonspecific immune defense systems (Gauthier, Pouliot, & Sauveur, 2006). Some peptides act as acetylcholine esterase inhibitor (Prasasty, Radifar, & Istyastono, 2018) and control blood pressure (Martı´nez-Maqueda, Miralles, Recio, & Herna´ndez-Ledesma, 2012), while some peptides act as neuroprotective agent to treat some neurologic disorders (Meloni et al.,

57

2015). Peptides also plays important vital role in diabetes control (Zhang et al., 2011), inflammation treatment and also act as analgesic (Binder et al., 2001). Peptides are also used as nutraceutical and functional aid to complete several food preparation processes (Binder et al., 2001; Zhang et al., 2011).

3.4.1 Therapeutics based on peptides Peptides with therapeutic actions have numerous merits compared to small organic compounds and other biomolecules-based therapeutics. Because of their smaller size, they exhibit the ability to permeate further into tissues, lower production cost, and they show lower immunogenic action. Furthermore, peptides do not have long life span, and this reduces the potential of systemic toxicity because of possible risk of accumulation of peptides in tissues. In this regard, the level of toxicity posed by peptides is also decreased because the breakdown of their products are amino acids (Vlieghe, Lisowski, Martinez, & Khrestchatisky, 2010). The reducing quantity of recommended therapeutic agents manufactured by the drug producing industries caused elevated costs for the manufacture of new drugs, and this has brought about different strategies to increase productivity in the pharmaceutical industries (Martinez et al., 2017). Due to the advantages of peptides over other therapeutic agents, there is an increasing interest in the use of these molecules as novel drug candidates (Vlieghe et al., 2010). Currently, the Food and Drug Administration (FDA) has approved more than 60 therapeutic peptides and this amount is proposed to elevate remarkably due to about 140 bioactive peptides in clinical trials and more than 500 in preclinical studies. In addition to all these features of peptides, they can serve as candidates for the manufacture of commercial agents (Fosgerau & Hoffmann 2015).

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Ferring Research Institute, a pharmaceutical industry based on R&D of bioactive peptides and other biomolecules-based therapeutics, was committed to forming a portfolio of new, innovative peptide-based therapeutics and, hence, they are investing significantly in the designing of new therapeutic agents (http://www.ferringresearch.com/research-development/research/). In addition, the Ferring Research Institute present the recent status in the development of bioactive peptides (Lau & Dunn 2016). Although peptide therapeutics have been highly successful when compared to other biomolecules-based therapeutics, they cannot be efficiently used in the treatment of all diseases and to all biological targets. In this respect, biomolecules-based therapeutics are normally not used for intracellular targets because of their inadequate membrane permeability. In particular, peptides exhibit intrinsic weaknesses such as poor physical and chemical stability, short life span in plasma, and inadequate cell permeability. These demerits should be tackled and an ideal plan should be put in place to improve peptide characteristics by converting them into nonnatural molecules and which could be stabilized by various means (Martinez et al., 2017; Waldmann et al., 2017).

3.5 Proteins Proteins are the basic structural components of cell organelles and involved in different physiological reactions (Tien, Berlett, Levine, Chock, & Stadtman, 1999). Involvement of proteins in these physiological processes is a significant indicator that protein-rich molecules might be used as effective biomedicines. Recently, several protein-rich drugs such as insulin, oxytocin, and vasopressin have been documented. These remarkable therapeutic agents showed that the precursors (amino acid) may display alleviate several disease

conditions if properly explored. Proteins are found in plants, animals, microbes and other sources (Nehete, Bhambar, Narkhede, & Gawali, 2013). The proper extraction and identification of these macromolecules might lead to discovery of various significant drugs for the treatment of different health problems. Researchers have reported that protein-based therapeutics display antibacterial, antifungal, antiviral, and anticancer activities (Levy, 2004). These macromolecules exhibit significant effects in the treatment of hepatoprotective and cardiovascular problems.

3.5.1 Therapeutics based on proteins (proteins and monoclonal antibodies) 3.5.1.1 Protein-based therapeutics Protein-based therapeutics are used in medicine for the management of different diseases (Claesson, Danielsson, & Svensson, 2005). These macromolecules are vital for several biological functions in humans, and the deficiency of proteins impairs normal tissue/cell processes. Protein-based drugs are usually large, characterized by peptidelike structures, and consist of more than one thousand amino acids bridged together by peptide linkages. Compounds containing over one thousand amino acids lead to high molecular weight, which in several cases can reach 150, 000 g/mol (Bayer, Small, & large molecules Internet, 2015). 3.5.1.2 Monoclonal antibodies Monoclonal antibodies (mAb) are produced by cloning a single white blood cell and consist of identical antibody molecules. These white blood cells are called leukocytes or leukocytes, which are cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. Leukocytes are found throughout the body, including the blood and lymphatic system

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3.5 Proteins

(Maton et al., 1997) The uniqueness of monoclonal antibodies has contributed to their broad use in the management of diseases such as Rheumatoid arthritis, kidney transplant rejection, cancer, and Crohn’s disease (Liu, 2014; Martinez et al., 2017; Smith, 2012). One of the most successful areas with the conjugated monoclonal antibodies is oncology. In this field, significant efficiency coupled with reduced detrimental effects as a result of high specificity of conjugated monoclonal antibodies were observed. Laboratorial and evolutionary monoclonal antibodies are the two classes of the antibodies. Furthermore, the evolutionary monoclonal antibodies has four main types, which are subgrouped into first, second, third and fourth generations (Scott, Allison, & Wolchok, 2012; Smith, 2017; The American Cancer Society medical & editorial content team, 2017). 3.5.1.3 First generation monoclonal antibodies The simplest type of antibody are the murine antibodies, which are made up of mainly one genetic information from a nonhuman source. Rats and mice are used as donor animals for the production of this type of antibodies. The first therapeutic mAb, muromonab-CD3 (Orthoclone OKT3), was approved by the United States FDA in 1986 (Ecker, Jones, & Levine, 2015) and comprises a murine mAb against T cell-expressed CD3 that functions as an immunosuppressant for the treatment of acute transplant rejection. In addition, mAbs are increasingly used for a broad range of targets; oncology, immunology, and hematology remain the most prevalent medical applications (Grilo & Mantalaris 2019). Most mAbs have multiple disease indications and at least one that is cancer-related (lymphoma, myeloma, melanoma, glioblastoma, neuroblastoma, sarcoma, colorectal, lung, breast, ovarian, head and neck cancers). The important outcome of applying this type of antibody is that to the human body, it is completely foreign. Based on

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this, the immune system will stimulate responses which could result in a deadly consequence (Martinez et al., 2017; Smith, 2017). 3.5.1.4 Second generation monoclonal antibodies These antibodies are genetic mixture of substances from nonhuman (e.g., mouse) and human origin (http://www.thechairmansblog. com/mabvax-therapeutics/david-hansen/ when-is-a-fully-human-antibody-really-fullyhuman; http://pdl.com/technology-products/ what-are-humanized-monoclonal-antibodies/). However, one third of genetic material from mouse and two thirds of genetic material from humans made up the chimeric monoclonal antibody (Martinez et al., 2017; Smith, 2017). The specific combination of two different genetic substances in the same amount have the characteristics of decreasing the immunogenicity (which is an estimation of antigens potential to activate the development and synthesis of specific antibody). The second generation monoclonal antibody have been found efficacious when utilized in the management of several autoimmune disorders (Smith, 2017). 3.5.1.5 Third generation monoclonal antibodies The third generation of mAb are also a genetic combination of two different genetic substances, similar to the second generation of monoclonal antibody. The difference between the two monoclonal antibodies is the number of genetic materials. The humanized monoclonal antibodies are synthesized in a controlled conditions and have about 90 per cent of human genetic material with small amount of nonhuman (10%) (Martinez et al., 2017). In this case, the nonhuman genetic material is from mouse. The nonhuman portion of humanized monoclonal antibody interacts with a particular antigen while the other portion is guided and not subject to binding with the immune system in the body. This reduces the possibility

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of elimination of the antibody (Duvall, Bradley, & Fiorini, 2011; Harding, Stickler, Razo, & DuBridge, 2010). The third generation antibody was reported to be effective in the management of different types of diseases (Martinez et al., 2017). Recently, an excellent review that deals with the development of therapeutic antibodies for the treatment of diseases was recently published by (Lu, Hwang, & Liu, 2020). 3.5.1.6 Fourth generation monoclonal antibodies The fourth generation of mAb are completely produced in the laboratory. Thus, these antibodies are synthesized from genes of humans and this implies that the fourth generation does not have any part of murine. Furthermore, the lack of murine part in this antibody decrease the risk of a stimulated immune stimulus. The first therapeutic agent under this class is adalimumab, also known as Humira designed by AbbVie Inc (Harding et al., 2010; Martinez et al., 2017).

3.6 Lipids Lipids are the fats, which are obtained from either animal or vegetal sources. These include lard, butter, vegetable oil, and waxes among others. Lipids are either synthetic or natural organic compounds composed of long hydrocarbon chains, and are esters of fatty acids. They are insoluble or sparingly soluble in water, and are impermeable because they are hydrophobic and nonpolar in nature, and that is why they are used for coating materials and films in the food substances (Pe´rez-Gago & Rhim 2014). Lipids are vital components of all living organisms and play a very important role in numerous biological processes. The structural configuration of all lipids are important factors to contribute in a biological process. Lipids also facilitate the process of drug absorption. In this respect, some parts of the

human body is densely coated with lipids such as blood brain barriers. Therefore it is very important to increase the lipid solubility of a drug to easily cross the blood brain barriers. Furthermore, the human body is approximately composed of 37.2 trillion cells, and each cell is surrounded by lipid bilayer; thus the body is more lyophilic. Therefore to improve the pharmacokinetic of a drug, a proper lipid solubility is needed, and hence, body lipids are important drugs targets. Within this context, arachidonic acid, prostaglandins, and leukotrienes, among others are the key eicosanoids responsible for different diseases. Accordingly, lipids are not only the biological components but also very important therapeutic agents (Charman, 2000). Due to these facts, lipids are very good excipient to improve the bioavailability of hydrophilic dosage forms (Nanjwade et al., 2011). Lipids can be used as biomedicines as well as in pharmaceuticals and cosmeceuticals. In this respect, lipid nanoparticles (LNs) are the most commonly used for dermal and transdermal application (Harding et al., 2010). LNs possess essential characteristics as biocompatibility, biodegradability, selfassembly, versatile particle size and low cost, making them attractive tools in cosmetics formulations (Lu et al., 2020).

3.6.1 Drug delivery-based on lipids Lipid-based agent delivery systems have received significant interest as a result of their remarkable characteristics. Lipids have several merits as a delivery system for drug including reduced levels of toxicity, enhanced modulated drug targeting, elevated drug release potential, and others (Puri et al., 2009). There are several methods for incorporating lipids to active molecules. Liposomes are spherical substances and have phospholipids bilayers for coating therapeutic agents. This spherical vesicles were first documented in the 60 s and in the mid-

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3.7 Nucleic acids and oligonucleotides

90s, first lipid delivery system was introduced into the market as Doxil. The first use of lipids as therapeutic agent delivery in pharmaceutics was in the management of cancer and lipid transporters are providing promising enhancement in the field (Martinez et al., 2017). Furthermore, LNs are the most commonly used for dermal and transdermal application (Neubert, 2011). LNs possess essential characteristics as biocompatibility, biodegradability, self-assembly, versatile particle size and low cost, making them attractive tools in cosmetics formulations (Duan et al., 2020).

3.7 Nucleic acids and oligonucleotides The most vital and important biological macromolecules are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are made of nucleotides. DNA is a macromolecule, which stores genetic information for long terms. There are approximately three billion nucleotides present in 23 pairs of chromosome in every nucleus of the cell. On the other hand, RNA is a more transient molecule found in the nucleus and cytoplasm and used in protein synthesis from the genetic information stored in DNA. RNA has a single strand whereas DNA is made up of two polynucleotide strands that are twisted together into a double helix shape. This structure was first described by Watson and Crick in their groundbreaking research (Watson & Crick 1953). Nucleotide monomers consist of nitrogen base, 5-carbon sugars, and a phosphate group. RNA contains ribose (C5H10O5) whereas, the deoxyribose (C5H10O4)is found in DNA. There are five types of nitrogenous bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), the genetic information is transported in nucleic acids in exact order of nucleotides. Importantly, thymine is only found in DNA, whereas uracil is only found in RNA (Gardner, Duprez, Stauffer, Ungu, & ClausonKaas, 2019).

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On the other hand, oligonucleotides are biomolecules with a particular general structure. These compounds have short deoxyribonucleic acid features. Oligonucleotides are short singlestranded RNA/DNA compounds, that is, siRNA and they have about 13
25 nucleotides. Oligonucleotides are often used as probes to investigate complementary sequences (Martinez et al., 2017).

3.7.1 Therapeutics based on oligonucleotides The aim of applying nucleic acids as therapeutic molecules was initially invented in the 1970s by introducing antisense oligonucleotides. Nucleic acids have some merits over small therapeutic agents when used as drugs. This includes using low doses, shunting several cascades that are used for small therapeutic agents, and cost effectiveness. In addition, they are specific, thus, they can minimize detrimental effects, which are observe with small therapeutic agents, and display longer period of action when compared to conventional agents. Therapeutics involving nucleic acid are grouped into four classes including RNA ribozymes, interfering RNA (RNAi), antisense oligonucleotides, and aptamers (Atyabi, Zahir, Khonsari, Shafiee, & Mottaghitalab, 2017). The first application of oligonucleotidesbased therapeutics was initiated in 1978, when Zamecnik and Stephenson proposed that nucleotides have the potential to abrogate Rous sarcoma virus replication. However, by 1986, when oligonucleotides were applied against HIV, industries initiated investment in the synthesizing of oligonucleotides as therapeutic agents. During this period, the procedure was challenging and understanding on molecular level was still speculative, which led to few industries surviving (Khvorova & Watts 2017; Martinez et al., 2017). It took about 40 years before oligonucleotide therapies could reach

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the clinical state (Khvorova & Watts 2017). Currently, therapies based on oligonucleotides are presumed to indicate great possibility in the management of different ailments. Oligonucleotides can be used to target anything via Watson-Crick base-pairing (Levin, 2017). The area of oligonucleotide-based therapeutics is quite comprehensive. Based on this concept, complementary sequence with a target RNA, which controls the expression of gene leads to inhibition of disease-inducing proteins. Consistently, the transportation of a target drug to the ideal cells and organs has been a main setback in the design of oligonucleotide-based therapeutics. Also, drug delivery-based on target is vital and procedures including several forms of drug conjugates are now being considered (Khvorova & Watts 2017; Levin, 2017; Waldmann et al., 2017). The different types of oligonucleotidebased therapeutics include antisense oligonucleotides, aptamers, small interfering RNA (siRNA) and others (Levin, 2017). Among the five FDA recommended oligonucleotide-based therapeutics, one is an aptamer and the remaining four are antisense oligonucleotides. Nevertheless, research is ongoing for therapeutic siRNA and significant success has been seen with targeting hepatocytes (Khvorova & Watts 2017). 3.7.1.1 Antisense oligonucleotides Antisense oligonucleotides are synthetic strings of RNA/DNA precursors that react with RNA-strain and reduce its action. Watson-Crick base-pairing is used to target RNA by antisense oligonucleotide. In addition, antisense oligonucleotide can target either messenger RNA (mRNA) or microRNA (miRNA), thus, preventing RNA degradation and promoting splicing (Waldmann et al., 2017). Compared to conventional therapeutic agents, which mainly react with proteins, antisense oligonucleotides change the expression of gene (translation of mRNA), which leads to

inhibiting the function of a particular protein. This causes a significant potential in the management of severe and heritable disorders, because they abrogate or slow the progression of diseases. In 1998, formivirsen (the first antisense oligonucleotide), received its FDA’s recommendation, however, it was withdrawn from circulation after few years (Isaacson & Evertts 2016; Martinez et al., 2017). Currently, four antisense oligonucleotides, including fomivirsen have the FDA’s recommendation and several other therapeutic agents are under clinical trials (Martinez et al., 2017). 3.7.1.2 Small interfering RNA Small interfering RNA (siRNA) is an RNA sequence, which is double-stranded and reacts with RNA-induced silencing complex (RISC). During the process of transcription, when argonaute protein binds with microRNA, the RISC complex forms and is responsible for the process of gene silencing, thus called RNA interference (RNAi). The capacity to influence silencing of gene has led to remarkable investment in the field of pharmaceutics (Waldmann et al., 2017). In this respect, it is worth mentioning that numerous siRNA-based therapeutic agents are under clinical trials, and this includes treatment for hepatitis B (Khvorova & Watts 2017). Drug delivery-based on tissue/ organ target has been a significant setback in the design of therapeutic siRNA. However, LNs have shown to be promising in the delivery of drugs to organs/tissues, and conjugation of lipids to siRNA were found to promote silencing of gene in tissues (Khvorova & Watts 2017; Waldmann et al., 2017). In addition, siRNA conjugation to Nacetylgalactosamine displayed significant results in suppressing gene expression when targeting liver mRNA. Along this line, Nacetylgalactosamine is a remarkable ligand for the receptor of asialoglycoprotein observed in liver. For this, conjugated siRNAs provides better stability to nucleases and displays greater

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3.8 Synthesis of macromolecules

pharmacokinetics (Khvorova & Watts 2017; Nair et al., 2014). Most of the documented clinical trials for siRNAs are for targeting cancer (Lundin, Gissberg, & Smith, 2015).

3.7.1.3 Aptamer Aptamers are oligonucleotide-based therapeutics and can bind to large and small molecules, mainly proteins, and are not like other therapeutic oligonucleotides that can react with RNAs (Waldmann et al., 2017). Moreover, they are characterized by remarkable affinity and specificity, which rely on their capacity to give 3-dimensional structures (Lundin et al., 2015). Their major function is to control a particular action of protein. Pegaptanib is the first aptamer to be recommended by the FDA in 2004. Other possible therapeutic agents are in clinical trials. Furthermore, this oligonucleotide-based therapeutic was utilized as a delivery system for drugs (Lundin et al., 2015; Waldmann et al., 2017).

3.8 Synthesis of macromolecules Synthesis of biological macromolecule is not as easy task and simple organic compounds are required. Merrifield method was developed for the chemical synthesis of biological macromolecules, which are more practical. These molecules can be used individually or combined together depending on their nature and the desired functionality of the target delivery system. Within this context, the practical synthesis of oligonucleotides, oligopeptides, and oligosaccharides were limited as their chain length was limited to solid phase method. Native chemical ligation, which is similar to oligonucleotide, ligation was used for the synthesis of polypeptides (Kent, 2009). In a similar fashion, ligation was used for the synthesis of more complicated

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macromolecules, which is also helpful in bioorthogonal chemistry. Numerous types of complex molecules were prepared including new synthetic polymer
bio macromolecular conjugates and biologically processed bio macromolecules. Stimuli-responsive polymers were used for medical and bio-industrial applications, by employing conjugation of biological macromolecules with poly(ethylene glycol) (PEG) (Velonia, 2010). For protein activation, the post-translational modification of proteins has been known for many decades. Several types of protein are classified according to their biological effects including ubiquitylation for proteolysis, glycosylation for extending protein half-life, targeting, and phosphorylation for signal transduction and cell
cell interactions. In this respect, Kumar and coworkers succeeded in obtaining the largest chemically synthesized polypeptide composed of 304 residues, which corresponds to folded K48-tetraubiquitin. The method employed coud be applied to any of the remaining tetraubiquitin chains, and should ultimately assist ongoing efforts to unravel how the remarkable diversity of ubiquitin signaling is achieved (Kumar et al., 2011). Similarly, Broncel and colleagues applied a semisynthetic strategy to generate a phosphorylated and biotinylated fully functional Alzheimer’s disease-related tau protein. The presented methodology allows for an unambiguous verification of individual phosphorylation sites on tau and significantly improves its purification (Broncel, Krause, Schwarzer, & Hackenberger, 2012). Protein glycosylation is one of the most complex post-translational modification process. In this respect, more than 50% of proteins in humans are glycosylated, while bacteria such as E. coli does not have this kind of modification. In addition, numerous small-molecule natural products require glycosylation to express their function. Syntheses like these will help tackling problems associated with

I. Background

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3. The importance of biological macromolecules in biomedicine

carbohydrate-mediated biological recognitions (Wong, 2005). To explain the role of glycoprotein it is very important to synthesize “glycoforms” in a homogenous pattern (Gamblin, Scanlan, & Davis, 2009). In this regard, Kajihara and coworkers used an adopted strategy to produce high yield of glycoprotein by employing extracted oligosaccharides instead of applying the general strategy of following different steps in sequence starting from monosaccharaides (Kajihara, Yamamoto, Okamoto, Hirano, & Murase, 2010). Furthermore, Kajihara and colleagues published a good review that describes progress related to the efficient method of oligosaccharide preparation and synthesis of glycoproteins including bioactive erythropoietin (Kajihara et al., 2010). Interestingly, the drug that is used for the treatment of renal anemia is known as erythropoietin (EPO), which is a heavily glycosylated protein. The drug consist of O-linked oligosaccharides, which contains 166 amino acids, where complex-type oligosaccharides are attached to the different sites such as Asn24, -38, and -83 and has 3 unlinked oligosaccharides. The half-life of EPO is increased by three complex-type sialyloligosaccharides, in blood they are present in three different structure like tri tetra-antennary-type (Lai, Everett, Wang, Arakawa, & Goldwasser, 1986).

3.9 Biomedicine The designation “Biomedicine” emphasizes the fact that this is primarily a biological medicine. In other words, it is a medicine based on the application of biological and physiological principles to clinical practice. The label Biomedicine was for these reasons conferred by (Gaines & Hahn 1985) on what had variously been labeled “scientific medicine,” “cosmopolitan medicine,” “Western medicine,” “allopathic medicine” and simply, “medicine” (George & Engel 1980). During the 1950s and

1960s a sociologist conducted a study related to biomedicine, which showed that biomedicine first came into the anthropological observation as a product of studies that required considering it as a professional medicine of other “Great Traditions” rather than the common or “ethnomedicines” of traditional in small-scale cultures. Biomedicine is the unique domain where knowledge is based on both features specialized knowledge and distinct practices (Gaines, 1979). The important factor in a medical system is the relationship between medical knowledge and medical practice (Gaines, 1982). In this respect, somatic interventions make sense when biomedicine defines the universe biologically and the action made is more reasonable and justified in the form of medical knowledge. In addition, biomedicine shows the rules and guidelines in its social and clinical trials also, and represents the order of division in labor. The orders of medicine are complex and multifaceted. In this regard, the intensive somatic interventions, which are naturallybased, are more highly valued. Therefore the surgeons have higher esteem and compensation than a family doctor and physiatrist. Similarly, less reliable treatment of women, children, and older people, is done by biomedicine (Gaines, 1992). In addition, some social structures, which are specific to the biomedicine domain, may include different categories such as principles, generative rules, and social identities, and the wider society includes sexual and gender identities (Hinze, 1999). Biomedicine is known as cohesive system; it is introduced by itself for studying the already established practices. Additionally, it is guided by mentors to pass it on to their students. Practitioners are taught to believe that biomedicine is science based, In this regard, the tradition of science is extremely resistant to changes but science adopts changes more quickly than biomedicine.

I. Background

References

3.10 Conclusions Biomolecules-based therapeutics such as carbohydrates, proteins, lipids, and oligonucleotides have acquired great attention from researchers as therapeutic agents in the field of biomedicine and other related areas. Their therapeutic application is attributed to their nontoxic, bio-degradable, and high bioavailability nature. Furthermore, isolation of naturally occurring pharmacologically active biological molecules is associated with high purity, maximum extraction yield, and stability of native structure. However, significant results to validate the application of these therapeutic biological macromolecules as novel agents in biomedicine will require a multidimensional approach from scientists of numerous arenas such as food science, healthcare, material science, organic chemistry, and engineering, among others.

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C H A P T E R

4 Modification techniques for carbohydrate macromolecules Ajay Vasudeo Rane1, Deepti Yadav2 and Krishnan Kanny1 1

Composite Research Group, Department of Mechanical Engineering, Durban University of Technology, Durban, South Africa 2Department of Biotechnology and Food Science, Durban University of Technology, Durban, South Africa

4.1 Introduction

with carbohydrate macromolecules. Let us now discuss the molecular structure of cellulose, hemicellulose, lignin, chitin, and chitosan before discussing the techniques used for modification of carbohydrate macromolecules.

Large molecules which are necessary for life and are built from smaller organic molecules are known as biological macromolecules. Four major classes of biological macromolecules which are listed in the Fig. 4.1. This chapter provides description of research related to modification of carbohydrate macromolecules specific to nonstarch polysaccharide carbohydrates, using physical and chemical process. Carbon, hydrogen, and oxygen are the important constituents of a carbohydrate with an atom ratio of 2:1 for hydrogen: oxygen. The term carbohydrate is synonym to saccharide. The three principal types of saccharides based on their degree of polymerizations (molecular weight) as shown in Fig. 4.2. This chapter is centered on the very timely topics of modification on macromolecules like cellulose, hemicellulose, chitin, chitosan, and lignin. Cell wall of plants have lignin in close association with cellulose and hemicellulose and forms an integral part of discussion along

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00004-X

4.2 Cellulose Cellulose is the most abundant naturally occurring biopolymer. Cellulose is partitioned in almost cell walls of plants, organisms and naturally occurring chemical compounds, with major partitioned towards plants. The primary cell wall of all the terrestrial and aquatic plants is a dynamic structure; and cellulose provides mechanical stiffness. Molecular structure of cellulose molecule is independent of the source from which cellulose is extracted. Cellulose is linear homopolymer organized in a crystalline state. Molecular representation of a cellulose molecule can be seen in Fig. 4.3. As seen from the Fig. 4.3, chemical structure of cellulose comprises of D-glucopyranose ring units

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FIGURE 4.1

4. Modification techniques for carbohydrate macromolecules

Biological macromolecules.

FIGURE 4.2 Principle groups of carbohydrates macromolecule.

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4.3 Hemicelluloses

FIGURE 4.3 Molecular structure of a cellulose molecule-Numbering system for carbon atoms in a cellulose molecule.

FIGURE 4.4 Molecular structures of (A) Hemicellulose and sugar units in a hemicellulose substrate (a) Glucose, (b) Galactose, (c) Mannose, (d) Glucuronic acid, (e) Rhamnose, (f) Arabinose, (g) Xylose.

linked by β-1,4-glycosidic bonds with three reactive hydroxyl groups exists in each anhydro glucose unit—a primary hydroxyl group at C6 and two secondary hydroxyl groups at C2 and C3 (Dufresne, 2017; Rojas, 2016). Reports confirm that hydroxyl group at 6 position reacts 10 times faster than hydroxyl groups at 2 and 3 positions, and hydroxyl group at 2 position reacts 2 times faster than hydroxyl group at 3 position. This characteristics of cellulose substrates facilitates the modifications. Approaches like grafting to, grafting from, siloxane formation, urethane formation, esterification, cationization, oxidation, physicochemical adsorption, hydrophobization, and oleophobization are used for surface modification. Partial esterification and partial

oxypropylation are used to carry out in-depth modification (Rojas, 2016).

4.3 Hemicelluloses Biosynthesized in nature by plants, and its close association to cellulose and lignin; hemicellulose forms the important constituents in cell wall of plants by holding together the cellulosic microfibrils and acting as an interphase within the lignin matrix (Gatenholm & Tenkanen 2003). Hemicelluloses are often branched and organized in a noncrystalline state. Sugar units of different types (see Fig. 4.4a-g) forms the major composition of hemicellulose. Hemicelluloses displays a

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FIGURE 4.5

4. Modification techniques for carbohydrate macromolecules

Molecular structures of lignin and its phenylpropane units (phenolic precursors/monolignols).

number of chemical structures depending on the sugar units in a hemicellulose substrate; molecular representation of a hemicellulose molecule can be seen in Fig. 4.4A. Chemical structure of hemicelluloses are characterized by the presence of β1, 4-linked-D-xylopyranosyl as the main chain with a number of monosaccharide substituents. Modification techniques such as oxidation, reduction, partial hydrolysis, esterification and etherification of the hydroxyl groups are well reported (Sataloff, Johns, & Kost, 2018) (Fig. 4.8).

molecular weight of lignin becomes difficult. Lignin’s molecular weight exceed 10,000 u. Lignin has a large number of aromatic units and hence hydrophobic. Types and structure of a lignin differs with respect to the method of isolation. Modifications such as acetylation and catalytic techniques with photo stabilization of lignin functional groups are reported (Glasser & Sarkanen 1989; Thomas, 2002).

4.5 Chitin and chitosan 4.4 Lignin Complex organic material that forms close association with cellulose and hemicellulose in the plant cell wall is the Lignin. Lignin forms the support tissue in the wood and bark region of the plant. Precursor lignols (see Fig. 4.5) crosslink in a number of ways to form a heterogenous macromolecule, that is, lignin. Para-coumaryl alcohol (4-hydroxyphenylpropane), coniferyl alcohol (4-hydrox-3-methoxyphenylpropane) and sinapyl alcohol (3, 5-dimethoxy-4-hydroxyphenylpropane) (see Fig. 4.5) are the three lignols that crosslink in a number of ways to form complex structure of lignin and hence determining

Chitin, a polysaccharide is one of the components found in the integument of a crustaceans. Acetylglucosamine as shown in Fig. 4.6 is the repeating unit of chitin linked together in β-1,4 manner. Chemical structure of chitin is closer to chemical structure of cellulose, wherein acetylamino group replaces one hydroxyl group on each repeating unit. Increased probability of forming a hydrogen bonding with chitin can be attributed to the presence of acetylamino group. Deacetylation of chitin produces chitosan; hence chitosan is a chitin derivative. Random alignment of deacetylated and acetylated units in a liner manner forms the chemical structure of chitosan. β-(1,

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4.6 Modification of carbohydrate biological macromolecules

FIGURE 4.6

Molecular structures of Chitin (left) and Chitosan (right).

FIGURE 4.7

Classification of modification.

4)-linked D-glucosamine is the deacetylated unit and N-acetyl-D-glucosamine forms a acetylated unit in a chitosan’s chemical structure (Eugene & Wan 2014; Muzzarelll, 1977; Pillai, Paul, & Sharma, 2011; Zikakis, 1984).

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4.6 Modification of carbohydrate biological macromolecules Environmental challenges associated with the use of petroleum-based products (synthetic

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4. Modification techniques for carbohydrate macromolecules

plastics) has prompted intensive research towards the naturally occurring biological macromolecules. Naturally occurring polymers (called biopolymers) are omnipresent in the form of plant biomass or microbial cell wall structure. Owing to their unique properties, biopolymers can be utilized in various applications such as drug delivery, composite, biofuels or packaging material. Despite the availability and being readily available, use of biopolymers is limited due to several challenges. In order to efficiently exploit the biopolymers modification is required which could potentially improve their solubility, compatibility, biodegradability etc. Biological macromolecules are abundantly

FIGURE 4.8

Modification techniques for hemicellulose.

available in nature. Their unique chemical composition can be efficiently exploited for valorization to produce novel functional materials. In this regard, this chapter discusses strategies (physical, mechanical and biological methods) for functionalization on biomaterials. Furthermore, functionalised biomaterials has shown its applicability in various fields such as catalysis, pollutant removal and supercapacitors. Further, in this chapter, we discuss and summarize the different biopolymers, their surface modification as well the applications of functionalised biopolymers. Additionally, the chapter also describes the developments made in this field along with issues and suggestions concerning with functionalization of biopolymers. Fig. 4.7 gives the classification of the modification based on the nature and the treatment process used for modifying carbohydrate biological macromolecules. Let us now further look into the different techniques used to modify the biological macromolecules, the Tables 4.1
4.8 describes the recent techniques to modify the biological carbohydrate macromolecules.

TABLE 4.1 Physical modification of cellulose. Physical treatment

Biopolymer

Properties imparted

References

Inert plasma

Cotton cellulose fibers

Change in hydrophilicity

(Kola´rˇova´, Vosmanska´, Rimpelova´, & ˇ Svorˇ c´ık, 2013)

O2-plasma pre-treatment

Cellulosic fabrics (linen, Improved fixation of Ag nanoparticles cotton, and lyocell) onto the tested fibers leading to enhanced antibacterial activities.

(Abdel-Aziz, Eid, & Ibrahim, 2014)

N2- or O2- plasma pre-treatment

Different cellulosic substrates namely cotton, linen, viscose, and lyocell

Improvement in antibacterial activity against gram positive (S. aureus) and gram negative (E. coli) pathogens

(Ibrahim, Eid, & Abdel-Aziz, 2017)

Oxygen plasma treatment

Coir fibers

Tensile strength increased along with enhanced fiber-matrix adhesion after plasma treatment

(de Farias et al., 2017) (Continued)

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4.6 Modification of carbohydrate biological macromolecules

TABLE 4.1 (Continued) Physical treatment

Biopolymer

Properties imparted

References

Plasma enhanced chemical vapor deposition (PECVD)

Coconut waste (CW) particles

Enhanced thermal, mechanical, wettability and flammability properties

(Kocaman, Karaman, Gursoy, & Ahmetli, 2017)

Air plasma treatment

Cellulose fibers

Improvement in matrix (thermoplastic starch)/cellulose fiber adhesion

(Fazeli, Florez, & Sima˜o, 2019)

DC air plasma along with cellulase enzyme

Cotton fabric samples

Increased hydrophilicity of the cotton fabrics

(Nithya et al., 2011)

Oxygen plasma treatment followed by Cu-acetate or Zn-acetate treatment exhibited best UV-protection or antibacterial activity respectively

(Ibrahim, Eid, Youssef, ElSayed, & Salah, 2012)

Different plasma gases (oxygen, air, Cellulose-containing and argon) followed by subsequent fabrics treatment with certain metal salts

TABLE 4.2 Chemical modification of cellulose (Bezerra et al., 2015). Mode of functionalization Functional group Chemical

Reaction conditions

Carboxymethylation Reaction of alkaline cellulose with chloroacetic acid or its sodium salt, resulting in sodium carboxymethylcellulose (CMC)

Applications Thickener in food industries, stabilizing emulsion, as drilling mud in petroleum industry, stabililization of silver nanoparticles, component of detergents, toothpaste, paper products

Phosphating

Homogenous reaction is performed in Adsorption of heavy metals from molten urea at 413 K. Reaction consists aqueous medium of cellulose, water and phosphoric acid to form disubstituted and trisubstituted form of celluloseHeterogenous reaction is performed in aqueous medium by adding 85% H3PO4 in cellulose. The reaction is performed at 373 K for 30 min.

Acylation

Reaction with cyclic organic anhydrides resulting into covalent incorporation of carboxyl groups

Increase in adsorption capacity of cellulose

Amination

Incorporation of nitrogen in cellulose

Raise in contaminant’s adsorption capacityEg modified cellulose 2aminomethylpyridine is effective in removal of divalent cations (Cu21, Co21, Ni21, and Zn21)

Sulfonation

Sulfur incorporation in cellulose structure (ethylenesulfide, sodium bisulfite, aminoethanethiol)

Contaminant removal of the aqueous mediumHigh capacity in extracting cations from aqueous medium

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4. Modification techniques for carbohydrate macromolecules

TABLE 4.3 Enzymatic modification of cellulose. Enzyme used

Functionality imparted

Properties

References

Laccase

Oxidation of cellulosic pulps

Increased water retention capacity and burst strength owing to the formation of inter-fiber hydrogen bonds

(Patel, Ludwig, Haltrich, Rosenau, & Potthast, 2011)

Grafting of phenolic compounds (Caffeic acid and isoeugenol) on cellulose

Developed antimicrobial cellulose packaging

(Elegir, Kindl, Sadocco, & Orlandi, 2008)

Grafting of lauryl gallate onto cellulose fibers

Improved hydrophobicity lead to reduced water penetration

(Cusola, Valls, Vidal, & Roncero, 2013; GarciaUbasart et al., 2012)

Hydrolases (Subtilisin, Lipase, esterase, cutinase)

Acylation led to the formation of cellulose esters

Applicability in the production of fibers, plastics, film, drugs, and cosmetics

(Karaki, Aljawish, Humeau, Muniglia, & Jasniewski, 2016)

Hexokinases

Phosphorylation reactions

Cellulose phosphates possess ability to bind calcium ions, therefore find its applicability in bone regeneration

(Karaki et al., 2016; Tzanov, Stamenova, & Cavaco-Paulo, 2002)

β-galactosidase

Catalyzed the transfer of galactose moiety to of HEC hydroxyl groups

Galactosylated HEC finds its applicability as thickening agent, rheology modifier and protective colloids

(Li, Cheng, Nickol, & Wang, 1999)

Finds its applicability as waterrepellent materials, oil adsorbents, and biodegradable detergents

(Kusumi, Lee, Teramoto, & Nishio, 2009; Li, Xie, Cheng, Nickol, & Wang, 1999)

Copolymerization reactions Lipases

Enzyme opened ε-caprolactone and catalyzed its polymerization onto hydroxymethyl cellulose film

Phosphorylases

Coupling of cellulose with amylose The copolymer was able to form films and stronger gels

(Kadokawa, 2012; Kaneko, Matsuda, & Kadokawa, 2007)

TABLE 4.4 Chemical modification of Lignin. Functionalization of

Functional group

Aromatic rings

Property/Application

References

Amination

Products with practical applications such as polychationic materials, slow-release fertilisers and surfactant chemicals

(Du, Li, & Lindstro¨m, 2014)

Hydroxy alkylation

Synthesis of lignin
phenol
formaldehyde (LPF) resin adhesives

(Yang, Zhang, Yuan, & Sun, 2015) (Continued)

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4.6 Modification of carbohydrate biological macromolecules

TABLE 4.4 (Continued) Functionalization of

Functional group

Property/Application

References

Nitration

Sustainable electro-catalyst for oxygen reduction

(Graglia, Pampel, Hantke, Fellinger, & Esposito, 2016)

Urea/ formaldehyde condensation

Lignin-urea-formaldehyde resin can reduce (Jiao, Xu, Cao, Peng, & She, 2018; Wang, Xue, Qu, & Liu, 2012) pollution from papermaking industries, slow-release fertilizer with potential application in agriculture and horticulture Thermoplastic applications, biobased composites, hydrogels (antimicrobial property and hydrophobic drug delivery), biobased polyesters or hydrophobic coatings

(Chung et al., 2013; Ding, Wu, Thunga, Bowler, & Kessler, 2016; Kim, Youe, Kim, Lee, & Lee, 2015; Koivu, Sadeghifar, Nousiainen, Argyropoulos, & Sipila¨, 2016; Larran˜eta et al., 2018; Xu et al., 2016)

Silylation

Enhanced thermal stability and solubility (in organic solvents) and hydrophobicity

(Buono, Duval, Verge, Averous, & Habibi, 2016; Li, Xie, Wilt, Willoughby, & Rojas, 2018)

Etherification

Bio-plastics with an enhanced thermal property, oxyethylated lignin (OEL) in soil improvement and rehabilitation

(Lee, Park, & Lee, 2017; Passauer et al., 2015)

Isocyanates

Synthesis of high-performance biobased materials

(De Haro et al., 2019)

Phenolic and Esterification aliphatic hydroxyl groups

Aliphatic hydroxyl groups

Propargylation Propargyl bromide replaced hydroxyl group of lignin produced UV-lightblocking filmsUV light blocker films.

(Sadeghifar, Venditti, Jur, Gorga, & Pawlak, 2017)

Tosylation

Fluorescent lignin

(Panovic et al., 2017)

Azidation

Fluorescent lignin

(Panovic et al., 2017)

Phenolation

Increases cross-linking resulting in (Yang, Wen, Yuan, & Sun, 2014) enhancing the reactivity of technical lignins

Acetalization

Prevents repolymerization reactions thereby preventing loss of functionality

(Yang et al., 2014)

TABLE 4.5 Enzymatic modification of lignin. Lignin

Enzyme/source

Functional molecule grafted

Guaiacylglycerol β-guaiacyl ether and syringylglycerol β-guaiacyl ether (lignin model compounds)

LaccaseTrametes hirsuta

Fluorophenols

Property

References

Improvement in hydrophobicity

(Kudanga et al., 2010)

(Continued) I. Background

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4. Modification techniques for carbohydrate macromolecules

TABLE 4.5 (Continued) Functional molecule grafted

Property

References

LaccaseT. hirsuta

Long chain alkylamines

Improvement in hydrophobicity

(Kudanga, Prasetyo, Sipila¨, Guebitz, & Nyanhongo, 2010)

Syringylglycerol β-guaiacyl ether

C. antarctica lipase

Oxirane

Increase in contact (Kudanga, Prasetyo, water angle, thereby Sipila¨, Nyanhongo, & Guebitz, 2010) increasing hydrophobicity

Kraft pulp

LaccaseAspergillus oryzae

Methyl syringate

Improvement in wet (Liu, Shi, Gao, & strength (wet tensile Qin, 2009) index doubled)

Wood (spruce) chips

LaccaseT. villosa

4-hydroxy-3methoxybenzylamine (HMBA) or 4hydroxy-3methoxybenzylurea (HMBU)

Increase in internal bond

(Fackler, Kuncinger, Ters, & Srebotnik, 2008)

Kraft pulp

LaccaseT. villosa

4Hydroxyphenylacetic acid, 4hydroxybenzoic acid, gallic acid, syringic acid, vanillic acid

Improving strength properties (increase in burst, tear and tensile indexes)

(Chandra & Ragauskas, 2002; Chandra, Felby, & Ragauskas, 2004; Chandra, Lehtonen, & Ragauskas, 2004; Schroeder, Aichernig, Guebitz, & Kokol, 2007)

Jute fibers

LaccaseTrametes versicolor

Octadecylamine (OA) Increase in surface hydrophobicity of jute fibers

(Dong, Fan, Wang, Yu, & CavacoPaulo, 2015)

Jute fibers

LaccaseAspergillus species

Dodecyl gallate

(Dong, Yu, Yuan, Wang, & Fan, 2014)

Jute fibers

LaccaseMycelioophtorathermophila 4-[4-(Trifluoromethyl) Increase in surface phenoxy] phenol hydrophobicity of (TFMPP) and 1 H,1H- jute fibers Perfluorononylamine (PFNL)

(Wu et al., 2016)

Coconut fibers

LaccaseT. versicolor

Syringaldehyde

Improved mechanical and antibacterial properties

(Thakur, Kalia, Kaith, Pathania, & Kumar, 2015)

Coconut fibers

LaccaseT. versicolor

p-coumaric acid

Improvement in hydrophobic and antibacterial properties

(Thakur, Kalia, Sharma, & Pathania, 2015)

Lignin

Enzyme/source

Lignin models and lignin monomers

Increase in surface hydrophobicity of jute fibers

(Continued) I. Background

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4.6 Modification of carbohydrate biological macromolecules

TABLE 4.5 (Continued) Lignin

Enzyme/source

Functional molecule grafted

Coconut fibers

LaccaseT. versicolor

Property

References

Ferulic acid

Improvement in thermal stabilities, hydrophobic and antibacterial properties

(Thakur et al., 2016)

Sinapic acid and the Lipase (Candida antarctica)/ H2O2, laccase (Trametes hirsuta) dimer syringylglycerol β-guaiacyl ether (lignin model substrates representing hard wood lignin substructures)

Methyl linoleate (fatty acid)

Increase in the water contact angle from 58.3 to 93.5 , therefore resulting in increased hydrophobicity

(Greimel et al., 2017)

Alkali lignin (AL)

Horseradish peroxidase (HRP)

Anhydrous sodium sulfite

Modified AL was used as a dopant and dispersant

(Yang, Huang, Qiu, Lou, & Qian, 2017)

Alkali lignin (AL)

HRP

Sulfite

Improved adsorption capacity of lignin

(Yang, Wu, Qiu, Chang, & Lou, 2014)

Kraft Lignin

Laccase fromT. versicolor

Sulfanilic acid (SA) and p-aminobenzoic acid (PABA)

Modified lignins suitable for commercially concrete dispersing agents

(Jankowska et al., 2018)

Hard wood Kraft lignin

Laccase from Galerina sp. HC1

Polyethyleneimine, chitosan and soy protein

Composite adhesives

(Ibrahim, Mamo, Gustafsson, & HattiKaul, 2013)

Kraft lignin

laccase/TEMPO

Soy protein

Lignin-protein adhesives exhibited strong elastic modulus, thermal stability and increased wet adhesion performance.

(Pradyawong, Qi, Sun, & Wang, 2019)

Kraft lignin

Laccase from Coriolus versicolor

Enzymatic polymerization of Kraft lignin

pH-sensitive anionic (Brzonova et al., lignin-based 2017) hydrogels

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TABLE 4.6 Physical treatments for chitin/chitosan. Physical Treatment

Reaction conditions

References 2

Ultrasonication

Sonication at 35.2 W/cm , 30 min

(Baxter, Zivanovic, & Weiss, 2005)

Gamma radiations

2% CS in 2% acetic acid, 200 KGy

(Choi, Ahn, Lee, Byun, & Park, 2002)

1% CS, 0.1% Tween 80 irradiation 50 kGy

(Garcı´a et al., 2015)

Autoclave



1% CS, 1% acetic acid, 121 C, 60 min, 1 bar

(No, Nah, & Meyers, 2003)

TABLE 4.7 Chemical modification of chitin/chitosan. Derivative

Examples

Potential applications

References

Thiolated chitosan derivatives

Chitosan-thioglycolic acid conjugates

Improved mucous adhesion (10 times)

(Kast & Bernkop-Schnu¨rch 2001)

2-iminothiolane

Promising mucous adhesive polymers

(Kafedjiiski, Krauland, Hoffer, & BernkopSchnu¨rch, 2005)

Chitosan-2-iminothiolane

Controlled drug releasing and mucous adhesive properties were also highly improved

(Bernkop-Schnu¨rch, Guggi, & Pinter, 2004; Kafedjiiski et al., 2005; Roldo, Hornof, Caliceti, & BernkopSchnu¨rch, 2004)

Alkylated chitosan derivatives

Thiolated methylated N-(4-N, Improved solubility and N-dimethylaminobenzyl) delivery properties of chitosan chitosan. no significant cytotoxicity against Hek293 cell line in comparison to chitosan, a suitable compound for biomedical purposes

(Hakimi et al., 2017)

Quaternized N-alkyl chitosan Enhanced antibacterial activity against E. coli as compared to the control chitosan

(Jia, Shen, & Xu, 2001)

N- substituted chitosan derivatives from variously functionalized aromatic aldehydes

Fluorescence, some were hydrophobic molecules and some were antimicrobial

(Jatunov, Franconetti, Go´mez-Guille´n, & CabreraEscribano, 2012)

Trimethyl chitosan chloride (TMC)

Improved the intestinal permeability of hydrophilic macromolecular drugs.

(Thanou et al., 2000)

N,N,N-trimethyl chitosan (TMC)

Improved bioadhesion potential in addition to enhanced Mw, swelling, viscosity, and solubility

(Pardeshi & Belgamwar 2016)

(Continued)

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4.6 Modification of carbohydrate biological macromolecules

TABLE 4.7 (Continued) Derivative

Examples

Potential applications

References

N-diethylmethyl chitosan (DEMC)

Enhanced antimicrobial (Avadi et al., 2004) activity against Escherichia coli than control chitosan

Series of methylated derivatives with degree of methylation

N-quaternization was found to be affecting the antibacterial activity against Staphylococcus aureus at pH 7.2 in contrast to acidic conditions

(Ru´narsson et al., 2007)

Quaternized chitosan conjugated with gallic acid/ caffeic acid

Improved antioxidant properties

(Ren, Li, Dong, Feng, & Guo, 2013)

N-Deacetylated chitin/ chitosan derivatives (Prepared by ring opening reactions with cyclic acid anhydrides in a lithium chloride/N,Ndimethylacetamide (LiCl/ DMAc) system)

Derivatives of partially NWater soluble DAC deacetylated chitin (DAC) derivatives were obtained were prepared by reacting with succinic, glutaric, maleic and pthalic anhydrides

(Shigemasa et al., 1999)

N-phthaloylated chitosan derivatives

N-phthaloyl-chitosan

Imparted solubility in polar organic solvents

(Dutta, Duta, & Tripathi, 2004)

Adsorbent for copper ions

(Baba, Noma, Nakayama, & Matsushita, 2002)

Achieved high viability reduction against E. coli and S. aureus, making it potentially applicable in water purification or biomedical applications

(Correia, Ferraria, Pinho, & Aguiar-Ricardo, 2015)

Biomedical applications

(Terbojevich et al., 1989)

N-sulfated chitosan derivative

Biomedical applications

(Holme & Perlin 1997)

Chitosan-glutaraldehyde cross-linking reactions

Poly(vinyl alcohol)/ chitosan (PVA/CS) using glutaraldehyde as a crosslinking reagent

Degradation rate of blend was found to be proportional with increase in chitosan content in PVA/CS blend

(Pokhrel, Adhikari, & Yadav, 2017)

Chitosan-epichlorohydrin cross-linking reactions

Epicholorhydrin crosslinked chitosan
clay composite beads

Biosorbent for the removal of Ni(II) and Cd(II) ions from aqueous solution

(Tirtom, Dinc¸er, Becerik, Aydemir, & C ¸ elik, 2012)

Methylthiocarbamoyl and phenylthiocarbamoyl chitosan Oxazoline grafted chitosan

Oligo(2-methyl-2-oxazoline) quaterinized with N,N-dimethyldodecylamine grafted on chitosan (CHT-OMetOxDDA)

Sulfated chitosan derivatives O-sulfated derivative

(Continued)

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TABLE 4.7 (Continued) Derivative

Examples

Potential applications

References

Chitosan-genipin crosslinking reactions

Result in colored and fluroscent product

Useful in microcapsule research and other biomedical applications

(Chen, Ouyang, Lawuyi, Martoni, & Prakash, 2005; Mi, Shyu, & Peng, 2005)

Crown ether bound chitosan Crown

N-benzylidene chitosan (CTB) from, Chitosandibenzo-18-crown-6 crown ether (CTBD), chitosandibenzo-18-crown-6 crown ether (CTSD)

(Wan, Wang, & Qian, 2002) CTBD and CTSD exhibited better adsorption properties for Ag1, Cu21, Pb21 and Ni21 ions than CTB.

Cyclodextrin linked chitosan

CD-linked chitosan

(Martel et al., 2001) Crosslinked polymer exhibited sorption rate and efficiency in decontaminating water containing textile dyes as compared to parent chitosan

Hydroxyalkyl chitosan

N, N-dicarboxy methyl chitosan (DCMC)

Good chelating ability with calcium phosphate thereby interfering with the precipitation of insoluble calcium salts

Hydroxypropyl chitosan (HPCS)

Showed antimicrobial activity (Peng, Han, Liu, & Xu, 2005) against pathogenic fungi

Quaternized carboxymethyl chitosan

Enhanced antimicrobial activity

(Sun, Du, Fan, Chen, & Yang, 2006)

O-carboxymethyl chitosan

Showed antibacterial and adhesive properties

(Zhu & Fang, 2005)

N-carboxyalkyl chitosan

Antigenotoxic activity against (Kogan et al., 2004) the tested mutagens

Thiosemicarbazone chitosan derivatives

Phenyl aldehyde thiosemicarbazone chitosan (PHTCNCS), ohydroxyphenyl aldehyde thiosemicarbazone chitosan (o-HPHTCNCS) and pmethoxyphenyl aldehyde thiosemicarbazone chitosan (p-MOPHTCNCS) derivative

Exhibited fungal activity against common cropthreatening pathogenic fungi

Thiadiazole-functionalized chitosan derivatives

1,3,4-thiadiazole, 2-methyl1,3,4-thiadiazole and 2phenyl-1,3,4-thiadiazole

Improved fungal activity and (Li et al., 2013) solubility in water

Catechol-functionalized chitosan iron oxide nanoparticle composite

Chitosan-catechol

Enhanced gastrointestinal retention leading to a possible application in mucosal drug delivery

O- and N- Carboxyalkyl chitosan

(Muzzarelli et al., 1998)

(Qin et al., 2012)

(Kim, Kim, Ryu, & Lee, 2015)

(Continued)

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4.6 Modification of carbohydrate biological macromolecules

TABLE 4.7 (Continued) Derivative

Examples

Potential applications

Catechol-chitosan hydrogel derivative crosslinked by genipinCat9-CS/GP and Cat19-CS/GP hydrogels having 9% and 19% catechol conjugation

Promising mucoadhesive and (Xu, Strandman, Zhu, biocompatible hydrogel Barralet, & Cerruti, 2015) system for buccal drug delivery

Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle Composite

Nanocomposites exhibited improved stability at high temperatures, acid/base conditions and physiological Ph.

(Zvarec, Purushotham, Masic, Ramanujan, & Miserez, 2013)

Benzoylation of chitosan

O-benzoyl chitosan

Exhibited solubility in organic solvents

(Lee et al., 2012)

Chitin/chitosan derivatives with hexamethylene diisocyanate

Chitin/chitosan reacted with 1,6-hexamethylene diisocyanate (HMDI) yielded derivatives

Improved thermal and (Gallego, Arteaga, Valencia, & Franco, 2013) rheological properties of the derivatives as compared to traditional lubricating greases

Ethylene diamine tetra acetic Chitosan-EDTA acid (EDTA) grafted onto chitosan

Succinylated and azidated chitosan

Oxychitin and fluorinated chitins

Therefore exhibited better bacteriostatic property than unmodified chitosan

References

(Netsomboon, Suchaoin, Laffleur, Pru¨fert, & Bernkop-Schnu¨rch, 2017)

Preactivated thiolated chitosan-EDTA (Ch-EDTAcys-2MNA) conjugates

Had enhanced mucoadhesive (Netsomboon et al., 2017) and chelating properties

Zwitterionic chitosan (ZWC)

Provided local (Hyun et al., 2017) antiinflammatory effect in the intestinal lumen.

O-succinyl-chitosan

Exhibited prolonged drug release

N-azidated chitosan

Exhibited solubility in one of (Kulbokaite, Ciuta, the few solvents for chitin i.e. Netopilik, & Makuska, 2009) 5% LiCl solution in Nmethyl-2-pyrrolidone

Oxychitin derivative

Retards the drug release, (Genta et al., 2003) exhibited biocompatibility towards human keratinocytes

fluorinated chitin derivatives

Showed better cell viability of (Chow & Khor 2002) 80%
100% for human fibroblast thereby have implications in biomedical applications

(Aiedeh & Taha 2001)

(Continued)

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TABLE 4.7 (Continued) Derivative

Examples

Potential applications

References

Chitin-methacrylate

CM-hydrogel

Water soluble, noncytotoxicity against three cell clines (NCTC clone 929, IMR-90 and MG-63)

(Khor, Wu, Lim, & Guo, 2011)

Sugar modified chitosan

Lactose-modified chitosan

Potent applicant in repairing of articular cartilage

(Donati et al., 2005)

Phosphorylated derivatives of chitosan

Chitosan diethyl phosphate

Potent insecticide

(Ca´rdenas, Cabrera, Taboada, & Rinaudo, 2006)

Ureidyl-chitosan derivatives

Ureidyl-chitosan derivative hydrogel

Highly selective and effective (Franconetti, Domı´nguezRodrı´guez, Lara-Garcı´a, catalyst for Knoevenagel Prado-Gotor, & Cabreracondensation reaction Escribano, 2016)

Thiourea chitosan derivatives

Thiourea chitosan—Ag 1 (TU-CTS-Ag 1 ) complex

Exhibited wide spectrum of antimicrobial activity

(Chen, Wu, & Zeng, 2005)

Chitosan-dendrimer hybrid

Grafting of hyperbranched dendritic polyamidoamine grafted onto chitosan

Wettability of chitosan powder surface was controlled

(Tsubokawa & Takayama, 2000)

Chitosan-dioxime derivative

Vic-dioxime chitosan (vDOCS)

Potential application as a heterogenous catalyst as they are insoluble in common solvents, therefore v-DOCS can be easily separated from reaction mixtures.

(Demetgu¨l & Serin, 2008)

TABLE 4.8 Enzymatic modification of chitin/chitosan. Enzyme

Grafted phenol

Property imparted

References

Tyrosinase (Mushroom tyrosinase)

Arbutin(phenolic acid found in pear)

Generation of biodegradable gel with enhanced strength along (proportion to the arbutin concentration in the reaction mixture)

(Aberg, Chen, & Payne, 2002)

Hexyloxyphenol

Altered structural and physiochemical properties of chitosan

(Chen, Kumar, Harris, Smith, & Payne, 2000)

Caffeic acid

Film formation with enhanced elastic properties, potential application in bioelectronics

(Liu et al., 2014)

(Continued)

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4.6 Modification of carbohydrate biological macromolecules

TABLE 4.8 (Continued) Enzyme

Tyrosinase from Trichoderma reesei

Grafted phenol

Property imparted

References

Chlorogenic acid (secondary metabolite in coffee seeds)

Exhibited solubility under acidic and basic condition, insoluble at near-neutral pH

(Kumar, Smith, & Payne, 1999)

Para-cresol

Removal of phenol from vapors

(Wu, Chen, Wallace, VazquezDuhalt, & Payne, 2001)

4-hydroxybenzoic acid, 3,4dihydroxybenzoic acid, 3,4dihydroxyphenyl-acetic acid and hydrocaffeic acid

Adsorbed cationic dyes (Crystal violet and Bismarck brown Y)

(Chao, Shyu, Lin, & Mi, 2004)

Flavonols, flavone, flavanone, Exhibited antioxidant and isoflavone antimicrobial activity

(Sousa, Guebitz, & Kokol, 2009)

Octyl gallate, dodecyl gallate (derived from plant tannins)

(Vartiainen, Ra¨tto¨, Lantto, Na¨ttinen, & Hurme, 2008)

Chitosan/gallate derivatives exhibited antimicrobial activity

Peroxidases (EC 1.11.1.x) use hydrogen peroxide/chloroperoxidase/organic peroxide as an oxidant (Itzincab-Mejı´a, Lo´pez-Luna, Gimeno, Shirai, & Ba´rzana, 2013; Pasanphan, Buettner, & Chirachanchai, 2010; ZavaletaAvejar, Bosquez-Molina, Gimeno, Pe´rez-Orozco, & Shirai, 2014)

Horseradish Peroxidase (HRP)

Gallate ester, gallic acid

Chitosan/gallate conjugates exhibited an enhanced antioxidant and antimicrobial activity along with better water solubility

Cloroperoxidase (CPO)

Quercetin,

Quercetin-chitosan conjugate (Torres et al., 2012) coating inhibited the browning of Opuntia ficus indica stems.

Laccases (EC 1.10.3.2) copper containing enzymes Laccase from Myceliophtora thermophila

Ferulic acid, ethyl ferulate

(Aljawish et al., 2012; Aljawish Novel conjugates with dual et al., 2014; Aljawish et al., 2014) functionalities (color and antioxidant) were obtained which could be exploited as food colorant or additives (for food preservation), improved cell adhesion on chitosan derivative films

Laccase (Trametes versicolor)

Caffeic acid, Gallic acid

Homogenous conjugates of CA/ chitosan and GA/chitosan exhibited antioxidant and antimicrobial properties

LaccaseTrametes hirsuta

Catechol caffeic acid, and 2,5- Displayed iron-chelating dihydroxybenzoic acid properties

Recombinant Bacterial Laccase [Bacillus vallismortis fmb-103 (fmb-rL103)]

Gallic acid (GA)

(Bozˆiˆc, Gorgieva, & Kokol, 2012)

(Brzonova, Steiner, Zankel, Nyanhongo, & Guebitz, 2011)

Chitosan grafted with GA (GA-g- (Zheng et al., 2018) CS) extended the shelf life of chilled meat (Continued) I. Background

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4. Modification techniques for carbohydrate macromolecules

TABLE 4.8 (Continued) Enzyme

Grafted phenol

Property imparted

References

Recombinant Bacterial Laccase [Bacillus vallismortis fmb-103 (fmbrL103)]

Ferulic acid (FA)

FA-g-CS coating reduced the incidence of disease and improved storage quality of mangoes

(Yang et al., 2018)

Laccase from Pleurotus ostreatus

Cinnamic acids (CADs)

CTS-g-CADs exhibited antibacterial activity against Ralstonia solanacearum.

(Yang et al., 2016)

Owing to the high viscosity of chitin/chitosan, it is rendered “difficult to use” for commercial application. Therefore, reducing the molecular weight is essential in order to reduce the viscosity and expand the biological applications of chitosan (Brasselet et al., 2019). Physical treatments processes include sonication, radiation and or autoclave.

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Thakur, K., Kalia, S., Pathania, D., Kumar, A., Sharma, N., & Schauer, C. L. (2016). Surface functionalization of lignin constituent of coconut fibers via laccase-catalyzed biografting for development of antibacterial and hydrophobic properties. Journal of Cleaner Production, 113, 176
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C H A P T E R

5 Biological macromolecules as nutraceuticals Ireri Alejandra Carbajal-Valenzuela, Nuvia Marina Apolonio
Hernandez, Diana Vanesa Gutierrez-Chavez, Beatriz Gonza´lez-Arias, Alejandra Jimenez-Hernandez, Irineo torres-Pacheco, Enrique Rico-Garcı´a, Ana Angelica Feregrino-Pe´rez and Ramo´n Gerardo Guevara-Gonza´lez Biosystems Engineering Group, School of Engineering-Campus Amazcala, Autonomous University of Quere´taro (Me´xico), Quere´taro, Me´xico

5.1 History of the applications of nutraceutical compounds in health care

of medicinal and aromatic plants to improve their health (Inoue, Hayashi, & Craker, 2017). The main distinction between foods and drugs is determined by the primary goal of nutrition, to promote health and growth through the correct supply of nutrients (Andrew & Izzo, 2017). Moreover, the nutraceutical revolution began until the 1980s, driven by distinguished clinical studies supporting the actual or potential benefits of calcium, fiber and fish oil. The term “nutraceutical” is constituted by the words “nutrition” and “pharmaceutical,” and it refers to any substance from food which possess demonstrated health benefits, including the prevention and management of diseases, it was first used in 1989 by Stephen DeFelice (1995). Due to the definition of nutraceutical proposed by DeFelice is ambiguous and could be

The role of the daily diet in public and individual health has been of significant interest since long before the development of nutrition as a scientific discipline. Ancient medicine was based mainly on herbal knowledge, with diseases were treated using natural remedies. Hippocrates (460
377 B.C) claimed that “differences in disease depend on nutrition,” showing the importance of the relationship between food and health (Andlauer & Fu¨rst, 2002). In fact, nutrition has been a key element in many traditional forms of medicine; there is evidence that shows that the first civilizations of the world, native from China, Greece, the Middle East and India had made use

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00001-4

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confused with other terms, such as food supplements, functional foods, or pharmaceuticals; a new definition was proposed in order to clarify the difference between concepts by their origin: the phytocomplex is the nutraceutical for every active ingredient from vegetal origin; and for dietary food of animal origin, a nutraceutical is mainly secondary metabolites (SM) (Santini, 2018). Nutraceuticals include an extensive diversity of compounds such as bioactive peptides, phenolic compounds, carotenoids, lipids, vitamins, etc. Ting, Jiang, Ho, and Huang (2014). One of the main differences from pharmaceuticals is that nutraceuticals do not function in isolation and have effects on multiple organs; moreover usually their effects act more slowly and are subtler than those of drugs. Also, nutrients generally show U-shaped concentrationeffect while the drug-receptor interaction commonly exhibit a sigmoid dose-response curve (Witkamp & van Norren, 2018), and last but not the least, nutraceuticals have very few side effects (Vishwakarma et al., 2018). Noncommunicable diseases (NCDs) are influenced by various factors, such as genetic, physiological, and environmental and habits like unhealthy diets and a lack of physical activity. Some examples of these conditions are chronic respiratory diseases, metabolic syndrome, type 2 diabetes, cardiovascular diseases and cancer. One important characteristic of the NCDs is that they tend to be of long duration and are so are so harmful that they are responsible of 41 million of deaths each year in the world (WHO, 2013). For most of the 20th century, nutrition was focused on tackling malnutrition problems by ensuring a safe diet that provided the adequate amount of energy, macro and micronutrients to meet the needs, and prevent deficiencies in the population. Lately, due to the increasing incidence of NCDs, academic and research bodies around the world have made it possible to gain new knowledge about the effects of

nutrition on health, some of the most important milestones are: the better understanding of metabolism, identification of nutrients related to chronic diseases, the discovery of new bioactive substances, the improvement on understanding the effects of the microbiome on immunity, obesity and cognitive function, as well as the development of the “omics” sciences and epigenetic (Shao et al., 2017). Some clinical studies have shown that nutraceuticals’ consumption can produce beneficial effects that improve the state of health, preventing diseases and increasing life expectancy; therefore, even though nowadays medical therapy is mainly based on pharmaceuticals, nutraceuticals are gaining importance in the medicine, as they are considered to be used not only to prevent illnesses or support pharmacological therapy, but also to be used as part of therapies for those who cannot be treated with drugs (Durazzo, Lucarini, & Santini, 2020). At the present time some nutraceuticals are recognized for their potential antiinflammatory and antioxidant activities, those characteristics are very attractive in medicine to prevent and treat major NCDs. Moreover, there is little information available about the effects of these active compounds in communicable diseases (CDs), particularly on viral infections such as COVID-19 (Alkhatib, 2020). Nutraceutical compounds have shown to have not one but numerous effects in the body so commonly they can be used to treat or prevent different pathologies. For example, curcumin has been considered as the golden nutraceutical thanks to its safety, efficacy and for its multiple and powerful beneficial effects such as antimicrobial agent as well as for being useful against chronic diseases, obesity, and neurological and autoimmune diseases (Kunnumakkara et al., 2017). SM are chemical compounds resulting from responses to both biotic and abiotic stressors; those metabolites are not essential for the life of organisms, however they are helpful in their environmental adaptation and interaction with

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5.2 Alkaloids

other entities, for example, attraction of pollinators, allelopathy and defense versus herbivores or plagues. SM can be classified in three groups: alkaloids, phenolic compounds and terpenes (Chomel et al., 2016). In addition to the relevance of primary metabolites such as lipids, carbohydrates and amino acids; scientific and industrial interest on SM is wide because due to their pharmacological effects they can be used for different purposes: drug production, the creation of cosmetics, agrochemicals, biopesticides, food additives, supplements and of course as nutraceuticals in the human and animal nutrition (Guerriero et al., 2018; Tapas, Sakarkar, & Kakde, 2008; Thakur, Bhattacharya, Khosla, & Puri, 2018). The natural production of these biocompounds by plants is reduced, with approximately 1% of the dry weight; therefore, elicitation is currently one of the biotechnological strategies used to stimulate biosynthesis and increase its concentration. Elicitors can be classified as biotic or abiotic; biotic elicitors, as their name suggests, are of biological origin and include proteins, carbohydrates, and plant growth promoting rhizobacteria, fungi, and hormones. While abiotics are made up of heavy metals, light, salinity, temperatures, and drought (Thakur et al., 2018). The biosynthetic pathways of secondary metabolism depend on molecules derived from primary metabolism, such as amino acids, shikimic acid, and acetate (Garcı´a, 2004). Many aromatic compounds such as cinnamic acids, some polyphenols, and aromatic amino acids come from the metabolism of shikimic acid. Fatty acids and polyketides are obtained from acetate by the acetate-malonate route, and some terpenes by the acetate-mevalonate route. While the inherent terpenes of plastids are synthesized from pyruvate and glyceraldehyde-3-P. Peptide alkaloids and antibiotics such as penicillins are synthesized from amino acids. Finally, various metabolic routes, in this particular case, form some metabolites such as stilbenes or flavonoids; said polyphenols are obtained through the shikimic

acid and acetate-malonate routes (Azco´n-Bieto & Talo´n, 2014). A general overview of main secondary/specialized metabolites with nutraceutical potential is shown in Fig. 5.1.

5.2 Alkaloids Alkaloids are nitrogenous compounds derived from amino acids, and benzylisoquinoline alkaloids, monoterpene indole alkaloids and glucosinolates are some of the most known alkaloids (Wang, Guleria, Koffas, & Yan, 2016). According to their chemical structure they can be classified as heterocyclic and nonheterocyclic, the group of heterocyclics is subdivided into 14 different groups depending on the structure of the ring. More than 18,000 alkaloids are known and have been identified in different animal, plant and microbial species (Othman, Sleiman, & Abdel-Massih, 2019). These compounds are being useful in the pharmaceutical industry for the creation of drugs. In fact, there are already some highly used drugs whose active compound are alkaloids, for example: berberine, vincristine and vinblastine, which are applied in cancer treatments; morphine and codeine, which act as analgesics; aconitine regulates heart rate and cocaine, which functions as local anesthetic (Mondal, Gandhi, Fimognari, Atanasov, & Bishayee, 2019). Additionally multiple alkaloids have shown an antiangiogenic activity as well as antiproliferative and cytotoxic activity against cancer cell lines (Alasvand et al., 2019).

5.2.1 Caffeine Caffeine (also known as trimethylxanthine) is formed by xanthine (Fig. 5.2). This compound results from purine nucleotides, therefore it is known as an alkaloid (Ghadimi, Ab Ghani, & Amiri, 2017). Although coffee is the main source, it can be extracted from some different plants like cacao, yerba matte, and

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Secondary metabolites

Alkaloids

Phenolic compounds

Caffeine, Capsaisine Theobromine,

Terpenes

Curcumin, Resveratrol, Quercetin Anthocyanines

Luteolin, Catechines, Naringenin

FIGURE 5.1

Lycopene α, β-carotene Lutein Zeaxantine

Overview of secondary/specialized metabolites with nutraceutical potential.

guarana´. Currently, a consumption limit of 200 mg has been established in healthy adults, not pregnant or lactating, due to the controversy about its possible adverse effects (van Dam, Hu, & Willett, 2020). The consumption of caffeine has become popular due to the effects that it produces in the central nervous system, through its action on dopamine receptors and stimulation for the secretion of neurotransmitters (dopamine, serotonin, norepinephrine) with a dose of 75 mg it manages to generate a feeling of energy, wellbeing and alert (dePaula & Farah, 2019). Antioxidant, antiinflammatory, antibacterial, neuroprotective, hepatoprotective effects of caffeine have also been reported. It eliminates hydroxyl radicals and some of its metabolites (1methylxanthine, 1-methyluric acid, and 1-methylurate) have shown in vitro antioxidant activity. On the other hand it modify the production of

cell signaling, inhibiting the action of phosphodiesterase, stimulating the release of antiinflammatory cytokines and as antagonist of adenosine receptor. It has an antibacterial capacity against Streptococcus mutans and Escherichia coli; regular intake reduce the risk of development of Parkinson, Alzheimer and some chronic liver diseases like cirrhosis (dePaula & Farah, 2019). Caffeine is being used as an ergogenic resource; it affects positively the endurance and performance in physical exercise. Besides, caffeine works favorably in weight loss treatments because it stimulates lipid oxidation while reducing carbohydrate use, increases energy expenditure, and presents thermogenic effects (Ruiz-Moreno et al., 2020). Higher than 2 g intakes of caffeine can cause intoxication, with varied symptoms ranging from headaches and anxiety to tachycardia, vomiting and diarrhea (dePaula & Farah, 2019).

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5.2 Alkaloids

Alkaloids

FIGURE 5.2

Caffeine

Capsaicin

Curcumin

Resveratrol

Quercetin

Luteolin

Naringenin

Catechins

Theobromine

Phenolic compounds

Chemical structure of some alkaloids and phenolic compounds with nutraceutical activity.

5.2.2 Capsaicin Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) (Fig. 5.2), comes from homovanillic acid and is the responsible of the pungency present in the genus Capsicum, which involves 30 different species of which only 5 are domesticated: Capsicum annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens. The unit of measure to determine the pungency level is the Scoville Heat Units (HUS), the C. chinense Jacq.cv. Naga King Chili from India is the hottest chili in the world, with 1,001,304 SHU (Chapa-Oliver & Mejı´aTeniente, 2016). Clinical investigations show that capsaicin is an effective drug for relieving pain and itch, currently it is in phase III clinical trials for treating diseases like rheumatoid arthritis, chronic neuropathic, musculoskeletal and post operatory pain. It has also exposed favorable

behavior in studies linked to blepharospasm, prostatic cancer, lung cancer, leukemia (Patowary, Pathak, Zaman, Raju, & Chattopadhyay, 2017), it reduces the risk of hypertension and chronic kidney disease, improves insulin sensitivity and also has valuable effects as antiobesity, bacteriostatic and antiinflammatory (Li et al., 2020).

5.2.3 Theobromine Theobromine (3, 7-dimethylxanthine) (Fig. 5.2), is obtained by the methylation of xanthine. Along with theophylline and caffeine constitute the principal methylxanthines present in tea, coffee, cocoa and chocolate; and are recognized for being antagonist of the adenosine receptor and inhibitors of the phosphodiesterase (Iaia et al., 2020). Theobromine can be found in a major amount in the leaves of cocoa

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plants, its concentrations declines as the leaves mature. Most of the results published about possible benefits of this nutraceutical come from animal and vitro studies, and they propose a possible cognitive improvement, protecting function on nerve cells as well as an antiinflammatory activity. Human studies are limited by the duration and size of the sample; however intakes of 1 g of theobromine perform safely (Cova, Leta, Mariani, Pantoni, & Pomati, 2019). Some of its uses are bronchodilator, antitussive, neurostimulator, vasodilator, muscle relaxant, diuretic, antiinflammatory, antitumoral as well as cardiovascular protector (Iaia et al., 2020); its adenosine receptor antagonistic properties is linked to the generation of the aforementioned effects (Cova et al., 2019). Recently, it has been shown that Theobromine promotes lipid oxidation and white adipocyte browning through AMPK signaling and the β-adrenergic pathway; suggesting the possible use of Theobromine in the treatment of obesity (Jang, Kang, Mukherjee, & Yun, 2018). Additionally, it has been identified as a strong inhibitor of uric acid crystallization in humans and combined with citrate, it may be effective to avoid the development of uric acid kidney stones (Hernandez et al., 2020).

5.3 Phenolic compounds Polyphenols (or phenolic compounds) are plant SM that contain at least one aromatic ring containing a hydroxyl group, in their chemical structure (Martı´n Gordo, 2018). Polyphenols are usually classified in flavonoid compounds, which include flavones, isoflavones, and anthocyanins; and nonflavonoid compounds, consisting of phenolic acids, stilbenes, lignans, lignins, and tannins (Tsao, 2010). Until today, more than 8000 phenolic compounds have been discovered (Tungmunnithum, Thongboonyou, Pholboon, & Yangsabai, 2018) and around 3000 of them correspond to flavonoids (Martı´n Gordo, 2018). Green tea, cocoa, grape, and

berries are some sources of polyphenols in human diet (Bianconi, Mannarino, Sahebkar, Cosentino, & Pirro, 2018). Phenolic compounds have shown to be beneficial for human health; they have been linked with antioxidant and anticancer effects, as well as their antibacterial potential, antiinflammatory and cardioprotective functions and their promoting effects on the immune system (Tungmunnithum et al., 2018). Moreover there are some disadvantages with phenolics: as they are photo and heat sensitive, and tend to rust easily, its gastrointestinal processing becomes complicated due to its instability. Hence, encapsulated forms of phenolic compounds represent a suitable solution to enhance their bioavailability and bioactivity (KhoshnoudiNia, Sharif, & Jafari, 2020).

5.3.1 Curcumin Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)1,6-heptadiene-3, 5-dione] was isolated from turmeric in 1870. Its chemical structure is made up of two rings with hydroxyl and methoxyl groups linked by a keto-enol chain of seven carbons (Fig. 5.2). Curcumin is an unstable compound in alkaline pH and in presence of oxygen; it also has low solubility in gastrointestinal fluid. Nonetheless, curcumin is known for its potential therapeutic in heart diseases, diabetes, obesity, cancer, it has antiinflammatory and neuroprotective properties, among other biological activities of interest within medicine (Ashrafizadeh et al., 2020). This polyphenol acts as a protective drug against cardiac diseases like arrhythmias, myocardial ischemia and cardiomyopathies through diminishing inflammatory processes, oxidative stress and apoptosis (Jiang et al., 2017). Its antiinflammatory activity occurs through various mechanisms, such as the reduction of the expression of TNF-alpha, interleukin-1 (IL1) and IL6 by the down regulation of the nuclear factor-kB (NF-kB) (Fadus, Lau, Bikhchandani, & Lynch, 2017). In diabetes, curcumin increases the

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5.3 Phenolic compounds

production of antioxidant enzymes to protect the pancreatic β-cells and decreases inflammation. These activities attenuate insulin resistance and consequently glucose tolerance (Thota, Acharya, & Garg, 2019). Curcumin intake is highly safe, oral administration of 12 g showed very little toxicity. However, due to the chemical instability and insolubility of curcumin it is important to consider different formulations and new technologies like nanoparticles to improve the bioavailability (Xue et al., 2020).

5.3.2 Resveratrol Two phenol rings interconnected by an ethylene bond constitute resveratrol (3,5,40-trihydroxy-trans-stilbene) (Fig. 5.2) (Salehi et al., 2018). It is a polyphenol and can be obtained by the consumption of grapes, mulberry and peanut (Li, Xia, Hasselwander, & Daiber, 2019). This compound is mostly known because of its high antioxidant power; multiple studies have evidenced a potential to inhibit carcinogenesis as well as other important effects like neuroprotective (Salehi et al., 2018); it also positively influences blood pressure and vascular function through various mechanisms such as stimulation of NO production, reduction of oxidative stress, inflammation, and hardening of arterial walls (Li et al., 2019). Transresveratrol form stimulates cell differentiation, induces apoptosis and limits the proliferation of cancer cells (Salehi et al., 2018).

5.3.3 Quercetin Quercetin is a water-soluble flavonoid, chemically composed by 3 rings and 5 hydroxyl groups (Fig. 5.2). It is distributed in multiple foods, some of them are: apples, berries, grapes, onions, broccoli, tomatoes, seeds, nuts, black and green tea. High concentrations of quercetin can be obtained from onion peel (Kumar, Vijayalakshmi, & Nadanasabapathi, 2017). There are several studies

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that have analyzed the behavior of quercetin in areas of interest for biomedicine; favorable results have been found: it has antiinflammatory activity; it can relieve asthma symptoms by easing the airway smooth muscle and diminishing histamine levels, it is an effective antiviral against viruses like herpes simplex type I, and parainfluenza type 3, also has vasodilatory properties (Kumar et al., 2017). This compound is able to diminish oxidative stress by mopping free radicals, and subsequently decreases the risk of heart disease and stroke (Jain, Buttar, Chintameneni, & Kaur, 2018) and generates an anticancer response (Upadhyay, 2006).

5.3.4 Anthocyanins Anthocyanins are water-soluble plant pigments, responsible for the color of red, blue and violet vegetables and fruits like berries, cherries, grapes, red onion, red cabbage and black beans (Smeriglio, Barreca, Bellocco, & Trombetta, 2016). Its chemical structure is characterized for having a 2-phenylchromenylium as base; depending on the flavylium B-ring they can be classified into cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin. Also, they can be found sugars linked to the main structure (Lee et al., 2017). The bioactivities of anthocyanins are multiple and complex; in addition to the powerful antioxidant ability that they have shown, they can act positively in multiple diseases such as diabetes, obesity, fatty liver, hypertension, cardiovascular disease and some others (Jiang et al., 2019). These active compounds satisfactorily regulate triglycerides and cholesterol (Jiang et al., 2019), inhibite NF-κB, reduce insulin resistance, suppress the development of different cancer cell lines (Lee et al., 2017), stimulates vasodilatation, prevent thrombus formation, improved visual function, inhibit the formation of adipose tissue, limitate body weight gain and have antimicrobial properties against Enterococcus faecium, Staphylococcus aureus, E. coli, Listeria innocua and so on (Khoo, Azlan, Tang, & Lim, 2017).

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5.3.5 Luteolin Luteolin (3,4,5,7-tetrahydroxy flavone) belongs to flavonoids group; its chemical structure gives it bioactive potential, specifically thanks to its double bond between carbons 2 and 3, as well as its hydroxyl residues (Fig. 5.2) (Sangeetha, 2019). Various plants contain important quantities of luteolin; cabbage, celery, carrot, chrysanthemum flowers, onions, sweet bell peppers, broccoli, artichoke, apple and parsley are examples of specimens of which it can be extracted (Imran et al., 2019; Sangeetha, 2019). Luteolin is an active and permeable chemical compound which also is able to modify some signaling routes: AKT/GSK 3β, AKT/PKB and NFκB-AF1 have been identified and associated to its capacity as antioxidant, immunomodulatory, insulin sensitivity enhancer, lipid modulator, antihistaminic, effectiveness against nervous disorders and so on (Sangeetha, 2019). Luteolin has also important antiviral activity; it acts by suppressing DNA replication of some viruses such as Japanese encephalitis, Epstein Barr and hepatitis B (Manzoor et al., 2019). Due to the ability of this compound to inhibit the cellular proliferation of malignant cells, induce apoptosis (Imran et al., 2019), reduced cancer tumors it could be an important element in chemotherapy. But also its role as a protector against DNA damage has made it an attractive agent for the treatment of Alzheimer’s (Manzoor et al., 2019).

5.3.6 Naringenin Naringenin (C27H32O14) is a flavanone glycoside, soluble in organic solvents (Fig. 5.2); it is part of citrus foods such as lemon, orange, clementine, grapefruits, tomatoes, bergamot (Karim, Jia, Zheng, Cui, & Chen, 2018; Salehi et al., 2019; Zaidun, Thent, & Latiff, 2018). Naringerin antibacterial and antiviral properties have been highlighted; currently its action

against COVID-19 is been studied. So far, it has been identified to inhibit the protease (3CLpro) and limit the activity of ACE2 receptors, both elements are implicated in the viral pathogenesis of SARS-CoV-2. Therefore, together with its antiinflammatory and antioxidant activities, it is considered a possible alternative for the treatment of this infection (Tutunchi, Naeini, Ostadrahimi, & Hosseinzadeh-Attar, 2020). This phytochemical is able to inhibit apoptosis, NF-kB pathway and oxidative stress, it suppress the activity of genes involved in lipogenesis and triglyceride synthesis, and stimulates glucose transporters, NK cells of the immune system as well as the reproduction of pancreatic B cells. Therefore, this element has been attributed various pharmaceutical uses, for example as analgesic, antihypertensive or antiinflammatory. Moreover its antioxidant activity has been more studied (Salehi et al., 2019) and it has been associated with mitochondrial protection, seeing promising results as an anticancer and antiaging agent (Da Pozzo et al., 2017).

5.3.7 Catechins Catechins are flavanoids (Fig. 5.2) found in various edible plant species such as legumes, cocoa, pear, apple, grapes, cherries and green tea (Gadkari & Balaraman, 2015; Mangels & Mohler, 2017). They act through different ways in the body generating positive responses for health; antihypertensive activity of catechins is very strong, they perform control over high blood pressure and act as inhibitor of platelet aggregation and anticoagulant (Mangels & Mohler, 2017). These bioactive compounds have a potential as antimetabolic syndrome agents; though the elimination of free radicals and stimulation of catalase and superoxide dismutase, catechins are able to improve lipid oxidation and diminish fatty acids synthesis, having as result a decrease in blood lipid concentration and body fat (Chen et al., 2016).

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5.4 Terpenes

5.4 Terpenes

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sarcinaxanthin, decaprenoxanthin and sarprenoxanthin have been discovered (Trost & Min, 2020).

Terpenes (or terpenoids) are the leading group of natural organic compounds from secundary metabolism, with more than 55,000 molecules isolated so far (Trost & Min, 2020). Their chemical structure derives from isoprene (C5H8) and they are categorized for the number of isoprene units that comprise them as mono-, sesqui-, di-, tri-, tetra- and polyterpenes (C10, C15, C20, C30 and C40 respectively) (Trost & Min, 2020). Despite the fact that terpene biosynthesis occurs from two cyclopentanes derived from acetyl coenzyme A: dimethylallyl pyrophosphate and isopentenyl pyrophosphate; chemically terpenes can be very different from each other because they are synthesized from different types of cells and metabolic pathways (Cho et al., 2017; Trost & Min, 2020). Terpenes have multiple applications in industry; currently they serve as biofuels, agrichemicals, fragrances, medicines, and nutraceuticals (Mewalal et al., 2017). As nutraceuticals, these phytonutrients obtained from foods like grains, soybeans, and green vegetables, have shown to have positive performances against cancer, infectious and inflammatory processes. Carotenoids such as β-carotene, lutein and lycopene have obtained additional uses in industry as natural food colorants and supplements, principally (Trost & Min, 2020). Carotenoids are biosynthesized by some types of bacterias, algae, fungi, yeasts, and plants. Animals only obtain carotenoids by eating these organisms as food. More than 700 different carotenoids have been isolated from leaves, flowers, and fruits, but only α- and β-carotene, β-cryptoxanthin, lutein, lycopene, and zeaxanthin can be found in major quantities (Grabowska et al., 2019). To enhance the production of some of these chemical compounds, particularly of carotenoids, metabolic engineering has made use of hosts like E. coli and S. cerevisiae. By using this process, not only large-scale production has been promoted, but also new carotenoids such as 4ketozeinoxanthin and C50 carotenoids like

5.4.1 Lycopene Lycopene (C40H56) is an unsaturated and lipophilic carotenoid (Fig. 5.3) soluble in organic solvents. It is responsible of the red color of fruits and vegetables, therefore it can be found in tomatoes, watermelon, papaya, apricots, red grapefruits, pumpkin, carrots, and pink guava. The state of maturation as well as temperatures above 35 C while ripening, can affect the amount of lycopene in vegetables. Blakeslea trispora, specie from the fungi kingdom, and marine haloarchaea can also produce lycopene (Grabowska et al., 2019; Mozos et al., 2018). Due to its multiple double bonds and unsaturated olefin structures, lycopene is susceptible to oxidation and isomerization, practically all environmental factors (light, heat, pH) can affect its stability. Lycopene delivery systems like emulsions, liposomes and nanostructured lipid carriers, play a very important role to guarantee the bioavailability of the component (Liang, Ma, Yan, Liu, & Liu, 2019). As a class A nutrient, lycopene has gained use in many fields like food industry, agriculture, medicine and nutrition (Liang et al., 2019). Lycopene acts as an antioxidant by stimulating the secretion of antioxidant enzymes and neutralizing reactive oxygen species (ROS) and RNS, reduce arterial stiffness (Mozos et al., 2018), improves glycolipid metabolism, acts against oxidative stress, suppresses the production of inflammatory cytokines (Crowe-White, Phillips, & Ellis, 2019); therefore it has shown to be helpful in the treatment of different types of cancer, hypertension, metabolic syndrome, cardiovascular, visual and neurological diseases (Liang et al., 2019; Mozos et al., 2018).

5.4.2 β-Carotene β-carotene (C40H56) is a polyene compound, vitamin A precursor and an orange pigment

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FIGURE 5.3 Chemical structure of some terpenes with nutraceutical activity.

Lycopene

β –Carotene

Lutein Zeaxanthin

synthesized by plants (Fig. 5.3). Humans can obtain it though diet and some of its more important functions in the human body are the action as provitamin A source, antioxidants properties, it is helpful to ensure proper growth, good eyesight (Bogacz-Radomska & Harasym, 2018; Larroude et al., 2018) and has significant benefits in the cognitive status, lumbar spine bone mineral density as well as immune system enhancer and antiinflammatory (Eggersdorfer & Wyss, 2018). This SM is principally found in yellow and orange fruits and vegetables (Larroude et al., 2018); orange carrot is the main source of β-carotene but kale, parsley lear, spinach and paprika are other important sources (Bogacz-Radomska & Harasym, 2018). Using β-carotene-producing microorganisms Blakeslea trispora, Xanthophyllomyces dendrorhous, Dunaliella salina, S. cerevisiae and E. coli this chemical compound can be produced on a large scale (Larroude et al., 2018). The European Food Safety Authority reported 15 mg as safe (Eggersdorfer & Wyss, 2018).

5.4.3 Lutein Lutein (C40H56O2) (Fig. 5.3) is a fat-soluble antioxidant and one of the main constituents of macular pigment (MP) (Eisenhauer, Natoli, Liew, & Flood, 2017), a component that protects the retina from oxidative species and acts as a blue light filter (Lima, Rosen, & Farah, 2016). It can be obtained through different foods, specifically from those yellow or orange, such as cantaloupe, corn, carrots, orange/yellow peppers, salmon and eggs (Abdel-Aal, Akhtar, Zaheer, & Ali, 2013). As it is an important element for eye health, it has been largely studied regarding its role in sight; recent results have shown that through the consumption of the three carotenoids that constitute the MP (lutein, zeaxanthin and meso-zeaxanthin) it is possible both to prevent and also to retard the evolution of age-related macular degeneration (AMD) (Eisenhauer et al., 2017). Daily supplementation with these carotenoids for at least 1 year increases MP optical densities (MPOD) (Ma et al., 2016).

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5.6 Proteins and peptides with biological activity of medical interest

5.4.4 Zeaxanthin Zeaxanthin (C40H56O2) is an oxygenated carotenoid with a molecular structure that contains 18 conjugated double bonds (Fig. 5.3); it is an abundant natural carotenoid present in vegetal food like corn and goji berries, but also from food of animal origin like egg yolk (Liu et al., 2019). There are reports of Zeaxanthin identified as an antioxidant, sight, and cardioprotector, as an immune system enhancer, and recently as an activator of the AMPK pathway and promotes the formation of brown adipocytes, suggesting alternatives in the treatment of obese patients (Liu et al., 2019). A summary of the information regarding the three categories of SM mentioned is shown in Table 5.1.

5.5 Future views Growing consumer interest in diets with health benefits and the continuous increase of geriatric population, have boosted the positioning of the actual nutraceutical industry. According to a recent report by Grand View Research, Inc. (2020), the global nutraceutical market was valued at USD 382.51 billion in 2019 and according to statistics it is projected to reach USD 578.23 billion by 2027. Recently, scientific interest in nutraceuticals and their biological roles has grown exponentially. Moreover, currently the main challenge consists on supporting with clinical evidences the safety and efficacy of the nutraceuticals compounds (Daliu, Santini, & Novellino, 2018). It must exist a strong justification for their use in a pathological condition, and by obtaining the necessary information, professionals and patients can know the adequate pharmaceutical form and concentration for their administration (Daliu et al., 2018). As we know, the efficacy of nutraceuticals highly depends on

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their bioavailability; as a result, the study of delivery systems is gaining more importance in the industry. Some areas of opportunity are: achieve exclusive use food-grade ingredients, control the release of the component and increase intestinal permeability (Gonc¸alves, Martins, Duarte, Vicente, & Pinheiro, 2018). In the following sections of this chapter, an overview possibility of another biological macromolecules (nucleic acids, lipids, proteins, carbohydrates) as nutraceuticals are described and discussed.

5.6 Proteins and peptides with biological activity of medical interest Proteins are essential macromolecules for the growth and maintenance of body structures (Toldra´, Reig, Aristoy, & Mora, 2018). Additional to the contribution of metabolic energy and essential amino acids, proteins have biological functionality with a direct effect on the physiological processes of the body. Peptides, like proteins, possess biological functionality and can be present as an independent sequence or latent state within a protein (Mast, Checco, & Sweedler, 2020). Their beneficial effect on one or more functions of the body has been successfully demonstrated. In recent decades there has been an exponential increase in the identification of peptides and proteins with a great variety of relevant biological activities in the biomedical field, which reflects the great chemical diversity of these biomolecules. Some examples are antibiotic, antioxidant, antihypertensive, anticancer peptides, or immunomodulatory activities (Rivas-Morales et al., 2016). The fundamental structure of proteins is relatively simple; are long chains of amino acids linked together by amide bonds, also called peptide bonds, between the carboxyl group of one amino acid and the amino group of another (Toldra´ et al., 2018). These chains are called polypeptides. A protein can be made up of a single polypeptide chain or several associated with

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TABLE 5.1 Secondary/especialized metabolites with nutraceutical activity. Compounds groups

Nutraceutical (bioactive compound)

Sources

Main applications in biomedicine

Alkaloids

Caffeine

Cacao, yerba matte and guarana´ (van Dam et al., 2020).

Neurostimulator, antioxidant, antiinflammatory, antibacterial, neuroprotective, hepatoprotective, ergogenic (dePaula & Farah, 2019; Ruiz-Moreno et al., 2020).

Capsaisine

Chilli (Capsicum) (Chapa-Oliver & Mejı´aTeniente, 2016).

Analgesic, antiinflammatory, anticancer, antiobesity (Li et al., 2020; Patowary et al., 2017).

Theobromine

Tea, coffee, cocoa, chocolate (Iaia et al., 2020).

Neuroprotective, antiinflammatory, bronchodilator, vasodilator, diuretic, antitumoral, antiobesity, antilithiasic (Cova et al., 2019; Hernandez et al., 2020; Iaia et al., 2020; Jang et al., 2018).

Curcumin

Turmeric (Ashrafizadeh et al., 2020).

Antioxidant, antiinflammatory, antidiabetic, neuroprotective, anticancer, cardioprotective (Ashrafizadeh et al., 2020; Fadus et al., 2017; Jiang et al., 2017; Thota et al., 2019).

Resveratrol

Mulberry, peanut and grapes (Li et al., 2019).

Antioxidant, anticancer, neuroprotector, antihypertensive, antiinflammatory (Li et al., 2019; Salehi et al., 2018).

Quercetin

Apples, berries, grapes, onions, broccoli, tomatoes, seeds, nuts, tea (Kumar et al., 2017).

Antiinflammatory, antihistaminic, antiviral, vasodilator, antioxidant, anticancer (Jain, Buttar et al., 2018; Kumar et al., 2017; Upadhyay, 2006).

Phenolic compounds

Anthocyanines Berries, cherries, grapes, red onion, red cabbage and black beans (Smeriglio et al., 2016).

Antioxidant, statin, antidiabetic, antihypertensive, cardioprotector, eye protector, antimicrobial (Jiang et al., 2019; Khoo et al., 2017; Lee et al., 2017).

Luteolin

Cabbage, celery, carrot, onions, sweet bell peppers, broccoli, artichoke, apple and parsley (Imran et al., 2019; Sangeetha, 2019).

Antioxidant, antidiabetic, antihistaminic, statin, antimicrobial, antiviral, anticancer (Imran et al., 2019; Manzoor et al., 2019; Sangeetha, 2019).

Naringenin

Lemon, orange, clementine, grapefruits, tomatoes, bergamot (Karim et al., 2018; Salehi et al., 2019; Zaidun et al., 2018).

Antibacterial, antiviral, antioxidant, analgesic, antihypertensive or antiinflammatory, antiaging (Da Pozzo et al., 2017; Salehi et al., 2019; Tutunchi et al., 2020).

Catechines

Cocoa, pear, apple, grapes, cherries and green tea (Gadkari & Balaraman, 2015; Mangels & Mohler, 2017).

Antioxidant, statin, antihypertensive, anticoagulant (Chen et al., 2016; Mangels & Mohler, 2017). (Continued)

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TABLE 5.1 (Continued) Compounds groups

Nutraceutical (bioactive compound)

Sources

Main applications in biomedicine

Terpenes

Lycopene

Tomatoe, watermelon, papaya, apricots, red grapefruits, pumpkin, carrots, and pink guava (Grabowska et al., 2019; Mozos et al., 2018).

Antioxidant, anticancer, antihypertensive, antiinflammatory, cardioprotector, eye protector, neuroprotector (Crowe-White et al., 2019; Liang et al., 2019; Mozos et al., 2018).

β-Carotene

Antioxidant, provitamin A, eye protector, Orange carrot, kale, parsley lear, spinach and paprika (Bogacz-Radomska & Harasym, antiinflammatory, immunostimulant 2018). (Bogacz-Radomska & Harasym, 2018; Eggersdorfer & Wyss, 2018; Larroude et al., 2018).

Lutein

Cantaloupe, corn, carrots, orange/yellow peppers, fish and eggs (Abdel-Aal et al., 2013).

Antioxidant, eye protector (acts agains AMD) (Eisenhauer et al., 2017; Lima et al., 2016; Ma et al., 2016).

Zeaxanthin

Corn, goji berries and egg yolk (Liu et al., 2019).

Antioxidant, immunostimulant, antiobesity (Liu et al., 2019).

each other and are made up of about twenty different amino acids (Anzai et al., 2018). Proteins are organic macromolecules, basically made up of carbon (C), hydrogen (H), oxygen (O) and nitrogen (N); although they may also contain sulfur (S) and phosphorus (P) and, to a lesser extent, iron (Fe), copper (Cu), magnesium (Mg), iodine (I), among others (Jorge Ruiz-Ruiz, 2016). These molecules correspond to the primary structures within the metabolism of living beings, along with carbohydrates and lipids. In general, proteins can be classified by different characteristics: 1. Physical and/or chemical properties: groups of proteins based on their size, structure, solubility, or degree of basicity. 2. The type of molecules to which they can bind: lipoproteins (lipid prosthetic group), nucleoproteins (protein nucleus prosthetic group), chromoproteins (pigment prosthetic group), metalloproteins (metallic prosthetic group) and glycoproteins (carbohydrate prosthetic group).

3. Their function in cells: they can be grouped into three general classes based on their function, enzyme, structural proteins (membrane proteins, cell walls or cytoskeletons), and storage proteins. Bioactive or functional peptides have been as sequences of inactive amino acids within the precursor protein, which exert certain biological activities after their release by chemical or enzymatic hydrolysis (Garcı´a-Cano et al., 2019). However, a few years later, they took up the concept to define it as specific protein fragments that have a positive impact on body functions or conditions and that can finally influence health (Minkiewicz, Iwaniak, & Darewicz, 2019). Generally, are small in size ranging from 3 to 20 amino acids, although sometimes they can exceed that length (Kim & Kini, 2017). It is even known that when administered orally to humans, these molecules can exert effects on various systems, such as the circulatory, digestive, immune and nervous systems (Garcı´a-Cano et al., 2019). In this sense, there are scientific reports that these molecules can cross the

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intestinal epithelium and reach peripheral tissues via the systemic circulation, being able to exert specific functions at the local level, and at systemic level; therefore, it is said that bioactive peptides could influence human cell metabolism and act as vasoregulators, growth factors, hormonal inducers, and neurotransmitters (Aslam, Shukat, Khan, & Shahid, 2020). Later, some authors suggested that bioactive peptides form part of the innate defense response elicited by the sense of danger in cells. Most of the bioactive peptides produced in plants have microbicidal properties, they are also part of cell signaling. They mention that the biological role of bioactive peptides begins with the binding to the target membrane followed by permeabilization of the membrane and rupture (Gull, Shamim, & Minhas, 2019). Due to the great importance of peptides, multiple biotechnological protocols have been developed for their isolation (Aslam et al., 2020) and their modification focusing on increase their biological role activity of interest (Mast et al., 2020). In Table 5.2, we describe different processes that functional peptides have demonstrated to cause effects on, such as cellular metabolism, hormone-receptor interaction and signaling cascades; they can also exert their action on the

regulation of metabolites controlled by the exceedance glands, adjusting blood pressure, exerting effects on sleep, memory, pain, appetite and the effects of the stress pathways on the central nervous system, exerting its effects locally or in various organs once they have entered the circulatory system (Gull et al., 2019; Sharma & Patel, 2005). It is relevant that any protein, independent of its functions and nutritional quality, can be used to generate peptides with biological activity, so it is highly recommended the use of proteins of unconventional origin or underutilized as proteins from wild sources, fish waste, by-products of oil extraction, etc. (Ronˇcevi´c et al., 2019). Considering the link between nutrition and health, peptides with biological activity could help reduce the current epidemic of degenerative diseases identified as chronic that affects a wide sector of world population. The future of functional foods is foreseeable, as concern for health leads to increased demand for new functional products based on measurable health beneficial effects (Anzai et al., 2018). Some strains of pathogenic microorganisms have developed resistance to conventional and currently available antibiotics, this represents a great threat to people’s health (Alsaadi & Jones, 2019).

TABLE 5.2 Biologically active peptides and their effects on the body. Peptides

Effect on the body

Immunomodulators

They stimulate the immune response

Angiotensin-I converting enzyme inhibitors

Reduce the risk of cardiovascular disease

Antioxidants

They prevent degenerative diseases and aging

Regulators of intestinal transit

They improve digestion and absorption

Regulators of intestinal proliferation

Reduce the proliferation of cancerous tumors

Antimicrobial

Reduce the risk of infections

Hypocholesterolemic

Reduce the risk of cardiovascular disease

Anticoagulants

Reduce the risk of thrombi

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5.6 Proteins and peptides with biological activity of medical interest

As is the case of coronavirus (CoV), which has infected many species and caused many different diseases (Seraphim & Houry, 2020). Cell membranes represent both an obstacle and an aid to CoV replication and consequently structural and nonstructural viral proteins have the ability to bind specific membranes. Structural proteins find the cell membranes both at virus entry and exit, while nonstructural proteins reorganize cell membranes to benefit virus replication (Watson, Phipps, Clark, Skylaris, & Madsen, 2018). Coronaviruses (CoVs) are enveloped positive sense RNA viruses; they are considered the largest of the RNA viruses with genomes ranging between 27 and 32 kb CoVs belong to the Coronaviridae family and contain two subfamilies Orthocoronavirinae and Letovirinae (Sa´nchez-Zu´n˜iga & Carrillo-Esper, 2020). The Coronaviridae are phylogenetically subdivided into four genera, α, β, γ, and δ. CoVs include members classified as emerging viruses, viruses that can cross the species barrier and cause pathology in a new target species. CoVs

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are structurally complex (Fig. 5.4) with purified virus particles that consist of four or five structural proteins along with a variety of minor components including host cell-derived and nonstructural proteins. All viruses have nucleocapsid (N), spike (S), envelope (E) and membrane (M) structural proteins, and some also encode a hemagglutinin esterase (HE) protein. The proteins of CoVs identified as structural occupy only about one third of the coding capacity of the genome. A much larger section of the genome, about two-thirds located at the 5 end encode two long open reading frames 1a and 1b that together encode the viral nonstructural proteins (McBride & Fielding, 2012). First, the virus proteins fuse in the membrane, the capsid proteins that clump together under the membrane patch where the viral glycoproteins are embedded. Second, the membrane protrudes outward to form a yolk decorated by the viral transmembrane proteins and enclosing the capsid, proteins and genome. Third and last, the yolk is separated from the rest of the membrane by cleavage, a pinch at the base FIGURE 5.4 Structural components of a Coronavirus particle.

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that releases the virion into an intracellular vesicle as in the case of CoV or directly outside the cell (Griffiths et al., 2005). CoVs are diverse, complex and adaptable viruses that have a significant impact on human health and animal productivity. Despite their diversity, there are common features, including the formation of membrane organelles that are driven by virus-encoded membrane-binding proteins (Griffiths et al., 2005). Both viral structural and nonstructural proteins contribute to membrane reorganization and viral protein. Interaction with membranes occurs at various stages of the virus replication cycle (McBride & Fielding, 2012). Finally, peptide nutraceuticals are probably the most versatile displaying functionalities as: antioxidant, antiproliferative/anticancer, antimicrobial, antihypertensive, antiinflammatory, among others. Despite the origin of peptide nutraceuticals is different their main role in the contribution of nutrients and prevention of diseases is highly recognized. Generally, the bioactive peptide nutraceuticals are isolated from protein precursors by digestive enzymes, during food processing, storage, or by in vitro hydrolysis by several proteolytic enzymes. Further future research will be necessary to better understand and find another sources of these compounds having nutraceutical properties.

5.7 Nucleic acids and their nutraceutical properties used in biomedicine 5.7.1 Nucleic acids overview Nucleic acids are important biomacromolecules in every living organism. The most known nucleic acid molecule is DNA, the master heredity molecule. Together with RNA has the biological function of store and transmit the needed information to produce all proteins presents in any cell (Watson et al., 2014). In structure, the nucleic acids are biopolymers composed of blocks called nucleotides. Each

nucleotide contains a pentose sugar, a phosphate group, and a nitrogenous base. The backbone of nucleic acids structure is made of the sugars of each nucleotide (ribose for RNA and deoxyribose for DNA), bonded together by the phosphate group. Nitrogenous bases are what dictate the ultimate characteristics of these molecules. There are five most common nitrogenous bases in nucleic acids, the purine bases: adenine (A) and guanine (G) and the pyrimidine bases: cytosine (C), thymine (T) (in DNA) and uracil (U) (in RNA) (Wang, Lim, & Son, 2014). Because of its physicochemical properties, each base is complementary with another, forming base pairs. C pairs with G through three hydrogen bonds and A with T in DNA and with U in RNA through two hydrogen bonds. When DNA is synthetized, it is doublestranded, this means that it consists of two long chains of nucleotides bound by the complementary nitrogenous bases of each nucleotide (Watson et al., 2014). The bound between complementary bases confers DNA properties to be a strong but flexible molecule, the hydrogen bonds between complementary bases can be modified by environmental changes as pH, heat, the presence of some chemicals, sonication, etc. When the hydrogen bonds break, separating the strands of the DNA is called denaturation. The two strands may reassociate when environmental conditions get to original parameters; this process is called renaturation (Smith, Schu¨ller, Engst, Ra¨dler, & Liedl, 2013). Denaturation and renaturation are very important processes that form the basis of multiple techniques for nucleic acids manipulation. The unique self-assembly properties of nucleic acids enable the development of programmable molecular nanostructures that have been useful in several disciplines, especially in biomedical applications. Two DNA molecules with complementary base sequences bind with each other and form numerous structures used depending on the application. One of the major advantages of DNA nanostructures is their

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5.7 Nucleic acids and their nutraceutical properties used in biomedicine

origin. In every living organism nucleotide material is constantly present. From self-DNA released from biological processes to DNA belonging to any other near organism. This makes nucleotide materials not inherently toxic to cell, as they are crucial native constituents in their inner environment (Keum & Bermudez, 2009). Still is important to monitor in each case that the material does not trigger a secondary response, a pathway interference or activation by its presence and detection. Avoiding the nucleic acids degradation mechanisms is imperial for its use in biomedical applications to assure the entrance of a medical treatment into the cell or tissue marked as a target. In vitro assays assessing the stability of small nanoconstructs consisting of a few nucleotides, demonstrated drastic differences to their linearized double-stranded analogs. Digestion of a DNA tetrahedra containing internal nicks by the nonspecific nuclease DNAse I occurred at a rate several times slower than its linear double-stranded equivalent. Having an increase of estimated half-life from 30 s to close to 2.5 min, at enzyme concentrations more than 25 times typical physiological levels (Gaˇcanin, Synatschke, & Weil, 2020). In the text below nucleic acid nanostructures that have been developed will be described to further applied examples. 5.7.1.1 DNA/RNA nanostructures The incorporation of branched junctions between three or more DNA strands allows the design and implementation of 2D and 3D structures and lattices with nanometer-sized features. This enables DNA to serve as a highly programmable structural building block. Some nanostructures are based on scaffolds with hydrophilic properties linked by short DNA sequences that act as cross-linking molecules. Such structures uses several programmable features of DNA as chemical flexibility and stability (Assi, Garavı´s, Gonza´lez, & Damha, 2018).

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Also, DNA molecules can function as crosslinkers in DNA motifs. Some examples of this are DNA i-motif: four-stranded DNA structure held together by hemi-protonated and intercalated cytosine base pairs (Liu et al., 2018), guanine quadruplex units: four guanines arranged in a plane stabilized by hydrogen bonds (Goodman et al., 2005) and Y-shaped or Xshaped DNA, enzymatic cross-linked oligonucleotide with sticky ends in each branch of the structure (Assi et al., 2018). These structural connectors provide the capacity to form flexible but strong and bigger net-like structures. Goodman created one of the most useful DNA-based structures and collaborators in 2005 consist of a family of DNA tetrahedra. It was designed to self-assemble in a single step in only a few seconds (Kavita, 2017). This mechanically robust structure is formed by four oligonucleotides that hybridize to form each of the four faces, and then the unions are enzymatically ligated. Some of its characteristics are high production yield and optimal size for encapsulation of individual protein elements up to approximately 60 kDa. DNA molecules are strong hydrophilic polyelectrolytes, this way, they absorb large water volumes resulting in generation of hydrogels. This property provide advantages in terms of molecular structure and functions as biocompatibility, controlled biodegradability, permeability for nutrients, stability against proteases, and ability of programmability for self-reconstruction and deconstruction (Assi et al., 2018). Several works have used the unique properties of molecular nucleic acids to develop specifically designed DNA devices for each application. The main function used in biomedical applications are the enlisted below. 5.7.1.1.1 Biosensing

The capacity of detection of specific analytes is vital in many biomedical fields such as disease diagnosis. Some characteristics adds

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practical value to biosensors, they offer visual detection of the target, making diagnosis faster and simpler. In general, biosensors are small devices that take advantage of highly specific biological recognition properties for the detection of a given molecule. Such devices rely on the coupling of a natural recognition mechanism with a physical transducer to develop a signal interpreted as the concentration of analytes (Tuerk & Gold, 1990). This kind of diagnosis techniques offers high reproducibility because if the structure of a disease-related protein is known, effective binders or inhibitors can be generated. In this case, DNA-based nanostructures can serve as highly specific, sensitive, and low-cost biosensors. A very clever biosensor design employ DNA cross-linkers as the sensors, this way, recognition of the analyte results in structural changes on biosensor molecule giving place to a report event such as release of an encapsulated cargo (Assi et al., 2018). In biosensors of nucleic acids, sensing elements are oligonucleotides and the biological recognition mechanism rely on complementary DNA base pairing. An in vitro method of ligand screening for a selected analyte from random nucleic acids molecules was developed since 1990. The set of DNA or RNA molecules that specifically bind to a specific analyte are isolated from a random sequence library and they were termed aptamers. The iteration of the evolution, selection, and replication steps in the procedure was named SELEX (Ellington & Szostak, 1990; Yu¨ce, Kurt, Hussain, & Budak, 2018). Nowadays, aptamers are perceived as short DNA or RNA antibody alternatives, with three-dimensional structures selected from large-scale random libraries through various in vitro iteration processes with a specific target from a broad range of biological molecules (Ng & Adamis, 2006). Recently, an antivascular endothelial growth factor (VEGF) aptamer is FDA-approved for use

in the treatment of macular degeneration. It has been named Pegaptanib and binds to VEGF inhibiting its actions as promotion of endothelial cell proliferation and survival and vascular permeability (Mor-Vaknin et al., 2017). Several other specific aptamer applications are in clinical trials for the treatment of diseases as acute myeloid leukemia, thrombosis (thrombotic microangiopathies), carotid artery disease, multiple-myeloma, non-Hodgkin’s lymphoma, type 2 diabetes and diabetic nephropathy (Gaˇcanin et al., 2017). 5.7.1.1.2 Drug delivery

One major limitation in drug administration is the capacity of control how much of the active compound will be delivered and when and where will be delivered. Often this limitation causes a reduction in the effectiveness of the treatment biological activity and an increase of off-targets side effects. Especially when the treatment is considered cytotoxic and nonspecific as chemotherapy. For this purpose, a highly sensitive and labile material is needed, just as DNA-based nanostructures and hydrogels. This technology has been used with highly positive results in the treatment of some diseases as osteoporosis (Song et al., 2015) and cancer (Ma et al., 2018) by the development of ingenious controlled delivery mechanisms. Anticancer drugs as floxuridine, a cytotoxic nucleoside analog, can be used as real nucleoside incorporating them into DNA strands by synthesis or incorporated into RNA by transcription. Then this nucleic acid strands can be employed to construct nanostructures thanks to their self-assembly property. Once assembled the DNA structures resemble the spherical nucleic acid architecture with excellent capability of rapid entering different cell lines without any transfection agents (Liao et al., 2017). The release of the cytotoxic drug is achieved by the hydrolysis of the nucleotide strands by DNases II within tumor cells.

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Some mechanisms have been explored to achieve hydrogel-to-solution transitions in DNA-based materials. Using DNA hairpin structures with an anti-ATP aptamer sequence within the hydrogel structure, the drug can be released when it enters in contact with ATP. This approach could be escalated to many other particular molecules, improving the specificity of the spatial and temporal drug release (Wang, Xia et al., 2015; Yata et al., 2017). Another already used strategy includes metal nanoparticles integrated in the DNA matrix by its positive charge. This way the hydrogel denaturation and drug release can be achieved by photo-thermal treatment and the nanoparticles reaction (Ma et al., 2018). There are some limitations about using allDNA nanostructures as sensitivity to heat and enzymatic digestion; some researchers integrate other compounds to the nanostructure to avoid these limitations. DNA-acrylamide copolymer hydrogels (Wang, Xia et al., 2015), protein-DNA hydrogels (Song et al., 2015), gold nanostructures (Capek, 2019), etc. Effective carrier systems aimed at delivering intracellular therapeutics must be able to cross the protective plasma membrane of cells, either by passive or active uptake. In general, particles on the order of tens or hundreds of nanometers in size are more readily taken in by cells’ active machinery than individual small molecules, an effect that has been confirmed for DNA nanostructures also falling within this size range (Wu & Zhao, 2020). Plus DNA nanostructures are proved to possess high cell-penetrating ability by endocytosis, even though without the help of transfection reagents (Burns, Lamarre, Pyne, Noble, & Ryadnov, 2018). Burns and collaborators designed an equilateral 25 nm DNA box held together by single strands staples that lock lid and bottom faces orthogonally via one side. The lid side is hinged on short i-motif staples that undergo conformational transition in response to

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lowered pH inducing the opening of the box. Thereby facilitating the egress of encapsulated cargo (Halley, Mcwilliams, & Patton, 2017). Boxes effectively transfected HeLa cells promoting Green Fluorescent Protein (GFP) release into the cytoplasm. Particulate spreads of fluorescent DNA were apparent in the cytoplasm within the first hours of incubation suggesting an excellent endocytic uptake. DNA nanoparticles serve as well as an efficient binding platform for intercalating anthracyclines doxorubicin and daunorubicin for acute leukemia treatment. In 2016, Halley y collaborators reported the development of a rodlike DNA origami used as a Trojan horse nanostructure that can be controllably loaded with daunorubicin. This nanostructuremediated delivery leads to increased drug entry and retention in cells relative to free drug at equal concentrations (Li et al., 2018). Some DNA nanostructures are called nanoscale robots because of their great potential as intelligent drug delivery systems that respond to molecular triggers. An autonomous DNA robot programmed to transport payloads and present them especially in tumors was developed in 2018. In the outside it has a DNA aptamer that binds with nucleolin, a protein that has a high expression patron on tumorassociated endothelial cells. Once recognized the nucleolin molecule, the aptamer triggers the opening of the DNA nanorobot, releasing thrombin, and a coagulation factor that induces intravascular thrombosis, thus resulting in tumor necrosis and inhibition of tumor growth (Nu¨rnberger & Brunner, 2002). 5.7.1.1.3 Immunomodulatory

The use of vaccination has improved the way we confront world diseases, with this strategy we train the immune system to get ready for the new pathogens by showing to the recognition system, highly conserved parts of the infectious agent. This way the immune system is prepared for the pathogen if it comes for

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real. The molecules used in the vaccines are called pathogen associated molecular pattern (PAMP) and there are molecules that resembles a danger signal (Krieg et al., 1995). Since 1995 a new immunomodulatory treatment arises by the recognition of a specific dinucleotide that has shown PAMP properties. The CpG genome motif is sensed by a protein called toll-like receptor 9 (TLR9) and is identified as a PAMP for being highly common in bacterial genomes (Nehete et al., 2020). This recognition releases a set of responses to activate the immune system including a variety of cell types spanning B cells, natural killer cells and antigen presenting cells (Nomura et al., 2018). Nowadays there are some experimental therapies ongoing. The addition of the CpG motif to vaccines has been reported with high rates of efficiency (Umeki, Saito, Takahashi, Takakura, & Nishikawa, 2017) when loaded with multiple antigens as ovalbumin (Umeki et al., 2018) or even immune cells (Mathiyalagan & Sahoo, 2017). Moreover, due to the DNA hydrogels physical properties allow even intranasal vaccination and excellent improvement to avoid repeated subcutaneous injections. Some diseases have been linked with immune system malfunctions, generally this happens when immune system, as a response to environmental or endogenous factors, triggers severe immunological mechanisms leading to an autoimmune disease. The immune system could start the destruction or disruption of the body’s own tissues. In this case it is necessary to down regulate the immune system by the inhibition of the response mechanisms. Recently, a DNA aptamer specific to DEK, a nuclear protein that has been identified as an autoantigen crucial for the development of inflammation in juvenile idiopathic arthritis and other autoimmune diseases. Is secreted by macrophages and released by apoptotic T

cells and then function as a chemoattractant for neutrophils and other inflammatory cells (Mathiyalagan & Sahoo, 2017). Using the ZIA mouse model with induced arthritis, we found that single-stranded anti-DEK DNA aptamers markedly attenuate inflammation in WT mice. The total white blood cell, including neutrophil, recruitment was significantly reduced after treatment with anti-DEK aptamers (Mathiyalagan & Sahoo, 2017). 5.7.1.2 DNA/RNA functional sequence 5.7.1.2.1 Non-mRNA sequences

Before the sequencing techniques become a common practice in the genome research, genes were, in general, delimited as the coding region, and it was believed that the gene diversity between species were in major part due to differences in the small portion of each genome that was coding for a protein. The rest of the sequences in the genome were called “junk DNA.” Nowadays it has been found the great importance that gene regulation has in every organism phenotype. Later, several cases of DNA sequences that were classified as noncoding DNA (ncDNA) or junk DNA are transcribed into ncRNA with functions else than produce a protein. RNA types other than mRNA and rRNA most of them having a major participation in gene transcription regulation have been described. Recently, two kind of ncRNA have been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders. MicroRNAs (miRNAs) are critical regulators of gene expression at the posttranscriptional level in higher eukaryotes. Consequently micro interfering RNAs are regarded as potential pharmacological candidates for therapeutic treatment for several disorders, as cardiovascular diseases (Martier et al., 2019) and neurodegenerative disorders (Scherman, Rousseau, Bigey, & Escriou, 2017). On the other hand, small interfering RNA (siRNA) is a class of short double-stranded RNA molecules,

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which can mobilize the RNA interference pathway. This is a sequence-specific gene-silencing process and offers a powerful platform for biomedical applications (Gonc¸alves & Paiva, 2017). 5.7.1.2.2 Gene therapy

As genetic studies revealed more information about how genes shape every part or our organism and its function, the medical research has had the objective to make local modifications in the human genome. Gene therapy is understood as the ability of genetic improvement through the correction of altered genes or site-specific modifications that target therapeutic treatment (Nun˜ez-Rivera et al., 2020). Currently, gene therapy-based most advanced treatments are experimental procedures. The potential treated diseases are the ones caused by recessive gene disorders, acquired genetic diseases and certain viral infections (Nun˜ ez-Rivera et al., 2020). One of the most significant challenges in this treatment development is the way that the gene will be released into the stem cell. The gene molecular carrier is called a vector, and in order to be accepted, it should be very specific, display efficiency in the release of the gene and not be recognized by the immune system and it should be viable to be produced and purified in large quantities and high concentrations (Nun˜ez-Rivera et al., 2020). There are two main approaches for application of gene therapy: virus-mediated and physical mechanisms. The first one, consist in introducing a human gene known to cause a genetic disease, into the genome of a virus. This virus must be nonpathogenic for humans or the pathogenic genes should be deleted from its genome. With this approach, the natural processes of the virus would achieve the injection of the genetic material into the targeted cell and the gene could be transcribed and translated into a functional protein, reducing the damage of the disease.

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The use of plant viruses for anticancer therapy has gained an increased interest. Recently, Nun˜ez-Rivera and collaborators demonstrated important properties of biomedical interest of the plant virus brome mosaic virus (BMV), such as biocompatibility, tumor cell internalization and their efficiency as nanocarriers for siRNA delivery, as well as the capacity of the BMV virus to modulate the immune response in vitro was also analyzed (Ding et al., 2018). This carrying system did not activate macrophages in vitro suggesting its low immunogenic properties. The second one main gene therapy approach includes a high diverse of structures obtained by advanced nanotechnology techniques; these are deeply evaluated to assure the injection of the genetic material into the cell and that it will not cause any adverse reaction. The major advantage of this approach is that the size of the nanostructures allows them to enter the cell without being recognized as a damage associated particle (Choi, Zhang, & Choi, 2018; Ding et al., 2018). With the purpose of reducing the toxicity of nanoparticles due to excess cationic polymers or lipids, researchers have developed a diversity of noncationic siRNA carriers. These systems are increasingly being used to silence genes in antitumoral therapy (FDA, 2020). Some authors employed siRNAs that specifically silence one gene as polo-like kinase 1 (PLK1) gene, whose inhibition induces cell apoptosis (Choi et al., 2018). In this strategy, the noncationic carriers are designed with a targeting ligand, so the nanostructure binds to specific tumor cells and the treatment is more effective. Some products currently approved in US and EU for gene therapy are shown in Table 5.3. 5.7.1.2.3 CRISPR

One of the most relevant biotechnological tools, Nobel prize winner in 2020 is the CRISPR system for gene edition. It is based on a biological mechanism found in procaryotes, it is an immuneadaptive system that recognizes an invading

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TABLE 5.3 Gene therapy products approved in the United States and EU (January 2020). Approved product

Country Indication

Development institution

Imlygic

United States and EU

Genetically modified, live, attenuated, herpes BioVex, Inc. simplex virus type 1 that promote an antitumor response in tumor cells

Ali et al. (2020), Greig (2016)

Kymriah (tisagenlecleucel)

United States and EU

Patient’s own T-cells genetically modified with a chimeric gene that directs the T-cells to kill leukemia cells

Novartis Pharmaceuticals Corporation

Ameri (2018), Greig (2016), Shahryari et al. (2019)

Luxturna

United States and EU

Recombinant adeno-associated virus 2 genetically modified with the human gene RPE65. Treatment for mutation-associated retinal dystrophy associated with mutation in RPE65 gene

Spark Therapeutics, Inc.

Ameri (2018), Anassi and Ndefo (2011), Greig (2016)

PROVENGE (sipuleucel-T)

United States and EU

Patient’s Antigen Presenting Cells modified with a recombinant antigen protein highly expressed in most prostate cancer cells

Dendreon Corp.

Greig (2016), Voelker (2020)

TECARTUnited States (brexucabtagene autoleucel)

US

Patient’s own T-cells genetically modified with a gene that directs the T-cells to kill lymphoma cells

Kite Pharma, Inc.

Greig (2016), Jain, Bachmeier, Phuoc, and Chavez (2018)

YESCARTA (axicabtagene ciloleucel)

US and EU

An anti-CD19 chimeric antigen receptor T-cell therapy for lymphoma cells fight

Kite Pharma, Inc.

Ameri (2018), Greig (2016), Mahajan (2019)

ZOLGENSMA (onasemnogene abeparvovec-xioi)

US

Adeno-associated virus that delivers a fully functional copy of human SMN gene into the motor neuron cells, mutations in this gene results in spinal muscular atrophy

AveXis, Inc.

Ameri (2018), Greig (2016), StirnadelFarrant et al. (2018)

Zalmoxis

EU

Allogeneic T-cells genetically modified with a retroviral vector encoding for a form of the human low affinity nerve growth factor receptor

MolMed, Inc.

Ameri (2018)

Strimvelis

EU

Patient’s CD341 cells modified with retroviral vector encoding for the human ADA gene. For severe combined immunideficiency due to adenosine deaminase deficiency

GSK

Ameri (2018), Scott (2015)

Glybera (alipogene tiparvovec)

EU

Adeno-associated viral vector expressing lipoprotein lipase. Low production of this enzyme results in severe abdominal pain, repetitive colicky pains and episodes of pancreatitis

UniQre, Inc.

Ameri (2018), Doudna and Charpentier (2014)

genetic material, cleaves it into small fragments, and integrates it into its own DNA. This way, if the same agent presents again the procaryotic cell

Reference

has a genetic “memory” and its able to transcript the CRISPR locus, creating small fragments of the generated mRNA that form complexes with

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double strand-nucleases (Cas9) (Cong et al., 2013). The complexes can recognize the exogenous genetic material and destroy them (Monteiro, Martins, Reis, & Neves, 2014). After the elucidation of this biological process, Cong and collaborators managed to demonstrate that Cas9 nucleases could be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. The development of this technique has open a numerous genome editing possibilities with powerful applications across several scientific areas, specially biomedicine (Monteiro et al., 2014). Sickle cell disease (SCD) is caused by a single nucleotide polymorphism in the gene for one of two globins that make up the major adult form of hemoglobin. This nonsynonymous mutation renders hemoglobin prone to polymerization under hypoxic conditions, producing characteristic “sickle” shaped red blood cells. Recently genome editing has provided a promising avenue to treat affected hematopoietic cells. Using a preassembled ribonucleoprotein complex, composed of recombinant Cas9 protein and unmodified guide RNA to introduce healthy gene versions into human adult hematopoietic cells, focusing on the SCD mutation. These reagents efficiently induce templated homology-directed repair-mediated editing of the SCD mutation in affected hematopoietic cells with minimal genic off-target activity. Corrected hematopoietic cells from patients produce less sickle hemoglobin and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts (Monteiro et al., 2014).

assess the risks of new procedure and the moral implications involved. For nucleic acid new applications, we can start seeing the DNA molecules as any other molecules with physical and biochemical properties that we can use in any way we need. This idea has been developed to construct a lot of nanodevices for several different purpose, but it gets to a closed door In the moment we recognize that DNA molecule have defined every specie and every living organism in the world, including human beings. If we manage and get to control the DNA, the sense of unique natural shaping of each person that conforms the great human diversity, gets lost. In the next years, these bioethical questions will be highly discussed, and choices will have to be taken to really apply all the scientific developments that are currently being explored. Meanwhile, manufacturers are investing more in this field, and an increasing number of products are under clinical development, mostly in early stages (Cong et al., 2013). The only medical products approved for its sell and use are listed in Table 5.1 for United States and the European Union. Although the approved treatments have no safety issues, there are some cases as Glybera that has been withdrawn, the reason for this is the high cost of the treatment (average of 1 million dollars per treatment), this is an important issue to add in the bioethical discussion. Almost no one have actually access to this kind of therapies (Cong et al., 2013).

5.8 Introduction of lipids

5.7.2 Perspectives Even though all the experimental treatments described in this section has promising results in several different diseases, the progress of research in this area is relatively slow. There are a lot of ethical issues surrounding nucleic acid- based treatments, especially about gene therapy. This has been the object of heated discussion in the field of science for a long time to

Lipids are small biological macromolecules that classifies mainly by their hydrophobic or amphiphilic properties. Therefore, lipids contains a large number of biological molecule groups as fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides. The amphiphilic nature of some lipids allows them to form organized

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structures such as vesicles or membranes, when immersed in an aqueous environment (Ayala, Mun˜oz, & Argu¨elles, 2014). Lipids are divided into two groups: apolar and polar. Triglycerides (apolar), stored in various cells, but especially in adipose (fat) tissue, are usually the main form of energy storage in mammals. Polar lipids are structural components of cell membranes, where they participate in the formation of the permeability barrier of cells and subcellular organelles in the form of a lipid bilayer (Balsinde, 2015). These compounds play a vital role in physiological and pathophysiological events of living systems (Balsinde, 2015). Lipids are key substances for intercellular communication; nowadays we know that lipids connect the external simulation of cells and tissues via receptor with the execution of specific responses (Schneider et al., 2017). Lipids and/or their precursors derived from food are taken up by the organism and used for the maintenance of general cell function. In particular, the integrity of cell membranes and signaling through membranes depend on lipid homeostasis in the brain. Studies published in recent years have provided new insights into how lipid homeostasis, most notably of phospholipids, sphingolipids, and endocannabinoid lipids, affects mental functions (Elmasry, Ibrahim, Abdulmoneim, & Shabrawey, 2018). Lipids and their metabolites are not only components of cellular membranes but also act as signaling molecules and are thus known as “bioactive lipids.” Among the key activities exerted by bioactive lipids are angiogenesis, regulation of inflammation and maintenance of homeostasis.

5.8.1 Polyunsaturated fatty acids Bioactive lipids cover a large number and variety of compounds and the most commonly ones are those derived from polyunsaturated fatty acids (PUFAs) (Hashimoto, 2019). These fatty acids contain multiple double bonds. Long chain PUFAs, which are more than 20 carbons in length,

are derived from two independent and nutritionally essential fatty acids (EFA), α-linolenic acid and linoleic acid (Elmasry et al., 2018). Evidence suggests that PUFAs plays a highly important role in normal physiology maintenance and function and structure regulation of multiple cell lines as neurons, glial cells, and endothelial cells in the brain (Sokola et al., 2018). Fatty acids are considered to be a fundamental building material for the structural components of cells, tissues, and organs as well as for the synthesis of certain biologically active substances (Su, Matsuoka, & Pae, 2015). They can be further classified in various groups due to their chemical structure. Omega fatty acids are classified according to the location of the first double bond (Su et al., 2015). Two main compound groups are distinguished (Table 5.4; Fig. 5.5). It has been established that PUFAs are required for the normal development and functioning of the brain and heart, and also for the equilibrium of all tissues and organs (Su et al., 2015). PUFAs are nowadays-desirable components of “specialty oils,” oils with special dietary, and functional properties that are used as nutraceuticals or cosmeceuticals. Due to the better understanding of their biological and functional properties, and their health benefits, PUFAs, specially omega-3 PUFAs are of great importance for health system, due to their potential applications in disease prevention (Mori et al., 2017). Deficit of omega-6 linoleic acid leads to poor growth, fatty liver, skin lesions, and reproductive failure, while the symptoms of omega-3 fatty acids deficiency include reduced vision or abnormal electroretinogram results (Su et al., 2015). Nutrition is a modifiable environmental factor that might be important in prevention medicine, the use of omega-3 PUFAs in the secondary prevention of heart disease with this latter prevention medicine mentioned (Balic, Vlasic, Zuzul, Marinovic, & Mokos, 2020). It has been established that PUFAs are required for the normal

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TABLE 5.4 Most important groups of PUFA. PUFA

Description

Sources

Omega-3 PUFAs

Essential macronutrients and must be obtained from dietary sources because the body cannot synthesize them effectively. May provide a range of neurobiological activities via modulation of neurotransmitters, antiinflammation, antioxidation and neuroplasticity, which could contribute to their psychotropic effects (Balic et al., 2020)

Fatty fish, such as salmon, and in fish-oil supplements Meat of free-ranged animals (herbivores and carnivores) (Su et al., 2015)

Omega-6 PUFAs

Represented by the parent compound linoleic acid and Corn, soybean, and sunflower oil, as well as nuts, constitutes another essential fatty acid that has to be including coconut together with coconut oil, delivered with diet (Su et al., 2015) almonds, pine-nuts, and hazelnuts (Balic et al., 2020)

Elcosapentaenoic acid (EPA) Omega-3

Linoleic acid (LA) Docosahexaenoic acid (DHA)

PUFA Arachidonic acid (AA) α-Linolenic acid (ALA)

Omega-6

Docosapentaenoic acid (DPA)

FIGURE 5.5

Precursors and types of each PUFA.

development and functioning of the brain and heart, and also for the equilibrium of all tissues and organs. Studies concerning the nutritional deficiency of omega-3 fatty acids as well as the particular roles of omega-6 and omega-3 have become the focus of numerous research groups around the world (Mori et al., 2017; Su et al., 2015).

5.8.2 The role of Omega-3 PUFAs in some disorders The omega-3 fatty acids group has been evidenced to help reduce inflammation (Su et al.,

2015). As we know, several psychiatric and neurological conditions are largely affected by inflammation, some of the most common are described in Table 5.5. Several lines of evidence support the importance of omega-3 PUFAs in brain disorders. The omega-3 PUFAs series include docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which are EFA that cannot be efficiently synthesized by the human body and have to be obtained through dietary intake. EPA and DHA have an antiinflammatory action via inhibition of free radical generation and oxidant stress, and have also been shown to regulate neurotransmitter and immune functions via the modulation of lipid rafts signaling platforms

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TABLE 5.5 Most common psychiatric and neurological disorders. Disorder

Description

Role of omega-3 PUFA

Depression and bipolar disorders

Depression, one of the most common disorders in the world, is a major psychiatric disorder with a high rate of relapse. The World Health Organization (WHO) estimates that more than 320 million individuals of all ages suffer from depression (Su et al., 2015). Psychiatric disorders based on current diagnostic systems are clinically and biologically heterogeneous. The heterogeneity is also reflected by current classification systems for antidepressant drugs (Balic et al., 2020).

Interestingly, two common pathways, neuroprotection and antiinflammation, have been found to be associated with all the antidepressant drugs. More importantly, these two common mechanisms link to antidepressant effects not only for drugs but also for nonpharmacological treatment omega-3 PUFAs (Balic et al., 2020). Several reports using meta-analysis demonstrated that omega-3 PUFAs could reduce depressive symptoms beyond placebo. Dietary intake of omega-3 PUFAs is known to be associated with lower risk of depression. Importantly, EPA-rich omega-3 PUFAs could be recommended for the treatment of depression (Sokola et al., 2018).

Inflammation in Parkinson’s disease (PD) is characterized by Parkinson’s deterioration of the nigrostriatal system and associated Disease with chronic neuroinflammation (Delattre et al., 2017). Evidence suggests that idiopathic Parkinson’s disease (PD) is the consequence of a neurodevelopmental disruption, rather than strictly a consequence of aging (Delattre et al., 2017).

Although the precise pathogenesis of PD is unknown, the pathological hallmark of PD involves the progressive loss of dopaminergic neurons in the substantia nigra (SN). Omega-3 PUFAs appear to be neuroprotective for several neurological disorders. It is reported that dietary intake of PUFAs is associated with lower risk of PD (Sokola et al., 2018).

Eating Disorders and ADHD

Anorexia nervosa (AN) is a serious eating disorder characterized by the persistent restriction of energy intake, intense fear of gating weight, and distribution in self-perceived weight or shape (Sokola et al., 2018). Attention deficit hyperactivity disorder (ADHD) is one of the most common psychiatric disorders affecting children (Delattre et al., 2017). ADHD has been suggested to be a disorder involving an impaired inhibition control system and a disrupted feedback of the rewarding and motivational system (Chang et al., 2018).

Deficiency in omega-3 polyunsaturated fatty acids has recently been investigated as a potential pathogenetic mechanism in ADHD. Omega-3 PUFAs have been associated with cognitive function and learning, including in patients with ADHD. Hence, omega-3 PUFAs may be considered one of such novel treatments (Chang et al., 2018).

Anxiety disorders

Anxiety, the most commonly experienced psychiatric symptom, is a psychological state derived from inappropriate or exaggerated fear leading to distress or impairment. Anxiety is often comorbid with depressive disorders and is associated with lower health-related quality of life and increased risk of allcause mortality (Su et al., 2018).

Some preclinical data support omega-3 PUFA as an effective treatment of anxiety disorders. For example, an EPA-rich diet could reduce the development of anxiety-like behaviors in rat as well as normalizing dopamine levels in the ventral striatum (Sokola et al., 2018). A number of trials have found that omega-3 PUFAs might reduce anxiety under serious stressful situations. Case-controlled studies have shown low peripheral omega-3 PUFA levels in patients with anxiety disorders (Su et al., 2018).

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5.9 The potential use of bioactive lipids in cancer stem cells and coronavirus disease (COVID-19)

on the cell membrane (Chang, Su, Mondelli, & Pariante, 2018; Su et al., 2018).

5.9 The potential use of bioactive lipids in cancer stem cells and coronavirus disease (COVID-19) 5.9.1 Bioactive lipids in cancer According to Begicevic et al. (2019), cancer progression is characterized by a continuous changeable state generating a very complex and heterogeneous multitude of cells with different morphology, genotype, and phenotype. Two main models explain this heterogeneity: The clonal evolution model and the cancer stem cell (CSC) model. CSCs present an elevated tumorigenic potential and an increased resistance to conventional and targeted therapy. Tumors share a common phenotype of uncontrolled cell proliferation and for this they must efficiently generate energy and biomass components in order to expand and disseminate. Hence, cancer cells show an expanded metabolic repertoire that affords the flexibility to withstand and grow in this harsh tumor environment (Djefaflia, Vasseur, & Guillaumond, 2016). Lipid dysfunction has been observed as a trait of more aggressive cancers that have adverse survival outcomes. Research is highlighting the specific alterations occurring in pathways involving lipids and cholesterol. An emerging concept is that CSCs are highly dependent on enzymes associated with lipid metabolism, even though the precise reason for this reliance is not completely understood (Das, 2020). Excessive lipids and cholesterol in cancer cells are stored in lipid droplets (LDs), and high LDs and stored-cholesteryl ester content in tumors are now considered as hallmarks of cancer aggressiveness (Ghaffari et al., 2020). Fatty acid synthesis and oxidation are indispensable components in the maintenance of the adult stem cell and CSC populations from various

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organs. Both anabolic and catabolic pathways are closely controlled in CSCs and are essential for self-renewal activity (Ghaffari et al., 2020).

5.9.2 Bioactive lipids in COVID-19 The current epidemic of COVID-19 (coronavirus) has been declared by WHO as a global epidemic. This COVID-19 epidemic is somewhat similar to the previous severe acute respiratory syndrome (SARS-CoV-2). Like other CoVs, SARSCoV-2 particles are spherical and have proteins called spikes protruding from their surface that can latch onto human cells, which then undergo a structural change allowing them to fuse with the cell membrane. This facilitates the viral genes to enter the host cell to be copied, producing more viruses (Aachary & Prapulla, 2011). The novel coronavirus (COVID-19) has led to the economic disruptions and global health concerns due to its sustained human-to-human transmission and rapid spread. Along with other endemic human coronaviruses, it seems that the SARS-CoV-2 will become the fifth endemic coronavirus in the human population. Nowadays, discovering therapeutic options from currently available agents appear to be essential for the treatment and prophylaxis of this pandemic (Aachary & Prapulla, 2011). Some unsaturated fatty acids can be served as endogenous antiviral compounds, and their deficiency make humans more susceptible to certain viral infections including SARS-CoV-2, SARS and MERS. The bioactive lipid amide OEA (Oleoylethanolamide) is synthesized in the gastrointestinal tract, and is related to several distinctive homeostatic properties, including antiinflammatory activities, immune response, stimulation of lipolysis and fatty acid oxidation (Aachary & Prapulla, 2011). In view of the fact that SARS-CoV-2 infection leads to increased release of the proinflammatory cytokines, including interleukin-6 (IL-6) and IL1β in COVID-19 patients via binding to the TLRs, it is assumed that OEA inhibits this

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pathway through its antiinflammatory properties (Aachary & Prapulla, 2011; Ghaffari et al., 2020).

5.10 Carbohydrates as nutraceuticals 5.10.1 Brief overview of carbohydrates Carbohydrates are one of the most abundant biological macromolecules in nature, commonly known as sugars or polysaccharides and their structure consists of branched or linear chains of monosaccharides (Yang & Zhang, 2009). Regularly, sugars are classified by their molecular charge: cationic, anionic or nonionic polysaccharides (Funami et al., 2008; Korakli & Vogel, 2006); by their chemical composition, depending on the functional groups present in their molecules or by the number of different monosaccharides that conforms them. In this case, if the molecule is composed of multiple copies of the same monosaccharide it is classified as homopolysaccharides (Amid & Mirhosseini, 2012), as cellulose that is composed by glucose monomers only, on the other hand, if the molecule contains two or more different monosaccharides are called heteropolysaccharides (Hurst, Poon, & Grifth, 1983). Together, polysaccharides constitute the basis of

life by the mediation of several biological signals allowing most of the biological mechanisms in every living organism (Englyst, Anderson, & Cummings, 1983). In Table 5.6 we describe the principal forms in which carbohydrates are presents in every large phylogenetic group. It is often difficult to characterize and classify polysaccharides because of their highly diverse molecular properties, but this had allowed a development of highly diverse functions and biological activities (Poletto, Ornaghi, & Zattera, 2014). The recent increase of bioanalytical techniques enables researchers to understand the structure of many biological molecules as polysaccharides. Every day increase in these knowledges promote the application of polysaccharides in several human development areas principally biomedicine (Frantz, Stewart, & Weaver, 2010). In this section, we describe the current developed biomedical applications of carbohydrates in their principal forms.

5.10.2 Role of polysaccharides in extracellular membrane Polysaccharides are present in large amounts and diversity of forms in the extracellular matrix

TABLE 5.6 Various polysaccharides in nature. Polysaccharides

Source

Functions

Starch

Plants

Storage, drug adjuvant

Cellulose

Plants

Cell structure, food additives

Pectin

Plants

Food additives

Alginate

Microorganism

Drug adjuvant

Carrageenan

Microorganism

Food additives

Heparin

Animals

Animal tissue structure, therapeutic agents

Hyaluronan

Animals

Animal tissue structure, therapeutic agents

Chondroitin sulfate

Animals

Animal tissue structure

Chondroitin sulfate

Animals

Animal tissue structure

Chitin and chitosan

Animals

Tissue scaffolds

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5.10 Carbohydrates as nutraceuticals

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(ECM) in animal tissues. As we know, ECM is a structure composed by networks of different molecules mainly heteropolysaccharides and proteins. Here, polysaccharides plays crucial roles in providing the unique physical properties to ECM such as cell adhesion, fluidity, flexibility and porosity for the diffusion of nutrients and oxygen (Rusnati & Presta, 1996). As researchers characterized these properties, new ideas in polysaccharide practical application as their use in structure constructions have been developed (Bohn & BeMiller, 1995).

environmental signals. In the case of TLR4 receptor several evidence confirms its relation with polysaccharide signaling (Zhao, Mancini, & Doria, 1990), for example in vitro tests performed in mice macrophages with mutations in the TLR4 gene caused inhibition of macrophage activation by polysaccharide presence (Kim & Kim, 2017). Several polysaccharide effects on biological signaling pathways are described in Table 5.7.

5.10.3 Immunostimmulatory effect of carbohydrates

Polysaccharides in plants form different structures than those found in animals, when ingested by animals, these molecules play new roles in animal metabolism. One of the main forms of polysaccharide found in plants is called dietary fiber. This classification includes cellulose, pectin and gums and refers to nondigestible polysaccharides with beneficial health properties to digestive system better described later in this section. Plant polysaccharides also have shown immunomodulatory properties regulating macrophages by signal recognition (Schepetkin, Faulkner, Nelson-Overton, Wiley, & Quinn, 2005) A vegetal recognized plant polysaccharide with these properties are glycans, known principally to phagocytic activation (Wong, Yuan, & Luk, 2012), increase cytotoxic activity on tumor cells and ROS.

Another important role of polysaccharides is the signal molecule role. This role has been confirmed mainly in the immunological system of animals (Xiao et al., 2011). Polysaccharides have been related to modulatory effect of cytokines and kinases pathways (Baek et al., 2010; Li et al., 2016, 2017; Niu et al., 2017; Wang, Liu, Li, Wang, & Wang, 2015; Xu, Xia, Wang, & Wang, 2008), specifically activities like production and transport of several cytokines have been seen modulated by some carbohydrates (Hamman, 2008; Hsu et al., 2004; Liao et al., 2015; Luk, Lai, Tam, & Koo, 2000). Immunomodulatory effects of polysaccharides as hematopoiesis (Xiao et al., 2011) and increase cellular number in vivo (Hollmig, Ariizumi, & Cruz, 2009) has been observed in mouse macrophages (Song et al., 2002). Two molecules have been suggested as molecular receptors responsive for the interactions between polysaccharides and macrophages: are toll-like kind receptors (Zamze et al., 2002) and mannose receptor (Ando et al., 2002). These receptors are activated by their union with polysaccharides leading to the activation of transduction cascades conformed mainly by proinflammatory factors until a change in cell metabolism is reached as response of

5.10.4 Carbohydrates from plants with nutraceutical activity

5.10.5 Cellulose and hemicellulose These polysaccharides are composed of β-Dglucan units connected by (1-4) glycosidic bonds (O’Riordan, Yi, Gonzales, Lee, & Portnoy, 2002), this structure provide physical unique properties to these molecules, this allows cellulose and hemicellulose to form part of the dietary fiber, having directly shown stimulation of intestinal movements. As researchers characterized physical properties of these polysaccharides, innovative materials based on cellulose structure

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TABLE 5.7 The polysaccharides derived from herbs in nature. Polysaccharides

Composition

Source

Physiological effects

Cellulose

β-(1
4)-Linked-glucopyranose

Grains, fruit, vegetables

Cell structure, food additives, regulate bowel movement

Juniperus scopolorum polysaccharide

β-Galactopyranose, and α-arabinofuranose Juniperus scopulorum

Immunomodulatory effect to the murine macrophages

Konjac glucomannan β-(1
4)-Linked-glucose, β-(1
4)-linkedpolysaccharide mannose

Amorphophallus konjac plant

Cholesterol lowering and immunoregulation

Chelidonium majus polysaccharide

Chelidonium majus

An effective antitumor immunostimulator

Reishi polysaccharide Arabinose, rhamnose, xylose, mannose, glucose at the different ratios

Ganoderma lucidum

Stimulating the expression of inflammatory cytokines

Ginseng polysaccharide

(1
4)-Linked homogalacturonan backbone

Ginseng, the root of Anti-rotavirus activity Panax ginseng

Bletilla striata polysaccharide

α-Mannose, β-mannose, and β-glucose at the ratio of 2.4:1

Bletilla striata

Modulating the function of macrophages

Eucommia ulmoides polysaccharide

Mannose, galactose, glucose, arabinose, rhamnose, and galacturonic acid

Eucommia ulmoides

Binding PDGF-BB growth factor and anti-infammatory effect

Astragalus polysaccharides

Rhamnose, arabinose and glucose in a molar ratio of 1:6.25:17.86

The roots of Astragalus

The effect of immunomodulatory

Pectin

α-(1
4)-D-Galacturonic acid and rhamnose

Plant primary cell wall

Food additives

Galactose, mannose, glucose in the molar ratio of 5:4:1

have been developed for different applications, mainly medical, some examples of this are adsorbent beads, filters and artificial tissues including engineered tissues as vascular, structural and heart tissues (Truman et al., 2008). Also, cellulose-based materials have been used to build drug carriers (Rabinovich & Toscano, 2009).

Glycosaminoglycans (GAG), a family of biological polymers are found principally in animal metabolism (DeAngelis, 2012), those include hyaluronic acid (HA), heparin and heparan sulfate between several others and have been evaluated for their great biological roles.

5.10.7 Heparin

5.10.6 Animal derived carbohydrates with nutraceutical activity Regarding the high similarity human metabolism have with multiple animals, the bioactive roles of polysaccharide observed in animals are largely important to development of new medical treatments. Here we mention the principal polysaccharide with an observed effect in biological processes in animals.

Formed by multiple units of sulfonated hexuronic acid (1 - 4) D-glucosamine bonded between each other, it is known that when metabolized, different versions of their residues are produced as unmodified (GlcN), N-sulfonated (GlcNS), or N-acetylated (GlcNAc). These residues present many biological functions (Casu, Naggi, & Torri, 2015). Heparin is present mainly on the cell surface and ECM, actually, it has been widely

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5.10 Carbohydrates as nutraceuticals

used in medicine, identified as one of the oldest drugs administrated. It is linked to multiple natural processes as angiogenesis, cell adhesion, cell growth and inflammation (Kadomatsu, Kishida, & Tsubota, 2013; Nikitovic, Mytilinaiou, Berdiaki, Karamanos, & Tzanakakis, 2014; Peysselon & Ricard-Blum, 2014). Studies suggests that heparin regulate biological processes by binding to proteins with important regulator roles such as growth factors (You et al., 2013), forming complexes to stabilize other molecules and prolong their functional life. These desired properties have been applied in the development of synthetic structures, for example a hydrogel system based on heparin molecules that released hepatocytes growth factor continuously (Chen et al., 2017; Feizi & Haltiwanger, 2015). In other studies, heparine-based structures as hydrogels (Howard, Ebert, Bloom, & Sloan, 1959) and coacervates (Friedman, 2016) have been used to treat skin wound and skull defects (Friedman, 2016) repair in mice models. Additionally, heparin present anticoagulant activity, induced by its interaction with antithrombin III, an inhibitor of serine proteases (Mulloy, Hogwood, Gray, Lever, & Page, 2016). The application of this molecule still presents some limitations, including the potential risk of heparininduced thrombocytopenia (Papakonstantinou, Roth, & Karakiulakis, 2012). Therefore, another version of heparin with low molecular weight has been obtained from unfractionated heparin and have shown reductions on clinical side effects (Jordan, Racine, Hennig, & Lokeshwar, 2015).

5.10.8 Hyaluronic acid Another natural component of ECM is HA, a polysaccharide composed of alternating units of D-glucuronic acid and N-acetyl-D-glucosamine of high molecular weight (Elmorsy et al., 2014; Menzel & Farr, 1998). It is found in the eyes and joints and in almost every connective tissues, its role as cartilage and articular protector has been suggested. Recently, HA

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has been commonly used in certain eye diseases treatments as well as surgeries, some examples are cataract removal and corneal transplantation (Chan & Tayama, 2002). In the same way, exogenous application of HA promotes the synthesis of proteoglycans, regulate the function of immune cells, and reduce the activity of proinflammatory cytokines (Lam, Truong, & Segura, 2014). Other role of HA is the structural, because of its great capacity of water retention it affects tissue hydration and overall osmotic balance in the body, this unlocks a variety of applications mainly in skin maintenance. Also, HA is generally considered as a signal molecule by its interaction with cell surface receptors, acting in many biological processes (Croisier & Je´roˆme, 2013; Younes & Rinaudo, 2015). Among these signals, HA binds to immune cells as CD44, studies have identified a CD44-dependent mechanism for HA guiding gene expression in mice (Anitha et al., 2014). Although the functional mechanisms of HA are not completely cleared, it has been successfully used worldwide due to its biocompatibility and biodegradability with great progress in its application in the biomedical field. However, the mechanism of HA-cell interaction needs to be further explored.

5.10.9 Chitosan and chitin Chitosan is derived from chitin, and both are linear polysaccharides, composed of N-acetyl-2-amino-2-deoxy-D-glucose (N-acetyl) and 2-amino-2-deoxy-D-glucose residues (N-deacetylated, amino). As a heteropolysaccharides, chitosan structure includes linear β-1,4-linking units. These structural polysaccharides are highly present in arthropods (Mukhopadhyay et al., 2014). Many studies have shown that chitosan and chitin can be used for various applications inside biomedicine area as in tissue engineering, wound healing and drug delivery (Ragelle et al., 2014). They can be

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5. Biological macromolecules as nutraceuticals

found and engineered into many physical forms, such as gels, membranes, nanofibers, nanoparticles and sponges (Zhang, Sun, & Chen, 2014). Recently, researchers are trying to develop chitin scaffolds in tissue engineering, and great progress has been made (Mendoza et al., 2013). These chitosan scaffolds show significant effects in supporting and assisting the production of ECM rich in proteoglycans in vivo. In addition, inside the field of tissue engineering, chitosan and chitin have been widely used as skin substitutes mainly attributed to the outstanding properties of chitin, including hemostasis and biocompatibility (Martins et al., 2015). Chitosan has also been shown to be auxiliar in wound healing process by infiltration of polymorphonuclear cells promotion at the needed site (De Leon-Peralta, Brudno, Kwee, & Mooney, 2016).

5.10.10 Carbohydrates with nutraceutical activity from microorganisms Other major type polysaccharides that exist in nature are derived from microorganisms. In essence, microbial polysaccharides can be neutral (e.g., dextran, scleroglucan) or acid (xanthan gum, gellan gum) and are often used as storage compounds in treatments similar to wound dressings, in the development of biomaterials and as auxiliar in tissue regeneration.

5.10.11 Alginate Alginate is a type of naturally occurring anionic polymer that can be extracted from brown algae, and it is present mainly in the cell walls of species as microcystis, purple algae, and seaweed. Alginate has a liner molecular structure composed of repeating units of 1,4-linked β-mannuronate (M) and 1,4-α-1-gulonate residues (G). Common alginate has a high degree of different physical and chemical properties heterogeneity, which

affects its quality but also triggers different practical applications (Jain & Bar-Shalom, 2014), one of the most studied is alginate application in biomedical area (Tutunchi et al., 2020), some interesting properties of alginate are biocompatibility, low toxicity, low cost, and moderate induction of gelation by divalent cations. Alginate gels has been largely evaluated and used for wound healing, therapeutic agents and drug delivery and as auxiliar in cell transplantation (Jain & Bar-Shalom, 2014). Other than structural, wound dressings made of alginate have functional properties as the maintenance of a moist environment in ECM. This has had great results in reducing the bacterial infection and promotion of wound healing. Alginate has been used as macromolecular structure bases, in order to develop bioactive agents delivery systems with a controlled release. Alginate gel is also used for cell transplantation in tissue engineering (Pipeleers & Keymeulen, 2016). It can deliver cells to designated sites and provide an artificial matrix for the formation of new blood vessels (Venkatesan, Bhatnagar, Manivasagan, Kang, & Kim, 2015).

5.10.12 Dextran Dextran is a high molecular weight polysaccharide composed by α-1,6 glucose bond to the main chain and α-1,4 glucose bond to the side chain. The structures of glucans are different depending on the origin microbial strains (Choudhary, Reck, Carr, & Bhatia, 2018). After dextran is cross-linked, it can usually be used to separate and purify biological macromolecules. Dextran possess a high biocompatibility, and it can be used as a plasma-bulking agent for biomedical applications (Siddiqui, Aman, Silipo, Qader, & Molinaro, 2014).

5.10.13 Bacillus striatum polysaccharide Bacillus striatum polysaccharide (BSP), composed of α-mannose, β-mannose, and β-glucose

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are widely used for tissue regeneration. Luo found that after treating wounds with BSP gel, it controlled inflammation and accelerated wound closure (Liang et al., 2019). Later, cotton gauze coated with BSP demonstrated a better healing effect (Friedman, 2016; Howard et al., 1959). In addition, BSP has been studied for regulating the biological activity of macrophages by binding to mannose receptors and regulating downstream signals (Friedman, 2016). This polysaccharide can regulate cytokines secretions, and hence it is highly used in tissue regeneration.

5.11 Credit All authors agree to Master in Sciences and Doctorate in Biosystems Engineering programs, for permit the participation of all the authors in this chapter. Additionally, Ireri Alejandra CarbajalValenzuela; Nuvia Marina Apolonio
Hernandez; Diana Vanesa Gutierrez-Chavez; Beatriz Gonza´lezArias; Alejandra Jimenez-Hernandez extend thanks to CONACyT for grant support for their post-graduate studies.

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C H A P T E R

6 Biological macromolecules as antioxidants T. Madhujith1, N.E. Wedamulla2 and D.A.S. Gamage3 1

Department of Food Science and Technology, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka 2Department of Export Agriculture, Faculty of Animal Science and Export Agriculture, Uva Wellassa University, Badulla, Sri Lanka 3Department of Chemical and Process Engineering, Faculty of Engineering, University of Peradeniya, Peradeniya, Sri Lanka

6.1 Introduction Free radicals can be defined as molecules or molecular fragments containing one or more unpaired electrons. Free radicals exhibit a high level of reactivity due to the presence of unpaired electrons. Free radicals carrying oxygen in the structure together with compounds that can easily generate such free radicals are known as reactive oxygen species (ROS). These ROS have been identified as one of the most important classes of such species (Valko, Rhodes, Moncol, Izakovic, & Mazur, 2006). ROS are produced by living organisms as a result of normal cellular functions (AlDalaen, 2014). Mitochondria peroxisomes, and inflammatory cell activation sites serve as potential endogenous sources of ROS. On the other hand, various exogenous sources also contribute to generation of ROS (Valko et al., 2006). Smoking, exposure to pollutants, ozone, heavy metals, and radiation are identified as exogenous sources (Al-Dalaen, 2014). Cigarette smoke contains high amounts of

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00006-3

free radicals, organic compounds, and many oxidants. Thus, smoking enhances the production of ROS which ultimately leads to oxidative stress (Fig. 6.1) (Abdalla, Saad-Hussein, Ibrahim, ElMezayen, & Osman, 2015). Besides, heavy metals also contribute to inducing the generation of ROS. The ROS generated through the metal-catalyzed reactions possess the ability to modify the bases of the DNA molecule and cause cellular damage. Moreover, exposure to ozone also enhances lipid peroxidation (Al-Dalaen, 2014). Elevated levels of ROS may cause damages to cellular structures including proteins, lipids, and nucleic acids. These harmful effects are mediated by the action of nonenzymatic antioxidants and antioxidant enzymes. Therefore the cellular antioxidant defense system protects against oxidative damage by ROS and controls the adverse changes of cellular components (Valko et al., 2006). Once the balance between the antioxidant defense system and ROS shifts toward the ROS generation, oxidative stress develops (Al-Dalaen, 2014). Oxidative

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6. Biological macromolecules as antioxidants

Cell function Immunity function

In vivo

Smoking Environmental stimuli

Radiation

Antioxidant defense

ROS

Pollutant

Exogenous Antioxidants

ROS Cancer

Oxidative Stress

Declined antioxidant capacity

Damage to DNA, protein, lipid and other macromolecules

Aging

FIGURE 6.1

Diabetes

Neurodegenerative diseases

Relationship between reactive oxygen species, oxidative stress and antioxidants.

stress can be defined as a lack of balance between the occurrence of reactive oxygen/nitrogen species (ROS/RNS) and the organism’s capacity to counteract their action by the antioxidative protection systems (Pisoschi & Pop, 2015). Fig. 6.1 illustrates the relationship between ROS, oxidative stress, and antioxidants. A large body of evidence has proven the correlation between oxidative stress and various pathogenesis including neurodegenerative diseases, cardiovascular and inflammatory diseases, cataract, and cancer. Furthermore, this free radicalmediated oxidative damages also contribute to aging and reducing lifespan (Pisoschi & Pop, 2015). Antioxidants can mitigate oxidative stress by direct scavenging of ROS and thereby limiting the harmful effects resulting from excessive free radicals. An antioxidant can be defined as a molecule stable enough to donate an electron to free radicals and neutralize it and thereby reducing its capacity to damage (Lobo, Patil, Phatak, & Chandra, 2010). Antioxidants can be classified

into three categories: first-line defense antioxidants, second-line defense antioxidants, and third-line defense antioxidants. First-line defense antioxidants include superoxide dismutase (SOD), catalase, glutathione reductase, and minerals like Se, Cu, Zn. Second-line defense antioxidants consist of glutathione, vitamin C, albumin, vitamin E, carotenoids, flavonoids, among others. Enzymes such as lipase, protease, DNA repair enzymes, transferases, methionine sulphoxide reductase fall into the third-line defense antioxidants. The surge of scientific studies has proven the beneficial health effects of antioxidant supplementation in many situations including stress, aging, pathogen infestation, apoptosis, and neurological diseases (Sindhi et al., 2013). Though endogenous antioxidants play a pivotal role in regulating normal cell functions by scavenging ROS, under certain conditions this protective mechanism becomes inefficient once the condition promotes oxidative

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6.2 Types and sources of biological macromolecules

stress. In such conditions, dietary antioxidants are equally important in regulating normal cellular functions (Fig. 6.1) (Al-Dalaen, 2014). Considering the adverse health effects associated with synthetic antioxidants, research has been directed toward the exploration of natural antioxidants. Currently, natural bioactive micromolecular compounds have drawn considerable attention owing to their promising features. Furthermore, number of bioactive macromolecular compounds also have been identified to exhibit excellent antioxidant properties. These compounds demonstrate good stability in the gastrointestinal tract than that of bioactive micromolecules. These macromolecules mainly consist of proteins, peptides, polysaccharides, and glycoproteins which are derived from plant, animal, and microbial sources (Song, Wu, Yun, & Zhao, 2020). Owing to the health promising characteristics of these antioxidative biomacromolecules there is an emerging trend toward the exploration of biological macromolecules with antioxidant properties. Thus, the chapter explains the sources, properties, and applications of biological macromolecules as a potent source of antioxidants.

6.2 Types and sources of biological macromolecules Bioactive macromolecules are mainly derived from plant, animal, marine, and microbial sources. Table 6.1 summarizes the sources and bioactivity of biological macromolecules.

6.2.1 Polysaccharides Polysaccharides are natural biopolymers that can be isolated from the plant, animal, marine, and microbial sources. These macromolecules contain several monomer units called monosaccharides which are joined to each other by glycosidic bonding. These polymers exhibit a large variety of structural

141

diversity and a variety of biological functions and properties (Delattre, Fenoradosoa, & Michaud, 2011). Recently, the influx of studies has directed toward the discovery of the number of antioxidative polysaccharides from plant, animal, marine, and microbial sources (Nemzer, Kalita, Yashin, Nifantiev, & Yashin, 2019). 6.2.1.1 Plant-derived polysaccharides Among natural polysaccharides, a significant number of polysaccharides are derived from plant sources. Moreover, several studies have proven the antioxidant activity of polysaccharides isolated from plant bases (Jiang, Zi, Pei, & Liu, 2018; Tian, Hao, Xu, Yang, & Sun, 2017). In the study of (Tian et al., 2017), polysaccharides were isolated from the rhizome of Angelica sinensis with ultrasound-assisted extraction. The study reported that Jiang et al. (2018) have extracted two novel polysaccharides from Plumula nelumbinis which is the embryo of the seed of Nelumbo nucifera. Moreover, the study further reported that these two novel polysaccharides possessed exclusive monosaccharide composition and molecular weight. Besides, the polysaccharides extracted from Zagros oak (Quercus brantii) using ultrasonic-assisted extraction techniques are also documented in the literature. The study further confirmed the antioxidant and antimicrobial activity of extracted polysaccharides (Tahmouzi, 2014). Polysaccharides are the major active constituents of Radix astragali, the dried root of Astragalus membranaceus apart from saponins and isoflavonoids. The study of Yin et al. (2012) has isolated the soluble hyperbranched heteroglycan with the average molecular weight of 1.33 3 106 Da from R. astragali. This polysaccharide exhibited significant immunomodulating effects (Yin et al., 2012). Recently, polysaccharide was isolated from peel of Trichosanthes kirilowii, and bioactivity was assessed. Trichosanthes peel has been widely

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6. Biological macromolecules as antioxidants

TABLE 6.1 Sources and bioactivities of macromolecules. Macromolecule

Source

Species

Bioactivity

References

Protein-Turmerin

Plantbased

Curcuma longa (Turmeric)

Antioxidant activity Antidiabetic agent

Lekshmi et al. (2012)

Antioxidant protein (TCPIII)(B16 kDa)

Plantbased

Terminalia chebula Retz (Hirda fruit)

Antioxidant activity

Srivastava, Raut, Wagh, Puntambekar, and Kulkarni (2012)

Protein (35 kDa)

Plantbased

Phyllanthus niruri

Antioxidant activity Cytoprotective activity

Sarkar, Kinter, Mazumder, and Sil (2009)

Protein

Plantbased

Semen Allii Fistulosi (seed of Allium fistulosum L.)

Antioxidant activity

Zuo et al. (2018)

Lectin protein

Plantbased

Lablab purpureus (Hyacinth bean)

Antioxidant activity

Saha et al. (2014)

Casein

Animal- based

Antioxidant activity

Khan et al. (2019)

Whey protein

Animal- based

Antioxidant activity

Khan et al. (2019)

Protein

Animal- Rhopilema esculentum based Kishinouye (Jellyfish)

Antioxidant activity

Yu et al. (2006)

Lectin protein

Algalbased

Bryothamnionsea forthii (Red algae)

Antioxidant activity Antibacterial activity

Alves et al. (2020)

Phycobiliproteins

Algalbased

Bangia atropurpurea (Red filamentous alga)

Antioxidant activity

Punampalam et al. (2018)

Protein

Algalbased

Ulva sp. and Gracilaria sp.

Antioxidant activity

Kazir et al. (2019)

Phycobiliproteins

Algalbased

Spirulina sp

Antioxidant activity

Pan-utai and Iamtham (2019)

P. nelumbinis Plantbased polysaccharide I and P. nelumbinis polysaccharide II

Plumula nelumbinis (embryo of the seed of Nelumbo nucifera)

Antioxidant property

Jiang et al. (2018)

Polysaccharides

Plantbased

Angelica sinensis Diels (rhizome) Antioxidant property Antiproliferative effects

Tian et al. (2017)

Polysaccharides

Plantbased

Quercus brantii Lindl (Zagros oak) (leaves)

Tahmouzi (2014)

Antioxidant activity Antimicrobial activity

(Continued)

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143

6.2 Types and sources of biological macromolecules

TABLE 6.1 (Continued) Macromolecule

Source

Species

Bioactivity

References

Polysaccharides

Plantbased

Semen cassia (Dry and mature seed of Cassia obtusifolia L. or Cassia tora L.)

Antioxidant activity

Liu et al. (2014)

Polysaccharides

Plantbased

Trichosanthes kirilowii Maxin (peel)

Hypoglycemic activity

Chen, Mao, et al. (2016), Chen, Zhang, et al. (2016)

Novel water-soluble polysaccharide

Plantbased

Periploca laevigata (Root barks)

Antioxidant activity Antimicrobial activity

Hajji et al. (2019)

Water-soluble polysaccharide (MWP-2)

Plantbased

Malus micromalus Makino fruit wine

Antioxidant activity Antiaging activity

Hui et al. (2019)

Water-soluble arabinoxylan

Plantbased

Andrographis paniculata (Green stem)

Antioxidant activity

Maity et al. (2019)

Polysaccharide

Plantbased

Hippophaerhamnoides L. (Seabuckthorn berries)

Antioxidant activity

Wei et al. (2019)

Polysaccharide

Plantbased

Polygonatum sibiricum

Antioxidant activity

Zhang et al. (2019)

Polysaccharide

Plantbased

Aloe barbadensis(Aloe vera)

In vitro and in vivo antioxidant activities

Kang et al. (2014)

Polysaccharide

Animal- Hyriopsis cumingii based

Neuroprotective effect

Hu et al. (2010)

Novel polysaccharides

Animal- Misgurnus anguillicaudatus based

Antioxidation and antiglycation effect

Zhang et al. (2011)

Chitosan

Animal- Chrysomya megacephala (Blowfly) Antioxidant activity based larvae

Song et al. (2013)

Chitosan

Animal- Musca domestica (Larvae of based housefly)

Antioxidant activity

Ai et al. (2008)

Chitin and Chitosan

Animal- Zopho basmorio (Larvae of based superworm)

Antioxidant activity

Soon et al. (2018)

Fucoidans

Marine- Brown seaweed based Undaria pinnatifida

Antioxidant activity

Mak et al. (2013)

Crude polysaccharide

Marine- Brown algae based Chnoospora minima

Antioxidant activity

Shanura Fernando et al. (2017)

Fucoidans

Marine- Brown seaweeds based Dictyota ciliolata, Padina sanctaecrucis, Sargassum fluitans

Antioxidant activity

Chale-Dzul et al. (2017)

Fucoidans

Marine- Brown seaweed-Laminaria based japonica

Antioxidant activity Anticancer activity

Zhao et al. (2018) (Continued)

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144

6. Biological macromolecules as antioxidants

TABLE 6.1 (Continued) Macromolecule

Source

Fucans

Species

Bioactivity

References

Marine- Brown algae based Lobophora variegata

Antioxidant activity antiangiogenic effect and anticoagulant activity

Castro et al. (2015)

Fucoidan

Marine- Brown algae based Nemacystus decipients

Antioxidant activity

Li, Luo, et al. (2017)

Fucoidan

Marine- Brown seaweed Sargassum based crassifolium

Antioxidant activity neuronal protective properties

Yang, Chen, and Huang (2017)

Fucoidan

Marine- Brown seaweed based Sargassum thunbergii

Antioxidant activity cytotoxic activity

Somasundaram, Shanmugam, Subramanian, and Jaganathan (2016)

Crude sulfated polysaccharides

Marine- Brown seaweed based Turbinaria ornata

Antioxidant activity

Saravana Guru, Vasanthi, and Achary (2015)

polysaccharides

Marine- Red algae based Pyropia yezoensis

Antioxidant activity

Lee et al. (2016)

Sulfated polysaccharides

Marine- Green algabased Ulva fasciata Red algaGloiopeltis furcate Brown algaSargassum henslouianum

Antioxidant activity Antitumor activity

Shao et al. (2013)

Sulfated polysaccharides

Marine- Gracilaria rubra based

Antioxidant activity Immunostimulating activity

Di et al. (2017)

Sulfated polysaccharides

Marine- Red algae based Pyropia yezoensis

Antioxidant activity

Lee et al. (2016)

Polysaccharides

Marine- Sarcodia ceylonensis, Ulva lactuca based Gracilaria lemaneiformis, Durvillaea antarctica

Antioxidant activity

He et al. (2016)

Sulfated polysaccharide

Marine- Red algae based Solieria filiformis

Antioxidant activity

Sousa et al. (2016)

Polysaccharide

Marine- Green algae Enteromorpha linza based

Antioxidant activity

Zhang et al. (2013)

Sulfated polysaccharide

Marine- Green algae based Ulva intestinalis

Antioxidant activity

Peasura et al. (2015)

Sulfated polysaccharide

Marine- Green algae based U. intestinalis

Antioxidant activity

Peasura, Laohakunjit, Kerdchoechuen, Vongsawasdi, and Chao (2016) (Continued)

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6.2 Types and sources of biological macromolecules

TABLE 6.1 (Continued) Macromolecule

Source

Species

Bioactivity

Chrysolaminarin

Marine- Odontella aurita (Marine diatom) Antioxidant activity based

Xia et al. (2014)

Aqueous extracellular polysaccharides

Marine- Graesiellasp (Micoalgae) based

Trabelsi et al. (2016)

Water-soluble polysaccharides

Marine- Pavlova viridis and Sarcinochrysis Antioxidant activity based marina Geitler

Sun et al. (2014)

Sulfated polysaccharides

Marine- Navicula sp. based

Antioxidant activity

Fimbres-Olivarria et al. (2018)

Extracellular polysaccharide

Marine- Aspergillus terreus (Marine based fungus)

Antioxidant activity

Wang et al. (2013)

Extracellular polysaccharide AS2
1

Marine- Alternaria sp. SP-32 (Marine based fungus)

Antioxidant activity

Chen, Mao, et al. (2016), Chen, Zhang, et al. (2016)

Extracellular polysaccharide

Marine- Fusarium oxysporum (Marine based fungus)

Antioxidant activity

Chen et al. (2015)

Bacterial exopolysaccharide

Marine- Bacillus amyloliquefaciens based 3Ms 2017 (Marine bacteria)

Antioxidant activity Antitumor activity

El-Newary et al. (2017)

Extracellular polysaccharides

Marine- Bacillus based thuringiensis RSK CAS4 (Marine bacteria)

Antioxidant activity Anticancer activity

Ramamoorthy, Gnanakan, S. Lakshmana, Meivelu, and Jeganathan (2018)

Exopolysaccharides

Marine- Polaribacter sp. SM1127 based

Antioxidant activity

Sun et al. (2015)

Sulfated polysaccharides

Marine- Holothuria fuscogliva (Sea based cucumber)

Antioxidant and anticoagulant activity

Li, Luo, et al. (2017), Li, Yu, et al. (2017)

Fucosylated chondroitin sulfate

Marine- Apostichopus japonicas, Stichopus based chloronotus, Acaudinamol padioidea (Sea cucumber)

Antioxidant activity Antiinflammatory

Mou, Li, Qi, and Yang (2018)

Fucoidan

Marine- Thelenota ananas (Sea cucumber) Superoxide radicals based scavenging ability

Yu et al. (2014)

Polysaccharides

Fungal- Hericium erinaceus (Mushroom) based

Antioxidant and neuroprotective activities

Cheng et al. (2016)

Polysaccharides

Fungal- Suillellus luridus (Mushroom) based

Antioxidant activity

Zhang et al. (2018)

Water-soluble and alkalisoluble crude polysaccharides

Fungal- Inonotus obliquus based

Antioxidant activity

Mu et al. (2012)

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Antioxidant activity antiproliferative activity

References

146

6. Biological macromolecules as antioxidants

used in traditional Chinese medicine to treat various diseases (Chen, Mao, et al., 2016; Chen, Zhang, et al., 2016). Water-soluble polysaccharides have drawn much attention due to their potential as a source of natural antioxidants and biologically active compounds. Hence, much research focuses on the discovery of these water-soluble polysaccharides. These were isolated from semen cassia and the study used water for extraction while ethanol was used for deposition. The mature seed of Cassia obtusifolia L. or Cassia tora L. is well-known in traditional Chinese medicine (Liu, Liu, Sun, Jiang, & Yan, 2014). Hajji et al. (2019) reported that hot water extraction was used to extract polysaccharides from root barks of Periploca laevigata. The study investigated the monosaccharide composition and has proven the presence of glucose, galactose, mannose, and glucuronic acid. However, arabinose, rhamnose, and galacturonic acid were not detected (Hajji et al., 2019). In another study, a novel water-soluble polysaccharide (MWP-2) was isolated from Malus micromalus Makino fruit wine using fractionated extraction, alcohol precipitation, macroporous resin purification, cellulose column purification, running water dialysis, and vacuum freeze-drying. The relative molecular mass of extracted polysaccharides containing galactose, glucose, mannose, and fructose, was found to be 1 3 105 Da (Hui, Jun-Li, & Chuang, 2019). Maity et al. (2019) revealed Andrographis paniculata a medicinal plant as a natural source to isolate bioactive polysaccharides. Water-soluble arabinoxylan having the molecular weight B1.49 3 105 Da was successfully isolated from the A. paniculata. Currently, Seabuckthorn (Hippophaerhamnoides L.) is used to isolate polysaccharides with promising bioactive properties using water as extraction solvent and microwave-assisted extraction as an extraction technique. Moreover, Seabuckthorn berries are believed to be rich in various bioactive components and used as a traditional medicine for a long period. Compositional analysis of

monosaccharides revealed the presence of rhamnose, mannose, glucose, and galactose (Wei et al., 2019). A recent study has isolated polysaccharides from Polygonatum sibiricum, traditional herbal medicine, and food widely used in China, Korea, and Japan. The study isolated four fractions of polysaccharides using water extraction and ethanol precipitation (Zhang et al., 2019). 6.2.1.2 Animal-derived polysaccharides Animal polysaccharides are produced by liver and muscles during glycogenesis and are used to store energy. Glycogen is the major animal polysaccharide composed of α-1,4-glycosidic bonds linked with branched α-1,6 bonds present at approximately every tenth monomer (Nemzer et al., 2019). Moreover, sulfated polysaccharides such as glycosaminoglycans have been found in large quantities in vertebrate tissues (Delattre et al., 2011). Hyriopsis cumingii belongs to the freshwater pearl mussel family which is widely cultivated in China and well-known for high-quality pearl production. The polysaccharides extracted from these H. cumingii were well accepted for their bioactive properties including antitumor, immunity-enhancing, antiinflammatory, neuroprotective effects and antioxidant activities (Hu, Li, Yang, Pan, & Wan, 2010). Misgurnusanguilli caudatus is an edible fish widely inhabiting in China, Japan, and Korea. This edible fish inhabits rivers, lakes, ponds, swamps, and rice fields. The polysaccharides extracted from M. anguillicaudatus possess several functional properties including antiinflammatory, antihypoxic, hypoglycaemic. Furthermore, they are reported to improve immunity, scavenge ROS protect DNA (Zhang, Wang, & Dong, 2011). Among animal-based polysaccharides, chitin and its derivative chitosan play a pivotal role. These biopolymers have randomly distributed β-(1
4)-linked d-glucosamine and N-acetyl glucosamine units. Crustacean shells are the main source of these polysaccharides. Waste residues of crustacean exoskeletons generated in the seafood

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6.2 Types and sources of biological macromolecules

industry such as shrimps, crabs, squids, and lobsters were used for the extraction of chitin (Kaur & Dhillon, 2014). Furthermore, insects are promising sources to extract chitin owing to the extraction methods, chemical consumption, time, and yield compared to other sources (Mohan et al., 2020). Several studies have isolated chitin and its derivatives from insects. The study by Ai, Wang, Yang, Zhu, and Lei (2008) has extracted chitosan from the larvae of houseflies (Musca domestica). Another study provides evidence on the extraction of chitosan from cicada sloughs, beetle, silkworm, and bumblebee. The study of Ma, Xin, and Tan (2015) revealed that chitosan extracted from Catharsius molossus L. was superior to commercial medical-grade chitosan from shrimp. Similarly, the study of Soon, Tee, Tan, Rosnita, and Khalina (2018) used superworm (Zopho basmorio) to extract chitin and chitosan. Moreover, Song, Yu, Zhang, Yang, and Zhang (2013) have discovered larvae of blowfly (Chrysomya megacephala) as a novel source of insect chitosan with promising antioxidant properties. Another study revealed that isolated chitin from Melolontha serves as a more promising source than Oniscus asellus (Crustacea) (Kaya et al., 2014).

6.2.1.3 Marine-derived polysaccharides Marine organisms serve as an excellent source of nutrients and bioactive compounds. Recently, several studies have directed the isolation and identification of bioactive polysaccharides from marine organisms. Fig. 6.2 illustrates the major sources of marine-derived polysaccharides. These polysaccharides are isolated from marine algae followed by marine microorganisms and marine animals. A large volume of literature is available on algal-derived polysaccharides with bioactive properties (Zhong et al., 2019). Alginates, fucans, laminarans, cellulose, and sulfated galactan are identified as prominent algal polysaccharides. Fucoidan is identified as a natural sulfated polysaccharide with promising health-promoting properties (Nemzer et al., 2019). The study of (Chale-Dzul, Freile-Pelegrı´n, Robledo, & Moo-Puc, 2017) extracted fucoidans from the tropical brown seaweeds and measured the protective effect against oxidative stress. The results reported that fucoidan extracted from Dictyotaciliolate possessed the highest ROS scavenging activity followed by Padinasanctaecrucis fucoidan and Sargassum fluitans fucoidan. Besides, fucoidans from Laminaria japonica has been

Microalgae Brown Algae

Fucan Alginate Laminarin

Fungi

Algae

Sources of Marine Polysaccharides

Microbes

Red Algae Bacteria

Agar Green Algae

Ulvan

Animal

FIGURE 6.2

Sources of marine polysaccharides.

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6. Biological macromolecules as antioxidants

extracted with excellent antioxidant activity. Furthermore, the reported study has developed a novel extraction process to extract fucoidans from L. japonica effectively (Zhao, Xu, & Xu, 2018). Another study assessed the fucans extracted from the brown alga, Lobophora variegate, which has rexhibited excellent antioxidant activity with strong inhibition on hydroxyl and peroxide radicals (Castro et al., 2015). Polysaccharides derived from red algae are also well-known for their promising bioactive properties. Several studies have been carried out on red algae as a major source of agar. The sulfated polysaccharides were isolated from three algae: green alga (Ulva fasciata), red alga (Gloiopeltis furcate), and brown alga (Sargassum henslouianum) by ultrasonic extraction and radial flow chromatography (Shao, Chen, & Sun, 2013). In another study by Di et al. (2017) sulfated polysaccharides were isolated from Gracilaria rubra with the aid of hot water extraction and chromatographic purification. The results of the study showed that the presence of excellent antioxidant activities against ABTS radical, superoxide anion radical, and lipid peroxidation inhibitory activity. He, Xu, Chen, and Sun (2016) extracted polysaccharides from four seaweed species Sarcodia ceylonensis, Ulva lactuca, Gracilaria lemaneiformis, and Durvillaea antarctica. Ulvan is a green algae-derived polysaccharide that gained considerable attention recently owing to its exhibited antioxidant properties. Zhang, Wang, Zhao, Yu, and Qi (2013) extracted polysaccharides from Enteromorpha linza with water and alkali and assessed the antioxidant activity. Structural analysis of extracted polysaccharides reported that they were composed of rhamnose, mannose, xylose, glucose, and galactose. A recent study has extracted polysaccharides from a tubular green seaweed, Ulva intestinalis which is well documented for its nutritional benefits (Peasura, Laohakunjit, Kerdchoechuen, & Wanlapa, 2015). Microalgal polysaccharides are also wellknown for their radical scavenging properties. Several pieces of evidence support the

antioxidant activity of polysaccharides extracted from microalgae. Studies have shown the promising antioxidant properties of novel chrysolaminarin extracted from marine diatom, Odontella aurita. The structural analysis of extracted novel polysaccharides revealed the presence of glucose that was linked by the main chain (β-D-(1-3)) and side chain (β-D-(1-6)) glycosidic bond (Xia et al., 2014). Moreover, extracellular polysaccharides released by microalgae Graesiellasp has also been studied (Trabelsi, Chaieb, Mnari, AbidEssafi, & Aleya, 2016). In another study, watersoluble polysaccharides from Pavlova viridis and Sarcinochrysis marina had been isolated and the antioxidant activity of their degradation products had been assessed (Sun, Wang, Li, & Liu, 2014). The results revealed that the low molecular weight fragments have displayed higher antioxidant capacities after degradation. The sulfated polysaccharides from Navicula sp. were isolated and revealed the presence of glucose, galactose, rhamnose, xylose, and mannose as main neutral sugars (Fimbres-Olivarria et al., 2018). Marine fungi are considered a promising source of novel antioxidant polysaccharides. Wang et al. (2013) isolated an extracellular polysaccharide from the fermented broth of marine fungus Aspergillus terreus. The study reported that the isolated polysaccharideis composed of mannose and galactose. A recent study has obtained extracellular polysaccharide AS2
1 from the fermented broth of the marine sponge endogenous fungus Alternaria sp. SP-32 and stated that the AS2
1 was composed of mannose, glucose, and galactose (Chen, Mao, et al., 2016; Chen, Zhang, et al., 2016). Recently, several studies have reported the promising antioxidant activity of exopolysaccharides obtained from marine bacteria. Wang et al. (2018) isolated marine bacteria that secrete exopolysaccharides from Aerococcus uriaeequi HZ strains. The study revealed that the isolated polysaccharide was composed of glucose and a small amount of mannose. Isolation of bacterial exopolysaccharides from

II. Bioactivity

6.2 Types and sources of biological macromolecules

marine Bacillus amyloliquefaciens 3Ms 2017 is also well documented (El-Newary, Ibrahim, Asker, Mahmoud, & El Awady, 2017). Besides marine algae, marine animal sources also greatly contributed as the sources of antioxidant polysaccharides. Marine invertebrates have drawn considerable attention in this regard. Polysaccharides isolated from these invertebrates showed that the excellent in vitro and in vivo antioxidant activities, thus could effectively alleviate free radicals-induced diseases. Table 6.1 summarizes the bioactive properties of few marine animals. The study of (Li, Luo, Yuan, & Yu, 2017; Li, Yu, et al., 2017) has reported the extraction of polysaccharides from sea cucumber (Holothuria fuscogliva)and the study has confirmed the antioxidant and anticoagulant activity of polysaccharides. 6.2.1.4 Bacterial and fungal polysaccharides The major components of fungal cell walls are β-glucan, chitin and mannans, which contain glucose, N-acetylglucosamine, and mannose as repeating units. The influx of studies into bioactive properties of these polysaccharides acknowledged mushrooms as a promising source of antioxidant polysaccharides. For an instance, the study by Cheng, Tsai, Lien, Lee, and Sheu (2016) has extracted polysaccharides from Hericium erinaceus and revealed the presence of antioxidant and neuroprotective properties. In the study, watersoluble and alkali-soluble crude polysaccharides were isolated from mushroom Inonotus obliquus and exhibited the ability of I. obliquus-derived polysaccharides in scavenging ROS (Mu et al., 2012). Moreover, isolation of a nonsulfated polysaccharide, fucogalactomannan from Tylopilusballouii mushroom is also well-acknowledged. The results of the study have shown that the extracted fucogalactomannan inhibited superoxide and hydroxyl radicals (Lima et al., 2016). The cellulose-assisted extraction method has recently been used to extract polysaccharides

149

from Tricholoma mongolicum Imai and indicated the in vitro antioxidant activities of extracted polysaccharides (Zhao et al., 2016). Bacterial polysaccharides have existed as extracellular polysaccharides, peptidoglycans, lipopolysaccharides, capsules, and exopolysaccharides. Staphylococcus epidermidis, Staphylococcus aureus, Bordetella bronchiseptica, and Escherichia coli like bacterial strains contain high levels of polysaccharides as extracellular polysaccharides (Nemzer et al., 2019). In the study, an extracellular polysaccharide has purified from the bacterium Paenibacillus polymyxa SQR-21. The study revealed that the isolated extracellular polysaccharide consists of mannose, galactose, and glucose. Furthermore, this extracellular polysaccharide exhibited excellent superoxide scavenging activity (Raza, Makeen, Wang, Xu, & Qirong, 2011).

6.2.2 Proteins The antioxidant properties of proteins in controlling lipid oxidation are well-known. An introduction of antioxidant proteins by genetic engineering can be used to increase the oxidative stability of foods (Elias, Kellerby, & Decker, 2008). The proteins which exhibit antioxidant activity may be derived from plant, animal, and algal sources. 6.2.2.1 Plant-derived proteins The surge of literature has proven the promising antioxidant properties of plantderived proteins. The study of (Szerszunowicz & Kłobukowski, 2020) has announced wheat, rice, buckwheat, rye, sorghum, barely, and oat grains as excellent protein sources characterized by a rich profile of antioxidant activity. The study of (Lekshmi, Arimboor, Raghu, & Nirmala Menon, 2012) also exhibited the potent antioxidant activity of turmerin, an antioxidant protein derived from turmeric (Curcuma longa). This water-soluble protein has exhibited promising DPPH and superoxide radical scavenging capacities with

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150

6. Biological macromolecules as antioxidants

moderate ABTS radical scavenging and Fe(II) chelation capacities. Saha et al. (2014) has isolated protein from Lablab purpureus seed and the antioxidant activity has been assessed. The study has revealed that the seeds of hyacinth bean (L. purpureus) contain lectin protein which has demonstrated antioxidant property compared to that of ascorbic acid. Moreover, oil seeds are also considered as a promising source of bioactive proteins. A recent study has reported chia seeds as an excellent source of protein. The protein content in chia seeds is estimated to be approximately18 g/ 100 g therefore it has been recommended to use slimming diets, vegetarian diets as a complementary source of protein. The study of (KoteckaMajchrzak, Sumara, Fornal, & Montowska, 2020) reported that globulins (52%) and albumins (17.30%) have been identified as the major fractions extracted from chia seed flour while glutelins (14.5%) and prolamins (12.7%) have been considered as minor fractions. Similarly, the seeds of pumpkin (Cucurbita sp.) also serve as a valuable source of proteins. The oil cake remains after the extraction of oils accounts for upto 65% of proteins which can be utilized as a functional food ingredient. The studies have shown the antioxidant potential of globulin fraction of C. moschata when performing radical and hydrogen peroxide scavenging, lipid peroxidation, and reducing power assays. In addition, the antioxidant activity of proteins isolated from sunflower is also extensively studied. Globulins account for the majority of sunflower proteins which range from 40%
90% whereas 10%
30% of proteins account for albumin. Other proteins present in sunflower are glutelins, especially prolamins (Kotecka-Majchrzak et al., 2020). Furthermore, studies have reported coconut cake as an excellent source of edible protein. Antioxidant activity of albumin, globulin, prolamine, glutelin-1 and glutelin-2 protein fractions of coconut cake is well-known. The results revealed that prolamine, glutelin-1, and glutelin-2 have good radical scavenging

activity and reducing power while globulin and prolamine have shown high ion chelating ability. This coconut cake is a by-product of the coconut milk and oil processing industry that contains approximately 10%
16% protein. Albumin and globulin are identified as major constituents of coconut proteins which contain 21 and 40 g/100 g, respectively (Li, Zheng, Zhang, Xu, & Gao, 2018). 6.2.2.2 Animal-derived proteins Proteins derived from animal sources are widely accepted to exert promising bioactivity. Among casein and whey protein derived from milk play a major role. Whey proteins are wellacknowledged as a valuable food component with excellent nutritional and functional properties. Moreover, many studies have proven the antioxidant activity of whey protein. Thus, this can be effectively utilized to control lipid oxidation. The antioxidant activity of whey protein is attributed to the lactoferrin and sulfur-containing amino acid. Tyrosine which scavenges the free radicals also equally responsible for the antioxidant activity of whey protein. On the other hand, β-lactoglobulin accounts for 50 to 55% of the total whey proteins also extensively studied for the antioxidant potential (Liu, Chen, & Mao, 2007). Casein is a major protein of bovine and ovine milk which occurs in the form of macromolecular aggregates. Different casein fractions are present in milk owing to the difference in phosphate content and these phosphates govern the exert antioxidant activity of caseins (Khan et al., 2019). Egg white is also identified as a promising source of bioactive proteins. The protein content of egg white is nearly 11%. The major protein present in egg albumin is ovalbumin (54%) followed by ovotransferrin (12%), ovomucoid (11%), lysozyme, (3.5%) and ovomucin (3.5%). However, egg white contains avidin, cystatin, ovomacroglobulin, ovoflavoprotein, ovoglycoprotein, and ovoinhibitor in minute quantities.

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6.2 Types and sources of biological macromolecules

Some of the proteins mentioned above possess excellent antioxidant activities. However, it is widely acknowledged that the peptides derived from these proteins exhibit higher antioxidant activity. Ovalbumin has been identified as the most abundant protein in egg white which contains disulfide bonds and four free sulphydryl groups and these are mainly involved in redox regulation when acting as metal chelators. Lysozyme also an egg white protein which able to inhibit generatin of ROS. Similar to other proteins derived from egg white, lysozyme also demonstrates increase antioxidant activity when combine with other compounds such as polysaccharides. Phosvitin has been identified as a major protein present in egg yolk. The comparatively high chelating ability for cations exert by the phosvitin protein may greatly responsible for the antioxidant properties. Moreover, tryptophan and tyrosine like free aromatic amino acids present in egg yolk also equally contributed to exhibited antioxidant activity (Benede´ & Molina, 2020). The study reported the superoxide anion radical scavenging, hydroxyl radical scavenging, total antioxidant activity, reducing power, and metal chelating activity of protein extracted from jellyfish (Rhopilema esculentum) against butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol, vitamin C, and mannitol. The results indicated that protein extracted from jellyfish possesses promising antioxidant activity to compare to that of standards (Yu et al., 2006). 6.2.2.3 Algal-derived proteins An extensive search of the marine environment results from the discovery of several bioactive compounds with proteinaceous. This includes proteins, linear peptides, cyclic peptides and depsi peptides, peptide derivatives, and amino acids. Among them, bioactive proteins have gained much attention. Some species of macroalgae have been reported to contain significant levels of proteins. Depend on the species, the protein content of macroalgae may range from

151

30%
40% (w/w dry weight). Generally, red algae contain high amounts of protein compared to green and brown algae. Lectins and phycobili proteins have been identified as major algal proteins (Harnedy & Fitzgerald, 2011). The studies have shown protein from marine red algae Bryothamnionsea forthii can reduce free radicals, thus exhibit antioxidant activities (Alves et al., 2020). Phycobiliproteins are another group of bioactive proteins extracted from algae that have water-soluble, colored, and highly fluorescent compounds with promising antioxidant properties. Phycoerythrin, phycocyanin, allophycocyanin, and phycoerythrocyanin are considered major phycobili proteins (Harnedy & Fitzgerald, 2011). The surge of studies has directed to study of the antioxidant activity of phycobiliproteins extracted from marine algae. Punampalam, Khoo, and Sit (2018) extracted phycobili proteins from the red filamentous alga, Bangia atropurpurea, and confirmed the antioxidant activity. In the study antioxidant activity of protein extracted from macroalgae which include phylum Rhodophyta, Chlorophyta, and Phaeophyta was assessed and the results revealed that total protein content and antioxidant activity varied among the extracts (Putri, Dewi, Handayani, Harjanto, & Chalid, 2018). The study also revealed that there was a positive correlation between the total protein content of the extract and the antioxidant activity. Furthermore, the phylum Rhodophyta showed the highest protein content and antioxidant activity (Putri et al., 2018). Research findings have confirmed green marine macroalgae Ulva sp. and Gracilaria sp. as a sustainable source of bioactive proteins. Ulva sp. contains a significant number of proteins which accounts for 7%
24% on a dry weight basis. Moreover, this Ulva sp. also contains significant quantities of essential amino acids. These proteins are identified as potential antioxidant agents as they contain amino acid residues with antioxidant ability. Besides, basic nutritional properties demonstrated by amino acids, assessment of antioxidant activity of

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6. Biological macromolecules as antioxidants

algal proteins has confirmed the additional potential health benefits of these proteins (Kazir et al., 2019). Arthrospira (Spirulina sp.) also well documented as a nutrition supplement and food additive owing to its’ high protein content (60%) and other reported healthbeneficial properties. Phycobiliproteins have been identified as the major protein present in Spirulina sp. and studies have confirmed the excellent antioxidant activity of this protein extract (Pan-Utai & Iamtham, 2019).

6.2.3 Other antioxidative macromolecules 6.2.3.1 Nonextractable polyphenols A large number of studies have focused on the phenolic compounds which can be extracted from food matrix with aqueous or organic solvents. This extractable polyphenol is extensively studied concerning their health-promoting properties for long period giving little attention to the high number of polyphenols remains in the residue of these extracts. This ignored fraction of dietary antioxidants is named nonextractable polyphenols or macromolecular antioxidants. This fraction is consumed when foods are ingested and microbial action makes them partially bioavailable, thus relate to health-beneficial properties (Gutie´rrezDı´az et al., 2021). Owing to these healthpromoting properties, emerging trends have created to research and explore the health aspects of these nonextractable polyphenols. Polymeric polyphenols or single polyphenols are nonextractable polyphenols linked to macromolecular food constituents. Common aqueous or organic extraction procedures are not sufficient to extract these fractions, and they are only available and bioavailable in the large intestine. These nonextractable polyphenols are not significantly released from the food matrix after ingestion either by mastication, or the action of digestive enzymes (Pe´rez-Jime´nez, Dı´az-Rubio, & Saura-Calixto, 2013). However, this fraction also exhibits significant antioxidant activity. Hydrolyzable polyphenols and

nonextractable proanthocyanidins are two major macromolecular antioxidants. Low molecular weight phenolic compounds which are strongly associated with polysaccharides or proteins are named hydrolyzable polyphenols while high molecular weight structures belong to nonextractable proanthocyanidins. Studies have directed the extraction of nonextractable polyphenols from commonly consumed fruits and vegetables. The reported study revealed that the nonextractable polyphenols have contributed to 57% of the total antioxidant activity of the selected fruits and vegetables. Further, the study revealed that significantly high hydrolyzable polyphenols content ranged from approximately 32 mg/100 g (watermelon) to approximately 1450 mg/100 g dry weight basis (lettuce). Moreover, apple, banana, grape, peach, and pear showed higher nonextractable proanthocyanidins than extractable polyphenols (Pe´rez-Jime´nez & Saura-Calixto, 2015). Studies have revealed that proanthocyanidins have significantly high antioxidant potential compared to natural antioxidants like vitamin C and E (Hashemi Gahruie & Niakousari, 2017). Another study reported that apple, banana, cranberry pomace, and cocoa powder as good sources of proanthocyanidins while onion, black olive, apple, medlar, mandarin, acerola, cashew apple, black currant pomace, and red ginseng serve as the sources of hydrolyzable phenolics. These hydrolyzable phenolics have different classes of phenolic compounds which include ferulic acid, caffeic acid, sinapic acid, and others. Nonextractable polyphenols that can be abundantly found in several nuts such as brazil nut and heart nut which are hydrolyzable tannins (Pe´rezJime´nez et al., 2013).

6.3 Macromolecules as antioxidants 6.3.1 Polysaccharides as antioxidants of

Several studies have proven the bioactivity polysaccharides derived from diverse

II. Bioactivity

6.3 Macromolecules as antioxidants

sources. Antioxidant properties of polysaccharides extracted from the rhizome of A. sinensis were well documented in the study of (Tian et al., 2017). The study has further directed toward the determination of scavenging activity of superoxide radicals which can damage the cell DNA and destroy the function of the human body. The findings of the study have exhibited that the polysaccharides isolated from A. sinensis have the potential to scavenge superoxide radicals. Hydroxyl radical can cause many adverse effects including cell membrane and DNA damage, killing of red blood cells, among others. Accordingly, the reported study has measured the scavenging activity of hydroxyl radicals and has proven the remarkable ability of A. sinensis polysaccharides in scavenging hydroxyl radicals at high concentrations (Tian et al., 2017). Polysaccharides derived from leaves are identified as important classes of antioxidants owing to their wide availability and low toxicity. Studies have proven that the polysaccharides extracted from oak have bioactive properties. They are anticoagulant and hydroxyl radical scavenging capacities. The presence of polyanionic charges is a great influence on these activities. The antioxidant activity of these extracts controlled by many factors such as plant origin, molecular weight, chemical structure, and arrangement of the active polysaccharide. Similarly, polysaccharides isolated from Q. brantii exhibited excellent hydroxyl radical scavenging activity compared to positive control vitamin C. To explain the hydroxyl radical scavenging of Q. brantii polysaccharides, two models of antioxidation mechanisms are proposed. They suppress the generation of hydroxyl radical and scavenges the hydroxyl radicals produced (Tahmouzi, 2014). The study of polysaccharides extracted from Semen cassia, showed that the scavenging effect of polysaccharides on the hydroxyl radical was better than that of vitamin C at the same concentration (Liu et al., 2014). The antioxidant activity of polysaccharides extracted from root barks of P. laevigata was

153

assessed and the results indicated that DPPH• assay measured the ability of antioxidants to scavenge the free radicals as free radical scavenging ability. It is identified as one of the main mechanisms exhibited by antioxidants in delaying the oxidation process. The study revealed that the extracted polysaccharides showed excellent DPPH• scavenging activity in a dosedependent manner. The DPPH• scavenging activities increased with the increasing concentration of P. laevigata polysaccharide. ABTS radical scavenging activity measures the ability of antioxidants to donate a hydrogen atom by converting it to the nonradical species. The results revealed the activity of P. laevigata polysaccharide against ABTS radical in a concentrationdependent manner. Fe (III) reduction reflects the electron-donating ability of antioxidants which is assumed an important mechanism of polysaccharide antioxidant action. Furthermore, the reducing power of extracted polysaccharides was increased with their concentration (Hajji et al., 2019). Hydroxyl radical, DPPH• and superoxide anion radical systems were mused to analyze the scavenging ability of polysaccharides extracted from M. micromalus Makino fruit wine (Hui et al., 2019). The results reported that the extracted polysaccharides showed significant antioxidant activity in the low concentration range which indicates that polysaccharides isolated from the wine are an excellent free radical scavenger (Hui et al., 2019). The in vivo antioxidant profiles of polysaccharides extracted from seabuckthorn berries (Hippophaerhamnoides L.) with few antioxidant biomarkers including malondialdehyde (MDA), SOD, glutathione (GSH), and protein carbonyls was studied. The results revealed a decrease in MDA and PCO as well as an increase in SOD and GSH (Wei et al., 2019). Moreover, the study claimed that the high amount of galactose present in the seabuckthorn berry polysaccharides may greatly be contributed for exhibited promising bioactive properties (Wei et al., 2019).

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Previous studies also support the positive correlation between the galactose content and DPPH radical scavenging capacity and reducing power. The study further described that the antioxidant activities of the polysaccharides. They are greatly influenced by their monosaccharide composition, molecular weight, and anomeric configuration (Zhang et al., 2018). The study revealed that the polysaccharides extracted from M. anguillicaudatus had an inhibitory effect on oxidation and glycation (Zhang et al., 2011). These activities exhibited a dosedependent response. The study has proven the presence of hexuronic acid, sulfate, neutrophilic sugar, and glucosamine in polysaccharides extracted from M. anguillicaudatus. Moreover, the content of glucosamine is comparatively high. The study claimed that the antioxidative ability of extracted polysaccharides may be attributed to the content of glucosamine (Zhang et al., 2011). Chitosan is another animal-based polysaccharide with potent antioxidant and antimicrobial activities. Nevertheless, the studies on the antioxidant activity of chitosan are still scanty. The study of (Sun, Zhou, Xie, & Mao, 2007) havestudied that the antioxidant activity of chitosan is governed by the molecular weight. The antioxidant activity increase with the decreasing molecular weight. Moreover, the study by Ai et al. (2008) also reported that the chitosan extracted from the larvae of a housefly can scavenge free radicals effectively. Therefore this extract can be used as a natural antioxidant to protect the human body from free radicals damages and finally retard the progression of free radical-induced chronic diseases (Ai et al., 2008). Recently, a number of bioactive polysaccharides extracted from green, red, and brown algae. Moreover, many studies have shown the ability of antioxidative polysaccharides in controlling oxidative stress-mediated diseases such as liver injury, diabetes, obesity, neurodegenerative disease, colitis, and breast cancer. These studies have suggested three distinct mechanisms for the above effect. They are scavenging the ROS,

regulating the antioxidant system, and oxidative stress-mediated signaling pathways (Nemzer et al., 2019). Fucoidan is one of the well-known heterogeneous sulfated polysaccharides found in marine invertebrates and brown seaweed with excellent biological activities. These activities are antioxidant, anticoagulant, antiviral, antitumor, antiinflammatory, and antithrombotic properties. Molecular weight, degree of sulphation, and monosaccharide composition of fucoidans are greatly influenced by the bioactivities. One of the recent studies isolated fucoidans from brown algae Undaria pinnatifida and measured the antioxidant activity by DPPH scavenging. As per the results of the study, fucoidans have exhibited strong antioxidant activity (Mak, Hamid, Liu, Lu, & White, 2013). DPPH and alkyl radical scavenging activities of crude polysaccharide extracted from brown algae Chnoospora minima and reported the excellent DPPH and alkyl radical scavenging activities (Shanura Fernando et al., 2017). Furthermore, the study reported that fucoidan is the major polysaccharide constituent present in the crude polysaccharide extract of brown algae. Thus, linked the degree of sulfation of fucoidans to exhibited antioxidant properties (Shanura Fernando et al., 2017). Sulfated polysaccharides were extracted from three algal species and revealed the presence of antitumor and antioxidant activity (Shao et al., 2013) (Table 6.1). Furthermore, this study reported that the polysaccharide with relatively lower sulfate content and the highest uronic acid content, displayed excellent antioxidant activities in superoxide radical assay, ABTS assay, and DPPH assay while exhibited minimal inhibitory effects on the growth of MKN45 gastric cancer cells and DLD intestinal cancer cells. However, a polysaccharide with the highest sulfate content and the lowest uronic acid content showed low activity on radical scavenging assays. The findings of the study claimed that the combined effect of sulfate content and uronic acid content may influence their exhibited antioxidant and antitumor activities (Shao et al., 2013).

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6.3 Macromolecules as antioxidants

The marine environment has become one of the most prominent sources of microbial extracellular polysaccharides. The antioxidant activity of these microbial extracellular polysaccharides extracted from marine microorganisms is well documented. Potential applications of chrysolaminarin extracted from the marine diatom, O. aurita in the food industry used as a natural antioxidant (Xia et al., 2014). The study further documented the stronger hydroxyl radical scavenging activity of novel chrysolaminarin extracted from a marine diatom. However, exopolysaccharides released by diverse species of microalgae also serve as excellent sources of bioactive with antioxidant, antiinflammatory, and antiviral properties (Trabelsi et al., 2016). On the other hand, polysaccharides derived from fungal bases also exhibited promising antioxidant activities. The study reported that the antioxidant activities of polysaccharides derived from thirteen boletus mushrooms and confirmed the presence of antioxidant polysaccharides (Zhang et al., 2018). Further, the in vitro antioxidant activities of these polysaccharides were related to their structural characteristics. DPPH radical scavenging activity exhibited strong correlations with arabinose and galactose content while galactose contents strongly correlate with ferrous ion reducing power (Zhang et al., 2018).

6.3.2 Proteins as antioxidants Recently, the influx of research has directed toward the bioactive proteins derived from various natural sources. Owing to suspected health adverse effects of synthetic antioxidants, natural antioxidants have attracted a great deal of attention. Protein fractions with antioxidant activity have exhibited greater potential in food industrial applications to extend the shelf life of the product (Li et al., 2018). Bioactive proteins majorly involve in controlling lipid oxidation by biologically defined or nonspecific mechanisms and these antioxidative

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proteins mainly contribute to the endogenous antioxidant capacity of food products. The antioxidant activity exerted by these proteins perhaps attributed to complex interactions between their ability to inactivate ROS. This can be done by scavenging free radicals, chelating prooxidative transition metals, reducing hydroperoxides, eliminating specific oxidants enzymatically, or by changing the physical properties of the food system which allows the separation of reactive species. Compared with other antioxidants, proteins are unique owing to their ability to function as multifunctional antioxidants. Therefore, they can inhibit several different lipid oxidation pathways. Moreover, these antioxidant activities of proteins can be enhanced by different techniques such as the Maillard reaction, increasing the accessibility of antioxidative amino acids by thermal processing, hydrolysis (Elias et al., 2008). However, disruption of tertiary structure leads to an increase in the overall antioxidant capacity of proteins. This helps further scavenging of free radicals and chelating the prooxidative metals by increasing the solvent convenience for amino acid residues (Sohaib et al., 2017). It is accepted that the antioxidant activity of the protein is determined by the composition and sequence of amino acids. The ability of the protein to chelate metals, scavenge free radicals, and reduce lipid hydroperoxides is governed by the composition and distribution of several key amino acids. Nevertheless, antioxidant activity exhibited by these amino acid residues seems to be narrow in the tertiary structure (Elias & Decker, 2010). A recent study has proven the potential antioxidant activity of turmerin, a protein isolated from turmeric. This water-soluble protein has exhibited promising DPPH and superoxide radical scavenging capacities with moderate ABTS radical scavenging and Fe(II) chelation capacities (Lekshmi et al., 2012). In another study reported that the antioxidant activity of coconut cake protein fractions: albumin,

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globulin, prolamine, glutelin-1, and glutelin-2 (Li et al., 2018). The results indicated that the prolamine, glutelin-1, and glutelin-2 have shown high free radical scavenging activity while globulin and prolamine have demonstrated excellent chelating ability which may be attributed to high amounts of sulfurcontaining amino acids. All protein fractions have exhibited higher reducing power which may be attributed to the high frequency of nonpolar side chains and high amounts of aromatic amino acids. Moreover, the highest •OH scavenging activity has been found in prolamine and glutelin-2 and this may be due to the high frequency of nonpolar side chains and average hydrophobicity. In consideration of increased hydrophobicity allows the peptide to reach hydrophobic targets, this contributes to increase the antioxidant activity. Prolamine and glutelin-1 have exhibited higher superoxide radical scavenging activity which may be characterized by the high content of histidine (Li et al., 2018). The utilization of whey proteins in food and nonfood application has become important across the globe owing to its’ bioactive properties. The addition of whey protein in soybean oil emulsion increased oxidative stability and it is widely accepted that fermented whey-based foods have demonstrated excellent antioxidant activity. Moreover, the oxidative stability of salmon oil emulsion increased with the addition of whey protein (Khan et al., 2019). Casein also possesses promising antioxidant activity. However, antioxidant mechanisms of action of casein are complex. It is believed that casein controls lipid oxidation by autoxidation of iron. Furthermore, to scavenge the free radicals the primary structure of casein molecules have to act as scavengers (Khan et al., 2019). Additionally, Ovalbumin isolated from egg white also demonstrated good antioxidant potential. Numerous studies have reported that the antioxidant activity of ovalbumin increased after covalent binding. Moreover,

glycosylation of ovalbumin with glucose under heat moisture treatment or microwave heating also helps to increase the antioxidant activity of ovalbumin. It is documented that the antioxidant activity of ovalbumin can be increased by combined with the polyphenol rutin or the mineral selenite. That is due to the formation of a molten globule conformation of the protein which enhances the surface hydrophobicity and its solubility (Benede´ & Molina, 2020).

6.3.3 Nonextractable polyphenols as antioxidants With the discovery of macromolecular antioxidants or nonextractable polyphenols number of studies have directed toward the assessment of biological activities of this unique fraction with promising characteristics. The study determines the macromolecular antioxidant content of selected fruits and vegetables (Pe´rez-Jime´nez & Saura-Calixto, 2015). Results of the study have identified nonextractable polyphenols as the major contributors of the total antioxidant content of the selected fruits and vegetables which accounts for the mean value of 57%. Sanz-Pintos et al. (2017) analyzed the macromolecular antioxidants present in several edible seaweeds viz. Gracilaria chilensis, Callophyllis concepcionensis, Macrocystis pyrifera, Scytosyphonlomentaria, Ulva sp. and Enteromorpha compressa. Similarly, macromolecular antioxidants have been recognized as the major polyphenol fraction which accounts for 42% of total polyphenol content. This fraction was also revealed that excellent antioxidant activity.

6.4 Applications 6.4.1 Food-based applications 6.4.1.1 Functional foods Foods that provide other pro-health benefits along with nutritional value are defined as functional foods. Generally, one or more bioactive

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6.4 Applications

compounds are incorporated into the food products which are not naturally present in sufficient quantities. Recently, functional and enriched foods have gained more popularity among food technologists and consumers. Biologically active proteins and peptides having antioxidant, antihypertensive, or neuroprotective properties have become a potential functional component in food products. However, the profile of exogenous amino acids with sulfur-containing amino acids provides excellent balance in newly formulated foods. Chia seed proteins as a potential bioactive ingredient and supplement in functional foods as allergens derived from chia seeds have not been confirmed to date (Kotecka-Majchrzak et al., 2020). Lately, marine resources have gained much attention from food technologists as a promising source of bioactive compounds to design and develop food supplements. The study by Zhang et al. (2010) has acknowledged brown, red, and green algal polysaccharides as an excellent source of antioxidants. Thus, suggested to utilize it as a possible supplement in food and pharmaceutical industrial applications. Moreover, functional polysaccharides derived from P. yezoensis have been used in food industry applications for a longer period due to their beneficial biological effects. Antioxidant, anticancer, antihyperlipidemia, and antifatigue effects are major biological properties attributed to P. yezoensis polysaccharides (Lee et al., 2016). Phlorotannins, phenolic compounds formed because of the polymerization of phloroglucinol also used as a potential ingredient to be used as functional foods with potential health effects. Phlorotannins have shown significant radical scavenging activities against superoxide and DPPH radicals compare to that of ascorbic acid and α-tocopherol. Therefore, used as a potential antioxidant in food industry applications. However, sulfated polysaccharides also reported possessing promising biological activities which include anticoagulant, antiviral and immunoinflammatory activities,

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thus, exhibit growing demand in functional foods, cosmeceutical, and pharmaceutical applications. A wide range of biological activities of marine-derived natural compounds, marine food sources have demonstrated the potential to expand the health-promoting properties toward the pharmaceutical and cosmeceutical industries without limiting them to food industrial applications (Zhang, Li, & Kim, 2012). 6.4.1.2 Food packaging Consumer demand-driven toward the use of natural food additives thus limits the use of synthetic additives. Similarly, the use of synthetic antioxidants has become questionable due to the potential toxicity associate with the synthetic antioxidant along with the possibility of migrating into food products. Therefore, the use of biopolymer-based packaging materials originates from natural sources such as proteins and polysaccharides to replace synthetic materials. These biopolymer-based materials are renewable, biodegradable, and inexpensive. Macromolecules, including proteins and polysaccharides, have been identified as prominent natural sources of biopolymers. Chitosan, starch, agar, cellulose, furcellaran, and carrageenan like polysaccharides and gelatin, alginate, collagen, gluten, and whey protein-like proteins are used as alternatives to synthetic packaging materials. The antioxidant properties of these macromolecules were able to control the oxidation of foods and help to extend the shelf life of food products (Jamro´z & Kopel, 2020). Algal polysaccharides have been extensively studied for potential use as edible films. Interestingly, polysaccharides derived from U. fasciata provides better mechanical strength apart from the excellent hydroxyl free radical scavenging activity. Antioxidative polysaccharides derived from green algae have been used to preserve the meat along with its potential use as food packaging material. Furthermore, these edible films derived from seaweed polysaccharides

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properly match with lipid-based food products due to the presence of natural antioxidants (Ramu Ganesan, Shanmugam, & Bhat, 2018). Browning has become one of the major deteriorating reactions associated with many foods including fruits and vegetables. Casein-based coatings are used in controlling oxidationinduced browning reactions of fruits and vegetables. Several studies have explored the potential of calcium caseinate and whey powder in controlling the enzymatic browning in slice potatoes and apples. The results of the study revealed that the ability of milk protein-based edible coatings delayed the enzymatic browning reaction. The study further stated that the antioxidant activity of whey proteins is higher than the calcium caseinate (Khan et al., 2019). 6.4.1.3 Food additives Macromolecules originated from natural sources are extensively used as food additives in controlling the number of deteriorative reactions in food products. Many studies have shown the ability of proteins in controlling lipid oxidation in foods. Proteins originating from milk, blood plasma, and soy protein can be used in muscle foods as apotent antioxidant source. Serum albumin and transferrin-like antioxidant protein were found to delay the formation of thiobarbituric acid reactive substances in both salted ground pork and cooked ground beef. Studies have shown the potential of whey and soy proteins in controlling lipid oxidation in cooked pork patties. Furthermore, whey protein also involves in controlling lipid oxidation in oil-in-water emulsions (Elias et al., 2008). Chitosan is identified as an approved food ingredient in Japan, Korea, the United States, and Europe, it has been used in the food industry as a food preservative to prevent spoilage. Chitosan is also used as a natural antioxidant due to metal ion chelation in lipidcontaining foods, thereby demonstrating the ability to extend the shelf life (Abd El-Hack et al., 2020). Lactoferrin is a glycoprotein that is

closely related to transferrin. It is also identified as a potent endogenous antioxidant in dairy foods. This can be effectively used as an antioxidant additive in processed lipid foods. For an instance, lactoferrin effectively controls the lipid oxidation in infant formula, milk, and mayonnaise (Elias & Decker, 2010).

6.4.2 Other applications Antioxidant activity of bioactive macromolecules is important in regulating the redox state of the body and thereby, reducing the damage caused by diseases or drugs. Several studies have confirmed the antioxidant activity and immunostimulatory activities of polypeptides extracted from natural sources. These biological properties exhibited in macromolecules claimed their potential applications in drug development (Song et al., 2020). Chitooligosaccharide derivatives which are derived from chitosan also exhibit potential application in the pharmaceutical and medicinal industry owing to their beneficial biological effects viz. antioxidant, antimicrobial, anticancer, and antidiabetic properties (Zhang et al., 2012). On the other hand, chitin and chitosan possess unique biochemical properties including biocompatibility, biodegradability, nontoxicity, and ability to form films. These properties greatly contribute to chitin and chitosan being potential candidates in biomedical applications (Elieh-Ali-Komi & Hamblin, 2016). Furthermore, antioxidant peptides are significant attention in the skincare industry owing to their ability to remove ROS and free radicals to protect skin cells, slow down aging, and inhibit melanin production(Song et al., 2020).

6.5 Limitations of biological macromolecules Though antioxidative macromolecules are widely used in many industrial applications owing to their positive biological properties,

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References

still few drawbacks need to be addressed carefully to improve the functionality of these macromolecules. It is widely accepted that proteins are not a suitable candidate to replace synthetic antioxidants in all food applications regardless of their proven antioxidant efficacy as proteins are well-known for allergens. Among, dairy, soy, nut, and egg exhibit greater potential as allergens. Contrary, incorporation of these proteins and peptides into food products may lead to undesirable rheological changes. They have increased viscosity of aqueous solutions or gelation under some conditions. Furthermore, Maillard reaction products lead to unavoidable color changes which is not a desirable characteristic for all foods (Elias & Decker, 2010).

6.6 Future trends Multifunctional natural antioxidants can be effectively utilized to produce oxidatively stable foods. Thus, increasing the effectiveness of native antioxidative macromolecules has become a priority research area. Antioxidant activity of native proteins can be improved by changing the tertiary structure by heating, enzymatic hydrolysis, and Maillard reaction. The greater antioxidant potential of peptides compared to that of native proteins from which they are derived has been studied well. Nevertheless, there is an urgent need of expanding future research in correlating peptide activity with amino acid composition and sequence (Elias & Decker, 2010). However, antioxidative macromolecules exhibit poor bioavailability and instability in the gastrointestinal environment after oral administration. Consequently, the expected antioxidant efficacy is hardly achieved. Therefore many researchers are in the urge to develop innovative solutions to improve the efficacy of antioxidant activity. These studies reveal that beneficial biological properties can be enhanced when bioactive micromolecules and polypeptides are prepared

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into nanoparticles by nanotechnology (Song et al., 2020). Thus, a new trend has been created toward nanotechnological applications in antioxidative macromolecules. Though the evaluation of antioxidant activities of bioactive macromolecules is vitally important, the availability of proper standards is still scanty, and therefore results in limitations in comparison and market standardization of antioxidative macromolecules. Therefore there is an urgent need for the development of evaluation standards to assess the antioxidant activity of bioactive macromolecules (Song et al., 2020).

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C H A P T E R

7 Biological macromolecules as antimicrobial agents Md. Shahruzzaman1, Shafiul Hossain1,2, Tanvir Ahmed1, Sumaya F. Kabir1, Md. Minhajul Islam1, Ashiqur Rahman1,3, Md. Sazedul Islam1, Sabrina Sultana1,4 and Mohammed Mizanur Rahman1 1

Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh 2Department of Chemical Engineering and Polymer Science, Shahjalal University of Science and Technology, Sylhet, Bangladesh 3National Institute of Textile Engineering and Research (NITER), Dhaka, Bangladesh 4Department of Arts and Sciences, Ahsanullah University of Science and Technology, Dhaka, Bangladesh

7.1 Introduction In the history of mankind, in 2020, now we are facing a mortal microbe, which is spreading rapidly and affecting people across the whole world. The pathogenic microbes are inevitably increasing for the last decades due to the unconsciousness of administrating antibiotic drugs. The abuse and misuse of the antibiotic drugs act as a back force for the rapid increase of the drug-resistant microorganism. The replication and mutation of the drug resistant pathogens are the main cause of multidrug resistant (MDR) microbes, which are known as superbugs (Khan & Khan, 2016). The superbugs show resistance to several antibacterial

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00007-5

agents by reducing the uptake and action of drugs. The MDR microbes have become a challenge for the modern world in terms of sustainable treatment against infectious diseases. The researchers are continuously trying to develop novel drugs to fight against the threat of microbes, but they cannot reach the expected level. For minimizing the microbial effect, the infection from clinical settings must be ceased. In this condition, the development of medical accessories such as implants and surgical devices having antimicrobial action have achieved the great interest. For the fabrication of these antimicrobial materials, the biodegradable, biocompatible, and nontoxic biological macromolecules can be the best choice.

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Biological macromolecules are very attractive for the development of antimicrobial materials because of their natural abundance, chemical robustness, and environmental compatibility properties. These macromolecules are important body elements that perform a variety of functions necessary for living organisms to keep them alive. The well-known natural macromolecules such as cellulose, chitosan, heparin, chondroitin, hyaluronic acid (HA), collagen, gelatin, keratin, fibrin, soy protein and various fatty acids are originated from different plants and animals. The natural macromolecule possesses reduced side effects compared to the small molecule. Moreover, the natural macromolecules show more advantages such as eco-friendly, costeffective and renewability properties compared to the synthetic molecules. The two or more natural macromolecules are blended with each other for the synthesis of biodegradable composite materials. The composite is formed between two polysaccharide molecules such as chitosancellulose (Bajpai, Chand, & Ahuja, 2015) chitosan-alginate (Bakhsheshi-Rad et al., 2020) and sometime between polysaccharide and protein such as gelatin-chitosan (Jridi et al., 2014), keratin-chitosan (Tan et al., 2015) and collagenalginate (Zhang et al., 2018). The synergistic effect of the composite materials have make them more attractable than the individual. In a research, Pereda and his co-workers (Pereda et al., 2011) evaluated the antimicrobial action of gelatin and chitosan-gelatin film forming solution against Escherichia coli and Listeria monocytogenes and found better result in composite film forming solution compared to the gelatin solution which indicates the better plausibility of composite materials. The superior physical, chemical and biological properties of these materials have make them capable for prevention of deleterious effect of microbes. The increased infectious diseases caused by the microorganism have augmented the researcher’s awareness toward the development of alternative approaches to mitigate the effect.

The nanotechnology based macromolecule composites have attracted the special interest because of their effectiveness against microorganism. It may reduce the side effects of several drugs, intake of dose amount and required time of curing. Nanoparticle based macromolecules can perform outstanding mechanism to combat against MDR bacteria. The major advantage of nanoparticle is the targeted drug delivery to the infected tissue to confirm the permanent inhibition of the microbes. In a research, (Irwin et al, 2010) prepared a keratin-capped nanoparticle and showed its excellent antimicrobial action against Staphylococcus aureus and Salmonella typhimurium and E. coli bacteria which proved the performance of nanoparticle based macromolecule. The multi-functional nanoparticle based macromolecule composites exhibit outstanding antimicrobial activities against pathogenic microbes. The rapid action of these composites are capable of destroying the microbial colony. A macromolecule composite of collagen [collagen/(ZnTiO3/SiO2) composite] has inhibited the microbial effect of S. holeresius, Bacillus cereus and C. lucitania bacteria (Vladkova, Staneva, Albu-Kaya, Martinov, & Ivanova, 2020). Really, the nanotechnology based macromolecule composite is a blessing for the complete inhibition of pathogenic bacteria and fungus. Numerous antimicrobial matrices such as fiber (Cremar et al., 2018), film (Abral et al., 2020), membrane (He et al., 2018), bandage (Dai, Tegos, Burkatovskaya, Castano, & Hamblin, 2009) and hydrogel (Heedy, Marshall, Pineda, Pearlman, & Yee, 2020), etc. are prepared from biological macromolecules that can be used in versatile fields. These matrices are very promising for application in biomedical field such as drug delivery (Vivek, Babu, Thangam, Subramanian, & Kannan, 2013) and wound dressing (Ding et al., 2017). The matrices of nanoparticles incorporated antimicrobial macromolecules are very potential for direct administration of drug to the infected body site which is known as targeted

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drug delivery. The wound dressing based on biological macromolecule with excellent antimicrobial activity and rapid re-epithelial capability are very prospective for treatment of wound infection in an expedite way. The development of biodegradable packaging film based on natural macromolecules having good antibacterial performance is an excellent replacement of nonbiodegradable polymer based packaging materials (Ramos, Valdes, Beltran, & Garrigo´s, 2016). These matrices encapsulate agrochemicals and genetic materials to ensure the controlled release of pesticides in order to protect the environment from the direct exposure of these toxic chemicals. Moreover, the antibacterial properties of these matrices assist to stabilize seafood-based products which expanded their application in sea food industry and aquaculture (Sruthy & Sailesh, 2018). The multidisciplinary applications of the antimicrobial biological macromolecules have made them a promising field of research.

7.2 Classification of biological macromolecule 7.2.1 Carbohydrate Carbohydrates are the most abundant biological macromolecules found in living organism on earth that are composed of polyhydroxy aldehydes or ketones or substances that yield such compounds on hydrolysis. These are very essential part of the food items which provide the necessary energy in the form of glucose. The

carbohydrate is more special than other biological macromolecules because of its ability to be associated with protein and lipid to form glycoprotein and glycolipid respectively. Furthermore, carbohydrates such as pentose sugars ribose and deoxyribose are important components of RNA and DNA respectively. Most of the carbohydrates have the empirical formulae of (CH2O)n where n is the number of atom. However, some carbohydrates may contain nitrogen, phosphorous or sulfur in their chemical structure. Depending on the size of the molecule carbohydrates are divided into four classes which are monosaccharide, disaccharide, oligosaccharide and polysaccharide. 7.2.1.1 Monosaccharide Monosaccharides are the simplest form of carbohydrates that are easily absorbed in intestine compared to the disaccharide, oligosaccharide and polysaccharide. The monosaccharide consists of single unit which contains carbon chain of three to six carbon. They can combine through glycosidic bonds to form larger carbohydrates. The main function of monosaccharide is to produce and store energy. Glucose and fructose are the most available monosaccharide in nature. Fig. 7.1A and B show the chemical structure of glucose and fructose respectively. 7.2.1.2 Disaccharide Disaccharide consists of two monosaccharide units linked together by a glycosidic bond which is formed by condensation reaction FIGURE 7.1 Chemical structure of (A) glucose and (B) fructose.

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between hydroxyl group of one monosaccharide with the hydrogen of another monosaccharide. The most abundant disaccharides are sucrose, lactose and maltose which are known as table sugar, milk sugar and malt sugar respectively. Sucrose is composed of glucose and fructose which is shown in Fig. 7.2. 7.2.1.3 Oligosaccharide Oligosaccharide is the short chain of monosaccharide unit which composed of 3-10 molecules connected by glycosidic linkage. The most common example of oligosaccharide is raffinose. It is a trisaccharide found in many plants and composed of galactose, glucose and fructose. The chemical structure of raffinose is shown in Fig. 7.3.

7.2.1.4 Polysaccharide The polysaccharides are ubiquitous biopolymers composed of long chain of monosaccharide units. It contains almost hundred to thousand monosaccharide units that may either straight or branched. Depending on the type of monosaccharide the polysaccharides are divided into two classes that are homopolysaccharides and heteropolysaccharides. The homopolysaccharides are composed of one type of monosaccharide such as cellulose, starch and glycogen are made up of glucose unit and fructan, xylan, galactan and chitin are made up of fructose, xylose, galactose and Nacetyl glucosamine respectively. The heteropolysaccharides are composed of two or more type of monosaccharides such as HA is formed FIGURE 7.2 Chemical structure of sucrose.

FIGURE 7.3

Chemical structure of raffinose.

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7.2 Classification of biological macromolecule

by thousand units of N-acetyl glucosamine and glucoronic acid and heparin is made up of glucosamine, glucoronic acid and iduronic acid. The major function of polysaccharide is energy storage or structural support of organism. The starch and glycogen are used for energy storage and cellulose and chitin are used for structural support of plant and animal respectively. The chemical structure of homopolysaccharide cellulose and heteropolysaccharide HA are shown in Fig. 7.4A and B respectively.

7.2.2 Protein Proteins are one of the most abundant biological macromolecules in living organism which perform a wide range of functions. A living system may contain thousands of different proteins having unique characteristics and functionalities. Proteins are long chain of chemically distinct 20 amino acids that are linked by peptide bonds. Based on essentiality

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amino acids are classified into two groups that are essential amino acid such as histidine, isoleucine and leucine etc. and nonessential amino acid such as alanine, arginine and cysteine, etc. Unlike nonessential amino acids the essential amino acids are not synthesized in our body for this it must be taken through the diet. There are four level of protein structures that are primary, secondary, tertiary and quaternary which provide necessary information about the final shape of protein. Each protein has its unique shape which may be disrupted if the protein’s environment such as pH or temperature is changed or exposed to any chemical. Proteins are available throughout the animal body such as bone, skin and fish scales are source of collagen, feathers and hairs are source of keratin and ligaments are source of elastin (Ferraro, Anton, & Sante´Lhoutellier, 2016). Protein performs a large range of functions in the body which are listed in Table 7.1.

FIGURE 7.4 Chemical structure of (A) cellulose and (B) hyaluronic acid.

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TABLE 7.1 Types and functions of protein. No. Type

Function

Example

1.

Enzyme

Perform almost thousands of chemical reactions that take place in cells and expedite the digestion process

Lipase, Amylase, Pepsin, Trypsin

2.

Hormone

Manage the activity of different body system

Insulin, Estrogen, Prolactin, Testosterone

3.

Antibody

Fight against foreign pathogens and protect the body

Immunoglobulin

4.

Transport

Carry atoms or small molecules throughout the body

Albumin, Hemoglobin, Ferritin

5.

Structural

Provide structure and support for cell

Keratin, Actin

6.

Storage

Serve as biological reserve of metal ion and amino acid

Albumin, Ferritin

7.

Contractile Mediate sliding of contractile fibers

Myosin, Actin

7.2.3 Lipid Lipids are a chemically diverse group of biological compounds that are naturally nonpolar. They are hydrophobic but soluble in organic solvent such as alcohol, ether and chloroform. The diversity in their chemical properties has made them capable for multi-functional activity. They perform several roles in the body which are very important for carrying out the biological functions of living organism. They serve as structural materials such as phospholipids and sterols are the major structural element of biological membranes. They act as energy storehouse whereas fat and oil are the stored form of energy in living organism. Furthermore, they act as an insulator for plants and animals to keep them safe from the adverse environment. The three major types of lipids are triglycerides, phospholipids, and sterols. 7.2.3.1 Triglyceride Triglyceride is the simplest lipid composed of three fatty acids that are connected with ester linkage with a glycerol unit. The main function of triglyceride is to store energy for later use. When any calorie more than the body

requirement is consumed it is converted to tryglyceride and stored in fat cells. A common example of triglyceride is tristearin which is composed of three stearic acid attached to a molecule of glycerol. The chemical structure of triglyceride is shown in Fig. 7.5A. 7.2.3.2 Phospholipid Phospholipid is amphiphilic compound consists of a glycerol molecule containing a polar head group and two fatty acid molecules acting as hydrophobic tail. Due to the amphiphilic property they can arrange themselves in a certain pattern in water to form cell membrane. Two important examples of phospholipid are phosphatidylcholine and phosphatidylserine that are found in plasma membrane. The chemical structure of phospholipid is shown in Fig. 7.5B. 7.2.3.3 Sterol Sterol is different from others lipids due to its multiple ring structure. The main function of sterol is to act as hormone or intrinsic component of cell membrane. Cholesterol is the familiar sterol that is found in regular food items such as milk, meat, egg yolk, etc. The cholesterol is the main constituent of some

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FIGURE 7.5 Chemical structure of (A) triglyceride, (B) phospholipid, and (C) cholesterol.

hormones including sex hormone. The chemical structure of cholesterol is shown in Fig. 7.5C.

7.2.4 Nucleic acid Nucleic acid is the naturally occurring biological macromolecule that carries out the intrinsic characteristics of a living organism.

The main functions of nucleic acid is to store and transmit genetic information from one generation to the next and to regulate the process of protein synthesis. The two major classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are composed of phosphoric acid, a fivecarbon sugar and mixture of nitrogen containing bases. Both DNA and RNA contain two

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purine bases and two pyrimidine bases among which the purine bases (adenine and guanine) and one pyrimidine base (cytosine) are same for both but another pyrimidine base is different. The second pyrimidine base of DNA is thymine and RNA is uracil.

7.3 Antimicrobial activity of biological macromolecules 7.3.1 Polysaccharides 7.3.1.1 Chitosan Chitosan is biocompatible, biodegradable and naturally abundant polymer. It is less toxic toward mammalian cell and can hinder the growth of bacteria (Franklin & Snow, 2013). Electrostatic interaction between positively charged chitosan and negatively charged different component of the cell membrane of bacteria results into antimicrobial activity of chitosan. Combining with DNA, chitosan can hinder the production of mRNA and DNA transcription (Sudarshan, Hoover, & Knorr, 1992). High molecular weight chitosan forms an impermeable membrane around the cell wall to alter the bacterial cell permeability (Leuba & Stossel, 1986) or to resist the transportation of essential nutrients inside the cell (Choi et al., 2001; Eaton, Fernandes, Pereira, Pintado, & Malcata, 2008). Chung et al. (2004) confirms that the mechanism of antibacterial activity of chitosan includes hydrophilicity and adsorption of positively charged chitosan on the cell membrane of bacteria (Choi et al., 2001; Eaton, Fernandes, Pereira, Pintado, & Malcata, 2008). The above stated process is more suitable against Grampositive bacteria rather than Gram-negative. But the mechanism of chitosan adsorption is pH dependant. In acidic condition chitosan is positively charged so the adsorption favors below pH 7. On the other hand it is evident that chitosan also inhibits the growth of

Gram-negative bacteria (Jeon, Park, & Kim, 2001; Muzzarelli et al., 1990; Rhoades and Roller, 2000). Higher degree of deacetylation means high rate of chitosan adsorption into the cell wall of bacteria at low pH as there will be more positively charged deacetylated group (Zheng & Zhu, 2003). They also found that an increase in molecular weight results into high rate inhibition of Gram-positive bacteria and vise versa for Gram-negative bacteria. They illustrated two mechanisms for inhibition of bacterial growth. High molecular weight chitosan blocks the transportation of cell nutrients by forming an impermeable membrane and low molecular weight chitosan enters into the cell wall through pervasion. Benhalbies et al. discussed the effect of MW of chitin and chitosan. The antimicrobial activities were compared between seven Gramnegative and four-Gram-positive bacteria for chitin and chitosan (Benhabiles et al., 2012). The analysis shows that chito-oligomers are more advantageous to be used as an antimicrobial agent because of its high activity and low water solubility. Mycelial growth, sporangial production, and release of zoospores and germination of cysts of phytophthorainfestans in tomato plants can be reduced by the application of chitosan. Atia, Buchenauer, Aly, and Abou-Zaid (2005). Direct interface of fungal growth and several defense process activation are two proposed function of chitosan after the formation of an impermeable membrane at cell membrane surface (Bai, Huang, & Jiang, 1988). The defense mechanism includes the accumulation of chitinases, synthesis of proteinase inhibitors, lignification, and induction of callous synthesis (El Ghaouth, Arul, Grenier, & Asselin, 1992). These activities on a particular fungal species like F. oxysporum and Alternaria solani depends on the molecular weight and degree of deacetylation of chitosan respectively but no dependency of molecular weight and degree of deacetylation was found in case of Aspergillus niger.

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7.3 Antimicrobial activity of biological macromolecules

7.3.1.2 Cellulose Cellulose is a β-1,4 linked polymer of Dglucose. Depending on the source the number of monomer units may vary from 10,000 to 15,000 units. Cellulose nanocrystals (CNC), bacterial cellulose (BC) micro fibrillated cellulose are mostly used forms of cellulose. The surface of CNC modified with quaternary ammonium salt (carbon more than 10 in alkyl chain) can hinder the growth of both positive and negative strains of bacteria. The positively charged modified surface of CNC agglomeration on the negatively charged cell membrane of bacteria inhibits the growth of bacteria (Bespalova, Kwon, & Vasanthan, 2017). Freeze-dried bacterial cellulose with benzalkonium chloride can act as a stable antibacterial material. Terpyridine modified metal complex cellulose nanofibril can inhibit the growth of S. aureus, E. coli and Saccharomyces cerevisiae (yeast). The cellulose-Cu complex showed a maximum inhibition rate against all the microbes stated above (Hassan & Hassan, 2016). 7.3.1.3 Alginate The basic component of a natural polymer named alginate is 1,4 β-D mannuroic acid and α-1 guluronic acid. Water barrier, mechanical, cohesiveness and antimicrobial properties of the polymer can be changed through crosslinking the polymer with polyvalent cations like calcium. Incorporation of oregano essential oil in the crosslinked polymer can inhibit the growth of positive bacteria (S. aureus and L. monocytogenes) more than Gram-negative bacteria (E. coli and Salmonella enteritidis) bacteria. Altering and changing crosslinking the release of OEO can be controlled and thus the antimicrobial efficacy (Benavides, Villalobos-Carvajal, & Reyes, 2012). Na-alginate with antimicrobial agent shows better antimicrobial efficacy for all indicator microorganisms than k-carrageenan based films. This study also shows that alginate film with EDTA shows stronger antimicrobial effects against Gram-negative bacteria

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especially found in processed food packaging (Cha, Choi, Chinnan, & Park, 2002). Strawberry decay is a common phenomenon during storage. The microbial attack is responsible for this decay. But the application of edible alginatebased electrostatic spraying can reduce the microbial attack to 5.6% (Peretto et al., 2017). 7.3.1.4 Heparin Heparin is a natural polysaccharide found in animals commonly used as an antithrombic coating in implant devices for antimicrobial activity (Junter, Thebault, & Lebrun, 2016). In tunneled hemodialysis catheter is the last option for vascular access. To prevent infection of the catheter the outer surface must be coated with antimicrobial film. For intensive care unit patients with nondialysis settings antimicrobial and antithrombic coatings are found to be efficient. This kind of coatings includes heparin that shows efficacy in catheter-related infection prevention. Helicobactor pylori bacterium is responsible gastrointestinal diseases like gastric ulcers, cancers and so on. Heparin with chitosan and berberine can prevent the growth of Helicobacter pylori (Chang et al., 2011). Heparin acts as a binder and propagates faster healing. Drug loaded chitosan-heparin complex is another material that also can prevent the growth of Helicobacter pylori in some specific sites of gastrointestinal tracts. This is stable in between pH of 1.5
2.5 (Lin et al., 2015). Ciprofloxacin works against bacterial infection in case of typhoid fever, intestinal infection, pneumonia. Ciprofloxacin linked with genuine crosslinked chitosan-heparin works against enteropathogenic bacteria in the gastrointestinal tract which was confirmed by the FESEM images (Fig. 7.6) (Kumar, Su, & Velusamy, 2016). 7.3.1.5 Chondroitin Chondroitin, an important part of cartilage and a dietary supplement. A type of chondroitin is chondroitin sulfate (CS). Loading of

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FIGURE 7.6 FESEM images of CIPRO-GP-CS/Hep NPs treated E. coli MTCC 443. Source: Reprinted from Kumar, G. V., Su, C.-H., & Velusamy, P. (2016). Ciprofloxacin loaded genipin cross-linked chitosan/heparin nanoparticles for drug delivery application. Materials Letters, 180 119-122. With permission from Elsevier.

different antimicrobials with chondroitin shows efficient antimicrobial activity against different bacterial strains like Staphylococcus, Enterococcus, Escherichia, and Pseudomonas (Mu¨ller & Kramer, 2000). A recent study showed that a nanogel prepared from poly(llactic)
graft-CS co-polymers can act as an antimicrobial carrier for infection treatments (Ghaeini-Hesaroeiye, Boddohi, & VasheghaniFarahani, 2020). Green synthesis of CS stabilized Silver nanoparticles is another advancement in the case of antimicrobial chondroitin materials. Antibacterial susceptibility was measured by the broth dilution method for A. baumannii E. coli, Pseudomonas aeruginosa, and S. aureus. The material is nontoxic and bactericidal against all the strains stated above (Young et al., 2018). Magnesium mineralized nanofiber made of CS is another electrospun fibrous structure that was found to be applicable in burn injury and can heal faster with an improved antimicrobial efficiency against the bacterial colony at the wound site (Leung et al., 2020).

7.3.1.6 Hyaluronic acid HA, a major constituents of extracellular matrics. Due to its biological activities, it can be used in ophthalmology, dermatology, dentistry, esthetic surgery, etc. Recently it is evident that HA can act against bacterial growth. But the intensity of antimicrobial property is molecular weight and concentration dependant. HA is an alternative antibiotic in case of chronic urinary tract infections. It is also evidently proved that the combined application of HA and CS can prevent the growth of bacteria in case of female recurrent urinary tract infections (Damiano et al., 2011). In a study of HA, dose-dependent growth inhibition was observed for different strains of different species and the same species like Staphylococci, Enterococci, two E. coli strains (Ardizzoni et al., 2011). In orthopedic surgery- HA, collagen TypeI, polylactic glycolic acid is used as bacteriostatic agents. HA shows better bacteriostatic effects and inhibits the growth S. aureus, Staphylococcus epidermidis, β-hemolytic streptococcus, and P. aeruginosa up to 76.8% (Carlson et al., 2004).

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7.3 Antimicrobial activity of biological macromolecules

7.3.2 Proteins 7.3.2.1 Collagen A natural biopolymer was recently used in biomedical materials like wound dressing, drug delivery. Though collagen has no antimicrobial activity but the incorporation of thymol in collagen films can act as an antimicrobial film. Collagen/thymol film was prepared by a solvent evaporation method. Antimicrobial studies of the collagen/thymol thin film on E. coli, P. aeruginosa, S. aureus, Bacillus subtitlis, Enterobacter erogenous, Candida albicans shows that the material can inhibit the growth of S. aureus most. It was also evident that the film shows more efficacy through direct contact with pathogens rather than diffusion of thymol from the film (Michalska-Sionkowska, Walczak, & Sionkowska, 2017). A hydrogel prepared from silica-collagen type-I loaded with antibiotics is another antimicrobial material for wound dressing application. Collagen concentration regulates the release of antibiotics and inhibits the growth of S. aureus (Alvarez et al., 2014). Collagen hydrolysate with industrial essential oil is an antimicrobially active material which is used in cosmetic formulation to prevent microbial growth (Yorgancioglu & Bayramoglu, 2013). 7.3.2.2 Gelatin Hydrolysis of collagen results into a biodegradable polymer named gelatin. It is widely used in tissue engineering and as an antimicrobial film or membrane because of its less toxicity, and adhesion properties. A recent study shows that fish skin gelatin loaded with different amounts of cinnamon essential oil works against some specific strains of bacteria. The film shows a brilliant inhibitory effect against E. coli, S. aureus, A. niger, Rhizopus oryzae and also shows a better antifungal activity. Concentration of CEO and the rate of bacterial growth inhibition is directly proportional to each other in case of all the bacterial strains, but nearly 60 mm inhibition zone was obtained for R. oryzae with 6% of

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CEO in gelatin film (Wu, Sun, Guo, Ge, & Zhang, 2017). Blend of gelatin and Zataria multiflora essential oil is antibacterially active all bacterial strains. Antimicrobial and antioxidant properties of gelatin makes it a potential material for food packaging (Kavoosi, Rahmatollahi, Dadfar, & Purfard, 2014). Another excellent gelatin film loaded with carvacrol also shows better antimicrobial and antioxidant properties. The material shows antimicrobial properties against all bacterial strains. The material is potential for use in antibacterial packaging (Kavoosi, Dadfar, Mohammadi Purfard, & Mehrabi, 2013). 7.3.2.3 Keratin Keratin is an important component of our hair, skin, and nails. It is also found in the internal glands of the human body. Keratin is biocompatible, easily available, renewable and sustainable. That is why keratin-based materials are mostly used now a days. Antimicrobial functionalization of keratin-based material with chlorination to obtain a positively charged surface that will show strong bactericidal activity. Chlorinated regenerated cellulose/keratin composite can kill E. coli and S. aureus both but shows a better killing rate in case of S. aureus (Dickerson et al., 2013). Another group of researchers found a composite made of metal ions adsorbed in keratin nanoparticles. The prepared material shows enhanced antimicrobial activity due to the positively charged metal ion and depending on the metal ion concentration the killing rate varies according to the nature of bacteria. It is mostly used as some eco-friendly antimicrobial nanomaterials (Shankar & Rhim, 2019). Another study proved that keratin film with a different dose of methylene blue (photosensitized dye) is a biodegradable and biocompatible antimicrobial material. Upon visible light irradiation, the material shows photo bacterial activity with a killing rate of 99.99% in the case of S. aureus (Aluigi et al., 2015).

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7.3.2.4 Soy protein Soy protein is an isolated protein from soybean. Major components of soy proteins are glycinin and β-conglycinin. Glycinin peptide shows better antimicrobial activity (except E. coli) against most of the bacterial strains than β-conglycinin. The material is potentially used for therapeutic uses (Vasconcellos, Woiciechowski, Soccol, Mantovani, & Soccol, 2014). Antimicrobial peptide kills microbes by inserting into and damaging the cell membrane of microbes. Soy protein is one of the antimicrobial peptides. A bilayer formed by soy protein with 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol can mimic the cell membrane of bacteria and thus shows antibacterial activity toward L. monocytogenes and E. coli (Xiang, Lyu, Zhu, Bhunia, & Narsimhan, 2017). A nanofibrous membrane prepared by ultraviolet reduction of soy protein isolate/ polyamide-6 silver nitrate shows 95% air filtration efficiency. The material also shows better antimicrobial activity against E. coli than Bacillus subtilis due to a difference in the cell structure of B. subtilis (Jiang et al., 2018). On the other, it was also evident that the concentration of silver nitrate and the rate of bacterial growth inhibition is inversely proportional. Post-harvest treatment of Persian lime with soy protein was found to be effective against blue mold with a 20% reduction in fungal decay of the lime (Gonza´lez-Estrada, Chalier, RagazzoSa´nchez, Konuk, & Caldero´n-Santoyo, 2017). 7.3.2.5 Fibrin A fibrous and nonglobular protein which is a part and parcel of blood clotting process is called fibrin. The bacterial colony grows in the fistula tract of the fistula patient. To reduce or kill the bacterial colony fibrin glue is a new invention. The bactericidal effect of Plateletleukocyte fibrin and Platelet-rich plasma was found to be much better than Platelet poor

plasma against Gram-negative bacteria. On the other hand, PLF showed enhanced antibacterial activity which is better than commercially available materials (Wu et al., 2013). Another study showed the effect of leukocyte
PRF in periodontal pathogens. The antimicrobial activity of L-PRF against P. intermedia, F. nucleantum, A. actinomycetemcomitans, and especially against P. gingivalis shows that the material is antibacterially active against P. gingivalis only but it is dose dependant on the planktonic solution (Castro et al., 2019). Advanced-PRF is a potential surgical adjunct for the previous infection or left exposed in the microbial rich environment or oral cavity. Advanced-PRF showed antimicrobial activity against C. albicans, Streptococcus mutans, Staphylococcus aureus, and Enterococcus faecalis (Bhamjee, 2017). 7.3.2.6 Lactoferrin Lactoferrin, an iron binding-binding antimicrobial protein found in milk, and potential antimicrobial peptide lactoferricin (LFcin) release from the milk through hydrolysis of pepsin. A dedicated team of researchers investigated the effect of LF and LFcin against Gram-positive and Gram-negative bacteria and fungi. The effect was investigated with a minor fraction of milk protein with a molecular weight of 15 kDa (Mr15). An increase in Mr15 concentration increases the antimicrobial activity of both LF and LFcin. This represents the antimicrobial properties of LF and LFcin with Mr15 in milk (Murata, Wakabayashi, Yamauchi, & Abe, 2013). Newborn babies are very prone to infections and intestinal pathogens. lactoferrin extracted from bovine milk was found to be a potential antibacterial agent against infections of preterm infants. It is also potential for the reduction of newborn morbidities worldwide. Bovine lactoferrin is antifungus and inhibits Gram-positive bacteria also can reduce lateonset-sepsis (Embleton, Berrington, McGuire, Stewart, & Cummings, 2013). Lactoferrin obtained from polymorphonuclear leukocytes is also active against bacteria. The mechanism of

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7.3 Antimicrobial activity of biological macromolecules

action is that lactoferrin is to binds with iron and makes unavailable for bacteria. Lactoferrin also a part and parcel of our immune system. Antibacterial study shows that the inhibition zone in case of Staphylococcus epidermidis is greater than Campylobacter jejuni and Salmonella (Jahani, Shakiba, & Jahani, 2015).

7.3.3 Fatty acids In natural fats and edible oils, fatty acids are commonly present, and they are considered vital compounds as they possess antibacterial and antifungal properties. Carboxylic acids with elongated, unbranched carbon chains are fatty acids, some of which may have double bonds. Most of the naturally available fatty acids consist of an unbranched chain of carbon atom, from 4 to 28. Moreover, these compounds are commonly not present in their isolated form in species but occur as separate esters groups instead (Moss, Smith, & Tavernier, 1995). Fatty acids are well-known for their bactericidal and antifungal characteristics for a very long period (Kabara, Swieczkowski, Conley, & Truant, 1972). They are fascinating as antibacterial mediators for different functions in medical speciality, agricultural science, and foodstuff preservation due to their comprehensive variety of function, inexplicit method of activity and protection, particularly where the use of traditional antibiotics is objectionable or forbidden (Desbois & Smith, 2010). The cell membrane is the prime target for antibacterial action of free fatty acids which is shown in schematic presentation (Fig. 7.7). The effectivity of fatty acids as antibacterial and antifungal agents mostly dependent on the structure, number of carbon atoms, chain length, and saturation properties of these compounds. Gram-positive bacteria are more vulnerable than Gram-negative bacteria to fatty acids. Short-chain fatty acids at higher concentrations can affect the activity of Gramnegative bacterias. On the other hand,

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elongated chain fatty acids are more effective in Gram-negative bacterias at lower concentrations. Sucrose fatty acid esters exhibit antimicrobial activity against Gram-negative bacteria, but to a far lower degree than those seen for Gram-positive bacteria. Fatty acid stereochemistry also has an influence as antimicrobial, with the (natural) cis-forms of unsaturated fatty acids showing better antibacterial activity than the corresponding trans-isomers. The location of unsaturation also influences biological activity. As opposed to esterification, unsaturation is less effective with low-chain fatty acids than with higher-chain fatty acids and increases activity against Gram-positive bacteria (Kabara et al., 1972; Marshall & Bullerman, 1994; McGaw, Ja¨ger, Van, & Staden, 2002; Nieman, 1954). The following section will discuss the antibacterial and antifungal performance of different fatty acid macromolecules against specific microbes. 7.3.3.1 Fatty acid methyl ester Fatty acid methyl ester (FAME) can be produced by transesterification of fats using methanol and different alkaline catalysts such as sodium hydroxide, sodium methoxide, or potassium hydroxide (Vyas, Verma, & Subrahmanyam, 2010). The unique FAME profile of every microorganism can be employed as a microbial source tracing technique to classify abnormal strains of bacteria and to describe new bacterial groups (Duran, Haznedaro˘glu, & Zitomer, 2006). FAME extracts of various halophytic plants have been investigated for antimicrobial activity against different strains of Gram-positive and Gram-negative bacterias, human pathogenic yeast type fungi and mold type fungi by Chandrasekaran et al. (Chandrasekaran, Kannathasan, & Venkatesalu, 2008). With the extracts of all the plants screened, an effective antibacterial and a mild anticandidal activity were reported. The S. brachiata extract demonstrated the strongest antimicrobial activity against B. subtilis and all other microorganisms examined, as it showed the lowest minimum

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FIGURE 7.7

Schematic representation of possible cell targets and mechanisms of antibacterial action of free fatty acids. Source: Reprinted from Desbois, A. P. & Smith, V. J. (2010). Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Applied Microbiology and Biotechnology, 85(6), 1629
1642. With permission from Springer Nature.

inhibitory concentration (MIC) against maximum strains. The investigation of Davoodbasha, Edachery, Nooruddin, Lee, & Kim (2018) examined the effectiveness of FAME isolated from microalgae (S. intermedius) as an antimicrobic agent against Gram-positive, Gram-negative bacteria and fungi. The FAME profile indicated that a higher proportion of active pharmacological compounds, such as palmitic acid methyl ester (C16:0), can be found to account for bioactivity. The FAME exhibited better inhibitory impact against Gram-negative bacteria than Gram-positive bacteria. Intermedius is a good candidate for the

development of pharmacologically active molecules among organisms which are not much active against microorganisms such as E. coli and S. aureus. Similar research by using freshwater microalgae was conducted by Suresh et al. and observed potent to moderate antimicrobial activity against pathogenic microorganisms (Suresh et al., 2014). A novel study of FAME extracts from fruits of white Oak (Q. leucotrichophora) and their uses as antimicrobial has been presented by Sati, Sati, Sati, & Sati (2017). Analysis of FAME fruit extract by GC
Ms showed a higher saturated fatty acid level than unsaturated fatty acids. They had greater

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7.3 Antimicrobial activity of biological macromolecules

antibacterial activity against Gram-positive bacteria than Gram-negative bacteria. These variations in the susceptibility to fatty acids between Grampositive and Gram-negative bacteria may be due to the Gram-negative bacterial outer membrane’s impenetrability since the Gram-negative bacterial outer membrane is an efficient barrier to hydrophobic elements (Agoramoorthy, Chandrasekaran, Venkatesalu, & Hsu, 2007). Their study demonstrates the capability of extracts as an antibacterial for therapeutic use in the cosmetics and medicinal industries. 7.3.3.2 Oleic acid Oleic acid can be found in various naturally occurring fats and oils present in animals and vegetables. Antibacterial activity of this is detected, especially in preventing the growth of respective Gram-positive species of bacteria. Dilika, Bremner, & Meyer (2000) isolated oleic acid from leaves of H. pedunculatum by using dichloromethane and studied its antibacterial efficiency against several Gram-positive and Gram-negative bacteria. Extracted oleic acid inhibited the development of Gram-positive bacteria stains but was not effective against Gram-negative bacteria. A lower MIC makes this a very efficient bioactive material in wound dressing applications in conventional male circumcision as an antiinflammatory and antibacterial mediator. Oleic acid can be incorporated with other compounds to improve the performance of it as an antimicrobial. In this regard, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction was used to synthesize oleic acid-grafted chitosan oligosaccharide (CSO-OA) (Huang et al., 2009). The use of oleic acid and oligosaccharide improves the ability of in vivo uses. The antibacterial activity of CSO-OA nanoparticles was reported against different bacterial strains. It was found that the antibacterial activity increased as the concentration and DS of CSOOA increased. It makes this nanoparticle to be a great candidate as drug and gene carriers. A

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paper was documented describing a feasible process for synthesizing silver nanoparticles (Ag NPs) colloidal solutions with oleic acid as a surfactant to stabilize the solution (Le et al., 2010). The use of oleic acid as a stabilizer showed better antibacterial characteristics compared to other fatty acid stabilizers. It showed inhibition against the growth of both Grampositive and Gram-negative bacteria. Even at low silver loads, their stable antimicrobial solution in environment-friendly industrial applications. Oleic acid was used as stabilizer in another study to synthesize magnetite and cobalt-doped magnetite nanoparticles and their in vitro repressive activities were measured against pathogenic bacteria and pathogenic fungi and molds (Rahdar, Beyzaei, Saadat, Yu, & Trant, 2020). The magnetite nanoparticles were marginally successful against all pathogens assessed, but the action of the cobaltdoped nanoparticles was slightly lower, likely due to the disruption of the bacterial membrane by Fenton reaction. 7.3.3.3 Linoleic acid Linoleic acid is a fatty acid containing two classes of cis alkenes, and it usually appears as a triglyceride rather than a free fatty acid in nature. Longer chain unsaturated fatty acids, such as linoleic acid, are the main components in antimicrobic food essences and certain antibacterial herbs as they exhibit antibacterial action (Zheng et al., 2005). P. aeruginosa raw extract of biotransformed linoleic acid was tested for antibacterial action against foodborne morbific Gram-positive and Gramnegative bacteria (Shin et al., 2005). A lower range of MIC confirms their antimicrobial activity against most of the strains tested. These findings indicated the potential use of bio-converted natural vegetable linoleic acid for research of food safety management. A similar study of linoleic acid’s inhibitory action against microorganisms that causes food poisoning exhibited similar results (Lee, Kim, &

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Shin, 2002). The acid efficiently hindered the growth of Gram-positive bacteria but not much effective against Gram-negative strains, as Gram-negative bacteria’s outer membrane has lipopolysaccharides, which constantly resists the penetration of acid into the cell. Although the incorporation of linoleic acid with emulsifiers (glycerol laurate and glycerol myristate) significantly improves the inhibition by lowering MIC and introducing a remarkable synergistic impact. Antibacterial activities were demonstrated in the ethanol fraction of fermented soybean produced using R. oligosporus and free fatty acids, monoglycerides, and fatty acid ethyl esters were included in the ethanol fraction (Kusumah et al., 2020). Among these elements, linoleic and α-linolenic acid exhibited the highest antifungal and antibacterial activity against bacterial strain by showing lower MIC and can be used as a food preservative. Linoleic acid can also be employed as a stabilizing agent with metallic (copper) nanoparticles to control the formation of nanoparticles and protect them from aggregation (Das, Gang, Nath, & Bhattacharjee, 2010). The antibacterial assessment of copper nanoparticles indicates that these are efficient growth inhibitors against various bacterial strains and are highly promising as cost-effective antimicrobials.

human host (Beavers et al., 2019). Via a lipid peroxidation process, this acid can eradicate S. aureus, through which acid is oxidized to reactive electrophiles that alter the macromolecules, causing toxicity. For the production of potential therapeutics to cure bacterial contagions, this lipid peroxidation process may be modified. In a dose-dependent manner, this fatty acid can stimulate neutral sphingomyelinase action of leukocytes and this activity allows acid to boost ceramide production in cells, which is believed to have tumoricidal action (Robinson, Hii, Poulos, & Ferrante, 1997; Shakor et al., 2014).

7.3.3.4 Arachidonic acid Arachidonic acid belongs to the group of polyunsaturated fatty acid which contains four cis-double bonds and found in the phospholipids of the cell membranes of the human and animal body. The antimicrobial ability of arachidonic acid against S. pneumoniae was investigated in vitro and cell culture assays (Eijkelkamp et al., 2018). The analysis showed that arachidonic acid induces antimicrobial properties toward the microorganism by modifying the pneumococcal layer at the cellular scale. Arachidonic acid was reported to be work against the toxicity of S. aureus strains, which can attack every single place of the

Chitosan is one of the most plentiful natural polymers with favorable biocompatibility properties and nontoxicity making it very desirable in medical applications as an antimicrobial. Another naturally abundant polymer, alginate, possesses low cell adhesiveness due to its inadequate protein adsorption for the hydrophilic character (Rowley, Madlambayan, & Mooney, 1999). Blends of chitosan and alginate can overcome these limitations. Alginate fiber was developed by using a conventional wet spinning method and was tested for its antibacterial activity (Dumont et al., 2018). But pure alginate fibers do not have any substantial antibacterial activity. When the fibers were

7.4 Antimicrobial activity of macromolecule composites Naturally found macromolecules, which have antimicrobial activity, can be fabricated with another material to form a composite. This incorporation can surprisingly enhance the biocompatibility and antibacterial action. This part will discuss the antimicrobial activity of several such macromolecule composites.

7.4.1 Chitosan-alginate

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FIGURE 7.8 Reaction schematic illustration of the composite OAlgCMCS hydrogel (Gel) integrated with gelatin microspheres (GMs) containing tetracycline hydrochloride (TH) via the Schiff base reaction. Source: Reprinted from Chen, H., Xing, X., Tan, H., Jia, Y., Zhou, T., Chen, Y., et al. (2017). Covalently antibacterial alginate-chitosan hydrogel dressing integrated gelatin microspheres containing tetracycline hydrochloride for wound healing. Materials Science and Engineering: C, 70 287295. With permission from Elsevier.

coated with chitosan, the antibacterial properties were significantly enhanced for both Gram-positive and Gram-negative strains. Chitosan and alginate hydrogel dressing composites integrated with gelatin microspheres were produced, and the schematic reaction was illustrated (Fig. 7.8) and both Grampositive and Gram-negative bacteria were screened for antibacterial activity (Chen et al., 2017). A good reticence of bacterial protrusion against E. coli and S. aureus has demonstrated that the gel has a promising potential to control bacterial contagions. With a continuous release of silver sulfadiazine in a regulated manner to deter bacterial infections, a sponge like chitosan-alginate polyelectrolyte hydrogel network was formed to employ it as a wound dressing (Yu et al., 2005). Tests against many bacterial strains confirmed that this hydrogel could successfully control bacterial replication to shield the wound from bacterial attack. In medicinal applications, alginate-chitosan biopolymer film incorporated with Aloe vera gel and silver nanoparticles can be greatly beneficial (Go´mez Chabala, Cuartas, & Lo´pez, 2017). The integration of silver

nanoparticles’ antimicrobial properties with the curing and softening properties of Aloe vera gel enables this film to improve its antimicrobial properties against multiple strains of bacteria and possible wound dressing for trivial wounds.

7.4.2 Gelatin-chitosan Gelatin-chitosan scaffolds have been extensively used owing to their cell-associated signaling activities, such as propagation, movement, and survival. Gelatin is an animal protein and derivative of collagen that is one of the critical components of the human extracellular matrix, commonly used to produce hydrogels for wound dressings (Liu et al., 2018; Sell et al., 2010). Chitosan-gelatin composite biofilm can inhibit bacterial growth of E. coli and L. monocytogenes. In contrast, neat chitosan and neat gelatin film did not produce substantial inhibition for any of these pathogens (Pereda et al., 2011). Park, Clark, Lichtensteiger, Jamison, & Johnson (2009) analyzed vital fibroblast growth factor loading chitosan scaffold found in gelatin microspheres

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FIGURE 7.9 SEM image of (A) intact and (B) dead E. coli. Source: Reprinted from Zhao, J., Wei, F., Xu, W., & Han, X. (2020). Enhanced antibacterial performance of gelatin/ chitosan film containing capsaicin loaded MOFs for food packaging, Applied Surface Science, 510 145418. with permission from Elsevier.

in lasting ulcers of elderly mice and they found this hydrogel was an important substance for the distribution of growth factor and quicker curing. Gelatin-chitosan matrix was modified by incorporating an antibacterial agent Capsaicin into iron-doped hollow metal-organic structures (Zhao, Wei, Xu, & Han, 2020). The SEM images of the intact and dead E. coli (Fig. 7.9) indicate that the film efficiently resisted the growth of E. coli, which opened a useful method to obtain propitious antibacterial biofilms for food packaging. The addition of sodium nitrite and garlic essential oil into the edible film of gelatinchitosan can significantly improve the antibacterial properties, which can be used as food coating and to extend the shelf life of food without cancer-causing hazards (Handayasari, Suyatma, & Nurjanah, 2019). Edible gelatin extracted from different kinds of fish and then incorporated with chitosan to form biofilms showed effective antimicrobial activity (Matiacevich, Celis Cofre´, Schebor, & Enrione, 2013; Yao, Ding, Shao, Peng, & Huang, 2017). These films can be used as bioactive edible food layer for food packaging.

7.4.3 Keratin-chitosan Chitosan, derived from chitin, is the most abundant polymer after cellulose. It is made of two monomeric units, glucosamine and

N-acetylglucosamine, linked through β-(1 2 4) glycosidic linkages. Chitosan has three nucleophilic functional groups in the structure, a NH2 group (C-2), a secondary OH group (C-3), and a primary OH group (C-6). Chitosan demonstrated to have antimicrobial activities against both Gram-positive and Gram-negative bacteria (Sarwar, Katas, & Zin, 2014). Chitosan exhibits antimicrobial property only in acidic medium when it has a net positive charge (No, Park, Lee, & Meyers, 2002). As chitosan has pKa of 6.3, chitosan -NH2 groups of chitosan become protonated when stronger acid is added to the solution. The positively charged -NH31 plays a vital role in destroying microbial cells. The activity of chitosan’s antimicrobial action is three folds. First, it brings change in cell permeability of the microbes, which incites internal osmotic imbalance and impedes cell growth (Tsai, Zhang, & Shieh, 2004). Second, it starts hydrolysis of peptidoglycans in the microbes’ wall which causes seeping of electrolytes from the cell and destroy it (Chen, Liau, & Tsai, 1998). Third, the chelation of protonated chitosan with metals stops the binding of elements with nutrients that inhibits the microbial cell growth (Cuero, Osuji, & Washington, 1991). However, to make chitosan effective antimicrobial agents even in nonacidic medium, derivatization of chitosan is carried out. It helps to improve the solubility of chitosan in aqueous medium as well as enhance antimicrobial properties. Keratin is the main constituent material of hair, horns, wool, feathers, and nails. It is a type of fibrous protein that is rich in cysteine forming the intermediate filaments of cytoskeleton and epidermal appendageal in hair, horns and wool (McLellan, Thornhill, Shelton, & Kumar, 2019). It mainly consists of cystine and serine which contains hydroxyl amino acid. The polypeptide chains of different amino acids forms the backbone of the structure of keratin. Inter and intramolecular bonding, originating from disulfide, hydrogen and ionic

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7.4 Antimicrobial activity of macromolecule composites

bonds, imparts strength and stability of the structure. Keratin shows lower solubility in water and other solvents due to the presence of cysteine disulfide bonds. But the existence of different ionic, hydrogen and hydrophobic bonds effected the characteristics of keratin in a greater manner (Tonin, Aluigi, Varesano, & Vineis, 2010). As a result, keratin does not show any antimicrobial activity either. For this reason, keratin is added with another polymer or nanoparticles with innate antimicrobial activity. A keratin-chitosan hydrogel containing nano zinc oxide showed antimicrobial action against Gram-positive S. aureus as well as Gram-negative E. coli in colony forming unit (CFU) methods (Zhai, Xu, Zhou, & Jing, 2018). In another work, a biocomposite based on cellulose and keratin was developed and investigated for antimicrobial activity (Tran, Prosenc, Franko, & Benzi, 2016). Ag nanoparticles was added to one sample of the composite to make a comparative study. First, the culture medium was developed in a sterile condition for overnight at 37 C and 150 rpm. The composites samples having size 3 3 20 mm were thermally sterilized at 121 C and 15 psi for 20 min. Then, the samples were kept in a diluted culture for the whole night. After that, they were incubated for 24 h at 37 C and 200 rpm. Bacteria were plated in serial dilutions onto sterile nutrient agar plates at time 0 and after 24 h. They were incubated at 37 C for the whole night. The composites containing chitosan, keratin and Ag nanoparticles of 3.5 mmol showed the maximum antimicrobial activity against all selected bacteria up to 99.9% growth reduction. Unsurprisingly, when Ag nanoparticles concentration was low at 0.48 mmol, the composite still showed about a 68% growth reduction for most of bacteria. But chitosan-keratin biocomposite showed no considerable antimicrobial activity. It is clear from this work that, though chitosan exhibits antimicrobial activity, it requires the presence of an antimicrobial agent like Ag nano particles to eliminate bacteria to a significant number.

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7.4.4 Collagen-alginate Collagen, one of the most abundant animal polymers, is a fibrillar structural protein in the extracellular matrices of connective tissues in mammalians. It is prevalent in skins, tendons, bones and cartilages. Collagen contains glycine, proline and hydroxyproline, which is the reason of its valuable properties like biocompatibility and nontoxicity. More importantly, this protein interacts with cells and instigates cell adhesion, cell anchorage, migration and proliferation (Moura et al., 2014; Zhang et al., 2018). Alginate is an anionic copolysaccharide originated from brown algae such as Laminaria hyperborea, Laminaria digitata, Laminaria japonica. Its structure is made of β-D-mannuronic and α-L-guluronic acid monomers linked by 1
4 linkages. However, collagen and alginate do not exhibit antimicrobial activity. As a result, it is a common practice to functionalize collagen
alginate-based materials with antimicrobial agents like silver, iodine, zinc oxide, antimicrobial peptides (AMPs) (Lin et al., 2019; Zhang et al., 2018). Though the mechanism of antimicrobial activity of AMPs is not established but it is predicted that they attack the bacterial cell wall and form ion channels across the cell membrane. The ion channels cause leakage of the contents of the cell and disrupts the structural integrity of the cell membrane. This eventually kills the bacteria (Roy, Lomakin, Gagnon, & Steitz, 2015). A collagenalginate wound dressing material was prepared by combining with HA and AMPs (Lin et al., 2019). The antimicrobial activity was conducted with inhibition zone method. Gramnegative bacteria E. coli and Gram-positive bacteria S. aureus and MRSA were taken as experimental microbes as they are the most widespread bacteria that causes skin wounds (Qu et al., 2018). The diameters of the inhibition zones for the collagen, alginate-HA containing AMPs wound dressings against E. coli, S. aureus, and MRSA were 19.4 6 0.8, 30.3 6 1.1,

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and 22.1 6 0.7 mm respectively. The results showed dressings containing AMPSs were lot better at inhibiting bacterial growth than dressing with it. In another work, an Ag nanoparticle doped collagen
alginate biocomposite, showed strong antimicrobial activity (Zhang et al., 2018). The antibacterial action of the composite was investigated against S. aureus and E. coli using the agar diffusion method. The results showed that chitosan and alginate composite without Ag nanoparticles exhibited nonexistent antibacterial activity. But when the chitosan and alginate composite was doped with Ag nanoparticles, it showed impressive antimicrobial action. Moreover, this action increase with increasing Ag nanoparticles concentrations. The composite with Ag nanoparticles concentration to 50 micromolar exhibited inhibition zones of S. aureus and E. coli of 1.9 and 2.7 mm respectively. As the concentration of Ag nanoparticles was increased to 400 micromolar, the inhibition zones of S. and E. coli were increased to 7.0 and 7.7 mm respectively. This result implied that Ag nanoparticle doped collagen
alginate biocomposite had effective antibacterial activity against both Gramnegative and Gram-positive bacteria. Ag nanoparticles has the ability to kill bacteria by producing reactive oxygen species. They can also induce the phosphorylation of the tyrosine and disrupt the signaling pathway inside the bacteria (Gopinath & Velusamy, 2013).

7.4.5 Chitosan-cellulose Cellulose, the major component of plant cell wall, is the most abundant biopolymer on earth. Cellulose is a linear polymer having anhydroglucose molecules connected by a β-1,4-glucosidic bond between C1 carbon of one molecule and C4 carbon of the another. The hydroxyl groups in the cellulose structure can create hydrogen bonds between two

adjacent chains (interchain) and between two glucan units within the same chain (intrachain) which forms the fibrillar packing (Nishiyama, Langan, & Chanzy, 2002). Though cellulose does not show any antibacterial activity, CNC have a special type of morphology like antimicrobial agents like Ag and Au nanoparticles. They have a rigid, narrow and rod like structure. This irregular morphology can induce damage to the microbe’s cell membrane and cause osmotic imbalance inside the cell leading to cell apoptosis (Ferrer et al., 2017). But antimicrobial activities can be introduced to cellulose by modifying its surface, loading metal/ metal oxides nanoparticles and blending with materials having antimicrobial properties. Chitosan was investigated for antimicrobial activity against several microbes such as Gram-positive bacteria (such as L. monocytogenes, B. megaterium, B. cereus, S. aureus, L. plantarum, L. brevis, L. bulgaris, etc.) and Gramnegative bacteria like E. coli, P. fluorescens, S. typhimurium (Goy, B, & Assis, 2009). Chitosan and cellulose are structurally comparable. The big difference is the presence of acetyl amino group in chitosan on C-2 carbon, on the other hand, cellulose contains hydroxyl group on C-2 carbon. As a result, it is possible to mix chitosan (CS) and cellulose (BC) each other to produce composite materials like, semi-IPN hydrogels, that exhibit antimicrobial properties (Wahid et al., 2019). In the work, the inhibition zones for S. aureus and E. coli decreased cellulose was increased in the ratio to chitosan in hydrogels. The reduction was from 15.4 to 11.3 mm and 15.2 to 11.0 mm respectively which was represented in Table 7.2. It was clear that cellulose exhibited no antimicrobial activity but when combined in a composite with chitosan, the composite could be useful in antimicrobial applications. The colony forming unit (CFU) method strengthened this notion further by showing an 88% reduction of bacterial population when cellulose content was reduced to 20 percent, while it was about 98% reduction from pure chitosan.

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7.5 Nanotechnology based antimicrobial macromolecule

TABLE 7.2 The antibacterial inhibitions zones of BCCS hydrogels. Reprinted from (Wahid et al., 2019) with permission from Elsevier. Samples

Inhibition zones (mm) S. aureus

E. coli

BC-0

15.4 6 0.5

15.2 6 0.5

BC-20

14.6 6 0.6

14.3 6 0.4

BC-40

13.7 6 0.4

12.7 6 0.5

BC-50

13.3 6 0.6

12.5 6 0.4

BC-60

11.3 6 0.5

11.0 6 0.5

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A lactoferrin and oleic acid complex was prepared and investigated for antimicrobial activity (Abd El-Baky, 2018). The results showed antibacterial as well as antifungal activities against 10 bacteria, 1 yeast, and 2 fungi. The lactoferrin and oleic acid complexes exhibited inhibition zone of maximum 42 mm against Gram-positive bacteria S. aureus at concentration 0.5 mg/mL. But the complex was not effective when applied at lower concentration of 0.0625 mg/mL. The test with Gram-negative bacteria showed poorer results as the lactoferrin and oleic acid complex failed to show considerable antimicrobial activity against them.

7.4.6 Lactoferrin-Oleic Acid Lactoferrin is an iron binding glycoprotein belonging to the transferrin group. It consists of a single-chain polypeptide chain of about 690 amino acids with molecular mass of about 78 kDa. Lactoferrin is found in human and bovine milk but also distributed in saliva and secretory fluids of mucous membranes of the upper respiratory, gastrointestinal, and urogenital tracts. Lactoferrin has some other important applications such as antibacterial, antifungal, antiparasitic, antiviral, antiallergic and catalytic functions. It exhibits antimicrobial activity due to its iron binding properties, which suppresses bacterial iron acquisition and uptake a proteins that are vital for microbial growth (Plaut & Geme, 2013). Oleic acid is an unsaturated monocarboxylic acid. It has a long chain and exhibits potent antimicrobial activity against numerous Grampositive bacteria Staphylococcus aureus (Farrington, Brenwald, Haines, & Walpole, 1992). This free fatty acid is a type of lipids typically found in human skin, breast milk, and bloodstream. It is considered an essential part of the natural immune system. However, their mechanism to fight against microbes is not clear. It is assumed that it can disrupt bacterial membranes and increase membrane permeability. As a result, the bacterium loses it vital components and is killed (Chamberlain et al., 1991).

7.5 Nanotechnology based antimicrobial macromolecule 7.5.1 Chitosan based nanocomposite It has been reported that, chitosan possesses antimicrobial activity due to the presence of certain functional groups (Mohandas, Deepthi, Biswas, & Jayakumar, 2018; Wang et al., 2006). For instance, the presence of NH31 functional group causes chitosan to interact with the negatively charged cell membrane (Khorasani, Joorabloo, Adeli, MansooriMoghadam, & Moghaddam, 2019). However, the antibacterial activity of chitosan not only depends on functional group but also is influenced by various factors, such as the concentration, pH, species of bacteria, solvent and molecular mass (Tripathi, Mehrotra, & Dutta, 2011). It has been reported that, low molecular weight chitosan can penetrate into bacterial cells whereas high molecular weight chitosan enclose bacteria cells and block passage of nutrients (Vrana, Liu, McGuinness, & Cahill, 2008). In addition, chitosan demonstrates better antibacterial activity in low pH environment and this property makes chitosan ideal for wound dressing since the wound environment is acidic. However, chitosan’s antibacterial activity can be further improved with the incorporation of metallic nanoparticles (Mohandas et al., 2018). Chitosan possesses strong

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affinity toward metal ions due to the presence of numerous amine and hydroxyl groups (Wang et al., 2006). So, inclusion of metallic nanoparticles (such as gold, silver, copper and ZnO) into chitosan can significantly improve the antimicrobial efficacy of chitosan. For example, ZnO nanoparticles (ZnO NP) have excellent antifungal and antibacterial activity. Thus, to exploit the antimicrobial property, ZnO NP was reported to be incorporated into gelatin for wound dressing (Mohandas et al., 2018). The antimicrobial properties of metallic nanoparticles also depends on several factors, such as, surface modification, particle morphology, size, concentration and larger number of polar facets, etc. (Shankar, Jaiswal, & Rhim, 2016) (Fig. 7.10). Rahman, Mujeeb, Muraleedharan, and Thomas (2018) prepared chitosan/nano zinc oxide composite film. They examine the antimicrobial activity of prepared nanocomposite for both Gram-positive (S. aureus) and Gramnegative (E. coli) bacteria and observed lower antibacterial activity of composites toward S. aureus bacteria in comparison to E. coli.

They explained that for Gram-negative bacteria the outermost membrane consists of thin peptidoglycan whereas for Gram-positive bacteria the outer membrane consists of thick peptidoglycan (Rahman et al., 2018). Moreover, it has been reported that, for Gram-negative pathogen, there is periplasmic region presents in between outer and inner membrane which can be easily be solubilized and leading to enter the inhibitory substances. On the other hand, the Gram-positive bacteria lacks periplasmic region and have lipoteichoic acids in cell wall which also restricts the entry of inhibitory substances (Malini, Thirumavalavan, Yang, Lee, & Annadurai, 2015). The detailed mechanism of the antimicrobial effect of ZnO is still under dispute and can be broadly divided into two categories: (1) dissolution (2) ROSInduction of reactive oxygen species. In dissolution method, release of Zn ions from ZnO NPs causes formation of active free radicals on the surface of the ZnO which plays an important role to destroy microbial cells (Wu et al., 2010).

FIGURE 7.10

Mechanism of antibacterial activity of gold nanoparticles released from chitosan scaffolds. Source: Reprinted from Mohandas, A., Deepthi, S., Biswas, R., & Jayakumar, R. (2018). Chitosan based metallic nanocomposite scaffolds as antimicrobial wound dressings. Bioactive Materials, 3(3), 267
277.

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7.5 Nanotechnology based antimicrobial macromolecule

The second methodology describe that the ZnO NPs liberate reactive oxygen species (ROS) (Malini et al., 2015). The liberated ROS and Zn21 ions attack the negatively charged cell walls, causes leakage and finally death of bacteria. Moreover, inclusion of ZnO in chitosan also increase the positive charge on the amino group of chitosan and causes easier interaction with negatively charged components on composite films and cell walls (Rahman et al., 2018). Incorporation of Ag nanoparticles (AgNP) into chitosan also enhance it’s antimicrobial activities. Tripathi et al. (2011) produced chitosan
silver oxide nanocomposite film to use for antimicrobial food packaging. They concluded that the prepared composite was effective for inactivating bacteria, which might be due to synergistic effect of both the Ag2O NPs and chitosan in the composite. The mechanism of bactericidal action of Ag can be explain in terms of the AgNP’s capacity to enter easily into the bacterial cell and form a less dense region in the center of the bacteria. The silver ions are proved to be reactive with thiol or sulphydryl groups. Therefore once AgNP enters a cell, it causes cell death by interacting with thiol containing important enzymes (Mamonova et al., 2015; Prabhu & Poulose, 2012). The second proposed mechanism is that, Ag tends to react with the weak acids in the genomic material, such as phosphate and thus causes disruption of DNA/RNA and therefore prevents the translation of proteins.

7.5.2 Alginate-based nanocomposite Alginate is a biocompatible, nontoxic biopolymer which possesses good mechanical properties and biodegradability (Mohandas, Sudheesh Kumar, Raja, Lakshmanan, & Jayakumar, 2015; Zahran, Ahmed, & El-Rafie, 2014). Till to date, alginate has been widely used in wound healing treatments in different forms of such as films, hydrogels, nanofibers etc. Alginate wound dressings are able to absorb excess wound fluid and

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thus reduce microbial infections at the wound site (Aderibigbe & Buyana, 2018). For instance, the alginate fibers absorb water and swell up, thus any bacteria presents in the wound exudates get entrapped in the wound dressing (Seo et al., 2012) (Fig. 7.11). As a low-priced, abundantly available, environmentally friendly and biocompatible biopolymer, alginate has also been used in the food and biotechnology industries as a nontoxic food additive, thickening and gelling agent, antimicrobial packaging material to maintain food quality and also to extend shelf life of packaged foods (Shankar, Wang, & Rhim, 2016). To enhance the antimicrobial properties of alginate, metallic nanoparticles possesses antimicrobial properties are dispersed as nanofillers into alginate. Mohandas et al. (2015) produced alginate hydrogel/zinc oxide nanoparticles (ZnO NP) composite bandage. They explained the antimicrobial activity of ZnO was due to the production of ROS by the released zinc ions from the composite bandages which caused damage of microbial cell wall and finally death of the organism. Ag or Ag ions have also known to exhibit powerful antimicrobial activity and are commonly used for wound healing since they are effective in killing a wide range of bacteria (Zahran, Ahmed, & El-Rafie, 2014). Shankar et al. (2016) prepared silver/alginate composite films and evaluated the effect of different forms of silver particles. They explained that the antibacterial activity of AgNPs depends on the size, shape and the electrostatic charge of the nanoparticles. The interaction between the nanoparticles and bacterial cell wall is usually determined by the electrostatic charge of AgNPs. For example, the electrostatic interaction between the positively charged AgNPs and negatively charged bacterial cell wall is very strong.

7.5.3 Cellulose based nanocomposite BC is a natural biopolymer synthesized by various strains of Acetobacter species and

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FIGURE 7.11 Transmission electron microscope image of the AgNPs synthesized in 1.0 wt.% sodium alginate solution. Source: Reprinted from Seo, S. Y., Lee, G. H., Lee, S. G., Jung, S. Y., Lim, J. O., & Choi, J. H. (2012). Alginate-based composite sponge containing silver nanoparticles synthesized in situ. Carbohydrate polymers, 90(1), 109
115. With permission from Elsevier.

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7.5 Nanotechnology based antimicrobial macromolecule

strains of pseudomonas, Achrobacter, Alcaligene, Aerobacter, and Azotobacter. High water content (98%
99%), high wet strength, good sorption of liquids, high chemical purity, etc. are important properties of BC. All these special properties have made BC suitable for biomedical applications. As biomaterials, one of the most important application of BC is in the therapy of burns, wounds and ulcers. However, BC itself possesses no antimicrobial activity. In this regard, antimicrobial BC membranes are developed by incorporating metallic nanoparticles, especially silver nanoparticles. silver nanoparticles (AgNPs), in particular, have been studied because of its strong antimicrobial actions toward different fungi, viruses, bacteria, etc. (Wu et al., 2014). Silver exhibits strong cytotoxicity toward a wide range of bacteria and causes a bacteriostatic (growth inhibition) or a bactericidal (antibacterial) effects. Barud et al. produced antimicrobial composite membranes of cellulose-silver nanoparticles. They concluded that the prepared composite membranes demonstrated a strong antimicrobial activity against specific Grampositive (Staphylococcus aureus) and Gramnegative bacteria (P. aeruginosa and E. coli). These are the bacteria commonly found in contaminated wounds. Wu et al. (2014) prepared BC/ silver nanoparticles composites for wound dressing. They examine the antibacterial activities against Gram-negative (E. coli and P. aeruginosa), Gram-positive (S. aureus).

7.5.4 Gelatin based nanocomposite Gelatin is a polypeptide which can be obtained from the controlled hydrolysis of collagen (Bakravi, Ahamadian, Hashemi, & Namazi, 2018). Gelatin is a biomaterial highly used as a wound dressing since it can absorb wound exudates, provides moist environment to a wound and accelerates wound healing. However, gelatin itself possesses no antimicrobial activity to prevent wound infection (Xu &

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Zhou, 2008). So, metal nanoparticles are incorporated in gelatin to impart antimicrobial activity. For instance, Galdopo´rpora et al. (2019) synthesized AgNp/gelatin nanocomposite with improved mechanical properties and observed that the nanocomposite with controlled silver delivery result in an efficient wound healing process. Besides wound healing, gelatin is also vastly used in antimicrobial food packaging since it has good film forming ability, high availability, low cost, biodegradable in nature and a good candidate for carrying the antimicrobials and antioxidants (Kanmani & Rhim, 2014a). Inorganic nano-size metallic particles, such as gold, silver, copper, zinc etc. are widely used with gelatin in food packing due to their excellent antimicrobial effects against food-borne pathogens. Among the metal nanoparticles, inclusion of AgNPs into gelatin for food packaging have attracted special attention due to their extraordinary antimicrobial effect against food-borne microorganism. For instance, Kanmani & Rhim (2014b) synthesized AgNPs- gelatin nanocomposite films and reported the antimicrobial activity of produced nanocomposite films against the food-borne microorganism; such as E. coli, L. monocytogenes, S. typhimurium, S. aureus ATCC, and B. cereus. As previously described, they also found that the Gramnegative bacteria were more susceptible to AgNPs than Gram-positive bacteria and attributed this susceptibility to thin peptidoglycan layer present in the cell wall of Gram-negative bacteria in comparison to thick peptidoglycan for Gram-positive bacteria. Moreover, negatively charged outer membrane of Gramnegative bacteria also enables the diffusion of AgNPs inside the cell. Amongst metallic nanofillers, ZnO nanoparticles (ZnO NP) had found to have strong antimicrobial activity against food-borne microorganism (Arfat, Benjakul, Prodpran, Sumpavapol, & Songtipya, 2014; Espitia et al., 2013). Arfat et al. (2014) prepared antimicrobial gelatin -ZnO

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nanocomposite films and suggested that this film could be used in food packaging to prevent growth of microorganism in foods. Shankar, Teng, Li, and Rhim (2015) prepared ZnO/ gelatin NPs nanocomposite films and reported that the ZnO/ gelatin nanocomposite films were more active against Gram-positive bacteria in comparison to Gram-negative bacteria. As previously described antibacterial activity of ZnONPs depend on the cell wall structure of bacteria. However, it has also been suggested that, the antimicrobial activity of ZnONPs is determined through its interaction with specific cell compounds; such as surface proteins, teichoic acids and lipoteichoic acids, which may be found in Gram-positive instead of Gram-negative bacteria (Tayel et al., 2011).

7.5.5 Collagen based nanocomposite The development of collagen based nanocomposite has been increased due to its excellent antimicrobial activity and easy preparation method. Different types of nanomaterials are embedded with collagen protein to synthesize novel antimicrobial biomaterials. Vladkova et al. (2020) prepared a collagen/(ZnTiO3/SiO2) composite using ZnTiO3 embedded in a SiO2 matrix and evaluated its antimicrobial activity against bacteria and fungus. The result demonstrated that the collagen/ (ZnTiO3/SiO2) composite at ratio of 2:1 showed outstanding antimicrobial activity against Gramnegative bacteria (S. holeresius), Gram-positive bacteria (B. cereus) and fungus (C. lucitania) presented as a sterile zone of 17.9 6 1.9, 24.3 6 0.6 and 19.9 6 2.1 mm respectively. The outstanding antibacterial and antifungal performance indicate that collagen/(ZnTiO3/SiO2) composite is a promising antimicrobial biomaterial. In another studies, different collagen based composites such as collagen/(Ag/RGO), collagen/(Ag/RGO/SiO2) (Vladkova et al., 2017) and collagen/(RGO/ZnO/ TiO2/SiO2) (Staneva, Albu-Kaya, Martinov, Ivanova, & Vladkova, 2020) were developed to use as an antimicrobial biomaterial.

7.5.6 Keratin-based nanoparticle The high content of cysteine residue has increased the suitability of keratin protein for the stabilization of metal nanoparticle. In a research, keratin was used as a biocompatible macromolecular capping agent for development of the spherical silver nanoparticles and evaluated its antimicrobial activity (Irwin et al., 2010). The result confirmed that 10
20 μL keratin-capped Ag nanoparticles (0.3
3 μM) totally stopped the development of an equivalent volume (103
104 CFU/mL) of one Gram-positive (Staphylococcus aureus) and two-Gram-negative bacteria (E. coli O157:H7 and S. typhimurium). Furthermore, the presence of bacteria was demonstrated after one week at 37 C and no further growth was observed. The excellent antibacterial property indicates that keratincapped silver nanoparticle is a prominent biomaterial.

7.5.7 Oleic acid based nanoparticle Oleic acid is used as a surfactant for preparation of the colloidal solutions of functional nanoparticle. Le et al. (2010) synthesized an oleic acid stabilized silver nanoparticle and analyzed its antibacterial activity. The result demonstrated that the oleic acid stabilized Ag nanoparticle is highly active against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. Interestingly, the nanoparticle effectively inhibited the growth and multiplication of the bacteria at very low concentration of silver only about 2.0 μg/mL. The low silver loading has made it a highly cost-effective antimicrobial solution for medical, microbiological and industrial applications.

7.6 Applications 7.6.1 Food packaging Food spoilage and quality deterioration caused by food-borne pathogenic microorganisms is a

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7.6 Applications

serious problem in food industry and preserving food and maintaining its quality has become a concern nowadays. Food packaging can play a vital role to keep food fresh, safe and extend its shelf life. However, commonly used nonbiodegradable plastic-based packaging materials are associated with health hazards and environmental pollution. Biological macromolecules based packaging materials can solve these problems as they are biodegradable, have sufficient mechanical properties like flexibility, stiffness and strength, can protect food from microbial attack and can act as a barrier to oxygen and moisture (Huang, Qian, Wei, & Zhou, 2019; Malhotra, Keshwani, & Kharkwal, 2015). Packaging systems with antimicrobial property can inhibit the growth of microorganisms that are contaminating foods and thus enhance safety and prolong the shelf life of the food. Among various biological macromolecules, chitosan is the most popular biopolymer investigated in food packaging research. Chitosan has good film forming ability, antimicrobial activity, antioxidant activity barrier property, mechanical property, and thermal stability which are particularly suitable for developing food packaging material (Wang, Qian, & Ding, 2018). Pure chitosan films along with its derivatives and composites with other biopolymers and inorganic materials have found applications in food packaging. TiO2 nano-powder incorporated chitosan film showed excellent antimicrobial activity against food-borne pathogenic microorganisms and better mechanical properties which made it a promising food packaging material (Zhang et al., 2017). The film was effective against four tested bacteria E. coli, S. aureus, C. albicans, and A. niger with 100% sterilization in 12 h. Tested on red grapes, this packaging material showed its ability by preventing microbial infection and extending their shelf life. In another research, a packaging film was developed by coating chitosan-ZnO nanocomposite on polyethylene film. The resulting film reduced the viability of Salmonella enterica, E. coli and

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Staphylococcus aureus pathogenic bacteria by 99.9% which showed its effectiveness to be used in active food packaging (Al-Naamani, Dobretsov, & Dutta, 2016). Sulfur nanoparticles incorporated alginate film was prepared by Priyadarshi, Kim, Rhim (2021) to investigate its prospect as active food packaging material. The film displayed good UV-barrier and mechanical properties. The composite film was tested against food-borne microorganisms E. coli and L. monocytogenes to evaluate its antimicrobial property. It showed bacteriostatic activity against E. coli (60% of bacterial colonies) but bactericidal activity against L. monocytogenes (100% bacterial elimination in 12 h) which indicated its possible use as food packaging material. In addition, polylactic acid/chitosan composite film (Niu, Liu, Song, Han, & Pan, 2018), essential oil-doped chitosan/poly(ε-caprolactone) hybrid nanofibrous mats (Ardekani-Zadeh and Hosseini, 2019), apple peel polyphenols incorporated chitosan film (Riaz et al., 2018), chitosan/ε-polylysine bionanocomposite film (Wu et al., 2019), capsaicin-loaded gelatin/chitosan film (Zhao et al., 2020), clove oil incorporated chitosan/ β-cyclodextrin citrate/oxidized nanocellulose biocomposite film (Adel, Ibrahim, El-Shafei, & AlShemy, 2019), TiO2 modified zein/chitosan films (Qu et al., 2019), cellulose nanofibril/silver nanoparticle composite (Yu, Wang, Kong, Lin, & Mustapha, 2019), ZnO nanoparticles incorporated gelatin/β-glucan nanocomposite film (Azari, Alizadeh, Roufegarinejad, Asefi, & Hamishehkar, 2020), copper sulfide nanoparticle loaded carrageenan films (Li et al., 2020), corn starch/chitosan biocomposite films (Jha, 2020), etc. have been investigated for their capability to be utilized as food wrapping material in recent times.

7.6.2 Drug delivery Various biological macromolecules such as carbohydrates, proteins, nucleic acids, lipoproteins, polyphenols and lipids have been investigated in

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drug delivery for decades to utilize their specific physicochemical property. Besides biodegradability, biocompatibility, nontoxicity, low immunogenicity, some biological macromolecules possess antimicrobial property and are especially useful in developing drug delivery systems. Biomacromolecules like chitosan, alginate, cyclodextrin, HA, heparin, collagen, albumin, transferrin, silk fibroin, keratin and their derivative are widely used in delivery of ocular drugs (Krishnaswami, Kandasamy, Alagarsamy, Palanisamy, & Natesan, 2018), anticancer drugs (Zhang, Sun, & Jiang, 2018), antimalarial drugs (Mhlwatika & Aderibigbe, 2018), insulin (Luo et al., 2016), antiviral drugs (Joshy et al., 2017), antibiotics (Stebbins, Ouimet, & Uhrich, 2014), cardiovascular drugs (Desbrieres et al., 2019), nonsteroidal antiinflammatory drugs, etc. (Tang, Guan, Yao, & Zhu, 2014) and many of these delivery systems have been approved for clinical use. In drug delivery, antimicrobial property of biological macromolecules based drug carrier can impart synergistic effect with loaded drug. This can even help to control antibiotic resistance, which is a major concern in recent years. In a research, localized delivery of an antibiotic drug, gentamicin was investigated with chitosan/poly (ethylene glycol) based delivery system (Masud et al., 2020). The zone of inhibition was found highest in case of drug loaded bocomposite than that of drug and biocomposite alone. This enhancement in antimicrobial activity was due to the combined effect of drug, chitosan and ZnO nanoparticles used in the biocomposite delivery system. Some recently developed biological macromolecules based drug delivery systems include hydrolyzed starch/chitosan nanocomposite (Shehabeldine and Hasanin, 2019), chitosan/poly (allylamine hydrochloride) blend films (Sarwar et al., 2020), chitosan coated halloysite nanotubes (Sharif et al., 2019), resveratrol loaded chitosangellan nanofiber (Rostami, Ghorbani, Delavar, Tabibiazar, & Ramezani, 2019), carboxymethyl

cellulose-alginate/chitosan hydrogel beads (Jeddi and Mahkam, 2019), starch/microcrystalline cellulose hybrid gels (Xu, Tan, Chen, Li, & Xie, 2019), lactose-crosslinked gelatin scaffolds (Etxabide, Long, Guerrero, de la Caba, & Seyfoddin, 2019), gelatin/gelatinized sago starch biomembranes (Wannaphatchaiyong et al., 2019), chitosan/alginate hydrogel encapsulated gelatin microspheres (Chen et al., 2019), folic acid conjugated chitosan (Islam et al., 2017), nanocellulose/ gelatin composite cryogels (Li et al., 2019), glucose crosslinked gelatin/zein nanofibers (Deng, Li, Feng, Wu, & Zhang, 2019), etc.

7.6.3 Wound dressing A wound is the disruption in the continuity of the skin that can be caused by physical or chemical means and are prone to microbial attack and infection. Moreover the healing of wound is a complex process and requires special attention for complete healing. Consequently, it is necessary to cover the wounded area with dressing material to prevent the infection caused by microorganism. Dressing materials having antimicrobial properties are particularly beneficial to prevent infection. Biological macromolecules like chitosan and its derivatives are well-known for their antimicrobial activities and dressing material prepared from them are effective against different microorganisms (Bano, Arshad, Yasin, Ghauri, & Younus, 2017). Not only chitosan but also other biological macromolecules like alginate, collagen, cellulose, HAs, sericin, fucoidan, etc. are extensively used in wound dressing materials research (Sahana and Rekha, 2018). In a research, wound dressing material prepared from chitosan and honey was found effective against S. aureus, B. cereus, E. coli, P. aeruginosa, and C. albicans due to the antimicrobial effect of chitosan (Movaffagh et al., 2019). Addition of inorganic nanoparticles like zinc oxide, copper oxide, graphene oxide, titanium dioxide, silver, gold, and copper nanoparticles, etc. to chitosan based matrix further enhance

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References

the antimicrobial activity of the dressing material (Mohandas et al., 2018; Venkataprasanna et al., 2020). Chitosan/alginate-based biocomposite dressing material loaded with ZnO nanoparticles showed excellent healing efficiency and wound treated with this biocomposite was completely healed within 10 days (Rahman et al., 2020). This biocomposite showed good antimicrobial property against E. coli and S. enterica due to the presence of chitosan and ZnO nanoparticles. Additionally, biological macromolecules based systems like alpha-tocopherol incorporated chitosan/alginate hydrogels (Ehterami et al., 2019), hesperidin loaded alginate/chitosan hydrogel (Bagher et al., 2020), cerium oxide nanoparticles incorporated chitosan/PVA hydrogels (Kalantari, Mostafavi, Saleh, Soltantabar, & Webster, 2020), Chitosan/glucan complex hollow fibers reinforced collagen wound dressing embedded with aloe vera (Abdellatif et al., 2020), cordycepin/ chitosan complex hydrogel (Song et al., 2019), thymoquinone loaded chitosan-lecithin micelles (Negi et al., 2020), alginate/CaCO3 composite microparticles (Shi et al., 2019), peptideconjugated alginate/hyaluronic acid/collagen wound dressings (Lin et al., 2019), silver nanoparticles loaded alginate/gelatine hydrogel (Diniz et al., 2020), collagen-hyaluronic acid hydrogel (Ying et al., 2019), cellulose nanocrystal/alginate/ gelatin scaffold (Shan, Li, Wu, Li, & Liao, 2019), etc. have been reported as potential wound dressing materials in recent times.

7.7 Conclusion Biological macromolecules have attracted significant interest over the last few decades. The use of biological macromolecules is inevitable because they provide exceptional antimicrobial properties. These macromolecules are unavoidable as they are important body elements that perform a variety of functions necessary for living organisms to keep

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them alive. These unique properties of biological macromolecules make them effective in many applications including food packaging, drug delivery, wound dressing, etc. We expect that the biological macromolecules and their composites are promising antimicrobial materials for future development by emerging products on the market.

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C H A P T E R

8 Biological macromolecules from algae and their antimicrobial applications Natanamurugaraj Govindan1,2, Gaanty Pragas Maniam1,2, Mohd Hasbi Ab. Rahim1,2, Ahmad Ziad Sulaiman3 and Azilah Ajit4 1

Algae Culture Collection Center & Laboratory, Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Kuantan, Malaysia 2Centre for Research in Advanced Tropical Bioscience, Universiti Malaysia Pahang, Kuantan, Malaysia 3Faculty of Bio-Engineering & Technology, University Malaysia Kelantan Kampus Jeli, Kelantan, Malaysia 4Faculty of Chemical & Natural Resources Engineering, Universiti Malaysia Pahang, Kuantan, Malaysia

8.1 Introduction Algae are the dominated diverse group of the unicellular and multicellular organisms in the eukaryotic phylum. They have close resemblance with photosynthetic plants in the form of metabolic pathways, but the actual root systems are not similar (Delwiche, Andersen, Bhattacharya, Mishler, & McCourt, 2004). Microalgae can be cultured using ordinary or simulated lights using freshwater, marine water blackish water, and waste effluent that feed the species and at the same time removing nutrients and organic pollutants from the effluent (Garcia-Moscoso, Teymouri, & Kumar, 2015). Peptides (BP) are specific protein fragments that are involved in a wide range of therapeutic activities as antihypertensive, antioxidant, antitumoral, antiproliferative, hypocholesterolemic, and antiinflammatory (Sa´nchez & Va´zquez, 2017). Besides, they are also involved in triggering mechanisms to

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00008-7

stimulate healthy cell growth (Hartmann & Meisel, 2007). Microalgae antimicrobial peptides (AMPs) have several advantages including gene expression similar to higher plants and efficiently can be grown for numerous applications.

8.2 Bioactive macromolecules 8.2.1 Terpenoids Culturing of algae has been exponentially increasing along course of decades due to the exploration of unique bioactive compounds available in various forms of algae species. Terpenoids are the class of bioactive components present in several marine algae species. Based on the number of isoprene units, terpenoids are further classified as monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids

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possessing a structure varying from linear, cyclic forms. Antimicrobial property exhibited by terpenoids varied in accordance with the nature of algae and type of terpenoids (Ebada, Lin, & Proksch, 2010; Ludwiczuk, Skalicka-Wo´zniak, & Georgiev, 2017) (Table 8.1). The cytotoxicity and antimicrobial effect of various bioactive terpenoids present in green algae from the class Udoteaceae whereas, few terpenoids existing in particularly prevalent are leaders of the Udotea, Penicillus, Avrainvillea and Halimeda genera. Chemical analyses of these prominent plants also resulted in the isolation of a remarkable variety of associated sesquiterpenoid as well as diterpenoid metabolites, which are mostly linear or noncyclical (Lee et al., 2008). The increasing frequency of the conjugated bisenol acetate molecular structure, that was unnoticed among natural products until previous surveys of these algal components, is a collective characteristic of these residues. This feature is a “shrouded” constellation of dialdehydes to which significant nutrient activity is normally credited. Interestingly, very strongly linked terpenoids, also possessing the properties of bisenol acetate, were also observed to be widespread but not omnipresent components of certain green algae of the genus Caulerpa (Caulerpaceae) (Sun et al., 2007).

A review of existing available chemical evidence shows that the key groups of antimicrobial and antiviral terpenoids present in the aquatic ecosystem are sesterpenoids, sesquiterpenoids, and meroterpenoids. (Fig. 8.1) The three R. strains. In comparison to the development of haslenes, setigera was reported earlier only in the benthic diatom Ostearium ostrearia. In addition to Caulerpa genus, diatoms are also one of the class of compounds which contribute high branched isoterpenoid (HBI) (Du et al., 2008). Particularly in HBI are frequently found in diatoms and exhibit prominent bioactivities, including antimicrobial and antiviral properties. Mostly the chemicals were found to be good inhibitors of isocitrate lyase, and active sterile effects against Bacillus subtilis and Proteus vulgaris were also seen. Hyrtiosal, isolated from the marine sponge Hyrtios erectus, which prevents the binding of HIV integrase to viral DNA through a new enzyme binding site, is another bioactive sesterpenoid (Xu et al., 2009). Furthermore, molecular dynamic analysis correlated with a site-directed mutagenesis simply assigned that such viral DNA binding inhibition induced by hyrtiosal was triggered by the fact that hyrtiosal could bind the HIV N-terminal domain at Ser17, Trp19 and Lys34.

TABLE 8.1 Classification of terpenoids (Garcia-Moscoso et al., 2015). Name

No. of isoprene units

No. of carbon atoms

General formula

Hemiterpenoids

1

5

C5H8

Monoterpenoids

2

10

C10H16

Sesquiterpenoids

3

15

C15H24

Diterpenoids

4

20

C20H32

Sesterterpenoids

5

25

C25H40

Triterpenoids

6

30

C30H48

Tetratepenoids (carotenoids)

8

40

C40H64

Polyterpenoids

.8

. 40

(C5H8)n

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8.2 Bioactive macromolecules

FIGURE 8.1

205

Isomers of terpenoids (Garcia-Moscoso et al., 2015).

Since hyrtiosal was recently discovered as an inhibitor of protein tyrosine phosphatase 1B, this study can also provide multiple target details for this natural marine product (Qiu & Wang, 2008). The 1,4-benzoquinone moiety is a typical structural feature possessing wide range of bioactive compounds, including antimicrobial and antiviral properties, have received considerable attention. In this regard, the Laurencia genus is specifically prolific, but this genus has identified 512 sesquiterpenes and 133 diterpenes of different structures. Some red algae species, such as Chondrococcus hornemanni, Hypnea pannosa, Plocamium cornutum, and Portieria hornemannii, are also developing terpenes, but less studied (Lane et al., 2010). In addition, also identified these kinds of compounds from aquatic algae. Novel sesquiterpene hydroquinones, Peyssonoic acid A and B, have been isolated from the crustose red algae Peyssonnelia sp (de Silva et al., 2009; Vairappan et al., 2008). Both compounds reduced the formation of Pseudoalteromonas bacteriolytica, a bacterial

pathogen of marine algae, at ecologically realistic concentrations, and Lindra thalassiae, a fungal pathogen of marine algae. One novel carbon skeleton was comprised of peyssonoic acids and revealed the value of scientific activities in the exploration of natural materials. Halogens such as tiomanene and acetylmajapolene A and B are occasionally formulated into antimicrobial sesquiterpenoidhydroquinones obtained from Malaysian Laurencia sp. Ji et al. (2007), and the latest 10-hydroxykahukuene B brominated metabolite isolated from Laurencia mariannensis red marine algae (Zhang, Khalil, & Capon, 2011). Meroterpenoids have also been used in studies were extracted from marine sponges. During an investigation to discover antimicrobials from marine species. A sequence of new acid induced hydrolysis/cyclization components of fascioquinol A are fascioquinols B, C and D. Fascioquinol A and B, both of these molecules, showed promising Gram ( 1 ) specific antibacterial activity against Staphylococcus aureus [0.9
2.5 μM concentration when compared 50 IC50 and Bacillus subtilis (0.3
7 μM IC50)] 2w

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206

8. Biological macromolecules from algae and their antimicrobial applications

(Desoubzdanne et al., 2008). Two enzymatic pathways of importance for malaria transmission, Pfnek-1 plasmodial kinase as well as farnesyl transferase enzyme, as well as numerous Plasmodium falciparum chloroquine-sensitive and resistant strains, demonstrated μM range activity in the materials. Diterpene and diterpene iso-nitriles from the tropical marine species Chlamydomonas reinhardtii (Wright et al., 2011) were also used as reports of terpenoid from marine sponge source which has antibacterial properties. Kamei et al., tested 342 specimens of marine algae extracts collected from Japan coast for antimicrobial effects against propionibacterium acnes and described a potential antimicrobial agent, diterpene sargafuran, from Sargassum macrocarpum, a sea brown algae methanolic fragment. Sargafuran were bactericidal but was totally destroyed by cell lysis bacterial cells by propionibacterium acnes (Nguyen et al., 2007).

8.2.2 Steroids As far as is understood, marine algae are divided as follows with respect to their sterols: Phaeophyceae probably have fucosterol, which often occurs in some Chlorophyceae and Rhodophyceae. Sargasso weed Sargassum ringgoldianum were shown to provide the fucosterol (sargasterol) isomer C-20. (Fig. 8.2) Cholesterol is available in 15 species of Chodophyceae, commonly considered as the signature animal sterol but not yet identified in any terrestrial plant. Chlorophyceae can comprise a combination of the sort of sitosterol contained in green land plants (Haslewood, 1960). Normally steroids in treating heart complications and atrial arrhythmias, several cardiac glycosides have been used therapeutically, and several glycoside derivatives demonstrate cytotoxic, antimicrobial, hypocholesterolemic, as well as other biological behaviors (Ivanchina, Kicha, & Stonik, 2011). Bacterial activity of the

FIGURE 8.2 Structure of fucosterol (Zhang et al., 2011).

glycosides mediated steroids, extracted from green alga Codium iyengarii. Further examination on bacterial activity on various bacteria, steroidal glycosides shows an effective inhibitory concentration against Corynebacterium diptheriae, Escherichia coli, Klebsiela pneumonia, Snigella dysentri, Staphylococcus aureus. In addition, various steroidal compounds such as cholesterol, β-sitosterol, campesterol and stigmasterol are also available in Eucheuma cottoni (red algae) (Ali, Saleem, Yamdagni, & Ali, 2002) (Fig. 8.3). Eurysterols A and B are two new steroidal sulfates extracted from an unexplored marine species acquired in Palau from the genus Euryspongia (Boonlarppradab & Faulkner, 2007). The molecule demonstrated antifungal efficacy toward Candida albicans amphotericin B-resistant and wild-type strains, respectively MIC values of 15.6 and 62.5 μg/mL in turn. The bioassay-guided fractionation of Topsentia sp. extract. Two additional sulfate sterols, geodisterol-3-O-sulfite as well as 29-demethylgeodisterol-3-O-sulfite, have been reported as ingredients in reverse efflux syringe-mediated fluconazole resistance (DiGirolamo, Li, Jacob, Clark, & Ferreira, 2009). Both agents improved fluconazole behavior in a strain of Saccharomyces cerevisiae transgenic the MDR1 Candida albicans efflux pump, and also in a clinical isolate of fluconazole-resistant Candida albicans reported to inhibit MDR1. Steroid-related bile acid additives are also examples of many other aquatic plant antimicrobial steroids against bacterium Psychrobacter sp (Li et al., 2009). The steroids which are of aromaticity from the Colletotrichum sp., demonstrating antimicrobial properties to

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8.2 Bioactive macromolecules

207

FIGURE 8.3 Various forms of steroids present in Eucheuma cottoni. (A) Cholesterol, (B) -sitosterol, (C) Campesterol, (D) Stigmasterol. (Fasya et al., 2019).

Escherichia coli and Bacillus megaterium bacteria (Li et al., 2009).

8.2.3 Phenolics Being one of the largest constituents of secondary metabolites in plants, phenolic compounds play vital roles in antimicrobial activities. These compounds are found in terrestrial as well as marine species and varying in structure; from an aromatic to multi-aromatic structures. They have significant role as an antioxidant for humans. The high content of phenolic especially in marine algae makes the source as one of the primary route for phenolic. Oxidative stability can be achieved with these phenolic compounds that in turn assist in maintaining health profile in humans. In addition, these phenolic compounds have been added to the diet of animals for the same purpose. Oxidative stress need to be managed well as it is one of the factor for health deterioration in

humans and animals, including neuron disease, ocular disease and Alzheimer’s disease. As such, the phenolic compounds suit well in countering the oxidative stress by containing those harmful oxidants. The benefits of these phenolic compounds extend to assist in preventing the forming of cancerous tissues as the phenolic can prevent cell and tissue damage through antioxidant activities (Kattappagari et al., 2015; PhamHuy, He, & Pham-Huy, 2008; Topdag, Aslaner, Tataroglu, & Ilce, 2005). As shown in Table 8.2, an array of few microalgae species can deliver phenolic activity from 1.23 mg GAE/g EW to as high as 32 mg GAE/g EW. Among the listed species, Nannochloropsis gaditana exhibits the highest phenolic activity and the other species with significant activity in term of phenolic are Tetraselmis sp. and Dunaliella salina with 25.5 mg GAE/g EW and 19.3 mg GAE/g EW, respectively. Among the reported phenolic compounds from algae that associated with antioxidant

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208

8. Biological macromolecules from algae and their antimicrobial applications

TABLE 8.2 Phenolic activity of selected microalgae species (Becker, 2013; Koyande et al., 2019; Pulz & Gross, 2004). Microalgae species

Phenolic activity (mg GAE/g EW)

Nannochloropsis gaditana

32.0 6 0.57

Tetraselmis sp.

25.5 6 1.50

Dunaliella salina

19.3 6 0.70

Phaeodactylum tricornutum

16.8 6 0.33

Dunaliella sp.

14.0 6 0.43

Isochrysis sp.

13.4 6 0.16

Chaetoceros sp.

11.9 6 0.28

Chlorella sp.

8.1 6 0.16

Botryococcus brraunii

1.99 6 0.17

Scenedesmus obliquus

1.94 6 0.16

Haematococcus pluviallis

1.23 6 0.06

fragilamide, martensine, martefragine, denticine and almazolone. Alkaloids that includes halogenated compound (bromo) that are isolated from red algae are Laurencia brongniartii, Laurencia similis, and Laurencia decumbens (Gu¨ven, Percot, & Sezik, 2010; Percot et al., 2009; Rolle et al., 1977).

8.2.5 Polysaccharides

activity are anthraquinones, coumarins, and flavonoids (Amaro, Guedes, & Malcata, 2011). As for seaweed, it was found that Himanthalia elongate has a significant amount of phenolic compounds at 151.3 mg GAE/g of extract as well as other useful macromolecules (Cox, Abu-Ghannam, & Gupta, 2010). Furthermore, it exhibits DPPH scavenging activity with EC50 level at 0.125 μg/mL of extract.

8.2.4 Alkaloids Alkaloids, group of chemical compounds that includes nitrogen atoms in a cyclic ring (halogens included in several alkaloids) and found in marine algae species such as Xestaspongia exigua and Agelas sp. Bajpai et al. (2016). Alkaloids have potential in antifungal activity, antibacterial activity, antibiotic activity and antiphotooxidative activity. Polysiphonia tripinnata and Scenedesmus acutus. Among the indole group alkaloids are caulerpin, caulersin,

Polysaccharides are structurally macromolecules composed with sugar molecules and its derivatives like uronic acid. In nature, the sugar ring is formed covalent bond with other sugar molecule. Micro or macroalgae are the main sources for variety of polysaccharides with potential antioxidant, antibacterial and antiviral activities (Jun et al., 2015; Ruocco, Costantini, Guariniello, & Costantini, 2016). One of the edible blue
green algae species Nostoc flagelliforme, producing nostoflan as one of the acidic polysaccharide shows antiviral activity (anti-HSV-1) (Kanekiyo, Hayashi, Takenaka, Lee, & Hayashi, 2007). Another microalga filamentous cyanobacterium Oscillatoria agardhii, inhibiting HIV replication in MT-4 cells (Sato, Okuyama, & Hori, 2007). Sulfated polysaccharides fucoidan F85 from algae Fucus vesiculosus showed growth inhibitory effects against all the dental plaque bacteria. The fucoidan F85 inhibited the growths of Listeria monocytogenes and Staphylococcus aureus lactic acid bacteria. The two Undaria pinnatifida and Kjellmaniella crassifolia shown excellent effect on Salmonella typhimurium bacteria (Jun et al., 2018). Fucoidan has antimicrobial activities against several cariogenic Streptococcus sp and the periodontopathogenic bacteria Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, and Porphylomonas gingivalis (Lee, Jeong, Choi, Na, & Cha, 2013). Fucoidan from a brown alga Sargassum wightii, having antibacterial properties against Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, Proteus proteus, Shigella sonnie, Pseudomonas aeruginosa,

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8.2 Bioactive macromolecules

S. typhimurium, and Klebsiella sp (Marudhupandi & Kumar, 2013). In Chaetomorpha aerea, a polysaccharide with sulfate group attached with, it provides a particular antibacterial property for the gram-positive Bacillus subtilis, Micrococcus luteus and S. aureus (Pierre et al., 2011). Seaweed carrageenans (λ, γ and κ) have bacteriostatic effects against S. enteritidis, S. typhimurium, Vibrio mimicus, Aeromonas hydrophila, E. coli (enterotoxigenic), and S. aureus (Yamashita, SugitaKonishi, & Shimizu, 2001). Studies on in vitro antioxidant activity of marine edible seaweeds Eucheuma cottonii and Padina sp. have a potential antioxidant activity (Foon, Ai, Kuppusarmy, Yusoff, & Govindan, 2013). Marine macroalgae Padina sp. and its macromolecules have a potential antibacterial activity towards pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa (Bhuyar et al., 2020). Algae Gracilariopsis longissima (Rhodophyta) polysaccharides have a potential antimicrobial activity against P. aeruginosa, Enterococcus sp. S. agalactiae, V. salmonicida, V. fluvialis, V. vulnificus, V. cholera, V. alginolyticus, C. albicans, C. famata and C. glabrata (Stabili et al., 2012). Brown alga Eisenia bicyclis have Inhibitory effects against Pseudomonas. aeruginosa and Streptococcus aureus (Lee et al., 2014). Lectins extracted from macroalga Hypnea musciformis exhibited antifungal activity against Trichophyton rubrum and Colletotrichum lindemuthianum (Singh & Walia, 2018).

8.2.6 Peptides Peptides in microalgae assist them for protection against many risks. To kill various microorganisms, AMPs have been involved more than 100 amino acids known in numerous species (Chu et al., 2015; Conlon & Sonnevend, 2010). In recent years, proteins research is intense in protecting against many ailments (Rasala & Mayfield, 2015). Microalgal peptides have higher antioxidant when compared with other microbial synthesis such as

209

E. coli, Pichia pastoris, Saccharomyces cerevisiae and many mammalian cells. The enzyme mediated peptides such as peroxidase, superoxide dismutase, glutathione peroxidase and even nonenzymatic factors eliminating the free radicals. The evidences indicating that the active oxygen and free radicals attacks some molecular DNA and protein that leads many degenerative diseases (Suja, Jayalekshmy, & Arumughan, 2004). Removal of free radicals to maintain pro-oxidant with antioxidant is one of the most preventive defense mechanisms against various diseases (Fazlul & Li, 2002). Recently the bioactive peptides from various diet products such as soy protein, whey protein, gelatine and wheat gluten exhibit a potential antioxiatuive activities (Elias, Kellerby, & Decker, 2008). However, recent attention of antioxidant peptides has been a new attention in recent years from marine food sources especially marine algae (Qian, Jung, & Kim, 2008b). Chlorella algae is one of the popular and well established for the production of antioxidant peptides as well as various nutritional supplements, dye and skin care applications (Spolaore, Claire, Elie, & Arsene, 2006). Nonribosomal peptides from marine Brazilian cyanobacteria isolates are antimicrobial (Silva-Stenico et al., 2011). From the preliminary reports revealed that the pepsin hydrolysate from the chlorella algae protein have a potential antioxidant peptides active as antioxidant as well as antibacterial potentials. Cyanobacteria Arthrospira platensis producing peptide through enzymatic hydrolysates (De Lucia et al., 2018) which is pharmacological and its antiproliferative effect in lung cancer cells (Czerwonkaa et al., 2018; Lu et al., 2010). Microalgae Chlorella pynenoidosa isolated peptide fraction inhibited antimicrobial activity (Chen et al., 2011; Sheih, Wu, & Fang, 2009). Lectins from Eucheuma serra and Galaxaura marginata, hindering Vibrio vulnificus and V. pelagicus (Smith, Desbois, & Dyrynda, 2010). The antimicrobial activity of lectins obtained

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210

8. Biological macromolecules from algae and their antimicrobial applications

from red algae was evident from report elsewhere (Vasconcelos et al., 2014). Peptides producing by microalgae Scenedesmus sp by flash hydrolysis methods for antimicrobial potential (Garcia-Moscoso, Obeid, Kumar, & Hatcher, 2013). There are numerous reports on bioactive compounds from various microbes were investigated and reported but the AMPs from algae have various means of oxidation in vitro.

8.2.7 Polyketide Polyketides have high biological activity and are complex organic compounds in terms of structure. The naturally occurring polyketides and their derivatives have considerable medicinal values and are either obtained from many drugs (Singh, Chaudhary, Shankar, & Prasad, 2019). An important source of novel metabolites with antimicrobial activities against different food-pathogenic microorganisms was found to be the seaweed-associated bacterial flora living on the surface of the host organism. Studies of surface associated seaweed microbial populations have demonstrated that the microbial species has chemical contacts with its host macroorganisms (Villarreal-Go´mez, Soria-Mercado, GuerraRivas, & Ayala-Sa´nchez, 2010). Seaweeds are recognized as a prolific source of numerous groups of biologically active compounds suitable for functional food and pharmaceutical lead production (Holdt & Kraan, 2011). Isolated two aryl polyketides which represent aryl substituted oxo-2H-pyranyl and oxa-cylododecadienyl macrolactone from brown seaweed Sargassum wightii. Polyketide analogs of α-tocopherol demonstrated similar antioxidant function. Firmicutes and gamma proteobacteria were significance among the various phyla of marine bacteria. In general, Shewanella algae and gamma proteobacteria have symbiotic associations with higher organisms (Kizhakkekalam, Chakraborty, & Joy,

2020; Timmermans, Paudel, & Ross, 2017). The extracts of red alga Gracilari adendroides found more efficient against the tested bacterial strain Escherichia coli strains followed by green alga Ulva reticulata and brown algae Dictyota ciliolate. Antibacterial polyketide compounds, used heterotrophic Bacillus amyloliquefaciens associated with edible red seaweed, Laurenciae papillosa (Chakraborty, Thilakan, Raola, & Joy, 2017). The isolation of highly interesting bioactive compounds has been from blue-green algae such as Alkaloids (lyngbyatoxin), polyketides (tolytoxin), cyclic peptides (tolytoxin), cyclic peptides (microcystin), depsipeptide, etc. All of these compounds have shown a flexible biological activity (Gamal, 2010). Its metabolites show very potent biological activity such as saxitoxin 19, neosaxitoxin and gonyautoxins produced by Alexandrium extremely selective, and some other genera of dinoflagellates are blockers of sodium channels.

8.2.8 Polyunsaturated fatty acids Omega-3 is an unsaturated essential fatty acid that cannot be synthesized by humans due to the absence of desaturase enzymes. Alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are three kinds of omega-3 fatty acids involved in human physiology (Harwood, 2019) (Table 8.3). ALA is the shortest N-3 chain present predominantly in vegetable oil and nuts; two other essential EPA and DHA derivatives are found in fish and many other microorganisms, such as microalgae and bacteria (Guedes, Amaro, Barbosa, Pereira, & Malcata, 2011). ALA can also be transformed in the body into EPA and DHA, but the conversion is very small and unsuccessful. Therefore omega-3 must be supplied in the form of a dietary supplement. These long chains of algae polyunsaturated fatty acid (PUFA) have profound health

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211

8.2 Bioactive macromolecules

TABLE 8.3 Product and key fatty acids of various microalgae species. Microalgae species

Product and key fatty acids

References

Chlorella vulgaris

Palmitic acid, linoleic acid, oleic acid, linolenic acid

(Palanisamy et al., 2020)

Selenastrum minutum

Palmitic acid, linoleic acid, oleic acid, linolenic acid, lignoceric acid

(Kudahettige, Pickova, & Gentili, 2018)

Nannochloropsis gaditana

Myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid

(Janssen, Wijffels, & Barbosa, 2019)

Botryococcus braunii

Palmitic acid, oleic acid and linoleic acid

(Dilia & Leila, 2018)

Chaetoceros muelleri

Myristic acid, γ-linoleic acid, arachidic acid, palmitic acid, stearic acid, oleic acid acid, eicosapentaenoic acid, docosahexaenoic acid

(De Jesu´s-Campos et al., 2020)

and medicinal advantages and functions (Khan et al., 2015). In metabolic and immune functions, DHA and EPA have various important roles and play a key role in health benefits linked to neuro and cardiovascular diseases. It also has many benefits and prevents tumor cells in diabetes mellitus (D’Eliseo & Velotti, 2016). The conversion of essential fatty acids as by several elongation and desaturation steps to their downstream products. Because the pathways n-6 and n-3 compete for enzyme activity with each other, the ratio of PUFAs n-6 to n-3 is very important for human health. An overabundance of fatty acids from one family would restrict the metabolic manufacturing of the other’s longer chain products. The standard Western diet includes a ratio of n-6 and n-3 PUFAs from 8:1 to 25:1 (Ander, Dupasquier, Prociuk, & Pierce, 2003). In stark comparison to the guidelines of the public health authorities, the values were about 4:1. The reduction in the n-6: n-3 ratio will minimize enzyme rivalry and promote the metabolism of more downstream ALA products. In addition, algae can be manipulated to produce more of the omega-3 fatty acids by

controlling their exposure to UV light, oxygen, sodium, glucose and temperature. It is also stated that the proportion of omega-3 fatty acids in microalgae will exceed values comparable to those of different fish species (Adarme-Vega et al., 2012). Long-chain polyunsaturated fatty acids (PUFAs) ($20 carbon atoms and .3 double bonds) are predominantly formed by phytoplankton in the aquatic food web and transmitted to herbivorous zooplankton. Thereby impacting the quality of food for species at higher trophic levels (Brett & Mu¨ller-navarra, 1997). In EPA, which has 20 atoms of carbon and five double bonds of cis, DHA has 22 atoms of carbon and six double bonds of cis (Stokes et al., 2020). As novel sources of ω-3 fatty acid, microalgae could replace many of the fish-related taste and odor complaints and discard the shortcomings of the fish a procedure focused on fish oil. It appears the commercial development of microalgal PUFAs be more practical than biodiesel production. Algae of the genus Schizochytrium, Ulkenia, Crypthecodinium and Crypthecodinium are used for the production of DHA. Phaeodactylum tricornutum, Nannochloropsis and Nitzchia are

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8. Biological macromolecules from algae and their antimicrobial applications

traditionally used for EPA processing. As a product of several enzymatic reactions, synthesis of TAGs in microalgae takes place primarily in subcellular compartments such as chloroplast and endoplasmic reticulum. The three key steps involved in the aggregation of lipids in microalgae are fatty acid synthesis in chloroplast, assembly of glycerolipids in the endoplasmic reticulum, and aggregation of TAGs into the oil bodies (Chen et al., 2018; Patel et al., 2020). Various stress conditions, such as physical, chemical, or environmental conditions, have been proven to facilitate the synthesis of high amounts of lipids individually or in combination. Microalgae can alter their metabolism under various stress conditions to shape and store neutral lipids in the shape of TAGs, which act as a form of carbon and energy storage (Arora, Patel, Pruthi, Poluri, & Pruthi, 2018). Phaeodactylum tricornutum marine algae diatom has demonstrated antibacterial activity due to specific (6Z, 9Z, 12Z)-hexadecatrienoic acid, polyunsaturated fatty acid, and (9Z)-hexadecenoic acid, monounsaturated fatty acid, both inhibiting gram-positive bacteria and Gram-negative marine pathogen Listonella anguillarum (9Z)-hexadecenoic acid kills bacteria at high pace. This showing potent activity against multidrug resistant strains of Staphylococcus Aureus. Phaeodactylum tricornutum produce EPA, which an antibacterial fatty acid inhibitory toward a range of gram-positive and gram-negative bacteria includes multidrug resistant Staphylococcus aureus (Desbois, MearnsSpragg, & Smith, 2009). Antibacterial compound isolated from microalgae Chlorella spp., was a mixture of fatty acids. This substance was active against both gram-positive and gram-negative bacteria (Pina-Pe´rez, Rivas, Martı´nez, & Rodrigo, 2017). As a protective mechanism against viruses, protozoa and bacteria, membrane-derived fatty acids from macro and microalgae organisms have been identified with microbicidal behavior (Leflaive & Ten-Hage, 2009; Vello et al., 2014).

Microalgae extract fatty acid methyl ester (FAME) has been found to be radially effective which inhibiting the radial growth of both bacterial and fungal pathogens. The antibacterial efficacy of the FAME extracts was demonstrated against three clinical pathogens, namely Escherichia coli, Salmonella typhi, and Enterobacter sp. The maximum inhibition range is 12.0, 12.0, and 11.0 mm, respectively (Suresh et al., 2014). Unsaturated fatty acid from red alga Tricleocarpa jejuensis and its derivatives algicidal (12-hydroxyoctadec-10-ynoic acid) exert antitumor and antibacterial activities in vitro (Zha, Kuwano, Shibahara, & Ishibashi, 2020). Microalgae contain a broad range of bioactive secondary metabolites, typically at the end of the exponential and stationary phases of development, which are deposited in the cell or excreted in the surrounding setting. Many of these metabolites, such as free saturated or unsaturated fatty acids, exhibit antibacterial activity. In order to reduce the misuse of commercial antibiotics, there is a research initiative to find natural molecules, since their repeated use may result in the creation of resistant bacterial strains. Antiproliferative or antifungal properties are present in other microalgal and cyanobacterial compounds, or are capable of inhibiting viral infection or replication (Dewi, Falaise, Hellio, Bourgougnon, & Mouget, 2018). The estimation of saturated and unsaturated fatty acids found that the largest proportion of palmitic acid was found in algal species of Ulva reticulata, Caulerpa occidentalis, Cladophora socialis, Dictyota ciliolata, and Gracilaria dendroides isolated from Red sea coastal waters of Jeddah, Saudi Arabia. The estimation of saturated and unsaturated fatty acids found that the largest proportion of palmitic acid was found in all the algal species studied. Marine algae fats and fatty acids may play a significant role in the development of certain other secondary bioactive metabolites, as certain fatty acids have been shown to have antibacterial activities (Al-Saif, Abdel-Raouf, El-Wazanani,

II. Bioactivity

References

& Aref, 2014). The antibacterial action was considered to be responsible for additional palmitic acid (Barbosa, Fleury, da Gama, Teixeira, & Pereira, 2007).

8.3 Conclusion Currently, marine algae have attracted rising consideration as a novel source for bioactive properties in addition to their nutritional worth because of their promising medicinal effects of algal constituents against various diseases or physiological concerns, such as antiallergy, anticancer, antihypotension, antiinflammation, antiobesity, antioxidation, and antithrombosis effects. Marine algae products have currently been recognized as the most promising source of bioactive substances for microbial activity. Seaweed has emerged from early aquatic environmental research as a large source of unusual structures and bioactive metabolites. The development of advanced nutraceutical and pharmaceutical industries focused on macroalgae and microalgae cultivation and their exploitation in the manufacture of bioactive substances has a strong and almost unrevealed potential. These marine algae sources are known as a rich source of antiviral, antibacterial, antifungal, and anticancer activities of biologically active compounds.

References Adarme-Vega, T. C., Lim, D. K., Timmins, M., Vernen, F., Li, Y., & Schenk, P. M. (2012). Microalgal biofactories: A promising approach towards sustainable omega-3 fatty acid production. Microbial Cell Factories, 11(1), 96. Ali, M. S., Saleem, M., Yamdagni, R., & Ali, M. A. (2002). Steroid and antibacterial steroidal glycosides from marine green alga Codium iyengarii Borgesen. Natural Product Letters, 16(6), 407
413. Al-Saif, S. S. A. L., Abdel-Raouf, N., El-Wazanani, H. A., & Aref, I. A. (2014). Antibacterial substances from marine algae isolated from Jeddah coast of Red Sea, Saudi Arabia. Saudi Journal of Biological Sciences, 21(1), 57
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Amaro, H. M., Guedes, A. C., & Malcata, F. X. (2011). Antimicrobial activities of microalgae: An invited review. Science Against Microbial Pathogens: Communicating Current Research and Technological Advances, 3, 1272
1284. Ander, B. P., Dupasquier, C. M., Prociuk, M. A., & Pierce, G. N. (2003). Polyunsaturated fatty acids and their effects on cardiovascular disease. Experimental & Clinical Cardiology, 8(4), 164. Arora, N., Patel, A., Pruthi, P. A., Poluri, K. M., & Pruthi, V. (2018). Utilization of stagnant nonpotable pond water for cultivating oleaginous microalga Chlorella minutissima for biodiesel production. Renewable Energy, 126, 30
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C H A P T E R

9 Biological macromolecules acting on central nervous system Dilipkumar Pal1 and Khushboo Raj2 1

Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, India 2School of Pharmacy, Arka Jain university, Tata, Jamshedpur, India

9.1 Introduction Around one billion people in the world is affected from the central nervous system (CNS) disorder. In the United States alone, mental illness has affected around 50 million people annually costing over 650 billions dollar. However, the exact reason of CNS disorders is still unknown. According to some service literature reviews and experimental analysis, biological macromolecules such as carbohydrates, lipids, and proteins may have a wide range of functions that can lead to CNS injuries, trauma ischemia, and various neurological disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) (Pal, Pahari, & Pathak, 2007). In addition, some of them also help to regulate the neurotransmitter receptors in postsynaptic membrane and provide energy in the form of glucose. Through the cell cycle process, a biological cell cycle protein cyclin and cyclin activated kinase (CDKs)controls various cell interaction in neurones during the embryonic development stage and also helps cells to enter different cyclic

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00009-9

phases (Pal, Dutta, & Sarkar, 2009). For example after the synthesis of D types-cyclins, it binds to CDK4 and CDK6 and helps to enter the cell into G1 phases whereas cyclin E helps in transition from G phase to S phase. In this section, we discuss the role of each biological macromolecules and their effect on CNS in a detailed manner (Pal, Sarkar, Gain, Jana, & Mandal, 2011).

9.1.1 Proteins 9.1.1.1 Amyloid-beta and tau protein These two proteins are recognized as the main reasons for the pathophysiology of AD due to their deposition, senile plaques, neurofibrillary, and increased solubility. Aß is derived from AßPP, a type one single pass transmembrane protein constructed by 639
770 amino acids in human, while toe is a microtubuleassociated protein. From past decades it is known that Aß deposition and clinical degree of dementia among suffered people has no correlation (Pal & Mazumder, 2014). Recent

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studies have suggested that every play a critical role by interfering in CNS with neuronal growth neurotransmitter release synaptic function and memory formation also the low concentration of amyloid- beta may alter the synaptic function and block the endogenous amyloid-ß in the healthy brain resulting impairment of synaptic plasticity and memory. Tau is a heat stable protein that is important for assembly of microtubule and for their stability it is found in six main isoforms derived from the alternate slice of axons 2, 3 and 10 of MPT genes (Gulisano, 2018). From the functional point of view toys involved in many subcellular functions such as in regulation of axonal elongation, maturation and transport participation in synaptic plasticity balancing the integrity of genomic DNA cytoplasmic and nuclear RNA etc. The various roles of in neuronal physiology have studied and found at a proper regulation is needed for the maintenance of its structure and functions due to dysregulated phosphorylation insoluble tower aggregates get deposited in NFT and from preclinical study it has been proved that the formation of top protein in the brain of Alzheimer patient in contrast to brain of healthy people occurring before NFT formation and clinical symptoms. In addition to oligomers have been also found in other taupathies (a wide range of neurodegenerative disorders) such as dementia, Lewy bodies, Huntington’s disease, etc. (Smith et al., 1995).

9.1.2 Cell cycle proteins Cell cycle proteins have a specific role in the case of CNS injury, and a significant increase in cell cycle protein expression can be seen during trauma or Ischemia. A progression of these proteins occurs after serious injury and attributes to apoptosis of postmitotic cell such as neurones and oligodendroglia. Cell cycle proteins impart a vital role in the case of

in vitro neuronal damage and death. Kianic acid is an example of such type of cell cycle protein. It persuades exit toxic cell death in cerebellar granule neurones also it elevates BrdU optic neurone after CNS injury following DNA replication (Byrnes & Faden, 2007). In addition cyclin D1 is available in very low concentration in the different part of brain such as hippocampus, piriform cortex and amygdale (Byrnes & Faden, 2007). In noninjured cortex cyclin D1 is coexpressed in map2 positive cells also p27 a class of CDK eye expression is elevated in the normal cortex. Microarray analysis have suggested that after traumatic brain injury there is neurone loss following inflammation induce injury and apoptosis occurred also a significant upregulation of cyclin D1 and CDK4 is observed at different time point. In addition after traumatic brain injury down-regulation is expressed p27 whereas p21 remain constant but in contrast Kobari et al. and Katan et al. showed that the label of p21 is increased within to 24 h after traumatic brain injury. p21 also impart a major role in spinal cord injury by improving both motor function and increase of number of axons extending beyond the lesson site. In Fig. 9.1, the role of different cell cycle proteins after CNS injury is shown (Pal, 2020).

9.1.3 Homer/vesl proteins These proteins were discovered in 1997. This protein is present in the mammalian brain at postsynaptic densities of excitatory synapses (Ehrengruber, Kato, Inokuchi, & Hennou, 2004). These homer protein binds to pyrrolidine rich sequences that are present on various glutamate receptors such as mGluRs, inositol (1,4,5)-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs) type 1 and 2, C-type transient receptor potential (TRPC) channels (Berke, Paletzki, Aronson, Hyman, & Gerfen, 1998). If we discussed about the function of homer

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Nucleus

Cyclin E and A

Activation of CDK4 through binding of cyclin D1 and CDK4

CDK4

CDK 2

Phosphorylation of Rb and transition into S phase.

CYCLIN D1

Cyclin B CDK2

Phosphorylati on of Rb

Translocaon to G1 phase

Release of E2F that activates DNA transcription.

FIGURE 9.1

Role of different cell cycle protein.

Translocation to M phase.

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proteins, it helps in regulation of neurotransmitter receptors in the postsynaptic membrane involves in intracellular single transduction cascades and alter synaptic strength (Kato, Ozawa, Saitoh, Hirai, & Inokuchi, 1997).

9.1.4 Central fatty hypothesis Central fatty hypothesis we all know that muscular fatigue is a result of impairments within the muscles which occurs due to failure of transmission of neural stimuli to the motor end plate of muscles some research suggests that fatigue can also occur due to changes that happens within the CNS and it is called as Central fatigue (Pal, Sahoo, & Mishra, 2005). Central fatigue theories have been proposed and it explained the role of different neurotransmitter likes serotonin (5-HT), norepinephrine and dopamine in Central fatty during exercise. Generally this hypothesis evidence that impairment of CNS activity occurs due to increase concentration of brain serotonin label which happens during prolong exercise and they decline the sports and exercise performances (Pal et al., 2005). The main reason of increase in 5-HT concentration is due to increase transmission to the brain of blood bone tryptophan and an amino acid precursor to 5-HT most of the tryptophan are loosely bound to albumin but generally they are unbound or present in free state and they are transported across blood brain barrier via a specific mechanism along with other large neutral amino acids like leucine isoleucine and valine (Jessell, Hynes, & Dodd, 1990). Animal studies where are also performed to prove the central factor hypothesis. Chaouloff et al. performed and animal experiment by using rat model in which he allowed 1
2 h of treadmill running of rats and it is observed that there was no effect on plasma total PRP concentration but there was a significant increase in plasma free TRP and 5-HIAA primary

metabolite of 5-HT (Chaouloff, Kennett, Serrurrier, Merino, & Curzon, 1986). Also it was observed that these to TRP and 5-HIAA increase in CSF during prolonged exercise does it suppose supports the hypothesis of Central fatigue you due to excessive exercise the synthesis of 5-HT in brain increases and causes Central fatigue (Crossin, Edelman, & Cunningham, 1984).

9.1.5 Carbohydrates The vertebrate system is made up of complex oligosaccharides spatially cellular interaction within the nervous system are mediated by carbohydrates that are present on the cell surface. The complex oligosaccharides are present in the association with ceramides in glycolipids or attached via N- or O-linkages to protein backbones (Bollensen & Schachner, 1987). A large number of endogenous proteins have been characterized that bind to distinct surface oligosaccharides on vertebrate cells. These carbohydratebinding proteins can be subdivided into several categories on the basis of their primary structure and biochemical properties (Gupta, Mazumder, Pal, Bhattacharya, & Chakrabarty, 2003). C-type lectins (calcium dependent): They refer to the lectins a class of carbohydrate that needed the presence of calcium to exert its binding activity. The structure of binding domains consists of a set of 18 amino acids that involves cysteine residue with disulfide bond and it is an important element which is needed to exert carbohydrate-binding activity. Lectins also consist the chicken hepatic-N-acetylglycosamine receptor that are soluble rat mannosebinding proteins, the pulmonary surfactant apoprotein, cartilage proteoglycan and lymphocyte homing receptor. S-lac type lectins (calcium independent soluble carbohydrate-binding proteins): They refer to low molecular weight soluble carbohydratebinding proteins that are isolated from a

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9.1 Introduction

variety of tissues and institute a separate class of lectins. They are also consists of a conserved set of amino acid which is necessary for carbohydrate-binding function but in comparison with c-type lectins they can be invited by oxidation and also lacks invariable cysteine residue although they have a significant similarity to that of the c-type lectins and also many of them show specific binding activity for beta-glycosides. They involved two class of proteins that are termed as RL 14.5 and RL 29 and they are known for their selectivity within developing nervous system Soluble lectins with mannose-binding properties have also been identified in neural tissues. Membrane bound or soluble glycosyltransferases: when there is no appropriate nucleotide sugar donors then glycosyltransferase exert carbohydrate-binding proteins activity. The consist of cDNA clones in their primary structure and do not have any striking sequence similarities with definite saccharide binding proteins (Cole & Akeson, 1989; Dodd & Jessell, 1985).

9.1.6 Role of carbohydrates on nervous system 9.1.6.1 In retino-tectal system It’s evident that carbohydrate provides biochemical support to the retino-tectal system and it was measured by the adhesion of dissociated dorsal or ventral retinal cells (Chou et al., 1986). Barbera et al. performed a treatment in which he treated ventral retinal cells or ventral tectum with proteus for it was observed that the adhesion of the retinal cells to their matching tectals halve was inhibited on the other hand when similar protease was treated with dorsal retinal cells or dorsal tectum did not show any alter adhesion. However, when the dorsal retinal or dorsal tectyl cells were treated with an N-acetylhexosaminidase or sialidase resulted in a decrease in specific retina tectal adherence activity (Barbera, 1975). Marchase

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(1977) proposed the existence of two opposing gradients of complementary molecules: a protease-insensitive molecule containing a terminal N-acetylgalactosamine residue that is more concentrated in dorsal retina and tectum, and a protease-sensitive molecule that is more concentrated in the ventral retina and tectum (Marchase, 1977).

9.1.7 In sensory organs From various literatures service it is evident now that specific complex oligosaccharide antigens expressions are involved in primary sensory neurones and peripheral sensory receptors cells that are present in olfactory, auditoriy and so sensory systems. Each distinct class of sensory afferents project to specific domains in the spinal cord that coincide with the laminar divisions originally defined on the basis of spinal cord neuronal cytoarchitecture (Edelman, 1986). A bunches of functional classes of dorsal root ganglion have been explained on the basis of their anatomy and their function. Carbohydrates epitopes are categorize in 2
3 class of oligosaccharides (1) globoseries (2) lactoseries and (3) ganglioseries. These all saccharides are expressed on subsets of dorsal root ganglion neurones. The expression of these oligosaccharides on subsets of DRG neurons correlates with the central projection sites of physiologically characterized subclasses of DRG neurons and defines subsets of DRG neurons during development (Dubois et al., 1986).

9.1.8 Glycans They are present in the form of glycoproteins, glycolipids, and amino glycans. Impart a major role in the CNS development learning memory and behavior. Any unbalanced biosynthesis of these glycans can become the major reason of various neurological disease or disorders such as Parkinson’s, Alzheimer’s, autism, etc. It consists monosaccharides that

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are bind together through O-glycosidic bond and can be bound to protein and lipid covalently N-linked glycans, Hyaluronic acid, glucose, aminoglycosides are the example of such glycoconjugates. During the neural development these glycans are associated with cellular changes in cells adhesion signal transduction molecular trafficking and differentiation. To visualize and target the glycan structures in the CNS, glycan binding protein and antibodies are used. Lectins are one of the most wellknown a glycan binding protein which is used to identify glycan residue such as sialic acid, fucose, galactose, mannose, etc. On the other hand IgM and IgM are the example of antibodies that are currently available as antiglycan antibodies (Tiemeyer, Yasuda, & Schnaar, 1989).

9.1.9 Role of glycan in neural development A glycan rich molecule plays a critical role in the development and maintenance of CNS. SSEA1 (stage specific embryonic antigen 1) and tumor rejection antigens (TRA1) are an example of glycan antigen. The mainly undergo

with dynamic changes over the period of time from differentiation to mature neurones and also glycans on the cell surface of neural stem cells also so they helps to identify the target cells during various development stages example of such glycoconjugates data observed in CNS are glycol-lipids and gangliosides. Glycosphingolipids are those glycans which are associated with ceramide liquid course structure and on the other hand gangliosides are defaults to sialic acid with which are present abundantly in the CNS and other tissues and body fluids. Gangliosides are divided into various classes and each has its own effect on CNS and they undergo with major changes in their expression during the development of brain from embryonic to adult. In Table 9.1 functions of different gangliosides are mentioned (Iqbal, Ghanimi Fard, Everest-Dass, Packer, & Parker, 2019).

9.1.10 Lipids 9.1.10.1 Phospholipids They are essential components that are present in mammalian cells and they are involved

TABLE 9.1 Different types of gangliosides and their functions. Gangliosides

Nature

Area

Functions

GD2

Simple

Neural stem cells, embryonic Act as active biomarker for tumor. brains

GM3

Simple

Neural stem cells, embryonic Precursor for the ganglioside species, neuritogenesis, brains immunologic role in Rheumatoid arthritis.

GD3

Simple

Neural stem cells, embryonic Act as biomarker to target and imaging neural stem cell, CNS brains signaling.

GM1

Complex Adult brain cell

Improve intellectual ability, modifies the process of differentiation, amplifies, response to neutrotrophic factors, protect against excitatory amino acid related neurotoxicity, reduces acute nerve cell damage.

GD1a

Complex Adult brain cell

Helps in synthesis and maintenance of GM1.

GD1b and GT1b

Complex Adult brain cell

protect against excitatory amino acid related neurotoxicity, reduces acute nerve cell damage.

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9.1 Introduction

in various biological activities such as formation of lipid bilayers as a precursor for different lipid second messengers. If any miss regulation occur in lipid metabolism it may become the reason of CNS disorders or may cause serious brain injury (Kirkland, Adibhatla, Hatcher, & Franklin, 2002).

9.1.11 Role of cPLA2 in cerebral ischemia Many in vitro studies have been performed to observe the upregulation of phospholipase A2 (PLA2) in various cerebral ischemia models first of rat hippocampus slices were subjected to oxygen glucose deprivation and reoxygenation. It was found that PLA2 activity increased by twofold after 30 min of OGD and remained in higher level at 24 h reoxygenation. When cPLA2 in which was used it causes low OGD induced neuronal death leading to involvement of cPLA2 in ischemia injury (Simmons, Botting, & Hla, 2004).

9.1.12 In the case of neurodegenerative diseases mRNA expression of proinflammatory sPLA2 IIA was up-regulated in AD brains compared to nondementia elderly brains. sPLA2 IIA immunoreactive astrocytes in AD hippocampus were associated with Aβ plaques. Aβ and NMDA induced cPLA2 phosphorylation and ArAc release via activation of nicotinamide adenine dinucleotide phosphate oxidase, ROS generation, and activation of ERK1/2 in cortical neuronal cultures. Aβ induced mitochondrial dysfunction through iPLA2 and cPLA2 in cultured cortical astrocytes. Free radical generation and lipid peroxidation play a significant role in PD. One of the factors responsible for this is believed to be phospholipases activation in substantia nigra, supported by the fact that cPLA2 deficient mice are resistant to 1-methyl-4-phenyl-1,2,3,6-tetrohydropyridine induced neurotoxicity, an animal model for PD (Liu et al., 2006). In Fig. 9.2

225

different CNS pathologiesin various disease/disorders of PLA2 is mentioned.

9.1.13 Lipid peroxidation It is a radical persuade chain reaction which is mainly responsible for cell destruction in the secondary injury process which is commenced by head and spinal injury and stroke (Simmons et al., 2004). Many studies have been done and it was observed that liquid peroxidation may lead to various neurodegenerative diseases or disorders such as AD, Parkinson disease and cerebral ischemia. In a recent study, it was observed that the patients suffered from mild cognitive disorder and early AD have high level of HNE and acrolein in the brain tissues which indicates that liquid peroxidation process is happening in the early in the pathogenesis of the AD. In addition reactive oxygen species such as hydrogen peroxide and superoxide radicals are product of cellular oxidative metabolism process which involves metabolism of ArAc by cyclooxygenase (COX) and lipoxygenases (LOX). These Reactive oxygen species have a major role in amyloid protein deposition, aggregation of Aß
peptides and tau proteins aggregation which leads to AD and many other disease like PD, Huntington’s Disease (Rosenberger, Villacreses, Contreras, Bonventre, & Rapoport, 2003). Transient forebrain ischemia in gerbil (which lacks circle of Willis and therefore collateral blood flow) is characterized by delayed hippocampal CA1 neuronal death. Neurodegeneration is evident by 3 days reperfusion and neuronal death culminates by 6 days. Five minutes of forebrain ischemia and 6 h reperfusion in gerbil resulted in significantly increased levels of MDA, HNE and lipid hydroperoxides in the cortex, striatum and hippocampus and thus preceded the onset of neuronal death. These increases persisted over 4 days reperfusion in the ischemia-vulnerable hippocampus. Lipid peroxidation was assessed

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Alzheimer’s disease: Up-regulaon leads to lipid peroxidaon

Parkinson’s disease: protect against MPTP toxicity

Wallerian degeneration: PLA2 plays an important role in myelin breakdown and phagocytosis. Upregulate cPLA2 in a positive feedback manner

Activity of PLA2

Spinal Cord Injury: PLA2 injected to spinal cord increase TNF-α, IL-1ß and HNE.

Regulates the patho -physiology of

Transient focal cerebral ischemia: increase cPLA2 experssion. PLA2 inhibitors reduces infraction size.

Multiple-Scerolosis Experimental Autoimmune Encephalomyelitis .

FIGURE 9.2

Various roles of PLA2 in CNS pathologies.

using thiobarbituric acid assay in rat brains after 30 min forebrain ischemia and up to 72 h reperfusion. Lipid peroxide levels were unchanged during ischemia and 1 h reperfusion but increased between 8 and 72 h of recirculation in the ischemia-vulnerable hippocampus, with the greatest increase at 48 h (Shelat et al., 2008).

or Nucleic acid (mRNA), and any of them can follow up or cause down-regulation and can modulate the pathophysiology of any neurodegenerative disorder/disease such as the role of cPLA2 (17) in ischemic injury. If more research work will continue, the functions and pathophysiology of these macromolecules on CNS injuries and disease can be revealed in a detailed manner in the nearby future.

9.2 Conclusion According to the published findings so far, biological macromolecules play a critical role in the modulation of CNS during injuries or any neurodegenerative disorder. It can be carbohydrates (glycans), lipids (PLA2), proteins,

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C H A P T E R

10 Biological macromolecules as antidiabetic agents Jaison Jeevanandam1, Caleb Acquah2 and Michael K. Danquah3 1

CQM—Centro de Quı´mica da Madeira (Madeira Chemistry Center), MMRG, Universidade da Madeira (University of Madeira), Campus da Penteada (Penteada campus), Funchal, Portugal 2School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada 3 Chemical Engineering Department, University of Tennessee, Chattanooga, TN, United States

10.1 Introduction Diabetes is the most common metabolic disorder with the potential to disrupt normal human life, decrease productivity, and can also lead to death (Oso´rio, Silva, & Ferreira, 2021). Cases of diabetes are globally widespread among different age groups, gender, and geographical region (Duarte & Golubnitschaja, 2020). There are three major types of diabetes viz type 1, type 2, and type 3 affecting almost 80% of the world’s population above the age of 35 (Alotaibi, Perry, & Gholizadeh, 2017). Type 1 diabetes occurs when the β-cells in the pancreatic islets fail to secrete insulin stemming from an autoimmune condition, while type 2 diabetes is as a result of poor insulin secretion due to the dysfunctioning of β-cells and/or insulin resistance. Meanwhile, type 3 diabetes is linked with chronic insulin resistance and insulin deficiency in the brain. Advancements in the medical field have resulted in several antidiabetic agents for the

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00010-5

treatment and cure of the major types of diabetes (He, Zhou, & Yang, 2015). Synthetic drugs such as metformin, acetohexamide, chlorpropamide, glibenclamide, and tolbutamide are being used extensively for several years to date in the treatment of diabetes (Harrower, 1996; Hidayat, Du, & Wu, 2019). The mechanism of action of the synthetic drugs involve one or more of the following: (1) inhibiting carbohydrases, (2) inhibiting dipeptidyl-peptidase-IV (DPP-IV), (3) enhancing glucagon-like peptide-1 (GLP-1) receptors, (4) inhibiting sodium glucose cotransporter 2, (5) prevent hepatic gluconeogenesis, (6) increase glucose uptake, (7) act as insulin secretagogues, and/or (8) decrease glucose production (Acquah, Agyei, & Obeng, 2020a; Acquah, Dzuvor, & Tosh, 2020b). Even though synthetic antidiabetic agents are beneficial in reducing the complications of diabetes they are fraught with severe side-effects and toxicity (Euglucon & GlibenLich, 2018; Kinoshita, Hosomi, & Yokoyama, 2020). As a

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result, biological macromolecules are recommended as superior potential antidiabetic agents for the treatment of the major types of diabetes. Generally, larger molecules that are made up of smaller organic molecules and necessary for living organisms are considered as biological macromolecules (Young, Hundt, & Cole, 2018). These macromolecules are widely classified into lipids, carbohydrates, proteins and nucleic acids. All these distinct classes of macromolecules are highly essential as cell components and in a wide range of functions (Krishnaswami, Kandasamy, & Alagarsamy, 2018). The building blocks of carbohydrates are simple monosaccharide sugars (Krishnaswami et al., 2018), whereas lipids are made up of fatty acids and glycerol (Liu, Kou, & Zhang, 2018). Likewise, the building blocks of proteins are amino acids while nucleic acids are made up of nucleotides (Freitag & Wagner, 2021). The building blocks of these macromolecules can be modified via enzymatic and chemical methods to form beneficial biological macromolecules for pharmaceutical applications (Pawar, Kamat, & Choudhary, 2015). Recently, these biological macromolecules have been utilized in different modalities as antidiabetic agents to increase insulin secretion, reduce blood glucose, beta-cell proliferation and inhibition of certain types of diabetes while reducing complications (Wang, Zhang, & Xu, 2013). For instance, the consumption of dry legumes which have been biologically processed using techniques such as germination and fermentation have been demonstrated in vitro to convert inherent macromolecules into potent bioactive compounds to inhibit the activities of DPP-IV and α-glucosidase (Di Stefano, Tsopmo, & Oliviero, 2019). Endogenous digestive enzymes such as DPP-IV, α-glucosidase, and α-amylase are essential in modulating postprandial glucose levels in the blood. As such, this chapter provides an overview of the different types of biological macromolecules

and their applications as potential antidiabetic agents. It also discusses the advantages, limitations and future perspectives of biological macromolecules as antidiabetic agents.

10.2 Types of biological macromolecules Carbohydrates, lipids, proteins and nucleic acids are the major types of biological macromolecules, as mentioned in the previous section. Generally, the main function of carbohydrates is to provide cells with short-term energy from food sources (McKeown, Meigs, & Liu, 2004). The common examples of carbohydrates as biological macromolecules are starch, glucose, cellulose, sucrose and chitin. Lipids, which include fats, waxes, phospholipids, oils, steroids and grease, provide long-term energy to the cells and are beneficial in the formation of biological membranes. Similarly, protein macromolecules such as keratin, enzymes, hormones and antibodies are essential for the maintenance of cell structure, send chemical signals and catalyze chemical reactions. Moreover, nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are useful in the storage and transfer of genetic information (Boodhun, 2018; Westhof, 1993). The method of extraction is the crucial process which influences the bioactivity and stability of these macromolecules (Liu et al., 2018). Enzymeassisted, supercritical-fluid and microwaveassisted extraction are the three significant types of approaches that are used to extract the macromolecules from biological sources as shown in Fig. 10.1. Enzyme-assisted extraction method is utilized to release the compounds that are bound in the interior side and are not accessible via conventional solvent extraction approaches (Rosenthal, Pyle, & Niranjan, 1996). This approach utilizes enzymes to facilitate the extraction and release of highly valuable as well as active biological macromolecules. The extraction of bioactive macromolecules in this approach

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10.2 Types of biological macromolecules

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FIGURE 10.1 Flow chart of (A) enzyme-mediated extraction, (B) Supercritical-fluid extraction and (C) Microwaveassisted extraction of bioactive macromolecules. Source: Reproduced with permission from Elsevier, Bilal, M., & Iqbal, H. M. N. (2020). Biologically active macromolecules: Extraction strategies, therapeutic potential and biomedical perspective. International Journal of Biological Macromolecules, 151, 1
18.

depends on the nature and source of the material, as well as the internally interlinked bioactive macromolecules with hydrogen or hydrophobic bonds (Singh, Sarker, & Kumbhar, 1999). Enzyme-assisted extraction possesses advantages such as greater selectivity, simple extraction procedure, induced efficacy, safer working conditions, recyclability, easy isolation and recovery of product, enhanced yield, minimal requirement of energy, no waste production or deprotection stage and less usage of toxic substance compared to conventional solvent-based extraction method (Bilal & Iqbal, 2020). Likewise, the supercritical fluid extraction approach is extensively utilized for the isolation and extraction of bioactive macromolecules, especially, fatty acid and pigments (Garcı´a-Pe´rez, Robledo-Padilla, & CuellarBermudez, 2017). This method requires only a few solvents and has a great impact on the

solubility of the extracted macromolecules. Notably, the size and concentration of the sample are the parameters to be optimized, which determines the yield of the extracted macromolecules and reduces the cost of the extraction process (Roodpeyma, Guigard, & Stiver, 2018). Further, this approach possesses advantages such as swift processing, enhanced selectivity and low degradability of the extracted biomolecules (Bilal & Iqbal, 2020). Furthermore, the microwave-assisted extraction approach is mainly employed in the extraction of polyphenolic molecules, biologically significant compounds and phytonutrients that are highly valuable in the pharmaceutical industry (Yuan & Macquarrie, 2015). In this method, the irradiation of microwave is repeated several times with uninterrupted cooling phases to avoid the thermal degradation of the extracted macromolecules

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and enhance the extraction yield (Ying, Han, & Li, 2011). Moreover, solvents with distinct polarities index such as ethanol, acetone, methanol, acetonitrile and dichloromethane were also used to extract the specific bioactive macromolecules from the biological fluid complex (Bilal & Iqbal, 2020).

10.3 Biological macromolecules It is noteworthy that not all biological macromolecules are utilized as antidiabetic agents. Only macromolecules that are biologically active or activate biomolecules are commonly used as antidiabetic agents. Also, these macromolecules can be immobilized on the surface of conventional synthetic antidiabetic drugs to improve their efficiency via targeted and controlled delivery (Tran, Pham, & Le, 2020).

10.3.1 Carbohydrates Recently, Zhang, Ma, and Wu (2020) utilized type 3 resistant starch from Canna edulis to exhibit antidiabetic activity. In this study, the resistant starch was injected in type 2 diabetic rats for the evaluation of their antidiabetic activity after 11 weeks of treatment. The study showed that the resistant starch altered the gut microbiota and the profiles of metabolism in serum. Compared to the synthetic antidiabetic drug, metformin, the resistant starch lowered blood glucose levels and enhanced the response of endogenous enzymes such as lipolytic enzymes to reverse insulin resistance and tolerance toward glucose for the treatment of type 2 diabetes mediated pathological damages in rats. Further, the starch exhibited improved efficacy to promote the diversity of gut microbiota, especially, Prevotella genera enrichment in diabetic rats (Zhang et al., 2020). Similarly, Varadharaj et al. (2019) fabricated 19.8 nm sized starch nanoparticles with an ellipsoidal morphology via Gymnema sylvestre using organic solvents.

The starch nanoparticles synthesized via a methanolic extract of G. sylvestre, as shown in Fig. 10.2, exhibited enhanced antidiabetic activity by inhibiting the activity of alpha-amylase enzyme (Varadharaj, Ramaswamy, & Sakthivel, 2019). According to Liu, Gao, and Tang (2017) the nonstarch polysaccharides from plants possess an enhanced ability to inhibit crucial enzymes such as DPP-IV, alpha-glucosidase, and alpha-amylase that are linked to type 2 diabetes. The polysaccharides from the pulp of Armeniaca sibirica, seeds of Plantago asiatica, corn silk, seaweeds, Citrus paradise, mulberry fruit, Camellia sinensis, and nonspecific binding effect of cellulose with starch have essential alphaamylase inhibitory effect. Further, nonstarch polysaccharides from Avena sativa, Cucurbita moschata, fucoidan from Sargassum wightii and Ascophyllum nodosum can inhibit the activity of alpha-glucosidase. Furthermore, polysaccharides extracted from the seeds of Plantago asiatica, cellulose, pectin and beta-glucan have been demonstrated to possess inhibitory activity toward lipase, which eventually reduces diabetes-related obesity, absorption of lipids and protects the pancreas (Liu et al., 2017). Chitosan is a carbohydrate polysaccharide that can be extracted from plants and animals and has an antidiabetic activity. Chitosan is beneficial in the formulation of insulin with controlled delivery functionality at the target site. Carboxymethyl-hexanoyl derivative and polyethylene glycol (PEG)-trimethyl complexes are some of the examples of chitosan as bioactive macromolecules for the effective delivery of insulin. That aside, the chitosan-based collagen complex sponges are beneficial in the healing of diabetic wounds (Islam, Rahman, & Ahmed, 2020). These chitosan complexes were reported to possess fat-lowering, fat-preventing property, hypocholesterolemic agent to decrease up to 50% of blood cholesterol level and increase insulin sensitivity in diabetic animal models by decreasing triglyceride levels and body weights. Moreover, administration of acetylated chitin in

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FIGURE 10.2 Scanning electron microscopic image of starch nanoparticles. Source: Reproduced with permission from Springer Varadharaj, V., Ramaswamy, A., Sakthivel, R. et al. (2019). Antidiabetic and antioxidant activity of green synthesized starch nanoparticles: An in vitro study. Journal of Cluster Science, 1
10.

diabetic models is reported to break down fats in adipocytes and decrease the accumulation of 50% fats. Likewise, chitosan and its associated oligonucleotides have been proven to protect pancreatic beta-cells while metal crosslinked chitosan complex reduces phosphorus levels in serum for chronic renal failure treatment associated with diabetes complications (Karadeniz & Kim, 2014). Furthermore, baicalin from Scutellaria baicalensis, pseudoprotoimosaponin AIII, prototimosaponin AIII from Anemarrhena asphodeloides, charantin from Momordica charantia, steroidal saponin of Ganoderma applanatum, choragin from Chlorophytum nimonii and elastosides G, H and I are the other carbohydrate-based antidiabetic agents that have been proposed to be beneficial for the treatment of diabetes

(Mishra, Singh, & Mishra, 2016). Recently, Han, Sheng, and Li (2019) fabricated novel berberine derivatives modified by carbohydrates to exhibit an antidiabetic property. The study showed that the mannose moiety conjugation with berberine derivative had 1.5 times lower cytotoxicity toward HepG2 cell lines compared to berberine with enhanced antidiabetic property by reducing glucose levels (Han et al., 2019).

10.3.2 Lipids Similar to carbohydrates, lipids have been recently utilized as potential antidiabetic agents. It has been reported that the fatty acid-derived lipids possess the ability to endogenously

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activate G protein-coupled receptors (GPCR) for the treatment of type 2 diabetes. It is worthy to note from literature that the specific agonist named fasiglifam (TAK-875), which is a GPCR, can target free fatty acid receptor 1 (FFA-1) to control glucose level in diabetic patients without leading to an increase in body weight and hypoglycemic condition when compared to synthetic antidiabetic agents (Tsujihata, Ito, & Suzuki, 2011). Generally, FFA is a signaling molecule to regulate several physiological and cellular processes with a carboxylic acid that is linked to an aliphatic tail. In particular, FFA-1 is reported to be expressed in pancreatic beta-cells that secrete insulin (Milligan, Shimpukade, & Ulven, 2017). Likewise, AMG-837, P-11187, JTT-851, ASP5034, LY-2881835, LY-2922470, LY-2922083, PBI4050, MK-8666 and SHR-0534 are the other fatty acid-derived lipids that can activate and modulate GPCRS for targeting FFA-1 to serve as antidiabetic agents. Further, KDT-501 (GPCR) that targets FFA4; oleoyl glycerol, APD-668, MBX2982, PSN-821, BMS-903452, APD-597, DA-1241, ZYG-19, DS-8500a and LEZ-763 that targets GPR119; and rimonabant, AZD-2207, AZD1175, TM-38837 and tetrahydrocannabivarin-9 to target CB1 are the other fatty acid-derived lipids (Gendaszewska-Darmach, Drzazga, & Koziołkiewicz, 2019). Recently, lipids were also fabricated into nanoparticles for the targeted and controlled delivery of insulin and other antidiabetic agents. Ahangarpour, Oroojan, and Khorsandi (2018) extracted a novel antioxidant named myricitrin from a plant and fabricated it into a solid lipid nanoparticle. These lipid nanoparticles were administered toward hyperglycemic myotube and type 2 diabetic mouse models that were induced by streptozotocin and nicotinamide to evaluate their effects. The results showed that the lipid nanoparticles regulated the body and pancreatic tissue weight, as shown in Fig. 10.3, reduced oxidative stress, hyperglycemia, insulin resistance, increase Glut-4 gene expression, index of bell cell

function and minimize pancreas apoptosis for the treatment of type 2 diabetes. Additionally, the study also emphasized that the lipid nanoparticles possess enhanced ability to improve antioxidant defense, glycogen amount and cellular survival of myotube cells to reduce the complications related to hyperglycemic condition (Ahangarpour et al., 2018). Similarly, Shah, Chavda, and Vyas (2020) formulated a novel antidiabetic drug named linagliptin using solid lipid nanoparticles for the improvement of oral bioavailability. The study showed that the enhancement of oral bioavailability was due to the prevention of permeability of glycoprotein (p-gp) efflux by targeting lymphatic tissue (Shah et al., 2020). Further, Mukherjee, Maity, and Ghosh (2020) assessed the antidiabetic activity of solid lipid nanoparticles made up of glyceryl monostearate that were loaded with glyburide (a class II sulfonylurea) to improve its oral glucoselowering effect. The results demonstrated that the lipid nanoparticles enhanced the glucose uptake ability in diabetic rats by normalizing blood glucose level and serum biochemical parameters such as level of insulin, glutamate pyruvate transaminase, glutamate oxalate transaminase, catalase, lipid peroxidase, hemoglobin (HbA1c) and glutathione (Mukherjee et al., 2020). Moreover, Sarker, Ali, and Barman (2018) synthesized novel solid lipid nanoparticles to load nifedipine for the enhancement of their antidiabetic activity via targeted dihydropyridine calcium channel block in diabetic rats induced by fructose. The study emphasized that the lipid nanoparticles improved the glucose lowering property of nifedipine by exhibiting antihyperglycemic activity, improving lipid profiles, reducing creatinine, uric acid levels, maintaining excellent cationic balance, regaining of hepatocyte structure, reducing bilirubin and serum glutamic-oxaloacetic transaminase with enhanced bioavailability when compared to free nifedipine (Sarker et al., 2018).

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FIGURE 10.3 Effect of myricitrin and solid lipid nanoparticles on the pancreatic tissue size. Source: Reproduced with permission from Hindawi Ahangarpour, A., Oroojan, A. A., Khorsandi, L. et al. (2018) Solid lipid nanoparticles of myricitrin have antioxidant and antidiabetic effects on streptozotocin-nicotinamide-induced diabetic model and myotube cell of male mouse. Oxidative Medicine and Cellular Longevity, 2018, 7496936.

10.3.3 Proteins It has been reported in literature that RNAbinding proteins are the significant regulators of RNA networks in the post-transcriptional stage that are dysregulated in diabetes conditions. These proteins are important for the function and survival of the cells as they are involved in the cell’s proliferation, differentiation and apoptosis. It is noteworthy that the RNA-binding

proteins such as Elav-like protein 1 (Elav— Drosophila neuron-specific protein), heterogeneous nuclear ribonucleoprotein K (hnRNP K), Y-box binding protein1 (YBX1), hnRNP F, LIN28 (encoded by LIN28 gene) and insulinlike growth factor 2 mRNA-binding protein 2 are dysregulated in diabetic nephropathy and diabetic kidneys. Furthermore, LIN28, RBP Fox1 homolog 2, fat mass and obesity-associated protein, eukaryotic initiation factor 4E and CUG

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triplet repeat binding protein ELAV-like family member 1 are the RNA-binding proteins that are identified to be dysregulated in diabetic conditions. Thus formulation of these proteins and delivering them to the diabetic cells via an efficient drug delivery system minimizes diabetes-related complications (Nutter & Kuyumcu-Martinez, 2018). Similarly, Niewczas, Pavkov, and Skupien (2019) evaluated about 196 circulating inflammatory proteins in three independent cohort type 1 and type 2 diabetic subjects. The study emphasized that some 17 proteins linked to extremely robust kidney risk inflammatory signature can contribute to inflammation in diabetic subjects, which eventually leads to renal disease (Niewczas et al., 2019). Hence, KRIS protein inhibitors such as kinases were proposed to be useful to reduce diabetic complications (Xiang, Ekinci, & MacIsaac, 2019). On another note, Moreno-Valdespino, LunaVital, and Camacho-Ruiz (2020) stated that bioactive proteins extracted from legumes can be highly beneficial in the prevention of obesity and type 2 diabetes as displayed in Fig. 10.4. Globular phaseolin protein and protein hydrolysates from Phaseolus vulgaris, black bean hydrolyzed protein lysate and protein concentrate

from legumes help reduce diabetes complications and control glucose level in in vitro and in vivo models (Moreno-Valdespino et al., 2020). Besides, Paula, Sousa, and Oliveira (2017) demonstrated that a protein isolate from the leaves of Moringa oleifera possess hypoglycemic effect in alloxan-induced diabetic mice. The study showed that the protein isolate possesses enhanced hemagglutinating property, zinc addition mediated precipitation and anti-insulin antibody facilitated cross-reaction. In addition, the study revealed that a single-dose protein isolate administration intraperitoneally can reduce 66.4% of blood glucose level in 5 h and 56.2% in 7 days (Paula et al., 2017). On another note, processing of diet embedded within dietary proteins are bioactive peptides and amino acids with various therapeutic functionalities which include modulation of endogenous enzymes involved in glucose regulation and prevention of obesity (Acquah et al., 2020a, 2020b; Gnasegaran, Agyei, & Pan, 2017). For instance, bioactive peptides generated from Amaranthus hypochondriacus, Oryza sativa, Glycine max, Lupinus albus and Chenopodium quinoa were shown to have dipeptidyl peptidase 4 (DPP 4) inhibitory activity, whereas peptides

FIGURE 10.4 Schematic of bioactive protein macromolecules from legumes with antidiabetic activity. Source: Reproduced with permission from Elsevier; Moreno-Valdespino, C. A., Luna-Vital, D., Camacho-Ruiz, R. M. et al. (2020). Bioactive proteins and phytochemicals from legumes: Mechanisms of action preventing obesity and type-2 diabetes. Food Research International, 130, 108905.

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from Cannabis sativa, Avena sativa, Phaseolus vulgaris, C. quinoa and Morus alba inhibited the activity of alpha-glucosidase (Patil, Goswami, & Kalia, 2020). Likewise, bioactive peptides extracted from P. vulgaris, Cuminum cyminum and C. quinoa were utilized as alpha-amylase inhibitors, while peptides from P. vulgaris were utilized for the inhibition of glucose transporter 2 (GLUT2) and sodium-glucose co-transporter2 (SGLT2) (Patil et al., 2020). Additionally peptides from G. max and Momordica charantia as hypoglycemic agents. Further, protein hydrolysates from the fruits of Juglans mandshurica, Momordica cymbalaria, Theobroma cacao, M. charantia and M. dioica, seeds of Hordeum vulgare, Triticum aestivum, Vigna angularis, Linum usitatissimum, Vigna unguiculata, Glycine max, Phalaris canariensis, Cucurbita pepo, Phaseolus vulgaris, Citrullus lanatus, Moringa oleifera, Chenopodium quinoa and Terfaira occidentalis were are also proven to possess antidiabetic activity (Patil et al., 2020). Furthermore, protein hydrolysates extracted from the leaves of Spinacia oleracea and whole plant of Lemna gibba G3 were reported to possess insulin mimicking property and the protein lysates from the rice bran of Oryza sativa were proven to improve insulin resistance (Patil et al., 2020).

10.3.4 Nucleic acids Numerous nucleic acids have also been employed as antidiabetic agents or to support conventional antidiabetic agents for the effective treatment of diabetes. Kaur, Bhatia, and Sethi (2017) demonstrated that chitosan nanoparticles loaded with probiotic DNA possessed hypoglycemic activity. In this study, diabetic Balb/c mice were treated with probiotic DNA extracted from the bacteria Lactobacillus acidophilus NCDC 343 to evaluate their antidiabetic potential. The results showed that probiotic DNA has hypoglycemic activity in the diabetic mice models and was improved by

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the chitosan nanoparticles via targeted and controlled delivery of the DNA (Kaur et al., 2017). Further, Cha, Lee, and Kang (2018) demonstrated that oral delivery of therapeutic DNA mediated by antibodies was effective for the treatment of type 2 diabetes mellitus. In this study, human immunoglobin G1 combined with neonatal Fc receptor, arginine and plasmid DNA was formed as a complex structure via electrostatic complexation reaction to improve its biodistribution and antidiabetic property as glucagon-like peptide-1 (GLP-1) gene delivery system in Balb/c mice or Lepdb/db diabetic mice model. The study showed that the oral administration of cationic therapeutic DNA complex with antibodies with a size of 40
60 nm prolonged the delivery of GLP-1 with less immunoactivity for the enhanced treatment of type 2 diabetes (Cha et al., 2018). Furthermore, Chang (2017) recently reported that the combination of berberine and noncoding RNA can be highly beneficial in the improvement of their antidiabetic activity. The author emphasized that the functional RNAs such as microRNA, transfer RNA (tRNA), piwiinteracting RNA and long noncoding RNA can exhibit a unique mechanism to activate certain complex intracellular enzyme systems for the treatment of diabetes. Thus these noncoding RNAs such as hepatic miR-122 is combined with berberine for the regulation of gluconeogenesis. Likewise, berberine was combined with adipose miR-30 for the regulation of diabetes-related autophagy and hepatic miR-29 to exhibit antidiabetic property. Besides, long noncoding RNA was combined with berberine for the identification of diabetes-mediated fatty liver disease induced by a high-fat diet (Chang, 2017). ¨ c¸eyler, and Soreq (2020) Recently, Meydan, U also demonstrated that noncoding RNA can be beneficial as a regulating agent of diabetic polyneuropathy. The authors showed that the miRNAs such as miR-146a, 106a, 9, 29b, 98 and 466a possess enhanced ability to regulate inflammation, pain and metabolic syndrome

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pathways to reduce the complications of diabetic polyneuropathy (Meydan et al., 2020).

10.4 Advantages, limitations, and future perspectives In general, bioactive macromolecules are highly beneficial in reducing several diabetic complications such as controlling glucose level, increasing insulin secretion and beta-cell proliferation (Patil et al., 2020). Also, the macromolecules possess therapeutic properties such as anticancer, cardio-protective, skin curative, ultraviolet ray protectant and immunomodulation (Bilal & Iqbal, 2020). Further, immobilizing the biological macromolecules on the surface of conventional antidiabetic agents improves the bioactivity, bioavailability and biocompatibility of the later (Unuofin & Lebelo, 2020). Even though bioactive macromolecules are beneficial as antidiabetic agents, they are poor in oral absorption, difficult to control their stability in biological fluids and maintain their structural integrity for a long time to exhibit their antidiabetic property (Tiwari, Melchor-Martı´nez, & Saxena, 2021). It is recommended that individual bioactive macromolecules are formulated with a biocompatible molecule or material to enhance their stability in a biological environment as antidiabetic agents. Recently, nanoparticles have been widely utilized to formulate bioactive macromolecules to improve their bioactivity, bioavailability and stability for delivering to target sites in a controlled approach (Martı´nez-Ballesta, Gil-Izquierdo, & Garcı´aViguera, 2018). Wong, Al-Salami, and Dass (2020) recently synthesized novel polymericoligonucleotide nanoparticles loaded with insulin with enhanced stability and bioactivity for the efficient treatment of diabetes (Wong et al., 2020). Similarly, complexes of mucinchitosan were synthesized as mucoadhesive nanoparticles loaded with insulin for orally enhanced the treatment of diabetes (Mumuni,

Kenechukwu, & Ofokansi, 2020). Moreover, polysaccharides extracted from natural resources were synthesized as both micro and nanoparticles and utilized for the formulation of insulin in oral administration (Meneguin, Silvestre, & Sposito, 2020). Besides, aptamers can also be used for the enhanced delivery of bioactive macromolecules as well as to improve their antidiabetic activity (Acquah et al., 2020a, 2020b). Aptamers are peptide or oligonucleotide molecules that molecularly interact with a specific target molecule (Jeevanandam, Sabbih, & Tan, 2021). These aptamer formulated bioactive macromolecules have been reported to be beneficial in enhancing their antidiabetic activity via targeted and controlled delivery approach (Ru¨tter, Miloˇsevi´c, & David, 2020). Thus nanoparticles, nanocomposites and aptamers or a combination of nanoparticles and aptamers can be a potential formulation and drug delivery system to improve the antidiabetic potential of bioactive macromolecules in the future. Similarly, virus nanoparticles have also been proposed as a potential macromolecule delivery system that can serve as a nanosized self-sustaining medicine to treat diseases including diabetes (Jeevanandam, Pal, & Danquah, 2018). However, the concept is still in its infancy stage and require extensive research for actualization.

10.5 Conclusion This chapter is an overview of various bioactive macromolecules that can be utilized as potential antidiabetic agents in lieu of conventional synthetic antidiabetic drugs. More so, existing synthetic antidiabetic agents can be modified with the biological macromolecules to enhance their applicability and reduction of severe complications linked with the synthetic drugs. The bioavailability, bioactivity, and biocompatibility of these macromolecules are highly beneficial with little to no known toxicity in the treatment of diabetes. However, poor

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oral absorption, low long-term stability, and structural integration are the major limitations with biological macromolecules. This can be improved by formulating biological macromolecules with aptamers or nanoparticles.

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20. Xiang, A. S., Ekinci, E. I., & MacIsaac, R. J. (2019). Inflammatory proteins in diabetic kidney diseasepotential as biomarkers and therapeutic targets. Annals of Translational Medicine, 7(Suppl 6), S243. Ying, Z., Han, X., & Li, J. (2011). Ultrasound-assisted extraction of polysaccharides from mulberry leaves. Food Chemistry, 127(3), 1273
1279. Young, G., Hundt, N., Cole, D., et al. (2018). Quantitative mass imaging of single biological macromolecules. Science (New York, N.Y.), 360(6387), 423
427. Yuan, Y., & Macquarrie, D. (2015). Microwave assisted extraction of sulfated polysaccharides (fucoidan) from Ascophyllum nodosum and its antioxidant activity. Carbohydrate Polymers, 129, 101
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C H A P T E R

11 Biological macromolecules as anticancer agents Himja Tiwari, Harshal Deshmukh, Nilesh Shirish Wagh and Jaya Lakkakula Amity Institute of Biotechnology, Amity University Maharashtra, Mumbai, —Pune Expressway, Bhatan Post—Somathne, Panvel, Mumbai, India

11.1 Introduction Cancer consists of a group of intricate and heterogeneous disease characterized by uncontrolled cell proliferation due to a myriad of biochemical, genetic, and epigenetic changes (Ju´nior et al., 2016). Cancer is the cause of 6 million deaths annually. Alone in 2018, approximately 1.1579 million cancer cases were reported out of which 0.784 million surrendered to the ailment. This is more in developing countries, where lack of medical facilities, basic diagnosis, and affordable treatments lead to mortality. This has led the scientists to develop synthetic drugs (Chemotherapeutics) which although are effective but also cause side effects minimizing the drug’s overall efficacy (Elansary et al., 2018). Since the treatment methods were not meeting the needs of the cancer patients. There was a shift to a major development in the cancer field which paved the way for finding natural compounds for cancer therapy. Application of such compounds have been widely researched due to their

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00011-7

astonishing properties like antimicrobial, antioxidant, carminative, hepatoprotective, anticarcinogenic and antiviral properties (Nasrollahi, Ghoreishi, Ebrahimabadi, & Khoobi, 2019). Especially flavonoids and monoterpenes are found to exhibit anticancerous properties both in vitro and in vivo. Recently the development of biological anticancerous drugs have gained enormous interest with the research community. Biological macromolecules have not only been subject to medicinal purposes, but also for skin care products indicating their safe and wide use. Like, essential oil from oregano (Origanum vulgare) was first popular in skin care products and later was used in scientific research (Han and Parker, 2017). Biological macromolecules include proteins, carbohydrates, essential oils, nucleic acids, fatty acids etc. the use of all these have proved their significance in the treatment of cancer. Not only from plant parts but algae, fungi and even weeds have been used to extract such macromolecules and have successfully proven to work against cancer. Determining the anticancerous properties

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11. Biological macromolecules as anticancer agents

of macromolecules is a tedious process, involving various phases and trial procedures. Not just testing the macromolecules but further finding the chemical compounds present in the extracted macromolecules and testing them individually for their anticancerous behavior. If found are proven to be of immense importance by acting as a natural drug. Some macromolecules are encapsulated in nanomaterials for better and targeted delivery for further enhancing their efficacy. These biological macromolecules have shown to have almost no toxic side effects and the treatment via these are not painful as in chemotherapy. So many botanical compounds like juice, peel and oil of Punica granatum has also been proven to possess anticancerous properties (Lansky and Newman, 2007). Similarly, another natural quinone containing compound β-lapachone extracted from the bark of lapacho tree (Tabebuia avellanedae) is said to have apoptosis inducing effects as it acts on DNA topoisomerase I and inhibits G1 or S phase hence inducing death in ovary, colon, lung, prostate, breast and many more cancers (Nobili et al., 2009). A compound from a family of stilbenoid, that is, Combretastatin A4 which is extracted from the stem wood of the South Africa tree Combretum caffreum also shows necrosis in cancerous cells. It’s Phase 1 trial has been successful and offers a favorable toxicological profile (Banerjee, Wang, Mohammad, Sarkar, & Mohammad, 2008). This chapter comprehensively focuses on the natural compounds or biological macromolecules used in cancer therapy and prevention (Table 11.1). Each section displays different sources for macromolecule extraction along with the various assays conducted.

11.2 Biological macromolecules for cancer therapy 11.2.1 Carbohydrates Carbohydrates are natural compounds which contain hydrogen and oxygen molecules in the

ratio 2:1. They yield energy when broken down, for example, sugars, starch and cellulose. We are becoming well aware of the role of carbohydrates in biological processes diseases. There are various carbohydrates found with bioactivities like anticancer, anti-inflammatory, anti-viral etc. Following are the carbohydrates which exhibit anti-cancer properties with respect to the type of cancer. 11.2.1.1 Cervical cancer The female stigma (Stigma maydis) from the dried maize known as corn silk is usually considered as a waste product but is known to have antioxidant and anti-cancer properties and used in France, United States, Turkey, and China. In this article, the anti-cancer properties of CSP (Corn silk polysaccharide) extracted from the corn silk obtained from Jilin province, China, were evaluated for cervical cancer. The elution curve of purified CSP showed that CSP-3 was present in very low amount compared to CSP-1 and CSP-2, hence only the latter were further studied. Composition of CSP-1 was rhamnose, xylose, arabinose, and galactose in the molar ratio 6.32:1.01:1.00:4.16 and CSP-2 was rhamnose, glucose, arabinose, and galactose in the molar ratio 1:0.169:3.58:8.71. Results of the DPPH (2,2-diphenylpicrylhydrazyl) revealed that CSP-2 had considerably greater radical-scavenging activity along with high molecular weight when compared to CSP-1 contradicting the general results. The results showcased for CSP-2 having significant inhibitory effect on the growth of HeLa cells. NMR (Nuclear Magnetic Resonance) analysis indicated that it had a triple helical conformation. It was reported by Wang et al. (2017) that triple helical conformation is associated with higher anti-cancer activities of polysaccharides. The findings suggest CSP-2 as a potential anticervical cancer therapy drug (Li et al., 2020). Plants of genera Ficus have been described for owning properties like antioxidant, neuroprotective and anti-cancer activity. In this study, a pectic polysaccharide extracted from Ficus pandurata

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TABLE 11.1 Natural compounds or biological macromolecules used in cancer therapy and prevention. Type of cancer

Type of macromolecule Macromolecule used

Source

Characterization and assays

Ref.

Breast cancer

Carbohydrates

(SPUP) Sulfated polysaccharide from Undaria pinnatifida

U. pinnatifida

MTT, Colony formation, Annexin V- FITC/PI, Immunofluorescence, wound healing, transwell, flow cytometry, Hoechst 33342 staining

Wu et al. (2019)

Chitosan

Procambarus clarkia (Crayfish)

FT-IR, UV, TEM, MTT, Zeta potential

Taher et al. (2019)

Arctigenin

Arctium lappa

SRB, western blot, colony formation, Annexin V-FITC/PI, flow cytometry, RT-qPCR

Zhu et al. (2020)

Yulangsan polysaccharide (YLSPS)

Millettia pulchra (Benth.)

CCK-8, flow cytometry, RT-PCR, western blot, VEGF expression, TUNEL, Annexin V-FITC/PI, IHC

Qin et al. (2019)

ACP1 (Aconitum coreanum polysaccharide)

A. coreanum

GC, FT-IR, MTT, Transwell, migratory behavior, F-actin content, immunoblotting, Rac1signalling

Zhang et al. (2017)

APS (Astragalus polysaccharide)

Astragalus membranaceus

SEM, NMR, HPLC, CCK-8, DAPI staining, flow cytometry, Annexin V-FITC/PI, immunofluorescence

Li et al. (2020)

miR-34

Marsupenaeus japonicus

Northern blot, flow cytometry, RT-qPCR, western blot, target gene prediction

Cui et al. (2017)

Schinus molle L. and Schinus terebinthifolius Raddi

GC-FID, GC-MS, DPPH, ABTS

Bendaoud et al. (2010)

Cedrelopsis grevei

DPPH, ABTS

Afoulous et al. (2013)

Nigella sativa L

DLS, UV-Vis, SEM

Periasamy et al. (2016)

PCP-I

Pholidota chinensis Lindl

HPSEC, GC, FT-IR, periodate oxidation-smith degradation, GC-MS, NMR, TEM, DPPH, MTT

Luo et al. (2018)

SPS-CF

Capsosiphon fulvescens

MTT, western blot, TUNEL, flowcytometry, fluorescence microscopy

Choi et al. (2019)

HEFP-2b

Hericium erinaceus

SEC, Ion chromatography, UV, FT-IR, GC-MS, NMR, SEM, MTT, western blot

Liu et al. (2020)

EPS-CP, EPS-SS and EPS-CS

Chlorella pyrenoidosa FACHB-9, Scenedesmus sp., Chlorococcum sp.

GPC, HPLC, UV, FT-IR, SEM, DPPH, Hydroxyl radical, CCK-8, colony formation

Zhang et al. (2019)

Nucleic Acids Essential Oils

Colon Cancer

Carbohydrates

(Continued)

TABLE 11.1 (Continued) Type of cancer

Type of macromolecule Macromolecule used Proteins

Source

Characterization and assays

Ref.

SPG-56

Sweet potato (Ipomoea batatas L.)

HPLC, colorimetry, mass spectrometry, MTT, Annexin VFITC/PI, western blot, IHC

Wang et al. (2017)

KRL (Kaempferia rotunda Lectin)

K. rotunda

SDS-PAGE, CCK-8, colony formation, phase-contrast microscopy, Annexin V-FITC/PI, flow cytometry, western blot

Islam et al. (2019)

Myrica gale L. (Myricaceae)

GC-MS

Sylvestre et al. (2005)

Croton flavens L. (Euphorbiaceae)

GC-MS

Sylvestre et al. (2006)

CP-4126 derivative of gemcitabine

Chemosensitivity tests, GC-MS

Bergman et al. (2004)

Protaetia brevitarsis larva (PBL)

NMR and mass spectrometry analysis, chemosensitivity assay

Yoo et al. (2007)

Polydatin

Polygonum cuspidatum

CCK-8, TMT, ITC, Human proteomic chip

Chen et al. (2020)

(PSP) Polygonatum sibiricum polysaccharide

P. sibiricum

Flow cytometry, qRT-PCR, western blot, CCK-8, ELISA

Long et al. (2018)

Engeletin

White grapes and white wine

MTT, flow cytometry, TUNEL, Annexin V-FITC/PI, Immunofluorescence, western blot, Co-IP, MMP, RT-qPCR, ELSA, IHC

Liu et al. (2020)

HSP-III (Hirsutella sinensis polysaccharide-III)

H. sinensis

MTT, TUNEL, MMP, western blot, flow cytometry, ROS

Liu et al. (2017)

SPS (Sulfated polysaccharide of Antrodia cinnamomea)

A. cinnamomea

SEC, western blot, MTT, flow cytometry, wound closure

Lu et al. (2017)

IOPs (Inonotus obliquus polysacchrides)

I. obliquus

HPGPC, HPAEC-PAD, CCK-8, colony formation, TUNEL Annexin V-FITC/PI, western blot, MMP, ATP

Jiang et al. (2020)

Essential Oils

Fatty Acids

Lung Cancer

Carbohydrates

(Continued)

TABLE 11.1 (Continued) Type of cancer Non-small cell lung carcinoma

Type of macromolecule Macromolecule used Nucleic acids

Source

miR-331-3p

Ref.

RT-qPCR, luciferase activity, western blot, CCK-8, transwell Tian et al. assay (2020) Curcuma zedoaria Roscoe

Essential oils

Characterization and assays

Western blot, MTT, GC-MS

Chen et al. (2013)

CSP-S-2 (Corn silk polysaccharides)

(Stigma maydis) Corn silk UV, FT-IR, DPPH, ABTS, hydroxyl radicals, IR, NMR

Li et al. (2020)

FP2

Ficus pandurata H

IR, NMR, MTT, Western blot

Lv et al. (2020)

PCHPs (Polygonatum cyrtonema Hua polysaccharides)

P. cyrtonema Hua

MTT, LDH, flow cytometry, RT-PCR, western blot, Annexin Li et al. (2020) V-FITC/PI

PRG1-1

Russula griseocarnosa

HPLC, GC
MS, UV, FT-IR, CCK-8, LDH, ROS, FACS, Annexin V-FITC/PI, flow cytometry, western blot

Liu et al. (2018)

Fucoidan

Undaria pinnatifida

SEC, PS, ZP, PDI, DLS, colorimetric assay, FT-IR, Annexin V-FITC/PI, Nuclear morphology, MTT, scratch assay, gelatin zymography

Etman et al. (2020)

ROH05

Ophiopogon japonicus

HPGPC, GC-MS, NMR, MTT, Nuclear morphology, western blot

Gu et al., 2018

Carbohydrates

SBPW3

Scutellaria barbata

HPGPC, GC-MS, IR, FT-IR, CCK-8, wound healing, transwell, western blot, metastatic assay

Li et al. (2019)

Nucleic Acids

miR-22

RT-qPCR, CCK-8, colony formation assay, transwell, western blot, luciferase reporter, IHC

Cong et al. (2020)

Hepatic cancer

Proteins

Albumen

SDS-PAGE, anti-bacterial, MIC, biofilm formation and inhibition assay, Cox inhibitory assay, MTT, PI staining

Sherly Carolyn et al. (2019)

Hepatocellular carcinoma

Nucleic acids

miR-133a-3p

RT-qPCR, western blot, CCK-8, colony formation, cell apoptosis, cell invasion assay, Luciferase activity

Han et al. (2020)

Liver cancer

Essential oils

Eupatorium adenophorum Spreng.

MTT, western blotting, FACS, TUNEL, IHC

Chen et al. (2018)

Aquilaria species (agarwood)

SRB, cell attachment assay

Hashim et al. (2014)

Cervical cancer

Pancreatic cancer

Colorectal cancer

Carbohydrates

Carbohydrates

Hen’s egg

(Continued)

TABLE 11.1 (Continued) Type of cancer

Type of macromolecule Macromolecule used

Ovarian cancer

Essential Oils

Skin cancer

Brain cancer

Essential Oils

Essential oils

Source

Characterization and assays

Ref.

Ocimum kilimandscharicum

DPPH,GC-MS

De Lima et al. (2014)

Rosmarinus officinalis L.

MTT, MIC and MBC values

Wang et al. (2012)

Salvia officinalis L.

MTT, GC-MS

Russo et al. (2013)

Pituranthos tortuosus

MTT, GC-MS

Krifa et al. (2015)

Bergamot

EE, zeta potential, MTT

Celia et al. (2013)

Elsholtzia ciliata (Lamiaceae)

MTT, GC-MS

Pudziuvelyte et al. (2017)

Prostate cancer

Carbohydrates

GTP (Green Tea polysachhride)

Camellia sinensis

qRT-PCR, MTT, colony formation, migration, invasion, wound healing assay, Bradford protein assay, flow cytometry, Annexin V-FITC/PI, western blot

Yang et al. (2019)

Murine sarcoma cancer

Carbohydrate

(1-3)-β-D-glucan

Saccharomyces cerevisiae

Western blot, flow cytometry

Mo et al. (2017)

Thyroid cancer

Essential oils

Syzygium aromaticum (clove bud)

Colony formation assay, V-FITC, MTT assay

Joyce Nirmala et al. (2019)

12 cancer cell lines

Essential oils

Cymbopogon flexuosus

SEM,TEM,LM

Sharma et al. (2009)

Colon and prostate cancer

Fatty acids

Ciprofloxacin fatty acids

LDH, MTT, FITC, IL-6 assay

Chrzanowska et al. (2020)

LL2, CT26 and CMT93 mouse cancer cell lines

Fatty acids

Linoleic acid (LA) and elaidic acid (EA)

Aldehyde dehydrogenase (ALDH) activity, GC-MS

Tanabe et al. (2017)

Breast cancer, Colorectal cancer

Proteins

Lyngabyal lectin

PCR, PAGE, SDS-PAGE, haemagglutination inhibition, MTT, plaque assay

El-Fakharany et al. (2020)

Lyngabya confervoides MK012409

11.2 Biological macromolecules for cancer therapy

(FP2) is characterized and evaluated for its anticancer properties. HPGPC (High Performance Gel Permeation Chromatography) analysis revealed that FP2 is a homogenous polysaccharide. IR (Infra-Red) and NMR analysis revealed that all residues in FP2 are α-glycosidically linked also that FP2 is linear and can possess special biological activities. The HeLa cells (cervical cancer cell line) were treated with FP2, 31.50 and 22.62 μg/mL were the IC50 values for 24 h and 48 h. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay results showed that FP2 had an anti-proliferative effect on HeLa cells in a dose and time dependent manner. A considerably high inhibition ratio was there at higher concentrations of 100
200 μg/mL at 48 h by around 30.48%
31.95%. To determine the mechanism behind the inhibitory effect on FP2, western blot analysis was done and indicated that FP2 could induce apoptosis in HeLa cells by up-regulating cleaved-PARP (poly-ADP ribose polymerase) protein and caspase-3 expression. The levels of Caspase-9 were measured to know if the apoptosis was induced via mitochondrial or intrinsic pathway. The results suggest FP2 as a potential natural therapeutic drug for cancer treatment (Lv et al., 2020). Polygonatum cyrtonema Hua (PCH) is a medicinal herb and polysaccharides extracted from it are known to have anti-cancer properties against lung and prostate cancer. Four types of polysaccharides have been successively extracted from P. cyrtonema (PCHPS)- CASS (concentrated alkali soluble solids), DASS (diluted alkali soluble solids), CHSS (chelating agent soluble solids) and HBSS (hot buffer soluble solids). The inhibitory properties of PCHPS were determined on HeLa cells. The results revealed that the strength of inhibitory effect of PCHPS was in the following direction: CASS . HBSS . CHSS . DASS. CASS showed maximum inhibitory effect (74.453% at 1100 μg/mL). The results revealed that all four of the PCPS inhibited proliferation in HeLa cells effectively to inhibit the tumor along with being concentration dependant. This difference is due

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to the differences in molecular weight and structure of the polysaccharides. CASS exhibited the strongest cytotoxicity effect from the others irrespective of the concentration. PI (Propidium iodide) staining and flow cytometry done for CASS on HeLa cells showed it can induce G2/M phase arrest in HeLa cells. The proposed mechanism for its anticancer effect is shown in Fig. 11.1. Data suggests that post PCHPS treatment, caspase-3 activity increased considerably which can be responsible for induction of apoptosis in HeLa cells. The expression levels of essential proteins were detected in the Death receptor pathway, it was found that by increasing protein and mRNA expression level of these genes, CASS could induce apoptosis in HeLa cells. These findings suggest PCHPS for treatment of cervical cancer (Li et al., 2020). Russula is a mushroom genus with greatly diverse ectomycorrhizal fungi. There have been reports of ergosterols, flavonoids, phenolics etc. extracted from Russula griseocarnosa to have antioxidant activity. Although there are only a few reports on the polysaccharides that exhibit biological activities. In this research, a novel crude polysaccharide derivative from wild R. griseocarnosa (PRG1-1), was obtained for activity on cervical cancer cell lines. PRG1-1 composed of fructose, xylose, mannose, galactose, and glucose in the molar ratio 0.447:0.663:3.17:29.2:66.5. CCK8 (cell counting kit-8) kit was used to assess the cell viability, and the results showed that the cell viability reduced in SiHa and HeLa cell lines. The IC50 values in HeLa and SiHa after 24 h of incubation were 190.4 μg/mL and 213.7 μg/mL, respectively. Flow cytometry was used to detect Reactive Oxygen Species (ROS) generation proving that PRG1-1 showed cytotoxic effect on HeLa and SiHa cervical cancer cell lines. Annexin V-FITC/ PI staining was done to determine association between reduction in cell viability and induction of apoptosis. The results indicated that PRG1-1 induced apoptosis in the cells. The results of western blot showed that the expression of cleaved caspase-3, cytochrome c and cleaved PARP in the

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11. Biological macromolecules as anticancer agents

FIGURE 11.1 The proposed mechanism responsible for the anticancer effect of CASS on apoptosis and HeLa cell cycle involving cascade of pathways. Source: Reprinted with permission from Li L., Thakur K., Cao Y.Y., Liao B.Y., Zhang J.G., Wei Z. J. (2020). Anticancerous potential of polysaccharides sequentially extracted from Polygonatum cyrtonema Hua in Human cervical cancer Hela cells. International Journal of Biological Macromolecules;148:843
50.

cytosol of tumor cells were increased after PRG11 treatment, which suggests that there is involvement of mitochondrial apoptopic pathway in the anti-tumor effect of PRG1-1. Thus PRG1-1 can be more studied as an anti-cervical cancer agent (Liu, Zhang, & Meng, 2018). 11.2.1.2 Colon cancer In this article, PCP-I, a major polysaccharide was extracted from the pseudobulbs of Pholidota chinensis Lindl, a plant known for its antiinflammatory, analgesic, antioxidant, and antitumor properties. To understand the relation between its biological activity and chemical

structure, the chemical structure of PCP-I was established by Luo, Wang, Li, and Yu (2018). Fourier transform infrared spectroscopy (FTIR) examination was done for PCP-I which showed characteristic polysaccharide peaks. TEM (transmission electron microscope) was done to determine the fine structure of the polysaccharide which revealed the entangled microstructure of the polysaccharide which can be observed in Fig. 11.2. It was reported that helix conformations and entangled chains played a crucial role to enhance the anti-tumor and antioxidant properties of the polysaccharides. Cytotoxicity test was done on human colon cancer cells Caco-2

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11.2 Biological macromolecules for cancer therapy

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FIGURE 11.2 TEM image of PCP-I in sodium dodecyl sulfate (SDS) solution (left) and the local amplification of the left image (right). TEM, transmission electron microscope Surce: Reprinted with permission from Luo D., Wang Z., Li Z., Yu X. (2018). Qiang. Structure of an entangled heteropolysaccharide from Pholidota chinensis Lindl and its antioxidant and anti-cancer properties. International Journal of Biological Macromolecules;112:921
928.

using MTT assay which showed IC50 value of 69.54 μg/mL for PCP-I. The biological activities of the polysaccharides might be relational to their glycosidic linkages and molecular weight. The anti-cancer effect was exhibited stronger because of 1 - 3 glycosidic linkage and low molecular weight (Luo et al., 2018). There are fungi which have also been studied for polysaccharides with anti-cancer properties. Hericium erinaceus, a medicinal mushroom is known to have various health benefits and its fruiting bodies have been used in China to treat digestive system associated disorders. There are many reports which indicate polysaccharides derived from H. erinaceus (HEPS) have shown anti-cancer activities against gastric cancer and liver cancer. Studies on the anti-colon cancer activities of the HEPS are limited, hence in one of study, Liu, Li, Sun, Tian, and Shi (2020) extracted and purified a novel polysaccharide obtained from H. erinaceus fruiting bodies (HEFP-2b) and investigated its structural properties along with

its anti-colon cancer activity. Ion chromatography was performed to determine the monosaccharide composition of HEFP-2b, results showed that it consisted of galactose, fructose, mannose, and glucose in the ratio 22.82:11.81:21.09:44.28. Results of FTIR analysis depicted major typical absorption peaks of polysaccharides and NMR spectroscopy indicated that α- and β- configurations were present. Scanning electron microscope (SEM) of HEFP-2b revealed that it had flat smooth shape and the particles present were lamellar. HEFP-2b effectively repressed the growth of colon cancer cell lines (HCT-116), with IC50 values of 0.345 6 0.026 mg/mL as studied using MTT assay. Flow cytometry analysis revealed that HEFP-2b arrests the colon cancer cells (HCT-116) at S-phase which causes the inhibition in the growth of colon cancer cell lines (Liu et al., 2020). Apart from plants and fungi, algae have also been found to have polysaccharides with anti-tumor activity. In this study, Choi et al. (2019) took an alga- Capsosiphon fulvescens (CF),

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11. Biological macromolecules as anticancer agents

which is known to have health benefits and used to treat hangover and stomach disorders since centuries. A sulphated glucuronorhamnoxylanSulphated Polysaccharide-Capsosiphon fulvescens (SPS-CF) was extracted and isolated from C. fulvescens to study its anti-cancer activity. The cytotoxicity of SPS-CF was evaluated in vitro using HT-29 human colon cancer cells which showed that the cell viability decreased dose-dependently- 93%, 90%, 80%, and 64% at the concentrations of 0, 50, 100, 200, and 500 μg/mL. The results of flow cytometry showed that SPS-CF causes cell death by G2/M arrest in the HT-29 human colon cancer cells. Fluorescence microscopy was used to monitor the SPS-CF treated cells stained with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay kit as well as the mitochondrial membrane potential (MMP). The results showed DNA fragmentation was induced by SPS-CF which led to apoptopic cell death and MMP monitoring results showed that SPS-CF causes mitochondria-dependent apoptosis mediated cell death in the HT-29 human colon cancer cells. Western blot and MTT assay were performed, it was found that SPS-CF induces dose-dependent cleavage of caspase-3 and caspase-9. Xenograft models of mice were intraperitoneally administered with 200 and 400 mg/kg SPS-CF significantly inhibited the growth of colon tumor without affecting the change of mice body weight. It was demonstrated that SPS-CF inhibited the growth of tumor in vivo by induction of apoptopic tumor cell death. It was found that the sulphate groups on SPS-CF are crucial factors for its anti-cancer activity against the HT-29 human colon cancer cells (Choi et al., 2019) Extracellular polysaccharides (EPSS) are biological macromolecules which are secreted by algae. Here, different EPSS have been extracted from three microalgae Chlorococcum sp.(EP-CS), Scenedesmus sp.(EP-SS) and Chlorella pyrenoidosa FACHB-9 (EP-CP), respectively. Their monosaccharide composition included rhamnose, fructose, glucose, xylose, mannose, galactose,

fucose and sugar derivatives. FTIR analysis revealed that they were acidic polysaccharides and indicated presence of glycosyl group. SEM images showed the three EPS to have different microstructures from one another. The IC50 values of EPS-CS, EPS-CP and EPS-SS were 1.38, 2.14 and 0.83 mg/mL, respectively. Cell viability assay was done to investigate the cytotoxicity of the EPSS on human colon cancer cell lines (HCT8 and HCT116). All the three EPSS showed similar repressive activities in HCT116 but better on HCT8 cells. EPS-CS, EPS-CP and EPS-SS showed cell viabilities of HCT116 as 81.3%, 82.8% and 80.8% respectively at 0.6 mg/mL. 77.1%, 64.1% and 61.4% were the cell viabilities showed by EPS-CS, EPS-CP, and EPS-SS respectively at 0.6 mg/ mL. 6-well plate assay was studied to examine the repressing activity on colony formation of colon cancer cells. The results disclosed that EPS-CS, EPS-CP, and EPS-SS inhibited the formation of colony of HCT8 by 35.25%, 11.5% and 29.3% respectively. This study indicated the three EPSS as potential anti-cancer agents for colon cancer and may be effective against other types of cancers as well (Zhang, Liu, Ren, & Chen, 2019). 11.2.1.3 Breast cancer Chitosan is a natural polymer which known to have various biological properties like immune-enhancing, antioxidant, anti-microbial along with anti-cancer activity. In this study chitosan (D1
D9) was prepared from the chitin which was extracted from Procambarus clarkia (red swamp crayfish) exoskeleton. The abdomen tergum of the crayfish had highest amount of chitin (85 g, 25%). Homogenous cubic shaped particles were observed using TEM images. The average zeta potential of D8 and D9 chitosan nanoparticles 41.5 and 26.6 mV, respectively. MTT assay was done to evaluate and compare the cytotoxicity of chitosan and its nanoparticles on breast cancer MDA-MB-231 and SK-BR-3 cell lines. All the

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11.2 Biological macromolecules for cancer therapy

samples exhibited cytotoxicity, specifically chitosan nanoparticles (D8 and D9) exhibited better cytotoxicity than the microparticles (D6 and D7). The cytotoxicity of the chitosan samples increased with the increase in drug DDA (degree of N-deacetylation) on MDA-MB-231 cell line. But the cytotoxicity of chitosan samples was found to be inversely proportion to drug DDA on SK-BR3 cell line. This contrast in results for both the cell lines have factors like electrostatic interaction, pH, differential expression of receptors and cell context. The highest cytotoxicity was exhibited by chitosan nanoparticles at 100 μg/mL. Chitosan and its nanoparticles are thus considered as a potential candidate for natural treatment of breast cancer (Taher et al., 2019). A bioactive lignan- Arctigenin (Arc) extracted from the seeds of Arctium lappa, is known for various medicinal properties like anti-inflammatory, immunomodulatory, antiviral along with anti-cancer activities against various cancers like glioblastoma, gastric cancer, lung cancer, bladder cancer and breast cancer. It was shown that Arc exerts antiproliferation in ER-positive human breast cancer cells in previous study, but the mechanism behind this was not completely explained. MCF7 and BT474 were the cell lines taken to determine the effect of Arc. Various assays were performed, and it was evident that Arc effectively inhibits ER-positive breast cancer cell line growth but doesn’t inhibit the ER-negative cell lines. Results of western blot analysis showed that Arc could not induce apoptosis in the ER-positive breast cancer cells. The results of flow cytometry indicated that G1 phase arrest was induced by Arc which caused the inhibition of growth of the ER-positive breast cancer cells. The key mechanism for the G1 phase arrest is Arc induced GSK3depependent cyclin D1 reduction. ER-positive breast cancers characteristically show high cyclin D1 expression. The reduction of cyclin D1 may have a crucial role in facilitating G1

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phase arrest in the cells. This reduction in cyclin D1 levels happens due to the reduction of mRNA levels and promotion of proteasomal degradation (Zhu et al., 2020). Brown kelps are known to have various health benefits as they protect against cardiovascular diseases, combat oxidative stress and are known to prevent cancer. Undaria pinnatifida, an important source of food in China and is known for its health beneficial properties like anti-viral activity, immunomodulation, and anti-cancer activities. It has also showed antitumor activity against HeLa (cervical cancer), HEPG2 (hepatocellular carcinoma), PC-3 (prostate cancer), and A549 (alveolar carcinoma) cell lines. Sulphated polysaccharide from U. pinnatifida (SPUP) was extracted to determine its anti-cancer properties. From the MTT assay results it was concluded that the inhibitory effect of SPUP was enhanced with increase in concentration and duration of the treatment on MCF7 cells. It was found that SPUP significantly inhibited the colony formation in MCF7 cells. Immunofluorescence analysis was done to assess PCNA (proliferating cell nuclear antigen) expression which is an indicator of cell proliferation. From the results it was inferred that SPUP downregulated PCNA which inhibited MCF7 cell proliferation. Would healing assay and transwell assay were done to evaluate the migration and movement of MCF7 cells. The results of both the assays demonstrated that SPUP inhibits the migration of cancer cells. Annexin V-FITC and PI were used to determine the stage of apoptosis, the results indicated that SPUP arrests the cells in S phase (Wu et al., 2019). Yulangsan polysaccharide (YLSPS) is extracted from the roots of Millettia pulchra (Benth) in one of the studies. It is a medicine in Zhuang, China with various properties like antifibrosis, immunity boosting, anti-inflammatory, anti-viral and anti-tumor activity. Previously done studies show that it has an inhibitory effect on A2780, BEL-7404, SGC-7901 and HEPG-2 cell

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lines. CCK-8 assay was used to evaluate 4T1 cell viability. The results indicated that proliferation of 4T1 cells was considerably repressed by YLSPS in a dose-dependent manner. To determine the role of drug-serum with YLSPS in apoptosis, flow cytometry showed that the apoptopic number of cells increased at higher concentration of YLSPS. Western blot and realtime PCR were used to measure protein and mRNA expression to investigate the YLSPS induced apoptosis mechanism. The results designated that YLSPS induced apoptosis in the 4T1 cells by inhibiting the Bcl-2 apoptopic pathway, increasing Bax and encouraging the cleavage of Caspase-3. YLSPS was administered in a 4T1 tumor-bearing mouse model group and it was observed that the content of antioxidant enzymes and non-enzymes increased, and vascular endothelial growth factor expression and MDA (mass drug administration) levels were lowered. TUNEL staining was done and results indicated that YLSPS applied a strong anticancer effect on the breast cancer model in vivo. An immunohistochemistry analysis of the breast cancer model indicated that YLSPS inhibits tumor angiogenesis efficiently. It can be said that YLSPS can be used in breast cancer therapy and treatment (Qin et al., 2019). Astragalus polysaccharide (APS), extracted from Astragalus membranaceous is known to have anti-tumor activity. High performance liquid chromatography (HPLC) analysis determined the monosaccharide composition of APS, it was comprised of arabinose (Ara), galactose (Gal), glucose (Glc), galacturonic acid (GalTA) and rhamnose (Rha). Out of which galactose, rhamnose and arabinose have an important role to play in improving the immunity of the host. Observations showed that the growth of 4T1 cells cannot be inhibited by APS activity. The non-cytotoxicity of APS toward 4T1 was confirmed by the results of CCK, showing it was mediated by macrophages (CM). Increased proportion of 4T1 cells were observed in G2 phase which indicated that CM

has inhibited cell growth via mitochondrial pathway by the disrupting G2 transition in the cell cycle. It was observed that APS supressed tumor cell proliferation in BALB/c mice over time. APS also seemed to improve anti-cancer and immunosuppressive effect of 5-FU (5-fluorouracil) and enhanced pinocytosis capacity of macrophages. The ability of APS to improve IFN-γ (interferon), TNF-α (tumor necrosis factor) and IL-2 (interleukin) expression may be linked to provoked immune response in 4T1 tumor models. Immunohistochemical assay was done to measure expressions of Bcl-2, Bax, and caspase-3. Caspase-3 expression was high in APS and APS along with 5-FU gave even higher expressions of caspase-3 and Bax. APS paired with 5-FU can enhance the anti-cancer activity, thus is suitable for chemotherapy as an immune adjuvant (Li et al., 2020). Acotinum coreanum is a herb found in China, Japan and Korea which has various medicinal properties and is used for treatment of epilepsy, rheumatic arthralgia, facial distortion, cardialgia, migraine headache, tetanus, infantile convulsion, and vertigo. In this study, of A. coreanum polysaccharide ACP1 and its sulphated derivative ACP1-s are evaluated for their anti-cancer properties. GC (gas chromatography) analysis showed that ACP1 was composed of glucose, mannose, and arabinose in the molar ratio- 3.23:1:0.24. MTT assay was used to assess the cytotoxic effect of ACP1 and ACP1-s on MDA-MB-435s. It was found that ACP1-s did not inhibit MDA-MB-435s cell viability below, when concentration was further increased, ACP1s considerably reduced MDA-MB-435s cell viability. It was observed that ACP1-s greatly reduced the number of migrated cells. It was also noted that ACP1-s showed better inhibitory activity than ACP1, from which we infer that sulphation can improve the inhibitory activity of polysaccharides. ACP1 and ACP1-s both were able to significantly decrease the F-actin content in MDA-MB-435s cells, ACP1-s nearly eliminated actin polymerization. ACP1 and ACP-s greatly reduced the VAV2

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phosphorylation levels in MDA-MB-435s cells and affected VAV2 signaling. ACP1 and ACP1-s reduced RAC1 activity. These findings show that APS1 and APS1-s can potentially be used in breast cancer treatment for metastasis (Zhang, Wu, Kang, Yu, & Liu, 2017). 11.2.1.4 Lung cancer Several medicinal plants included in the Chinese medicine are reported to have tumorsupressing polysaccharides that can be extracted. Polydatin (PD) is a bio-active compound, known for its bioactivities like hypolipidemic, anti-inflammatory and anti-cancer activities. PD is extracted from the rhizome and roots of Polygonum cuspidatum- Chinese herbal medicine. Polydatin has been reported to show cytotoxicity against nasopharyngeal carcinoma cell lines, OVCAR-8 cell line, MCF-7 and HeLa, A431 and A549 cell lines. Results of cytotoxicity tests show that polydatin significantly inhibited the growth of A549 cells. The effect of PD on tumor bearing mice was tested and it was observed that PD repressed the growth of tumor and increased the weight of mice considerably. PD has a characteristic green fluorescence property which was used to screen its protein targets by proteomic chip with the help of bioinformatics analysis. By MS (mass spectroscopy) and western blot Glutathione had drawn interest. PD drugs with high to medium concentration can maintain GSH (glutathione) levels at a low stable level, which can be the way it controls the growth of tumors and has low toxicity. It was observed that only high concentration of PD can have anti-tumor effect and low concentrations of PD and reservatrol may promote tumor growth. Tumor tissues have high GSH content. PD covalently binds to GSH to stabilize it at low levels, which reduces the high GSH content as well as protects normal tissues. These findings reveal Polydatin as potential cancer therapy drug and application

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of GSS as a potential anti-cancer target (Chen et al., 2020). Polygonatum sibiricum is a plant used in Chinese medicine used for cancer treatment. One of the major bio-active components of P. sibiricum is P. sibiricum polysaccharides (PSPs). HPLC analysis showed that PSP is composed of galactose, glucose, mannose, xylose, arabinose and rhamnose. This study was done to assess the therapeutic effect of PSP on lung cancer. Using LLC (Lewis lung carcinoma), tumor-bearing mouse model was established to explore the tumor inhibitory effect of PSP. The results showed that PSP has prominent anti-tumor effect on lung cancer. The cytokine and immune organ indexed were measured post PSP treatment to evaluate the effect of PSP on the immune system of tumor bearing mice. The results showed that PSP had a notable immunoregulatory effect on the tumor bearing mice. Flow cytometry results indicated that PSP could enhance the percentage of CD41 lymphocytes from which we can infer that by increasing CD41 lymphocyte percentage, PSP enhanced the immunological functions. To detect the proteins TLR4-MAPK/NFκb (Toll-like receptor 4- mitogen-activated protein kinase/nuclear factor) signaling pathway and mRNA, western blot, and qRT-PCR (realtime quantitative reverse transcription PCR) were used. From the results we infer that PSP regulated the downstream MAPK/NF-κb signaling pathways. It was found that PSP regulated the function of macrophages. These findings suggest PSP as a potential therapeutic agent against lung cancer (Long et al., 2018). Wine is known for its various health benefits as shown in this study. Engeletin (ENG) is a flavanol known to exhibit biological properties like anti-inflammatory and anti-cancer activity. It can be extracted from the skin of white grapes and is found in white wine. MTT analysis was done to assess the cytotoxicity of ENG on the tumor cells. For H446, H292, H460, A549 and H1299 cell lines the cell viability was reduced

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after treated with ENG. No notable effects were observed on normal lung cells. The results of flow cytometry indicated that ENG induced apoptosis in A549 and H460 cells is a dose dependent manner. TUNEL analysis results demonstrated that ENG can cause cell death in lung cancer cells. It was observed that XIAP (X-linked inhibitor of apoptosis protein) expression was drastically diminished which was confirmed by Immunofluorescence staining. It was found that reduction of XIAP has a role to play in the apoptosis induced by ENG in lung cancer which is controlled by ENG required caspasedependent cleavage. It was observed from the results of western blot that ER stress was promoted by ENG treatment. To ensure the effects anticancer effects of ENG, H460 cells were used to establish xenografts. The results indicate that ENG exhibited minimal toxicity along with effective and clinically applicable anti-tumor activities (Liu et al., 2020). Several fungi were found to show lung cancer-supressing activity. Hirustella sinensis (anamorphic, mycelial form of Cordyceps sinensis) is a part of traditional medicine in China. Polysaccharide extracted from H. sinensis (HSP-III) is known to have anti-tumor properties. MTT assay was done to evaluate the inhibitory effect of HSP-III on H1299 (lung cancer) and MRC-5 (normal) cell lines and was observed that HSP-III reduced cell viability of H1299 cell lines. TUNEL assay was done to assess the apoptosis effect of HSP-III on H1299, which designated that HSP-III encourages apoptosis and declined cell growth. It was found that MMP (mitochondrial membrane potential) was decreased dose dependently. Cytochrome c changes along with Bcl2 expression were examined. The interpretation of the data confirms that HSP-III induces intrinsic mitochondrial apoptosis in H1299 cells. ROS levels were evaluated in HSP-III treated H1299 cells. The results indicated that ROS production is involved in HSP-II induced apoptosis, and decreases mitochondrial respiratory complex

expression contributes to HSP-III induced apoptosis of H1299 cells. Nude mice experiment was conducted to evaluate the effect of HSP-III on cancer cells. It was observed that HSP-III reduced proliferation of cancer cells in vivo by inducing apoptosis. All these findings suggest HSP-III as a potential anti-cancer agent for lung cancer (Liu, Xie, Sun, Meng, & Zhu, 2017). Inonotus obliquus (Chaga mushroom) commonly found in China, Alaska, Russia, Japan and parts or Europe. It is used to treat diabetes and improve overall health in Russia and Asia. Polysaccharides extracted from I. obliquus (IOP) are known to have bioactivities like immunomodulatory, anti-diabetic, antioxidant and anti-tumor activity with low or no toxicity. The inhibitory properties of IOP is evaluated on lung cancer cell lines. HPAECPAD (high-performance anion-exchange chromatography) was used to determine the monosaccharaide composition, which showed majority of glucose (74.95%) and xylose, rhamnose, and galacturonic acid and arabinose in trace amounts. CCK-8 assay kit was used to evaluate the cell viability on LLC1 cells. The results indicated that colony growth was supressed dosedependently by IOP. LLC1 and A549-LKB1 cells were treated with IOP. From the results we can infer that the pro-apoptopic effect exhibited by IOP is affected by intact LKB1/ AMPK (Liver Kinase B1/AMP-activated protein kinase) axis. It was found that there were changes made in glucose metabolism by IOP in A549-LKB1 cells which were dependent on activation of AMPK by LKB1. The results of in vivo analysis supported the AMPK activation mediated effect of IOP. From the findings it was suggested that IOP causes AMPK activation which exerts the inhibitory effect on the lung cancer cell lines by reducing MMP, thus inducing apoptosis both in vitro and in vivo. Therefore this study suggests IOP as a supplementary or alternative lung cancer therapy agent (Jiang et al., 2020).

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Antrodia cinnamomea is a medicinal mushroom/fungus found in Taiwan known for having immunologic functions, immunomodulation, antioxidation, hepatoprotections and anti-cancer properties. A sulphated polysaccharide of A. cinnamomea (SPS) has been testified to exhibit antiinflammatory activity and its tumor inhibitory effect. SPS majorly has mannose, glucose, galactose and fucose. LLC1 and A549 cells were treated with SPS to analyze cell growth. The results show that only sulphated polysaccharides were able to considerably reduce tumor cell proliferation. It was observed that polysaccharides extracted from A. cinnomeaaa needs sulphated modifications so as to induce apoptosis and cell arrest. SPS inhibition of migration and cell viability along with apoptosis involves TGFβ1-mediated intracellular pathways. Effect of SPS on TGFRI/II protein expression was examined in various cell lines like LLC1 and A549 cells. The results SPS has a major role in TGFR (Transforming growth factor receptor) expression suppression. All of these findings, it suggests that SPS has a part to play in enhancing TGFR degradation in lung cancer cell lines (Lu, Lin, Chao, Hu, & Hsu, 2017). 11.2.1.5 Pancreatic cancer As mentioned earlier, brown kelps are known for their health benefits which include its anti-cancer activity. Fucoidan is a naturally occurring sulphated polysaccharide obtained from brown algae known for exhibiting bioactivities like anticoagulant, anti-viral, antimicrobial and anti-tumor activity. Fucoidan (FCD) extracted obtained from U. pinnatifida has been described to display cytotoxicity against lung, hepatocellular, colon and breast cancer. FCD based nanoparticles are used for enhanced drug delivery. In this study, antipancreatic cancer properties of FCD and its nanoparticles were investigated. The FCD nanoparticles were prepared by electrostatic interaction (polyelectrolyte complexation) with Lactoferrin (Lf- positively charged protein). It was found that the ratio of 1:1 of FCD:Lf was

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optimum for the yield of stable nanoparticles. TEM was used and sphere-shaped particles were observed with small particle size. Dialysis bag method was used to evaluate the release of FCD from FCD/Lf NP, the results indicated that the nanoparticles could sustain the release of the FCD, thus improving its receptor mediated cellular uptake. The results of MTT assay showed that FCD nanoparticles showed 2.3 times lower IC50 values on PANC1 cells, indicating that nanoparticles showed more cytotoxicity. The results of nuclear morphology revealed that the apoptosis is involved in the anti-tumor effect of FCD. Annexin/ Propidium iodide assay was done by flow cytometry which further confirmed the greater anti-cancer activity of FCD nanoparticles. The results of scratch assay supported previous results that the nanoform of FCD was responsible for its high uptake. All these findings highlight the potential of FCD nanoparticles for the treatment of pancreatic cancer (Etman, Abdallah, & Elnaggar, 2020). Besides algae, Mondo grass, a plant was found to have anti-cancer proteins. Ophiopogon japonicus is a medicinal plant whose roots are extensively used in Southeast Asia, Japan and China. O. japonicus has been known to have bioactive polysaccharides that show immunomodulation, anti-diabetic activity. In this study, ROH05 (galactan) obtained from O. japonicus is acetylated (ROH05A) to enhance its bioactivity and evaluated for its anti-cancer activity against pancreatic cancer. MTT assay was done to predict the cytotoxicity of ROH05 and ROH05A on pancreatic cancer cells Bxpc-3 and PANC-1. According to the results, ROH05A showed inhibitory ratios of as 60% on BxPC-3 and 26.2% on LO2 (normal liver cell). This indicates that ROH05A significantly inhibited proliferation in tumor cells but also showed little toxicity toward normal cells. DAPI (40 ,6-diamidino-2-phenylindole) staining indicated decrease in cell number. Crenulation and nucleus fragmentation after ROH05A

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treatment. Results of western blot revealed that ROH05A induces apoptosis leading to cell death via both extrinsic death receptor pathway and intrinsic mitochondrial pathway in BxPC-3 cell line. The findings lay a foundation for ROH05A as therapeutic agent for pancreatic cancer treatment (Gu et al., 2018). 11.2.1.6 Others There are polysaccharides that show anticancer activity against type of cancers other than the ones mentioned earlier. They are mentioned as follows: SBPW3 is a water-soluble polysaccharide extracted from Scutellaria barbata a plant used in traditional Chinese medicine known for its antiinflammatory, antioxidant, and anti-cancer properties. CCK-8 assay showed that SBPW3 at maximum concentrations had only a little impact on HT-29 (colorectal) cells. SBPW3 was found to supress metastasis and invasion of HT-29 cell lines. Transwell invasion and wound healing assay revealed that the inhibition of invasion and metastasis by SBPW3 was induced by TGF-β1. Epithelial-mesenchymal transition (EMT) is important for metastasis of tumor cells. The results showed that TGF-β1 could considerably inhibit the process of EMT. SBPW3 also inhibited metastasis in liver tumor cells in vivo. From the results of in vivo experiments, it was evident that SBPW3 also improves the immunity of mice which aided the inhibition of metastasis of tumor cells. These results suggest SBPW3 as a potential chemotherapeutic drug for preventing metastasis, and treatment of colorectal cancer (Li et al., 2019). Green tea is widely known for its great health benefits and its polysaccharides are also known to have anti-cancer properties. GTPpolysaccharide extracted from green tea was tested for its anti-cancer properties on prostate cancer cells. qRT-PCR analysis revealed that miR-93 was considerably upregulated in prostate cancer cells. On that basis the expression of miR-93 is suggested as a prognostic biomarker for prostate cancer. Cell propagation

and colony formation assays indicated that miR-93 plays a role in proliferation and formation of colonies in prostate tumor cells. It was found from the transwell and wound-healing assays that miR-93 is responsible for promotion of invasion and migration of prostate tumor cells. In in vivo experiments, upregulation of miR-93 was observed in tumor growth. The annexin V/PI staining assay showed that apoptosis is induced cell death by apoptosis in PC3 cells. All these findings propose miR-93 as a potential target by GTP for treatment of prostate cancer (Yang et al., 2019). Fungal polysaccharides have been studied a lot recently for their immunity boosting and anticancer activities. (1-3)-β-D-glucan is a polysaccharide extracted from yeast (Saccharomyces cerevisiae) and has been researched on for its antitumor activity. The immunoregulatory and antitumor activities of (1-3)-β-D-glucan in S180 (murine sarcoma) cancer bearing mice. It was found that (1-3)-β-D-glucan significantly supressed the proliferation of tumor cells but did not affect the metabolism of normal mice in any manner. PI/FITC-staining and flow cytometry analysis was done which indicated that (1-3)β-D-glucan induces apoptosis in S180 tumor bearing cells. It was found that Bcl-2 and Bax have a crucial role to play in the induction of apoptosis. These findings suggest (1-3)-β-Dglucan as a potential novel anti-cancer therapeutic agent (Mo et al., 2017).

11.2.2 Proteins and nucleic acid Proteins are macromolecules constituted by amino acids. Proteins play a decisive role in the biological processes. They serve as catalysts for various processes, participate in the immune response, act as transporters for molecules such as oxygen, facilitate transmission old messages from cell to cell etc. There are various biological proteins like lectins, glycoproteins etc which show anti-cancer properties.

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MicroRNAs are known for their role in post transcriptional regulation of gene expression by binding to the target mRNA on 3’UTR region. Thereby, regulating cell migration, cell events, cell proliferation and several pathways. These are endogenous, non-coding type RNAs and are about 18
25 nucleotide long. It has been found that they can influence about 60% protein coding expression of genes. It has also been found that various miRNAs can target single mRNA, while many mRNAs can be influenced by one miRNA. 11.2.2.1 Colon cancer Sweet potato, a tuberous plant was found to have glycoproteins with anti-cancer activities. Ipomoea batatas L. (sweet potato) is the seventh most important crop over the world. There have been studies done on sweet potato glycoproteins (SPG) and their biological activities. In this study, its anti-cancer properties against colon cancer cells were evaluated. SPG-56 was extracted from I. batatas by using water extraction, chromatography, isoelectric precipitation, and electrophoresis. HPLC analysis of SPG-56 revealed that it composed of arabinose, galactose, galacturonic acid, glucuronic acid mannose and xylose. SPG-56 exhibited IC50 values of 143.21 μg/mL on HCT-116 cells. SPG-56 inhibited proliferation of HCT-116 cells in a dose and time-dependent manner. After staining HCT-116 cells with Annexin V-EGFP/PI and flow cytometry was done, the results show that SPG-56 induces apoptosis in HCT-116 cells. Nude mice were injected with HCT-116 cells. The tumor volume and body weight were regularly monitored. It was observed increasing dose of SPG-56 reduced the growth of cancer cells. The results of the western blot indicate that the apoptosis is induced by the mitochondrial pathway. The outcomes of immunohistochemistry analysis suggested that SGPG-56 greatly enhances the inhibition of cancer cell proliferation. This study suggests

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SPG-56 as a therapeutic agent for colon cancer treatment (Wang et al., 2017). Kaempferia rotunda is a tuberous rhizome belonging to the ginger family is known for its medicinal like anti-microbial and anti-cancer properties. The lectins extracted from K. rotunda (KRL) have been reported to show anti-cancer activity. In this study the anticancer activity of KRL is evaluated against SW48 and SW480 (colorectal cancer) cell lines. KRL was extracted by using affinity chromatography. SW48 and SW480 cells were treated with KRL, it was found that KRL inhibited the proliferation of both the cells lines up to 3 days of treatment. KRL also significantly inhibited the colony formation capacity of SW48 and SW480 cell lines (Fig. 11.3). Using fluorescence microscopy, it was observed that KRL treated cells showed apoptopic phenotypes. The results of phase contrast microscopy further indicated induction of apoptosis in tumor cells. Flow cytometry showed that KRL inhibited cell proliferation in SW48 and SW480 by reducing cells at S phase and arresting at G0/G1 & G2/ M phase. PARP1 (tumor promoter) was deactivated after KRL treatment. Western blot was done to assess the expression of caspase-3 and caspase-9. The results designated that the apoptosis may be mediated via mitochondrial intrinsic pathway. This study indicates that KRL induces apoptosis in SW48 and SW480 colorectal cancer cell lines to inhibit their proliferation (Islam, Gopalan, Lam, & Kabir, 2019). 11.2.2.2 Colorectal and breast cancer Cyanobacteria are a great source of several bioactive compounds with activities like antiviral, anti-coagulant, immunosuppressant, antiinflammatory and anti-tumor activities. Lyngabya confervoides MK012409, a newly isolated strain was evaluated for its biological activities. Lyngbyal lectin- novel lectin extracted and purifies from L. confervoides MK012409. It has been reported to show anti-proliferative effects and viricidal activities. Lyngbyal lectin was

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FIGURE 11.3 Effect of KRL on the colony formation of SW480 and SW48 colon cancer cells. (A) SW480 and SW48 cells treated with KRL reduced the colony formation capacity considerably. (B) Bar graphs presented the number of colonies that have been generated from control and KRL treated cells. (C) Surviving fraction of cells obtained from control and KRL treated cells. Source: Reprinted with permission from Islam F., Gopalan V. Lam A.K.Y., Kabir S.R. (2019). Kaempferia rotunda tuberous rhizome lectin induces apoptosis and growth inhibition of colon cancer cells in vitro. International Journal of Biological Macromolecules;141:775
82.

found to be stable at a varied range of pH values and is also heat stable. Lyngbyal lectin reduced 12% Vero cell viability. It was observed that lyngbyal lectin repressed cell proliferation in a dose-dependent manner in Caco-2 and MCF-7 cell lines The Vero cell viability percentages for lyngabyal lectin for the concentrations 100, 200, 400 and 800 μg/mL were 99.85 6 0.64%, 95.2025 6 0.8%, 91.5425 6 0.4% and 88.7 6 0.33% respectively (El-Fakharany, Saad, Salem, & Sidkey, 2020). There were two separate studies done with involvement of nucleic acid as agents against both breast and colorectal cancer individually. Herein, the miRNAs (Micro RNAs) from Marsupenaeus japonicus shrimp (a marine invertebrate) have been reported as a probable

therapeutic agent for cancer treatment. The miRNAs (mir-34) have been observed to exhibit different patterns of expression under stress and virus infection. The anti-tumor and anti-viral activity properties of mir-34 were investigated. The real-time qPCR results showed that proliferation in mir-34 expressing tumor cells was considerably repressed. Flow cytometry analysis was done which revealed that mir-34 expression caused arrest of cell cycle at G0/G1 phase. Further mir-34 was able to induce apoptosis in the breast cancer cell lines. The negative role in the growth of tumor cells by Shrimp mir-34 was evident through these findings. Female mice (NOD/SCID) were injected with MDA-MB-231 breast cancer cells to determine tumor-supressing properties of

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shrimp mir-34 in vivo. The results indicated that shrimp mir-34 was able to inhibit tumor cell proliferation in vivo. Target gene prediction was done to determine the mechanism behind the suppression of tumor cell proliferation and metastasis. The results indicated that the shrimp mir-34 showed interaction with six human target genes- CDK6, E2F3, FOSL1, CCND1, CCNE2 and MET. It was concluded that shrimp mir-34 targeted these genes to inhibit cancer cell proliferation and metastasis. Cell metastasis assays revealed that shrimp mir-34 inhibited metastasis of tumor cells in vivo. In this study it was exposed that miRNAs from hosts like M. japonicus which are infected by a virus can prove to be as a good source for anti-tumor miRNAs (Cui, Yang, & Zhang, 2017). miR-22 has been reported to show tumor suppressive effects in breast and pancreatic cancers. In this study, miR-22 was evaluated for its tumor-suppressing activity on colorectal cancer cells and its relationship with NLRP3. CCK-8 assay was done to assess the expression levels of miR-22. From the results can infer that miR-22 plays a crucial role in regulating cell proliferation, migration, and invasion. It was found that miR22 has an important role in EMT regulation in HCT116 cells. From the results of western

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blotting and RT-PCR, it was indicated that NLRP3 was a target gene for miR-22 to regulate the proliferation, invasion, and migration in HCT116 cell line. From transwell assay (Fig. 11.4), it was found that the transfection of NLRP2 or miR-22 noticeably reduced the migration and invasion of HCT116 cells. Overexpression of miR-22 was found to inhibit the proliferation, migration, and invasion of HCT116 cells by regulation of NLRP3. By western blot, it was confirmed that the inhibition of miR-22 was weakened by overexpression of NLRP3. In vivo experiments were done which proved that overexpression of miR-22 supressed proliferation of tumor cells by NLRP3 (NLR family pyrin domain containing 3) regulation. The tumor-supressing activity of miR-22 was demonstrated in this study, suggesting it as therapeutic agent and a prognostic biomarker (Cong, Gong, Yang, Xia, & Zhang, 2020). 11.2.2.3 Hepatic cancer The albumen present in the egg has an important function to protect the eggs from microbes. It has several biological properties like anti-bacterial, anti-microbial activity, etc. Oil beetles (blister beetles), when disturbed, secrete a pungent smelling yellow oil from their leg joints. Cantharidin is the toxic material that

FIGURE 11.4 Transwell assay to examine migration and invasion of HCT116 cells. Source: Reprinted with permission from Cong J., Gong J., Yang C., Xia Z., Zhang H. (2020) miR-22 suppresses tumor invasion and metastasis in colorectal cancer by targeting NLRP3. Cancer Management and Research;12:5419
29.

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is present in this secretion which has been reported to exhibit anti-cancer properties against leukemia, hepatic, pancreatic, bladder, melanomas, colorectal and breast cancers. In this study, dried body of blister beetle (Massonia pustulata) is treated with hen’s albumen and its anti-proliferative and anti-inflammatory activities are evaluated. The albumen was observed to exhibit anti-bacterial properties similar that of ciprofloxacin which is a commercial antibiotic. COX inhibitory assay was used to determine the anti-inflammatory property of M. pustulata treated albumen extracts. The albumen extracts showed 84.91% inhibition at 100 μg/ mL. MTT assay was done to estimate the cytotoxicity of albumen extracts on Hep G2 cancer cells. The cell viability reduced by 23.61% in Hep G2 cells. To further investigate the antiproliferative effect of albumen extracts, the cells were stained with PI (propidium iodide) and were observed with help of a phase contrast microscope. Loss of membrane stability and morphological changes were observed. These findings reveal that albumen extracts were able to inhibit Hep G2 cell proliferation. Hence it is concluded that dried M. pustulata body soaked in hen’s albumen can be used for antiinflammatory, anti-bacterial, and anti-cancer treatment (Sherly Carolyn et al., 2019). Studies on nucleic acid, miR-133a-3p indicate that it has tumor-suppressing properties on gastric cancer, esophageal squamous cell carcinoma, prostate cancer, etc. In this article, its mechanism against hepatocellular carcinoma (HCC) will be determined. The UALCAN website and RTqPCR were used to investigate the expression levels of miR-133a-3p, it was found that in HCC tissues, miR-133a-3p showed decreased expression when compared to normal tissues. The results of CCK-8 analysis indicated that overexpression of miR-133a-3p inhibited proliferation in HCC cells. miR-133a-3p mimic supressed the ability to form colonies in HCC. Cell apoptosis promotion by the introduction of miR-133a-3p mimic was indicated by flow cytometry. The

results of Transwell invasion assay showed that miR-133a-3p mimic considerably inhibited the cell invasion ability in HCC cells. From the assays done it was also found that cell growth in HCC cells was stimulated by the knockdown of miR-133a-3p and it also promoted cell invasion. The results of western blot determined that miR133a-3p mimic decreased CORO1C protein level. In vivo studies also showed that miR-133a-3p supressed cell proliferation and reduced the weight and volume of the tumor. This study suggested the tumor-supressing activity of miR133a-3p (Han et al., 2020). 11.2.2.4 Non-small cell lung cancer In this study, the role of MMLLT10 and miR-331-3p was defined in non-small cell lung cancer (NSCLC) tumorigenesis. RT-qPCR was done and from the results we can infer that downregulation of miR-331-3p is largely associated with NSCLC development. It also showed overexpression of miR-331-3p in H1650, A549 and H1299 cells. It was demonstrated that H1299 cells repressed proliferation of NSCLC cells. From a dual-luciferase activity assay it was confirmed MLLT10 as a downstream target gene of miR-331-3p. By western blot analysis it was observed that miR-331-3p negatively modulated MLLT10 in NSCLC and that miR-331-3p induces EMT-mediated metastasis. From the results of transwell assay and CCK-8 analysis, it is indicated that MLLT10 relieves the inhibitory activity of miR-331-3p on the proliferation, migration, and invasion of NSCLC cells. This study suggests miR-331-3p/ MLLT10 axis as a therapeutic target and diagnostic marker for NSCLC (Tian, Xia, Zhang, Gao, & Wang, 2020).

11.2.3 Lipids Natural fatty acids are biocompatible, biodegradable and have shown augmented cellular uptake in cancer cells, therefore combining

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them with anticancerous drugs can be beneficial for cancer treatment. Similarly, essential oils are used for variety of applications such as in perfumes, sanitary products, and make-up products. They are also being used as food preservatives and additives and as natural remedies. Therefore there has been a focus on using the essential oil for biomedical applications. But an extensive study is required for better understanding and usage. Here, the possibilities of using lipids for anticancerous purposes has been highlighted. 11.2.3.1 Colon cancer Native and traditional plants are seen to be very effective as shown in the studies below. A native Canadian plant Myrica gale L. was used for extraction of essential oil by hydro distillation at different time intervals (30 and 60 mins) and its activity was compared. The 30 min EO contained 29.43% sesquiterpenes, with 5.25% oxygenated sesquiterpenes while 60 mins contained 53.10% sesquiterpenes with 20.96% oxygenated sesquiterpenes as identified from GC-MS. Alcohol constituents were present in 60 mins fraction whereas were completely absent in 30 mins fraction. Both the fractions were accessed on human lung carcinoma A-549 and colon adenocarcinoma DLD-1 cell lines. Results showed that 60 mins fraction was more active in both cell lines showing IC50 value of 88 6 1 μg/mL compared to 30 mins fraction having IC50 to be 184 6 4 and 160 6 3 μg/mL, respectively. The anticancer action of monoterpenes existing in the EO were assessed against DLD-1 tumor cell line showing inactive nature (data not mentioned) while the sesquiterpenes present in the EO showed some anticancer activity (Sylvestre, Legault, Dufour, & Pichette, 2005). A traditionally used plant Croton flavens L. from Caribbean area is used in this study. Muriel Sylvestre, Andre Pichette used the leaf of the same to extract the essential oil. The extracted oil was dark yellow and viscous in nature. GC-MS identified 47 compounds

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present in the oil of which viridiflorene (12.22%), germacrone (5.27%) were dominant. The anticancer tests were done on human lung carcinoma cell line A-549 and human colon adenocarcinoma cell line DLD-1. GI50 values were calculated showing 27 6 4 μg/mL value for A549 and 28 6 3 μg/mL value for DLD-1. Such low values of GI50 shows strong anticancer properties of EO. The anticancerous properties can be due to the presence of α-cadinol, β-elemene and α-humulene. It is suggested to determine the other properties of the EO for complete understanding of bioactivity (Sylvestre, Pichette, Longtin, Nagau, & Legault, 2006). With respect to fatty acids, there has been various studies as depicted below. Biologically active fatty acids were isolated from Protaetia brevitarsis L (PBL) for assessing their anticancer potential. First dichloromethane was extracted from PBL, this extract was then passed through silica gel column chromatography. Obtained four fractions (F2, F4, F5, and F7) contained three fatty acids namely (Z)-9-octadecenoic acid, palmitic acid, and octadecenoic acid. When tested on colon 26 cancer cell line, dichloromethane showed concentration dependant activity. Dichloromethane completely killed colon 26 cancer cell within 5 h at concentration 100 μm/mL and within 12 h at 50 μm/mL. Among the previously mentioned four fractions, the most effective was F2. The fractions showed 3-caspase and DNA ladder leading to apoptosis, furthermore when enzyme activity analysis was conducted for every fraction there was enhanced intracellular level of 3-caspase enzyme activity. The palmitic acid found in the fractions is said to cause apoptosis in the colon cancer cells. This study suggests that PBL contains fatty acids that show cytotoxic behavior (Yoo et al., 2007). In this study, the two major fatty acids linoleic acid (LA) and elaidic acid (EA) were studied for their effect on 5-fluorouracil (5-FU). Both in vivo (Male BALB/c mice) and in vitro tests on CT26 (colon cancer), LL2 (lung cancer) and CMT93 (rectal cancer) were conducted. LA

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individually showed decrease in cell viability in all the three lines whereas EA alone did not supress tumor viability in all the three cell lines. On additions of 5-FU with EA, there was no change or enhancement in the tumor reduction ability of 5-FU. The expression of 5-FU is more when added with LA or EA compared to when alone. But anti-chemotherapeutic activity of EA and LA was shown in using a mouse subcutaneous tumor model. Oral intake of LA or EA repelled the anti-tumor effects of 5-FU. Therefore the fatty acids mentioned here are effective in anticancerous treatments, but their anti-chemotherapeutic effects are to be also considered (Tanabe et al., 2017). Combining cancer drugs with fatty acids have opened new doors in cancer treatment. Herein, a drug Ciprofloxacin (CP) was combined with saturated and unsaturated fatty acids to examine its anticancerous behavior. The researchers tested human primary (SW480) and metastatic (SW620) colon cancer, metastatic prostate cancer (PC3) and normal (HaCaT) cell lines. When CP was combined with monounsaturated oleic acid the IC50 was found to be 7.7 6 2.1 μM whereas for diunsaturated sorbic acid the IC50 was 11.7 6 1.8 μM. The highest selectivity index (SI) was found in conjugates like oleic (17.8), sorbic (9.3) and elaidic (9.2) which showed the best cytotoxicity. The long chain monounsaturated geometrical isomers: oleic and elaidic showed late apoptosis in PC3 cells whereas late apoptosis in metastatic SW620 cells was not so spectacular. It was also noted that the CP-derived amides with the lowest lipophilicity displayed highest microbial activity. These conjugates reduced the secretion on IL-6 by cancer cells (Chrzanowska et al., 2020). Another drug, Gemcitabine is a deoxycytidine (dCyd) analog which is not readily taken up by the cell membrane. Therefore to increase it uptake, retention in the cell, a lipophilic prodrug was synthesized (CP-4126) with esterified fatty acid at 5’ position. In colon cancer cell lines, it was seen that the orally as well as

intraperitoneal administered CP-4126 showed the similar effects. CP-4126 was 1.7 times less active in the leukemia L5 cells while was two times more active than gemcitabine in BCLO cells and equally active in the solid tumor C26A cells. In conclusion, the most sensitive disease order was shown by leukemia. renal cancer. NSCLC. CP-4126 seems to be a promising new anticancer drug (Bergman et al., 2004). 11.2.3.2 Breast cancer The aim of the experiments presented in this section is to find various properties of essential oil from various sources. Herein, berries of Schinus molle L. and Schinus terebinthifolius are used for the same. The percentage of essential oil extracted from both S. molle and S. terebinthifolius were found to be 2.7 6 0.1% and 2.5 6 0.1% respectively. The main difference between both species being higher quantity of sesquiterpenes hydrocarbons in S. terebinthifolius (26.81%) than S. molle (3.69%). In total there were 57 and 62 components identified in both berries respectively. The ABTS assay showed IC50 of 24.1 6 0.8 mg/L for S. terebinthifolius whereas S. molle had IC50 of 270 6 12.0 mg/L indicating S. terebinthifolius to show better antioxidant properties. When tested against the cell line MCF-7, both species showed concentration dependant effect, S. terebinthifolius showed more cytotoxicity than S. molle. The higher cytotoxicity of S. terebinthifolius is attributed to its antioxidant activity along with the minor hydrocarbons that are found in it. This study showed a starting point for identifying certain compounds responsible for various activities (Bendaoud, Romdhane, Souchard, Cazaux, & Bouajila, 2010). Another plant Nigella sativa L. (NS) possess significant biological properties and has been used for almost 1400 years. In this study, polysorbate 80, water and ultrasonic emulsification is used to formulate N. sativa essential oil nano emulsion (NSEO-NE). Water to oil ratio was varied in the ratio 1:1, 1:2 and 1:3 (v/v). Nano

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emulsions with 1:2 (v/v) and 1:3 (v/v) ratios of oil and surfactant had a droplet diameter of 44.64 6 3.7 nm and 37.47 6 4.1 nm respectively. Therefore the results showed that the droplet size can be decreased by low oil: surfactant ratio. Same was found for turbidity at 600 nm, which increases with more surfactant. The IC50 value was found to be higher after 24 h (82 6 8.6 μL/ mL) than 48 h (59 6 4.5 μL/mL). MCF-7 cells were tested with NSEO, showing 44% cell death in 24 h whereas 28% of cells underwent a necrotic death showing cytotoxic activity (Periasamy, Athinarayanan, & Alshatwi, 2016). Herein, the essential oil of an endemic plant Cedrelopsis grevei from Madagascar has been used for study. Samia et al. and Hicham et al. for the first time reported the anticancer, antimalarial, anti-inflammatory and antioxidant properties of this plant. GC-MS quantified the essential oil in which 64 components were found, the major components being, (E)-b-farnesene (27.61%), d-cadinene (14.48%), acopaene (7.65%) and b-elemene (6.96%). When tested for cytotoxicity on MCF-7 cells, the IC50 value was found to be 21.5 6 2 mg/L whereas it was 17.5 mg/L against P. falciparum. But this essential oil showed poor antioxidant activity using assays, DPPH (IC50 . 1000 mg/L) and ABTS (IC50 5 110 mg/L). It was also found that this essential oil shows good 5-Lipoxy genase inhibition. The various tests conducted showed that the component 1, 4-cadinadiene (r2 5 0.61) is responsible for antimalarial activity whereas (Z)-b-farnesene (r2 5 0.73) showed significant activity against cancer. Further studies are required to determine the exact component responsible for various activities (Afoulous et al., 2013). 11.2.3.3 Brain cancer In this study, activity of BEO (bergamot essential oil) and bergapten-free BEO (BEO-BF) loaded in pegylated liposomes was tested against cell line SH-SY5Y (neuroblastoma cells). Previous studies have been conducted

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for the same but due to meager solubility of this compound an attempt has been made to encapsulate it in liposomes. The encapsulation efficiency of BEO and BEO-BF was 75 6 2.32% and77 6 1.94% respectively. There was a slight change in the zeta potential from 24.57 to 22.95 mV for BEO liposomes and 22.57 mV for BEO-BF liposomes. This change can be attributed to the partial distribution of BEO on the external layers of liposomes changing its surface charge. It was observed that the liposomal formulation exhibited more efficacy even in low concentration (0.01 and 0.02%v/v) within 24 h compared to the free components. But in the long run BEO-BF was found to be more effective in reducing cell viability (B20% reduction in 72 h) whereas BEO showed B60% reduction. This experiment highlighted the importance of liposomes in successfully delivering any formulation (Celia et al., 2013). Another study for brain cancer was conducted, which is the first study on Elsholtzia ciliata (Lamiaceae), an herb said to have medicinal properties. Herein, the essential oils of fresh, frozen, and dried herbal materials of E. ciliata are used and their different extraction methods are compared. GC-MS identified 46 compounds in SPME (dynamic headspace solid phase micro extraction) composition, majorly ketones (dehydroelsholtzia and elsholtzia) in dried samples while only Artemisia ketone was determined in fresh samples. While HD (hydro distillation) showed 26 compounds, the main compounds of this essential oil being dehydroelsholtzia ketone (78.28%) and elsholtzia ketone (14.58%). Both fresh and frozen extracts had the main compound as dehydroelsholtzia ketone: 45.74% and 58.47% respectively. The in vitro anticancer activity was checked on three cancer cell lines, namely pancreatic cancer (Panc-1), (human glioblastoma (U87), and triple negative breast cancer (MDA-MB231). The EC50 (half maximal effective concentration) for the three samples was in the range: 0.017%
0.021%. The results showed these extracts were not effective on the

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cell lines. Did not have any impact on U87 cell line, whereas they diminished the cell viability of MDA-MB231 cells up to only 76.4%. Therefore in conclusion, further study required for different extraction methods and their impact (Pudziuvelyte et al., 2017). 11.2.3.4 Liver cancer A non-invasive weed, Eupatorium adenophorum Spreng. (EA) belonging to genus Eupatorium is conventionally used for medical purposes in China. Herein, the aim was to explore the capability of the essential oil from the same (EAEO) against HCC cell. It was seen that EAEO had the ability to prevent apoptosis of HCC cells by arresting them at G1 phase. There were 18 components found in EAEO, majorly being torreyol (30.10%). IC50 values of HepG2, Hep3B, and SMMC-7721 cell lines after 48 h were 17.74 6 1.92, 49.56 6 5.01, and 39.20 6 3.37 mg/mL, respectively. EAEO showed time and dose dependant decrease of G2/M phase of HepG2 cells to G0/G1. In vivo analysis was done on HepG2-bearing nude mouse model, TUNNEL assay showed augmented expression of cleaved caspase-9 and 3. There was dose dependant action of caspase-12 (ERS-induced apoptosis) and caspase-7 in vivo. AKT (protein vital for cancer cell proliferation) appeared to be a major target for EAEO action. Western blotting further proved that EAEO lead to phosphorylation of AKT at Ser473 and Thr308 (residues required for AKT activation) but did not decrease the quantity of AKT. Therefore EAEO is a good candidate for anticancer purposes (Chen et al., 2018). Agarwood is a priceless forest product that was conventionally used for incense and traditional medicines. There has been no report about the use of Aquilaria spp. for cancer treatment, but China has previously used it for its anti-inflammatory properties. Herein, essential oil of agarwood obtained via distillation of resin was used for treatment on breast cancer cell line MCF-7. They added the extracted AEO into adherent cells for cell viability assay and

to the cells at the time of inoculation for cell attachment assay. More than 50% of the cells were killed when 0.2 mg/mL AEO was added depicting cytotoxic activity. At 44 μg/mL, AEO obtained 50% cell inhibition (IC50) proving anticancer activity against MCF-7 cells. In conclusion the occurrence of sesquiterpenoid compounds can be responsible for its anticancer activity (Hashim, Phirdaous, & Azura, 2014). 11.2.3.5 Ovarian cancer In this study, Rosmarinus officinalis L. essential oil and three of its main components namely α-pinene (19.43%), 1,8-cineole (27.23%), and β-pinene (6.71%) were assessed for their anticancerous properties. While comparing antimicrobial activities it was found that R. officinalis L. had the same activity as α-pinene whereas the lowest activity was found for 1,8cineole then β-pinene. Gram positive bacteria were more sensitive for the three except 1,8cineole than gram negative. MTT assay showed dose dependant increase in cytotoxicity for cell lines. When checked for SK-OV-3 (ovarian cell line) cell viability treated by R. officinalis L. essential oil, α-pinene, β-pinene and 1,8-cineole were 36.13%, 45.85%, 67.77% and 93.03%, respectively. The IC50 values for 1,8-cineole against these three cell lines (SK-OV-3, HO8910, Bel-7402) were much higher than R. officinalis L. essential oil, α-pinene and β-pinene. MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) tests were performed by the broth microdilution method. Bacteria were killed within 24 h by β-pinene. Therefore in conclusion the strongest cytotoxic activities were in the order: essential oil .α-pinene . β-pinene . 1,8-cineole (Wang, Li, Luo, Zu, & Efferth, 2012). Another plant known for its medicinal purposes, Ocimum kilimandscharicum Guerke, popularly known as “kilimanjaro,” is a semievergreen shrub found in East Africa is used in this experiment. Essential oil from fresh leaves of O. kilimandscharicum (EOOK) was 0.16% (v/w),

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43 components were found in the essential oil majorly camphor (51.81%). DPPH assay showed IC50 of 8.21 μg/mL, the high antioxidant activity shown by the essential oil can be credited to high percentage of monoterpenes. When verified on ten different human cancer cells, the cytotoxic effect was shown majorly on OVCAR-3 ovarian cells with value of GI50 was 31.90 mg/mL. Direct leukocyte migration pleurisy model of carrageenan showed that the EOOK when administered orally at doses 30 and 100 mg/kg inhibited leukocyte migration by 82 6 4% and 95 6 4%, respectively. In conclusion, this study for the first time showed properties of O. kilimandscharicum which can be attributed to the presence of limonene, camphor, and 1,8 cineole (De Lima et al., 2014). 11.2.3.6 Skin cancer In this study, the essential oil (EO) from the aerial parts of Pituranthos tortuosus was extracted via hydro distillation, to access its composition and immunomodulatory potential. As identified by GC-MS there were 17 compounds in P. tortuosus EO, majorly sabinen (24.24%). For in vitro test, Splenocytes from naı¨ve mice were secluded and cultured with EO. The concentration of 1.25 μg/mL led to splenocyte proliferation of 78.6 6 4.2% whereas with LPS the same dosage caused higher proliferation. 400 μg/mL reached a maximum of 91.3 6 3.6% after 48 h of incubation in B16F10 cells. Results for EO-LPS indicate increase in B cells proliferation, suggesting LPS to be a good mitogen. It is also possible that EO itself contains some components that act as mitogen. Due to the compound terpinen-4-ol present in EO, it acts as an anticancer agent and it is also speculated that it acts as an antimelanoma agent. Although further investigation is needed to confirm the same (Krifa et al., 2015). Similarly, the essential oil of Salvia officinalis L. has also been used for anticancer activity. It has been used traditionally as herbs for range of diseases. The essential oils were labeled

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from S1 to S18 depending on where they are collected from. The leaves of these samples were air dried and crushed then hydro distilled for 3 h which were then used for analysis. There were 14 compounds found in the majorly α-thujone (7.8%
20.1%), camphor (8.4%
20.8%) but in general oxygen containing monoterpenes (32.6%
59.8%). MTT assay was conducted on A375, M14 and A2058 human melanoma cell lines showing the essential oil (S1
S18) to be effective. But, the most effective ones were S6, S13 and S18 in which S13 had an IC50 value of 8.2, 12.1 and 11.7 μg/mL in M14, A375 and A2058 cells, respectively. When cancerous cells get disrupted LDH is released therefore here LDH levels were examined. There was no significant release in LDH secretion in melanoma cancerous cells when treated with S1
S18 at 50 μg/mL concentration. The shown anticancer activity of S1 to S18 samples were due to active α- and β-thujone isomers (Russo et al., 2013). 11.2.3.7 Others Several reports demonstrate the efficacy of various biological macromolecules against cancerous cells. A variety of lemon grass flexuosus were studied for its cytotoxic activity against 12 human cancer cell lines along with Ehrlich and Sarcoma-180 mice tumor models. Essential oil from the same were used in this study. The most pronounced effect was shown on three cancer cell lines namely 502713 (colon), IMR-32 (neuroblastoma), Hep G2 (liver) and SiHa (cervix) having IC50 values of 4.2, 4.7, 4.8 and 6.5 μg/mL, respectively. This may be due to different molecular characteristics of these cells. There was a dose dependant increase in cytotoxicity as shown by incubating HL-60 cells for 12 h in different concentrations of EO. SEM revealed the untreated HL-60 cells were to be of round shape and when treated with EO showed shrinking (by chromatin condensation and nuclear fragmentation). The reduction in tumor growth was significant in

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both the models, that is, at 200 mg/kg EO the inhibition was 57.83% in Ehrlich and 36.97% in S-180. This experiment showed that the EO of flexuosus is active against selected cancer cell lines (Sharma et al., 2009). Another report for the use of essential oil is presented by Chen and Chen. Herein, they used Curcuma zedoaria Roscoe, to extract essential oil (zedoary) for cytotoxic effect on nonsmall cell lung carcinoma (NSCLC) cell. Zedoary is used as spice and herb in India, Indonesia, and China. To examine the activity of Zedoary on NSCLC cells, H1299, A549, and H23 cells were exposed to different concentrations of Zedoary. The IC50 values were found to be dependent on the concentration of Zedoary and the best cytotoxic effect were expressed on H1299 cells. Flow cytometry analysis showed that zedoary induced cell death (sub-G1 phase) in a time dependant manner (Fig. 11.5A) which was proved when H1299 cells were treated with Zedoary for 72 h the sub-G1 cells increased from 11.4% to 53.3%. Fig. 11.5B shows DNA fragmentation, which is the hallmark for cell apoptosis further strengthening the effect of Zedoary. To determine the enzymatic activity of caspase (plays role in initiation and completion of apoptosis) western

blotting test was done. The results further proved that Zedoary is time dependent. The in vivo antitumor activity of zedoary essential oil was performed on H1299 cells of nude mice showing reduction in size of tumor. There were a lot of components found but the sesquiterpenes contributed up to 60% in the oil. It is said that further studies are required to determine the active compound (Chen et al., 2013). Herein, nano emulsion of essential oil from clove buds (Syzygium aromaticum) was assessed for its anticancer activity. To prepare nano emulsions, standard titration technique was used. Tween 20 and Tween 80 were used as surfactants; oil to surfactant ratio 1:4. A phase diagram (as shown in Fig. 11.6) was plotted for EO, surfactant and water which showed the hydrophilic lipophilic balance for Tween 20 and Tween 80 to be 16.7 and 15 respectively. The droplet size of essential oil was measured without dilution as it may alter the state via DLS. The average mean droplet size was found to be 11.73 6 0.32 nm. UV-Spectroscopy absorbance at 600 nm showed to be decreasing with increasing surfactant concentration possibility due to weak scattering. MTT assay was done for both cancerous (HTh-7) and non-cancerous cell lines (Hek-293). EO was found to exhibit no harm to non-cancerous cells

FIGURE 11.5

Effect of zedoary essential oil on caspase activity in H1299 cells. (A) H1299 cells were treated with various concentrations of zedoary essential oil for 48 h. Cells were then harvested and lysed for the detection of the expression of cleaved caspase-3, cleaved PARP, and β-actin. (B) H1299 cells were treated with 110 μg/mL zedoary essential oil for 6, 24, 48, and 72 h. Cells were then harvested and lysed for the detection of the expression of pro-caspase-3, cleaved caspase3, cleaved PARP, and β-actin. Source: Reprinted with permission from Chen C.C., Chen Y., Hsi Y.T., Chang C.S., Huang L.F., Ho C.T., et al. (2013). Chemical constituents and anticancer activity of Curcuma zedoaria roscoe essential oil against non-small cell lung carcinoma cells in vitro and in vivo. Journal of Agricultural and Food Chemistry;61(47):11418
27.

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FIGURE 11.6

Ternary phase diagram constructed using clove bud oil, Tween 20, and 80, respectively, and water showing oil/water microemulsion region varying oil:surfactant ratio from 1:1 to 1:9 (left), and from 1:9 to 9:1 (right) for both Tween 20 and Tween 80. Source: Reprinted with permission from Joyce Nirmala M., Durai L., Gopakumar V., Nagarajan R. (2019). Anticancer and antibacterial effects of a clove bud essential oil-based nanoscale emulsion system. International Journal of Nanomedicine;14:6439
50.

whereas the IC50 for HTh-7 was found to be 0.7 μg/mL which killed ,55% cells in 48 h. It was seen that Tween 20 was easily absorbed in the EO, also the surfactants are important for making the emulsion clear and small. This study meets all compliance requirements, but further tests are suggested for usage (Joyce Nirmala, Durai, Gopakumar, & Nagarajan, 2019).

11.3 Conclusion The clinical therapy of disease has made generous enhancements since the early long periods of present-day hostile to tumor drug research. The application of natural compounds in the therapy of malignancy, the extremely regular “plague” of our advanced occasions, has brought about expanded remedial viability. Their fruitful application has opened many doors in cancer treatment. Toward such an end, it is trusted that the present chapter will give some significant information to progressing investigations of various biological macromolecules.

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C H A P T E R

12 Biological macromolecules as immunomodulators Eduardo Costa, Manuela Machado, Manuela Pintado and Sara Silva Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal

12.1 Introduction Inflammation is a naturally occurring process required to maintain homeostasis and, therefore perceived as beneficial so long as it remains a tightly regulated acute process. When this alters, for example, the inflammatory process becomes chronic without need, the resulting altered immune state is frequently perceived as harmful to the body. In cases of imbalance, the most frequent therapy considered is the pharmacological manipulation of the immune response, that is immunomodulation (Gertsch, Viveros-Paredes, & Taylor, 2011). Immunomodulation, in a broad sense, is a term used to refer to an array of potentially therapeutic interventions that result in a specific immune response. Generally, two different types of response may be considered, one directed at stimulating an increase in immune response and another targeting its attenuation. The type of immunomodulatory approach to use is, therefore dependent on the problem itself with the first being required whenever the natural immune

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00012-9

response is diminished or inadequate and the second when it is excessive (Gea-Banacloche, 2006). Therapeutic macromolecules are characterized by their large molecular weights (MWs). Research on the potential of (bio)macromolecules conjugated with synthetic drugs is not recent, with pioneering works on targeted delivery, namely the conjugation of immunoglobulins (Ig) and drugs, going back to the late 50s (Mathe, Tran Ba, & Bernard, 1958; Yang & Kopeˇcek, 2014). However, in this case, the macromolecular component is frequently regarded as a delivery vehicle, that is while it accomplishes a specific task (frequently associated with drug delivery) by itself it has no perceived therapeutic value. This paradigm has shifted with the emergence of “drug-free” macromolecular therapeutics (polymer-based nanomedicine designs) which are based upon the idea that the direct recognition of naturally occurring motifs (e.g., protein antiparallel coiled coils) by cell surface receptors may result in the mediation of biological processes and the subsequent activation of signaling cascades triggering a desired therapeutic effect

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(Chu & Kopeˇcek, 2015; Wu, Liu, Johnson, Yang, & Kopecek, 2010; Yang & Kopeˇcek, 2014).

12.2 Immunomodulation As the body of work associated with immune cell communication grew, it became possible to devise strategies to manipulate these signaling cascades and, consequently manipulate the resulting immune response by targeting specific immune cell populations (such as neutrophils, macrophages, natural killer (NK) cell or lymphocytes) and up/down-regulating cytokine production. This intervention upon the immune system may be broadly referred to as immunomodulation with the approaches being classified as immunosuppressant or immunostimulant (Bascones-Martinez, Mattila, Gomez-Font, & Meurman, 2014; Tzianabos, 2000). From a different perspective, immunomodulatory compounds may also be classified, in accordance with their mode of action, as specific or nonspecific. Specific-action immunomodulatory compounds affect the cells depending on the presence of an antigen exhibiting a selective specific immune response, that is, stimulation results in a targeted immune reaction. Vaccines are the best example of this type of immunomodulation, as vaccine administration results in the immunization against specific antigens. Nonspecific-action immunomodulators alter the immune response without directing it toward a specific target and can be further classified into three different groups: (1) those that act upon a normal immune system, (2) those that act upon an immunosuppressed system, (3) and those that act on both (Bascones-Martinez et al., 2014) (Fig. 12.1).

12.3 Immunomodulation, biomolecules, and applications Vaccination is one of the most successful examples of the widespread use of a immunostimulatory

FIGURE 12.1

Immunomodulatory approach for different health conditions.

technique with its benefits in reducing the morbidity and mortality of disease being well-established (Greenwood, 2014; Nandi & Shet, 2020). Moreover, it is also a relatively unique application for immunomodulation as it aims not to ameliorate a response resulting from an existing pathology, but to minimize the risk should the individual encounter it. Vaccine production/development also presents itself as one good example of the use of biomacromolecules as they may be used as both the active components (e.g., Heamophillus influenza type b vaccine’s active principle is the outer coat’s polysaccharide attached to a protein to aid its identification by the immune system or tetanus vaccine whose active principle is the inactivated form of the tetanus toxin, a protein) or as adjuvants (e.g., CpG 1018 adjuvant, used in one hepatitis B vaccine, is comprised of a cytosine phosphoguanine motif mimicking genetic material from viral and bacterial sources resulting in an exacerbation of the immune response) (Aguilar & Rodrı´guez, 2007; Campbell, 2017; Hyer & Janssen, 2019; Jiang & Schwendeman, 2008; Schneerson, Barrera, Sutton, & Robbins, 1980). A different application for biomacromolecules is the control of inflammatory bowel diseases (IBD). These conditions (e.g., Chron’s disease and ulcerative colitis) occur when there is an uncontrolled immune response in the intestine. Therefore

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it stands to reason that one of the potential therapeutic strategies for the control of these pathologies is either the use of monoclonal antibodies to selectively block proinflammatory cytokines or the use integrin blockers that could aid in the control of lymphocyte migration to the gut, although that does come at a higher risk of infection. So far, the Food and Drug administration has approved for clinical use two different anti-Tumor Necrosis Factor (TNF) antibodies, Infliximab and Adalimumab, and one anti- Interleukin (IL)-12 and anti-IL-23 antibody, Ustekinumab. These may be administered to patients with moderate-to-severe active IBD but it requires multiple intravenous and/or subcutaneous injections (Hanauer et al., 2020; Papamichael et al., 2019; Sands et al., 2019; Wei & Claus-Michael, 2018). Several other examples of this type of approach may be reported: Sarilumulab is a recombinant humanized anti-IL-6 antibody used to reduce the inflammation process in patients with rheumatoid arthritis (Fleischmann et al., 2017; Huizinga et al., 2014). Siltuximab is recombinant human-mouse anti-IL-6 antibody approved for the treatment of patients with Castleman’s disease (an rare condition characterized by an atypical lymphoproliferation) (Van Rhee et al., 2010). Etanercept/Enbrel a recombinant anti-TNF and antilymphotoxin antibody approved for the treatment of an array of different inflammatory conditions like plaque psoriasis, rheumatoid, polyarticular juvenile idiopathic and psoriatic arthritis (Goffe & Cather, 2003; Moreland et al., 1999). Golimumab/Simponi is a fully humanized anti-TNF Ig G monoclonal antibody which has been approved for the used in the treatment of rheumatoid and psoriac arthritis, ulcerative colitis and ankylosing spondylitis (inflammatory rheumatic disease of the axial skeleton) (Lo¨wenberg & D’Haens, 2013; Mazumdar & Greenwald, 2009). These anticytokine antibody-based therapies are frequently associated with an array of side effects, some of which may become very severe. One of them stems from the rational of the therapeutic approach, that is the inflammatory response plays a major role in the defense against pathogens if it is

inhibited the susceptibility to pathogens is increased. In fact, an increase in the development of infections is a frequent side effect of these treatments (Antoni & Braun, 2002; Li, Zheng, & Chen, 2017; Pfeffer, 2003). Other examples are an increase of the risk of developing certain types of cancer, the exacerbation of some conditions (e.g., congestive cardiac failure and multiple sclerosis), cutaneous adverse events, or the emergence of new autoimmune diseases (Balakumar & Singh, 2006; Conrad et al., 2018; Guerra et al., 2012; Robinson, Genovese, & Moreland, 2001).

12.4 Polysaccharides The possibility of the use of polysaccharides as immunomodulators was proposed in the early 40s when a polysaccharide mix, known as Shear’s polysaccharide, was described as inducing tumor necrosis (Shear, Turner, Perrault, & Shovelton, 1943; Srivastava, Breuninger, Creech, & Adams, 1962). Polysaccharides are frequently regarded as T-cell-independent antigens and, therefore do not result in cell-mediated immune responses. Antigen Presenting Cells (APCs) have been reported as unable to process polysaccharidic antigens and are therefore unable to present them on their cell surface for posterior T-cell recognition resulting in a lack of activation of the innate immune. This could explain why exposure to bacterial polysaccharides typically stimulates only the production of IgM (lowaffinity Ig) and IgG. While this may translate into a lower immunostimulatory capacity, this is not true for all polysaccharides (Tzianabos, 2000; Weintraub, 2003).

12.4.1 Immunomodulatory polysaccharides Zwitterionic polysaccharides (ZPs) are a group of structurally diverse, charged (positively and negatively) capsular polysaccharides

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comprised of several repeating units (of 1
8 monosaccharides in length per repeating subunit) isolated from pathogenic bacteria. Unlike other capsular polysaccharides, they exhibit a zwitterionic charge motif (i.e. the presence of equal amounts of positively and negatively charged functional groups) which allows them to directly interact with immune cells resulting in the activation of CD4 1 T-cells. APCs capable of recognizing Major Histocompatibility Complex (MHC) II molecules have been reported as paramount for this process. ZPs have been shown to interact with APC’s Human Leukocyte antigen— Dr isotype receptor (HLA-Dr; a specific MHC II cell surface receptor). This interaction plays an essential role in T-cell activation that, in turn, is dependent on the interaction between MHC II and T-cell receptors (TCR), more specifically αβ-TCR, as well as in the activation of CD28-B7 (CD 86 ligand) T-cell co-stimulatory pathway. This results in the production of T-cell derived cytokines namely IL-2, interferon (IFN)- γ and IL10 (Tzianabos et al., 1999; Tzianabos, Wang, & Kasper, 2003; Tzianabos, 2000). B-glucans, which may be isolated from algae, yeast and fungi, are a diverse group of glucose homopolymers that have been linked with an array of different biological activities, one of which is immunostimulation (Bohn & BeMiller, 1995; Cleary, Kelly, & Husband, 1999; Novak & Vetvicka, 2008). Different β-glucan structural properties (e.g., helical conformation and branching degree) have been associated with differences in their immunomodulatory activity (Bohn & BeMiller, 1995; Cleary et al., 1999). For instance, higher MW glucans have been described as being more stimulatory of macrophage activation than their smaller counterparts, with the authors that carried out this particular study hypothesizing that the higher MW aided in the cross-linking of receptors (Cleary et al., 1999). Even though they have been described as influencing the activity of several different immune system cells from Band T-cells, to NK, eosinophils and

neutrophils, their major immunostimulatory effect has been associated with their interaction with macrophages (Cleary et al., 1999; Czop, Puglisi, Miorandi, & Austen, 1988; Di Renzo, Yefenof, & Klein, 1991; Hashimoto, Suzuki, & Yadomae, 1991; Mahauthaman et al., 1988). These cells are one of the elements involved in the humoral immune response. The innate immune response is dependent on the recognition of specific, highly conserved, molecules present in pathogenic microorganism named PathogenAssociated Molecular Patterns (PAMPs). As β-glucans belong to the PAMPs group, it stands to reason that they are directly recognized by macrophages through an array of different receptors like Dectin-1, Lactosylceramide, Toll-Like Receptor (TLR)-2 and integrin CD11b/CD18. This recognition results in the activation of an array of signaling cascades that trigger the immune response ranging from macrophage migration to the production of reactive oxygen species (ROS), leading to the activation of other phagocytes and proinflammatory cytokine expression (e.g., TNF-α and IL-1) (Novak & Vetvicka, 2008; Schepetkin & Quinn, 2006; Zipfel & Robatzek, 2010) Chitosan is a copopolymer (2-amino-2-dedoxydglucose and 2-acetamino-deoxy-d-glucose units linked with β-(1
4) bonds) that results from the alkaline deacetylation of chitin. During this process, amino groups are formed from chitin’s original acetamide groups with the overall number of amino groups present in the final molecule being characterized by its deacetylation degree (DD). DD and MW are perceived as the main decisive parameters behind chitosan’s biological activities. Davydova et al. (2016) reported that peroral administration of chitosan to mice resulted in an increase of serum IL-10, an antiinflammatory cytokine, with this stimulation being stronger with low MW chitosan than with high MW chitosan. Moreover, this administration conferred some protection against acetic acid-induced colitis as treated animals exhibited smaller injuries in the large intestinal wall and lower levels of myeloperoxidase (an enzyme that

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catalyzes the production of ROS that is found in higher levels in patients with IBD) (Chami, Martin, Dennis, & Witting, 2018; Davydova et al., 2016). This antiinflammatory potential has been widely reported in literature with several authors reporting decreases in proinflammatory cytokines (IL-1β; IL-4; IL-6, IL-12; IL-13; TNF-α) and increases in antiinflammatory ones (IL-2; IL-10; Transforming Growth Factor (TGF)-β1), using an array of different chitosans (e.g., carboxymethylated, different MW and DD), chitosan-based formulations (e.g., nanoparticles, films) and disease models (e.g., acne, asthma, colitis) (Chung, Park, & Park, 2012; Friedman et al., 2013; Oliveira, Santos, Oliveira, Torres, & Barbosa, 2012). The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)—stimulator of IFN genes (STING) pathway and the NLRP3 inflammasome are two of the pathways in which chitosan is perceived to modulate.

12.4.2 Gut microbiota modulation From a different perspective, polysaccharides immunostimulatory has also been strongly associated with not a direct effect upon the immune system but my interacting with gut microbiota, modulating its composition and consequently, its interactions with the host (Vieira, Fukumori, & Ferreira, 2016; Wang, Zhu, & Qin, 2019). In fact, the gut microbiota, through its constituents and its metabolites, has been reported as interacting with an array of different cells in an immunomodulatory manner [from intestinal epithelial cells (IECs) to lymphocytes]. It not only teaches the immune system to tolerate symbiotic bacteria and contributes to the integrity of the barrier function but dysbiosis has been strongly associated with an array of different immune and/ or inflammatory disorders (Agace & McCoy, 2017; Chiu et al., 2019; Guenther, Josenhans, & Wehkamp, 2016; Honda & Littman, 2016; Meisel et al., 2017). Short Chain Fatty Acids (SCFA) are a prime example of a class of

metabolites that have been demonstrated as exerting an antiinflammatory effect which is perceived as a consequence of their capacity to inhibit histone deacetylases (HDACs) and activating G protein-coupled receptor (GPR; namely GPR109A and GPR43) of IECs (Borthakur et al., 2012; Macia et al., 2015; Priyadarshini, Kotlo, Dudeja, & Layden, 2018; Thangaraju et al., 2009; Zhao et al., 2018). Moreover, they have also been described to interact with the innate immune system resulting in the release of prostaglandin E2 and the increase of IL-10 levels. SCFA, are of particular interest in this case because, while they are neither macromolecules nor polysaccharides, they may be the byproduct of their fermentation by the gut microbiota. Several different types of polysaccharides have been associated with an increase in SCFA producing bacteria and, overall SCFA levels. This effect has been extensively reviewed and is discussed later in the chapter (Flint, Bayer, Rincon, Lamed, & White, 2008; Harris, Morrison, & Edwards, 2020; Ho Do, Seo, & Park, 2020; Kim, Park, & Kim, 2014; Morrison & Preston, 2016; Singdevsachan et al., 2016; Van Den Munckhof et al., 2018; Xu, Xu, Ma, Tang, & Zhang, 2013). Regardless, dietary carbohydrate fibers have been regarded as contributing to the management of an array of immunological diseases, effect that, while the mechanisms are not completely elucidated, seems to stem mostly from their capacity to modulate gut microbiota and its metabolites although some other mechanisms may also be at play. Namely, pectins have been shown to bind to TLR2 consequently inhibiting TLR1TLR2 proinflammatory pathway while allowing the TLR2-TLR6 tolerogenic pathway to remain unaltered (Sahasrabudhe et al., 2018).

12.5 Lipids Lipids comprise a large group of compounds and the majority have fatty acids in

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your structure and plays important roles in cell structure and homeostasis. They are components of cell membranes and a source of energy (Aluko, 2016). Its metabolites act as intracellular and extracellular mediators, therefore can modulate the immune functions of cells, influencing both its structure and its metabolism (Kumar et al., 2019). The immunomodulatory properties of dietary fatty acids depend on their unsaturated or saturated nature and their degree of unsaturation (Radzikowska et al., 2019). The most studied lipids are SCFA and their impact on gut health, and the omega-3 and omega-6 polyunsaturated fatty acids and their important role on inflammation and immune function.

12.5.1 Immunomodulatory effect of lipids One of the most studied SCFA is butyrate and its important contribution to gut health and cell proliferation. Butyrate plays a regulatory role in transepithelial fluid transport (Kunzelmann & Mall, 2002). Besides, it has stimulatory effects on normal colonic cell proliferation and has been used as a substrate used for both growth and regeneration of cells in the large and small intestine (Aluko, 2016; Rowland et al., 2018). On the other hand, it can inhibit the growth and proliferation of colon cancer cell lines. As reviewed by Canani et al. (2011), its role in the protection against colorectal cancer has been widely supported. Indeed, some in vitro studies with carcinoma cell lines, have suggested that the anticolon cancer properties are achieved through enhanced apoptosis of mutant colonic cells and inhibition of proliferation (Selber-Hnatiw et al., 2020). The antiinflammatory potential shown by butyrate has been primarily attributed to its ability to inhibit nuclear factor κB (NFκB) activation in human colonic epithelial cells (Inan et al., 2000). In relation to the omega-3 and omega-6 fatty, they are essential fatty acids and play different roles in the human body. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids

are metabolic derivatives of α-linolenic acid and have antiinflammatory potential (Fig. 12.2) (Kumar et al., 2019). In contrast, omega-6 fatty acids like linoleic acid are converted to γ-linoleic acid and arachidonic acid (AA) and have distinct roles in inflammation (Calder, 2017; Kumar et al., 2019). Biochemically, higher concentrations of omega-3 fatty acids compete with AA for the synthesis of lipid mediators and can balance the inflammatory/proresolution phenotypes (Spite & Serhan, 2010). In addition, fatty acids can competitively modulate the signaling pattern recognition receptor and GPR40 on leukocytes and reduce the risk of inflammation disease progression (Dennis & Norris, 2015; Lee, Zhao, & Hwang, 2010; Simopoulos, 2008). Nevertheless, metabolites of long-chain fatty acids, also known as eicosanoids, can interact with GPRs and have been implicated in the development of atherosclerosis (Simopoulos, 2008). The intake of omega-3 fatty acids promotes the production of antiinflammatory lipids like leukotrienes (five series LTx5) or three series prostanoids (PGx3). On the other hand, an increase in omega-6 fatty acids promote the production of proinflammatory mediators like leukotrienes (LTA4, LTB4, LTC4, LTD4, LTE44) and prostanoids (PGx2) (Kumar et al., 2019). The metabolites produced, further regulate inflammation by feedback inhibition of biosynthetic enzymes (Kumar et al., 2019; Watanabe, Onozaki, Yamamoto, & Okuyama, 1993). In the human body, the initial inflammatory response, as represented by the production of LTx4, is important for the infiltration of neutrophils to the site of infection and thereby beginning the cascade for the production of proinflammatory cytokines (Sadik & Luster, 2012). The incorporation of these fatty acids into the cell membrane affects the fluidity and surface receptor expression and regulates the function of immune cells (Dridi et al., 2016). Studies show that the intake of EPA and DHA suppresses the T-cell proliferation and cytokine secretion (Pompos & Fritsche, 2002). The reduction of Tcell proliferation in the presence of EPA and

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12.5 Lipids

FIGURE 12.2

279

Essential omega-3 and omega-6 fatty acids biosynthesis and inflammation roles.

DHA can be explained by the reduction of IL-2 secretion or by the reduction of its receptor IL-2R (Collison, Collison, Murphy, & Jolly, 2005; Gorjao, Cury-Boaventura, De Lima, & Curi, 2007; Zeyda et al., 2003). These facts suggest that omega-3 fatty acids can interrupt the autocrine IL-2 pathway (Radzikowska et al., 2019; Zeyda et al., 2003). Other studies reveal that DHA has immunosuppressive effects due to its capacity to modulate the calcium concentration on T-cells (Aires, Hichami, Moutairou, & Khan, 2003; Bonin & Khan, 2000). This occurs by inhibition of mitochondrial translocation to the immunological synapse, thereby reducing Ca21 uptake by mitochondria. This blocks the phosphatase activity of calcineurin, consequently, these events suppress the genes involved in T-cells activation (Shaikh, Jolly, & Chapkin, 2012; Yog, Barhoumi, Mcmurray, & Chapkin, 2010). On contrary, the presence of saturated fatty acids like palmitic acid promotes the T-cells activation and their

differentiation into proinflammatory phenotypes (Radzikowska et al., 2019). Regarding B-cells the presence of saturated fatty acids suppresses activation by lipoapoptosis, in contrast, the presence of monounsaturated fatty acids or DHA prevents this effect (Rockett, Salameh, Carraway, Morrison, & Shaikh, 2010). An in vitro study demonstrated that treatment of Raji B cells with EPA and DHA (concentrations range between 12.5 to 70 μM) during 48 h, increase the cell proliferation. Another study shows a reduction in the production of cytokines such as IL-10, TNFα, and INF-γ, in Raji B cells exposed to EPA and DHA (Verlengia et al., 2004). The impact of lipids on T and B-cells increases research interest in the use of this macromolecule as adjuvants on vaccine development. Monophosphoryl Lipid A (a chemical derivative from lipopolysaccharide) are widely used in the production of vaccines, due to their safe immunostimulant action via alterations in

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280

12. Biological macromolecules as immunomodulators

TLR signaling (Casella & Mitchell, 2008; Sarti et al., 2011). More recently, lipids have been used as a vehicle for DNA and RNA-based vaccines, in the form of lipid nanoparticles (LNPs) (Mucker et al., 2020; Shirai et al., 2020). Moreover, studies demonstrated that the use of this kind of system, increased mice’s immune response particularly antigen-specific antibody and T-cell mediated responses. Moreover, LNPs have been described as being an efficient antigen-delivery vehicle, for example, their combined use with dengue and hepatitis B virus showed an increase in antigen-specific antibody, CD4 1 T-cell, and CD8 1 T-cell responses in animal models (Shirai et al., 2020).

12.6 Proteins

proteins required for the presentation of antigens to T-cells. Recognition of the MHC/antigen complex by T-cells with TCR will lead to the proliferation and differentiation into effector cells. For type II MHC/antigen complex TCRs recognition will occur on CD41 T cells, with effector cells differentiating into T helper (Th) or T regulatory (Tr) cells which will then control the proliferation of B and T cells thus modulating the immune response. On the other hand, when TCR binding and activation occurs with Type I MHC/antigen complex CD81 T cells will be differentiated to cytotoxic T-cells which are responsible for the elimination of infected cells (Murphy & Weaver, 2016; Tsoras & Champion, 2019) (Fig. 12.3).

12.6.1 Known immunomodulatory proteins

Off all biological macromolecules, proteins are the ones most commonly associated with immunomodulatory effects as they already play key roles in the normal functioning of the immune system. They are the antigens that Dendritic cells (DCs) and other APCs present on their surface, via MHC, and that promote the activation of cytokine and costimulatory surface proteins production which will lead to T-cells recognition and consequent proliferation/differentiation of the whole immune response cascade. Because of this capacity to modulate the human immune system response, proteins (and their peptides) have been used in vaccines development, in autoimmune and neurodegenerative diseases and in cancer treatments (Legastelois et al., 2017; Tsoras & Champion, 2019). From a mechanistic standpoint proteins upon entering the organism will be internalized by APCs, which in turn will process them and present cytosolic peptides on MHC surface proteins (Type I for pathogenic microorganisms’ proteins, Type II for the remaining foreign material). From here APCs will activate the production of cytokines, through direct recognition by B-cells, and costimulatory surface

When considering immunomodulatory proteins, two sources can be considered: manmade and natural. On the man-made side, the prime example of an immunomodulatory protein are monoclonal antibodies. Currently used for the treatment of several diseases monoclonal antibodies (mAbs) are IgG based and composed of the same basic structure—two heavy 50 kDa and two light (25 kDa) chains held together by disulfide bonds forming a Y-shaped molecule (Dimitrov, 2010; Ryman & Meibohm, 2017). First produced through a hybridoma technique in mice, mAbs were murine in nature and triggered strong immune reactions which led to the development of fully humanized mAbs, through phage display and transgenic mice, thus completely removing murine components and leading to reduced, but not complete, immunogenicity. Nevertheless, as mAbs have elevated target selectivity and low toxicity, their clinical value is high, approximately 30 mAbs have already been approved for treatment by the Food and Drug Administration and some examples of their possible applications can be seen in Table 12.1 (Shepard, Phillips, D Thanos, & Feldmann, 2017; Singh et al., 2018).

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12.6 Proteins

FIGURE 12.3

281

Overview of protein interaction with the immune system.

TABLE 12.1 Examples of already approved clinical uses of mAbs. Commercial name

Ab

Target Target disease

Alemtuzumab

IgG1κ

CD52

Multiple sclerosis, chronic lymphocytic leukemia

Daclizumab

IgG1

CD25

Transplant rejection

Tositumumab

IgG2aλ CD20

Rheumatoid arthritis

Pertuzumab

IgG1

HER2

Breast, gastric adenocarcinoma

Cetuximab

IgG1

EGFR

Squamous cell carcinoma of the head and neck, colorectal cancer, nonsmall cell lung cancer

Golimumab

IgG1κ

TNF-α Crohn’s disease, ulcerative colitis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis

Nowadays, two of the major fields of mAbs success are anti-TNF-α therapies to treat inflammatory diseases and their application in cancer treatment. In the anti-TNF-α therapy mAbs block of TNF-α expression reduces the production of other inflammatory cytokines (IL-1, IL-6, IL-8 among others) and has great

clinical success in the treatment of inflammatory diseases such as Rheumatoid arthritis, Crohn’s disease and juvenile Rheumatoid arthritis. When regarding cancer treatment one of the most important targets is the Epidermal Growth Factor Receptor (EGFR) family, especially when concerning colorectal, head and

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282

12. Biological macromolecules as immunomodulators

neck cancers, with mAbs binding to the extracellular domain of human EGFR, activating the tyrosinase kinase receptor and downregulating cell growth, matrix metalloproteinase production and promoting apoptosis (Chiavenna, Jaworski, & Vendrell, 2017; Pento, 2017; Ryman & Meibohm, 2017; Shepard et al., 2017). Natural immunomodulatory proteins can be obtained from various sources, but one prime example is Lectins. Lectins are carbohydratebinding proteins of nonimmune origin that can be obtained from plants, fungi and fish. They have been described as being capable of acting upon components of the innate and adaptative immunity through an interaction with glycan’s moieties present on immune cells. This interaction has been shown to lead to immunomodulatory activity through modulation of various parts of the immune system with the promotion of Th1 cytokines, induction of CD69 and CD25 activation molecules, promotion of IL-2 and decrease of TNF-α (among other factors) being some of the examples. (El Enshasy & Hatti-Kaul, 2013; Pereira, Correˆa, Vericimo, & Paschoalin, 2018; Souza, Carvalho, Ruas, RicciAzevedo, & Roque-Barreira, 2013; Sze, Ho, & Liu, 2004). An example of lectin is Tarin. This molecule has been shown to be capable of modulating the immune response with the promotion of the expression of IL-2, (associated with NK and T and B lymphocytes activation), IFN-γ (promotes differentiation of T-cells to Th1 and activates B and T lymphocytes), IL1-β and TNF-α (interleukins associated with endothelial cells inflammatory response), neutrophil activation (via modulation of TNF-α levels) and stimulation of hematopoietic cells. This immunomodulatory capacity can be ascribed to Tarin capacity to bind with the antigens CD173/H2 and CD174/Lewis Y which are present in CD34 1 progenitor cells, peripheral neutrophils and endothelial cells (among others) (Kang, Lee, Seo, & Park, 2019; Moehler et al., 2008; Pereira et al., 2015; Pereira et al., 2018). Other example are glycoproteins.

Hemocyanins are oxygen-transporting glycoproteins found in some mollusks which can enhance the innate and adaptative immune response. They have been shown to have high affinities, in a glycan-dependent manner, toward human mannose receptors (Mr) and DC-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This enables them to promote DC maturation, via up-regulation of class II MHC cell surface expression and costimulatory molecules, Th1 immune response, via prolonged antigen presentation to T or B lymphocytes and interaction with macrophages, and their interaction with APCs leads to increased secretion of proinflammatory cytokines. (Arancibia et al., 2014; Becker, Arancibia, Salazar, Campo, & Ioannes, 2014; Kang et al., 2019; Salazar et al., 2019).

Acknowledgments This work was supported by National Funds from FCT— Fundac¸a˜o para a Cieˆncia e a Tecnologia through project UID/Multi/50016/2019. The author Manuela Machado would also like to acknowledge FCT—Fundac¸a˜o para a Cieˆncia e a Tecnologia for her grant SFRH/BD/136701/2018.

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C H A P T E R

13 Biological macromolecules acting on gastrointestinal systems Dilipkumar Pal1 and Supriyo Saha2 1

Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, India 2School of Pharmaceutical Sciences and Technology, Sardar Bhagwan Singh University, Dehradun, India

13.1 Introduction Gastrointestinal system (GS) consists of two major parts such as alimentary canal and other associated organs. Alimentary canal is also known as gastrointestinal tract (Pal & Saha, 2012). Gastrointestinal tract is a long void channel observed from mouth to anus including other important organs such as esophagus, liver, stomach, pancreas, small and large intestines (Saha & Dimri, 2013). Biotransformation of food into simple chemical component and excretion of waste product are the two major works of GS. There are several common disorders/diseases are highlighted with GS such as irritable bowel syndrome (IBS), diarrhea, constipation, ulcer, inflammation and cancer of GS associated organs (Pal & Saha, 2019; Saha, Pal, & Kumar, 2016). In other hand, carbohydrate, amino acid, proteins, lipids, fatty acids, nucleic acids (deoxyribonucleic acid and ribonucleic acid) are included in the category of biological macromolecules (Fig. 13.1) (Nayak & Pal, 2013a; Pal & Nayak, 2012; Pal, Banerjee, & Ghosh, 2012). In this article, we focus

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00013-0

on the role of biological macromolecules on GS (Nayak & Pal, 2013b).

13.2 Role of carbohydrates in gastrointestinal system Carbohydrate is a molecule comprised of carbon, hydrogen, and oxygen atoms. Carbohydrates are also known as saccharides such as monosaccharide, disaccharide, oligosaccharide and polysaccharide. Glucose, fructose, galactose; sucrose, lactose, maltose and sorbitol, mannitol are the common examples of monosaccharide (Ms), disaccharide (DS) and polyols, respectively (Pal, Sahu, & Haldar, 2014; Reynolds et al., 2019; Sacchan, Chandra, & Pal, 2015a, 2015b). Maltodextrin, raffinose and amylose, amylopectin, glycogen, cellulose are the most familiar examples of oligosaccharides (OS) and polysaccharides (PS) respectively. Nowadays peoples are very much concern about the amount of carbohydrates intake, by considering these facts quinoa, oats, and buckwheat are the mostly used carbohydrates

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© 2022 Elsevier Inc. All rights reserved.

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FIGURE 13.1

13. Biological macromolecules acting on gastrointestinal systems

Types of biological macromolecules.

(https://www.healthline.com/nutrition/, 2020). A group of researchers confirmed the effects low concentration of fermentable OS, DS, Ms and polyols on the symbiotic and pathogenic microorganisms observed in gut region associated with IBS examined by 16S ribosomal deoxyribonucleic acid sequencing technique. In this matter ninety five persons with IBS were taken to evaluate the factor. The outcome showed that gram positive anaerobic Bifidobacteria creates a negative response on the protein level. Finally it was concluded that Saccharomyces, Lactobacillus and Streptococcus are the most relative strains and presence of low concentration of fermentable OS, DS, Ms and polyols synergistically creates a positive environment for the patients with IBS (Staudacher et al., 2020). In this situation another research showed that when a group of patients advised to take oligofructan rich almond inverse of wheatflour, the severity of IBS was surprisingly low. So the perfect ratio of saccharides with almond creates a visible change in disease severity (Darvishmoghadam et al., 2019). As we know ulcerative colitis (UC) is well known gastrointestinal disorder. In this category, the novel alkali soluble PS isolated from purple

sweet potato showed greater activity against dextran sulfate sodium (DSS) activated UC. In this case, the composition of microorganism, occurrence of inflammation was evaluated. It was also observed that the PS responsible for inhibition of proinflammatory factors (interleukins) with improvised features of gut microorganism (Sun et al., 2020) (Figs. 13.2 and 13.3). In the management of this condition, turanose (reducing DS) played an essential role to combat the severity of DSS induced UC and IBS. The treatment showed an improvised colon length and myeloperoxidase activity with lowered expression of micro ribonucleic acid and acetylated histone linked inflammatory mediators. These data confirmed the positive effects of turanose on IBS (Kim et al., 2019). Another study showed the importance of gluten free and low concentration of fermentable OS, DS, Ms and polyols (especially fructan) showed a positive response on celiac disease and problems associated with IBS (Muir, Varney, Ajamian, & Gibson, 2019). A double blind clinical test was performed on IBS patients to observe the effect of starch

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FIGURE 13.2

Effects of alkali soluble polysaccharide on colon length on UC patient. Source: Copyright permission granted from Sun et al. (2020) Elsevier Inc.

FIGURE 13.3 Effects of alkali soluble polysaccharide on short chain fatty acid concentration. Source: Copyright permission granted from Sun et al. (2020) Elsevier Inc.

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TABLE 13.1 Self-reported improvement in bowel habit after supplementation in participants consuming psyllium or starch-entrapped microspheres. Psyllium 12 g (n 5 10)

SM 9 g (n 5 12)

SM 12 g (n 5 14)

Constipation severity

5.0 (0.5)

5.0 (2.0)

5.0 (1.8)

Stool frequency

5.0 (0.5)

4.5 (2.0)

5.0 (1.0)

Bowel movement completeness

5.0 (0.5)

4.0 (2.0)

5.0 (1.8)

Stool consistency

5.0 (0.0)

4.5 (2.0)

5.0 (1.8)

Ease of defecation

5.0 (0.3)

5.0 (2.0)

5.0 (1.0)

Overall well-being

5.0 (0.3)

4.0 (2.0)

4.5 (2.0)

Copyright permission granted from Rasmussen et al. (2017) Elsevier Inc.

embedded microsphere against psyllium husk. Stool frequency, bowel movement, composition of familiar and pathogenic gut microorganisms and concentration of short chain fatty acids were significantly improved with starch microsphere intake than psyllium husk (Rasmussen et al., 2017) (Table 13.1). Along with IBS, constipation, ulcer and cancer are another pair of disorders of GS. In this way dietary fiber obtained from Auricularia polytricha was administered on constipation induced sprague-dawley pathogen free rats. A good normal stool needed improve water retention and fat adsorbing capacity. The results revealed 5% dietary fiber observed with increased amount of wet stool, modified peristalsis movement with greater adsorption of fluid and fat (Jia et al., 2020). Another study showed that chitosan oligosaccharide improved the quality of microorganism present in intestinal gut area in case of loperamide induced constipated mice. The intestinal movement, modulation of proinflammatory responses without any effect on intestinal bacterial flora as observed by 16S ribosomal deoxynucleic acid gene sequencing (Zhang et al., 2021). Gastrointestinal motility associated with acetic acid activated UC was Gracilaria fisheri OS (GFOS) was evaluated and other gastrointestinal track transition time, ex vivo propulsion, colonic microorganism composition with short chain

fatty acids were also observed. GFOS showed greater improved behavior of entire intestinal bacterial flora with reduced amount of pathogenic bacteria’s as well as enhance gastrointestinal motile behavior (K-da et al., 2020) (Fig. 13.4). In this category lotus seed OS also showed its effect to conquer constipation related problem. Here four monomer of lotus seed OS (LSOS 4, LSOS3
2, LSOS 3
1 and LSOS 2) were taken to prepare a unique combination with ratio 1.107:0.554:0.183:0.443 respectively. As well as lotus seed resistant starch formulation was prepared upon reaction between lotus seed starch, disodium citrate buffer incubated with alpha amylase enzyme. Then different doses of lotus seed OS mixture was administered to mice and quantify its effect on pathogenic microorganism and short chain fatty acid content. The presence of LSOS with lotus seed resistant starch improved the quality of feces and other intestinal movement linked with constipation (Su et al., 2019). Another study revealed the combination of saccharides (isomaltose-OS) on the severe constipated patients. After administration of 30 g of saccharides for 1 month period, the frequency of bowel, bowel movement and other gastrointestinal maladies were improved with lowered marked decreased in triglyceride and cholesterol contents by approximately 18% and 19%, respectively. This treatment

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13.2 Role of carbohydrates in gastrointestinal system

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FIGURE 13.4 Effects of Gracilaria fisheri OS on UC. ** means: P less than equals to 0.01, *** means: P less than equals to 0.001, ### means: P less than equals to 0.01. Source: Copyright permission granted from K-da et al. (2020) Elsevier Inc.

also creates a positive response toward cardiovascular diseases (Wang et al., 2001). A group of researcher also stated that OS (especially rhamnogalacturonan moiety) isolated from Panax ginseng showed marked effect on hydrochloric acid, ethanol, indomethacin and stress induced ulcerative gastric lesion (Kiyohara et al., 1994). Cancer of

pancreas are also related with problems associated with GS. A group of scientists confirmed the importance of different plant derived saccharides (citrus pectin, partially hydrolyzed citrus pectin, pectinase treated partially hydrolyzed citrus pectin, arabinogalactan-protein from Echinacea purpurea and arabinogalactan-protein from E. purpurea

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13. Biological macromolecules acting on gastrointestinal systems

partially hydrolyzed with trifluoroacetic acid) in the management of pancreatic adenocarcinoma using small interfering ribonucleic acid mediated galectin-3 expression system with greater liver cell adhesion mechanism (Naderer et al., 2020). In another experiment outcomes showed the greater antineoplastic effect of biologically degraded poly alpha malic acid conjugated 5-flurouracil against liver and cervical cancers (Ohya, Kobayashi, & Ouchi, 1991). As well as some researcher reported the importance of isolated PS from Bletilla striata against the occurrence and progression of gastric ulcer. Among isolated saccharides, mannose (70.59%) and glucose (29.41%) were exhibit its proportion with 2.4:1 molar concentration. The

antiulcer activity was evaluated with antioxidative efficacy assessment using 2,20 -azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and ferric reducing antioxidant power assay methods as well as toxicity behavior against human gastric epithelial cells. The outcomes revealed that after administration of 100 mg/kg body weight of isolated saccharide for a period of 3 days, the gastric lesion induced by ethanol was markedly decreased in related proinflammatory mediators (such as tumor necrosis factor, interleukin, etc.) using mitogenactivated protein kinase-nuclear factor kappa beta mechanism (Zhang et al., 2019) (Fig. 13.5). Another study of anticancer efficacy of saccharide complex was also studied. In this

FIGURE 13.5 Effects of BSP pretreatment on the macroscopic appearance of the gastric mucosa in the ethanol-induced gastric ulcer rat model. (A) Illustration of experimental design. (B) Quantitative analysis of gastric injury area was assessed by Image-Pro Plus software. *P , .05 versus normal control group, ¢P , .05 versus ethanol alone treatment group (n 5 6 per group). (C) The representative images of gastric tissues in normal control group (a), ethanol-induced ulcer model group (b), 100 mg/kg of BSP pretreatment group (d), and Suc pretreatment group (d). Source: Copyright permission granted from Zhang et al. (2019) Elsevier Inc.

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13.3 Role of proteins in gastrointestinal system

research, vanadium alginate PS and vanadium alginate OS were studied against hepatic cancer cell line (HL-7702). The formation of saccharidevanadium complex, alginate saccharide and vanadyl sulfate pentahydrate were reacted followed by efficiency tested against radical scavenging and cell proliferation of liver and breast cancer. Both poly and oligo saccharide-vanadium complexes were active against liver cancer (Liu, Liu, & Yi, 2015). As per the previous study, we came to know that glycosidase enzyme inhibition creates a positive impact on the treatment of viral manifestation, cancer progression and other metabolic disorders. In this category, a series of pseudo aza-disaccharide and pseudo azadisaccharide were evaluated against different alpha-D-glucosidase, beta-D-glucosidase, alpha-Dmannosidase and alpha-L-fucosidase; glycosidase enzymes. The outcomes revealed that 1,6dideoxy-1,6-di-N-(10,60-dideoxy-10,60-imino-Dmannitol)-D-mannitol showed greater effect on these enzymes (McCort, Saniere, & Merrer, 2003).

13.3 Role of proteins in gastrointestinal system Protein is one of the most common biological macromolecule composed of amino acids connected with peptide linkage. Amino acids are the basic units of protein. Proteins are mainly four types such as primary, secondary, tertiary and quaternary (Harold, 1951). Primary protein is mainly a straight chain of amino acids connected via peptide linkages, secondary protein structure is stabilized by hydrogen bond followed by formations of alpha-helix and beta-sheet, tertiary protein structure is stabilized by nonlocalized interaction with hydrophobic center linked by disulfide linkages and quaternary protein is structured by polypeptide linkages (Pauling & Corey, 1951; Perrett, 2007; Sumner, 1926). Albumin, globulin, alpha1 acid glycoprotein and lipoproteins are the main proteins associated with drug-protein binding. Also tight junction

295

proteins are responsible for prevention of GS from various gastrointestinal diseases (Zeisel, Dhawan, & Baumert, 2019). A group of scientist showcased the effects of high (1.5 g/kg body weight) and low dose (1 g/kg body weight) of albumin on liver cirrhosis (with and without bacterial growth) followed by assessing the level of serum albumin, plasma renin, blood circulation behavior along with level of inflammatory mediators. In this fashion, 78 patients were selected with bacterial growth followed by collecting their blood samples. The outcomes revealed the lower levels of inflammatory responses with improvement in cardiac function with improved serum albumin level (Fernandez et al., 2019) (Fig. 13.6). In relation with IBS, some proteins played an important role to study this correlation scientists investigated the tissue sample of colon region of IBS patients along with normal persons using immunohistochemical analysis. The outcomes showed that RunX3 protein and a polypeptide transforming growth factor beta-1 were present in abundance with definite correlation (Sun, Lan, An, & Sun, 2011). In response to treat pancreatic cancer, conjugates were prepared from monomethylauristatin E, beta-glucuronidase, gallic acid and maleimide using ramification technique. The conjugate administered into blood, then inside the body the conjugate linked with albumin and beta-glucuronidase enzyme directly traveled to location of tumor. These conjugates showed greater activity toward pancreatic cancer (Chatre et al., 2020). In another research on tight junction protein, a group of researchers investigated the importance of kaemferol (phenolic compound) on liver injury. In this experiment, three different doses such as 25, 50 and 100 mg/kg of kaemferol on mice (ethanol-induced liver cirrhosis). The intestinal data expressed through tight junction zonula occludens-1, occluding, butyrate transporter (SLC58A) and receptor (GPR109A) with marked increased in alanine and aspartate transaminase markers (Chen et al., 2020). There is a clear relation between GS and occurrence of breast cancer through abnormal eating behavior,

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FIGURE 13.6 Effects of high dose and low dose of albumin on cirrhosis.13.6 A: Changes in serum albumin concentration and PRA induced by treatment with HAlbD (blue color in all panels) and LAlbD (red color) in the 18 patients included in the PilotPRECIOSA Study. (A) Individual changes in serum albumin concentration among the 13 patients with baseline hypoalbuminemia (serum albumin concentration , 34 g/L). The horizontal lines indicate the upper and lower normal limits of serum albumin. All 6 patients with hypoalbuminemia treated with HAlbD developed a rapid increase (within 2 weeks) in serum albumin concentration up to normal levels, which persisted during the remaining 10 weeks of the study. In contrast, although all 7 patients with baseline hypoalbuminemia treated with LAlbD had increased serum levels of albumin during treatment, only 1 normalized the serum albumin concentration. (B
D) Two factors influenced the response to albumin treatment. 13.6 B The first factor was the albumin dosage: among the 13 patients with baseline hypoalbuminemia, the individual absolute median increase in serum albumin was almost double in patients receiving HAlbD vs those receiving LAlbD. 13.6 C: The second factor was the feedback mechanism by which baseline serum albumin

13.3 Role of proteins in gastrointestinal system

297

L

concentration influences the hepatic synthesis of albumin. In most patients without hypoalbuminemia, the inhibition of hepatic synthesis of albumin prevented the increase in the serum concentration of albumin to abnormal levels during albumin treatment. 13.6 D: This feedback mechanism was also reflected by the close inverse correlation between the baseline serum albumin concentration and the mean increase in serum albumin during treatment. The lower the baseline levels of serum albumin, the higher the absolute mean increase in the serum concentration of albumin in both the LAlbD and HAlbD groups. 13.6 E: Circulatory dysfunction was extremely unstable during albumin treatment in patients receiving LAlbD, with high peaks of PRA in 6 patients.13.6 F: Circulatory instability was significantly improved in patients receiving HAlbD, with only 1 patient presenting 1 peak of PRA throughout treatment. Source: Copyright permission granted from Fernandez et al. (2019) Elsevier Inc.

FIGURE 13.7 MyD88 is significantly upregulated in D-gal/LPS induced acute liver injury mice. Mice were exposed to D-gal/LPS for 12 h. (A) Total protein was extracted from the liver tissues and MyD88 expression were measured with western blot (B) Band densities were digitized and semiquantitated. Each lane density of GAPDH was divided by that of MyD88 (C) Total RNA was extracted from liver tissues and MyD88 mRNA levels were measured by real-time PCR. ** means: P less than equals to 0.01, ### means: P less than equals to 0.01. Source: Copyright permission granted from Ding et al. (2019) Elsevier B.V.

pancreatitis with other genetic mutations (lynch syndrome; peutz-jeghers syndrome, brca 1 mutation and chek 2 mutation). In this scenario, TJM2010
2 and TJ-M2010
5 were successfully worked against myeloid differentiation primary response 88 gene with susceptibly inhibited the occurrence, progression and invasion of breast cancer cell line (MCF-7 and MDA-MB-231) as well as galactosamine and lipopolysaccharide induced cirrhotic liver disease (Ding et al., 2019; Liu et al., 2020) (Fig. 13.7). Another important protein is heat shock protein. In this condition, heat shock proteins (60, 72 and 90 kDa) were induced with increased temperature (42.5 C for 25 min) in rat and western blot analysis revealed that heat shock protein (72 kDa) is abundantly expressed in rat esophagus associated with reflux esophagitis with marked suppression in tumor necrosis factor alpha and interleukin-1 beta inflammatory

responses (Izumi et al., 2009) and heat shock protein 90 also showed its efficacy to protect gastric epithelium lining against oxygen induced free radical generation process (Leung, Redlak, & Miller, 2015). Heat shock protein 70 was protected the injury due to inflammation. Cold stress condition influenced the injury in cecal tissue but the amount of heat shock protein was highly expressed (Liu et al., 2019). Among different proteins, serum C-reactive protein and albumin are the important proteins expressed in pancreatic inflammation. In this condition, Atlanta and ranson score was used to correlate between mortality due to pancreatitis. If the ratio between C-reactive protein and albumin was greater than 16.28, the rate of mortality was increased by 19 times. This score was directly related with pancreatic inflammation (Kaplan et al., 2017). A group of scientist stated that presence of antigladialin immunoglobulin G and

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immunoglobulin A without any gluten in food showed greater effect on diarrhea management with marked improvement in composition of symbiotic and pathogenic microorganism (Sanchez et al., 2020); as well as immunoglobulin G created a positive response on celiac disease with improved stool frequency and habit (Wahnschaffe, Schulzke, Zeitz, & Ullrich, 2007). Certain bacteria showed direct linked with UC and diarrhea. In this category, whey protein isolated from cow milk showed marked efficiency against diarrhea (Young, Munro, Taylor, Veldkamp, & Dissel, 2007). Furthermore another research showed that amino acid deficiency correlated with the size of pancreas. In this experiment, four groups of mice were fed with 20% protein (control category), 0% (zero protein), amino acid rich food and leucine free food, followed by assessment of mammalian target of rapamycin complex 1 expression (linked with pancreas size). The outcomes showed that no protein food and without leucine free food observed with partial to no effect on rapamycin1 complex. These information confirmed the direct effect of leucine on the health of pancreas (Sans, Crozier, Vogel, D’Alecy, & Williams, 2021).

13.4 Role of fatty acids in gastrointestinal system Fatty acid comprised with aliphatic acid chain with or without double bonds (total carbons ranged from 4 to 28). As per the chain length, four types of fatty acid are observed such as short chain fatty acid (butyric acid), medium chain fatty acid (triglyceride), long chain fatty acid (alpha linolenic acid) and very long chain fatty acid (arachidonic acid) (Goodhart & Shils, 1980). Depend upon the presence of unsaturation, fatty acids are divided into two categories such as saturated and unsaturated fatty acid. Saturated fatty acid contains no double bonds (e.g., caprylic acid, capric acid, palmitic acid, stearic acid); whereas depend upon the position and number of double bond,

unsaturated fatty acids are divided into cis and trans category (Whitney & Rolfes, 2008). Some common cis categorized unsaturated fatty acids are myristoleic acid, palmitoleic acid, sapienic acid and oleic acid whereas trans unsaturated fatty acids are elaidic acid, vaccenic acid, linoelaidic acid, etc. In GS, some fatty acid especially EFA (omega-3 and omega-6 fatty acid) played a crucial role in system management (Burr, Burr, & Miller, 1930). In view of GS disorders, fatty liver is one of the crucial disease. Fatty liver is basically two types such as alcoholic and nonalcoholic fatty liver. In the management of nonalcoholic fatty liver disease, hepatocytes were evaluated after EFA omega-3 fatty acid (O3FA) administration which induced the removal of cell debris linked with liver toxicity as well as minimized the accumulation of free fatty acid in liver (Chen, Xu, Yan, Yu, & Li, 2005). Another study showed the role of omega-3 fatty acid on liver and renal system toxicity. The experiment portrayed the minimization of the severity of paracetamol induced liver and renal system toxicity by omega-3 fatty acid. As we know paracetamol responsible for internal organ toxicity especially with gastrointestinal and renal systems using free radical generation. O3FA showed its effects through heme oxygenase-1nuclear factor erythroid 2-related factor 2 and the transcription factor BTB and CNC homology1 pathway (Eraky & Abo El-Magd, 2020) (Fig. 13.8). EFAs are also showed its activity profile to conquer metabolic syndrome and other mitochondrial dysfunctionality. In this context O3FA and omega-6 fatty acid showed a marked effect to minimize the disease severity through mammalian target of rapamycin complex 1 or mechanistic target of rapamycin protein complex 1 pathway (Liu et al., 2020). Also a related study on weight loss, nonalcoholic fatty liver disease with composition of EFA, in this regard it was concluded that amount of alpha linolenic acid was increased but other EFA such as eicosapentanoic acid and docosapentanoic acid markedly decreased (MarinAlejandre et al., 2020). Another group of

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299

Protective effects of ω 2 3 fatty acids on Alanine aminotransferase (A) and Aspartate aminotransferase (B) serum activities in APAP-induced toxicity in rats. *** means: P less than equals to 0.01, # means: P less than equals to 0.5, ### means: P less than equals to 0.01. Source: Copyright permission granted from Eraky et al. (2020) Elsevier Inc.

FIGURE 13.8

FIGURE 13.9 Serum Vascular Cell Adhesion Molecule levels for control and modified beef diet rats. * means: P less than equals to 0.5. Source: Copyright permission granted from Medeiros et al. (2007) Elsevier Inc.

researchers experimentally proved the role of alpha linolenic acid (obtained from flaxseed) on the amount of cholesterol and heart related problems as compare to normal corn oriented food. In this experiment, a group of cattles fed with beef enriched with flaxseed and beef with normal corn. The results showed that flaxseed rich beef food observed with marked increase in amount of docosahexanoic and arachidonic acid as well as total cholesterol and vascular

cell adhesive molecule were significantly increased (Medeiros et al., 2007) (Fig. 13.9). In this way, a group of scientists experimented on the effect of O3FA on inflammation, hyperglycemia and nonalcoholic fatty liver, to confirm the role forty four patients suffered from hyperglycemia and nonalcoholic fatty liver subdivided into two groups, 22 each and each group administered with and without O3FA followed by assessment of fasting blood

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sugar level, hemoglobin A1C, serum insulin, C-reactive protein, interleukin and tumor necrosis factor alpha. The outcomes showed improved pancreatic beta cell activity without any effects on insulin sensitivity and interleukin-6 but the expression of inflammatory mediators were markedly decreased (Orang, Mohsenpour, & Khosravi, 2020). The role of phospholipid creates positive environment in the management of liver condition. A bile acid-phospholipid ursodeoxycholyl lysophosphatidyl ethanolamide showed greater activity on tumor necrosis factor alpha induced inflammation associated with damage of parenchyma (Pathil, Warth, Chamulitrat, & Stremmel, 2011). Furthermore liver protective of phospholipid obtained from female gonads of squil on transgenic zebrafish was evaluated. The outcomes revealed that squil isolated phospholipid observed with lower accumulation of free fatty acids as well as minimized in vacuole present in liver (Xia et al., 2020).

13.5 Role of nucleic acids in gastrointestinal system Nucleic acid is the most important biological macromolecule composed of two nitrogenous bases (purine and pyrimidine) connected via phosphodiester linkage with ribose sugar backbone (Dahm, 2008). Nucleic acids are mainly two types such as deoxyribonucleic acid and ribonucleic acid. Nucleic acids played an important role in replication and protein synthesis (Cox & Nelson, 2008). Nucleic acids are also showed its activity toward different disorders related to GS (Lander et al., 2001). In this relation, long noncoding ribonucleic acid showed its greater efficacy against inflammation associated with celiac disease and IBS; in this scenario gluten free diet creates a positive response (Lashgarian, Karkhane, Marzban, Yazdi, & Shahzamani, 2020) (Fig. 13.10).

In the management of cancer related to GS, purine nucleoside as 8-hydroxy-deoxyguanosine, glutathione created a positive relation with oxidative stress linked with cancer (Gonenc, Hacısevki, Aslan, Torun, & Simsek, 2012). In relation with long coding ribonucleic acid to pancreatic cancer. The outcomes showed that nonpancreatic cancer patients showed greater expression of long coding ribonucleic acid and activated tumor growth factor beta with lesser chance of survival as well as the chances of tumor metastasis was reduced (Cheng, Fu, Li, & Jiang, 2020). A new GS cancer biomarker DDX39 showed its role in the diagnosis of GS tumor. DDX39 showed its activity through adenosine triphosphate dependent ribonucleic acid helicase enzyme as confirmed by immunohistochemical analysis (Kikuta et al., 2020). Visceral smooth muscle is the important feature of GS smooth muscle development. In this way, posttranscriptional modification of messenger ribonucleic acid and its related multiple splicing-2 are the critical steps for the development of visceral smooth muscle formation; for this way colon tissue of pediatrics with Hirschsprung’s disease and chronic pseudo obstruction syndrome showed greater expression of ribonucleic acid binding protein related to multiple splicing-2 and showed proper linked for GS visceral smooth muscle development (Notarnicola et al., 2012). Also addition of double stranded ribonucleic acid into the inner cell persuaded posttranscription of gene silencing mechanism associated with gastrointestinal track motility and cellular characteristics (Sklan & Glenn, 2007). Furthermore, a new ribonucleic acid binding LIN28 protein was expressed with gastric hepatoid and hepatocellular cancers as compare to other markers such as sallike protein 4, alpha fetoprotein, glypican-3, antihepatocyte specific antigen, carcinoembryonic antigen and cytokeratin 7. The outcomes showed that LIN28 was highly expressed in both the cancers but not effective as other markers (Zhao et al., 2018).

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13.6 Conclusion

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FIGURE 13.10 Comparison of lncRNA13 expression level in healthy individuals and celiac disease patients. Source: Copyright permission granted from Lashgarian et al. (2020) Elsevier Ltd.

13.6 Conclusion In a total perspective, biological macromolecules played an essential role in the management of GS and its related disorders. In this respect, saccharides and dietary fibers obtained from potato, Amygdalus dulcis L, A. polytricha, G. fisheri, P. ginseng, Lotus saccharide and B. striata; among proteins albumin, globulin, Runx3, tight junction protein, heat shock protein, C-reactive protein,

immunoglobulins; among fatty acids omega-3/6 fatty acid, alpha linolenic acid, docosapentanoic/ docosahexanoic acid, squid phospholipid and among nucleic acids noncoding ribonucleic acid, adenosine triphosphate dependent ribonucleic acid as well as gluten free diets portrayed its role in IBS, constipation, diarrhea and gastric system related cancers with improved symbiotic and pathogenic microorganism environment. So it was concluded that biological macromolecules

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showed greater applicability toward GS and its related disorders.

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C H A P T E R

14 Synthetic macromolecules with biological activity Stefania Racovita1, Marcel Popa2,3, Leonard Ionut Atanase4 and Silvia Vasiliu1 1

“Petru Poni” Institute of Macromolecular Chemistry, Iasi, Romania 2Faculty of Chemical Engineering and Environmental Protection, Department of Natural and Synthetic Polymers, “Gheorghe Asachi” Technical University of Iasi, Iasi, Romania 3Academy of Romanian Scientists, Bucuresti, Romania 4 Faculty of Dental Medicine, “Apollonia” University of Iasi, Iasi, Romania

14.1 Introduction Bioactive macromolecules are materials based on natural and synthetic polymers that exhibit a specific biological response. Proteins, nucleic acids, lipids, and carbohydrates are natural bioactive macromolecules and play an important role in our daily life. Bioactive compounds must meet several requirements in terms of their physicochemical properties (charge, solubility, hydrophobicity, molecular weight and stability) and biological characteristics (adsorption, distribution, metabolism, elimination, and toxicity). Starting from the years 1940 to 1950, when the first biologically active polymers such as, 2-vinyltiophene (1941), 2-vinylfuran (1946), Nvinylpyrrolidone (1952) and 2-vinylpyrrole (1954) have been mentioned, until today, the synthetic bioactive polymers represent an area in continuous expansion receiving a great

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00014-2

attention due to their huge potential as new therapeutic agents for treatment of various human diseases (Gebelein & Carraher, 1982; Smith, Pentzer, & Nguyen, 2007; Wang, Xu, Li, & Turng, 2020). Due to their versatility, these polymers have some advantages, such as: easy to process, low price, eco-friendly properties, low toxicity and do not develop resistance to microbes (Alfei & Schito, 2020). Synthetic macromolecules with biological activity represent a very interesting field of research relying on the knowledge from several disciplines, such as: chemistry, medicine, biology and engineering. The need to develop new materials with antimicrobial properties designed to limit or eliminate microbial development, due to the increasing number of infectious diseases caused by the pathogenic microorganisms, such as bacteria, viruses, parasites and fungi, is an important desideratum.

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Antimicrobial activity represents a complex process, which may have the effect of eliminating the microorganisms, inhibiting bacterial growth and preventing the formation of microbial colonies (Khameneh, Iranshahy, Soheili, & Bazzaz, 2019). Antimicrobial agents, like antibacterial, antiviral, antifungal and antiparasitic polymers, have different mechanisms of action in order to eradicate the infectious diseases. For example, the discovery, development and use of antibacterial polymers contributed to the decrease of death caused by bacterial infections. Antifungal polymers are used to arrest or inhibit the fungal growth. Antiviral polymers are designed to treat viral infections, while antiparasitic polymers are suitable for the treatment of infectious diseases like leishmaniasis, malaria and chagas diseases (Siedenbiedel & Tiller, 2012; Swiontek Brzezinska et al., 2019). Antioxidant polymers are another important class of bioactive polymers. Antioxidants are the substance that prevent and repair the damage caused by free radicals. The protein and DNA damage is the result of the mechanism of action of the free radicals and can be explained as follows: the unstable radical due to the unpaired electron has the tendency to stabilize by electron pairing with proteins, lipids or DNA founded in the human cells, leading to a weakening of cellular antioxidant defense systems. The role of the antioxidant polymer is to supply electrons to damage cells as well as to convert the free radicals into waste by-product in order to eliminate them from the body (Rahman, Islam, Biswas, & Khurshid Alam, 2015). Cancer is a generic term for many groups of diseases that affect the various organs of the human body. According to the World Health Organization cancer is the second cause of death, leading to the death of about 9.6 millions of people worldwide in 2018 (Bray et al., 2018). The first anticancer polymeric drug was a copolymer based on divinyl ether and maleic anhydride being used in clinical trial in the

early 1960s. Unfortunately, this polymer showed sever systemic toxicity but was the starting point for the next generation of chemotherapeutic agents (Duncan, 2003). Another very-known anticancer polymer is N-(2-hydroxypropyl)methacrylamide copolymer which was introduced in clinical trial in 1994 as a conjugate with doxorubicin (Duncan, Ringsdorf, & Satchi-Fainaro, 2006). Nowadays, the new polymerization routes [organocatalytic living ring-opening polymerization, ring-opening metathesis polymerization (ROMP), etc.] help to create new polymers with well-defined and well-controlled molecular structures. Also, the synthetic macromolecules can work as functional sequestrants for potentially harmful molecules from environment, diet or generated endogenously (Li, Yu, Chen, & Qupicky, 2015). The binding selectivity of polymers can be influenced by various parameters: structure and size of polymer chain, the presence and distribution of functional groups. This chapter reviews some classes of synthetic macromolecules with biological activity that have a great importance on the human comfort and health including, antimicrobial polymers, antioxidant polymers and polymeric sequestrants.

14.2 Synthetic macromolecules with antimicrobial activity The appearance of new strains of pathogenic microorganisms (antibiotic resistant tuberculosis, avian influenza A and viruses like Ebola, Zika and recently SARS-CoV-2) and infectious diseases as well as the increase of antimicrobial resistance represent serious global concerns in various fields of applications, such as hospitals, medical devices/surgery instruments, dentistry, health care products, water purification systems, textile and food industries (Avila, Said, & Ojcius, 2008; Jiao et al., 2017).

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14.2 Synthetic macromolecules with antimicrobial activity

In this context, the development of new synthetic macromolecular antimicrobials, known as antimicrobial polymers and surfaces with antimicrobial properties, which prevent bacterial adhesion and biofilm formation can be the solution to solve the problem of multidrug resistance. Antimicrobial polymers are materials capable to kill or inhibit the growth of microorganisms on a surface or surrounding environment (Jain et al., 2014).

14.2.1 History of antimicrobial agents and antimicrobial polymers Antimicrobial agent is a general term that includes antibacterials, antivirals, antifungals and antiprotozoans (Asif, 2017). The history of using antimicrobial agents began about 2000 years ago when the ancient Greeks and Egyptians used various mixtures with antimicrobial properties based on plant extracts in order to treat infectious diseases (Forrest, 1982). Later on, the work of L. Pasteur, a pioneer in microbiology (known as the “father of microbiology”), related to the discovery that specific organisms (yeasts) were involved in the fermentation and putrefaction processes, led to the appearance of the germ theory (Berche, 2012). The germ theory was applied by Pasteur in medicine to explain the infectious diseases. Based on the information obtained by Pasteur, the British surgeon J. Lister (Jessney, 2012) developed in 1865 the antiseptic medicine, by utilizing phenol as effective antiseptic for sterilization of surgical tools as well as to treat suppurated surgical wounds, drastically reducing the number of deaths caused by postoperative infections. The names of P. Ehrlich and A. Fleming were associated with modern “antibiotic era” (Aminov, 2010). The term “antibiotic,” introduced in 1942 by S. Waksman (inventor, biochemist and microbiologist), refers to a class of antibacterial drugs that kills bacteria or inhibits their growth

307

(Waksman, 1947). The discovery of arsphemanine, known as Salvarsan as well as the improved pharmaceutical form Nosalvarsan, to treat African trypanosomiasis and syphilis, the first sulfonamide, namely Protonsil that had effect against Gram-positive cocci and the penicillin as antimicrobial agents with good clinical results had opened interesting opportunities in medicine for the preparation of new synthetic antibacterial drugs (Domagk, 1935; Kardos & Demain, 2011; Williams, 2009). In the gold era of antibiotics, (1940
70), 12 new classes of small molecular weight antibiotics (aminoglycosides, tetracycline, amphenicols, lipopeptides, macrolides, glycopeptides, streptogramins, asamycines, lincosamides, etc.) had been introduced on the market, but after that a decline of the discovery rate was observed (Lei et al., 2019). The first antimicrobial polymers were discovered in 1965 and were represented by polymers and copolymers of 2-methacryloxytroponones (Cornell & Donaruma, 1965). Later on, the various synthetic macromolecule structures that showed antimicrobial activity were developed. Some examples are: polymerized salicylic acid (1970), polymers with quaternary ammonium groups and antimicrobial peptides that mimic the natural peptides (1980) (Panarin, Solovski, & Ekzemply, 1971; Vogl & Tirrell, 1979). The antimicrobial peptides or host defense peptides (AMPs) possess a large variety of antimicrobial activity including antibacterial, antiviral, antifungal, anticancer and overcome bacterial drug-resistance (Aminov, 2017). The vaccination principle was introduced by L. Pasteur, which helped the development of the vaccines for chicken cholera (1879) as well as for anthrax and rabies (1889), being one of the founders of the discipline of immunology (Scorpio, Blank, Day, & Chabot, 2006). Since 1947, an increasing interest for antiviral polymers has been known. Until now, the following directions have been developed: (1) polyanionic systems used in particularly for the treatment of HIV; (2)

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polymers as interferon-inducers; (3) polynucleotides and oligonucleotides as well as their derivatives; (4) polymers with pendant carbohydrates used for the inhibition of influenza virus (Bianculli, Mase, & Schultz, 2020).

14.2.2 Classification of antimicrobial polymers Generally, five types of antimicrobial polymers are known: 1. polymeric biocides are represented by the polymers that contain the bioactive repeating units with antimicrobial activity, like amino, carboxyl and hydroxyl groups (Ikeda, Yamaguchi, & Tazuke, 1984); 2. biocidal polymers characterized by the antimicrobial effect of whole polymer chain. Usually, these polymers contain cationic biocidal groups, such as: quaternary ammonium, phosphonium, sulfonium and guanidinium groups (Ahmed, Hay, Bushell, Wardell, & Cavalli, 2008); 3. biocide releasing polymers are represented by the nonactive polymers (carriers) that contain biocide which are covalently linked or physically entrapped (Ali et al., 2020); 4. bioactive peptide are protein fragments which have high tissue affinity, specificity, efficiency and positive impact on the human health (Daliri, Oh, & Lee, 2017). The bioactive peptides can be obtained by four strategies: enzymatic synthesis (Mojica & de Mejia, 2016), microbial fermentation (Rai, Kumari, Sanjukta, & Sahoo, 2016), chemical synthesis (Van Lancker, Adams, & De Kimpe, 2011) and recombinant DNA technology (Li, 2011). 5. antimicrobial surfaces. The antimicrobial surfaces can be: (a) natural surfaces (cicada wings, plant leaves, fish scale, shark skin, spider silk) are the surfaces found in nature and have the ability to resist or prevent bacterial attachment (Liu & Jiang, 2011); (b) synthetic surfaces are the surfaces that can be obtained by two main

strategies: (i) chemical approaches that include surface coatings and modification of the surface chemistry (functionalization, derivatization or polymerization); (ii) physical approach that include the modification of the structure architecture (mechanical and surface structuring processes) (Hasan, Crawford, & Ivanova, 2013). The biocidal synthetic surfaces are preferred to those which release biocides because they have some advantages, as mentioned by Jiao et al. (2017): (1) present improved and prolonged antimicrobial activity; (2) have nontoxic and nonirritant properties; (3) have a different mechanism of bactericidal action compared to the antibiotics; (4) do not develop the antibiotic resistance (Aminov, 2017). Also, the antimicrobial polymers can be classified in function of the sources of infections (bacteria, viruses, fungi and parasites) as: antibacterial, antiviral, antifungal and antiparasitic polymers (Fig. 14.1). Another classification of antimicrobial polymers, presented in Fig. 14.2, consist in: 1. polymers with inherent antimicrobial activity. These polymers are natural or synthetic and possess native antimicrobial properties. The following types of polymers fall into this category: (a) nitrogen containing polymers: polymers with quaternary ammonium groups, polymers with ring containing nitrogen, polyetyhylenimine (PEI), polyguanidine, cationic conjugated polyelectrolytes, polysiloxanes, hyperbranched and dendritic polymers, poly(ionenes), oxazolines, polymers mimic natural peptides, synthetic peptides and polynorbornene derivatives (Munoz-Bonilla & Fernandez-Garcia, 2012); (b) halogen polymers: fluorine containing polymers, chlorine containing polymers, Nhalamine compounds (Hui & DebiemmeChauvy, 2013); (c) polyzwitterions that contain in their molecules both negative and positive charges (Mi & Jiang, 2014). A

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14.2 Synthetic macromolecules with antimicrobial activity

309 FIGURE 14.1 Classification of antimicrobial polymers depending on the microorganism sources.

special class of polyzwitterions is represented by the polybetaines. Polybetaines are synthetic ionic polymers which bear both anionic and cationic groups in the same monomer unit, these being separated by an alkyl chain of different lengths. Depending on the anionic groups the polybetaines are: poly(carboxybetaines), poly(sulfobetaines) and poly (phosphobetaines) (Racovita, Vasiliu, & Neagu, 2010). Among them, poly (sulfobetaines) have the best antimicrobial properties. 2. chemically modified polymers. This type of polymers possesses the antimicrobial activity after their chemical modification by introduction of active pendant groups or by attachment of antimicrobial compounds. 3. polymeric composites. In this category the following types can be found: (a) polymeric composites with antimicrobial organic compounds that were obtained by noncovalent addition of low molecular weight antimicrobial compounds (polymeric

biocides with controlled release) or by mixing of antimicrobial polymers with nonactive polymers (polymeric biocides without controlled release); (b) polymeric composites with antimicrobial inorganic compounds that can be obtained by addition of metal particles (Au, Ag, Cu), insertion of oxides (titanium dioxide, zinc oxide, nitric oxide) or by inclusion of antimicrobial modified inorganic systems (Hui & Debiemme-Chauvy, 2013).

14.2.3 Preparation routes for antimicrobial polymers Many efforts have been performed to discover new polymeric materials with antimicrobial properties because it has been observed an increase in the resistance of pathological microorganisms, such as: bacterial, fungi, viruses and parasites to some of the existing antimicrobial polymers or antimicrobial agents. Two main synthesis routes are used to prepare the synthetic polymers with biological activity: (1)

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FIGURE 14.2

14. Synthetic macromolecules with biological activity

Classification of antimicrobial polymers depending on the synthesis routes.

polymerization of monomers containing the bioactive functional groups. To obtain antimicrobial polymers the conventional and new polymerization techniques can be applied: free radical polymerization, cationic and ionic polymerization, atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT) ring-opening metathesis polymerization and click chemistry (Caykara, Sande, Azoia, Rodrigues, & Silva, 2020; Dai & Zhang, 2019; Lienkamp, Madkour, Kumar, Nusslein, & Tew, 2009; Phillips et al., 2017; Zuo, Wu, & Fu, 2010). (2) Chemical modification of polymer structure. In order to achieve the polymeric materials with antimicrobial activity, the chemical modification of polymers can be realized by: covalent incorporation of low molecular weight antimicrobial compounds, coupling of antimicrobial peptides and by grafting

reaction of other antimicrobial polymers (Adlhart et al., 2018; Badrossamay & Sun, 2009; IzquierdoBarba et al., 2009). The antimicrobial polymers must be prepared considering the structure of the outer envelope of the cell of different microorganisms, but at the same time they should meet certain requirements (Kenawy, Worley, & Broughton, 2007; Matsuzaki, 2009): the synthesis must be done by simple, reproducible and low cost techniques; the polymer structure must be very stable as a function of time and temperature; should be nontoxic, nonirritating and without the formation of toxic byproducts after degradation; must contain sufficient hydrophilic moieties to realize the adhesion to the microbial cell envelope but also to have the appropriate hydrophobic part in order to avoid the toxicity of the polymeric material and loss of

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14.2 Synthetic macromolecules with antimicrobial activity

the selectivity; must be used for several operating cycles; must have a broad spectrum of antimicrobial properties in brief time contact. Current technologies offer the possibility to obtain synthetic macromolecules with antimicrobial activities in different presentation forms: nano and microparticles, fibers, hydrogels, films and membranes (Bshena, Heunis, Dicks, & Klumperman, 2011; Mukherjee & Du, 2018; Sofokleous et al., 2017; Yang et al., 2018).

14.2.4 Factors affecting the antimicrobial activity In Fig. 14.3 are presented the factors that can influence the antimicrobial activity.

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14.2.4.1 Hydrophilic/hydrophobic balance Hydrophilic/hydrophobic balance represents an important criterion in designing process of antimicrobial polymers because it affects both final antimicrobial properties as well as the interaction mode of polymers with the cell membrane. To develop a polymer with good antimicrobial activity and low toxicity to the host cell, an optimal balance between hydrophilic and hydrophobic parts must be found. The hydrophilic moieties, represented by the cationic groups (quaternary ammonium, pyridinium, imidazolium, thiazolium, triazolium, phosphonium, sulfonium or guanidinium groups), are directly related to the interaction between polymer and the bacterial surface, while the hydrophobic moieties, represented by the alkyl groups of varying length, are FIGURE 14.3 Factors that influence the antimicrobial activity.

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responsible for the toxicity of the polymeric materials (Rodriguez-Hernandez, 2017). For example, a large hydrophilic moiety leads to a better interaction between polymer and cell membrane, but a large hydrophobic moiety has a negative influence, the polymeric materials being toxic to all types of cells (Palermo, Sovadinova, & Kuroda, 2009). Three main strategies can be used to obtain an appropriate hydrophilic/hydrophobic balance: (1) “segregated monomer” approach which involved the copolymerization of nonpolar monomer and charged monomer; (2) “facially amphiphilic” approach that uses monomers which contain in their molecule both hydrophobic part and a cationic group; (3) “same centered” approach uses monomers that possess an alkyl chain with variable length attached to a positive charge (Engler et al., 2012). 14.2.4.2 Molecular weight Molecular weight is another important parameter that can have an influence on antimicrobial properties and hemolytic activity. Initially, it was believed that a high molecular weight is ideal for obtaining the polymeric materials with high antimicrobial activity. However, an increase of molecular mass has some limitations, such as: limited diffusion through the bacterial cell membranes due to poor solubilization, aggregation in biological media and the tendency of high molecular weight polycations to become toxic to the cells of the human body (Ergene & Palermo, 2018). Also, high molecular weight favors the increase of crystallinity degree leading to the decrease of the antimicrobial activity. A recent study, carried out by Santos et al. (2019), has shown that in case of controlled linear and star-shaped copolymers, based on hydrophobic poly(butyl acrylate) and cationic poly(3-acrylamidopropyl) trimethyl ammonium chloride moieties, obtained via supplemental activator and reducing ATRP, the increase of macromolecular weight led to increased antimicrobial activity. Another study followed the influence of molecular weight on

the antimicrobial activity of cationic polymetahcrylate with rosin as pendant group and it was observed that the lower molecular weight polymers have a great antimicrobial activity against both Gram-negative and Gram-positive bacteria compared with high molecular weight polymers (Chen et al., 2012). 14.2.4.3 Counter ion effect The effect of counter ions can be describes in terms of their solubility and ion-pair formation. The quaternary ammonium poly(propylenimine) dendrimers with bromide anions exhibit a better biocidal activity compared to those with chloride (Chen et al., 2012). Lienkamp et al. (2009) showed that the change of hydrophilic counter ions of a ROMP-derived diamine polymer with hydrophobic organic counter ions led to a drastically reduced of antimicrobial activity of that polymer. Another study carried out by Kanazawa, Ikeda, and Endo (1993) highlights that the antimicrobial activity of cationic polymers increases when tight ionpairing and hydrophilic anions were used. 14.2.4.4 Charge density The charge density and their spatial arrangement in the polymer chain structure are other factors that affect the antimicrobial activity. The positive charges of antimicrobial polymers are responsible for the electrostatic interactions with negatively charged bacterial cell walls. An increase of the amount of charges on the polymeric backbone has the effect of increasing antimicrobial and hemolytic activity (Jiang et al., 2008). Also, the localization of charges in the polymer structure represents an important factor that influences the antimicrobial and hemolytic activities. The importance of the charge place in the structure of the polymer was studied on amphipathic quaternary ammonium containing poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) copolymers with rosin moiety as pendant group obtained by quaternization reaction (Chen et al., 2012). The authors prepared

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14.2 Synthetic macromolecules with antimicrobial activity

two types of PDMAEMA-g-rosin polymers: first type of polymer was synthesized by direct polymerization of monomer DMAEMA-g-rosin, but the polymer was characterized by a very low antimicrobial activity; the second polymer was obtained by grafting rosin onto PDMAEMA, the quaternary ammonium groups being located at the periphery of the entire polymer. It was observed a difference in the antimicrobial activities depending on the charge location on the polymers, as follows: (1) when the cationic groups were located at the end of the pendant groups, the antimicrobial activity was higher because the polymer easily reacts with bacterial cell surface through electrostatic interactions and diffuses inside of the bacterium body leading to its destruction; (2) when the cationic groups are founded between PDMAEMA backbone and rosin moieties, the interaction with bacteria cell wall is hindered due to the steric hindrance effect resulting in a decrease of antimicrobial activity. Thus, a balanced quaternization exhibited higher antimicrobial activities. Also, Guo et al. (2018) synthesized imidazolium, quaternary ammonium and 1,4-diazobicyclo[2,2,2]octane-1,4 diium cation-based small molecule cationic compounds as well as their corresponding side chain/main chain cationic polymers in order to study the influence of the location of cationic groups onto antibacterial activity. These authors observed that the antibacterial activity decreased in the following order: main chain cationic polymers . side chain cationic copolymers . small molecule cationic compounds. Also, the main chain cationic polymers were exhibited extremely low hemolytic activity. 14.2.4.5 Polymeric architecture It is well-known that there are several important differences between macromolecular antimicrobial agents and low molecular weight antimicrobial agents such as, molecular weight, presence of functional groups and the complex architectures of macromolecular systems. The macromolecular antimicrobial agents can be

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synthesized by different synthesis routes resulting polymers with various architectures including homopolymers, telechelic polymers, random copolymers, block copolymers, branched polymers and ionic or zwitterionic polymers (Ganewatta & Tang, 2015). Some examples of synthetic macromolecules with antimicrobial activities and different polymer topologies are presented in Table 14.1. 14.2.4.6 Alkyl spacer Alkyl spacer of varying length can be found as pendant groups in side chains of the copolymer or as spacer group between polymer backbone and cationic groups (Ergene & Palermo, 2018). The influence of spacer length on the antimicrobial and hemolytic activities was studied by Palermo, Vemparala, and Kuroda (2012) on 2-aminoethylene, 4-aminobutylene and 6-aminohexylene. Among these copolymers, those that contain 4-aminobutylene side chains presented better antimicrobial activity and lower toxicity. The perfluoro-oxaalkylated end-capped-2-(3acrylamidopropyldimethylammonio) ethanoate has higher antimicrobial activity against both Staphiloccocus aureus and Pseudomonas aeruginosa compared to perfluoro-propylated end-capped-2(3-acrylamidopropyldimethylammonio) ethanoate, probably due to the longer length of the macromolecular chain (Sawada, Umedo, Kawase, Tomita, & Baba, 1999).

14.2.5 Synthetic macromolecules with antibacterial activity Antibacterial materials act on bacterial cells in two ways: either by keeping bacteria at distance, or by killing them directly. Therefore, according to the mechanism of antibacterial activity, the antibacterial materials are classified into: biopassive or bioactive materials (Huang et al., 2016). Biopassive materials prevent bacterial adhesion by ensuring minimal protein adsorption. This can be realized by coating the surface of the polymeric material with a layer of the

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TABLE 14.1 Examples of various structures of synthetic macromolecules with antimicrobial activity. Types of antimicrobial polymers

Chemical structure

Antimicrobial activity MIC or HC50 (μg/mL)

References

1. Homopolymers

42
63 (Staphiloccocus aureus)

Thoma, Boles, and Kuroda (2014)

2. Telechelic polymers

200 (S. aureus)

Waschinski and Tiller (2005)

3. Random copolymers

104 (Escherichia coli)

Mizutani et al. (2012)

4. Block copolymers

1.6
2.4 (E. coli)

Oda, Kanaoka, Sato, Aoshima, and Kuroda (2011)

5. Zwitterionic polymers

1250
1750 (S. aureus)

Ward, Sanchez, Elasri, and Lowe (2006)

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14.2 Synthetic macromolecules with antimicrobial activity

hydrophilic film that prevents the contact between the surface of the polymeric materials and the bacterial cells (Charnley, Textor, & Acikgoz, 2011). Some examples of passive polymers are: self-healing polymers—poly(dimethylsiloxane); uncharged polymers—poly(ethylene glycol) (PEG), poly(N-vinylpyrrolidone), poly(dimethylacrylamide); polyampholytes and zwitterionic polymers—phosphobetaine, sulfobetaine and phospholipid polymers. The disadvantage of this method consists in difficulty of perfect coating with hydrophilic film, the existence of cracks and defects of the coating layer leading to the bacterial contamination of the polymeric material. Due to the excellent properties (high chain mobility, large exclusion volume), PEG has been mostly used in the preparation of synthetic polymers with antibacterial activity. It was observed that the polymeric materials based on PEG exhibit high antifouling ability (Dalsin & Messersmith, 2005; Dong, Manolache, Wong, & Denes, 2011; Shahkaramipour, Tran, Ramanan, & Lin, 2017). Bioactive materials are functionalized polymers with antimicrobial agents that kill bacteria which adhere to the polymer surface. These

FIGURE 14.4

315

materials are classified into two categories: biocidal release materials and contact-killing materials. The mechanism by which the active polymer kills bacteria depends on the structure of the active agent and can be achieved by electrostatic interaction or by biocidal interaction. The cell wall of the bacteria containing phosphatidylethanolamine and phosphatidyglycerol without cholesterol is negatively charged and interacts by electrostatic interaction with antimicrobial polymers carrying positively charges (Ren, Cheng, Wang, & Liu, 2017). In this way, the cytoplasmic membranes is destroyed by forming pores in different mechanisms (barrel-stave, toroidal pores and carpet) followed by the attack on intracellular targets leading to the cell lysis (Tew, Scott, Klein, & De Grado, 2010; Timofeeva & Kleshcheva, 2011). The most widely used synthetic biocidal polymers are those containing either QAS or quaternary phosphonium salts (QPS) (Xue, Xiao, & Zhang, 2015). In Fig. 14.4 are presented various types of polymeric biocides with quaternary ammonium/ phosphonium salts. Polyurethanes containing quaternary ammonium groups have good antimicrobial activity against S. aureus and Escherichia coli. The

Types of polymeric biocides containing quaternary ammonium/phosphonium salts.

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bioactive polymer was obtained by incorporation of carbamate-group-containing QAS into polyurethane coatings through crosslinking with terpene-based polyol and polyisocianate (Liu, Wu, Jin, & Kong, 2015). Zhang, Liu, Lei, Hong, and Lin (2015) synthesized three series of acrylamide monomers/ polymers containing QAS in order to establish a relationship between polymeric structure and antimicrobial activity against two bacterial strains Staphylococcus albus and E. coli as well as against to phytopathogenic fungi Rhizotocnia solani and Fusarium oxysporum f. sp. cubense race 4. The methods used for the determination of antimicrobial activity were: minimum inhibitory concentration (MIC) and triphenyl tetrazolium chloride (TTC) coloration method for antibacterial activity as well as mycelia growth inhibition, MIC and minimum fungicidal concentration for antifungal activity. The results highlight that the polymers containing benzyl group attached to nitrogen atom showed better antimicrobial activities. To allow the control over structure and molecular weight of polymers advanced polymerization techniques such as reversible deactivation radical polymerization, reversible addition-fragmentation chain transfer were developed. Qin et al. (2015) prepared block copolymers based on poly(2-dimethylamino) ethyl methacrylate and poly(dimethylsiloxane) containing small fraction of 2-hydroxyethyl methacrylate by RAFT followed by the quaternization of the amino groups of PDMAEMA with n-octyl iodide. It was found that the antibacterial activity increases with increasing of cationic polymeric segment length. A very interesting new class of antibacterial polymers is represented by zwitterionic polymers. Ward et al. (2006) prepared poly(sulfopropyl)betaine by polymer analogous reactions starting from a series of statistical copolymers based on 2-(dimethylamino)ethyl methacrylate and four hydrophobic monomers, such as: ethyl, butyl cyclohexyl and octyl methacrylate.

For polymer analogous reactions the authors used 1,3 propansultone as betainization agent. The results obtained showed that the MIC values for S. aureus and E. coli were higher compared with those for the ampicillin and erythromycin. Grigoras et al. (2012) studied three poly(carboxybetaines) based on poly(Nvinylimidazole) with different spacer length (methylene, ethylene and propylene) between the positive and negative charges. Poly(carboxybetaines) with methylene and propylene spacers presented good antibacterial activity against S. aureus. The results suggest that the antibacterial activity depends on the type of bacteria as well as on the length of spacer between the positive and negative charges. Other polymers that have antimicrobial activity are poly(ethyleneimine), polyguanidine and N-halamine. Other examples of bioactive materials with antibacterial activities based on these polymers are: • poly(ethyleneimine) is a synthetic, cationic polymer that has in his structure primary, secondary and tertiary amino functions. Dental composites with strong antibacterial activity against Streptococcus mutans were prepared following different steps: (1) synthesis of cationic nanoparticles based on crosslinked PEI, followed by the quaternization with bromooctane and methylation with methyl iodide; (2) incorporation of the nanoparticles (NPs) into commercial dental resin composite during polymerization. The results indicated that the dental resin composites possess high biocompatibility (Beyth, Yudovin-Farber, Bahir, Domb, & Weiss, 2006). • polyguanidines are polymers characterized by excellent properties: high water solubility, nontoxicity, wide antimicrobial spectrum and excellent biocidal efficiency (Jain et al., 2014). Recent studies have focused on the preparation of new

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14.2 Synthetic macromolecules with antimicrobial activity

copolymer of 2,2-diallyl-1,1,3,3 tetraethylguanidinium chloride and 2hydroxyethyl acrylate, using free radical copolymerization. The guanidine-containing polymer was then chemically modified by two methods: (1) preparation of polymeric guanidine-containing conjugates by anion exchange reaction via hydroxide; (2) synthesis of biocide polymeric guanidinium salt by interaction of acrylate hydroxyl group belonging to the copolymer with biologically active acids in presence of dicyclohexylcarbodiimide and 4dimethylaminopyridine as dehydrating agent and esterification catalyst, respectively. New guanidinium polyelectrolytes present antibacterial activities against both Gram-positive and Gram-negative bacteria (Gorbunova, Lemkina, & Borisova, 2018). N-halamines represent the polymers that contain one or more nitrogen atom that is found in the form of imide, amide or amine groups and form covalent bond with halogen (Li, 2011). By chlorine bleach of the copolymer based on 2-vinyl-4,6-diamino-1,3,5 triazine and styrene, a copolymer with a powerful antimicrobial activity against S. aureus and E. coli was obtained. An interesting approach in order to improve antibacterial activity is to combine the antimicrobial polymer with nanomaterials and bioactive agents. This strategy can be realized by coadministration of two different antimicrobial polymers (combination between polymers based on Nhalamines and polycations) or by incorporation of metal oxides and metal NPs in antimicrobial cationic polymers (Yanez-Mocas et al., 2019).

14.2.6 Synthetic macromolecules with antiviral activity Viruses are submicroscopic pathogens with diameters of about 20 nm to over 300 nm, which

317

does not have the ability to self-reproduce, but are multiplied only inside the living cells of the host (Jarach, Dodiuk, & Kenig, 2020). The history of viruses began with development of the vaccines for chicken cholera (1879) and for anthrax and rabies (1889) by L. Pasteur and continued with discovery of many other viruses. Some examples are: tobacco mosaic virus (1898, M. Beijerinck), influenza (1931), poliovirus (1950), equine arteritis virus (1957), hepatitis B virus (1963, B. Blumberg), first retrovirus (1965, H. Temin), human immunodeficiency virus (HIV) (1983, L. Montag) and hepatitis C (1989, M. Houghton) (Goodpasture, Woodruff, & Buddingh, 1931; Mezner, 2006; Moss, Davey, Steigbigel, & Fang, 2010; Robiotti, Deresinski, & Pinsky, 2015). Viral replication involves several stages: attachment, penetration, uncoating, replication, assembly and release, each of these stages being potential targets for antiviral agents (Schneider-Schaulies, 2000). Influenza A virus causes severe diseases both in domestic poultry and in human population. To control the spread of this virus in present are used both vaccines and small molecular weight drugs (amantadine, rimantadine, zanamivir and oseltamivir). Vaccination offers limited protection and drugs have limited therapeutic window, side effects and high cost. Also, similar with bacteria, the most viruses are resistant to the M2 inhibitors (Lee et al., 2012). An alternative to the conventional antiviral agents is represented by the design of the polymeric inhibitors. The synthetic polymers can be used as antiviral agents by using the following strategies: (1) carriers for antiviral drug delivery; (2) polymers containing ions/metal particles; (3) polymers containing antiviral moieties (amines, carboxylic acid, sulfates and phenols). Linear poly(acrylamides) with pendant sialic acid groups have a higher binding affinity on the viral surface, inhibiting agglutination of erythrocytes by influenza virus (Sigal, Mammen, Dahmann, & Whitesides, 1996). Lee et al. (2012) developed a polymer-drug conjugate based on

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poly-L-glutamine and the active agent-zanamivir. The conjugate was linked to viral neuraminidase having as result the inhibition of both enzymatic activity and the release of virions from infected cells. By attaching of drug to the polymeric chain the authors obtained a multivalent antiviral that represents an alternative to conventional combination. Organotin polymers based on organotin dihalides, camphoric acid and lamivudine showed antiviral activity against Vaccinia virus and Zika virus (Roner et al., 2020). Polycationic superparamagnetic core-shell NPs with cores consisting of magnetite clusters and shell of functional silica covalently bound to poly(hexamethylene biguanidine), poly(ethylenimine) or PEI terminated with aziridine moieties were evaluated as antiviral agents. The core-shell NPs containing biguanidine and aziridine moieties were efficient against bacteriophage Ms2, herpes simplex virus HSV-1, nonenveloped infections pancreatic necrosis virus and enveloped viral hemorrhagic septicemia virus (Bromberg et al., 2012). Dendrimers based on N-alkylated 4,40 -bipyridinium units, known as viologen, sulfonated polymers, copolymers of styrene and maleic acid are polymers that have been explored in prevention and treatment of HIV (Asaftei & De Clercq, 2010; Clayton, Hardman, La Branche, & McReynolds, 2011; Pirrone et al., 2010; Witvrouw et al., 2000).

14.2.7 Synthetic macromolecules with antifungal activity Human health can be severely affected by fungal infections because they are difficult to treat (Brown et al., 2012). Fungal infections are caused by eukaryotic organisms called fungi. Two forms of fungal infections are known, namely local fungal infections and systemic fungal infections. Local fungal infections can be caused by both yeast and mold and are localized to the skin, nails and hair. Systemic fungal infections are caused by yeast and mold, fungi that reach the gastrointestinal tract (candidiasis), lungs (aspergillosis) and

even the brain. There are two types of organisms that cause systemic fungal infection (RautemaaRichardson & Richardson, 2017): (1) the opportunists (Aspergillus and Candida spp.) which take advantage of a weakness of the human body such as, weakness of the immune system, altered microbiota or breached integumentary barriers. The most well-known diseases caused by these fungi are: aspergillosis, candidiasis, cryptococcosis, mucormycosis, pneumocystosis and fusariosis; (2) true pathogenic fungi that have a restricted geographical distribution. The diseases caused by true pathogenic fungi are: blastomycosis, coccidioidomycosis, histoplasmosis and paracoccidioidomycosis. When the immune system is weakened, the fungi expand and create problems. The most affected people are diabetics, HIV-infected patients, patients with intestinal problems and people undergoing treatment with certain medications (such as antibiotics and cortisone). The most common fungi found are Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. C. albicans is a pathogenic yeast and is detected in the gastrointestinal tract, genital area and mouth. New studies indicate that C. albicans may cross the blood
brain barrier (Wu et al., 2019). A. fumigatus is a species of fungus in the genus Aspergillus that can cause major health problems in immunocompromised individuals such as AIDS patients, organ transplant recipients, bone marrow and patients diagnosed with leukemia. C. neoformans is an encapsulated fungal organism. It is widespread because it can live in both plants and animals and is often found in bird droppings. C. neoformans infections occur in the lungs (Tripathi, Mor, Bairwa, Poeta, & Mohanty, 2012), in most cases but can also occur as secondary infections such as fungal meningitis and encephalitis in AIDS patients. Systemic fungal infections have been found to be very dangerous because they cause a fairly high mortality rate. This may be due to both low efficacy and side effects of current antifungal drugs used in the therapeutic protocol. The body’s strategy to fight against

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14.2 Synthetic macromolecules with antimicrobial activity

microbial infection includes the production of peptides known as “host defense peptides.” These are produced by the eukaryote as an innate immune response and they activate in case of invasion by fungi, protozoa, bacteria and viruses (Hancock & Sahl, 2006). The use of synthetic polymers that mimic the important properties of AMPs has been the subject of research studies. Unlike the large number of antibacterial polymers, there are few reports of synthetic polymers with antifungal activity. Nylon-3 polymers have been studied to demonstrate their antifungal activity, being obtained by ring-opening polymerization of β-lactams (Liu et al., 2013). Mowery, Lindner, Weisblum, Stahl, and Gellman (2009) reported the preparation of a new β-lactam, namely NM (“no methyl”). The researchers choose this compound because it contains fewer saturated carbon atoms and therefore should have lower hydrophobicity than the cationic nylon-3 subunits derived from β-lactams, MM (“monomethyl”) and DM (“dimethyl”) synthesized by Liu et al. (2014). The polymers synthesized by Mowery et al. (2009) showed strong antifungal activity and favorable selectivity toward the fungal agent C. albicans. Rank et al. (2017) continued their studies regarding the antifungal properties of poly-βNM and identified nylon-3 polymers that are active against two other pathogenic fungi, namely A. fumigatus and C. neoformans. Lidani et al. (2019) synthesized the nylon3 MM-TM copolymer which has good activity against Candida. The MM-TM polymer together with the drug azole has a strong synergistic activity against C. albicans and A. fumigatus.

14.2.8 Synthetic macromolecules with antiparasitic activity Parasites are a class of pathogens that are much more dangerous to humans than bacteria. Parasites cause chronic diseases that are difficult to diagnose in time. The parasites that can cause diseases in humans are classified in three main classes: protozoa, helminths and cetoparasites.

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The most common parasitic diseases are malaria, the chagas disease and leishmaniasis. Malaria is an infectious disease caused by protozoan parasites of the Plasmodium group also called “parasites of malaria.” Five species of Plasmodium, namely Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi can infect the human body. P. falciparum causes the most severe form of malaria leading to most deaths. Chagas disease is a tropical parasitic disease caused by the Trypanosoma cruzi. This disease is spread to humans through insects known as Triatominae or “kissing bugs” that feed on blood (Lidani et al., 2019). Leishmaniasis is a disease caused by protozoan parasites, such as: Leishmania that spreads through the bite of some types of female sandflies. This disease in humans is caused by over 20 species of Leishmania. There are three types of Leishmaniasis, namely cutaneous Leishmaniasis, monocutaneous Leishmaniasis and visceral Leishmaniasis which is the most serious form of the disease and which if not treated properly can be fatal. Due to the long life cycles of parasites, in general the treatment of parasitic diseases requires frequent doses for a longer period of time. This is the reason why researchers have tried to find solutions for the improvement of the treatment of parasitic diseases for the comfort of patients. Recent studies have used polymeric NPs to release amphotericin B against Leishmaniasis (Asthana, Jaiswal, Gupta, Dube, & Chourasia, 2015; Kumar et al., 2017). Singh, Arish, Kumar, Rub, and Riaz (2020) used ultrasonic polymerization of azobenzene with aniline, 1-naphthylamine, luminol and o-phenylenediamine to obtain the polymers poly(aniline-azobenzene) (PANI-AB), poly(1-naphthylamine-azobenzene) (PNA-AB), poly(luminol-azobenzene) (PLu-AB), and poly(o-phenylenediamineazobenzene) (PPd-AB). The authors revealed that the polymers PANI-AB, PNA-AB and PPd-AB showed antileishmanial activity.

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Garcia-Valdiviaa et al. (2020) studied the antiparasitic and antiinflammatory activities of two multifunctional isomorphous (4,4) square-grid 2D coordination polymers based on 1H-indazole-5-carboxylic acid.

14.3 Synthetic macromolecules with antioxidant activity Initially, the general term antioxidant referred to any substance that prevented the consumption of oxygen. Many research studies, conducted in the late 19th and early 20th century, have expanded the definition of antioxidants (Halliwell, 1995). In the present, an antioxidant is a compound that delay and inhibit the oxidation process of other molecules and protect the body from the harmful effects of free radicals. Free radicals (reactive oxygen species and reactive nitrogen species), produced either by normal cellular metabolism or as an effect of pollution and exposure to other external factors, are responsible for premature aging of the body. Also, they cause the oxidative stress, a process that can lead to the cell damage and play an important role in triggering of some diseases, such as: heart diseases, cancer, arthritis, stroke, respiratory diseases, immune deficiency, emphysema, Parkinson and Alzheimer diseases, diabetes, cataracts, age-related macular degeneration, etc. For this reason, the use of antioxidants is beneficial in preventing the aging process of the body cells as well as the occurrence of serious diseases helping to maintain a good health. Also, the antioxidants bring many other benefits for the body: regulates cholesterol levels, protects the nervous system, protects eyesight, strengthens the immune system, prevents degenerative diseases and cardiovascular diseases and helps in the treatment of cancer. Depending on the pathways of synthesis, antioxidants are classified into (Pokorny, 2007):

• natural antioxidants (vitamin A, vitamin C, vitamin E, beta-carotene, lycopene, lutein, zeaxanthin, manganese, selenium, flavonoids, anthocyanins, resveratrol, hydroxytyrosol, quercetin, phenolic acid) that are synthesized by various microorganisms, fungi, animals and plants. They possess the ability to protect from oxidative damage and to prevent the appearance of some diseases (oxidative stress, inflammation, hypertension, hyperglycemia, dyslipidemia) (Calixho, 2017); • synthetic antioxidants that are obtained by synthesis or biosynthesis in the industry; • antioxidants similar to those from the nature are compounds that are found in foods, but are synthesized in the industry. Antioxidants are essential to protect the materials against oxidation being designed to improve the shelf life of finished products and materials. Synthetic antioxidant molecules are widely used in the food industry due to the limited possibility of using natural antioxidants. Moreover, the natural antioxidants present some disadvantages, such as: insufficient quantities, high costs and poor stability. On the other hand, the synthetic antioxidants with high degrees of purity can be obtained by means of chemical reactions. Furthermore, synthetic antioxidant used in the food industry can delay food lipid oxidation and thus improve the shelf life of processed foods with minimal nutritional loss (Saiga, Tanabe, & Nishimura, 2003). Generally, synthetic antioxidants with low molecular weight are represented by phenolic compounds and ascorbic acid derivatives as follows: • gallic esters: octyl gallate, propyl gallate and dodecyl gallate. Octyl gallate or octyl 3,4,5trihydroxylbenzoate is obtained by condensation of the carboxyl group of gallic acid with the hydroxyl group of octanols and is used in the pharmaceutical,

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14.3 Synthetic macromolecules with antioxidant activity

cosmetically and food industries. Propyl gallate (propyl 3,4,5-trihydroxylbenzoate) was obtained by condensation of gallic acid with propanol. This compound is used in foods, cosmetic, adhesives and lubricants industries. Dodecyl gallate or lauryl gallate is produced by the esterification of gallic acid with the dodecanol. The esterification is activated by the presence of p-toluene sulfonic acid (Xin, Ma, Lin, Xu, & Chen, 2013). Dodecyl gallate is an antioxidant added to foods and cosmetics to prevent their damage; • butylated hydroxytoluene (BHT) is a synthetic antioxidant composed of 4-methylphenil modified with tert-butyl groups in positions 2 and 6. The synthesis of BHT can take place in two ways: (1) first method consists by hydroxymethylation or aminomethylation of 2,6-di-tert-butylphenol followed by hydrogenolysis; (2) the second method is generally used at industrial scale and consists in the reaction of p-cresol (4-methylphenol) with isobutylene (2-methylpropene). This reaction is catalyzed by sulfuric acid, the butylated hydroxytoluene being used in elastomer, lubricants, cosmetically and food industries. This low molecular weight antioxidant may be found in the composition of gelatin capsules, tablets or another pharmaceutical dosage forms, as well as in gels and cre`me for pharmaceutical use. Because it assumes that may cause cancer, it has been replaced with butylated hydroxyanisole (BHA); • BHA is prepared by the chemical reaction between 4-methoxyphenol and isobutylene. In general, commercial BHA preparations are a mixture of two isomeric compounds, namely 2-tert-butyl-4-hydroxyanisole (15%) and 3-tert-butyl-4-hydroxyanisole (85%) (Whysner & Williams, 1996). BHA is used as an antioxidant and preservative in food, food packaging, cosmetics and petroleum products. Also, the BHA is used as

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excipients in the composition of some pharmaceutical dosage forms; • 2-tert-butylhydroquinone (TBHQ) is a derivative of hydroquinone and was synthesized by the alkylation reaction between hydroquinone and isobutene (Zhu & Yan, 2004). The reaction took place in the presence of the catalyst H3PO4 or H2PO4H3PO4. TBHQ is used in food industries; • sodium erythorbate is an antioxidant and preservative used in cosmetically and food industries and is obtained by a combination of bio- and chemical synthesis starting from D-glucose (Andersen, 1999); • isoascorbic acid is a synthetic low molecular weight antioxidant prepared by reaction between sodium methoxide and methyl 2-keto-D-gluconate and it is used an antioxidant in processed foods. Also, the ascorbyl acid esters represent a class of synthetic antioxidants used in the food industry. The ascorbyl acid esters, namely ascorbyl palmitate, ascorbyl dipalmitate and ascorbyl stearate were obtained from ascorbic acid and palmitic acid or stearic acid. Although conventional antioxidants provide protection against oxidative processes, these antioxidants present disadvantages regarding to their low molecular weight. For example, the loss of antioxidant efficiency is due to different processes, such as: evaporation, diffusion and leaching (Kuczkowski & Giliick, 1984). Moreover, low molecular weight antioxidants could diffuse into the surrounding medium and may contaminate the food, certain types of packaging or medical devices, leading to the release of potentially toxic by-product substances (Al-Malaika, 1991; Pospisil, 1992). Another disadvantage consists in the incompatibility between antioxidants and the materials they come in contact, leading to damage of the contact surfaces. The thermal stability of low molecular weight antioxidants decreases with increasing temperature. All these

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disadvantages have been combined with disappointing results obtained in the in vivo studies. Consequently, the use of high molecular weight antioxidants also known as macromolecular antioxidants would reduce these disadvantages. One of the advantages of macromolecular antioxidants consists in their mechanism of action. After ingestion, the macromolecular antioxidants pass intact through stomach and small intestine and reach the colon when by the interaction with colonic microbiota through fermentative process could lead to the production of antioxidant metabolites. Antioxidant metabolites reach the bloodstream about 8 h after ingestion, increasing and thus prolonging the antioxidant status (blood antioxidant concentration). After that the antioxidant metabolites are distributed to the cells and organs. In case of low molecular weight antioxidants, the passage of the antioxidant metabolites into the bloodstream occurs between 0.5 and 2 h after ingestion, because these types of antioxidants are rapidly absorbed in the small intestine. Thus, the macromolecular antioxidants offer an improvement of antioxidant status in both plasma and gastrointestinal tract (Calixho, 2017). Thus, to extend the shelf life of finished products and materials is necessary and important to improve the efficiency of antioxidants. For this purpose, the appearance of the new materials which possess a superior antioxidant activity and a better thermal stability led to the development of polymers with antioxidant properties. Consequently, the synthetic macromolecules with antioxidant properties can be more effective and may be a therapeutic alternative compared to the antioxidants with low molecular weight. Therefore, among the antioxidants mentioned above, in the following paragraphs one will review only the synthetic antioxidants. Synthetic macromolecules with antioxidant properties have been used in various fields as these materials combine the properties both of polymeric materials and antioxidant

components. Thus, synthetic macromolecules with antioxidant properties that possess superior antioxidant activity with improved mechanical strength and thermal stability have proven to be very useful in elastomer, lubricants and fuels industries. In the medical field, synthetic macromolecules with antioxidant properties can be used as active pharmaceutical ingredients and pharmaceutical excipients. Moreover, they can be used in anticancer therapies, wound healing and tissue engineering. It is known that the use of cosmetics is often limited by the short-term stability of their components, which leads to reduced efficiency and even at the appearance of toxic side effects. Thus, the cosmetics industry has shown interest in synthetic macromolecules with antioxidant properties both for their biological activity and for their ability to protect cosmetics from degradation. Furthermore, synthetic macromolecules with antioxidant properties have been studied from nutritional and industrial point of view, in terms of food or food packaging materials. The presence of antioxidant compounds in food influences human health as well as on the nutritional value, contributing to the preservation of food quality in storage conditions. The research has shown that during storage, a food can change its nutritional qualities as a consequence of the interaction with atmospheric agents or packaging materials. To solve this problem, the so-called “intelligent packaging” based on antioxidant polymers was developed. The free radical polymerization technique is the most used method to obtain synthetic macromolecules with antioxidant properties. This method takes place in two stages: (1) antioxidant molecules are functionalized with polymerizable groups such as vinyl, acryl, styrenic, maleimide groups, etc. (2) polymerization or copolymerization of functionalized molecules with antioxidant properties by using the conventional free radical polymerization techniques (Ortiz, Vazquez, & Roman, 1999). Nikulin,

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14.3 Synthetic macromolecules with antioxidant activity

Misin, Komarov, and Misin (2003) reported the synthesis of poly-2-vinylphenothiazine by bulk radical polymerization of 2-vinylphenothiazine at high temperatures and in the presence of azoisobutyronitrile as radical initiator. Poly-2vinylphenothiazine can be used as antioxidant in rubber industry. Singh and Kaplan (2003) synthesized synthetic macromolecules with antioxidant properties based on ascorbic acid and fluoro-vinyl monomer. The fluoro-vinyl monomer was obtained by esterification of 4-vinylbenzoic acid with trifluoroethanol. Ascorbic-acid-modified polymers for free radical scavenging were obtained in two stages: (1) enzymatic transesterification where primary hydroxyl group belonging to ascorbic acid was acylated by trifluoroethyl 4-vinylbenzoate to obtain L-ascorbyl-4-vinylbenzoate; (2) polymerization of L-ascorbyl-4-vinylbenzoate in presence of horseradish peroxidase as a catalyst. In the study of Coleman, Mock, and Painter (1999), oacetyl-2,6-diisopropyl-4-vinylphenol was copolymerized with styrene and then deacetylated by reaction with hydrazine monohydrate to finally obtain a polymer with antioxidant properties. Shehata, Nasr, and Farouk (2005) have synthesized an antioxidant polymer namely poly-N-(4-mercaptophenyl) acrylamide using the free radical polymerization method. This antioxidant polymer protects and improves the extraction and compression recovery properties of styrene-butadiene rubber under oxidative aging conditions. Oh et al. (2001) prepared monomers based on maleimide. In a second step, the antioxidant monomers were polymerized in presence of azoisobutyronitrile as initiator, in order to obtain the synthetic macromolecules with antioxidant properties. The authors also investigated the thermal behavior of these homopolymers by thermogravimetric analysis. The synthetic macromolecules with antioxidant properties showed a better thermal stability compared to the corresponding monomers. Also, synthetic macromolecules with antioxidant properties can be obtained by derivatization

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of a polymerizable monomer with an antioxidant molecule. For example, Puoci et al. (2008) synthesized antioxidant copolymers by radical polymerization of methacrylic acid with ferulic acid in the presence of water-soluble redox initiators. This strategy is a very simple approach for the preparation of macromolecular systems with high antioxidant activity. These polymeric materials can be used as preservatives in food and cosmetics. Graft polymerization is a well know method for the modification of the surface properties of the polymer to tailor for antioxidant applications. The grafting methods can be divided into two processes, namely grafting to- and grafting from processes (Atanase, Desbrieres, & Riess, 2017). Thus, in the case of the grafting to- method, performed polymer chains carrying reactive groups at the end or side chains are covalently coupled to the surface (Spizzirri et al., 2009). The grafting from method utilized the active species existing on the material surfaces to initiate the polymerization of monomers from the surface toward the outside bulk phase. For example, the phenolic compounds like catechin and gallic acid were grafted onto gelatin. It is known that polyurethanes are used in various medical devices. It has been found that the cracking of cardiovascular devices based on polyurethane is due to the oxidative degradation of their surface upon long-term contact with inflammatory cells in the human body. To solve this problem, Stachelek et al. (2006), synthesized the thiophenol-bound polyurethane by grafting method in two steps. Kimwomi, Kossmehl, Zeinalov, Gitu, and Bhatt (2001) reported the synthesis of new synthetic macromolecules with antioxidant properties, obtained by grafting method of three antioxidants [divernolin and trivernolin extracted from vernonia oil and a phenolic antioxidant namely 3-(3,5-di-tert-butyl4-hydroxyphenyl) propionic acid] onto polystyrene and polyurethane. The ring-

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opening metathesis polymerization method was used by Xue, Ogata, and Toyota (2007), in order to obtain synthetic macromolecules with antioxidant properties. Moreover, ringopening metathesis polymerization is a method that uses well-defined catalytic systems to obtain macromolecules with controlled architecture (Trnka & Grubbs, 2001). In the first stage of the study accomplished by Xue et al. (2007), they prepared the functional monomer based on norbornene derivative that possesses sterically hindered phenol. For the synthesis of the intermediate polymer, the ring-opening metathesis polymerization of the functionalized monomer was performed by using the bis(tricyclohexylphosphine)benzylidene ruthenium (IV) dichloride as catalyst. In order to obtain the desired synthetic macromolecules with antioxidant properties, the intermediary polymer was hydrogenated with p-toluenesulfonyl hydrazide. Another method for the preparation of the synthetic macromolecules with antioxidant properties is the condensation of aldehydes and phenols. Malshe, Elango, and Rane (2006) used the condensation reaction of phenols with formaldehyde in the presence of oxalic acid to obtain synthetic macromolecules with antioxidant properties based on pnonyl phenol formaldehyde and p-dodecyl phenol formaldehyde resins. Moreover, poly (catechin), a synthetic macromolecule with antioxidant properties was obtained by Chung, Kurisawa, Kim, and Kobayashi (2004) using the condensation reaction of acetaldehyde with a polyphenolic substance, namely catechin, in the presence of an acid catalyst (acetic acid). The antioxidant capacities of the samples (plant extracts, commercial antioxidants, etc.) can be analyzed using different methods of investigation. In general, the antioxidant capacities of the samples are influenced by several factors such as the test system and the mechanism of action.

In Table 14.2 are presented the main methods for evaluating the antioxidant capacity of different materials.

14.4 Polymer sequestrants Polymer sequestrants are a class of insoluble polymer drugs that are used for removal of harmful molecules, such as: inorganic ions (potassium, phosphate, iron, cadmium), bile acids, nucleic acids, peptides and proteins (toxins). High level of low-density lipoprotein cholesterol (LDL) called the “bad” cholesterol is associated with high incidence of atherosclerosis and coronary heart diseases (myocardial infarction and coronary death). LDL is the lipoprotein that contains the highest amount of cholesterol, about 60%
70% of total serum cholesterol being involved in the transport of cholesterol to the body tissues. The rest is represented by the high-density lipoprotein known as “good” cholesterol. There are two therapeutic options for lowering plasma total cholesterol and LDL: lifestyle intervention and lipid-lowering drug therapy (Hermankova et al., 2018). The second therapy method used statin for lowering cholesterol while for patients with statin intolerance the treatment is realized with niacin and bile acid sequestrants. Bile acids are amphipathic steroids alcohols that are synthesized from cholesterol in the liver. Bile acids are stored in gallbladder and after meal intake are released in gastrointestinal tract where they help in the adsorption process of nutrients, dietary fats, steroids, vitamins and drugs (Chiang & Ferrell, 2020). Polymeric bile acid sequestrants have been introduced on the market as well as used in clinical trial for many years (Connor, Lees, & Maclean, 2017). Some requirements must be considered, when designing the structure of polymeric bile acid sequestrants: (1) bile acids have anionic characters and in consequence the polymeric sequestrants must have cationic

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14.4 Polymer sequestrants

TABLE 14.2 Methods evaluation of antioxidant activity. Methods

Characterization

References

DPPH Radical Scavenging Assay

This method is fast, simple and inexpensive compared to Cespedes, El-Hafidi, Pavon, and other evaluation methods. The molecule 1,1-diphenyl-2Alarcon (2008) picrylhydrazyl (DPPH) can produce a stable free radical in ethanol solution and has a maximum adsorption in UV-VIS at the wavelength of 515 nm. The change in the optical density of DPPH radicals is followed to evaluate the antioxidant potential of the tested samples.

ABTS Radical Scavenging Activity

ABTS Radical Scavenging Activity is applied to evaluate both hydrophilic and lipophilic antioxidant activities. By the reaction between 2,20 -azino-bis [3-thylbenzothiazoline-6sulfonate] (ABTS) with potassium persulfate the radical ABTS 1 is generated. The radical cation ABTS 1 has a stable dark blue color and is reduced by an oxidant into colorless ABTS which can be determined by UV-VIS spectroscopy at the wavelength of 734 nm. Antioxidants reduce the color intensity of ABTS 1 depending on their antioxidant activity.



Shui and Leong (2002)





Phosphomolybdenum Assay

Phosphomolybdenum assay is a spectrophotometric method Prieto, Pineda, and Aguilar (1999) that is based on the reduction of Mo (VI) to Mo (V) in the presence of antioxidants leading to the formation of phosphate Mo (V) complex. This green colored complex at acidic pH can be determined spectrophotometrically (UV-VIS) at the wavelength of 695 nm.

Reducing Power Assay

Reducing Power Assay is based on the fact that the presence of antioxidants in the samples can form a colored complex with potassium ferricyanide, trichloroacetic acid and ferric chloride which can be determined spectrophotometrically at the wavelength of 700 nm.

Chou, Chiang, Chung, Chen, and Hsu (2006)

Hydroxyl Radical Scavenging Activity

The scavenging ability of hydroxyl radicals, which is one of the strongest species of reactive oxygen in the biological system, was determined by Kunchandy and Rao’s method. The hydroxyl radical was generated by Fenton reaction using Fe31-ascorbate-ethylenediaminetetraacetic acid-hydrogen peroxide system.

Kunchandy and Rao (1990)

β-Carotene Leaching Assay

β-Carotene leaching assay is a rapid method for detection of the antioxidants that is based on the principle that the peroxyl free radicals are produced from the autoxidation of linoleic acid by heating and under air atmosphere. Discoloration of the yellow solution of β-carotene is achieved when the peroxide radicals attack the C 5 C double bonds belonging to β-carotene leading to the rupture of the π conjugation. The absorbance of mixture solution is determined spectrophotometrically at the wavelength of 434 nm.

Kabouche, Kabouche, Ozturk, Kolal, and Topcu (2007)

(Continued)

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TABLE 14.2 (Continued) Methods

Characterization

References

Superoxide Radical Scavenging Assay

The superoxide anions can produce strong and dangerous Li, Jiang, Zhang, Mu, and Liu hydroxyl radicals, as well as singlet oxygen, two reactive (2008), Robak and Gryglewski oxygen species that initiate the oxidative stress process. (1988) Superoxide anions can be generated enzymatically using a xanthine-xanthine oxidase or nonenzymatically in a phenazine methosulfate-NADH system. To assay the superoxide radical scavenging potential of antioxidant, the antioxidant sample is incubated with phenazine methosulfate-NADH-nitro blue tetrazolium and the absorbance of the mixture is determined at 562 nm.

Metal Ion Chelating Assay

The metal chelation method uses the ability of iron to participate in chelation reactions and is based on the measurement of red ferrozine-Fe21 complexes absorbance at 562 nm.

Determination of Total This method for the determination of the total flavonoid Flavonoid Content content is based on the calorimetric method described by Wang et al.

groups at an appropriate charge density in order to establish the electrostatic interactions between both partners; (2) the existence of a hydrophobic moieties for attraction of the steroid skeleton of the bile acid. Two very important factors such as, the length of hydrophobic chain and their distribution must be taking into consideration (Chiang & Ferrell, 2020); (3) the degree of crosslinking; (4) flexibility of polymer chains; (5) swelling capacity under physiological conditions. Colestipol and cholestyramine were the first generation of polymer bile acid sequestrants that have been introduced on the market. Colestipol is an anion exchange resin based on diethylenetriamine or tetraethylenepentamine and epichlorohydrin being used since the 1970s. Moreover, the colestipol is used in the treatment of chronic diarrhea (Heel, Brogden, Pakes, Spight, & Avery, 1980). Cholestyramine (Amberlite, Dowex) is a strong anion exchange resin based on styrenedivinylbenzene copolymer possessing benzyl trimethyl ammonium chloride groups and it was used to treat hypercholesterolemia and diarrhea

Singh and Rajini (2004)

Wang, Yuan, Jin, Tian, and Song (2007)

resulting from bile acid malabsorption (Scaldaferri, Pizzaferrato, Ponziani, Gasbarrini, & Gasbarrini, 2013). Because both resins have low sequestration potency, the research studies have been directed to finding solutions to improve the binding capacity of bile acids. In this context, the second-generation of polymer bile acid sequestrants were developed. Compared to the first generation of polymer bile acid sequestrants, the second generation (colestimide and colesevelam) are characterized by the amphiphilic character and an enhanced binding affinity to bile acids. Colestimide is a copolymer of 2-methylimidazole and epichlorohydrine. Colesevelam is an amphiphilic polymer based on poly(allylamine) obtained in two steps: (1) crosslinking reaction of poly(allylamine) with epichlorohydrin; (2) chemically modification with bromodecan and (6-bromohexyl)trimethylammonium bromide, the bromide ions being replaced with chloride ions by washing (Connor et al., 2017). Also, poly(meth)acrylates, poly(meth)acrylamides, polyalkylamines and poly(allylamines) are considered as potential candidates for design

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14.4 Polymer sequestrants

the polymeric bile acid sequestrants (Cameron, Eisenberg, & Brown, 2002; Mendoca, Serra, Silva, Simoes, & Coelho, 2013; Wang et al., 2016). For example, Polomoscanik, Holmes-Farley, Petersen, Sacchiero, and Dhal (2012) prepared a series of amphiphilic cationic hydrogels bearing pendant quaternary ammonium groups based on poly (allylamine) backbone. The amphiphilic cationic gels exhibit cholesterol lowering properties and the preliminary in vivo studies showed that the increase of the chain length from C8 to C12 led to the increase of the binding capacity of hydrogels. These observations suggest that the hydrophobicity of the pendant alkyl group has an important role on the bile acid binding capacity of amphiphilic hydrogels. The same group has obtained the functional hydrogels having pendant amine and guanidinium groups. The polymers with pendant amine groups were obtained either by direct polymerization of monomer that contain amine groups or by chemical modification of suitable polymeric precursors. Polymers with pendant guanidinium groups were prepared in aqueous solution by polymer analogous reactions on crosslinked polymeric amines (primary and secondary) using an appropriate guanylating agent. It was found that the ammonium functionalized polymers possess higher bile acid sequestration capacity compared to guanidinium functionalized polymers (Huval et al., 2004). Chemically modification of cyclodextrins and their polymerization using different crosslinkers (epichlorohydrin and dininyl sulfone) led to the diversification of the structures of polymeric sequestrants (Baille, Huang, Nichifor, & Shu, 2000; Morales-Sanfrutos, Lopez-Jaramillo, Elremaily, Hernandez-Mateo, & Santoyo-Gonzales, 2015). Presence of high number of ions such as potassium, phosphate and iron have harmful effects on the human body leading to the lethal disorders like electrolytic disorders and heavy metal intoxication. This problem can be solved by using polymer sequestrants, the mechanism of ion binding being: coordination, ion-ion interaction, hydrogen bonding and ion-aromatic interactions (Schneider, 2009).

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Hyperkalemia is a severe disease that occurs due to high levels of potassium ions. Potassium is a chemical element that is important in the proper functioning of nerves and muscles, including the heart muscles. The normal amount of potassium in the blood is 3.6
5.2 mmol/L, but a blood potassium level higher than 6 mmol/L requires immediate treatment. For this purpose, various polymer sequestrants structures were developed. Kayexalate, a cation-exchange resin based on sodium polystyrene sulfonate administered by oral and rectal routes and used for the treatment of hyperkalemia was approved by the FDA in 1958. Kayexalate is obtained by suspension polymerization of styrene and divinylbenzene followed by functionalization to the sulfonic acid and then transformed to the sodium salt of poly(styrene sulfonate) (Connor et al., 2017). Calcium Resonium or Kalimate represents a calcium salt form of poly(styrene sulfonate) being indicated for the treatment of hyperkalemia associated with anuria and severe oliguria (Kao, Tsai, Chiang, Mao, & Kao, 2015). Clinical studies showed that Kayexalate has gastric side effects, including colonic necrosis. Patiromer is another medication used in treatment of hyperkalemia. Patiromer is a nonabsorbed polymer sequestrant obtained by suspension polymerization of methyl 2-fluoroacrylate in the presence of two crosslinking agents such as, divinylbenzene and 1,7-octadiene. In clinical trials, it was observed that patiromer induced a small increasing in urinary calcium excretion as well as a decrease in urine potassium, urine sodium, urine magnesium and urine phosphate leading to the conclusion that a small quantity of calcium ions in patiromer is available for absorption while some part of calcium released is binding to intestinal phosphate (Buslinsky et al., 2016). Hyperphosphatemia is another severe disease characterized by a high level of phosphate ions. Sevelamer hydrochloride, a crosslinked

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polymer based on poly(allylamine) containing primary and secondary aliphatic amine residues was the first polymeric phosphate sequestrant approved by the FDA in 1998 (Taniguchi & Kakuta, 2015). Bixalomer (Kiklin capsules) is a phosphate binding polymer obtained by suspension polymerization techniques as a spherical beaded hydrogel. Various structures of brushed and dendritic polymeric sequestrants terminated with hexadentate ligands based on hydroxypyridinone, hydroxypyranone and catechol showed high selectivity for iron (III) being used for the treatment of haemochromatosis (Zhou et al., 2006).

14.5 Conclusions Synthetic macromolecules with biological activities represent a special class of polymeric materials that have a great potential for water purification, textile, food, pharmaceutical and cosmetical industries, as well as for biomedical applications such as, antimicrobial polymers, antioxidant polymers, anticancer polymers, polymer sequestrants, etc. Nowadays, new and advanced synthesis methods are used to obtain polymeric materials with controlled architectures and welldefined properties. But the field of synthetic macromolecules with biological activity is very complex and when designing such materials many requirements in terms of their physicochemical properties and biological characteristics must be considered. Also, current technologies are used for the preparation of bioactive materials in various forms of presentation such as nano and microparticles, fibers, hydrogels, membranes and films. The development of novel strategies regarding the decrease of microbial resistance to antibiotics, the investigation of the structure-bioactivity relationship as well as preventing the biofilm formation represents

a challenge for the scientific world. Finally, the emergence of new infection diseases or new strains determines the scientists worldwide to find solutions to solve these problems.

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Further reading

Further reading Chen, C. Z., Beck-Tan, N. C., Dhurjati, p, Van Dyk, T. K., La Rossa, R. A., & Cooper, S. L. (2000). Quaternary ammonium functionalized poly(propylene imine) dendrimers as effective antimicrobials: Structure-activity studies. Biomacromolecules, 1(3), 473
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derived from 2-vinyl-4,6-diamino-1,3,5, triazine. Journal of Polymer Science Part A-Polymer Chemistry, 43, 4089
4098. Kamber, N. E., Jeong, W., Waymouth, R. M., Pratt, R. C., Lohmeijer, B. C. G., & Hedrick, J. L. (2007). Organocatalytic ring-opening polymerization. Chemical Reviews, 107(12), 5813
5840.

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C H A P T E R

15 Biological macromolecules in drug delivery Amit Kumar Nayak1, Md Saquib Hasnain2, Anindita Behera3, Amal Kumar Dhara4 and Dilipkumar Pal5 1

Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, India 2 Department of Pharmacy, Palamau Institute of Pharmacy, Daltonganj, India 3School of Pharmaceutical Sciences, Siksha “O” Anusandhan, Deemed to be University, Bhubaneswar, India 4 Department of Pharmacy, Contai Polytechnic, Contai, India 5Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, India

15.1 Introduction Biological macromolecules (including carbohydrates, proteins, peptides, nucleic acids, and lipids) are the biologically derived molecules of high molecular weight comprising polymerized forms of smaller units of monomers (Krishnaswami, Kandasamy, Alagarsamy, Palanisamy, & Natesan, 2018; Zhang, Sun, & Jiang, 2018). These biological macromolecules are abundantly derived/extracted from various natural renewable resources as these occur in plants, algae, fungi, microbes, insects, animals, etc. (Bilal & Iqbal, 2020; Yu, Shen, Song, & Xie, 2018; Zhang et al., 2018). Recently, a variety of macromolecules are being produced by biotechnological fermentation and microbiological culture process (Lynd, Weimer, van Zyl, & Pretorius, 2002; Mart˘au, Coman, & Vodnar, 2020). In the native settings, biological

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00015-4

macromolecules perform a diverse set of functions, for example, carbohydrates function in membrane components, cellular communications and as storage sites for energy sources, whereas proteins function as structural substances, catalysts, transport vehicles, nutrients, etc. (Slavin & Carlson, 2014; Watford & Wu, 2018). Lipids function as precursors of triglycerides and sterols, storage and source of energy, cell membrane components, chemical messengers between cells, absorption of nutrients, etc. (Corn, Windham, & Rafat, 2020; van Meer, Voelker, & Feigenson, 2008). Transport proteins function as carriers for delivery of nutrients and other biochemicals are of special interest in therapeutics (Baltaci & Yuce, 2018; Montalbetti, Dalghi, Albrecht, & Hediger, 2014). Almost all of these biological macromolecules are degraded by in vivo enzymes and their metabolites also possess very less or no

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toxicity, in vivo (Ulery, Nair, & Laurencin, 2011). Inspired by such examples of biological functions, scientists have started to utilize various biological macromolecules including carbohydrates, proteins, peptides, nucleic acids (RNA and DNA), and lipids in many biomedical applications including biomedicines, drug delivery, tissue regenerations, wound management, orthopedics, etc., due to their sustainable production, renewability, biocompatibility, biodegradability, longer blood circulation period, targeting capability, etc (Chandika et al., 2020; Chauhan, Shaik, Kulkarni, & Gupta, 2020; Daly, Reynolds, Sigal, Shou, & Liberman, 1990; Kapoor, Singh, Saldarriaga-Herna´ndez, ParraSaldı´var, & Iqbal, 2021; Krishnaswami et al., 2018; Oana, Adriana, Mircea, Dragos, & Monica, 2018; Schwartz, Schwartz, Mieszerski, McNally, & Kobilinsky, 1991; Sun, Ma, & Hu, 2021; Zhang et al., 2018). In addition, various functional groups like hydroxyl groups, amine groups, carboxyl groups, etc., present on the structures of biological macromolecules can be exploited for chemical/functional modifications, which make the chemically modified/ functionalized biological macromolecules more competent biomaterials for the uses in biomedical applications (Thompson and Kovall, 2019; Alkahtani, Hasnain, Nayak, & Aminabhavi, 2021; de Marco, 2018). The advancements in the physiological understanding of various biological macromolecules and their utilizations in drug delivery carrier systems have shown some potential advantages over the synthetic-made carrier systems in terms of ease of production, safety, stability, etc. (Frandsen & Ghandehari, 2012; Kandar, Hasnain, & Nayak, 2021; Krishnaswami et al., 2018; Mu & Holm, 2018; Nayak & Hasnain, 2019; Pandey & Kohli, 2018; Varanko, Saha, & Chilkoti, 2020; Wiraja et al., 2019). The uses of biological macromolecules-based drug releasing carriers have already shown to enhance the pharmacokinetics of loaded drugs with reduced systemic toxicity and immunogenicity (Zhang et al., 2018).

Since past few decades, a variety of biological macromolecules-based drug releasing carriers have been reported in forms of various dosage forms like injectables, tablets, capsules, gels, hydrogels, beads, microparticles, nanoparticles, patches and films, fibers, liposomes, transferosomes, scaffolds, etc. (Frandsen & Ghandehari, 2012; Hasnain et al., 2020; Kandar et al., 2021; Krishnaswami et al., 2018; Mu & Holm, 2018; Nayak & Hasnain, 2019; Nayak, Hasnain, Pal, Banerjee, & Pal, 2020; Nayak, Mohanta, Hasnain, Hoda, & Tripathi, 2020; Pandey & Kohli, 2018; Varanko et al., 2020; Wiraja et al., 2019; Zhang et al., 2018). The biological macromolecules for drug delivery should be chosen on the basis of their stability, biodistribution, degradation, clearance and their ability to penetrate into the target tissues (Carvalho et al., 2009; Liu et al., 2018). In the current chapter, we focus on the advancements in the in the uses of various biological macromolecules in drug delivery applications.

15.2 Drug delivery using various biological macromolecules The development of carrier-systems for delivery of therapeutics and/or imaging agents is the important most area to treat numerous diseases (Din et al., 2017; Etrych, Janouˇskova´, & Chytil, 2019; Jeswani, Paul, & Jha, 2018). The biomaterials used for the development of carrier-systems should be biocompatible, biodegradable and nonimmunogenic in nature (Din et al., 2017; Idrees et al., 2020; Nayak, Ansari, Sami, Balvir Singh, & Hasnain, 2020; Song et al., 2018). The ideal carrier-systems should be safe, efficient and possess capability to achieve optimal bioavailability (Nayak, Ara, Hasnain, & Hoda, 2018; Pal & Nayak, 2017). Various biological macromolecules like carbohydrates (such as chitin, chitosan, alginates, gellan gum, pectins, starches, gellan gum, locust bean gum, sterculia gum, tamarind gum, hyaluronic acid, agar, xanthan gum, dextran,

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15.2 Drug delivery using various biological macromolecules

etc) (Hasnain & Nayak, 2018; Milivojevic, PajicLijakovic, Bugarski, Nayak, & Hasnain, 2019; Nayak & Pal, 2016; Nayak et al., 2018; Nayak, Hasnain, & Pal, 2018; Nayak, Ansari, Pal, & Hasnain, 2019; Nayak, Hasnain, Dilipkumar, & Dilipkumar, 2021; Nayak, 2016; Nayak, 2017), proteins (such as albumin, gelatin, collagen, keratin, fibroin, transferin, lipoproteins, etc) (Frandsen & Ghandehari, 2012; Hasnain et al., 2020; Varanko et al., 2020), nucleic acids (RNA and DNA) (Buddolla & Kim, 2018; Tan, Jia, Wang, & Zhang, 2020; Wiraja et al., 2019), and lipids (such as fatty acids, triglycerides, cholesterol, phospholipids, bile acids, etc.) (Chauhan et al., 2020; Corn et al., 2020; Mu & Holm, 2018; Pandey & Kohli, 2018), are being used to formulate different kinds of drug delivery carriers. The uses of various biological macromolecules in drug delivery are described below.

15.2.1 Drug delivery using carbohydrates Carbohydrates are naturally occurring biocompatible and biodegradable macromolecular biopolymers (Mosaiab, Farr, Kiefel, & Houston, 2019; Ranjbari et al., 2018; Shukla & Tiwari, 2011). These are occurred as important components in various cell organelles and membranes involving in different physiological activities. Polysaccharides are the key candidates of carbohydrates and their molecular structures compromise of linear or branched polymeric chain depending upon the nature of monosaccharideunits related to occurrence of varying types and numbers of functional groups, such as hydroxyl, carboxyl, amine, etc., (Kandar et al., 2021; Maity, Hasnain, Nayak, & Aminabavi, 2021; Nayak & Hasnain, 2019; Nayak, Hasnain, Tabis, & Aminabhavi, 2021). These functional groups occurred in the polysaccharide-structures can be targeted for chemical modifications via various chemical reactions (Nayak, Bera, Hasnain, & Pal, 2018; Xie et al., 2020). Due to their inherent biodegradability, high biocompatibility, hydrophilicity, stability, adhesivity, etc., polysaccharides are

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chosen as the best category of natural biopolymeric excipients to formulate many kinds of drug delivery dosage forms (Kandar et al., 2021; Nayak & Hasnain, 2019; Pal & Nayak, 2017; Sun et al., 2021). In addition, ionic nature polysaccharides, both negatively charged (anionic natured) like sodium alginate, low methoxy pectin, gellan gum, heparin, etc., and positively charged (cationic nature) like chitosan possess the intrinsic ability to recognize the specific cells with targeting moieties via the specific receptors (Desai, 2016; Hasnain et al., 2020; Nayak & Pal, 2016; Nayak et al., 2021; Pal & Nayak, 2017; Patil, Kamalapur, Marapur, & Kadam, 2010; Racovita, Vasilu, Popa, & Luca, 2009). During past few decades, many naturally occurring polysaccharides are being widely used as carbohydrate excipients in the formulations of many kinds of drug delivery dosage forms, such as suspensions, emulsions, injectables, tablets, capsules, gels, hydrogels, beads, microparticles, nanoparticles, patches and films, fibers, scaffolds, etc (Hasnain & Nayak, 2018; Hasnain et al., 2020; Idrees et al., 2020; Kandar et al., 2021; Milivojevic et al., 2019; Nayak & Hasnain, 2019; Nayak & Pal, 2016; Nayak et al., 2018; Nayak et al., 2018; Nayak et al., 2019; Nayak et al., 2020; Nayak et al., 2020; Nayak et al., 2021; Nayak, 2016; Pal & Nayak, 2017; Sun et al., 2021). In a research, paracetamol suspensions were formulated employing sweet basil (Ocimum basilicum L.) seed mucilage as suspending agent (Avlani, Agarwal, Khattry, Biswas, & Majee, 2019). These paracetamol suspensions prepared using 1% w/v sweet basil seed mucilage demonstrated a flocculated action with improved stability. Starches from yam (Dioscorea sp.) and arrowroot (Maranta arundinacea) were used as suspending agents to formulate paracetamol suspensions (Piriyaprasarth et al., 2010). Formulation of sulphamethoxazole suspensions were reported, where moringa (Moringa oleifera) exudate gum was employed as a suspending agent (Rao, Prasad, Srinivas, & Rao, 2005). In two separate works, zinc oxide (20% w/v)

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suspensions were prepared using spinach (Spinacia oleracea L.) leaves mucilage and fenugreek (Trigonella foenum-graecum L.) seed mucilage as suspending agents (Nayak, Pal, Pany, & Mohanty, 2010; Nayak, Pal, Pradhan, & Ghorai, 2012). Suspensions prepared using both the suspending agent (spinach leaves mucilage and fenugreek seed mucilage) demonstrated enhanced degree of flocculation as well as suspension redispersibilty than those of bentonite, gum acacia and gum tragacanth. Using gum odina as an emulsion stabilizer, water/oil/water (w/o/w) multiple emulsions of lamivudine were formulated and these showed a sustained pattern of lamivudine releasing over a period of 6 h (Jena, Nayak, De, Mitra, & Samanta, 2018). The emulsion stabilizing capacity of gum odina was revealed to be comparable to that of Tween 80 in the preparation of w/o/w multiple emulsions. In a research, sweet basil (O. basilicum L.) seed mucilage (0.3%
0.5% w/v) was used to prepare stable sunflower oil emulsions (Avlani et al., 2019). The matrix tablets of metformin HCl and diclofenac sodium were formulated, where different hydrophilic biopolymers, namely xanthan gum, cashew gum, and hydroxypropyl methylcellulose (HPMC) were employed as drug releasing retardant agents by direct compression technology (Ofori-Kwakye, Mfoafo, Kipo, Kuntworbe, & Boakye-Gyasi, 2016). Albizia procera gum was employed as drug releasing retardant agent to prepare controlled drug releasing matrix tablets of paracetamol and these formulated A. procera gum-based matrix tablets exhibited a sustained paracetamol releasing profile over 12 h (Pachuau & Mazumdar, 2012). In another research, Hibiscus rosa-sinensis leaves mucilage was used as drug releasing retardant agent as well as matrixforming agent to prepare sustained releasing matrix tablets of ketoprofen (Kaleemullah et al., 2017). These H. rosa-sinensis leaves mucilage-based matrix tablets showed a sustained ketoprofen releasing profile up to 24 h.

In a work, starches extracted from various Dioscorea species were evaluated for their tablet binding properties to formulate chloroquine phosphate tablets (Okunlola & Odeku, 2011). A faster disintegration result was reported for the starches of Dioscorea dumetorum and Dioscorea oppositifolia; whereas starches of D. dumetorum and D. oppositifolia were reported for defects like tablet capping and tablet lamination in these starch-based tablets of chloroquine phosphate. In an investigation, gum odina was used to prepare controlled drug releasing matrix tablets of tolterodine tartarate and pioglitazone HCl, which exhibited controlled drug releasing pattern over a prolonged period (Sinha, AlAzaki, & Kumar, 2011). Acetaminophen releasing matrix tablets made of chitosan particles cross-linked with tripolyphosphate (ionic cross-linker) were developed and characterized (Pinto, Saripella, Loka, & Neau, 2018). The optimized matrix tablets of acetaminophen displayed a sustained profile of acetaminophen releasing over a longer period with the facility of avoiding the burst release of acetaminophen. Different hydrophilic biopolymeric-matrix tablets of theophylline were formulated using various chitosan derivatives, namely chitosan aspartate, chitosan lactate, chitosan glycolate, and chitosan glutamate (Huanbutta, Cheewatanakornkool, Terada, Nunthanid, & Sriamornsak, 2013). The chitosan glycolatebased matrix tablets of theophylline presented the utmost swelling in both neutral and acidic media in comparison with that of other chitosan derivatives (chitosan aspartate, chitosan lactate, and chitosan glutamate) and also, showed sustained theophylline releasing in the acidic media, in vitro, on account of thick swollen hydrophilic gel and lower matrix erosion rate. Hydralazine HCl floating matrix tablets made of cashew gum-HPMC was formulated in an investigation (Hasnain, Rishishwar, & Ali, 2017b). These floating tablets demonstrated sustained in vitro hydralazine releasing

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15.2 Drug delivery using various biological macromolecules

along with satisfactory in vitro buoyancy and ex vivo biomucoadhesivity in gastric pH. Gastroretentive swellable and mucoadhesive matrix-based tablets of milnacipran HCl were developed by Hussain et al. (2020) by modified solvent-based wet granulation using medium and low molecular weight chitosans with polycaprolactone (Hussain et al., 2020). In vitro biomucoadhesivity was evaluated for these milnacipran HCl gastroretentive matrix-based tablets and the results showed better biomucoadhesion period (12 h) onto the rabbit gastrointestinal mucosa. The optimized formulation exhibited in vivo release of milnacipran HCl in dogs with augmented elimination half-life of milnacipran HCl indicating the sustained release of milnacipran HCl from these gastroretentive matrix-based tablets of milnacipran HCl. Floating hydrodynamically balanced systembased hard gelatin capsules of moxifloxacin HCl were formulated using medium or low molecular weight chitosans-HPMC (K4M or K15M) or in combinations (Verma, Dubey, Verma, & Nayak, 2017). The formulated moxifloxacin HCl capsules contained medium or low molecular weight chitosans and their combinations revealed the failure to float on the medium of acidic pH (1.2). In contrast, formulated moxifloxacin HCl capsules contained HPMC (K4M or K15M) or in combinations with medium or low molecular weight chitosans exhibited excellent floatation in acidic pH (1.2) and these hydrodynamically balanced system-based hard gelatin capsules of moxifloxacin showed sustained release pattern of moxifloxacin HCl over a period of 9 h in acidic pH (1.2), in vitro. In a research, the retardation of theophylline releasing from hard gelatin capsules containing sodium alginate, HPMC and sodium carboxymethyl cellulose was investigated (Malakar & Nayak, 2012). The in vitro theophylline releasing from these capsules was found pH dependent. The calcium ions cross-linked sodium alginate and sodium carboxymethyl cellulose to form

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gelled polymeric matrices within the capsules, which showed prolonged patterns of theophylline releasing from these capsules. Aceclofeanc-releasing ispaghula husk mucilagezinc pectinate beads were formulated by ionic gelation technique using zinc sulfate as ionic cross-linker (Guru, Bera, Das, Hasnain, & Nayak, 2018). These biopolysaccharide-based beads exhibited a controlled aceclofenac releasing over a period of 10 h with a pH-responsive swelling pattern. Tamarind seed polysaccharide-pectinate mucoadhesive beads (Nayak, Pal, & Santra, 2014d), ispagula husk mucilage-pectinate mucoadhesive beads (Nayak, Pal, & Santra, 2014a), fenugreek seed mucilage-pectinate mucoadhesive beads (Nayak, Pal, & Das, 2013) and jackfruit seed starch-pectinate mucoadhesive beads (Nayak & Pal, 2013b) containing metformin HCl were developed. All these beads exhibited controlled sustained patterns of drug releasing, in vitro. These ionically-gelled biopolysaccharide-based beads also showed excellent mucoadhesion, ex vivo and antidiabetic activity in diabetic rats, in vivo. In another research, similar kinds of okra gumalginate mucoadhesive beads were developed and evaluated (both in vitro and in vivo) for oral delivery of glibenclamide (Sinha, Ubaidulla, & Nayak, 2015). These glibenclamide-loaded okra gumalginate mucoadhesive beads exhibited controlled aceclofenac releasing over a period 8 h, in vitro and excellent mucoadhesion onto goat intestinal mucosa, ex vivo. Metformin HCl-loaded tamarind seed polysaccharide-alginate beads were developed by ionic gelation technique using calcium chloride as ionic cross-linker (Nayak & Pal, 2013a; Nayak, Pal, & Santra, 2016). All these tamarind seed polysaccharide-alginate beads exhibited controlled patterns of metformin HCl releasing over a period of 10 h, in vitro. These mucoadhesive beads exhibited excellent mucoadhesion onto goat intestinal mucosa, ex vivo and significant antidiabetic activity in alloxan-indiced diabetic rats over a prolonged period, in vivo. The same group also developed the formulation of metformin HClloaded tamarind seed polysaccharide-gellan gum

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mucoadhesive beads for controlled drug releasing (Nayak, Pal, & Santra, 2014e). These ionically gelled beads demonstrated a controlled pattern of metformin HCl releasing, in vitro with excellent mucoadhesion, ex vivo. Similar kinds of controlled metformin HCl releasing mucoadhesive beads were developed using biopolysaccharidic blends of fenugreek seed mucilage-gellan gum (Nayak & Pal, 2014), ispaghula husk mucilage-gellan gum (Nayak, Pal, & Santra, 2014b) and jackfruit seed starch-gellan gum (Nayak, Pal, & Santra, 2014c). In an investigation, potato starch-sodium alginate blends were used to prepare potato starch-based beads containing tolbutamide, which showed a controlled drug releasing pattern, in vitro (Malakar, Nayak, Jana, & Pal, 2013). Diclofenac sodium-loaded biomucoadhesive beads of sodium alginate and Linum usitatisimum mucilage were formulated by ionic gelation technique (Hasnain, Rishishwar, Rishishwar, Ali, & Nayak, 2018b). In these L. usitatisimum mucilage-alginate biomucoadhesive beads, L. usitatisimum mucilage was used as matrix forming agent, release retarding agent and biomucoadhesive agent. These beads were of spherical-shaped with irregular surface morphology (Fig. 15.1). A controlled pattern of

drug releasing, in vitro (Fig. 15.2) and excellent mucoadhesion onto goat intestinal mucosa, ex vivo, was demonstrated by these L. usitatisimum mucilage-alginate biomucoadhesive beads of diclofenac sodium. In an investigation, diclofenac sodium-loaded groundnut oil-entrapped tamarind seed polysaccharide-alginate buoyant beads were prepared by emulsion-gelled based ionic gelation (Nayak, Pal, & Malakar, 2013). These beads exhibited low density with excellent floating and in vitro sustained releasing of loaded drug. Aceclofenac-loaded mineral oil-entrapped sterculia gum-alginate buoyant beads were developed and these buoyant beads displayed outstanding floating behavior with in vitro sustained releasing of aceclofenac in gastric pH (1.2) (Guru, Nayak, & Sahu, 2013). In a work, oil-entrapped alginate-HPMC based floating beads of salbutamol sulfate were formulated and these oilentrapped floating beads of salbutamol sulfate were then capsulated in the hard gelatine capsules, which displayed a prolonged sustained release of salbutamol sulfate in gastric pH, in vitro, because of their low density (Malakar, Dutta, Purokayastha, Dey, & Nayak, 2014). The FIGURE 15.1 Scanning electron micrographs of Linum usitatisimum mucilage-alginate biomucoadhesive beads of diclofenac sodium. Source: With permission from Hasnain M.S., Rishishwar P., Rishishwar S., Ali S., Nayak A.K. (2018b). Isolation and characterization of Linum usitatisimum polysaccharide to prepare mucoadhesive beads of diclofenac sodium. International Journal of Biological Macromolecules;116:162-172. Copyright r 2018 Elsevier B.V.

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FIGURE 15.2 In vitro diclofenac sodium releasing from Linum usitatisimum mucilage-alginate biomucoadhesive beads of diclofenac sodium. Source: With permission from Hasnain M.S., Rishishwar P., Rishishwar S., Ali S., Nayak A.K. (2018b). Isolation and characterization of Linum usitatisimum polysaccharide to prepare mucoadhesive beads of diclofenac sodium. International Journal of Biological Macromolecules;116:162-172. Copyright r 2018 Elsevier B.V.

in vivo X-ray imaging study results demonstrated good gastroretention within the gastrointestinal region of rabbits. In a work, aceclofenac-loaded tamarind seed polysaccharide-chitosan interpenetrating polymeric network (IPN) microparticles were prepared and evaluated for sustained drug releasing potential (Jana, Saha, Nayak, Sen, & Basu, 2013). These tamarind seed polysaccharide-chitosan microparticles displayed sustained pattern of drug releasing over a period of 8 h, in vitro, and a significant antiinflammatory action over a longer period in the carrageenan-induced rats, in vivo (after oral intake). In another work, isoxsuprine HCl-loaded carboxymethyl cashew gum-alginate microbeads were formulated by ionic gelation technique using zinc sulfate as ionic cross-linker (Das, Dutta, Nayak, & Nanda, 2014). The scanning electron micrographs of these isoxsuprine HCl-loaded carboxymethyl cashew gum-alginate microbeads demonstrated that these microbeads were found as spherical-shaped with a rough surface morphology (Fig. 15.3). In the study of in vitro drug releasing, it was found that these biopolysaccharidic microbeads were capable of releasing loaded drug in a sustained release manner over a longer period (Fig. 15.4).

Gum Arabic-chitosan based nanoparticles of curcumin were formulated by polyelectrolyte complexation, which presented an in vitro sustained curcumin releasing profile in the simulated gastrointestinal environment (Tan, Xie, Zhang, Cai, & Xia, 2016). In a research, methotrexateloaded de-esterified gum tragacanth-chitosan nanoparticles were formulated, which were reported for sustained methotrexate releasing for 9 days and found to be endocytosed via the asialoglycoprotein receptors (Sadrjavadi, Shahbazi, & Fattahi, 2018). Itraconazole-loaded pectin-based nanoparticles were prepared via the nanoemulsion templates, which were reported to be formed by a high-pressure homogenization procedure utilizing high methoxyl, low methoxyl, and amidated low methoxyl pectins (Burapapadh, Takeuchi, & Sriamornsak, 2012). These itraconazole-loaded pectin-based nanoparticles were reported to be produced satisfactory in vivo absorption of loaded itraconazole. In a research, lipase functionalized guar gum-based nanoparticles were synthesized by nanoprecipitation and cross-linking procedures (Soumya, Ghosh, & Abraham, 2010). The synthesized nanoparticles were loaded with an antihypertensive drug and the results of in vitro drug releasing revealed that

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FIGURE 15.3 Scanning electron micrographs of the surface of optimized isoxsuprine HCl-loaded carboxymethyl cashew gum-alginate microbeads at different magnifications (75 3 , 200 3 ,1500 3 , 2000 3 , 4000 3 and 10,000 3 ). Source: With permission from Das B., Dutta S., Nayak A.K., Nanda U. (2014). Zinc alginate-carboxymethyl cashew gum microbeads for prolonged drug release: Development and optimization. International Journal of Biological Macromolecules;70:505-515. Copyright r 2014 Elsevier B.V.

15.2 Drug delivery using various biological macromolecules

347 FIGURE 15.4 In vitro isoxsuprine release from carboxymethyl cashew gum-alginate microbeads of isoxsuprine HCl. Source: With permission from Das B., Dutta S., Nayak A.K., Nanda U. (2014). Zinc alginate-carboxymethyl cashew gum microbeads for prolonged drug release: Development and optimization. International Journal of Biological Macromolecules;70:505515. Copyright r 2014 Elsevier B.V.

the drug releasing rate and amount were found to be enhanced up to 24 h and afterward, decreased. Indomethacin-loaded acetylated cashew gum-based nanoparticles were prepared employing self-assembled method by dialysis (Pitombeira et al., 2015). The scanning electron micrographs of these nanoparticles revealed spherical shaped with an average size of 179 nm. In vitro indomethacin releasing results clearly presented an initial burst releasing of indomethacin from these acetylated cashew gum-based nanoparticles within the initial period of 2 h and subsequently, a controlled indomethacin releasing pattern was noticed up to 72 h. In a recent research, Dubey, Mohan, Dangi, and Kesavan (2020) developed brinzolamide-loaded chitosanpectin mucoadhesive nanoparticles for ocular use were formulated by polyelectrolyte complex coacervation methodology for ocular administration to treat glaucoma (Dubey et al., 2020). These brinzolamide-loaded mucoadhesive nanoparticles displayed a sustained release pattern of brinzolamide over a period of 8 h, in vitro, along with high corneal permeation rate of brinzolamide, ex vivo. In another study, surfactant (polysorbate 80)-coated ropinirole HCl-loaded chitosan nanoparticles were synthesized by conventional

emulsification cross-linking methodology using glutaraldehyde as a covalent cross-linker (Ray et al., 2018). The scanning electron micrographs of uncoated ropinirole HCl-loaded chitosan nanoparticles and polysorbate 80-coated ropinirole HCl-loaded chitosan nanoparticles are shown in Fig. 15.5, where both the nanoparticles were of irregular shaped. The in vitro ropinirole HCl release from these uncoated and coated chitosan nanoparticles displayed an initial burst releasing of loaded ropinirole HCl followed by a sustained ropinirole HCl release pattern over a period of 10 h (Fig. 15.6). In Wistar rats, in vivo ropinirole HCl biodistribution results exhibited an augmented ropinirole HCl concentration in the brain with lesser ropinirole HCl concentrations in various vital organs like liver, kidney, and spleen even after 1 h of intravenous administration for polysorbate 80-coated ropinirole HCl-loaded chitosan nanoparticles as compared to its corresponding uncoated nanoparticles, suggesting for prospective use surfactant-coated chitosan nanoparticles in the treatment of Parkinson’s disease. In a research, Cocculas hirsutus leaf mucilage was used to formulate topical gels of flurbiprofen and these showed improved in vivo antiinflammatory action than that of the marketed

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FIGURE 15.5 Scanning electron micrographs of uncoated ropinirole HCl-loaded chitosan nanoparticles and polysorbate 80-coated ropinirole HCl-loaded chitosan nanoparticles. Source: With permission from Ray S., Sinha P., Laha B., Maiti S., Bhattacharyya U.K., Nayak A.K. (2018). Polysorbate 80 coated crosslinked chitosan nanoparticles of ropinirole hydrochloride for brain targeting. Journal of Drug Delivery Science and Technology;48:21-29. Copyright r 2018 Elsevier B.V.

FIGURE 15.6 In vitro ropinirole HCl release from uncoated ropinirole HCl-loaded chitosan nanoparticles and polysorbate 80-coated ropinirole HCl-loaded chitosan nanoparticles [mean 6 S.D., n 5 3]. Source: With permission from Ray S., Sinha P., Laha B., Maiti S., Bhattacharyya U.K., Nayak A.K. (2018). Polysorbate 80 coated crosslinked chitosan nanoparticles of ropinirole hydrochloride for brain targeting. Journal of Drug Delivery Science and Technology;48:21-29. Copyright r 2018 Elsevier B.V.

flurbiprofen gel (Rao, Gnanaprakash, Badrinath, Chetty, & Alagusundaram, 2010). Dellinia (Dillenia indica L.) fruit gum was used to formulate topical gels of 4% lidocaine HCl

using Carbopol 940, propylene glycol and methyl paraben as gel-former, plasticizer and preservative, respectively with and without incorporation of 1% menthol as permeation

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15.2 Drug delivery using various biological macromolecules

enhancer (Hasnain et al., 2020). These 4% lidocaine HCl containing dellinia fruit gum-based topical gels demonstrated a sustained ex vivo lidocaine permeation pattern over 7 h when the porcine ear skin was used. The utmost ex vivo lidocaine permeation flux was calculated for the 4% lidocaine HCl topical gels contained 1% menthol. In another research work, the same research group developed 4% lidocaine HCl gels using cashew gum and HPMC (K4M) for topical uses (Hasnain, Rishishwar, & Ali, 2017a). These cashew gum-based topical gels exhibited good ex vivo lidocaine permeation across the skin. Another one similar kind of cashew gumbased topical gels of 4% lidocaine HCl were formulated, where Carbopol 940 was employed as blend with cashew gum (Das, Nayak, & Nanda, 2013). These topical gels exhibited a sustained pattern of ex vivo lidocaine permeation across the porcine skin over a period of 7 h. Topical gels of diclofenac potassium were prepared using fenugreek seed mucilage as gel-forming excipient and presented good results, in vitro (Mundhe, Pagore, & Biyani, 2012). In an investigation, hydrotrope potato starch-based topical gels of rofecoxib (1%) were formulated (Nazim, Dehghan, Shaikh, & Shaikh, 2011). The topical gels of rofecoxib (1%) exhibited a sustained pattern of in vitro rofecoxib releasing within a period of 6 h. In a research, amidated pectin-based patches were developed for transdermal delivery of insulin (Hadebe, Ngubane, Serumula, & Musabayane, 2014). These transdermal patches of insulin showed a sustained release of insulin over a prolonged period with satisfactory diabetic parameters in streptozotocin-induced diabetic rats. Biodegradable chitosan patches for transdermal glibenclamide delivery were formulated using nanocrystals of glibenclamide by casting procedures (Ali & Hanafy, 2017). The permeation from these transdermal patches exhibited good glibenclamide permeation. The results of in vivo study clearly exhibited that the better effectiveness of transdermal patches made of chitosan contained nanocrystals of glibenclamide to lower the blood glucose levels.

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Buccal patches were formulated using an atenolol-contained biomucoadhesive layer of dellinia fruit gum-HPMC and 1% ethylcellulose made backing layer without atenolol employing solvent-casting procedure enhancer (Hasnain et al., 2020). These dellinia fruit gumbased buccal patches containing atenolol demonstrated a sustained ex vivo atenolol permeation pattern over 12 h and satisfactory results of ex vivo buccoadhesion when the porcine buccal mucosal membrane was used. In a research, buccoadhesive buccal films were formulated using tamarind seed glucan and Carbopol 934P for buccal delivery of rizatriptan benzoate (Avachat, Gujar, & Wagh, 2013). The permeation of rizatriptan benzoate from tamarind seed glucan-Carbopol 934P made buccal films across the porcine buccal mucosa demonstrated satisfactory results of flux over a longer period. Aceclofenac (1%) containing dental pastes were prepared using cashe gum and proposed for periodontitis treatment (Hasnain, Rishishwar, Rishishwar, Ali, & Nayak, 2018a). These cashew gum-based dental pastes containing aceclofenac demonstrated a sustained pattern of aceclofenac releasing over 6 h, in vitro, and satisfactory ex vivo oral mucosal biomucoadhesion. In another study, 1% aceclofenac containing dental pastes were prepared using dellinia fruit gum and proposed for periodontitis treatment (Hasnain, Rishishwar, Ali, & Nayak, 2020). The formulation of dental pastes was carried out by conventional trituration and all these dental pastes containing aceclofenac exhibited a sustained pattern of aceclofenac releasing over 6 h, in vitro. Some recently developed drug delivery systems using carbohydrates are presented in Table 15.1.

15.2.2 Drug delivery using proteins and peptides Proteins are a promising class of biomaterial, which are the basic structural components

III. Functional Applications

TABLE 15.1 Some recently developed drug delivery systems using carbohydrates. Drug delivery systems using carbohydrates

Drug loaded for delivery Reference

Alginate-hydroxyapatite-polyvinyl pyrrolidone nanobioceramic composite beads

Diclofenac sodium

Hasnain et al. (2016)

Corn fiber gum-based gastro-retentive floating tablets

Cinnarizine

Mangla, Jain, and Malik (2020)

Sustained-release matrix tablets made of sucrose esters and oxidized konjac glucomannan-cassava starch

Bovine serum albumin

Liu, Li, Li, Xie, and Liu (2020)

Floating matrix tablets made of Brachystegia eurycoma gum

Metronidazole

Ovenseri, Clifford, and Uwumagbe (2018)

Delayed release tablets made of native Chapparada avare starch

Diclofenac sodium

Singh and Kumar (2020)

Cellulose-coated chitosan hydrogel beads

Verapamil HCl

Song, Pham, and Yun (2020)

pH sensitive gellan gum-based hydrogel beads

Quercetin

Dey, Ghosh, and Giri (2020)

Grafted Azadirachta indica gum-based hydrogel

Methyl prednisolone

Singh and Singh (2020)

Carboxymethylated Cassia obtusifolia galactomannanbased beads

Diclofenac

Verma, Rimpy, and Ahuja (2020)

Oxidized gellan gum/resistant starch hydrogel beads

Resveratrol

Wang, Luo, and Xiao (2021)

Carboxymethyl cellulose/Starch/zinc oxide nanocomposite hydrogel beads

Doxorubicin

Gholamali and Yadollahi, (2020)

Microparticles made of xylan (extracted from corn cobs)

Mesalamine

Urtiga SCDC et al. (2020)

Galactomannan microparticles

Fluoxetine

Josino et al. (2021)

Starch nanocapsules

Fluoxetine

Dos Santos et al. (2020)

Carboxymethyl β-cyclodextrin grafted carboxymethyl chitosan hydrogels-based microparticles

Insulin

Yang, Liu, Chen, Cheong, and Teng (2020)

In situ forming β-cyclodextrin gel and microparticle for periodontal uses

Meloxicam

Rein, Lwin, Tuntarawongsa, and Phaechamud, (2020)

Modified chitosan microparticles

Fluticasone propionate and salmeterol xinafoate

Ainali, Xanthopoulou, Michailidou, Zamboulis, and Bikiaris (2020)

Colon-specific chitosan nanoparticles into retrograded starch/pectin microparticles

5-fluorouracil

Dos Santos et al. (2021)

Coacervate thermoresponsive nanoparticles made of curdlan and hydroxypropyl cellulose

Piroxicam

Lachowicz et al. (2020)

Nanohydrogels made of cationic pullulan and anionic dextran derivatives

Piroxicam

Lachowicz et al. (2019)

Phthalated cashew gum for drug delivery systems

Benznidazole

Oliveira et al. (2021)

Nanoparticles loaded gellan gum transdermal film

Rasagiline mesylate

Bali and Salve (2020)

Poly(ε-caprolactone) grafted cashew gum nanoparticles

Epirubicin

Ribeiro et al. (2021)

Enteric coated guar gum nanoparticles

Amphotericin B and piperine

Ray et al. (2021)

15.2 Drug delivery using various biological macromolecules

of cell organelles involving in various physiological activities (Ekladious, Colson, & Grinstaff, 2019; Stevens, Kaur, & Klok, 2021). Naturally proteins are occurred in plants, animals, microbes, etc (Nayak, 2010). A number of biologically occurring proteins and peptides are already being explored and exploited for their therapeutic uses to treat many diseases (Frandsen & Ghandehari, 2012; Stevens et al., 2021; Varanko et al., 2020). Protein-based biopolymers consist of repetitive natural or engineered amino acid sequences. Recent years, proteins and peptides are also being used as biomaterials because of their favorable biochemical as well as biophysical characteristics over the synthetic materials like ease of extraction from the biological tissues and via genetic engineering, their purification, biocompatibility, biodegradability, ease of scaleup and processing (Chambre, Martı´n-Moldes, Parker, & Kaplan, 2020; Stevens et al., 2021; Varanko et al., 2020). Since past few decades, various proteins and peptides are also being employed as biomolecular excipients in the formulations of various kinds of drug delivery platforms. Recent years, an increasing interest as well as implementation of recombinant protein-based biopolymers like silk-like protein, silk-elastinlike protein, elastin-like protein polymers, etc., in the formulations of drug delivery systems has been noticed (Chambre et al., 2020; Varanko et al., 2020). The production of recombinant protein-based biopolymers with the advent of recombinant DNA technology has made feasible to design polypeptides de novo to be used as effective biomolecular excipients in the designing of various environmental stimuli-responsive drug delivery systems (Frandsen & Ghandehari, 2012; Price, Poursaid, & Ghandehari, 2014). Casein-coated diltiazem HCl tablets were developed and these tablets showed 80% of diltiazem HCl releasing at pH 1.2 within 2 h (Abu Diak, Bani-Jaber, Amro, Jones, & Andrews, 2007). The casein-coated diltiazem

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HCl tablets underwent the postcoating heat treatment at 100 C exhibited a sustained diltiazem HCl releasing pattern at pH 6.8. In a research, freeze-dried compacts of chlorothiazide and hydrochlorothiazide were prepared using sodium caseinate (Millar & Corrigan, 1991). These prepared compacts showed the increasing patterns of hydrochlorothiazide and chlorothiazide dissolution rates of 1.fivefold and 35-fold, respectively. The same research group further evaluated the dissolution rate of ibuprofen from these types of compacts and they reported that the ibuprofen dissolution rate of acidic casein-made freeze-dried compacts was half than the sodium caseinatemade freeze-dried compacts. Compressed buccal tablets of propranolol HCl were formulated using gelatin-chitosan microparticles prepared by spray drying technique (Abruzzo, Cerchiara, Bigucci, Gallucci, & Luppi, 2015). These gelatin-based tablets exhibited good mucoadhesion and found effective buccoadhesive drug delivery. A collagen matrix-based implant containing mitomycin-C was developed and evaluated its effectiveness in trabeculectomy followed by postoperation for 5 years (Cillino et al., 2016). The results demonstrated the efficacy of collagen matrix-based implant containing mitomycin-C for a long term use than that of mitomycin-C. Cross-linked collagen nanoparticles-based shields were formulated for ocular delivery of pilocarpine HCl to treat glaucoma (Agban, Lian, Prabakar, Seyfoddin, & Rupenthal, 2016). These collagen nanoparticles-based shields showed a sustained release of pilocarpine HCl and present highly efficacious than that of the normal eye drops. In a research, albumin nanoparticles loaded with an antiviral drug-acyclovir were formulated (Suwannoi, Chomnawang, Sarisuta, Reichl, & Mu¨ller-Goymann, 2017). These acyclovir-loaded albumin nanoparticles showed effective ocular permeation across the human corneal epithelial cells.

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15. Biological macromolecules in drug delivery

In a work, zein-sucrose acetate isobutyrate hydrogel was formulated to shrink the burst releasing pingyangmycin HCl (Gao et al., 2007). The formulated zein-based hydrogel exhibited a prolonged drug release pattern for a period of 4 days. Doxorubicin-loaded in situ zein-based hydrogel was formulated for intratumoral injection (Cao et al., 2012). The formulated injectable zein-based hydrogel exhibited an extended release profile of loaded doxorubicin, which resulted in improved antitumor effectiveness of the doxorubicin-loaded hydrogel and the doxorubicin was found to be influenced by the zein concentration in the hydrogel formula. Keratin-based hydrogels were prepared in a research, where the disulfide bond impacted on the drug releasing from these hydrogels (Cao et al., 2019). This approach also enhanced the mechanical behavior of these keratin-based hydrogels, and decreased gelation period, even using a lesser amount of keratin for gelation. Doxorubicin and ciprofloxacin releasing were found to be inversely proportional to the cysteine levels in these keratin-based hydrogels as demonstrated zero-order kinetics for drug releasing in phosphate buffer saline. In another research, elastin-like polypeptide hydrogel scaffolds were prepared for localized delivery of doxycycline (Amruthwar & Janorkar, 2012). A higher releasing pattern for doxycycline was noticed at 37 C temperature as compared to that of 25 C, which might be due to the alteration in porosity of the elastin-like polypeptide hydrogel scaffolds. Gelatin-based microspheres loaded with antitubercular drugs-isoniazid and rifampicin were developed employing a covalent crosslinking procedure and these gelatin-based microspheres proved the effectiveness as promising platform for pulmonary drug delivery (Manca et al., 2013). Zein microspheres of ciprofloxacin were developed for antibiotic therapeutics (Fu, Wang, Zhou, & Wang, 2009). These ciprofloxacin-loaded microspheres made

of zein as protein biomaterial exhibited antibacterial activity. In another work, prednisolone (one of the corticosteroids) was loaded within zein microparticles and this formulation was evaluated for predisolone delivery via oral route (Lau, Johnson, Mikkelsen, Halley, & Steadman, 2012). Ivermectin (a parasitic agent) was also loaded in corn protein zein-based microspheres (Liu, Sun, Wang, Zhang, & Wang, 2005). Bovine serum albumin nanoparticles loaded with salicyclic acid were formulated and these salicyclic acid-loaded nanoparticles presented a biphasic rapid releasing as well as pHsensitive releasing of salicyclic acid, in vitro (Bronze-Uhle, Costa, Ximenes, & Lisboa-Filho, 2017). In another research, human serum albumin nanoparticles were developed for the loading of curcumin and found the enhancement of curcumin solubility (Gong et al., 2015). These human serum albumin nanoparticles containing curcumin provided the site-specific localization of the loaded curcumin in tumor cellcytoplasm. Albumin nanoparticles were also prepared to encapsulate cabazitaxel and the developed nanoformulation exhibited improved uptake in the tumors to extend systemic circulation in the nude mice xenograft model of prostate cancer (Qu et al., 2016). In an investigation, gemcitabine-conjugated albumin nanoparticles were developed for the use in pancreatic cancer therapeutics (Kushwah et al., 2017). The developed albumin nanoparticles exhibited the capacity to induce the significant DNA damaging as well as apoptosis than that of free gemcitabine. In another work, gemcitabine-loaded gelatin nanoparticles were developed for lung cancer-targeted therapeutics (Youngren-Ortiz et al., 2017). The obtained results of in vivo study demonstrated a sustained drug-plasma level with enhanced mean residence time. In addition, the accumulation of gemcitabine-loaded gelatin nanoparticles was estimated in augmented level at the lungs, while a reduced in vivo biodistribution of

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15.2 Drug delivery using various biological macromolecules

gemcitabine was estimated in other organs. Moxifloxacin-loaded gelatin nanoparticles were formulated by a modified two step desolvation technique using type A gelatin for ocular delivery (Mahor et al., 2016). The shape and morphology of optimized moxifloxacin-loaded gelatin nanoparticles were visualized by scanning electron micrographs and tansmission electron micrographs (Fig. 15.7A and B,

353

respectively). The results of in vitro moxifloxacin releasing from these gelatin nanoparticles displayed an initial burst releasing profile, followed by a controlled releasing profile for the subsequent 12 h (Fig. 15.8). These moxifloxacinloaded gelatin nanoparticles exhibited antibacterial efficacy. In an investigation, 5-fluorouracil-loaded zein nanoparticles were prepared for liver

FIGURE 15.7 (A) Scanning electron micrographs and (B) transmission electron micrographs of optimized moxifloxacin-loaded gelatin nanoparticles. Source: With permission from Mahor A., Prajapati S.K., Verma A., Gupta R., Iyer A. K., Kesharwani P. Moxifloxacin loaded gelatin nanoparticles for ocular delivery: Formulation and in-vitro, in-vivo evaluation. Journal of Colloid and Interface Science. 2016;483:132-138. Copyright r 2018 Elsevier B.V. FIGURE 15.8 In vitro moxifloxacin releasing from moxifloxacin-loaded gelatin nanoparticles. Source: With permission from Mahor A., Prajapati S.K., Verma A., Gupta R., Iyer A.K., Kesharwani P. Moxifloxacin loaded gelatin nanoparticles for ocular delivery: Formulation and in-vitro, in-vivo evaluation. Journal of Colloid and Interface Science. 2016;483:132-138. Copyright r 2018 Elsevier B.V.

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delivery, which were labeled with rhodamineB and intravenously administered (Lai & Guo, 2011). These 5-fluorouracil-loaded zein nanoparticles were reported to be augmented the liver-targeting efficiency of 31% as compared to rhodamine B and 5-fluorouracil uptaking in the liver. Doxorubicin-loaded zein nanoparticles were formulated, which were reported to achieve high doxorubicin loading efficiency (90%) (Dong et al., 2016). The in vitro releasing of doxorubicin from these zein nanoparticles was found pH-responsive and a more rapid releasing of doxorubicin was noticed in the acidic pH. In another research, lovastatinloaded zein nanoparticles were prepared, which exhibited the antiproliferative action in HepG2 cells (Alhakamy et al., 2019). Carbazole-loaded gliadin nanoparticles were formulated for oral delivery and improved absorption of carbazole via the stomach mucosa was observed (Arangoa, Campanero, Renedo, Ponchel, & Irache, 2001). Cyclophosphamide was also loaded within gliadin nanoparticles and the developed formulation showed a remarkable enhancement of drug loading efficiency (72%) (Gulfam et al., 2012). In another research, clarithromycin was loaded within gliadin nanoparticles via a desolvation procedure and these clarithromycin-gliadin nanoparticles demonstrated a sustained pattern of clarithromycin releasing at acidic pH, suggesting more effectual in eradicating Helicobacter pylori, in vitro, than free clarithromycin (Ramteke & Jain, 2008). In another work, amoxicillin-loaded gliadin nanoparticles were formulated to eradicate H. pylori by increasing the gastrointestinal residence period of amoxicillin (Umamaheshwari, Ramteke, & Jain, 2004). The results of the work demonstrated that a lower dose can be effectual to eradicate H. pylori infection in comparison to that of free amoxicillin in a Mongolian gerbil model. Soy protein isolate nanoparticles of curcumin were designed via a desolvation procedure and the developed formulation showed a remarkable enhancement of drug loading efficiency (97%) (Teng, Luo, & Wang, 2012). A

biphasic trend of curcumin releasing was revealed from these curcumin-loaded soy protein isolate nanoparticles. Mitoxantrone-loaded β-casein nanoparticles were formulated, which were reported to be clustered to entrap mitoxantrone in-between micelles and within their cores (Shapira, Assaraf, & Livney, 2010). In another work, doxorubicin-loaded self-assembly silkelastin-like protein nanoparticles were prepared and evaluated for drug releasing potential (Xia, Wang, Lin, Xu, & Kaplan, 2014). To trigger silkelastin-like protein micelle formation, doxorubicin was used in this kind of formulation. An effectual cell uptake as well as apoptosis was revealed in HeLa cells, demonstrating that these doxorubicinloaded self-assembly silk-elastin-like protein nanoparticles can be used as a promising platform for tumor targeting delivery of anticancer agents. Cross-linked gelatin nanofibers were prepared by electro-spinning technique for controlled releasing of a hydrophobic drug, piperine (Laha, Yadav, Majumdar, & Sharma, 2016). For cross-linking, saturated glutaraldehyde vapor was used. A controlled piperine releasing pattern, in vitro, was noticed with the alteration of cross-linking period and pH. Curcumin (a natural antioxidant drug)-loaded gelatin-poly(acrylamidoglycolic acid) IPN nanogels were synthesized by free-radical emulsion polymerization methodology, where curcumin was loaded, in situ (Madhusudana Rao, Krishna Rao, Ramanjaneyulu, & Ha, 2015). The in vitro curcumin releasing from these gelatin-based nanogels were found pHsensitive. In a research, a gelatin-based semiIPN film was prepared for intestinal as well as colon specific release of diethyl carbamazine citrate and amoxicillin (Mehta & Kaith, 2018). To prepare the gelatin-based semi-IPN film, γ-radiation methodology was employed and the prepared film was found biodegradable as it evaluated by degradations of biocomposting and vermicomposting procedures. The maximum releasing of both the drugs-diethyl

III. Functional Applications

15.2 Drug delivery using various biological macromolecules

carbamazine citrate and amoxicillin was noticed in pH 9.2 with a sustained release pattern. In another research, glycerol gelatinbased pastilles containing rifampicin-loaded liposomes were developed for oral use (Lankalapalli & Vinai Kumar Tenneti, 2016). In a work, gentamicin-incorporated soy protein isolate film was prepared by solventcasting process and it demonstrated prolonged releasing of gentamicin over 4 weeks (Peles, Binderman, Berdicevsky, & Zilberman, 2013). However, an initial burst releasing of gentamicin was seen. Soy protein isolate-based film of rifampicin was prepared using formaldehyde as cross-linker and these rifampicin-loaded films demonstrated that the gastric enzymes influenced the drug releasing (Chen, Remondetto, Rouabhia, & Subirade, 2008). A gelatin-based membrane containing usnic acidloaded liposomes was developed and evaluated using a porcine model for the use in skin burn healing management (Nunes et al., 2016). In a research, collagen-based scaffold impregnated with gelatin microspheres loaded with ciprofloxacin was developed and the scaffold exhibited in vitro drug releasing in a controlled pattern (Kirubanandan, 2017). The developed collagen-based scaffold was found effective for epidermal and dermal tissue regeneration to be used in wound healing management. Some recently developed drug delivery systems using proteins and peptides are presented in Table 15.2.

15.2.3 Drug delivery using nucleic acids Recent years, nucleic acids are recognized as an emerging category of therapeutic agents as well as versatile drug targets (Buddolla & Kim, 2018; Tan et al., 2020). The important advantage for both the purposes (as therapeutic agents and drug targets) is that nucleic acids fold into the complex 3-D structures, which is able to perform vital enzymatic activities as well as to control the genetic information

355

transfer from DNA to proteins (Sundaram, Wower, & Byrne, 2012). However, the unmodified nucleic acids are excessively hydrophilicnatured for payload encapsulation as well as cell uptake. Even, this fact may lead to show undesirable responses like activation of immune system, maintenance of the blood coagulation pathway, etc (Tan et al., 2020). In general, nucleic acids are safe biological macromolecules. In the past, nucleic acids have mainly been developed as therapeutic agents to treat various rare-type diseases (Sehgal, Vaishnaw, & Fitzgerald, 2013; Shen & Corey, 2018). With the advancement of DNA/RNA nanotechnology and the stimuli (natural or engineered)-responsive capability of nucleic acids, many research groups have already considered to use nucleic acids for drug delivery with prospective therapeutic outcomes (Tan et al., 2015; Tan et al., 2020). Intrinsically, DNA complexes hold triggerresponsive qualities because of the stringent base-pairing characteristics, which are responsive to environmental issues. In a work, a selfassembled covalently-closed DNA nanocage was encapsulated with horseradish peroxidase via temperature-controlled encapsulation process (Burns, Lamarre, Pyne, Noble, & Ryadnov, 2018). The investigated 3-D DNA nanocage was truncated octahedron structured, which was assembled from 8 oligonucleotides after annealing as well as ligation. One end of the DNA nanocage was reported to be functionalized with a palindromic DNA sequence to shape a hairpin structure. The encapsulated horseradish peroxidase was found to be catalytically active within the self-assembled DNA nanocages. In another research, antiinflammatory glucocorticoid, dexamethasone, was conjugated with the 6 helix DNA nanotubes, which was assembled by means of single-stranded tile process, and 3 of the tiles were extended with i-motif sequence (Sellner et al., 2017). These dexamethasone-conjugated DNA nanotubes were revealed to be promptly localized

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15. Biological macromolecules in drug delivery

TABLE 15.2 Some recently developed drug delivery systems using proteins and peptides. Drug delivery systems using proteins and peptides

Drug loaded for Reference delivery

Gelatin/Fe3O4-alginate dual-layer magnetic nanoparticles

Doxorubicin

Huang, Chuang, Ke, and Yao (2020)

Carbon dot cross-linked gelatin nanocomposite hydrogel Cefadroxil

Bhattacharyya et al. (2020)

L-Dopa conjugated gelatin hydrogels

Doxorubicin

Pham, Su, Huang, and Jan (2020)

Mucoadhesive gelatin films for the vaginal delivery

Econazole

Dolci et al. (2020)

Electrospun gelatin nanocontainers

Piroxicam

Zhao et al. (2020)

Albumin-drug conjugates

Monomethyl auristatin E

Liu et al. (2020)

Bovine serum albumin/gold nanoparticles

Curcumin

Khodashenas, Ardjmand, Sharifzadeh Baei, Shokuhi Rad, and Akbarzadeh Khiyavi (2020)

Engineered bovine serum albumin-based nanoparticles

Doxorubicin

Yang et al. (2020)

Folate-receptor mediated pH/reduction-responsive albumin-based nanoparticles

Doxorubicin

Wang et al. (2020)

Casein nanoparticles

Curcumin

Barick, Tripathi, Dutta, Shelar, and Hassan (2021)

Calcium phosphate coated core-shell sodium caseinate nanocarriers

Curcumin

Wu et al. (2020)

Silk fibroin fibers

Diclofenac

ˇ skova´ et al. (2020) Opa´lkova´ Siˇ

Silk fibroin microneedle patches

Levonorgestrel

Yavuz et al. (2020)

Silk fibroin microneedle patches

Insulin

Zhu et al. (2020)

Surface modified silk fibroin nanoparticles

Doxorubicin

Pandey et al. (2020)

Silk fibroin H-fibroin/poly(ε-caprolactone) core-shell nanofibers

Rhodamine B

Wang et al. (2021)

Soy protein isolate\grafted[acrylic acid-co-4-(4hydroxyphenyl)butanoic acid] hydrogel

Ciprofloxacin

Mehra, Nisar, Chauhan, Singh, and Rattan (2020)

Soy protein films

Bupivacaine

Goder, Giladi, Furer, and Zilberman (2021)

PVA/anionic collagen membranes

Ciprofloxacin HCl

Daza et al. (2020)

Nanostructured dense collagen-polyester composite hydrogels

Spironolactone

Wang et al. (2021)

in the endosomes of MH-S macrophages, and the acidic endosomal milieu triggered i-motif formation and this allowed to the releasing of dexamethasone containing strand. The

mechanism of dexamethasone releasing at the acidic milieu is presented in Fig. 15.9A. Dexamethasone-conjugated DNA nanotubes were characterized as a diameter of B8 nm

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15.2 Drug delivery using various biological macromolecules

357

FIGURE 15.9 Characterization of dexamethasone-conjugated DNA nanotubes: (A) Scheme depicting the design of 6helix nanotubes with i-motif-dependent dexamethasone release. (B) Transmission electron micrograph of dexamethasone DNA nanotubes. Black arrows point out the individual DNA nanotubes, the inset depicts a single dexamethasoneconjugated DNA nanotube. Scale bar 40 nm. Source: With permission from Sellner S., Kocabey S., Zhang T., Nekolla K., Hutten S., Krombach F., . . . Rehberg M. (2017). Dexamethasone-conjugated DNA nanotubes as anti-inflammatory agents in vivo. Biomaterials;134:78-90. Copyright r 2017 Elsevier Ltd.

and a length of B30 nm. Transmission electron micrographs of dexamethasone-conjugated DNA nanotubes demonstrated the assembly into nanotube structure (Fig. 15.9B). Tumor-targeting magnesium-stabilized multifunctional DNA nanoparticles were developed for pH-sensitive delivery of an anticancer drugdoxorubicin (Zhao et al., 2018). In this work, rolling circle amplification was employed to fabricate a longer ssDNA containing repeating segments of the sgc8 aptamer for targeting of cancerous cells. The segments of i-motif-forming hairpin sequence were used for loading of doxorubicin and pH-

sensitive releasing of doxorubicin. In the unfolded condition at the physiological pH milieu, i-motifforming sequence in the hairpin loop was remained. This fact allowed doxorubicin to be intercalated with hairpin. The arrangement of imotif forced a conformational alteration of hairpin and this led the releasing of doxorubicin under the acidic milieu of tumor environment. In another study, a di-block conjugate was developed in form of DNA-block-poly(paclitaxel), where paclitaxel was polymerized and consequently conjugated to an antisense DNA strand (Tan et al., 2016). The paclitaxel releasing was

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358

15. Biological macromolecules in drug delivery

revealed upon the endocytosis process, which induced the high level of cytotoxicity and it is comparable to that of free paclitaxel. DNA nanotechnology presents a number of distinctive benefits, such as precise external and internal nanoarchitectures, stimuliresponsiveness, lacking of intrinsic toxicity (Mohri, Nishikawa, Takahashi, & Takakura, 2014; Zahid, Kim, Hussain, Amin, & Park, 2013). With remarkable structural features, DNA nanostructures are progressively being explored and exploited for drug delivery applications. Characteristically, DNA nanostructures are used to circumvent the renal clearance and can employ with enhanced permeability and retention (EPR) effect related to the growth of malignant tumors (Fang, Nakamura, & Maeda, 2011; Greish, 2010). This fact can be utilized for the delivery of small anticancer agents like doxorubicin. In fact, doxorubicin is reported to be capable of intercalating with GC-rich regions of duplex DNA structure (Carvalho et al., 2009). This also reported to inhibit the action of topoisomeraseII, which decreases the tumorous cells’ growth. The intercalation in-between GC-rich regions of DNA duplex and doxorubicin are reversible in particular under the lower pH milieu, which facilitates the DNA nanostructures to perform as drug carriers. Some DNA origamis were assembled from the phage DNA (M13mp18) as carriers for doxorubicin with two kinds of origamis (a tubular and a triangular) (Jiang et al., 2012). Both the tubular and triangular origamis demonstrated augmented cytotoxicity against drug resistant MCF 7 cells (human breast adenocarcinoma cancer cells) (MCF 7) with an enhancement of cell uptake. Origamis have already been evaluated for the combination delivery of doxorubicin and p53 tumor suppressor gene, in vivo (Liu et al., 2018; Zhang et al., 2014). The resultant doxorubicin-loaded DNA nanostructures showed a marked antitumor action in MCF-7R xenograft-model. However, in BALB/c mice, little systemic

toxicity and immune stimulation were observed. Very interestingly, the shape effect was noticed; when the triangular shape DNA origami demonstrated a high level of tumor localization and antitumor action in comparison with that of the tubular shape as well as square shape DNA origamis. In a research, the controlling of doxorubicin release rates via regulating the degrees of global twist in the double-helix structural feature of DNA molecules was explored, where a twisted nanotubelike design with a 12 base-pair per turn demonstrated 33% higher doxorubicin loading (Zhao et al., 2012). This exhibited doxorubicin release over an extended period as compared to that of the usual straight design with a 10.5 basepair per turn, in vitro. RNA is another nucleic acid macromolecule with distinctive characteristic features like structural flexibility, enzymatic activity functions, etc (Shu et al., 2018). Recent years, RNA nanotechnology has been emerged as a novel therapeutic area with the exploration and exploitation of nanometeric scale architectures of RNA molecules. In general, RNA nanoparticles demonstrate the fundamental attribute of DNA canonical base pairing, while containing the purposeful multiplicity of proteins (Jasinski, Haque, Binzel, & Guo, 2017). RNA nanotechnology advancements facilitate several potential benefits like controlled drug delivery, targeted drug delivery, biosensor systems, medical detection, etc. RNA nanostructures modified with ligands as well as aptamers have already been investigated as promising carrier in the targeted delivery of drugs (Li et al., 2015). In a recent work, rubber-like RNA nanoparticles were developed for targeted codelivery of MiRNA and paclitaxel to surmount the drug effluxion and chemoresistance for the treatment of hepatocellular carcinoma (Wang et al., 2021). The multivalent RNA nanoparticles were conjugated with different copies of hepatocyte targeting-ligands (24 copies of paclitaxel

III. Functional Applications

15.2 Drug delivery using various biological macromolecules

and one copy of miR122), which introduced the tumor specificity to the rubber-like RNA nanoparticles as these bound and internalized into the cancerous cells of liver, selectively. The results of in vivo evaluations using mice xenograft-model demonstrated the accumulation of these multivalent RNA nanoparticles in the hepatocellular carcinoma tumor sites, predominantly. After the multiple intravenous injections, these RNA nanoparticles also hampered the tumor growth, efficiently. Four-way junction based RNA nanoparticles loaded with paclitaxel were prepared for the uses in targeted cancer therapeutics (Guo et al., 2020). The paclitaxel loading within these RNA nanoparticles was based on the covalently loading process and this augmented the aqueous solubility of paclitaxel by 32,000-fold. The paclitaxel-loaded RNA nanoparticles were revealed to be rigid as well as stable enough. Intravenous administration of these paclitaxelloaded RNA nanoparticles modified with specific cancer-targeting ligand inhibited the growth of breast cancer cells. In another research work, RNA based micellar-type nanostructure, in which cholesterol was conjugated on a helical end of branched pRNA 3way junction motif, was developed for loading and delivery of an anticancer agent (Shu et al., 2018). The developed RNA micelles (amphiphilic in nature) comprising a covalentlylinked hydrophobic-natured lipid tail and a hydrophilic-natured RNA head assembled in an aqueous environment by means of hydrophobic interaction. Paclitaxel-N3 can react with end alkyne labeled RNA via Click chemistry and paclitaxel can be later released out from RNA strand by hydrolysis (Fig. 15.10). The assembled RNA based micellar-type nanostructure was found able to accompanying the manifold purposeful modules because of pRNA 3-way junction branched structural feature. Within the RNA micelles, paclitaxel was loaded, which was reported to enhance the aqueous solubility. Paclitaxel-loaded assembled

359

RNA micelles internalized into the cancerous cells and inhibited their proliferation. These paclitaxel-loaded micelles also induced the apoptosis of cancerous cells in a Caspase-3 dependent mode, which were targeted to the tumorsites devoid of any kinds of accumulation in healthy organs, in vivo.

15.2.4 Drug delivery using lipids Lipids are vital ingredients of all the living organisms (Corn et al., 2020). These are wellknown to play several significant roles in various physiological processes, which are related to their structural configuration (Corn et al., 2020; van Meer et al., 2008). Lipids are fats, which are naturally extracted from either vegetal and/or animal resources. These also include butter, vegetable oils, waxes, fatty acids, triglycerides, cholesterol, phospholipids, bile acids, etc (Corn et al., 2020; Zhang et al., 2018). Biologically derived lipids comprise of organic structures having long hydrocarbon chains with esters of various fatty acids (Mu & Holm, 2018; Pandey & Kohli, 2018). These are insoluble or sparingly soluble in the aqueous solvents, but soluble in organic solvents. Lipids are impermeable in nature due to their nonpolar and hydrophobic characters. Employing these two characters, lipids are used as coating agents and films in many foods and pharmaceutical products (Hassan, Chatha, Hussain, Zia, & Akhtar, 2018). Lipids also facilitate the absorption of drugs. Some important parts of the body are composed of lipids, such as blood brain barrier. Therefore it is vital to enhance the lipid solubility of some dugs to easily cross the blood brain barrier (Corn et al., 2020). Because of these facts, lipids are useful excipient to augment the bioavailability of various drugs. Since past few decades, lipids have shown several significant merits as drug delivery excipients for effective delivery of drugs like enhanced potential of drug releasing,

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15. Biological macromolecules in drug delivery

FIGURE 15.10 The rational design of RNA-paclitaxel conjugates (PTX: Paclitaxel). Source: With permission from Shu Y., Yin H., Rajabi M., Li H., Vieweger M., Guo S., . . . Guo P. (2018). RNA-based micelles: A novel platform for paclitaxel loading and delivery. Journal of Controlled Release: Official Journal of the Controlled Release Society;276:17-29., Copyright r 2018 Elsevier B.V.

improved modulated drug targeting, reduced toxicity or nontoxicity, etc (Mu & Holm, 2018; Pandey & Kohli, 2018). In a recent research, calcitriol tablets were formulated using hybrid lipid-based solid dispersions (Yue et al., 2020). These hybrid lipidbased calcitriol tablets exhibited high level of content uniformity and improved stability in comparison to that of the commercial calcitriol capsules (Rocaltrol). Directly compressed lipidbased matrix tablets of fixed dose combination (lumefantrine and artemether) were developed

using solid lipid dispersions via hot fusion process (Wilkins, du Plessis, & Viljoen, 2020). Lipid-based sustained drug releasing tablets were developed, where a single-step twinscrew granulation procedure was used for granulation employing Precirol ATO 5, Compritol 888 ATO and Geleol as sustained drug releasing excipients (Kallakunta, Tiwari, Sarabu, Bandari, & Repka, 2018). Lipid-based tablets manufactured by twin-screw granulation demonstrated a sustained pattern of drug releasing over a period of 24 h. In a work,

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15.2 Drug delivery using various biological macromolecules

sucrose stearate-based lipid matrix tablets for controlled release of etodolac were formulated by hot fusion process (Abd-Elbary, Tadros, & Alaa-Eldin, 2013). To prepare these etodolac matrix tablets, various lipid materials like cetyl alcohol, cetostearyl alcohol, stearic acid, Compritol ATO 888, Precirol ATO 5 and Imwitor 900K were used. The sucrose stearate D1805-based matrix tablets showed above 90% of etodolac releasing over a period of 12 h. Conjugation of drugs with lipids presents some important advantages like targeted delivery, better cellular uptake, reduced side-effects, etc. (Ding et al., 2012; Radwan & Alanazi, 2014a; 2014b). In a work, cholesterol was conjugated with 5-fluorouracil and the cholesterolconjugated 5-fluorouracil demonstrated improved anticancer actions as compared to that of free 5-fluorouracil (unconjugated) (Radwan & Alanazi, 2014a). In this conjugation strategy, hydroxyl groups of cholesterol molecules structure a carbonyl link with 5fluorouracil. Docosahexaenoic acid/10-hydroxycamptothecin conjugate was synthesized in a work and the conjugate demonstrated the inhibition of tumor growth in the treatments of various cancers like lung cancer and colorectal cancer (Wang, Li, Jiang, & Larrick, 2005). The conjugate of docosahexaenoic acid-paclitaxel was synthesized for the further enhancement of the tumor targeting capability of paclitaxel with the reduction of toxicity to the normal healthy tissues (Wang, Li, Jiang, Yang, & Zhang, 2006). Pharmacokinetics results indicated that docosahexaenoic acid-paclitaxel conjugate was well distributed in the plasma after administration by intravenous (i.v.) injection. The synthesized docosahexaenoic acidpaclitaxel conjugate was reported to maintain the higher paclitaxel concentrations in tumorsite as well as plasma for a prolonged period in comparison to that of free paclitaxel. In a work, cytarabine was conjugated with a lauric acid moiety and after oral administration in rats, the lauric acid-cytarabine conjugate

361

showed higher stability in the plasma with a 32.eightfold increment in cytarabine bioavailability (Liu, Zhao, Ma, & Luan, 2016). Lithocholic acid-tamoxifen conjugates were synthesized via the covalent attachment of lithocholic acid to its amine group (Yadav et al., 2015). Owing to positive charge and the occurrence of lipophilic group, cationic lithocholic acid-conjugated tamoxifen exhibited improved anticancer action in comparison to that of free tamoxifen. Zidovudine was conjugated with ursodeoxycholic acid and this enhanced the antiviral action against human immunodeficiency virus (HIV) because of its reduction of transport-mediated resistance and decreased hydrolysis rate in human plasma (Dalpiaz et al., 2012). In a group of reports, linker-based lecithin microemulsions were formulated and evaluated for lidocaine delivery (Yuan & Acosta, 2009; Yuan, Ansari, Samaan, & Acosta, 2008; Yuan et al., 2010). These lidocaine-loaded linker-based lecithin microemulsions were prepared using caprylic acid (hydrophobic) and sodium caprylate (hydrophilic) linkers, which were stabilized by using egg phosphatidylcholine. The obtained results of these reports presented that these lecithin microemulsions exhibited higher lidocaine permeation across the reconstructed human skin. Lipid-based microemulsion formulations of bupivacaine were formulated using medium chain triglycerides and glycerol monooleate Myverol 18
99K (commercial) (Yaghmur, Rappolt, Østergaard, Larsen, & Larsen, 2012). The influence of lipids was evaluated on the liquid crystalline phases as well as microemulsions. These lipid-based microemulsion formulations of bupivacaine were reported to modulate the drug release rate. An in situ forming lyotropic liquid crystal-based gel of bupivacaine HCl was developed for sustained release of bupivacaine HCl long-acting postoperative analgesia (Mei et al., 2018). At some point in the in situ gelation process, a lamellar-hexagonal-cubic

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15. Biological macromolecules in drug delivery

phase transition was noticed. The hexagonal and cubic phases contributed higher viscosity to the in situ gel systems, facilitating the sustained release of bupivacaine HCl. The effect of persistent analgesia was achieved with in situ forming gel of bupivacaine HCl, in vivo and the plasma concentration of bupivacaine was revealed steadier in comparison to that of the marketed bupivacaine injection. In a work, solid lipid nanoparticles loaded with a corticosteroid-betamethasone-17valerate were prepared using three different lipids, namely tripalmitate, cetylpalmitate and distearate (Jensen, Petersson, & Nielsen, 2011). The elevated quantity of betamethasone-17valerate in the skin and mainly, in the stratum corneum was measured after application of betamethasone-17-valerate-loaded solid lipid nanoparticles made of distearate. Aceclofenacloaded nanostructured lipid carriers were formulated using PEG-8 Miglyol 812 as liquid lipid and Compritol 888 ATO as solid lipid (Phatak & Chaudhari, 2013). The particle size of aceclofenac-loaded nanostructured lipid carriers was found to be reduced from 350 to 134 nm with the increasing of solid lipid to liquid lipid ratio. It was also found that the aceclofenac entrapment efficiency was affected by the stabilizer concentration and drug to lipids ratio. Medium-chain triglycerides, cetyl palmitate, and polyglyceryl-3 methylglucose distearate were used to formulate nanostructured lipid carriers, which were loaded with coenzyme Q10 (Chen et al., 2013). These coenzyme Q10-loaded nanostructured lipid carriers were found capable of targeting the epidermis and facilitating a sustained release pattern. Using coconut oil, stearic acid and soya lecithin, nanostructured lipid carriers were prepared to load flurbiprofen (Kawadkar, Pathak, Kishore, & Chauhan, 2013). These flurbiprofen-loaded, nanostructured lipid carriers were reported to permeate the deep layers of skin. In an investigation, nanostructured lipid carriers loaded with voriconazole were formulated via the hot-

melt emulsification procedure followed by ultra-sonication process using lipid materials like glyceryl monostearate and oleic acid (Waghule et al., 2019). The glyceryl monostearate to oleic acid ratio was maintained as 7:3. The voriconazole-loaded nanostructured lipid carriers were transferred to carbopol gels for topical delivery of voriconazole. The optimized formulation of voriconazole-loaded nanostructured lipid carriers presented voriconazole loading of 6.59% and voriconazole entrapment efficiency of 70.52 6 5% with an average particle size of 107.7 6 8 nm. The scanning electron micrographs (by FESEM analysis) of voriconazole-loaded nanostructured lipid carriers were presented in Fig. 15.11. The results of in vitro drug release demonstrated a prolonged voriconazole releasing pattern for 10 h (Fig. 15.12). The optimized topical gel containing voriconazole-loaded nanostructured lipid carriers exhibited improved voriconazole permeation (66.45%) as compared to free voriconazole, ex vivo. The voriconazole releasing from optimized topical gel was found to be sustained up to 11 h. Ethosomal formulation based on soya lecithin and ethanol were prepared and evaluated for topical delivery psoralen (Zhang, Shen, Zhao, & Feng, 2014). These psoralenloaded ethosomes containing optimized formulation exhibited a 6.56 fold-enhancement of psoralen permeation rate in comparison to that of psoralen tincture. The ethosomal formulation of psoralen prepared using 10% w/v soya lecithin and 45% v/v ethanol presented the lowest skin permeation of psoralen at 24 h. An ophthalmic liposomal gel for delivery of besifloxacin HCl was developed, where the liposomes were prepared by thin film hydration process using lipids like cholesterol and soya lecithin (Bhattacharjee et al., 2020). To prepare the besifloxacin HCl-loaded liposomes, soya lecithin to cholesterol ratio and lipid to besifloxacin HCl ratio on entrapment efficiency, drug loading and particle size characteristics of

III. Functional Applications

FIGURE 15.11 Scanning electron micrographs (by FESEM analysis) of voriconazole-loaded nanostructured lipid carriers. Source: With permission from Waghule T., Rapalli V.K., Singhvi G., Manchanda P., Hans N., Dubey S.K., . . . Nayak A.K. (2019). Voriconazole loaded nanostructured lipid carriers based topical delivery system: QbD based designing, characterization, invitro and ex-vivo evaluation. Journal of Drug Delivery Science and Technology. 52:303-315. Copyright r 2019 Elsevier B.V.

FIGURE 15.12 In vitro drug release from free voriconazole and voriconazole-loaded nanostructured lipid carriers. Source: With permission from Waghule T., Rapalli V.K., Singhvi G., Manchanda P., Hans N., Dubey S.K., . . . Nayak A.K. (2019). Voriconazole loaded nanostructured lipid carriers based topical delivery system: QbD based designing, characterization, in-vitro and ex-vivo evaluation. Journal of Drug Delivery Science and Technology. 52:303-315. Copyright r 2019 Elsevier B.V.

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15. Biological macromolecules in drug delivery

liposomes were analyzed by 32 factorial design. The optimized besifloxacin HCl-loaded liposomal formulation showed high encapsulation efficiency and loading. The ex vivo corneal permeation of the besifloxacin HCl-loaded liposomal gel revealed a significant increase of besifloxacin HCl permeation in comparison to that of the besifloxacin HCl-loaded carbopol gels. The histological evaluation of ophthalmic liposomal gel containing besifloxacin

HCl very clearly indicated that there was no sign of corneal drainage even after 8 h of application when tested using pig cornea (Fig. 15.13). In a work, insulin-loaded transferosomes (ultra-deformable liposomes) were formulated using lipids like cholesterol and soya lecithin (Malakar, Sen, Nayak, & Sen, 2012). The prepared insulin-loaded transferosomes were transferred to methylcellulose gel (5% w/v) to

FIGURE 15.13 (I): Representation of acute eye irritation test of besifloxacin HCl-loaded liposomal gel using slit microscope: (a) control 1 h (b) 1 h after treatment (c) control 24 h (d) after 24 h instillation (e) Control 48 h and (f) after 48 h instillation (g) Control 72 h (h) 72 h treatment. (II): Histological cross-section of excised pig cornea [(A) normal cornea and (B) optimized besifloxacin HCl-loaded liposomal gel treated cornea (magnification 100X)]. Source: With permission from Bhattacharjee A., Das., P.J., Dey S., Nayak AK., Roy PK., Chakrabarti S., . . . Mazumder B. (2020). Development and optimization of besifloxacin hydrochloride loaded liposomal gel prepared by thin film hydration method using 32 full factorial designColloids and Surfaces A: Physicochemical and Engineering Aspects 585: 124071. Copyright r 2019 Elsevier B.V.

III. Functional Applications

15.2 Drug delivery using various biological macromolecules

develop transferosomal gels for transdermal delivery of insulin. The effects of various formulation parameters like soya lecithin to cholesterol ratio, surfactants (Tween 80 to sodium deoxycholate) ratio and lipids to surfactants ratio were evaluated on the in vitro insulin permeation flux across the porcine ear skin were evaluated. For the formulation optimization in this work, 23 factorial design was employed. The optimized formulation of insulin-loaded transferosomes exhibited 56.55 6 0.37% insulin entrapment efficiency. The optimized insulinloaded transferosomes containing gel exhibited 13.50 6 0.22 μg/cm2/h of insulin permeation flux. The in vitro permeation of insulin from the optimized insulin-loaded transferosomes containing gel showed a sustained pattern of skin permeation over 24 h. In addition, in vitro permeation of insulin from optimized gel was evaluated employing iontophoresis (used current supply: 0.5 mA/cm2) and this enhanced

365

insulin permeation flux (17.60 6 0.03 μg/cm2/ h). After the transdermal application of optimized insulin-loaded transferosomes containing gels, a prolonged hypoglycemic action over 24 h in diabetic rats (induced by alloxan) was revealed. In another research, similar kind of transdermal gel containing risperidone (an antipsychotic drug)-loaded transferosomes (made of lipids like cholesterol and soya lecithin) was formulated (Das, Sen, Maji, Nayak, & Sen, 2017). The optimized formulation of risperidone-loaded transferosomes exhibited 61.54 6 2.14% risperidone entrapment efficiency with a mean vesicle diameter range of 589.5 nm and zeta potential of 220.90 mV. The optimized risperidone-loaded transferosomes containing gel exhibited 0.2387 6 0.0245 μg/cm2/h of risperidone permeation flux (Fig. 15.14). In the iontophoresis study, the risperidone permeation flux of 0.2753 6 0.0263 mg/cm2/h across the porcine

FIGURE 15.14 Cumulative percentage of ex vivo risperidone permeated through porcine skin from various transferosomal gels (F-O 5 optimized formulation) (Mean 6 SD, n 5 3). Source: With permission from Das B., Sen S.O., Maji R., Nayak A.K., Sen K.K. (2017). Transferosomal gel for transdermal delivery of risperidone: Formulation optimization and ex vivo permeation. Journal of Drug Delivery Science and Technology.;38:59-71. Copyright r 2017 Elsevier B.V.

III. Functional Applications

FIGURE 15.15 Cumulative percentage of ex vivo risperidone permeated through porcine skin from optimized transferosomal gel (F-O) in normal condition and inontophoretic condition (F-OI) with the influence of current supply, 0.5 mA/cm2 (Mean 6 SD, n 5 3). Source: With permission from Das B., Sen S.O., Maji R., Nayak A.K., Sen K.K. (2017). Transferosomal gel for transdermal delivery of risperidone: Formulation optimization and ex vivo permeation. Journal of Drug Delivery Science and Technology.;38:59-71. Copyright r 2017 Elsevier B.V.

TABLE 15.3 Some recently developed drug delivery systems using lipids. Drug delivery systems using lipids

Drug loaded for delivery

Reference

Lipid-drug conjugated nanoparticles

5-fluorouracil

Shinde et al. (2020)

S-Protected thiolated nanostructured mucoadhesive lipid carriers

Bergapten

Arshad et al. (2020)

Nanostructured lipid carrier for ocular use

Ibuprofen

Rathod, Shah, and Dave (2020)

Fucose-conjugated nanostructured lipid carrier

Paromomycin sulfate

Chowdhary, Chowdhary, Agrawal, and Jain (2021)

Solid lipid nanoparticles coated with vitamin B12-stearic acid conjugate

Amphotericin B

Singh et al. (2020)

Liposomes endowed with a cholesterol-conjugated indoximod prodrug in the lipid bilayer

Mitoxantrone

Mei et al. (2020)

Oral lipid-based drug delivery using mono-/di-glycerides as single component excipients

Celecoxib

Ilie et al. (2020)

Lipid-based formulations prepared by long-chain or medium-chain lipids

Ritonavir

Tanaka, Doi, Katano, and Kasaoka (2021)

Supersaturated lipid-based drug delivery systems

Cinnarizine

Ilie et al. (2021)

Self-microemulsifying drug-delivery systems

S-carvedilol

Zhang et al. (2020)

Lipid nanoparticles

Cyclosporine A

Varache, Ciancone, and Couffin (2020)

Solid lipid microparticles for pulmonary use

Salbutamol sulfate

Ignjatovi´c et al. (2021)

Nanostructured lipid carrier for topical use

Astaxanthin

Geng et al. (2020)

Lipid nanocarrier dispersions

Lidocaine

Guo, Wei, Lee, Maia, and Morrison (2020)

Directly compressed lipid matrix tablets

Artemether and lumefantrine

Wilkins, du Plessis, and Viljoen (2021)

Solid lipid microparticles

Ibuprofen

Ho, Xiang, Gopal, and Khan (2021)

Nanostructured lipid carriers integrated into in situ nasal gel

Flibanserin

Fahmy et al. (2020)

References

skin was measured, which was higher in comparision with that of the optimized transferosomal gel in normal condition without iontophoresis (0.2387 6 0.0245 mg/cm2/h) (Fig. 15.15). Some recently developed drug delivery systems using lipids are presented in Table 15.3.

367

to develop effectual biological macromoleculesbased drug delivery carriers in full measure to attain paramount therapeutic output. To date, a few biological macromolecules-based drug delivery carriers have been approved for their clinical applications. In summary, there is a promising future for biological macromolecules-based drug delivery carriers.

15.3 Conclusion Biological macromolecules including carbohydrates, proteins, peptides, nucleic acids, and lipids are abundantly derived/extracted from various natural renewable resources (plants, algae, fungi, microbes, insects, animals, etc.), biotechnological fermentation, and microbiological culture process. These encompass several advantages like sustainable production, renewability, biocompatibility, biodegradability, longer blood circulation period, targeting capability, etc. Owing to a variety of biological functions, different biological macromolecules (carbohydrates, proteins, nucleic acids, and lipids) are being used in many biomedical applications including biomedicines, drug delivery, tissue regenerations, wound management, orthopedics, etc. Since the past few decades, a variety of biological macromolecules-based drug releasing carriers have been reported in forms of various dosage forms like injectables, tablets, capsules, gels, hydrogels, beads, microparticles, nanoparticles, patches and films, fibers, liposomes, transferosomes, scaffolds, etc. The uses of biological macromolecules-based drug releasing carriers have already shown to enhance the pharmacokinetics of loaded drugs with reduced systemic toxicity and immunogenicity. However, there are still many challenges associated to extraction/ production, quality control, safety and storage of different biological macromolecules-based drug delivery carriers. For example, proteins and peptides as drug delivery carrier-biomaterials are not ideal for oral drug delivery owing to proteolysis-connected poor stability of the systems. Therefore further researches are necessary

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C H A P T E R

16 Biological macromolecules in tissue engineering Pandurang Appana Dalavi1, Sesha Subramanian Murugan1, Sukumaran Anil2 and Jayachandran Venkatesan1 1

Biomaterials Research Laboratory, Yenepoya Research Centre, Yenepoya (Deemed to be University), Deralakatte, Mangalore, India 2Department of Dentistry, Oral Health Institute, Hamad Medical Corporation, College of Dental Medicine, Qatar University, Doha, Qatar

16.1 Introduction Defective and diseased organs/tissues significantly reduce the quality of a person’s life. Tissue engineering is an emerging area of research that aims to develop artificial tissue/organs using materials, cells, and growth factors to treat the defective or diseased organ/tissue. Biological macromolecules are used in the place of materials to mimic the extracellular matrix of the native tissue. Tissue engineering is a broad area had their significance in the life sciences, biotechnology and engineering. It had its footprints and achievements in medical history. The transplantation of health care needs including organ transplantation, implants, sutures, medical textiles, and scaffolds are the main focused area in tissue engineering (Akbari et al., 2016). Grafting techniques of autograft and allograft were practiced for organ transplantation. The autograft techniques use the graft from the same donor to give beneficial results against the immune rejection but the

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00016-6

allograft fails. The main disadvantage is that it requires more grafting tissue if the organ damage or tissue damage is more. It leads to a shortage of tissues and organs. To overcome this challenging situation, tissue-engineered artificial organs and implants were used (Barker & Markmann, 2013). The transplanted implants must adhere to the native tissue in every way which is adapted to the local environment atmosphere. The biomacromolecules in the implants can have cell communication, cell, and hormone signaling to interact with the neighboring cells, epigenetic factors, and proteins to adhere on the surface for fast healing (Terheyden, Lang, Bierbaum, & Stadlinger, 2012).

16.2 Bone tissue engineering Bone loss may happen for several reasons including diseases, vehicle accidents, trauma, birth defects, etc. To restore, regenerate, and maintain damaged or diseased bone is still a

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challenging task in orthopedic treatments (Rose & Oreffo, 2002). Two main traditional grafting techniques are used to regenerate diseased bones are autograft and allograft. Autograft is still considered the gold standard. An autograft, patients own tissue i.e. from iliac crest will be used for the treatment. However, the autograft technique has several disadvantages. It is expensive and requires secondary operation at the harvested site. The patient may suffer from bleeding, scarring, morbidity, and chronic pain. Besides this, when there is the necessity of a large volume of bone autograft technique is not suitable. An allograft is another grafting technique in which grafting material will be taken from other sources like a cadaver. Hence, it is associated with the risk of immunogenic reactions and transmission of diseases (Amini, Laurencin, & Nukavarapu, 2012; Burg, Porter, & Kellam, 2000). To overcome shortcomings of the autograft and allograft techniques synthetic graft will be developed to regenerate damaged bone. To develop a synthetic graft synergistic combination of biological macromolecules, polymers (Liu & Ma, 2004), copolymers, metals, ceramics, cells, and growth factors will be used (Amini et al., 2012; Stevens, 2008). To construct the artificial bone tissue, both synthetic and natural materials are extensively studied. A technique used for the regeneration of damaged or diseased bone is called bone tissue engineering (BTE) (Mistry & Mikos, 2005). HA has the chemical formula Ca10(PO4)6(OH)2. HA is playing important role in BTE due to its important properties including nontoxicity, biocompatibility, osteoconductivity, biodegradability, and mechanical strength (Deville, Saiz, & Tomsia, 2006; Farokhi et al., 2018; Venugopal et al., 2010). Hydroxyapatite is an extensively studied biomaterial for BTE. Due to the brittleness of the hydroxyapatite, it is often combined with other polymeric substances including alginate, chitosan, carrageenan, fucoidan, ulvan, and other synthetic polymeric substances to mimic the natural functions of bone (Venkatesan, Anil,

Kim, & Shim, 2016). The naturally derived biomaterials show good biocompatibility and nontoxicity toward cells line and bone tissue. However, bone formation, mechanical strength, and uneven biodegradation lead to research to make biocomposite with other materials including beta-tricalcium phosphate, metals, polymers, and copolymers (Islam et al., 2017; Sergi, Bellucci, & Cannillo, 2020). In the book chapter, we aimed to discuss the composite of biological macromolecules with hydroxyapatite toward BTE applications.

16.3 Biological macromolecules in bone tissue engineering Recently biological macromolecules are playing a vital role in BTE which includes chitosan, fucoidan, alginate, carrageenan, ulvan, dextran, collagen, and gelatin due to their excellent biocompatible, biodegradable nature. Besides this, all these macromolecules have osteogenic properties. (Swetha et al., 2010). One of the problems, when we use these macromolecules in BTE, is that they all are having low mechanical strength. Hence, to achieve synergistic osteogenic properties and to get higher mechanical strength, a combination of hydroxyapatite with these macromolecules is the best choice.

16.3.1 Alginate Alginate polysaccharide is commonly isolated from brown seaweed. The divalent cations in the Ca, Na and Ba alginate was widely used in the biomedical sector mainly on drug delivery and tissue regeneration (Sahoo & Biswal, 2021). It can able to easily modify their structural compatibility by using crosslinkers which improvise the mechanical strength and cell affinity. This favors many researchers to choose the alginate for fabricating the constructs for the

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tissue engineering applications. Alginate spreads its applications on both soft and hard tissues. The soft tissues of the skin use the alginate composites of akermanite hydrogels for skin regenerations (Han et al., 2017). The hard tissues of the bone regeneration have achieved by the alginate graphene composites which induce the bone marrow stem cells into osteogenic differentiation (Liu et al., 2015). Alginate/ hydroxyethyl cellulose/hydroxyapatite composites show better human mesenchymal stem cell viability and attain the maximum mechanical strength of 23.9 MPa. This composite shows excellent physicochemical properties with good protein absorption for bone cell attachment and proliferation (Tohamy, Mabrouk, Soliman, Beherei, & Aboelnasr, 2018). Moreover, the alginate microspheres were used for the controlled drug delivery (Sun & Tan, 2013). Different types of scaffolding systems can be achieved by using the alginate including foams, microsphere, sponges, microcapsules, and fibers were used for tissue healing, cartilage, and bone repair.

16.3.2 Chitosan Chitosan is a cationic polysaccharide and it is commonly derived through the deacetylation of chitin. Chitosan is widely used in BTE due to its biocompatibility, biodegradability, antibacterial property, nontoxicity, and pH-dependent solubility (Costa-Pinto, Reis, & Neves, 2011; Di Martino, Sittinger, & Risbud, 2005; Kong et al., 2006; Li et al., 2019). Besides, the physicochemical and biological properties of the chitosan can be tailored (LogithKumar et al., 2016). Surfacemodified chitosan can play important role in BTE (Abinaya, Prasith, Ashwin, Viji Chandran, & Selvamurugan, 2019). Porosity on the chitosan was created by using gelatin and the role of porosity on the osteogenic differentiation of the adipose-derived stem cells (ADSCs) was checked. Biological cell assay proves that chitosan scaffold with lower porosity can promote osteogenic

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differentiation of stem cells (Ardeshirylajimi, Delgoshaie, Mirzaei, & Khojasteh, 2018). Sintered chitosan microsphere matrix was developed by using the ionotropic gelation technique. Developed microspheres were characterized by various analytical techniques. Further, the porosity of the microsphere was found to be 19% which can promote cell proliferation. Also, the compressive modulus of the microspheres was comparable with the human cancellous bone. These findings results suggest that chitosanbased sintered microspheres can be used as a bone implant (Abdel-Fattah, Jiang, El-Bassyouni, & Laurencin, 2007). Besides this, several chitosanbased nanofibers, sponges, injectable gels, nanocomposite scaffolds, microspheres have already been reported for bone regeneration applications (Balagangadharan, Dhivya, & Selvamurugan, 2017; Li, Zhang, & Zhang, 2018; Shavandi, Bekhit, Sun, & Ali, 2016; Sultana et al., 2015). Chitosan with hydroxyapatite is an ideal scaffold for bone regeneration applications. In vitro and in vivo bone formation ability of this scaffold was studied by Chesnutt, Yuan, Buddington, Haggard, & Bumgardner, 2009 (Chesnutt et al., 2009). An injectable scaffolding system containing chitosan, nano-HA, and collagen was developed. Biological assays with MC3T3-E1 prove that this nanocomposite scaffold is nontoxic to the cells. Besides this, enhancement in the alkaline phosphatase (ALP) and osteogenic gene expressions was observed. This finding result proves that the developed scaffold can promote osteogenic differentiation (Chen et al., 2012). Mirza and his coworkers have fabricated a monohybrid scaffold containing bael fruit gum, chitosan, and nanoHA. In vitro studies have proven that the scaffold can induce apatite formation and showing higher protein adsorption. Further, In vitro cell interaction with MG-63 cell lines shows cells are viable with the scaffold, and enhancement in the ALP activity was observed (Mirza et al., 2018). Wu et al. have studied the utility of strontium (Sr) substituted in HA with graphene oxide nanosheets added in a chitosan-containing

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scaffolding system for BTE. Finding results showing higher mechanical strength with excellent osteogenic properties (Wu et al., 2020). Chen et al. has fabricated chitosan, HA, and multiwalled carbon nanotubes containing scaffolding system by using in situ precipitation method. The mechanical strength of this scaffold was found comparable with the human bone and scaffolds show osteogenic properties (Chen, Hu, Shen, & Tong, 2013). Besides this, several scaffolding systems containing chitosan HA has reported for bone regeneration applications chitosan with nHA, gelatin-nano hydroxyapatite, polylactic acid-hydroxyapatite, alginate-hydroxyapatite, hydroxyapatite-genipin, hydroxyapatite-magnetite, montmorillonitehydroxyapatite, agarose-hydroxyapatite, fibroinhydroxyapatite, nanohydroxyapatite-PLGA loaded simvastatin, hydroxyapatite-marine sponge collagen, and carbon nanotube-chitosan-natural HA (Cai et al., 2009; Dan et al., 2016; Demirta¸s, Irmak, & Gu¨mu¨s¸ derelio˘glu, 2017; Huysal et al., 2017; Kar, Kaur, & Thirugnanam, 2016; Kazimierczak, Benko, Nocun, & Przekora, 2019; Kim et al., 2007; Li et al., 2018; Pallela, Venkatesan, Janapala, & Kim, 2012; Venkatesan, Qian, Ryu, Kumar, & Kim, 2011).

16.3.3 Carrageenan Carrageenan is a polysaccharide that contains D-galactose and 3,6-anhydrous-D-galactose structural units. Based on the presence of the chemical structures carrageenan has classified into six types Mu (μ)-, Nu (γ), Iota (l)-, Kappa (k)-, Lambda (λ), and Theta()- carrageenan. Carrageenan can be extracted from the red seaweeds and used in nutraceuticals, pharmaceutical and recently for biomedical application due to its biocompatibility, biodegradability, and nontoxicity (Berton et al., 2020; Campo, Kawano, da Silva, & Carvalho, 2009; Tytgat et al., 2019). K-carrageenan-based porous nanocomposite scaffold was developed by freeze-drying method. Biological assays with mouse preosteoblast MC3T3-E1 cell lines have

proven the bone regeneration capacity of the scaffold (Khan et al., 2020). Santo et al. have developed carrageenan-based hydrogels for the sustainable delivery of the platelet-derived growth factor (PDGF). An experimental study reveals incorporation of the PDGF in the hydrogels and controlled delivery of the PDGF. This finding result reveals that carrageenan-based hydrogels have potential applications in BTE (Santo et al., 2009). Yegappan et al. have proven the utility of the injectable carrageenan-based hydrogels for bone regeneration applications (Yegappan, Selvaprithiviraj, Amirthalingam, & Jayakumar, 2018; Yegappan et al., 2019). Besides, sprayable and injectable carrageenan-based hydrogels have been reported for bone regeneration applications (Tavakoli, Kharaziha, Kermanpur, & Mokhtari, 2019). To achieve better osteogenic properties, biocomposite contains carrageenan and hydroxyapatite will be ideal candidates for bone tissue construction (Gonza´lez & Ossa, 2017). Recently, k-carrageenan and nano-HA containing injectable composite scaffold were developed for bone regeneration applications. In vitro studies on the osteoblast cells shows that developed scaffold can induce mineralization. Besides this, enhancement in the alkaline phosphate activity was observed (Ocampo et al., 2019). Mirza et al. have developed a nanocomposite scaffold containing gum Arabic, K-carrageenan, and nanoHA. Slow degradation and higher protein adsorption were found on the composite scaffold. Further, the alizarin red S staining assay demonstrates the apatite forming capacity of the scaffold. Besides, enhancement in the ALP activity and osteogenic genes including osteocalcin, and osteopontin was observed (Mirza et al., 2020). Besides this, there are several biocomposites containing carrageenan and HA that has already well-proven for bone regeneration applications which include collagen-k-carrageenanHA, carbon nanotube-carrageenan-chitosanstrontium (Sr) and cerium (Ce) substituted HA, carrageenan-HA-graphene oxide, collagen-iota

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carrageenan-HA, and k-carrageenan-HA-maleic anhydride-casein-doxorubicin, etc (Chen et al., 2020; Feng et al., 2017; Liu et al., 2014; Nogueira, Maniglia, Bla´cido, & Ramos, 2019; Praphakar et al., 2019; Sumathra, Rajan, Amarnath Praphakar, Marraiki, & Elgorban, 2020).

16.3.4 Fucoidan Fucoidan is a sulfated polysaccharide, obtained from marine brown seaweed algae. Fucoidan is a complex structure of the L-fucose and sulfate ester group. It has been found that the biological activity of the fucoidan is mainly dependent on the degree of sulfation and the molecular weight (Li, Lu, Wei, & Zhao, 2008; Rao, Saptami, Venkatesan, & Rekha, 2020; Wang et al., 2019). Fucoidan has many biomedical applications including antiviral, antiinflammatory, antithrombotic, and antioxidant, etc. (Cho, Jung, Kim, Choi, & Kim, 2009; Venkatesan et al., 2015). Fucoidan is playing a vital role in BTE due to its important properties including its nontoxic, biocompatible, and biodegradable nature. In addition to this, it has been proven that fucoidan can induce osteogenic differentiation (Dinoro et al., 2019; Kim, Yang, You, Shin, & Lee, 2018; Pajovich & Banerjee, 2017). In vitro cell interaction with fucoidan was carried out with human adiposederived stem cells (hADSCs). Enhancement in the ALP was observed. Besides, enhancement in the osteogenic gene markers including osteocalcin (OCN), osteopontin (OPN), type I collagen, and Runt-related transcription factor 2 (run-2) was observed. These results reveal that fucoidan can promote osteogenic differentiation (Park, Lee, Lim, & Lee, 2012). Fucoidan was isolated from Undaria pinnatifida brown algae was evaluated for BTE applications. (3-(4, 5-Dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide) (MTT) assay it is clear that cells are viable with the isolated fucoidan. Further, enhancement in the ALP activity was observed.

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Besides this, Alizarin red staining assay demonstrates that isolated fucoidan can induce mineralization (Cho et al., 2009). Besides, low molecular weight (LMW) fucoidan isolated from Sargassum hemiphyllum can promote in vitro and in vivo osteogenic differentiation (Hwang et al., 2016). Fucoidan-HA nanocomposite scaffold was developed to check its efficiency in BTE. Further, from the alizarin red S staining assay, it is cleared that, developed nanocomposite scaffold can induce higher content of mineralization compared to only HA (Jeong, Venkatesan, & Kim, 2013). Ahn et al. have proven osteogenic differentiation of the nanoHA-fucoidan composite scaffold. Initially, in vitro cell interaction studies were done with the ADSCs, they found an enhancement in the osteogenic gene markers. Further, in vivo studies on the rabbit model demonstrates that scaffold can form new bone (Young et al., 2016). To achieve synergistic osteogenic properties, a composite scaffold containing chitosan, natural nano-HA, and fucoidan was fabricated. Further, biological assays with periosteumderived mesenchymal stem cells proves that developed scaffold can promote osteogenic differentiation (Lowe, Venkatesan, Anil, Shim, & Kim, 2016). Lu et al. have reported osteogenic properties of the genipin cross-linked with fucoidan, nano-HA, and hydroxypropyl chitosan biocomposite scaffold (Lu, Lu, Chen, & Mi, 2019). Venkatesan et al. have proven the bone regeneration potential of the chitosan, alginate, and fucoidan-containing scaffolding system (Venkatesan, Bhatnagar, & Kim, 2014).

16.3.5 Ulvan Ulvan is a sulfated polysaccharide usually found in the cell walls of green algae. Similarly, like fucoidan, its biological properties are dependent on the degree of sulfation and molecular weight. Ulvan is widely used in

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the biomedical field due to its antidiabetic, antioxidant, antimicrobial, antihyperlipidemic, anticancer, and antifungal properties. Ulvan has attracted interest in BTE due to its nontoxicity, biocompatibility, and biodegradability. Besides this, physicochemical properties of the ulvan can be tailored and ulvan can be molded into various shapes which include sponges, hydrogels, spheres, matrices, etc., (Alves, Duarte, Mano, Sousa, & Reis, 2012; Dash et al., 2018; Kidgell, Magnusson, de Nys, & Glasson, 2019; Pangestuti & Kurnianto, 2017; Sudha, Gomathi, & Kim, 2020; Tziveleka, Ioannou, & Roussis, 2019; Tziveleka et al., 2020). Ulvan based biofunctionalized scaffold was developed by a photo-crosslinking method. In vitro cell interaction with MC3T3-E1 cell lines reveals that the scaffold can enhance ALP activity. Besides, the mineral formation was observed. This finding result illustrates that ulvan can be used as a bone implant (Dash et al., 2014; Toskas et al., 2012).

16.3.6 Gelatin Gelatin is a natural water-soluble polymer, which is obtained from animal collagen. Acid treatment and alkaline hydrolysis are the main techniques to obtain gelatin. Gelatin is used in BTE applications due to its biodegradable, nontoxic, low immunogenic, and biocompatible properties (Echave, Sa´nchez, Pedraz, & Orive, 2017; Hoque, Nuge, Yeow, Nordin, & Prasad, 2015). Several scaffolding systems including highly porous electrospun nanofibres, 3D micro, and nanoscaffolds, hydrogels, sponges, films, and microspheres of gelatin is already utilized for bone regeneration application (Aldana & Abraham, 2017; Cakmak et al., 2020; Echave et al., 2017; Fayyazbakhsh et al., 2017; Gentile et al., 2010; Ghorbani, Nojehdehian, & Zamanian, 2016; Isikli, Hasirci, & Hasirci, 2012; Jaiswal, Kadam, Soni, & Bellare, 2013; Kavya, Jayakumar, Nair, & Chennazhi, 2013; Lee et al.,

2016; Maji, Dasgupta, Pramanik, & Bissoyi, 2016; Nabavinia, Khoshfetrat, & NaderiMeshkin, 2019; Zuo et al., 2015). Poor mechanical strength is one of the issues when the utilization of gelatin for bone regeneration application. Hence, gelatin with hydroxyapatite composite scaffold was developed by using the solvent casting method. Finding results reveals that, the mechanical strength of the developed scaffold is comparable with the mechanical strength of natural bone (Hossan, Gafur, Karim, & Rana, 2015). Also, a controlled pore structure of the scaffolds containing HA and gelatin was developed for BTE applications by using combined solvent casting, freeze-drying, and lamination processes (Azami, Samadikuchaksaraei, & Poursamar, 2010). Research study has proven that gelatin and HA containing fibers can enhance collagen and calcium production (Salifu, Lekakou, & Labeed, 2017). Collagenderived gelatin and HA nanocomposite containing scaffold was developed by a conventional method. Further, In vitro cell interaction studies with MG-63 cells on the composite scaffold shows enhancement in the ALP activity and osteocalcin production than only gelatin material, suggests that biomimetic gelatin and HA composite scaffolds can be an ideal bone implant material (Kim, Kim, & Salih, 2005). In vivo studies done on the rabbit have proven that a scaffolding system containing double layered hydroxide with gelatin and HA can form new bone (Fayyazbakhsh et al., 2017). To achieve better osteogenic properties and higher mechanical strength microcapsules of nanoHA, alginate and gelatin were developed. From the biological assays, it is clear that the hydrophilic nature of the gelatin is enhancing the osteogenic properties of the scaffold (Nabavinia et al., 2019). Cakmak et al. has fabricated a composite scaffold containing polycaprolactone, gelatin, bacterial cellulose, and HA by using 3D printing technology. The average pore size of the scaffold was found to be nearly

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300 μm, which can easily facilitate cell proliferation (Cakmak et al., 2020). Dan et al. have researched the utility of scaffolding system containing chitosan, gelatin, and nano-HA for bone regeneration applications. Further, biological assays with MC3T3-E1 preosteoblast cell lines proves that, scaffold is nontoxic and viable with the cells. Besides this, scaffold has capacity to induce mineralization, hence developed scaffold is an ideal source for bone implantation (Dan et al., 2016). In addition, There are several biocomposite containing gelatin and HA has proven for bone regeneration applications which includes alginate-HAgelatin, chitosan-gelatin-alginate-HA, gelatincarboxymethyl chitosan-nano-HA, gelatin-HAchitosan-hMSCs, gelatin-HA-bioactive glass, PLGA-gelatin-HA, HA-gelatin with dexamethasone loaded PLGA, PLGA Nanofibres with gelatin-nano-HA, niobium pentoxide and HA loaded in polycaprolactone-gelatin (Gentile et al., 2010; Ghorbani et al., 2016; Lee, Kim, Heo, Kwon, & Choi, 2010; Li et al., 2014; Maji, Agarwal, Das, & Maiti, 2018; Marins et al., 2019; Sharma, Dinda, Potdar, Chou, & Mishra, 2016; Yadav & Srivastava, 2019; Yan et al., 2016).

16.4 Conclusion Biological macromolecules play an important role in the medical and food industries. Alginate, chitosan, fucoidan, carrageenan, ulvan, collagen, and gelatin-derived products are some on a commercial scale. However, several problems and research gaps still exist, which need to be identified and conduct the research (physicochemical properties, biocompatibility, mechanical strength, bioavailability, toxicity, and biodegradation). Different kinds of modern methods to prepare the artificial tissue using electrospinning and 3D bioprinting are an emerging area of research. Different formulation methods including microsphere,

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hydrogels, films, sponges, and capsules were adopted and optimized according to their biological applications. In BTE application, the foremost importance of bone marrow cell interaction, proliferation, differentiation, and adsorptions were studied in detail with different compositions of the biomacromolecules. Hence, biological macromolecules in the form of composite biomaterials will be the potential candidates for tissue engineering applications.

Acknowledgment The work was supported by a seed grant (YU)/Seed Grant from Yenepoya Research Centre, Yenepoya Deemed to be University, Mangalore, Karnataka.

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1002. Tytgat, L., Van Damme, L., Arevalo, M. d P. O., Declercq, H., Thienpont, H., Otteveare, H., . . . Van Vlierberghe, S. (2019). Extrusion-based 3D printing of photocrosslinkable gelatin and κ-carrageenan hydrogel blends for adipose tissue regeneration. International Journal of Biological Macromolecules, 140, 929
938. Tziveleka, L.-A., Ioannou, E., & Roussis, V. (2019). Ulvan, a bioactive marine sulphated polysaccharide as a key constituent of hybrid biomaterials: A review. Carbohydrate Polymers, 218, 355
370. Tziveleka, L.-A., Sapalidis, A., Kikionis, S., Aggelidou, E., Demiri, E., Kritis, A., . . . Roussis, V. (2020). Hybrid sponge-like scaffolds based on ulvan and gelatin: Design, characterization and evaluation of their potential use in bone tissue engineering. Materials, 13, 1763. Venkatesan, J., Anil, S., Kim, S. K., & Shim, M. S. (2016). Seaweed polysaccharide-based nanoparticles: Preparation and applications for drug delivery. Polymers (Basel), 8. Venkatesan, J., Bhatnagar, I., & Kim, S.-K. (2014). Chitosanalginate biocomposite containing fucoidan for bone tissue engineering. Marine Drugs, 12, 300
316. Venkatesan, J., Lowe, B., Anil, S., Manivasagan, P., Kheraif, A. A. A., Kang, K. H., & Kim, S. K. (2015). Seaweed polysaccharides and their potential biomedical applications. Starch-Sta¨rke, 67, 381
390. Venkatesan, J., Qian, Z.-J., Ryu, B., Kumar, N. A., & Kim, S.-K. (2011). Preparation and characterization of carbon nanotube-grafted-chitosan
natural hydroxyapatite composite for bone tissue engineering. Carbohydrate Polymers, 83, 569
577. Venugopal, J., Prabhakaran, M. P., Zhang, Y., Low, S., Choon, A. T., & Ramakrishna, S. (2010). Biomimetic

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hydroxyapatite-containing composite nanofibrous substrates for bone tissue engineering. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 368, 2065
2081. Wang, Y., Xing, M., Cao, Q., Ji, A., Liang, H., & Song, S. (2019). Biological activities of fucoidan and the factors mediating its therapeutic effects: A review of recent studies. Marine Drugs, 17, 183. Wu, T., Li, B., Wang, W., Chen, L., Li, Z., Wang, M., . . . Zhang, T. (2020). Strontium-substituted hydroxyapatite grown on graphene oxide nanosheet-reinforced chitosan scaffold to promote bone regeneration. Biomaterials Science, 8, 4603
4615. Yadav, N., & Srivastava, P. (2019). In vitro studies on gelatin/hydroxyapatite composite modified with osteoblast for bone bioengineering. Heliyon, 5, e01633. Yan, J., Miao, Y., Tan, H., Zhou, T., Ling, Z., Chen, Y., . . . Hu, X. (2016). Injectable alginate/hydroxyapatite gel scaffold combined with gelatin microspheres for drug delivery and bone tissue engineering. Materials Science and Engineering: C, 63, 274
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400. Yegappan, R., Selvaprithiviraj, V., Amirthalingam, S., Mohandas, A., Hwang, N. S., & Jayakumar, R. (2019). Injectable angiogenic and osteogenic carrageenan nanocomposite hydrogel for bone tissue engineering. International Journal of Biological Macromolecules, 122, 320
328. Young, A. T., Kang, J. H., Kang, D. J., Venkatesan, J., Chang, H. K., Bhatnagar, I., . . . Kim, S.-K. (2016). Interaction of stem cells with nano hydroxyapatitefucoidan bionanocomposites for bone tissue regeneration. International Journal of Biological Macromolecules, 93, 1488
1491. Zuo, Y., Liu, X., Wei, D., Sun, J., Xiao, W., Zhao, H., . . . Zhang, X. (2015). Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon. ACS Applied Materials & Interfaces, 7, 10386
10394.

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C H A P T E R

17 Biological macromolecules for drug delivery in tissue engineering Marcel Popa1,2 and Leonard Ionut Atanase2 1

“Apollonia” University of Iasi, Faculty of Dental Medicine, Iasi, Romania 2Academy of Romanian Scientists, Bucuresti, Romania

17.1 Introduction Tissue engineering, associated with the term regenerative medicine, is a relatively new field that uses a combination of cell biology, polymer and organic chemistry, medicine and pharmacology to prepare three dimensional (3D) systems in order to mimic the architecture of extracellular matrix (ECM) in order to induce growth, proliferation, and differentiation signals for the promotion of tissue repair or functional regeneration. Moreover, these systems are able to include, transport and release drugs or other active ingredients in the affected area. An effective system for tissue engineering requires appropriate component selection and therefore a suitable system is composed of three major elements: cells, drugs, engineered biomaterials and signaling molecules (growth factors) (Yousefzade, Katsarava, & Puiggali, 2020). This chapter aims to present some aspects for investigating the physicochemical and biological characterization of engineered biomaterials, based on biological macromolecules (polysaccharides and proteins), as scaffolds which are capable of

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00017-8

supporting the physiological activities of cells and are also capable of being loaded with biologically active principles so that can act as drug delivery systems (DDS) for tissue engineering and wound healing. The use of biodegradable polymers, for the preparation of these scaffolds, is preferred because they do not usually require surgical removal after the end of the treatment increasing thus the patient’s compliance. Depending on their nature, biodegradable polymers can be also classified into two categories: natural and synthetic. Natural polymers are biologically suitable materials in tissue engineering applications but the lack of purity and molecular compositional homogeneity has led, in numerous cases, to their combination with synthetic ones, as these polymers have satisfactory biocompatibility, lack of toxicity, and suitable mechanical properties for drug encapsulation and controlled delivery (Rey et al., 2020). DDS were designed in various shapes and sizes offering multiple drug release profiles. The most common DDS are particulate systems including micro and nanoparticles. However, for tissue engineering applications

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© 2022 Elsevier Inc. All rights reserved.

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17. Biological macromolecules for drug delivery in tissue engineering

are preferred other DDS which have the ability to mimic ECM. In this sense, fibrous scaffolds are interesting examples of nanosystems that are actually under investigation in tissue engineering as they exhibit a similar structure to protein nanofiber in the ECM. Generally, these fibers can be fabricated by three methods: electrospinning, self-assembly and phase separation. Among these, the electrospinning process has been particularly successful used, in recent years, for the preparation of fibers which best mimics the natural ECM in terms of micro to nanoscale topography due to a high porosity similar to it, a very important feature that decisively influences the release kinetics of the loaded principles (Koyyada & Orsu, 2020). Hydrogels are another category of intensively used biomaterials as DDS and cell delivery in tissue engineering (Spicer, 2020; Zhao, Liu, Zhang, Yin, & Pei, 2020). Obtained from hydrophilic polymers, they consist of threedimensional networks of polymer chains built by connecting linear chains by covalent bridges or by ionic or physical interactions (hydrogen bonds, Van de Waals forces, etc.). Their naturally strong hydrophilic and reticulated structure allows them to absorb large amounts of water without dissolving. The biopolymers, polysaccharide and proteins, are preferred as precursors for the preparation of hydrogels for tissue regeneration and drug delivery as their unique properties include: biocompatibility, biodegradability, low cytotoxicity, the possibility to tailor the hydrogel into an injectable gel and their similarity to physiological environment. However, biopolymer-based hydrogels have some limitations regarding especially the mechanical properties and thus their combination with synthetic polymers is often experimented. Among all types of prepared hydrogels, injectable hydrogels are the most suitable for practical applications in tissue engineering and therefore only this type will be reviewed in this chapter.

17.2 Drug-loaded electrospun fibers used in tissue engineering applications and drug delivery In the electrospinning process, an electrical field is applied to a polymer solution in order to produce electrospun micro/nanofibers. Different types of electrospinning techniques exist: (1) basic electrospinning; (2) blend electrospinning; (3) coaxial electrospinning; (4) emulsion electrospinning; (5) melt electrospinning; and (6) gas jet electrospinning (Stojanov & Berlec, 2020). Electrospun fibers (ESF) can be produced from various polymeric materials, which can be biopolymers, synthetic, or a combination of both and have several practical applications. They can serve as DDS or carriers of cells for tissue engineering. Also, because it protects the wound from external harmful effects, mainly microbial infection and keep proper moisture and gas exchange at the wound site, the ESF are suitable as wound dressings. For these applications, the ideal properties of the ESFs are: good mechanical strengths, biocompatibility, high drug loading efficacy, tunable porosity, ECM mimicking, and high ratio between surface and volume. In the literature are numerous studies concerning the preparation of ESFs which were reviewed very recently (Aavani, Khorshidi, & Karkhaneh, 2019; Bhattarai, Bachu, Boddu, & Bhaduri, 2019; Bose, Koski, & Vu, 2020; Catoira, Fusaro, Di Francesco, Ramella, & Boccafoschi, 2019; Contreras-Caceres et al., 2019; Ding et al., 2018; Iacob et al., 2020; Jain, Shetty, & Yadav, 2020; Kalantari, Afifi, Jahangirian, & Webster, 2019; Najafiasl, Osfouri, Azin, & Zaeri, 2020; Rahmati et al., 2020; Sun et al., 2019; Ye, Kuang, You, Morsi, & Mo, 2019). The high number of review articles concerning this subject is a direct proof of the fact that this field of research has an actual and practical interest. Generally, the researchers were interested by the optimization of the electrospinning parameters in order to obtain fibers with the smallest diameter or with the highest mechanical properties. Therefore in

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17.2 Drug-loaded electrospun fibers used in tissue engineering applications and drug delivery

this section, it was taken into account only the studies concerning the preparation of drugloaded ESFs, fabricated from pure biopolymers (polysaccharides and proteins) or in combination with synthetic polymers, with practical applications as DDS or for tissue engineering.

17.2.1 Drug-loaded polysaccharidesbased electrospun fibers Single nozzle electrospinning technique was used for the preparation of dual drug composite nanofibers based on chitosan (CS), poly (caprolactone) (PCL) and sodium tripolyphosphate, as crosslinking agent (Wang et al., 2011). Both Rhodamine B and Naproxenloaded CS-based nanoparticles (NPs), with diameters in the range of 300
500 nm, were dispersed in drug-loaded PCL-based fibers at a proportion of 40%. As expected, a more controlled drug release was noticed for the composite fibers in comparison with the bare NPs. Another type of composite ESFs were fabricated from CS, crosslinked with genipin, with dispersed hydroxyapatite (HA) NPs for bone tissue engineering (Frohberg et al., 2012). The crosslinking process increased the fiber diameters from 227 to 335 nm. It was demonstrated that such scaffolds facilitate the proliferation, differentiation and maturation of osteoblast-like cells being therefore suitable for bone tissue engineering applications. Similar composite fibers were prepared by coaxial electrospinning of a mixture of CS, PCL, gelatin and HA NPs (Ahmadi, Modaress-Pezeshki, Irani, & Zandi, 2019). The average diameters of these nanofibers were between 200 and 260 nm. Moreover, a high biocompatibility for osteoblast cells was observed leading to the conclusion that these scaffolds can be promising materials for bone tissue engineering. Topsakal et al. (2018) have also prepared composite nanofibers based on CS, polyurethane (PU) and β-tricalciumphosphate (β-TCP)

395

for bone tissue engineering applications. Amoxicillin was loaded in these fibers with an efficacy of around 62% and the cumulative release after 24 h was around 60%. Also for bone tissue engineering, Aidun et al. (2019) have recently prepared composite nanofibers based on CS, PCL, collagen and graphene oxide (GO). GO had no influence on the fiber’s diameter but the hydrophicity, bioactivity, attachment and proliferation of cells increased with increasing the GO content. A DDS based on CS, PCL and dipyridamole, as a model anticoagulant agent, was prepared by electrospinning (Repanas, Glasmacher, & Mavrilas, 2016). Smooth cylindrical submicronic fibers, with an average diameter of around 560 nm, were obtained. Concerning the drug release study, an initial burst release was noticed during the first 10 h which are suitable for clinical cardiovascular applications. Another type of DDS prepared by electrospinning was based on a mixture of CS with poly (vinyl alcohol) (PVA) and loaded with curcumin (Thien, Quyen, Tri, Thoa, & Tham, 2016). The average diameter of the ESF was in the range of 100
250 nm, as a function of the molecular weight of the CS. Moreover, the authors have demonstrated that the molecular weight of the CS had also a direct influence on the drug release rate, the cumulative release being smaller for the 200 kDa CS than for 150 kDa. A more complex DDS was studied by Li, Wang, Yang, Liu, and Zhang (2018). These authors have obtained by electrospinning double layer nanofibers having a first layer composed of a mixture of PCL and mupirocin and a second layer based on lidocaine HCl-loaded CS. This double layer structure led to an initial burst release of lidocaine HCl whereas mupirocin had a controlled release. Moreover, these nanofibers had highly effective antibacterial properties and were nontoxic. However, further in vivo tests are necessary in order to establish their applicability as wound dressing material.

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Mucoadhesive nonwoven fibers based on CS, poly(ethylene oxide) (PEO) and cysteine were loaded with vancomycin and amphotericin B, as antibacterial and antifungal agents, respectively (Karimi, Moradipour, Azandaryani, & Arkan, 2019). The average diameter of the fibers was around 200 nm and the drug encapsulation decreased this value. In vitro drug release studies showed that vancomycin was released at a high rate in the first 24 h, whereas a more controlled release was noticed for the antifungal drug. Moreover, these drug-loaded fibers had good antibacterial and antifungal properties being suitable for tissue engineering applications. In a more recent study, berbeine, a plant extract, was loaded in CS/PEO fibers obtained by electrospinning (Jafari, Tabaei, Rahimi, Taranejoo, & Ghanimatdan, 2020). An average diameter of around 97 nm was noticed by SEM. The water uptake ranged from 55.4% to 93.5%. This DDS showed a good in vitro anticancer effect against breast and cervical cancer cells. Metformin-loaded PCL/CS, crosslinked with glutaraldehyde, fibers were prepared by electrospinning with possible application in bone regeneration (Zhu, Ye, Deng, Li, & Wu, 2020). Beads-free fibers with smooth surface and diameters under 500 nm were obtained. The drug encapsulation reduced the diameter of these nanofibers. Moreover, it appeared that these membranes could boost the osteogenic mineralization of BMSCs. The same starting system, CS/PCL was used for the preparation of fibers loaded with a all-trans retinoic acid (atRA), as a signaling molecule, with possible application in tracheal tissue engineering (O’Leary et al., 2020). The obtained fibers had average diameters in the range of 181
197 nm. The atRA release from these scaffolds has as effect an increase in mucociliary gene expression which is an advantage for tissue engineering. Very recently, a coaxial electrospinning method was applied for the preparation of chitin-lignin fibers coated with PCL (Abudula et al., 2020). Penicillin and streptomycin were

both loaded in these core-shell nanofibers. The presence of PCL in the composition of nanofibers has as effect a better control of drug release and the drug-loaded fibers exhibited a superior antibacterial effect against both grampositive and -negative bacteria without observable cytotoxicity. These features make these nanofibers god candidates for wound dressing applications. Drug-loaded core-shell nanofibers, based on poly(vinylpyrrolidone) (PVP) shell and ethyl cellulose (EC) core, were fabricated using a coaxial electrospinning procedure (Li, Wang, Zu, & Williams, 2014). Quercetin, a natural polyphenol, was used as a model drug. By SEM it was possible to observe the submicronic size and also the smooth surface of these fibers. An initially fast drug release was noticed, due to the hydrophilic PVP shell, followed by a sustained release controlled by the EC core. In another study, ESFs of EC, poly(N-isopropylacrylamide) (PNIPAM) and a mixture of them were prepared in the absence and in the presence of ketoprofen (Hu et al., 2016). Highly uniform and cylindrical fibers, with no beads, were obtained. The diameter of the pure ECbased fibers was 343 nm whereas an average diameter of 679 nm was noticed for pure PNIPAM fibers. Moreover, the drug encapsulation had no effect on the morphology of these fibers but their diameters increased from 400 to 532 nm for a PNIPAM/EC ratio of 1:2. Drug release rate was higher at 25 C than at 37 C indicating a thermoresponsiveness behavior due to the presence of PNIPAM. This sensitivity can be of interest in tissue engineering applications. Hydroxypropyl cellulose (HPC) was mixed with PU in order to obtain electrospun nanofibers which were further loaded with domepezil HCl having possible application as transdermal DDS (Gencturk et al., 2017). Pure HPC fibers could not be obtained, independent of the solution concentration. The PU/HPC fibers at rations of 10:1; 10:2 and 10:4 exhibited

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17.2 Drug-loaded electrospun fibers used in tissue engineering applications and drug delivery

smooth surfaces with submicronic diameters. The drug loading led to an increase of the fiber diameter but also to the apparition of some beads on the surface of the fibers. The drug release was controlled by a diffusion mechanism and followed the Korsmeyer-Peppas kinetic model. The Korsmeyer-Peppas kinetic model describes well the release process and the value of the exponential factor n proves the diffusion mechanism of transport of the drug through the polymer matrix. Carboxymethyl cellulose (CMC)-based fibers, containing Ag NPs, with average diameters around 20 nm, were prepared by electrospinning in the presence of PEO (Shi, Wang, Lan, Zhang, & Shao, 2016). Ag NPs were uniformly distributed on the surface of fibers and the obtained membranes exhibited excellent antimicrobial activity. ESFs were also fabricated from CMC-graft-poly(methyl acrylate) copolymer in the presence of PEO and loaded with tetracycline HCl (Esmaeili & Haseli, 2017). A sustained drug release was noticed for 72 h and these drug-loaded nanofibers exhibited an excellent bactericidal activity. More recently, a waterbased and needless electrospinning method was used for the preparation of drug-loaded crosslinked fibers based on CMC, poly(ethylene glycol) (PEG) and diclofenac, as a model drug (Kureˇciˇc et al., 2019). Butanetetracarboxylic acid, as crosslinking agent, and sodium hypophosphite as catalyst, were studied. As expected, the drug release was affected by the increase of the crosslinking density. Moreover, it appeared that the concentration of the crosslinking agent had an influence also on the fiber diameters. The controlled and prolonged drug release, over 48 h, is an indication that these nanofibers can be useful for the treatment of chronic wounds and in regenerative medicine. Tetracycline HCl was loaded in cellulose acetate (CA)-based fibers (Sultana & Zainal, 2016). At a concentration of 10% CA, nanofibers with beads were produced whereas the increase of this concentration led to beads-free nanofibers. These

397

drug-loaded fibers showed antibacterial activity and had high water uptake properties therefore they can be suitable for wound healing applications. In another study, coaxial electrospinning was used for the fabrication of core-shell CAbased fibers as DDS (Mehrabi, Shamspur, Mostafavi, Saljooqi, & Fathirad, 2017). Pure CA was used for the shell of these fibers whereas the core was formed by the mixture of CA with either acetaminophen or morphine. As expected, a controlled drug release was noticed for these fibers. Other drug-loaded fibers, based on CA and gelatin (Gel), were prepared by a wet-electrospinning method (Farzamfar et al., 2018). The model drug was gabapentin, which is an anticonvulsant controlling the neuropathic pains. Based on their morphology, the fibers containing 6% drug were studied for the in vivo sciatic nerve defect model. The obtained results demonstrated the applicability of these fibers for neutral tissue applications. Very recently, other CA/Gel ESFs, loaded with berberine in this case, were prepared for the treatment of diabetic foot ulcer (Samadian et al., 2020). The average diameter of these fibers was around 500 nm. Moreover, they exhibited antibacterial activity against gram-positive and -negative bacterium. Furthermore, the in vivo studies demonstrated that these drug-loaded fibers enhanced the wound healing process. CA was also mixed with carboxylated cellulose nanocrystals (CCNCs) in order to obtain composite fibers as DDS of tetracycline HCl (Hu, Qin et al., 2018). It appeared that the introduction of CCNCs increased the thermal stability, mechanical properties and drug delivery behavior. In fact, a drug cumulative release of around 94% within 420 h was noticed indicating that these fibers are promising materials for controlled drug delivery. Tetracycline HCl was also loaded in CA/PVA fibers obtained by a waterbased electrospinning process (SouriyanReyhanipour, Khajavi, Yazdanshenas, Zahedi, & Mirjalili, 2018). Fibers with a uniform shape and a narrow diameter of around 160 nm were observed by SEM. Excellent antibacterial activity was also noticed indicating the applicability of

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17. Biological macromolecules for drug delivery in tissue engineering

these scaffolds as wound dressing with controllable drug delivery. PVA solutions were also blended with sodium alginate (SA) in order to obtain fibers by electrospinning (Arthanari et al., 2016). An optimum ratio SA/PVA of 3:7 led to gatifloxacin HCl-loaded uniform and smooth fibers. The release of this third generation antibiotic was sustained for 24 h which can be an advantage for wound healing applications. In another study, PVA was combined with SA, with different degrees of sulfation, for the preparation of electrospun scaffolds capable of delivering heparinlike growth factors (Mohammadi et al., 2019). The thermal crosslinking of these fibers increased the mechanical properties. Moreover, the sulfation process improved the binding and entrapment of the growth factor which is a clear advantage for tissue engineering. Recently, SA/ PVA ESFs, at a volume ratio of 20/80, were also obtained and loaded with herbal extracts of

Calendula Officinolis which is used in both wound healing and as antiinflammatory agent (Tahami, Nemati, Keshvari, & Khorasani, 2020). A series of fibers was obtained by varying the applied potential. The loading of the herbal extract led to an increase of the fiber sizes. From the SEM micrographs it appeared that the fibers had high porosity and high surface to volume ratio which are appropriate features for tissue engineering and wound dressing. Ciprofloxacin HCl was used as model drug for the preparation of alginate nanofibers in the presence of PEO 1000 kDa and Pluronic F127, as surfactant (Kyziol, Michna, Moreno, Gamez, & Irusta, 2017). Beads-free fibers were obtained only for 3 and 4wt.% alginate in combination with 2% PEO 1000 kDa, 1% Pluronic F-127. The evolution of the fiber’s morphologies as a function of the alginate, PEO 1000 kDa and Pluronic F-127 concentrations is illustrated in Fig. 17.1.

FIGURE 17.1 SEM images of electrospun alginate fibers: (A) 3.0wt.% AL, 1.5wt.% PEO 1000 kDa, 1.0wt.% Pluronic F127, (B) 3.0wt.% AL, 2.0wt.% PEO 1000 kDa, 0.5wt.% Pluronic F-127, (C) 3.0wt.% AL, 2.0wt.% PEO 1000 kDa, 1.0wt.% Pluronic F-127, (D) 4.0wt.% AL, 1.5wt.% PEO 1000 kDa, 1.0wt.% Pluronic F-127, (E) 4.0wt.% AL, 2.0wt.% PEO 1000 kDa, 0.5wt.% Pluronic F-127, (F) 4.0wt.% AL, 2.0wt.% PEO 1000 kDa, 1.0wt.% Pluronic F-127. Source: Reprinted from Kyziol, A., Michna, J., Moreno, I., Gamez, E., & Irusta, S. (2017). Preparation and characterization of electrospun alginate nanofibers loaded with ciprofloxacin hydrochloride. European Polymer Journal, 96, 350
360 with permission of Elsevier.

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17.2 Drug-loaded electrospun fibers used in tissue engineering applications and drug delivery

Moreover, the drug loading increased the size of these cylindrical fibers from 109 to 161 nm. Around 24% of the drug was released within 20 h in PBS at pH 7.4 and it appeared that these DDS are promising candidates for biomedical applications. In another study, PEO was mixed with alginate and soy protein isolated (SPI) for the preparation of smooth and uniform vancomycin-loaded fibers with diameters around 200 nm (Wongkanya, Chuysinuan, Pengsuk, & Nooeaid, 2017). A sustained drug release was noticed during 48 h of immersion in PBS. These drug-loaded fibers, having also antimicrobial activity against Staphylococcus aureus for 24 h, could have a promising application in tissue engineering. An antiseptic drug, cephalexin, was loaded in halloysite nanotubes (HNT) reinforced alginate-based nanofibrous scaffolds (De Silva & Dissanayake, 2018). The diameter of these ESFs, which mimic the natural ECM, ranged from 40 to 522 nm. The incorporation of HNT increased the mechanical properties of the nanofibers. Moreover, they exhibited excellent antimicrobial properties. These features indicate the great potential of these scaffolds to be used as artificial ECM for tissue engineering applications. Electrospinning was very recently used for the fabrication of a multilayer membrane based on PCL, as an external layer, and ZnO NPs dispersed in physically crosslinked alginate, as internal layer (Dodero, Alloisio, Castellano, & Vicini, 2020). This alginate layer can promote the cell viability, allow exudate removal and gas exchanges. In order to study the ability of this membrane as DDS, two model molecules, such as methylene blue and methyl orange, were loaded. The release studies showed that these membranes can be used as simple and cost-effective wound healing patches. Dextran (Dex)/poly(lactic-co-glycolic acid) (PLGA) fibers were obtained by a coaxial electrospinning method and loaded with vascular endothelial growth factor (VEGF) for vascular

399

tissue engineering (Jia et al., 2011). PLGA was the shell of these fibers whereas bovine serum albumin (BSA) was loaded as an active principle in the Dex core. From the in vitro release profiles, it appeared that VEGF was released during 28 days. Moreover, these membranes promote spreading and proliferation of cells which is a potential advantage for vascular tissue engineering. In another study, fucoidan was incorporated in Dex/pullulan ESFs and then VEGF was loaded in order to promote angiogenesis (Rujitanaroj, Aid-Launais, Chew, & Le Visage, 2014). Trisodium trimetaphosphate was used as a chemical crosslinker and the average diameter of the fibers was around 500 nm. In this case, a sustained release of VEGF up to 7 days was noticed and the release rate was influenced by the crosslinking degree, as expected. In vivo subcutaneous implantation of these scaffolds showed enhanced angiogenic response. Electrospun acetalated Dex fibers were encapsulated with resiquimod, an immunomodulatory tall-like receptor agonist (Borteh et al., 2013). Fibers had a ribbon-like morphology but without beads. Almost 100% of drug was released after 60 h in PBS at pH 7.4 from the last degrading fibers. On the contrary, only 60% of the drug was released after 6 days from the low degrading scaffolds. This controlled temporal drug release makes these scaffolds excellent candidates for tissue engineering applications. Another DDS, based on tetracycline HCl loaded in ESFs was fabricated starting from a mixture of Dex, CA and PCL (Liao et al., 2015). Submicronic smooth fibers, with average diameters around 700 nm, were obtained for the drug-loaded fibers. A high antibacterial activity was assessed and the presence of Dex enhanced the cell’s proliferation and adhesion which is a clear requirement for wound dressing and skin engineering applications. Acetalated Dex-based scaffolds, loaded with saquinovir, were also prepared by electrospinning and further processed into

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17. Biological macromolecules for drug delivery in tissue engineering

microconfetti (Collier et al., 2016). The drug release was tuned via polymer degradation tests. An in vivo drug release of over one week was noticed. This injectable microconfetti DDS can be used for the long acting and release of different hydrophobic drugs. Coaxial emulsion electrospinning was used for the preparation of Dex/PVA fibers loaded with ciprofloxacin (Moydeen, Padusha, Aboelfetoh, Al-Deyab, & El-Newehy, 2018). The core-shell structure of the nanofibers,

with diameters in the range of 400
600 nm, was determined by TEM. As both polymers are hydrophilic, a physical crosslinking step, in oven at 120 C for 6 h, was necessary. As a consequence, a sustained drug release following a non-Fickian diffusion mechanism was noticed. Several other polysaccharide-based ESFs were fabricated by electrospinning and the principal components of these DDS are provided in Table 17.1.

TABLE 17.1 Electrospun DDS based on miscellaneous polysaccharides. Components

Drug

Fiber’s diameter (nm)

Application

Reference

Hyaluronic acid PLGA

Ginsenoside Rg3

910
1220

Wound healing

Cheng et al. (2013)

Pullulan B-cyclodextrin

R-( 1 )-limonene

370

DDS

Fuenmayor et al. (2013)

Pullulan Gel

Adipose-derived stem cells

n.d

Tissue engineering

Nosoudi et al. (2020)

Pullulan Gel PHB

BSA

470
590

DDS

Sun, Guo, Liu, and Yu (2020)

Guar gum PVA

Acalypha indica

100
350

Wound healing

Jenifer, Kalachaveedu, Viswanathan, and Gnanamani (2018)

Pectin CS PVA

Tetracycline HCl

100
200

Skin tissue engineering

Lin, Chen, Chang, and Ni (2013)

Xanthan gum CS oligomeres

Diclofenac

100
500

DDS

Mendes, Strohmenger, Goycoolea, and Chronakis (2017)

Xanthan gum CS

Curcumin

910

DDS

Shekarforoush, Ajalloueian, Zeng, Mendes, and Chronakis (2018)

Starch PVA/PEO

Ampicilin

100
500

DDS

Tang et al. (2016)

Rice starch PVA

Chlorpheniramine 191

DDS

Jaiturong et al. (2018)

Corn starch

Carvocrol

15
3400

DDS

Tampau, Gonza´lez-Martinez, and Chiralt (2017)

Native saga starch PVA

Paracetamol

90
150

DDS

Tuah, Chin, and Pang (2021)

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17.2 Drug-loaded electrospun fibers used in tissue engineering applications and drug delivery

17.2.2 Drug-loaded protein-based electrospun fibers Two different classes of drugs, aceclofenac and pantaprozole, were simultaneously loaded in zein/eudragit S 100 fibers (Karthikeyan, Guhathakarta, Rajaram, & Korrapati, 2012). Uniform and smooth fibers, with sizes in the range of 50
200 nm, were fabricated by a single nozzle electrospinning process. An in vitro sustained release was noticed during 8 h. Moreover, in vivo test confirmed that the coadministration of these drugs reduced the gastrointestinal toxicity. In another study, coaxial electrospinning was used for the fabrication of core-shell drug-loaded fibers (Yu et al., 2013). The external layer was generated by pure zein whereas the core of the fibers was produced by a mixture of zein with ketoprofen, as a model drug. These fibers had a ribbon-like morphology, a smooth surface and average diameters between 680 and 860 nm, as a function of the sheath-to-core flow rate ratios. In vitro drug release studies indicated a sustained release over a period of 16 h via a diffusion mechanism, without any initial burst effect. Zein was also mixed with PCL in order to obtain composite fibers by using an oriented electrospinning technology (Hu, Yang et al., 2018). Poly (ε-lysine) (EPL) was added to enhance the antibacterial properties of the obtained fibers. For a zein/PCL ratio of 70/30, the fiber average diameter was 460 nm and the increase of the PCL amount led to a decrease of fiber’s diameter. The addition of EPL at a concentration of 2%
4% decreased the fiber’s diameter to 220 nm but a further increase up to 20%, led to an increase up to 530 nm. Good antibacterial effects against Listeria monocytogenes was noticed which is favorable for wound dressing. Another type of drug-loaded composite nanofibers was based on zein, GO and tetracycline HCl (Asadi, Ghaee, Nourmohammadi, & Mashak, 2020). The fiber diameters decreased from 230 to 137 nm with increasing the GO

401

content from 0 to 1.5wt.%. By TEM it was possible to notice that GO particles were homogenous distributed inside the zein nanofibers leading thus to improved mechanical properties. A sustained drug release was also observed. Moreover, these fibers had good antibacterial properties and led to quicker wound healing. Very recently, composite fibers based also on zein and inorganic bioactive glasses were prepared by electrospinning (Mariotti, Ramos-Rivera, Conti, & Boccaccini, 2020). The average diameter of these fibers, with good mechanical properties, was around 800 nm. Cell biology studies confirmed that the cell’s proliferation was enhanced due to the presence of bioactive glasses. Moreover, these fibers showed antibacterial properties and therefore they can represent a novel system suitable for tissue engineering applications. Pure zein fibers, loaded with α-bisobolol (BIS), were obtained after crosslinking with citric acid (El-Lakany et al., 2019). Beads-free fibers, with average diameters of around 181 nm, were noticed by SEM. The crosslinking process had an expected influence on both swelling degree and drug release. By increasing the BIS concentration, the cell’s adhesion was improved. Moreover, the antiinflammatory and proliferation effects of these scaffolds could be regarded as important features for tissue regeneration and wound healing processes. Silk fibroin (SF)-based patches were prepared for the loading of two growth factors PDGF and TGF (Pignatelli et al., 2018). By SEM, it appeared that the average diameter of the fibers was around 530 nm and they had a smooth surface. The crystallinity of fibroin controlled the release kinetics of these two growth factors. In another study, SF and PLGA were used for the preparation of nanofiber scaffolds, loaded with human bone morphogenetic protein (rhBMP2) and dexamethasone (DXM), by a coaxial electrospinning process (Yao et al., 2019). These core-shell nanofibers had an average diameter around 600 nm. Moreover, an

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early burst effect of DXM was noted followed by a sustained release of rhBMP2 which might be of interest for bone regeneration. An interesting study was carried out by Qian et al. (2020) regarding the preparation of SF fibers grafted onto surface with surface-animated liposomes encapsulating leptin for oral mucosa regeneration. SEM images showed a smooth surface and a fiber’s diameter around 800 nm. Moreover, the liposomes grafted did not affect the morphology of the fibers but increased both stability and mechanical properties. Furthermore, these fibers had angiogenic abilities and promoted the oral mucosa regeneration. Beads-free fibers based on SF and Gel (10%) were loaded with thyme essential oil (TEO) and doxycycline monohydrate (DCMH), as antibacterial agents (Chomachayi et al., 2018). The addition of Gel increased the fiber’s diameter from 182 to 380 nm. The release studies showed a burst release of TEO in the first 3 h whereas DCMH had a sustained release during 48 h indicating that these fibers are suitable for application in skin tissue engineering. Gel/poly(epichlorohydrin-co-ethylene oxide) (PECO) fibers were loaded with diclofenac were prepared by forcespinning (Mamidi et al., 2017). Beads-free fibers, with average diameters around 300 nm, were obtained at a Gel:PECO ratio of 4:1. Gel addition increased the pore size of these fibers. Moreover, they showed a prolonged drug release and had good cellular viability, features which are necessary for wound healing and skin tissue engineering. The same research group had prepared, using the same procedure, fibers based on Gel and zein, at a 1:4 ratio (Mamidi, Romo, Gutieres, Barrera, & Elias-Zuniga, 2018). In this case, berberine was used as a model drug. Very recently, triaxial electrospinning technique was used for the preparation of drug-loaded fibers based on PCL (core layer), PLGA (sheath layer) and Gel (intermediate layer) (Nagiah, Murdock, Bhattacharjee, Nair, &

Laurencin, 2020). Dual drug, rhodamine (RhB) and fluorescein isothiocynate BSA, encapsulation and release were studied. The addition of these two drugs decreased the mean diameter of the fibers from 1.88 μm to around 1.2 μm. The presence of small molecule RhB improved the release of BSA-FITC. These tri-axial fibers promoted the growth of mesenchymal stem cells (MSCs) and therefore may suitable for regenerative engineering applications. Collagen was added to poly(lactic acid) (PLA) and the obtained fibers were loaded with two drugs, such as: irgasan (antibacterial) (IRG) and levofloxacin (antibiotic) (LEVO) (Barrientos et al., 2017). The addition of collagen decreased the diameter of the fibers from 2.73 to 1.67 μm. Moreover, the loading of IRG has no influence on the fiber’s diameter whereas the LEVO decreased by half the mean diameters. The SEM micrographs of these fibers are given in Fig. 17.2. Furthermore, collagen had an effect on the drug release rate. A high antibacterial efficacy was also showed by the scaffolds. In another study, collagen/PCL fibers loaded with gentamicin were fabricated by elctrospinning for skin tissue engineering (Khodir, Razak, Ng, Guarino, & Susanti, 2018). The incorporation of drug did not affect the average diameters of the fibers which was around 140 nm. An initial burst effect was noticed for the drug release followed by a sustained release during 72 h. Emulsion electrospinning was used for the preparation of core-sheath fibers based on PCL and SF (Li et al., 2011). Phallaidin conjugated with fluorescein isothiocyanate was studied as active principle. The fiber diameters decreased from 562 to 274 nm with the increasing the SF concentration of the electrospinning solution. These scaffolds promoted the cell’s adhesion and proliferation which is a clear advantage for tissue engineering applications.

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17.3 Drug-loaded injectable hydrogels used in tissue engineering applications and drug delivery

403

FIGURE 17.2 SEM images of: (A) PLA Unloaded, (B) PLA-IRG, (C) PLA-LEVO, (D) PLA-Collagen, (E) PLA-CollagenIRG, and (F) PLA-Collagen-LEVO. Source: Reprinted from Barrientos, I. J. H., Paladino, E., Szabo, P., Brozio, S., Hall, P. J., Oseghale, C. I., . . . Lamprou, D. A. (2017). Electrospun collagen-based nanofibres: A sustainable material for improved antibiotic utilization in tissue engineering applications. 531(1), 67
79, article in open access.

Very recently, keratin/PLLA fibers loaded with 5-fluorouracil (5-FU) were fabricated by electrospinning (Zhang, Li et al., 2020). The drug encapsulation led to a decrease of the average diameters from 1.5 to 0.81 μm. By TEM it was showed that the keratin and 5-FU are mainly located in the inner part of the fibers. Moreover, these fibers showed a pH-sensitive behavior which led to a difference on the drug release amount between acidic and neutral environment.

17.3 Drug-loaded injectable hydrogels used in tissue engineering applications and drug delivery Polymeric hydrogel-like scaffolds are very attractive materials for tissue engineering applications being effective in repair and regeneration of a large variety of tissues and organs, but also

being able to transport therapeutic agents in the affected areas (Zhang, Yu et al., 2020). Those based on biopolymers (polysaccharides, proteins), as well as those based on synthetic polymers are biocompatible, permeable to oxygen and nutrients, have physical properties similar to the characteristics of the native ECM, and these characteristics can be modeled depending on the desired application: repair and regeneration treatment of tissues, such as: bone, cartilage, muscle, and skin, as well as delivery of active principles (drugs, cells) for the treatment of inflammatory and infectious diseases and cancers (Baumann et al., 2009; Lee, 2018). Two ways of introducing hydrogels for tissue engineering have emerged: (1) the implantation of preformed hydrogels at a desired site in the body and (2) injection of a formulation to a targeting site where the in situ gelation will occur.

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17. Biological macromolecules for drug delivery in tissue engineering

The first route of administration demands, however, an invasive surgical procedure that can cause the patient’s pain and discomfort and suppose cost and time. As a consequence, the clinical uses of preformed hydrogels are less preferred. Contrary, the injectable hydrogels have the benefits to overcome such drawbacks because they can be injected with minimal invasiveness into target sites and can be used for irregularly shaped sites where gelling occurs in situ (Moreira et al., 2014). As a consequence, this type of hydrogel administration are preferable because it is more comfortable, involve less pain, have a faster recovery period, lower costs, fewer complications and side effects (Mellati & Akhtari, 2018). In the literature specific to this field, several reviews have appeared in the last decade that describe different methods to obtain injectable hydrogels, their applications, aspects regarding injectability and their biomaterial properties (Lee, 2018; Liang, Bae, & Kurisawa, 2019; Liu et al., 2017; Mellati & Akhtari, 2018; Sun, Nan, Jin, & Qu, 2020; Upadhyay, 2017). Injectable hydrogels were formulated to serve as a temporary and artificial ECM, so it should mimic the structure and composition of natural ECM, which assumes that it must ensure a favorable microenvironment for cell survival and growth, to determine a specific cellular response and to conduct the repair of affected tissue. An injectable hydrogel must satisfy some criteria, such as: (1) the sol-gel transition should take place under mild conditions; (2) the gelation of the formulations is preferred to occur in time in order to avoid the out flow to the surrounding tissue; (3) the gelation and the degradation of the hydrogels should not require harmful stimulus, toxic reagent or produce toxic byproducts (Sun, Nan et al., 2020). The mechanisms involved in the in situ formation of hydrogels are diverse, involving chemical processes (ionic or covalent crosslinking), either physical gelling due to changes in temperature, pH or solvent exchange, and even simply thickening upon removal of the

injection shear. Fig. 17.3 illustrates in vivo forming injectable hydrogels, as well as the crosslinking and potential applications of the obtained systems (Mathew, Uthamanb, Choc, Chod, & Parke, 2018). Injectable hydrogels derived from biopolymers, especially polysaccharides, are intensively used in tissue engineering. The carbohydrate polymers impart tailorability of desired physicochemical properties and versatile injectable chemistry due to presence of modifiable functional groups. These are basic considerations (in addition to their intrinsic biocompatibility properties and lack of toxicity) that have recently led to the development of highly potent biomimetic biomaterials which can be formed in situ (Upadhyay, 2017). The most used polysaccharides for bioapplications, implicitly for the production of injectable hydrogels are chitin, CS, carageenan, hyaluronan, alginate, agar and less often fucoidan or ulvan. Due to its remarkable properties (biocompatibility, biodegradability, mucoadhesivity, antimicrobial activity etc. . . .) CS thus appears as a relevant candidate for the preparation of biomaterials, which can repair or even replace damaged tissue and organ, may include, transport and release of drugs, cells and growth factors and allow cell attachment and proliferation (Dutta, Rinki, & Dutta, 2011). An excellent literature review on the use of CS in tissue engineering was carried out by Croisier and Je´roˆme (2013). The gelation occurs easily in conditions compatible with the human body, without the need for the use of toxic crosslinkers, because injectable formulations exhibit a sol-gel transition upon injection into the body and the gel can take the desired shape in the affected tissue so that scaffold is perfectly adjusted to the defect. Injectable gels are mostly of the “physical” type, so they are formed due to the interactions that can occur between polymer chains, such as: electrostatic and hydrophobic interactions or hydrogen bonds, which in turn depend on several parameters, such as: pH, concentration, temperature. However, there are some disadvantages: it is

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17.3 Drug-loaded injectable hydrogels used in tissue engineering applications and drug delivery

405

FIGURE 17.3 Crosslinking as well as the mechanism of interaction and application in both tissue engineering and drug release of an injectable hydrogel system. Source: Reprinted from Mathew, A. P., Uthamanb, S., Choc, K. H., Chod, C. S., & Parke, I. K. Injectable hydrogels for delivering biotherapeutic molecules. International Journal of Biological Macromolecules, 110, 17
29 with permission from Elsevier.

difficult to accurately control the size of the hydrogel pores, sometimes uncontrolled partial dissolution of the gel can occur, and the mechanical resistance/strength of the hydrogels is quite limited (Chenite et al., 2000). For pH-sensitive, water-soluble polymers, like CS, a sol-gel transition can occur with the pH variation. CS is a polycation that shows a sol
gel transition when pH changes from slightly acidic to neutral (around pH 5 6.5). By raising the pH in the basic medium, it becomes nonionic, generating a three-dimensional network by performing hydrogen bonding interactions between chains. An injectable in situ forming system, for example, was developed from acidic CS solution with the combination

of sodium bicarbonate (NaHCO3) (Liu, Tang, Wang, & Guo, 2011). A thermosensitive composite injection system based on CS and apatite was similarly prepared, using Na2CO3, as an ionic crosslinker, usable for bone MSCs loading (Li, Liu, Ding, & Xie, 2014). Such systems are promising for applications in tissue engineering but also in drug delivery. Injectable gels can also be obtained by in situ gelation of CS through ionic interactions with anionic, nontoxic crosslinkers (Liu, Gao, Lu, & Zhou, 2016). A rapid in situ gelation system based on CS and guanosine 5’-diphosphate (GDP), as an anionic crosslinker, was reported by Mekhail, Daoud, and Almazan (2013). High gelling speed prevents undesirable flow to

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17. Biological macromolecules for drug delivery in tissue engineering

surrounding tissues as injectable carriers. These hydrogels showed a porous morphology, good cytocompatibility and mechanical properties resembling those of human soft tissue. A biomimetic injectable hydrogel capable to encapsulate MSC with applications in spinal cord injury treatment, having a rapid gelation upon temperature increase from 0 C to 37 C, was obtained, by Boido et al. (2019), starting from CS by crosslinking with β-glycerophosphate disodium salt. The hydrogel not affects MSC viability, is able to release MSC and maintain the antioxidant properties of CS. The preliminary in vivo tests on mice showed good handling during injection of the hydrogel and high encapsulated MSCs survival after 7 days. Another paper reports the encapsulation of chondrogenic factors or human MSC in a chitosan-β glycerophosphate-hydroxyethyl cellulose injectable hydrogel (Naderi-Meshkin et al., 2013). During the 28-day investigation, the system provided suitable conditions for chondrogenic differentiation of the encapsulated human MSCs. By in situ injection, the hydrogel is able to fill the cartilage tissue defects. Dipotassium hydrogen orthophosphate can neutralize the acidic CS solution and, at body temperature, induce its gelation; the gelation time, of several minutes, depended on CS anionic crosslinker concentration. These noncytotoxic gels present an in vitro sustained release of β-lactoglobulin (up to 10 days) (Ta, Han, Larson, Dass, & Dunstan, 2009). Composite thermosensitive CS-based hydrogels crosslinked with β-glycerophosphate and reinforced with β-tricalcium phosphate by physical interactions were reported by Dessi, Borzachiello, Tawheed, Wafa, and Ambrosio (2013) and proved to be a promising candidate for injectable in situ gelling bone analogs. CS-based injectable hydrogels can be made in situ by covalent bonds between chains. To avoid the use of covalent crosslinkers, often toxic, one way is to cocrosslink modified CS by introducing new functional groups,

complementary to another reaction partner polymer, or to native CS with a polymeric partner with functional groups complementary to amines or hydroxyls groups from CS, with which they can react. For example, Schiff’s reaction between the amino and aldehyde groups can be used to form, in situ, pHsensitive hydrogels for protein delivery or as wound dressing biomaterials. N- and Omethyl carboxylated CS and oxidized alginate were cocrosslinked to obtain an injectable hydrogel with potential application in tissue engineering and as DDS (Li et al., 2012). Similarly, carboxymethyl CS and partially oxydized CMC based hydrogels intended for the treatment and repair of skin burns was tested in vivo, finding that the wound coated with the hydrogel was completely filled with new epithelium within two weeks, without any significant side effects (Fan et al., 2013). Hydrogels consisting of oxidized alginate, PEG and carboxymethyl CS or gelatin were synthesized, too, by Naghizadeh, Karkhaneh, and Khojasteh (2018). They demonstrated the ability to survive and proliferate MSCs, so they could be considered as an appropriate candidate for injectable self-crosslinking application in tissue engineering. CS-based injectable hydrogels can be obtained by chemical crosslinking with di/ polyfunctional agents, which, however, must be nontoxic and biocompatible; genipin meets these requirements. Injectable hydrogels based on CS and hyaluronic acid (HA) cocrosslinked with β-glycerophophate and genipin present excellent mechanical properties, fast in situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions. All these advantages make them ideal candidates for tissue engineering, especially for cartilage regeneration (Jalani et al., 2015; Muzarelli, Mehtedi, Bottegoni, Aquili, & Gigante, 2015). Thiol-ene click reactions are also used in order to form CS based hydrogels owing to

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17.3 Drug-loaded injectable hydrogels used in tissue engineering applications and drug delivery

their high efficiency in mild reaction conditions (Chen, Wang, Deng, Hu, & Dong, 2013; Jo et al., 2013). The duration to obtain the hydrogels can be varied from minutes to hours by controlling the temperature and pH of the precursor solution. Controlled architectures and materials with enhanced mechanical properties can be obtained in this way for soft tissue engineering applications. A pH-responsive injectable hydrogel formed by carboxymethyl CS and PEG di-acrylate was obtained, under physiological condition via in situ amino-yne click reaction (Huang & Jiang, 2018). The gelation step, mechanical and swelling behaviors and the degradation kinetics of the obtained hydrogels were optimized by varying the concentrations of diacrylic crosslinker and precursors. A possibility to produce crosslinking in situ is enzymatic catalysis. An in situ gel-forming system composed of rutin- and tyramine-conjugated CS derivatives was prepared under the action of horseradish peroxidase and hydrogen peroxide for dermal wound repair. In vitro study showed that rutin significantly improved cell proliferation. In vivo wound healing study was carried out by injecting the rat dorsal wounds with hydrogels and the obtained results indicated that these formulations might be a promising injectable gel-type wound dressing (Tran, Joung, Lih, & Park, 2011). CS-based injectable hydrogels can also be obtained by combining it with other biopolymers, which bring an additional contribution to their properties. Shen et al. (2015) have obtained, for example, a tough biodegradable and biocompatible CS
gelatin hydrogel, with improved mechanical properties, for potential cartilage tissue via an in situ precipitation method. A hydrogel based on CS, collagen, hydroxypropylc-cyclodextrin and PEG was obtained by another group (Naghizadeh et al., 2018). The hydrogels demonstrated the ability to survive and to proliferate MSCs, presents acceptable compressive properties and degradation time,

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so they could be considered as an appropriate formulations for injectable self-crosslinking application in tissue engineering. Another hybrid hydrogel based on CS, collagen, hydroxypropyl-c-cyclodextrin, PEG and nanoHA was obtained by Pe´rez-Herrero et al. (2019). The in vitro culture of rMSC in hydrogel showed no signs of intolerance or toxicity, and an intense proliferation of the cells after seven days. A composite thermosensitive injectable hydrogel based on CS, k-carageenan, poly(Nisopropyl acrylamide) and gold NPs was reported by Pourjavadi, Doroudian, Ahadpour, and Azari (2019). Crosslinking occurs at a temperature of 37 C, so it is possible to perform in situ after injection. The gold NPs enhance the conductivity of the obtained scaffold. The hydrogel has the ability to improve the attachment and proliferation of MG-63 human cell. Very interesting double crosslinked injectable networks based on catechol-modified methacryloyl chitosan (MC), and methacryloyl chitosan (CMC) were recently reported by Wang et al. (2019). The double crosslinking is ensured by the light curing (blue light) of the methacryloyl macromers of CS, respectively by catecholFe31 chelation, as schematically presented in Fig. 17.4. The hydrogel present a good tissue adhesion, as demonstrated with a hemorrhaging liver model, a good hemostatic performance so it can remarkably induce the healing of bacteria-infected wound. Alginate-based hydrogels are characterized by a soft nature, which makes them identical to the most native tissues, and have a set of advantages regarding the cell encapsulation and entrapment. They can be formed in situ, in physiological conditions (temperature and pH), in the absence of toxic solvents. An important advantage is that mechanical properties can be tuned, depending on the nature of the tissue to be rebuilt (Bidarra, Barrias, & Granja, 2014). Because alginate is considered to be nonimmunogenic it has a great

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17. Biological macromolecules for drug delivery in tissue engineering

FIGURE 17.4 Schematic illustration on the fabrication of a photocrosslinkable, injectable, and tough chitosan hydrogel with enhanced tissue adhesion and antibacterial activity via double-crosslinkingdouble-network design for promoting wound healing. (A) Schematic for a novel design of hydrogel formation by crosslinking CMC and MC via simultaneously carbon
carbon double bonds and catecholFe3 1 chelation. CMC, Carboxymethyl cellulose; MC, methacryloyl chitosan. Source: Redrawn from Wang, L., Zhang, X., Yang, K., Fu, Y. V., Xu, T., Li, S., . . . Lee, C. S. (2019). A novel double-crosslinking-doublenetwork design or injectable hydrogels with enhanced tissue adhesion and antibacterial capability for wound treatment. Advanced Functional Materials, 30(1), 1904156.

potential as a cell delivery vehicle (Lee & Mooney, 2012). Injectable alginate hydrogels with potential applications in tissue engineering can be obtained by many methods, including ionic gelation thermal gelation (based on phase transition), cell-crosslinking, « click » reactions, double crosslinking, free radical polymerization. Alginate is an excellent example of ionic interaction crosslinking. In order to produce the in situ gelling of alginate, divalent cation salts of low solubility are used, which slow down the gelling speed and allow a good control of the process time. Both calcium carbonate and sulfate, hardly soluble salts, gradually release Ca21 ions in the alginate solution thereby allowing gradual gelation (Fonseca, Bidarra, Oliveira, Granja, & Barrias, 2011). It is very important that cell viability is not affected by crosslinking conditions. Injectable and degradable composite hydrogels based on alginate combined with HA and

gelatin microspheres were obtained by in situ crosslinking with Ca21 ions (Yan et al., 2016). The preparation method is easy, it proceeds in mild conditions, it generates hydrogels with good mechanical properties and has potential applications in bone tissue engineering, drug delivery and other related biomedical fields. Injectable covalently crosslinked alginate hydrogels can be prepared, by different crosslinking methods, with a large range of mechanical properties. For example, injectable poly(Lglutamic acid)/alginate hydrogels were obtained by self-crosslinking of hydrazide-modified poly (L-glutamic acid) moderately oxidized alginate to the formation of carbonyl groups (Yan et al., 2014). The hydrogels are highly injectable, manifested a fast in vivo formation and mechanical stability, proved a good biocompatibility (effect due to the fact that it does not use bifunctional crosslinkers often toxic) and ensures the viability

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17.3 Drug-loaded injectable hydrogels used in tissue engineering applications and drug delivery

of the entrapped rabbit chondrocytes. They can be applied in a variety of pharmaceutical formulations, especially for cartilage tissue engineering. Using borate bonding, injectable hydrogels were also obtained. For example, alginate was modified by boronic acid and the derivative obtained generates boronate-type ester bonds in reaction with neighboring pyranose ring diols belonging to a neighboring macromolecule, at pH . 7. This hydrogel had a shear-thinning behavior and thus it was injectable using 18G or 21G needles (Pettignano et al., 2017). Another interesting way to obtain hydrogels is photocrosslinking. Injectable photocrosslinkable alginates with potential applications in tissue engineering have been designed in order to allow an enhanced control over the mechanical properties, swelling ratios and degradation behaviors than ionically crosslinked alginates (Rouillard et al., 2011). The method first involves obtaining a polymerizable alginate derivative, for example a methacrylic macromer. After injection, it is light-cured at short exposure to UV light and a photoinitiator, under mild conditions, which causes the process to be performed in direct contact with cells. K-carrageenan is appropriate suitable biopolymer for tissue engineering, as it mimics the biomimetic property and resembles the natural glycosaminoglycan structures (Lokhande et al., 2018). Different types of carrageenan, such as κ-carrageenan (kappa) in presence of potassium ions and ι-carrageenan (iota) with calcium ions, can lead to the preparation of thermoreversible gels. In the case of carrageenans, both physical and chemical methods can be used for crosslinking. The properties of hydrogels can be greatly improved by combining other polymers with carrageenan (gelatin, locust beam) or by making composite gels (eg with nanosilicate). A hydrogel based on ionic crosslinked kcarageenan with K1, was obtained in order to encapsulate and release human adipose-derived stem cells, being intended for cartilage tissue-

409

engineering applications (Popa, Caridade, Mano, Reis, & Gomes, 2015). Stem cells encapsulated in hydrogels remain viable, proliferate and differentiate into the chondrogenic lineage. The mechanical properties of hydrogels, especially at compression, are similar to those observed for native cartilage. By ionotropic gelation (Ca21 ions) was recently obtained an injectable hydrogel based on iota and kappa carrageenan, locust bean gum and gelatin, with potential applications for wound healing and tissue regeneration (Pettinelli et al., 2020). The hydrogel exhibit shear-thinning behaviors and might be injected for noninvasive applications. Injectable hydrogels based on mixtures of kcarrageenan and PVA covalently crosslinked with 3-(aminopropyl) triethoxysilane were recently reported (Rasool, Ata, Islam, & Khan, 2019) and these hydrogels can be degraded by various enzymes into small chain polysaccharides. Cephadrine was used for encapsulation, and his in vitro release rate was investigated in simulated intestinal fluids. The polymerdrug system manifests a strong antibacterial activity against S. aureus and weaker against Escherichia coli. Covalent injectable hydrogels were also obtained by photocrosslinking methacrylated kcarrageenan (Tavakoli, Kharaziha, Kermanpur, & Mokhtari, 2019). The polysaccharide macromer can be polymerized under visible-light action. This sprayable hydrogel can be used for the healing of skin lesions or to be injected as a bioprinting material for the in situ healing of soft tissues. Hialuronic acid (HA) and its sodium salt are largely used for the preparation of formulations used for tissue engineering as they are capable of producing highly reproducible and affordable biomaterials (Burdick & Prestwich, 2011). A relatively recently published paper reviews the physical and chemical methods of making hydrogels based on this polysaccharide and its derivatives (Khunmanee, Jeong, & Hansoo, 2017).

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Most of the injectable hydrogels based on HA, reported so far in the literature, are obtained by chemical crosslinking. A brief review of the most important chemical methods, specifying the reaction partners which may be HA or its derivatives, and other natural or synthetic polymers, is given in Table 17.2. In this table are also indicated the types of bonds formed by crosslinking or the type of reaction leading to these specific bonds are specified, as well as the medical application for which the obtained systems were prepared. An injectable in situ
forming clickcrosslinked HA hydrogel, for example, having a chondrogenic differentiation factor (cytomodulin), was obtained for the first time by Park et al. (2019). The crosslinking reaction is based

on a click-reaction between tetrazine-modified hialuronic acid and transcyclooctene-modified hialuronic acid. This injectable formulation induces an easier healing of the affected articular cartilage. Agar-based injectable hydrogels or combinations with other polymers are also reported in the literature. A agar and gelatin based hydrogel, crosslinked with genipine, was developed and specifically designed for the insertion into the lumen of hollow of the nerve guidance channels through a syringe during surgery. The in vitro tests showed that all the cells in contact with the hydrogel had morphology, viability and proliferation patterns similar to those of the positive control (Tonda-Turo et al., 2017). Because the hydrogel is obtained in mild conditions, close to

TABLE 17.2 Some examples of injectable hydrogels based on hyaluronic acid or chemically obtained derivatives. Components

Type of reaction/ Active principle formed bond

Application

Reference

HA 1 aldehyde-functionalized fourarm PEG

Imine

Metformin and 5-fluorouracil

Colon carcinoma

Wu, He, Wu, and Chen (2016)

Aldehyde modified HA 1 genipine

Imine




Tissue engineering

Tan, Li, Rubin, and Marra (2011)

Aldehyde modified HA 1 hydrazide modified HA

Hydrazone

Bone morphogenic Bone augmentation for protein-2 orthopedic applications.

Thiolated carboxymethyl HA 1 oxidized glutathione

Disulfide

Adipose-derived stem cells

Ophthalmic applications Zarembinski (posterior pole of the et al. (2014) eye)

Thiolated HA

Disulfide

Fibroblasts cells (L929) and chondrocytes.

Tissue engineering and regenerative medicine

Bian et al. (2016)

Thiolated HA 1 p(HPMAm-lac (1
2))-PEG-p(HPMAm-lac(1
2)) triblock copolymer

Michael addition

Stromal cell

Regenerative cell matrix and controlled drug delivery

Dubbini et al. (2015)

HA 1 PEG

Diels Alder « click » chemistry

ATDC-5 cells

Cartilage tissue engineering

Yu et al. (2014)

HA 1 methacrylated glycol CS

Photocrosslinking Chondrocytes

Cartilage repair

Park, Choi, Hu, and Lee (2013)

III. Functional Applications

Martı´nez-Sanz et al. (2011)

References

the physiological ones (temperature, pH), it can be advantageous for the inclusion of biologically active compounds avoiding their denaturation, and by the morphology obtained they can ensure their controlled release for a longer period or even weeks. The combination of agar with synthetic polymers also allows the production of hydrogels capable of including and releasing some biologically active compounds, an example being the hydrogel based on agar and carbomer reported by Santoro et al. (2011). Scarcely used for the preparation of injectable hydrogels is chitin, which often needs to be chemically modified before use. Recently, hydrogels based on acrylamidemodified β-chitin and alginate dialdehyde were synthesized, revealing excellent biocompatibility, biodegradability, injectability and self-healing properties. Also, by in vivo tests on zebrafish it has demonstrated that these hydrogels have wound healing properties (Balitaan, Hsiao, Yeh, and Santiago, 2020). Fucoidan is a polysaccharide that is becoming more and more used in obtaining injectable hydrogels. Recently, it has been reported an injectable hydrogel based on fucoidan and gelatin, with the genipine as crosslinking agent, capable of including and releasing cells and growth factors. This hydrogel can be used for the treatment of cartilage defects without potentially having side effects (Lu et al., 2019). The hydrogel showed adequate injectability, strong adhesive ability, and high strength to enzymatic degradation. In vivo tests on rabbits have shown that the hydrogel loaded with platelet-rich plasma induced cartilage regeneration, leading to an enhanced PRP therapy of the cartilage.

17.4 Conclusions

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polymers were developed for a wide range of practical applications such as bone, cartilage, cardiac, ophthalmic, neural, wound healing, and skin tissue engineering. These systems were prepared with specific features in order to promote cell adhesion, proliferation, tissue growth, and regeneration but also to release drugs with a sustained and controlled rate. The ideal system for tissue engineering must be a multifunctional scaffold that mimics tissue architecture and releases multiple types of encapsulated molecules (drugs, cells, growth factors) under the action of specific triggers in order to perform localized and functional tissue healing and regeneration. Moreover, it should be resorbable, bioactive if possible and, once implanted into the body, would help the patient heal itself. The need for these biomaterials to be primarily biocompatible limits the range of polymers that can be used for this purpose, directing the research to natural ones—polysaccharides and proteins—and a small number of synthetic polymers that fulfill this condition. However, the preparation methods are varied, being chosen depending on the concrete chemical structure of the polymer and the final application or destination of the biomaterial. The interest for the development of such systems is very high, fact proved by the large number of studies published especially in the last decade. It is obvious that a great variety of polymeric biomaterials intended for tissue engineering applications has been designed and tested until now, but only a few systems are scaled up for their use in clinical applications. Therefore there is an urgent requirement to deepen these studies in order to develop clinically available devices for tissue regeneration.

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C H A P T E R

18 Biological macromolecules for growth factor delivery M.D. Figueroa-Pizano Biopolymers-CTAOA, Research Center for Food and Development (CIAD), Hermosillo, Sonora, Mexico

18.1 Introduction Growth factors (GFs) are endogenous polypeptide molecules essential for cell survival able to stimulate and regulate several vital processes throughout cell life (Niu, Li, Ding, Dong, & Wang, 2019; Park, Hwang, & Yoon, 2017). They are a broad group of soluble glycoproteins categorized into several families (Fig. 18.1) depending on their structure and function (Caballero Aguilar, Silva, & Moulton, 2019; Park & Kim, 2017). GFs are produced in many types of cells in the body (muscular, endothelial, neurons, hepatic, stem, epithelial, and others) at different stages (Burgess, 2015). They are secreted in the extracellular matrix (ECM) and can act in autocrine, paracrine, and a few cases in an endocrine way (Caballero Aguilar et al., 2019; De Witte, Fratila-Apachitei, Zadpoor, & Peppas, 2018). Sometimes, immature GFs remain joined to the cellular membrane acting in a juxtacrine way with the surrounding cells. All GFs are recognized by specific external receptors at the cellular membrane level (transmembrane proteins),

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00018-X

containing an internal tyrosine kinase domain (Wang et al., 2017a). The activation of GFsreceptors implies the internal domain’s action as a tyrosine kinase enzyme, which triggers tandem reactions inside the cell to promote modification of gene expression and finally direct the cell fate (Niu et al., 2019). Their natural functions are crucial for cells because they mainly regulate fundamental cellular processes as differentiation, proliferation, migration, and communication (Subbiah & Guldberg, 2019). GFs are indispensable for regenerating damaged organs and tissues and perform vital roles during the inflammation stage, immune response, and wound healing, either by activating signaling pathways or regenerative processes (Mina, 2015). Moreover, most GFs present pleiotropic roles because the same GF can be produced by different cells and exert alternative stimuli; it all depends on the GF concentration, the kind of receptor cell, and its maturity. (Burgess, 2015; Qu et al., 2020). GFs are considered a potential tool for the biomedical area because they have been used with great success as exogenous therapeutic

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FIGURE 18.1

18. Biological macromolecules for growth factor delivery

Classification of growth factors. Leading families and their representants

agents to treat several injuries and diseases. They have been isolated from natural tissues or produced as recombinant proteins in labs, evidencing effective and suitable accelerators of healing processes for tissue engineering and wound management (Bruggeman et al., 2017; Caballero Aguilar et al., 2019; Qu et al., 2020). Bone morphogenetic proteins (BMPs) such as BMP-2 and BMP-7 have shown reliable results for bone tissue reparation, while transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) are used for cartilage engineering restoration (Sivaraj & Adams, 2016; Venkatesan, Anil, Kim, & Shim, 2017). In the case of different kinds of wounds, a set of GFs as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF),

epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin like growth factor (IGF), and TGF-β are proved to cause a synergetic effect to enhance or improve the healing (Niu et al., 2019; Park & Kim, 2017; Park et al., 2017). They also are a promising strategy to treat ailments related to the nervous tissue due to the BMP-9, BDNF, IGF-1, IGF-2, nerve growth factor (NGF), and glial cell derived neurotrophic factor (GDNF) have had a favorable therapeutic implication on Alzheimer’s disease (Lauzon, Daviau, Marcos, & Faucheux, 2015; Sivashankari & Prabaharan, 2016; ZilonyHanin et al., 2019). Some NGFs like GDNF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4, and NT-5 have acted as neuroprotective molecules for

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18.2 Delivery systems for growth factors

glaucoma illness (Nafissi & Foldvari, 2016). Coronary or cardiovascular diseases have been treated with a combination of VEGF, IGF, EGF, TGF-β, or PDGF, which can stimulate resident cardiac stem cells and repair the tissue and organ function (Madonna & De Caterina, 2011). GFs can be used to regenerate any part of the body because they can induce angiogenesis, the building of ECM, the formation of bone, and the assembly of granular tissue (Gainza, Villullas, Pedraz, Hernandez, & Igartua, 2015). However, their clinical application is limited because some intrinsic-GFs issues are necessary to resolve, and only a few products are commercially available. Many limitations of GFs implementation in the biomedical area are related to their structures and administration. Owing to their protein nature, GFs are labile to different physicochemical factors, presenting a short half-life and low stability either in the process or manufacturing and administration (Wang et al., 2017a). Usually, the GFs are injected directly or incorporated into creams, solutions, gels, lotions, sprays, and ointments applied on the interest site (Yamakawa & Hayashida, 2019). However, the GFs are quickly inactivated by enzymes inside the body, reducing their healing or regenerative action, which causes consecutive dose application making not very cost-effective treatments (Subbiah & Guldberg, 2019). Besides, the use of exogenous GFs requires special care in the concentration of applied dose since slightly high amounts can result in side effects (Niu et al., 2019). Some scientific studies have focused on GFs modification, seeking more excellent stability and reducing their susceptibility to physical, chemical, or enzymatic degradation. The structural alterations of GFs include the formation of cycles through the union of amino-terminal extreme with the carboxyl-terminal extreme and the graft of polyethylene glycol (PEG) onto GFs chains (Braun, Gutmann, Mueller, Lu¨hmann, & Meinel, 2018; Lu¨hmann et al.,

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2020). These modifications keep the GFs stable against temperature and hide the cleavage site to avoid enzymatic action (Mitchell, Briquez, Hubbell, & Cochran, 2016). Beyond the chemical structural modifications, several genetic changes have been made in the genes of GFs, trying to form these proteins with extended half-life and stability (Jones, Tsai, & Cochran, 2011; Mitchell et al., 2016). These modifications involve the production of smaller GFs, and frequently, they are without cleavage sites for proteases but with the same functional capacity as endogenous GFs.

18.2 Delivery systems for growth factors Alternatively, the delivery systems represent a more efficient way to protect GFs from degradation and extend their healing properties. These systems can control, maintain, and direct the release of GFs to achieve their biological activity in a specific way. A crucial advantage of use delivery systems is that they can spacetemporally regulate the release of GFs, allowing them to achieve better therapeutic results (Li et al., 2020). Besides, sustained release of GFs avoids the application of constant doses and maintains efficient local concentrations without a burst effect. Both properties help reduce inappropriate amounts of GF and, consequently, its harmful effects (Park et al., 2017; Wang et al., 2017a). These systems have been designed in various shapes, sizes (ranging from nano to macroscopic scale), and based on different types of materials, obtaining positive release and therapeutic results. The aim has recently been to get materials capable of releasing more than one GF with a therapeutically active and programmable pharmacokinetics profile. It is intended to reach any part of the body and respond to specific stimuli to meet healing needs (Laiva, O’Brien, & Keogh, 2018). However, its design still faces several problems related to low encapsulation efficiency, the

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preservation of GF properties in the manufacturing process, and the activation of immune responses after a long installation time. The accuracy of GFs delivery systems implies a good loading-delivery strategy since the amount of GF loaded will determine the amount released and directly affect the therapeutic results. This strategy depends on the biological, biochemical, and physical properties of target cells, GFs, and carrier material, respectively (Caballero Aguilar et al., 2019). It is central to recognize the anatomy and functions of target tissues and the molecular weight and roles of selected GF. They determine the kind of encapsulation material which must have an excellent capacity to join and break away the GF, be biocompatible and biodegradable, and present good mechanical properties (Subbiah & Guldberg, 2019; Vasita & Katti, 2006). Other essential factors are the shape, size, composition, microstructure, and hydrophilicity of the delivery matrix. They also define the loading efficiency, the releasing kinetic, and capacity to react to the target site (Vasita & Katti, 2006). Finally, the loading step requires carefully manipulating the GFs to avoid denaturalization and estimate the precise amount for the expected results (De Witte et al., 2018). Different advanced approaches have been used to immobilize and incorporate of the GFs in the delivery systems, including physical, chemical, and affinity methods (Qu et al., 2020; Wang et al., 2017b). Physical processes are considered the simplest and safest to maintain the biological properties of the GFs and the integrity of the encapsulating material because they do not use extreme conditions. The GFs are entrapped physically into delivery systems and interact through hydrogen bonds, van der Waals forces, hydrophobicity, or electrostatically (Ren, Zhao, Lash, Martino, & Julier, 2020). Incorporating of GFs can be as simple as adding drops or a more extensive GF solution to the material explanation, which is in

preparation. Then the GFs are encapsulated or entrapped once the carrier matrix is formed. Another form uses a preformed material that the GFs solution will be absorbed on the materialz, or it can diffuse into the interior. Besides, the almost ready material can be submerged in the GF solution allowing its infusion. The process could be realized manually or in specialized equipment that maintains the controlled temperature and movement conditions. Most of these procedures have the disadvantages of not ensuring a uniform distribution of GFs inside the material. It requires large volumes of GF solutions. The amount of encapsulated or diffused GFs in the material is uncertain. Sometimes they may need highly porous materials (DiStefano et al., 2018; Kanda et al., 2016). However, every day successful improvements are made to these systems that have proven highly effective in the controlled release of GFs. Regarding the chemical methods, they are considered more precise strategies to directly incorporate the GFs into the material structure that will form the carrier system. Generally, this type of approach is based on chemical or enzymatic reactions to graft the GFs on functional groups available in the material’s chemical structure (Niu et al., 2019). In this way, the amount of GF retained in the system and, its distribution is better known, allowing a safer release. An important consideration is the risk that the incorporated GFs could be modified during chemical procedures, and therefore, they may not perform a proper function. Another effective way of incorporating GFs into delivery systems is by using materials that are remarkably like the components of the ECM, where GFs normally interact. In this case, the retention of GFs is carried out by the affinity between them and the materials. In nature, in ECM, many polymers protect the enzymatic degradation factors and regulate their binding with receptors (Bruggeman, Williams, & Nisbet, 2018). The union is

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18.3 Materials for delivery systems of growth factors

undoubtedly given by physical or chemical interactions between the GFs and the encapsulating matrix; however, it occurs spontaneously without the necessity of chemical procedures. This strategy is increasingly used, especially when more than one GF will be incorporated into the system. However, mimicking the conditions that the ECM provides for GFs is not always easy. Each of these methodologies has some advantages and disadvantages to achieve a safe loading and release of GF. It is essential to consider specific conditions such as the target tissue, the type of GF to be incorporated, and the encapsulation material properties.

18.3 Materials for delivery systems of growth factors Choosing the ideal material to prepare of GFs delivery systems requires evaluating and considering all its properties since the release efficiency will depend significantly on them. Generally, its fabrication has been with synthetic, natural origin, or inorganic compounds and their combination (Zhang, Lu, Mamidwar, Wang, & Iii, 2020). Synthetic polymers such as PLGA, polyvinyl alcohol (PVA), and PEG have been investigated to release GFs because their intrinsic characteristics are very stable and allow safe design. However, not all of them are biodegradable and can generate some immune responses. On the contrary, specially biomacromolecules such as polysaccharides, proteins, lipids, and even nucleic acids, represent an option with a lower risk of toxicity (Kowalczewski & Saul, 2018). Besides, they are quickly metabolized, create the natural environment, stimulate ECM synthesis, facilitate cellular adhesion, and enhance the bioactivity of GFs. Some disadvantages are their deficient mechanical properties and low uniformity or control of their intrinsic characteristics. Still, they have continuously been improved through modifications and combinations to

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maintain their beneficial properties and achieve a good delivery system design. Depending on the physical structure of the carrier matrix, delivery systems are classified as hydrogels, scaffolds, or particle systems (nanoscopic or macroscopic) (Gainza et al., 2015; Park et al., 2017). Hydrogels are 3D polymeric matrices with the ability to absorb a large amount of water and highly porous structures where GFs can be loaded and diffused as in the ECM (Gyles, Castro, Ota´vio, Silva, & Ribeiro-Costa, 2017; Sharma, Sharma, & Kumar, 2019). These materials can be adapted to the site of interest since they are semi-solid and possess great flexibility. Sometimes they could be injectable, presenting a sol-gel transition at 37 C to solidify or stimuli-responsive, taking the shape of the place where they have been implanted after detecting an environment change (Sood, Bhardwaj, Mehta, & Mehta, 2016). They are frequently used for wound healing because they help maintain moisture and absorb the fluids being leached during the GFs release, making treatment more effective. In the case of scaffolds, they are natural or polymeric matrices, like hydrogels, that work as 3D cell cultures, arranged in a mesh form where the GFs can be trapped (Nyberg, Holmes, Witham, & Grayson, 2016). They are widely employed for tissue engineering because they are strong enough to support high loads and promote tissue renewal. The GFs are incorporated in scaffolds to promote the in vitro differentiation of stem cells and the growth and propagation of several cell lines, and after that, implant the scaffold. Moreover, these systems can contain the GFs when inserted into the body to improve tissue formation and healing in the damaged areas. However, they are limited by their low flexibility and can only be used in specific human body areas. Finally, their size on the nano or microscopic scale, allows them to reach deeper places in the organism and encapsulate the GFs safer than hydrogels and

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scaffolds (Gainza et al., 2015). They have been formed mainly by lipids and different polymers under several strategies and categories. The loaded GFs in these systems are found in different ways. For example, they can be in the core, spread the entire particle, or inside several layers on the surface. Depending on the design of the particle, components with hydrophobic, hydrophilic, or both natures can be loaded onto them. In addition to these structures, composite materials have widely been implemented for the release of GFs. They combine two or more of the mentioned structures (even contain inorganic materials) to joint their best properties as delivery systems and enhance the healing benefits of GFs. They can be classified as metallic, polymeric, or ceramic, depending on the highest part of the material (Zhang et al., 2020). GFs delivery can be used polymeric hydrogels or scaffolds containing particulate systems of any nature (metallic, ceramic, lipidic, protein, polymeric, nucleic acid, etc.). They can be injectable or stimuli-responsive (Jiang et al., 2018; Rohit & Dixit, 2016). All these combinations open the possibility of incorporating two GFs or several bioactive compounds/drugs with different natures into the delivery system to obtain a controlled release. It is essential to consider the properties of each material to build a suitable composite material, hydrogel, scaffold, or particulate system. A versatile group of materials to create any of these kinds of procedures are biological macromolecules.

18.4 Biological macromolecules for delivery systems of growth factors In the last years, biological macromolecules such as proteins, polysaccharides, lipids, and nucleic acid, have continuously been used to create delivery systems for drugs and bioactive substances, including GFs (Chen, Miao, Campanella, Jiang, & Jin, 2016; Krishnaswami,

Kandasamy, Alagarsamy, Palanisamy, & Natesan, 2018). As mentioned before, these molecules have the advantage of being of natural origin, and therefore they are nontoxic, highly biocompatible, and generally do not evoke immune responses. Many of them maintain the stability of the GFs and interact with them to modulate their functions. All macromolecules are characterized by possessing high molecular weight, linear or branched structures composed of select monomeric units for each category, and many functional groups along with them (Krishnaswami et al., 2018). Generally, their functional groups are highly reactive, and they are used to join with other molecules or chemically modified, leading to newly available properties in the structure. The long chains of proteins and polysaccharides in liquids form very viscous solutions, semi-solid or concrete materials. Furthermore, their highly reactive groups interact with each other or with other polymers to form complex structures such as particles, hydrogels, or scaffolds used for delivery systems (Gull, Khan, Islam, & Butt, 2019; Khansari et al., 2017). The lipids are usually used to create particulate carrier systems such as liposomes because they tend to be arranged in particular ways due to their amphiphilic characteristics. While the nucleic acids, explicitly the aptamers, are employed as a complement to bind the GFs in the delivery system. These aptamers, also called chemical antibodies, are short and folded sequences of about 70
100 nucleotides of RNA or single-stranded DNA, which can bind with high affinity and specificity target molecules GFs (Yoon & Rossi, 2018; Zhou & Rossi, 2017). The design of delivery systems for GFs requires an appropriate selection of biological macromolecules based on their nature, molecular structure, and kind of GF. Everything will depend on the functional characteristics that are sought in the final carrier. There are many systems built-in base only in one type of

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18.4 Biological macromolecules for delivery systems of growth factors

biological macromolecule. However, they can be made based in two or more macromolecules of the same or different nature. The following sections describe several delivery systems for GFs formed with biological macromolecules and the processing, efficiency, and characteristics.

18.4.1 Protein-based materials for growth factor delivery Among the main used biological macromolecules to construct GFs delivery systems are proteins. They are considered excellent materials as carriers for GFs because they are biodegradable, have emulsification properties, low immunogenicity, and come from renewable sources (Chen et al., 2016). Furthermore, proteins present a three-dimensional microstructure that encourages cell proliferation. Usually, they are extracted from animal and plant tissues, which result in waste products after industrial processes. Proteins are characterized by having amine acid residues with superior properties such as hydrophobic/hydrophilic or cationic/anionic character, conferred to the entire molecule. Materials as hydrogels, scaffolds, and particles are commonly produced from proteins including gelatin, keratin, elastin, collagen, albumin, and fibrinogen/fibrin. Recently, keratin has been used to produce hydrogels as GFs delivery systems. Keratin is an intermediate filament protein found in wool, nails, hooves, horns, and hair (Shavandi, Silva, Bekhit, & Bekhit, 2017). It is considered a promising biomacromolecule for tissue engineering due to its self-assembled capacity leading to structures that regulate cellular recognition and behavior (Rouse & Van Dyke, 2010). An essential characteristic of keratin is the high cysteine residues and the polypeptide structure, capable of spontaneously crosslinking the chain. Depending on the extraction method, keratin presents changes in its chemical structure. An oxidative extraction can be

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carried out to obtain a keratose molecule that contains unavailable cysteine residues because of the sulfonic acid content. The latter prevents the formation of disulfide bonds between cysteines. However, if the isolation is reductive, only one molecule of keratin is obtained, and the cysteine residues contain sulfhydryl groups able to establish spontaneously crosslinking through disulfide bonds (Shavandi et al., 2017). The two types of keratin, mainly derived from human hair, have been tested as GFscarrier hydrogels to implement as part of the Volumetric Muscle Loss (VML) treatment (Tomblyn et al., 2016). Both keratin and keratose-based hydrogels promote in vitro proliferation of muscle myoblast cells and better adhesion than the control. However, they showed different degradation behavior. Significantly, keratose hydrogels were degraded faster than keratin hydrogels due to the presence/absence of crosslinking points. The IGF-1, FGF, and VEGF have successively been loaded due to their essential roles in the viability of muscle cells. The releasing profile of GFs from hydrogels was distinct. FGF was quickly released while the delivery of VEGF was slow, and the IGF-1 release was minimal. These differences attributed to the change between the rate of keratin and keratose degradation. However, the lowest release of IGF-1 is also attributed to its high affinity with these proteins. The in vivo test proved an increase in the wound site muscle mass due to combined GFs action. Combinations of 70:30 keratose
keratin to produce hydrogels for release of IGF-1, FGF, or IGF-1 1 FGF, have been demonstrated better than the individual hydrogels and a significantly greater functional recovery of VML injuries (Passipieri et al., 2017). The mixture of keratose and keratin at different ratios (30:70, 50:50, and 70:30) resulted in tunable erosion rates, the high the keratose content, the increased erosion rate (Ham et al., 2016). The hydrogels with more keratin

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presented a greater elastic modulus than hydrogels with high keratose content due to the formation of more crosslinking points in the former. The release rate of IGF-1 was also tuning with the variation on keratin
keratose ratio, leading to a slower release when keratin content is highest. Collagen and gelatin are other promising protein-origin biomacromolecules to construct GFs delivery systems. Collagen is the most abundant fibrous structural protein of ECM in animals; it is found in the skin, tendons, blood vessels, cartilage, bone, ligaments, and other tissues (Beata & Olha, 2020; Bhagwat & Dandge, 2018). This protein has been reported as a material carrier for several kinds of molecules. It can increase the bioavailability of bioactive compounds. Moreover, the collagen molecule has a high affinity for some proteins present in the ECM since they contain specific binding domains. These binding domain sequences have been conjugated with VEGF to form collagen-binding VEGF (CB-VEGF) (Park et al., 2018). This approach was directly identified from a random library to produce the highest affinity CB-VEGF and direct them to the damaged tissues since they are rich in collagen. The CB-VEGF promotes tissue regeneration by enhancing endothelial cell growth and angiogenesis in skin wounds and infarcted myocardium. The atelocollagen-1 (collagen with low immunogenicity produced by removing the amino and carboxyl-terminal ends) was integrated into natural bone mineral (NBM). The NBM-collagen enhances the absorption of BMP-2 and BMP-9 and increases the in vitro osteoblast differentiation (Fujioka-Kobayashi, Schaller, Saulacic, Zhang, & Miron, 2017). The collagen hydrolysis, acid or alkaline, produces the gelatin, which presents similar properties (Siburian, Rochima, Andriani, & Praseptiangga, 2020). Gelatin can form stable polymeric matrices ranging from microstructures to membranes or films with the potential to be used in tissue engineering or

wound healing. The modification of gelatin with alkali groups has allowed forming physical crosslinks hydrogels through hydrophobic interactions. This gelatin-modified hydrogel is negatively charged and forms an electrostatic complex with the FGF (positively charged), promoting angiogenesis when it is implanted in rats’ wounds (Takei et al., 2020). Gelatin microspheres produced by the water-in-oil emulsion method and cross-linked with genipin were used to encapsulate TGF-β1. The microspheres loaded with TGF-β1 were incorporated into a culture of smooth muscle cells that self-assemble to form ring-shaped vascular tissues. Microspheres with TGF-β1 were uniformly distributed in self-assembled vascular tissue without adverse effects on ring structure or mechanical properties and stimulate the expression of smooth muscle contractile proteins in tissue rings (Strobel et al., 2017). Elastin, the second most abundant structural component in the ECM, is commonly used in biomaterial fabrication due to its biomechanical properties. It is found in the same human tissues like collagen, but its primary functions are to provide elasticity and resistance (MirandaNieves & Chaikof, 2017). Due to these properties, elastin has been considered a potential component of bioprosthetic heart valves, helping flexibility and movement. A VEGF-loaded elastin-based hydrogel was recently used to hybridize glutaraldehyde-fixed pericardium (a kind of valve bioprostheses). The hybrid VEGFloaded elastin-based hydrogel enhances the cell adhesion in human umbilical vein endothelial tissue while decreases proliferation, platelet adhesion, and calcification. All these properties improve the endothelialization, which is critical for the excellent performance of this device (Lei, Tao, Xie, & Hong, 2019). Fibrinogen protein and its derivative, fibrin, are commonly used to form highly biocompatible hydrogels that readily promote cell growth. Additionally, these proteins have been functionalized with aptamers to be able to recognize GFs.

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FIGURE 18.2 Schematic of the extraction processes of two types of keratins: keratose and keratin. Source: From Tomblyn, S., Pettit Kneller, E. L., Walker, S. J., Ellenburg, M. D., Kowalczewski, C. J., Van Dyke, M., et al. (2016). Keratin hydrogel carrier system for simultaneous delivery of exogenous growth factors and muscle progenitor cells. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 104, 864
879. https://doi.org/10.1002/jbm.b.33438.

A hybrid aptamer-fibrinogen macromer was developed, which exhibited dual functions of protein assembly and molecular recognition under physiological conditions of VEGF (Zhao et al., 2019). The hydrogels were constructed through the hydrolysis of aptamer-functionalized fibrinogen into fibrin with the thrombin enzyme’s action. The assembled hydrogels were loaded with VEGF and implanted in vivo. They showed angiogenesis promotion and healing of skin wounds. The angiogenic and osteogenic potential of an aptamer-functionalized fibrin hydrogel loaded with VEGF was evaluated (Fig. 18.2). The functionalization of fibrin with the aptamer did not change its gelation capacity or biocompatibility. These hydrogels showed efficient incorporation and retention and better release kinetics of VEGF than the original fibrin hydrogels, extending the release for several days. The VEGF released from the aptamer-functionalized fibrin hydrogel allows a high in vivo vascularization (angiogenesis) and osteogenesis of the murine critical-sized cranial defect (Juhl et al., 2019). The formation of delivery systems for GFs between several proteins or other polymers has been considered an ideal strategy to enhance the system properties. In that way, hydrogels of albumin-PEG and fibroin-PEG were formed to deliver BMP-2 (Kossover et al., 2019). These hydrogels are biocompatible, and they proved to modulate the release of BMP-2 by the PEG content. The lesser the PEG content, the slower the BMP-2 release. Their functional

effectiveness was demonstrated because, at 13 weeks after implantation, a large bone was detected on a critical size tibial defect.

18.4.2 Polysaccharide-based materials for growth factor delivery Polysaccharides are commonly used in drug delivery applications due to their excellent complex network properties and intrinsic characteristics (Vasita & Katti, 2006). Members of this group are widely distributed in nature and are relatively easy and inexpensive to obtain. Most of them present highly biocompatible and biodegradable structures that, on many occasions, have intrinsic biological activities that are beneficial for the biomedical area. Polysaccharide structures can form networks under physiological conditions where other functional molecules can become entrapped (Chen et al., 2016). Due to polysaccharides’ versatility, frames with different shapes and sizes can be developed, which favors adjusting the kinetic pattern of release. Several delivery systems like hydrogels, scaffolds, nano, and microparticles have been produced with polysaccharides. Chitin, chitosan, carrageenan, pectin, alginate, starch, cellulose, hyaluronic acid, and different gums are some of them. Specifically, for GFs delivery, many polysaccharides have been used, and they have shown very favorable results.

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The discovery of non-animal polysaccharides containing galacturonic acid has allowed the production of materials with a high affinity for GF capable of stimulating angiogenesis. (Li et al., 2016). A new polysaccharide named EUP3 contains mannose, galactose, glucose, arabinose, rhamnose, and acid galacturonic. An in vivo assays proved the high affinity of EUP3 for FGF-2 and BDGF-B, promoting blood vessel production and maturation. Alginate, a very used polysaccharide, was cross-linked with CaCO3 to form sustainable delivery system as microparticles with FGF. Generally the particulate systems are preferable to reach or penetrate different small and difficult-access parts in the body. The burst effect and the release rate of FGF from these microparticles decrease as the crosslinker concentration increases. The release evokes cell proliferation and migration, and during the in vivo evaluations, they showed the capacity to enhance angiogenesis, epithelium regeneration, and collagen deposition, which promote wound healing (Shi et al., 2019). The use of polysaccharides for delivery systems demands additional aspects to its chemical structure, such as the molecular weight and the ratio used. All these elements can affect the performance of the system, including the loading and releasing efficiency. An alginate bioink was prepared using alginate with different molecular weights and varying the ratio of the crosslinking agent to tuning the mechanical properties and its capacity to determine the cell fate. In addition to these results, the study revealed that the molecular weight of alginate could influence these hydrogels’ loading capacity since the lower the molecular weight, the lower the encapsulation efficiency (Freeman & Kelly, 2017). Besides, it is critical to consider the interactions between the components. Several combinations of GFs, drugs, cells, other kinds of active biomolecules can change the performance. The in vivo application of a hyaluronic acid hydrogel loaded with

the chondrogenic factor TGF-β3 led to a positive increase of staining of collagen type II as part of the new cartilage formation. However, the combination of TGF-β3 and mesenchymal stem cells in the same hydrogel and placed in vivo did not produce synergistic effect (Fisher et al., 2016). The concentration of GFs is another crucial factor for healing purposes. Several concentrations of VEGF loaded into nanoparticles heparin/poly-l-lysine, immobilized on cardiovascular stents surfaces, were evaluated for optimum performance. The results indicated that the VEGF-loaded nanoparticles were blood compatibles and promoted endothelial cell adhesion, proliferation, migration, and biological activity, at an intermedia VEGF concentration (3550 ng/ mL). Apoptotic cells appeared at lower concentrations, while a shrinkable cell shape occurred (Tan et al., 2020). Probably, the most used polysaccharide for biomaterial fabrication is chitosan. This polysaccharide comes from the chitin’s deacetylation process, which is extracted from invertebrate organisms such as crustaceans and some fungus classes. The chitin- and chitosan-based materials are characterized by their high and wellinterconnected porosity, facilitating the loading and releasing of drugs and GFs. A few works report the use of chitin to design carrier. Chitin or chitin gel was produced in the lab through the acetylation of chitosan. The result was chitin gel susceptible to enzymatic degradation, especially by lysozyme and fetal bovine serum, representing a potential system for controlled release. Additionally, chitin gel was fused with a chitin-binding domain, which, at the same time, it was bounded to an FGF-2 for its release. The system showed a time-dependently releasing. Interestingly, it does not present the initial burst effect, suggesting that in additional to lysozyme degradation, the repetition of the cycle of binding could be influenced by the releasing (Tachibana, Yasuma, Takahashi, & Tanabe, 2020).

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On the contrary, there are many works about the fabrication of chitosan-based materials. In solution, chitosan acquires the particular property of having a positive charge due to its amino groups’ protonation. Chitosan presents mucoadhesive, antibacterial, antifungal, and hemostatic activity, and pro-angiogenic activity is watersoluble (Sivashankari & Prabaharan, 2016). Besides, the positive charge allows the chitosan to easily bind with the cell membranes and any other negatively charged molecules. Riederer, Requist, Payne, Way, & Krebs, 2016 engineering microspheres of chitosan chemically cross-linked with genipin, loaded with two isoforms of VEGF contrarily accused to chitosan molecules. On the other hand, BMP-2 was loaded on glycolchitosan hydrogels, which showed high osteogenic activity. After four weeks of implantation of the BMP-2-loaded hydrogel, a critical size bone defect was complete closure due to the enhancement of osteogenic activity from the hydrogel. This activity’s spatial localization was followed by a transgenic fluorescent reporter mouse model at the cellular level, which confirms the diffusivity of BMP-2 (Gohil, Wang, Rowe, & Nair, 2018).

18.4.3 Polysaccharide combinations for growth factor delivery Different combinations between polysaccharides to form delivery systems are described below. Similarly, the xanthan-galactomannan microsphere was constructed using the layer-bylayer method, where a surfactant liposome was used as the initial template. In this system, the xanthan gum presents an anionic charge and the galactomannan, a neutral polysaccharide, acts as a polycation. Both polysaccharides are alternated until eight layers. The increase in layers in the microsphere contributed to increasing the sustained release of EGF up to five times more. EGF delivery was attributed to the polymer chain relaxation (Kaminski, Sierakowski, Pontarolo,

429

Santos, & Freitas, 2016). Other polysaccharides, such as carboxymethylcellulose (CMC), have been structurally modified with sulfated groups to develop the anionic molecules to facilitate the GFs binding. The sulfated CMC mimics natural sulfated polysaccharides found in the ECM, such as heparin, and can easily bind to positively charged GFs (Ko¨witsch, Zhou, & Groth, 2018). The new sulfated CMC was blended with normal CMC and gelatin to fabricate an injectable scaffold, demonstrating its capacity to bind with high affinity the TGF-β and enhance chondrogenesis after 28 days (Waghmare, Arora, Bhattacharjee, & Katti, 2018). Another modified polysaccharide, the alginate-sulfate, was blended with PVA to construct scaffolds that can deliver TGF-β. These composite scaffolds showed a great attraction by the TGF-β, binding it better than hydrogels been built with natural alginate and displaying a lesser releasing concentration of the TGF-β, which is desirable for GFs delivery (Mohammadi et al., 2019). On the other hand, the naturally sulfated polysaccharide chondroitin sulfate was used to form a hybrid hydrogel system with the tailoring capacity of the GF delivery and the cellular responses. Chondroitin sulfate was functionalized with transglutaminase factor XIII, which is employed for its crosslinking with PEG. Depending on the functionalization degree and the polymer ratio, the hydrogels properties can be tuned, including the binding of BMP-2. The hybrid hydrogels could release the BMP-2 to mediate the osteogenic differentiation as proof that they promote bone tissue regeneration (Anjum et al., 2016). The unique polycationic character that chitosan possesses has also allowed production delivery systems based only on physical electrostatic interactions without using chemical crosslinkers. Chitosan-alginate is a typical combination of contrary charged polyelectrolytes. They have been merged to build hydrogel matrices, which have resulted in very porous networks (characteristic in chitosan materials) and presented an inherent chondrogenic

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property. Besides, the structure was loaded with TGF-β, and cell viability, proliferation, and cartilage matrix production were improved. In this case, the chitosan-alginate hydrogel extended the TGF-β release and minimized the burst effect (Reed & Wu, 2017). Microspheres of three bilayers of chitosan-alginate have also been engineered by a layer-by-layer method to release of BMP-2. This last system activated the chemotaxis of preosteoclast cells and increased the mineralization after two weeks (Xu et al., 2018). Another common polyelectrolyte combination is chitosan with heparin. The latter is a natural glycosaminoglycan widely used to mimic the GFs union with the ECM in vivo. A multilayered matrix based on chitosan-heparin was created with TGF-β, which was used to deliver the GF on the monoculture of primary human hepatocyte cells. The complex chitosan-heparin reduces the time and concentration of the releasing of TGF-β, accelerating the healing times (Lin et al., 2018). Furthermore, scaffolds based on chitosanheparin were used for NGF delivery and nerve regeneration improvement due to the NGF stabilizing the heparin. Finally, they enhance the attachment and proliferation of Schwann cells, and therefore, the present potential for healing the nerve (Li, Xiao, Zhang, Zhao, & Yang, 2017). Alternatively, mixtures of nonionic polysaccharides can be prepared through other physical interactions such as hydrophobic interactions. Hyaluronic acid and its combination with methylcellulose are used to encapsulate IGF-1. In this approach, the chains of methylcellulose were chemically modified with one binding peptide to achieve the entrapping of IGF-1, which was genetically produced to give it a high affinity with the binding protein. The system demonstrated tunable affinity-based release and promoted the survival of pigment epithelium cells (Parker, Mitrousis, & Shoichet, 2016). The combination of hyaluronic acid with several proteins has been another commonly used strategy for GFs delivery. This polysaccharide belongs to the glycosaminoglycans group and was mixed with

silk fibroin to produce films without crosslinking agents. Their characteristics and releasing capacity are modulated only through the variation of hyaluronic acid content and synthesis temperature. The results showed that the degradation rate and the releasing profile of VEGF are accelerated by increasing hyaluronic acid in the film. In contrast, the increase in the formation temperature decreases the releasing speed of VEGF (Zhou et al., 2016).

18.4.4 Composites materials for growth factor delivery Polymeric composites are an excellent approach for designing GF delivery systems with application in tissue engineering, especially for bone and cartilage repair. These systems are characterized by the content of two or more materials. One of them is found as a higher proportion (matrix), and the others are incorporated as reinforcements to a lesser extent, distributed in the matrix. Composite materials are fabricated since they have improved capabilities compared to their components. In this way, composite materials have better mechanical, swelling, and degradation properties. Most importantly, they offer a perspective to design strategies for the sequential, sustained, or responsive release of various active molecules, such as GFs. The composition ranges from combinations of polysaccharides or protein with micro or nanopolymeric particles and polysaccharides or protein combination with minerals.

18.4.5 Protein-based composite for growth factor delivery Several composite materials have been created from silk fibroin, a fibrous protein, combined with different minerals compounds for bone regeneration. The formation of a hybrid scaffold of silk fibroin and hydroxyapatite

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FIGURE 18.3

Schematic of the functionalization synthesis of fibrinogen with the aptamer. Source: From Juhl, O., Zhao, N., Merife, A. B., Cohen, D., Friedman, M., Zhang, Y., ... Donahue, H. (2019). Aptamer-functionalized fibrin hydrogel improves vascular endothelial growth factor release kinetics and enhances angiogenesis and osteogenesis in critically sized cranial defects. ACS Biomaterials Science & Engineering, 5, 6152
6160. https://doi.org/10.1021/acsbiomaterials.9b01175.

(HA) enables a sequential and controlled release of BMP-2 and the stromal cell-derived factor (SDF) (Fig. 18.3) (Shen et al., 2016). The dual spatial-temporal release was achieved owing to the composite contained microspheres of silk fibroin loaded with BMP-2, and they were embedded on the silk fibroin-HA matrix, where SDF was dispersed. The releasing profiles showed a fast SDF released in the first days, while the BMP-2 release was maintained for up to 3 weeks. The composed scaffold loaded with two GFs promotes cellular recruitment and osteogenic differentiation helping in bone regeneration. An electroresponsive composite with potential application in neural tissue engineering was constructed (Fig. 18.4). It consisted of silk fibroin and graphene oxide for electrochemically controlled delivery of NGF-β (Magaz et al., 2020). The NGF-β was electrostatically loaded in the graphene oxide structure. It was released by slow diffusion in a passive form, reaching to deliver around 10%. In contrast, an active release was achieved by the electric stimuli application to release almost wholly the content of NGF-β at the same time. A hydrogel of silk fibroin with poly

(ethylene glycol dimethacrylate (PEGDMA)poly(lactic-co-glycolic) microspheres (PLGA) could present a dual release profile of FGF and TGF (Fathi-Achachelouei, Keskin, Bat, Vrana, & Tezcaner, 2020). Combining these physically and chemically cross-linked polymers makes it possible to adjust the systemmechanical properties and the rate of swelling and degradation of the system. The sequential release of these two GFs from nanoparticles creates a microenvironment for chondrogenic differentiation due to increased amounts of DNA and glycosaminoglycans. These findings demonstrated the synergistic effect of the dual release of FGF and TGF. HA is commonly used for bone tissue implants because it is the primary mineral component of vertebrate bones and presents considerable biocompatibility, bioactivity, and mechanical strength (Szcze´s, Hołysz, & Chibowski, 2017). The combination of this mineral component in scaffolds and hydrogels transforms these materials into composites. Several arrangements between proteins and HA have been proved as delivery systems of GFs to be used as graft materials in tissue reparations. Collagen-HA composites for bone

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FIGURE 18.4 Schematic of composites preparation. Source: From Shen, X., Zhang, Y., Gu, Y., Xu, Y., Liu, Y., Li, B., & Chen, L.(2016). Sequential and sustained release of SDF-1 and BMP-2 from silk fibroin-nanohydroxyapatite scaffold for the enhancement of bone regeneration. Biomaterials, 106, 205
216. https://doi.org/ 10.1016/j.biomaterials.2016.08.023.

reparation, including BMP-2 and VEGF, were reported. One of them evidenced HA’s ability to regulate in a sustained manner, releasing both GFs over 30 days (Walsh et al., 2019). In another work, in addition to the mineral component, microspheres of o-carboxymethyl chitosan loaded with BMP-2 were embedded in the collagen-HA matrix, where VEGF was also loaded (Dou et al., 2019). These compounds exhibit a gradual release of the GFs, which is beneficial because it helps get vascularized bone regeneration relatively fast. Collagen has been used to engineering a novel strategy to spatially control the rapid diffusion of BMP-2 in the sponges from this protein. A sink of poly-ε-caprolactone nanofibers with physically entrapped heparin microparticles around the collagen sponge was created. These composite materials bind with high affinity the heparin-loaded microparticles, decreasing the release of BMP-2 avoiding its dispersive diffusion (Hettiaratchi et al., 2017). Another innovative collagen composite was also used for BMP-2 releasing. In this case, was used porous biphasic calcium phosphate microspheres constructed by a water-in-oil

emulsion technique. The BMP-2 was loaded by immersion, and they are remaining joined to the surface and inside the pores. Finally, the microsphere was repeatedly dip-coated with collagen protein to fill the pores and retain the BMP-2. This design proved to maintain a sustained release of the GF and a positive effect on cellular differentiation (Seong et al., 2020).

18.4.6 Protein-polysaccharide composites for growth factor delivery Many times, the chitosan and gelatin have been blended to be used as scaffolds. In some cases, nanoparticles can be incorporated into these systems to control the GFs, releasing them better. Chitosan nanoparticles, synthesized by ionic gelation technique, were loaded with FGF into the chitosan-collagen matrix. The incorporation of these nanoparticles changes the physical properties of scaffolds. Still, its performance was a sustained FGF release, improving the fibroblast proliferation when the cells were setting in the scaffold (Azizian, Hadjizadeh, & Niknejad, 2018). A new approach reported nanodiamonds first

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References

433

FIGURE 18.5

Schematic of electroresponsive biohybrid composites based on silk fibroin and reduced graphene oxide. Source: From Magaz, A., Ashton, M.D., Hathout, R.M., Li, X., Hardy, J.G., Blaker, J.J. (2020). Electroresponsive silk-based biohybrid composites for electrochemically controlled growth factor delivery. Pharmaceutics, 12, 1
12. https://doi.org/10.3390/pharmaceutics12080742.

used to bind VEGF with high affinity, embedded in a collagen-chitosan hydrogel (Fig. 18.5). VEGFnanodiamonds presence improves the mechanical properties of hydrogels, but they do not affect the thermosensitive gelation property. The composite material was biocompatible, and the use of VEGF joined to nanodiamonds allows a sustained release of this exogenous GF (Pacelli et al., 2017).

18.4.7 Polysaccharide-polysaccharide composites for growth factor delivery A chitosan composite loaded with two different microparticles was used for controlled and sequential IGF-1 and BMP-6. Specifically, IGF-1 was loaded into alginate microparticles and BMP-6 in poly(lactic-co-glycolic acid). The results showed how IGF-1 is quickly released in the early stages while BMP-6 release was maintained longer. The stepwise pattern of releasing these composite materials induced the proliferation and osteoblastic differentiation of cementoblasts (Duruel, C ¸ akmak, Akman, Nohutcu, & Gu¨mu¨s¸ derelio˘glu, 2017). An alginate/gellan gum-calcium phosphate cement (CPC) composite with VEGF has been created by the advanced technique for constructing polymeric materials, the 3D plotting. The composite material preserves the VEGF bioactivity because its release stimulates in vitro endothelial cell proliferation and angiogenesis. Besides, it encourages the

differentiation of rat mesenchymal stromal cells into osteoblasts, and the in vivo evaluations support the formation of new bone tissue after 12 weeks. The composite parts acted synergistically because the CPC component induces osteoconductive activity, and the VEGF molecule enhances the vascularization in the defect region (Ahlfeld et al., 2019). A complex biocompatible of heparin, and ethylene arginyl aspartate diglyceride (PEAD), with TGF-β3 and interleukin-10, were quickly prepared to reduce scar formation during wound healing. They were coacervated structures where the GFs were protected and later released in a sustained manner. The expected results were resolved due to the dual GF delivery enhance the proliferation and migration of skin-related cells, and gene expression studies showed upregulation of ECM formation at early stages. At the same time, the scar-related genes were down-regulated. Additionally, damaged skin in a rat demonstrated accelerated wound closure and skin regeneration after three weeks, and the immunohistochemical staining revealed the formation of the epidermal layer. The coacervate platform augments the quality and quantity of regenerated skin tissues without scar formation (Park et al., 2019).

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C H A P T E R

19 Biological macromolecules for growth factor delivery in bone regeneration Aristeidis Papagiannopoulos and Eleni Vlassi Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece

19.1 Introduction

19.2 Bone regeneration

This chapter presents the application of biomacromolecules as vehicles for successful encapsulation, release and delivery of growth factors targeted for treatment in bone injury and bone regeneration. Additionally, it demonstrates how new formulations of growth factors by the aid of natural polymers can be synthesized using the experience and knowledge of the field of nano-drug delivery. The chapter begins with a description of the process of bone tissue regeneration in heal injuries and arthritic conditions, introduces the main ideas through the scope of allogenous and autogenous transplantation and describes the role of growth factors in these processes. In the next sections special categories that are important in current growth factor delivery for applications in bone regeneration are presented. Examples include hydrogels, nanoparticles (NPs) and polysaccharide complexes in order to illustrate the methodologies for the investigation of loading and release of growth factors and characterization of the carrier systems at the nanolevel.

Bone is a rigid organ that supports body weight, protects other organs and transfers forces from muscles in order to make motion possible (Bates & Ramachandran, 2007). It is a superstructure that contains soft collagen protein and stiff mineral. These two assemble into mineral fibrils and can be considered as the building blocks of the variety of bone tissues. The relative content of collagen and mineral defines the stiffness of a bone to a high degree in a sense of a bionanocomposite material. Morphology of the bone at the micro- and macro-level, rate of bone remodeling, microdamage, cellular apoptosis, collagen structure and cross-linking, crystal size and mineralization affect the quality of the tissue (Tzaphlidou, 2008). Overhydroxylation and cross-linking variation have been reported to increase the fragility of the bone. Moreover, hydroxylation may be related to the density and trabecular volume of the bone. There is evidence that osteogenesis imperfecta is connected to impairment of triple helix formation of type I collagen which is overmodified posttranslationally. Osteoarthritis and osteoporosis, as diseases that cause complications

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00019-1

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19. Biological macromolecules for growth factor delivery in bone regeneration

in connective tissues, are also related to mutations of structural genes (Rubin et al., 2003). In addition, no significant difference between collagen fibril apatite crystals at the nanolevel was found between normal and osteoporotic human bone. Bone regeneration is a fascinating physiological procedure (Cho, Gerstenfeld, & Einhorn, 2002) that is found throughout a person’s life. Osteogenesis starts in the sixth or seventh week of the development of the embryo and completes at around the 25 years of age. A precursor mesenchymal tissue transforms directly into bone (intramembranous ossification, flat bones of the skull) or after a cartilage intermediate (endochondral ossification, axial skeleton and long bones). In healing of normal bone fractures, acute inflammatory response triggers the formation of hematoma. A model for callus formation is created on the fracture ends and in the medulla by blood and bone marrow cells. Mesenchymal stem cell recruitment, proliferation and differentiation into are the basis of bone regeneration. Granulation tissue which is rich in fibrin follows the hematoma formation with the assistance of cartilaginous tissue which provides mechanical stability to the fracture. Supply of blood to the bone repair zone is achieved by angiogenic pathways, apoptosis of chondrocytes and degradation of cartilage. The cartilaginous soft callus is subsequently replaced by bone callus. Bone callus at this stage provides adequate stability and strength however, remodeling toward a bone morphology of lamellar structure is needed. This process although it is faster in young patients it may take several years. Clinical conditions that result from injuryinduced bone defects, infection, resections after operation or inadequate regenerative progress entail enhanced bone regeneration. Other conditions include atrophic nonunions and osteoporosis. Autologous grafting of bone (transplantation within the same patient) has osteoconductive, osteoinductive and osteogenic properties and therefore can be incorporated faster and in

higher degree. Autogenous cancellous bone graft is considered as “the gold standard” for filling spine and bone defects as it involves the necessary components for bone healing such as bone matrix protein, osteoconductive hydroxyapatite collagen matrix and osteoprogenitor cells (Dimitriou, Jones, McGonagle, & Giannoudis, 2011). Except from providing the support for formation of bone and wound healing, grafts are containers of minerals. Bone grafting replaces bones that are missing by substitutes by surgical procedure. Natural growth of the existing bone in the body of the patient takes the place of the graft bone. Bone grafts depend on the material that is used and many times there are complications regarding limited supply and donor site acceptance (Wang & Yeung, 2017). Allogenic bone graft (transplantation from another individual) offers a very good alternative and has been successfully used in clinical practice, in particular for patients with low potential for healing. Allografts are immunogenic and have a failure rate that is higher than the ones observed in autografts. Histocompatibility complex antigens are generally considered as the underlying cause. It has been found that allografts with small histocompatibility differences lead to increased acceptance of transplants. Strategies for effective bone regeneration include synthetic substitutes and scaffolds in combination with active molecules. In this case, growth factors interfere with the bone progenitor cells and trigger bone development. Additionally, nanoparticle drug delivery is applied for wound and defect healing of damaged tissue with the scope to prevent bacterial growth and infection. Treatments that are based on delivered cells such as osteoblasts are used in bone transplants (Omrani, Ansari, Kordestani, Kiaie, & Salati, 2019). Adult mesenchymal stem cells have capacity for unlimited differentiation including osteoblasts and chondrocyte and have been utilized in regeneration of bone fractures. Bionanocomposites with mineral compounds provide mechanical stability and induce osteoconduction to the scaffolds

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19.3 Growth factors in tissue and bone regeneration

(He et al., 2009). Bone tissue engineering is supported by the high surface area provided by nanofibers for interaction with hydroxyapatites. Biodegradation properties and controlled bioaction can be controlled by polymer-based compounds, where ability for deformation is one of their most important characteristics. Biopolymers such as polysaccharides and proteins and synthetic biocompatible polymers such as polylactic acid are usually employed in macromolecular substrates. Bone grafts are designed to fit in defects within osseous tissue and applied in 3D printing in order to create personalized substitutes using stem cellladen hydrogels (Ansari, 2019). The mechanisms under the complex process of physiological fracture healing incorporates bone induction and conduction and are based on molecular signaling both intra- and extracellularly while it is still not fully understood (Einhorn, 1998; McClung et al., 2014). It has been shown that the process of fracture repair replicates formation of tissue at the stage of embryonic tissue development. Growth and differentiation factors interact with cartilageforming primary cells and bone-forming cells at the fracture site. Conditions at the fracture site that depend on hormones, nutrients, pH, mechanical stability and electrical environment and the role of periosteum and the bone marrow all contribute to the healing process. In particular, periosteum responds when stabilization and fixation of the wound are absent by stimulating a larger callus with endochondral ossification (Ghanbari, Vakili-Ghartavol, Zorzi, & Miranda, 2016).

19.3 Growth factors in tissue and bone regeneration Communication at the intercellular level has long been known and demonstrated (Cross & Dexter, 1991). Cellular processes are affected by external parameters such as physical environmental variables, molecules of the

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extracellular matrix, molecules adhesive to cells and multiprotein complexes of the intercellular bridges (Lee, Silva, & Mooney, 2011). Culture and engineering of tissues has been based on particular soluble factors. These polypeptides are termed “growth factors” and are able to trigger and guide cellular activities. Cell-signaling is a complex and multidimensional series of events which begins with the secretion of a growth factor by a producer cell. Cell surface receptors receive signals from the extracellular matrix by binding of growth factors or other molecules (hormones, cytokines, etc.). An integrated response of the cell is initiated when the signal reaches the cell nucleus and includes ion flux, metabolism changes, protein synthesis and gene expression (Cohen, Ren, & Baltimore, 1995). Owing to the short half-life and low diffusion coefficient within the extracellular matrix, growth factors have short-ranged action. A growth factor can transport different signals depending on the number of target cells, receptor type and signal transduction within the particular cell. The ability of the growth factors to bind to the extracellular matrix, degradation of extracellular matrix and other external conditions may also affect the cellular response. The ability of a tissue to restore its structure and function after being injured or inflamed is crucial for the survival of an organism. Regeneration may be achieved by some tissues as they can restore their damaged parts. In this case proliferation of uninjured cells occurs. For example, skin and intestine epithelial cells are proliferating very fast. In parenchymal organs as the liver the restoration capability is remarkable. Stem cells may also support tissue repair. Mammalian organisms do not have an extended capability of full tissue restoration (Maddaluno, Urwyler, & Werner, 2017). A scar is normally formed in such cases while the incapacity of the tissue for complete restoration leads to deposition of fibrous tissue. Scar formation is by no means a restoration of the

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tissue but it provides structural integrity for the tissue to be able to function. In fibrosis conditions deposition of collagen, for example, in the liver, kidneys, and lungs originates from chronic inflammation (Maddaluno et al., 2017). Cell proliferation which is the central process for tissue repair is driven by growth factors. Vascular endothelial growth factor (VEGF) has been demonstrated to increase multipotential stromal cell proliferation platelet-derived growth factor facilitates osteogenesis (Rodrigues, Griffith, & Wells, 2010) and nerve growth factor reduces neurological deficits caused by brain injury in animals (Aloe, Rocco, Bianchi, & Manni, 2012). Skin regeneration is a good example of the action of growth factors. At the early stages of wound healing pro-inflammatory growth factors and cytokines are released which stimulate the expression of other growth factors. Fibroblasts regulate fibroblast growth factor (FGF) 7 at the site of the wound and keratinocytes in combination with macrophages produce VEGF which is the major angiogenic regulator (Xie et al., 2013). Subsequently, the wound area is gradually filled by the granulation tissue, a network of collagen, hyaluronic acid and fibronectin. Inflammatory cells decrease in number while platelet-derived growth factor and transforming growth factor attract fibroblasts to the wound site (Park, Hwang, & Yoon, 2017). In chronic wounds, for example, diabetic foot ulcers, a complication that arises often is inflammatory cells that are responsible for matrix metalloproteinases and other proteases are at increased expression. This leads to cytokine deficiency which compromises cellular signaling related to healing. Risk of bone fracture is related to the accumulation of bone mass. In more detail, bone mass reaches a plateau which lasts for several years after the adolescence. Bones become more fragile and osteoporotic at older ages because bone mass decreases and therefore risk of fracture becomes greater. Acquisition of peak bone mass by adolescent individuals is very crucial for the future condition of the

bones. Body composition is regulated by endocrines which are controlled by the insulin-like growth factor 1 (IGF-1) along with the conservation of mineral density in the bones. It has to be noted that IGF-1 and bone mass are correlated and in addition IGF-1 can be used as a predictor for osteoporosis and fracture risks (Ohlsson et al., 2009). It plays a critical role in fetal organogenesis and regeneration of tissues including bones, nerves and muscles. IGF-1 has been proposed as an alternative to none morphogenetic protein which although stimulates bone formation it has shown controversial effects in clinical and experimental studies. On the other hand, IGF-1 is correlated well with bone regeneration and has shown promising results in osteogenic differentiation of mesenchymal stem cells (Reible, Schmidmaier, Moghaddam, & Westhauser, 2018). The success of therapies that are based on growth factors in clinical practice (Lee et al., 2011; Lorentz, Yang, Frey, & Hubbell, 2012; Mitchell, Briquez, Hubbell, & Cochran, 2016) can be increased by the use of delivery methods and especially technologies at the nanoscale. Carriers that include biomacromolecules (Kuroda, Kawai, Goto, & Matsuda, 2019) such as hyaluronic acid, fibrinogen, gelatin, collagen and others are suitable for producing NPs, nanocomposite scaffolds and hydrogels able to carry and deliver growth factors in a controlled and potentially stimuli-responsive manner. Carrier materials design should focus on biocompatibility, non-cytotoxicity and nonimmunogenicity in order for side effects on neighbor tissues and involved cells to be minimized (Yun et al., 2012). Cell adhesion and cell proliferation would have to be enhanced by the carrier material. Finally, the materials should be degradable by enzymes and biological fluids. Control on dose administration in terms of amount and time of delivery is another perquisite for the development of efficient carriers of growth factors. Synergistic and antagonistic effects may take place in multiple growth

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19.4 Biomacromolecules as carriers of growth factors

factor administration and therefore the complexity of bone repair and regeneration has to be taken into account (Schmidmaier et al., 2003; Vonau, Bostrom, Aspenberg, & Sams, 2001).

19.4 Biomacromolecules as carriers of growth factors Absorbable collagen sponge has been termed as a standard carrier for delivery in bone regeneration. Recombinant human bone morphogenetic protein-2 (rhBMP-2) doses at high levels are needed in this case because of the leakage and burst release from this sponge material. Microbeads based on alginate have been proposed as carriers of rhMBP-2 with scope of controlling the release of the growth factor and eventually to be able to function with low dose levels (Abbah, Liu, Goh, & Wong, 2013). The beads were prepared by electrostatic encapsulation allowing alginate droplets to gel in SrCl2 solutions. The microbeads were coated by two successive polyelectrolyte layers taking advantage of polyelectrolyte complexation. Initially a layer of the cationic natural polyelectrolyte poly-l-lysine was formed on the beads’ surface by immersion in the polyelectrolyte solution. Subsequently, a layer of the anionic biomacromolecule heparin is formed by immersing in the corresponding solution, after washing. Posterior spinal fusion was performed on forty rats. Implantation of the scaffolds was followed by micro-computed tomography and histological analysis (Wang et al., 2013). Spinal fusion rate was comparable to the one of absorbable collagen sponge however achieved at lower dose. Additionally, it was pointed out that a significant reduction of seroma rate and size, heterotopic ossification and swelling of soft-tissue inflammation occurred. Weakening of the side effects of the treatment, which was a perquisite of the investigation, was related to the localization of the growth factor rhBMP-2 within the microcarrier and the incorporation of the growth factor modulator, heparin.

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The capacity of rhBMP-2 for osteoinduction is greatly increased when it is administered by the aid of a carrier. The carrier may also function as a matrix for the invasion of cells and immobilizing the growth factor at the desirable tissue site (Uludag, D’Augusta, Palmer, Timony, & Wozney, 1999). Solid-liquid phase separation was used to prepare porous collagen matrices. Subsequently, chondroitin sulfate was added in aqueous medium and a crosslinked chondroitin sulfate/ collagen matrix was formed by the aid of glutaraldehyde (Wang et al., 2010). The scaffolds were loaded with solutions of rhBMP-2 in appropriate volume and concentration and equilibrated for 12 h at 4 C. Release kinetics was measured in vitro and in vivo (male Sprague-Dawley rats) by radioiodination and radioactive counting. Addition of chondroitin sulfate increased the hydrophilic character of the matrices significantly. The authors proposed that this effect would enhance absorption of water. The initial burst growth factor release was enhanced because of its easier dissociation from the matrix. In the presence of the polysaccharide, the higher amount of absorbed rhBMP-2 resulted to increased levels of growth factors at the implant site. Additionally, a hydrophilic surface favors the adhesion of cells, their proliferation and differentiation. All these factors contributed to the acceleration of bone regeneration. Bae, Choi, Joung, Park, and Han (2012) proposed a titanium-based surface as a template with both osteoconductive and osteoinductuve properties with the aid of nanocomplexes of BMP-2 with chondroitin sulfate. Electrostatic complexation between the growth factor and the polysaccharide took place in 10 mM Tris buffer at pH 7.4. The resulting NPs had a size in the range 150
250 nm while their surface charge was affected by the added amount of chondroitin sulfate. The nanocomplexes were freeze-fried and kept in vacuum. Titanium disks were coated with hydroxyapatite by an oxidization protocol in order to obtain a biomimetic surface. The solution of the nanocomplexes was precipitated on

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the hydroxyapatite-modified surface by the aid of calcium/phosphate solution. A gradual release without any initial burst was observed in a period of four weeks. Fast proliferation of mouse osteoblasts was achieved. Nanocomplexes were responsible for increased upregulation of bonespecific markers gene expression. This system was put forward as capable of having both osteoinductive character due to the growth factor and osteoconductive property because of the underlying hydroxyapatite and was proposed for implants in dentistry and orthopedics. Succinylated type I atelocollagen was used as a carrier of rhBMP-2 by chemical bonding. Loaded carriers were added to the growth medium of seeded ST2 cells in 10-cm petri dishes. Cells were analyzed in terms of gene expression by quantitative reverse transcription polymerase chain reaction after being collected from incubation in fixed time intervals. RNA was spectrophotometrically tested in order to quantify its quantity and purity after its extraction and purification. The study revealed two patterns of expression of rhBMP2 downstream genes. In the first pathway BMPR-IA gene, Smad 1 gene and Smad 5 gene revealed high level gene expression that lasted for a long time period in the immobilized group. In the next stage a decline occurred. The nonimmobilized group was characterized by an initial sharp decrease in gene expression and a subsequent increase that followed the increase of BMP-4 expression. Low basic expression was observed in the second pattern for Smad 6, 7, and 8 genes and it was followed by an initial increase and a decrease at the final stage upon addition of rhBMP. For the nonimmobilized case gene expression was effectively absent in the beginning while a delayed gene expression was found which accompanied the expression of BMP-4. This work demonstrated a promising method for effective bone regeneration (Tsujigiwa et al., 2006). Keratins have been used in the delivery of pharmaceutical substances for conditions in skin,

bone, nerve and hemostasis (Han, Ham, Haque, Sparks, & Saul, 2015). In particular they have been used in the delivery of rhBMP-2, antibiotics and model drugs. Reconstituted keratin is itself a promising raw material for orthopedic applications. Osseointegration and biocompatibility of keratin was investigated in vivo on an ovine study (Dias, Peplow, McLaughlin, Teixeira, & Kelly, 2010). Keratin biomacromolecules were modified in several ways including compact and porous constructs, acidic or neutral regeneration, hydroxyapatite addition. The materials were formulated in the shape of small rods and introduced in round defects inside sheep femur and tibia. Histological tests were performed after a minimum fixation of 48 h. Tissues were processed and immersed in paraffin wax so that they would be cut in the transverse plain. Morphological investigations proved that the introduced materials did not cause any immune or toxic bone response. The keratin-based implants did not show any degradation and functioned as an inert material. The addition of hydroxyapatite improved the formation of new bone tissue. It is evident from both histological and morphological analysis in this study that these keratin-based materials do not produce an immune or toxic response in bone. The keratin implants did not seem to have degraded to any extent after 8 weeks. In the 12- to 24-week period, there was peripheral resorption and infiltration of bony trabeculae with regard to the porous constructs only. Indeed, the tissue reaction, as stated earlier, appeared to model that of a fairly inert material, and the introduction of hydroxyapatite seems to improve new bone formation. Histological profiles illustrated formation of granulation tissue, new bone formation originating radially from the bone that already existed. Bone implants were fabricated in vitro from mesenchymal stem cells that were previously transfected by basic FGF. Tricalcium phosphate (β-TCP) ceramics with biodegradable properties and pore size between 200 and 500 μm and 78%

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19.5 Hydrogels and sponges

porosity were used as a scaffold for cell seeding (Guo et al., 2006). These scaffolds were proposed for large segmental bone defect repair and were applied as allografts on New Zealand White Rabbits. Basic FGF except from accelerating bone repair by inducing proliferation and assisting cell differentiation, it promotes angiogenesis which is crucial in the initiation of endochondral and intramembranous ossification in fracture healing (Huang, Kaigler, Rice, Krebsbach, & Mooney, 2005). The mesenchymal stem cells could adhere to the TCP scaffolds and effectively generate new bone tissue. The in vivo studies proved that 12 weeks after transplantation was better in comparison to the control group. In more detail, animals had a quick recovery and regained their movement within three weeks without any sign of infection or limitation. Examination by X-rays revealed calluses that formed around the defects in 2 weeks whereas in 8 weeks the defects were united and in 12 weeks osteotomies were found with bony union. In 2 weeks capillary vasculatures were observed within the porous structure of the TCP including hemocytes. Large amounts of bone were formed and distributed evenly within the porous structure in 12 weeks.

19.5 Hydrogels and sponges Bone morphogenic proteins (BMPs) have high osteoinductive capabilities and as growth factors they are widely used in bone regeneration. They stimulate mesenchymal stem cells to migrate to sites where bone formation is needed and to differentiate in order to regenerate the bone tissue (Gautschi, Frey, & Zellweger, 2007). A disadvantage of BMP-2 in clinical treatment, which is otherwise very efficient, is its short half-life. This may lead to significant decrease of its bioactivity in solution and this way require large amount of administered dose. Therefore a technology to prolong the release of BMP-2 is beneficial for treatment of injured bones. Fibrinogen conjugated by heparin was synthesized by carbodiimide

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chemistry (Yang et al., 2010). Upon mixing with thrombin an injectable hydrogel was prepared. An enzyme-linked immunosorbent assay was run in order to quantify the release kinetics of BMP-2 from the hydrogels. Bioactivity was quantified in vitro on cultured rat calvarial osteoblasts by the ability of released BMP-2 to stimulate alkaline phosphatase activity. Implantation of BMPcontaining fibrin in Sprague-Dawley rats and soft X-ray radiography, computed tomography and histological analysis were used to characterize bone formation. The release of BMP-2 was prolonged from 3 to 13 days with the heparinconjugated fibrin in comparison to normal fibrin in regard to about 80%
90% of the released growth factor. Additionally, expression of alkaline phosphatase was found higher. Effective bone formation was achieved at significantly lower doses of BMP-2 in comparison to unconjugated fibrin and other delivery systems such as collagen sponge (Yang et al., 2012). Poloxamines are combined with cyclodextrins in order to obtain better gelling properties at lower concentration of polymeric material. For example, α-cyclodextrins (Simo˜es et al., 2013) create threads along poly(ethylene oxide) chains (polypseudorotaxanes) in aqueous media reducing the hydrophilic in comparison to pristine poloxamine blocks. The threaded structures selfassemble into nanocylinders that are stacked in crystalline channels. This hierarchical morphology allows the formation of a shear-thinning fluid able to perform as an injectable gel (del Rosario et al., 2015). Except from BMP2, simvastatin was also loaded to the produced hydrogels because it also assists angiogenesis and osteogenesis. The tetronic gels in the absence of cyclodextrin showed shear-thinning and predominantly viscous properties at room temperature (20 C) and transformed to strong hydrogels at body temperature (37 C). In the presence of cyclodextrin the viscosity was enhanced at room temperature but the viscoelasticity was compromised at body temperature. Nevertheless, simvastatin weakened viscoelasticity in cyclodextrin-containing gels at

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37 C because of preferable interaction of cyclodextrin with simvastatin instead of tetronic. The drug release profiles from the gels without and with cyclodextrin revealed full release within 3 weeks when the drug was at low dose. At high dose the release was not completed in this time period. The supramolecular gels increased osteogenic and vascular response while cyclodextrincontaining hydrogels had better performance. Synergistic effects of BMP-2 and simvastatin was not observed, however the required BMP-2 dose could be significantly reduced. The role of the extracellular matrix in guiding tissue repair and in the delivery of growth factors has been investigated on extracellular matrix with removed cells. Decellularized matrices maintain the natural environment of the tissues and therefore promote cell proliferation and stimulate cell proliferation. As a result the interest on their use as therapeutic agents is rising (Kim, Majid, Melchiorri, & Mikos, 2018). Hydrogels from porcine menisci decellularized matrices with thermoresponsive properties have been loaded with chondrocytes (Wu et al., 2015), in another case matrices were obtained from cartilage showing cytocompatibility and no inflammatory response (Kwon et al., 2013). A major drawback of this kind of hydrogels is weak mechanical properties. Mixing with biopolymers (Smith et al., 2014; Smith et al. 2014) and cross-linking (Beck et al., 2016; Rowland, Lennon, Caplan, & Guilak, 2013) has been suggested as a way to enhance their strength. The content in growth factors may be decreased because of the removal of the cells, an amount sulfated glycosaminoglycans and extracellular matrix proteins are preserved. Regeneration of ulna bone at the segmental bone defect of rabbits was investigated by Yamamoto, Takahashi, and Tabata (2006). BMP-2-containing hydrogels based on gelatin were illustrated to function differently in relation to their water content. This parameter could be tuned by controlling the gel structure

via the amount of added gelatin and crosslinker (glutaraldehyde). Gelatin hydrogels were implanted after 4 cm length incisions and dissecting of the tissue above the diaphysis in order to prepare the segmental defects. BMP-2 release in the sites of implantation was maintained for extensive time periods. The swelling ratio of the hydrogels could control the release rate which was faster as the swelling decrease was higher. BMP-2 retention (5
30 days) and hydrogel degradation profiles were correlated pointing that hydrogel degradation defined release kinetics (Yamamoto, Takahashi, & Tabata, 2003). Photographs with soft x-rays on 6 weeks ulna defects with implanted BMP-2containing hydrogels gave clear evidence of the bone regeneration in contrast to blank gelatin hydrogels. Gelatin hydrogels with water percentage 97.8 wt% maximized the value of bone mineral density of the defect than the cases of lower and higher water content suggesting an interplay between induction of bone and release kinetics.

19.6 Scaffolds and fibers A system for the delivery of bone marrow protein growth factors has been designed to incorporate two kinds of nanocapsules in 3-D scaffolds with the aim of sequential release. Scaffolds based on chitosan were fabricated by wet spinning in order to have two functions that is to act as scaffolds for tissue growth and to deliver growth factors for bone tissue regeneration (Yilgor, Tuzlakoglu, Reis, Hasirci, & Hasirci, 2009). Chitosan and chitosan mixed with poly (ethylene oxide) were used to make 3D fiber mesh scaffolds. Solutions of the components in acetic acid resulted to fibers after injection to Na2SO4 coagulation bath. A double emulsionsolvent evaporation method (Yilgor, Hasirci, & Hasirci, 2010) was used to prepare nanocapsules of poly(lacticacid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

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19.6 Scaffolds and fibers

(PHBV) in order to encapsulate BMP-2 and BMP-7. The release from these nanocapsules is sequential as it consists of an early release of BMP-2 and a late release of BMP-7. The underlying mechanism is based on the difference between the degradation rates of the two polymers and can be used to prolong the release of one of the components in comparison to the other. The reported encapsulation efficiencies were adequate and resulted to satisfactory release rates. Mesenchymal stem cell differentiation into osteoblasts increased while proliferation rate decreased. The hierarchically constructed system (fibers/nanocapsule/growth factors) and the sequential process led to better performance in alkaline phosphatase activity (induced differentiation). Sequential release was considered more effective as it mimics natural healing process (Yilgor et al., 2009). Nanofiber scaffolds were prepared for dual delivery of the proliferation/angiogenesis stimulator FGF2 and the osteogenic. FGF18. Hollow core-shell fibers were produced by polycaprolactone as an outer layer and poly(ethylene oxide) as an outer layer using coaxial electrospinning. Mesoporous bioactive glass nanospheres were used for the delivery of FGF18. FGF2 was intended to be the molecule for fast release whereas FGF18 was intended for slow release. The hollow core of the fibers contained FGF2 and FGF18-loaded mesoporous bioactive glass nanospheres. Nanospheres of welldefined shape, monodisperse size (120 nm), mesopore size at 7 nm and negative ζ-potential were obtained. The hollow fibers had a diameter of about 1 μm with mechanical properties (tensile strength and elastic modulus) where of the order of several MPa and increased as a function of bioactive glass content. At the same time however, maximum strain decreased. Hydrolytic degradation was tested in phosphate buffered saline (PBS) at 37 C for the nanocomposite scaffolds. It was found that the degradation was stronger in the presence of the bioactive glass NPs by observation of

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morphology (SEM), weight loss and compromise of the mechanical properties (Kang, Kim, Singh, Jang, & Kim, 2015). In another dual delivery system the synergy of BMP2 with FGF2 or BMP2 with VEGF increased neovascularization and formation of new bone. The growth factors FGF and VEGF were not effective when they were used alone (Kuttappan et al., 2018). Guided bone regeneration was evaluated on Go¨ttingen miniature female pigs using collagen/hydroxyapatite/β-TCP scaffolds and collagen matrix from jelly fish with incorporated cocktail of growth factors. The first and second bicuspid teeth were removed from both lower jaw quadrants. Cleaning and rinsing of the teeth were done in PBS with the support of antibiotics to stop bacterial infection. Periodontal ligament stem cells were isolated and characterized by flow cytometry. Life-death assay was run for the quantification of stem cell survival in collagen powder. Implants were placed in the premolar positions after three months of healing. New bone was observed in a time period of 120 days. Additional increase in osseous tissue was negligible by employing the growth factor cocktail or periodontal ligament stem cells (Ka¨mmerer et al., 2017). The use of peptides that self-assemble in physiological conditions of pH and salt content are attractive candidates for delivery applications using hydrogels. Cartilage repair and tissue engineering is one example. The peptide (KLDL)3 has been proposed for the delivery of biotinylated IGF-1 and transforming growth factor-1 for the stimulation of chondrocytes and bone marrow stromal cells to produce proteoglycan respectively (Miller, Kopesky, & Grodzinsky, 2011). This work showed that the versatility in peptide sequence functionalization has to be combined with the proper mode of delivery to maintain bioactivity. In more detail, tethered growth factors were not adequately effective in stimulating production of proteoglycans.

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19.7 Nanoparticles and nanoassemblies Growth factor delivery is a challenging task because of the short half-life, poor stability, and rapid deactivation. In order to enhance their bioavailability and biocompatibility, enhanced encapsulation into NPs is a promising path as it provides targeted delivery, low dose administration, and minimum side effects. Gelatin methacryloyl hydrogels have been successfully applied in cardiac tissue engineering (Navaei et al., 2016; Shin et al., 2016) and bone tissue vascularization (Kazemzadeh-Narbat et al., 2017). Gelatin methacryloyl in combination with chitosan were used for the production of nanocarriers of bFGF (Modaresifar, Hadjizadeh, & Niknejad, 2018). Chitosan of low molar mass was dissolved in acetic acid and mixed with bovine serum albumin (BSA) and bFGF at pH 5.5. Tripolyphosphate was used as a crosslinker leading to the formulation of the three components into NPs. Blank chitosan NPs had a size of about 270 nm whereas the loaded NPs were about 420 nm. Release of BSAbFGF from the NPs had an initial burst release (about 2 h) where about 80% of the loaded substances were unloaded whereas the rest of release continued for several days. Hydrogel scaffolds were prepared by UV cross-linking gelatin methacryloyl. Hydrogels with encapsulated NPs were prepared by introducing the NPs prior to the exposure to UV light. The release of BSA-bFGF from the NPs-containing hydrogel was characterized by a 75% release within 4 days and 90% at 7 days. Normal human dermal fibroblast cell proliferation and viability increased in hydrogels with BSA and the growth factor. The results from MTT assay were also encouraging and additionally the cell viability was retained. These nanodelivery systems can find application in angiogenesis and bone regeneration. NPs from polysaccharide complexes between chitosan and chondroitin sulfate were incorporated in biphasic calcium phosphate scaffolds. A burst release of the growth factor

that lasted 6 days was followed by a sustained release that lasted 6 weeks. The system showed good osteoconductivity and mineralization of rabbit ectopic bone (Wang et al., 2015). Poly-llysine NPs incorporated in fibrin networks with encapsulated BMP-2 showed an increased release kinetics rate whole the burst release was weak. Human mesenchymal stem cells were encapsulated in the fibrin hydrogel and stimulated by the released growth factor. Their osteogenic differentiation was enhanced and histological examination showed homogeneous formation of bone (Park, Kim, Moon, & Na, 2009). Gelatin microspheres were incorporated in poly(propylene fumarate) scaffolds had an initial 24 h burst and a 27-days sustained release of VEGF. However, no significant increase in bone development was observed (Young et al., 2009). Another VEGF loaded system, that is spheres of PLGA in a gelatin hydrogel with sustained release up to 50
60 days showed enhanced formation of ectopic bone (Kempen et al., 2009). Similarly, alginate microcapsules embedded in collagen scaffolds improved osteogenesis in umbilical mesenchymal stem cells and enhanced formation of bone and vascularization in calvarial defects (Subbiah et al., 2015). Chitosan scaffolds that contained microspheres of gelatin presented increased activity of alkaline phosphatase in stromal cells (Kim et al., 2012). Finally, silk fibroin microparticles incorporated in hydroxyapatite scaffolds showed promising results in regenerated bones in rat calvarial defect (Jun et al., 2013). A fusion protein with the ability to induce osteoblastic differentiation was demonstrated to be rapidly purified and be able to self-assemble in nanoaggregates (McCarthy, Yuan, & Koria, 2016). Therefore it was proposed as a potential delivery system for BMP-2 in hydrogel matrices. Excision of BMP-2 gene from plasmid pUC57 was performed with enzymes in order to prepare the gene encoding the fusion protein BMP-2-V40C2.

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References

The fusion protein transforms into the aggregated state at 32 C in a gradual temperature dependence. The transition was investigated by dynamic light scattering. At 4 C a mean diameter of 11 nm was observed while at 37 C the size was again monomodal although the diameter was at 417 nm. C2C12 cells were treated with the fusion protein which was found to maintain its functionality. The differentiation of the cells to osteoblasts was confirmed by phosphatase activity although a fusion affected BMP-2 activity. Glycosaminoglycans, such as heparan sulfate, are the major constituent of the extracellular matrix and they have a key role in immobilizing growth factors (Bernfield et al., 1999). The related growth factors, that is, heparin-binding growth factors associate with glycosaminoglycans with electrostatic interactions. The amino acid residues of bFGF interact with the carboxyl groups of the heparan sulfate not only by electrostatic but also by van den Waals interactions (Raman, Venkataraman, Ernst, Sasisekharan, & Sasisekharan, 2003). Platelet-derived growth factor, BMP, VEGF and others are heparin-binding and hence the extracellular matrix is considered as a container of growth factors (Ashikari-Hada et al., 2004). Zandi et al. proposed a biomimetic system based on proteoglycans motivated by the role of sulfated glycosaminoglycan polymer chains which immobilize growth factors by electrostatic interactions in native tissues (Zandi et al., 2020). NPs were synthesized by complexation of oppositely charged polyelectrolytes. Two anionic/cationic pairs were produced with dermatan sulfate/poly-l-lysine and tragacanth gum/poly-llysine. Poly-l-lysine solution was added dropwise into the anionic polysaccharide solution in order to prepare the complexes. NPs of size between 100 and 200 nm in the case of dermatan sulfate and between 200 and 400 nm for tragacanth were obtained. Size increase as the percentage of polyl-lysine increased. The spherical shape of the NPs was confirmed by SEM and TEM. VEGF in PBS was mixed with NPs solutions in order to encapsulate the growth factor in the NPs. Loading

efficiency was reported at 93% for dermatan sulfate-based and 80% for tragacanth-based nanocarriers. VEGF mitogenic activity was maintained significantly in dermatan sulfate/poly-l-lysine complexes contrary to unbound VEGF. NPs were compatible with human bone marrow stromal cells. Enhanced metabolic activity was observed for VEGF in both kinds of NPs and the system was overall considered good candidates for nanodelivery of growth factors and cytokines for tissue engineering.

19.8 Concluding remarks Biological macromolecules for delivery in medical science, food industry, and tissue engineering are thoroughly investigated because of their biocompatibility, nontoxicity, and biodegradability. They can be formulated in many morphologies such as NPs, intermolecular complexes, nanostructured hydrogels, nanocomposite fibers, and scaffolds. Their application in the delivery of growth factors seems a very promising field for the regeneration and healing of bone tissues as administered dose can be optimized and side effects can be diminished. Additionally, great versatility of design of nanocarriers with controllable and stimuli-responsive release of growth factor comes from the ability of biomacromolecules such as polysaccharides and protein to interact nonspecifically via electrostatic, hydrophobic, and hydrogen bond interactions with the loaded molecules. Powerful noninvasive methods based on scattering of radiation (light scattering, small angle X-ray and neutron scattering) play a key role in elucidating these promising nanostructure materials.

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4), 350
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Aloe, L., Rocco, M. L., Bianchi, P., & Manni, L. (2012). Nerve growth factor: From the early discoveries to the potential clinical use. Journal of Translational Medicine, 10(1), 239. Ansari, M. (2019). Bone tissue regeneration: Biology, strategies and interface studies. Progress in Biomaterials, 8(4), 223
237. Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., & Kimata, K. (2004). Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library. The Journal of Biological Chemistry, 279(13), 12346
12354. Bae, S. E., Choi, J., Joung, Y. K., Park, K., & Han, D. K. (2012). Controlled release of bone morphogenetic protein (BMP)-2 from nanocomplex incorporated on hydroxyapatite-formed titanium surface. Journal of Controlled Release, 160(3), 676
684. Bates, P., & Ramachandran, M. (2007). Bone injury, healing and grafting. In M. Ramachandran (Ed.), Basic orthopaedic sciences the Stanmore guide (pp. 123
134). London: Hodder Arnold;. Beck, E. C., Barragan, M., Tadros, M. H., Kiyotake, E. A., Acosta, F. M., Kieweg, S. L., & Detamore, M. S. (2016). Chondroinductive hydrogel pastes composed of naturally derived devitalized cartilage. Annals of Biomedical Engineering, 44(6), 1863
1880. Bernfield, M., Go¨tte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., & Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annual Review of Biochemistry, 68, 729
777. Cho, T. J., Gerstenfeld, L. C., & Einhorn, T. A. (2002). Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. Journal of Bone and Mineral Research: The Official Journal of the American Society for Bone and Mineral Research, 17(3), 513
520. Cohen, G. B., Ren, R., & Baltimore, D. (1995). Modular binding domains in signal transduction proteins. Cell, 80(2), 237
248. Cross, M., & Dexter, T. M. (1991). Growth factors in development, transformation, and tumorigenesis. Cell, 64(2), 271
280. del Rosario, C., Rodrı´guez-E´vora, M., Reyes, R., Simo˜es, S., Concheiro, A., E´vora, C., . . . Delgado, A. (2015). Bone critical defect repair with poloxamine
cyclodextrin supramolecular gels. International Journal of Pharmaceutics, 495(1), 463
473. Dias, G. J., Peplow, P. V., McLaughlin, A., Teixeira, F., & Kelly, R. J. (2010). Biocompatibility and osseointegration of reconstituted keratin in an ovine model. Journal of Biomedical Materials Research Part A, 92A(2), 513
520. Dimitriou, R., Jones, E., McGonagle, D., & Giannoudis, P. V. (2011). Bone regeneration: Current concepts and future directions. BMC Medicine, 9(1), 66.

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C H A P T E R

20 Biological macromolecules for nutrients delivery Long Chen1,2, Zhongyu Yang1, David Julian McClements3, Zhengyu Jin1,2 and Ming Miao1,2 1

School of Food Science and Technology, Jiangnan University, Wuxi, P.R. China 2State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, P.R. China 3Department of Food Science, University of Massachusetts, Amherst, MA, United States

20.1 Introduction Consumer awareness of the link between food and health is increasing as they become more affluent. As a result, there is significant interest in the food industry in fortifying foods with health-promoting bioactive substances (nutrients) such as carotenoids, curcuminoids, polyphenols, probiotics, fatty acids, and peptides (Liu, Zhang, Li, McClements, & Liu, 2018). However, the incorporation of these nutrients into foods is often challenging because of their low solubility, poor chemical stability, poor food matrix compatibility, adverse flavor profile, or low bioavailability (Ezhilarasi, Karthik, Chhanwal, & Anandharamakrishnan, 2013; Rashidi & Khosravi-Darani, 2011). For this reason, there has been a considerable research and development effort to design and fabricate delivery systems for these nutrients, such as hydrogels, microcapsules, and nanoparticles. The human body is an extremely complex and

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00020-8

dynamic system, which has to be taken into account when designing these delivery systems. This chapter focuses on the types of nutrients that need to be delivered, the biological macromolecules that can be used to construct edible delivery systems, the most common delivery systems currently used for this purpose, and some of the major challenges that must be addressed in the future.

20.2 Nutrients Nutrients are edible substances that ensure the growth, development, reproduction and maintenance of the human body (Gonc¸alves, Martins, Duarte, Vicente, & Pinheiro, 2018). The nutritional requirements of individuals depend on their specific needs, which is determined by their age, sex, health status, and lifestyle. For example, pregnant women need to take nutrients such as vitamin D, folic acid and

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iodine during pregnancy (Mo et al., 2018). Many children also need vitamin D supplements during their growth and development, since lack of this essential micronutrient leads to rickets and other serious health problems (Lips, 2001). For the elderly, studies have found that vitamin B supplementation can effectively improve their cognitive ability (Cheng et al., 2016). Both water-soluble and oil-soluble nutrients need to be incorporated into fortified food products. Water-soluble nutrients, such as vitamin C and vitamin B, often have poor chemical stability and low bioavailability, which reduces their efficacy. Similarly, oilsoluble nutrients, such as vitamins A, D, and E, phytosterols, curcuminoids, and carotenoids, may also have poor efficacy due to their low water solubility, poor chemical stability, and low bioavailability (McClements, 2015). Several common nutrients are summarized in Table 20.1, along with their claimed health benefits and practical limitations.

20.2.1 Water-soluble nutrients In this section, we provide a few specific examples of water-soluble nutrients to provide some insights into their functional properties and potential challenges. 20.2.1.1 Ascorbic acid Ascorbic acid (C6H6O6), also known as vitamin C, is a water-soluble molecule that is necessary for human growth and development (Linster & Schaftingen, 2006). It is a relatively small (Mw 5 174.108 g/mol) and strongly hydrophilic (log D 5 2 8.2 at pH 3 and 210.9 at pH 7) molecule that has a carboxyl group that dissociates at higher pH values (pKa 5 4.3) leading to a net negative charge. Chemically, it is a strong reducing agent that it is susceptible to chemical transformations, thereby altering its bioactivity (Garcia et al., 2018). The natural

configuration of ascorbic acid is L-type, but Dtype ascorbic acid is usually added to foods as an antioxidant (Garcia et al., 2018). In addition to its function as a micronutrient, ascorbic acid also plays a role in improving iron deficiency anemia, mainly because it can be used as an electron donor to participate in the interaction between iron and ferritin, thereby promoting the absorption of iron by the human body (Traber & Stevens, 2011). Ascorbic acid can also be used as an antioxidant to scavenge free radicals in the body and thereby protect DNA and protein from loss. However, ascorbic acid is also a highly unstable vitamin, which is easily degraded by heat, oxygen, enzymes, pH, light, and other factors (Taguchi et al., 2014). In particular, ascorbic acid is rapidly decomposed under high temperatures and alkaline conditions, which makes it difficult to incorporate into many foods. 20.2.1.2 Riboflavin Riboflavin (C17H20N4O6), also known as vitamin B2, is another example of an important water-soluble vitamin (Casadey, Challier, Altamirano, Spesia, & Criado, 2021). It is a fairly small (Mw 5 376.369 g/mol) and moderately hydrophilic (log D 5 2 0.92 at pH 3 and 21.94 at pH 7) molecule that has a functional group that dissociates at higher pH values (pKa 5 6) to form a negatively charged molecule. Riboflavin is present in many kinds of food, including egg, kidney, liver, meat, milk, green vegetables and fortified foods (Powers, 2003). In nature, it exists mainly in the form of a phosphate ester of flavin mononucleotide and flavin adenine dinucleotide. Riboflavin has been shown to improve immune function and reduce antimyocardial ischemia (Tong & Xu, 2003). Riboflavin is stable in acidic or neutral solutions, but is rapidly decomposed in alkaline solutions, especially at higher temperatures. Furthermore, it is easily damaged by visible and ultraviolet light.

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20.2 Nutrients

TABLE 20.1 Claimed health benefits and limitations of several common nutrients. Category Name

Claimed function

Limitations

References

FatVitamin A soluble nutrients Vitamin D

Anticancer, antioxidation

Water insoluble, low bioavailability

Katouzian and Jafari (2016)

Prevent cancer, heart disease

Chemical instability, water insoluble, low bioavailability

Katouzian and Jafari (2016)

Vitamin K

Promote blood clotting

Water insoluble, easily destroyed by light and alkali

Katouzian and Jafari (2016)

β-Carotene

Anticancer and antioxidation

Chemical instability, crystalline state exist, high melting point, water insoluble, low bioavailability

Chen, Li, Li, McClements, and Xiao (2017)

Lutein

Antioxidation, antiatherogenic, antiinflammatory

Chemical instability, water insoluble, low bioavailability

Qv, Zeng, and Jiang (2011)

Lycopene

Anticancer, antiinflammatory, antioxidation

Susceptible to oxidation and isomerization, water insoluble, low bioavailability

Hernandez-Espinoza, PinonMuniz, Rascon-Chu, SantanaRodriguez, and Carvajal-Millan (2012)

Stigmasterol

Antiinflammatory, anticancer, antioxidation

Chemical instability, water insoluble, low bioavailability

Mari et al. (2013)

Resveratrol

Anticarcinogenic, antiinflammatory, antiobesity

Chemical instability, water insoluble, low bioavailability

Das and Ng (2009)

Curcumin

Antibacterial, antiinflammatory, Chemical instability, water anticancer insoluble, low bioavailability

Quercetin

Cardiovascular diseases prevention, antiaging, anticancer, antidiabetic and antioxidant activities

Bordoloi (2018)

Water insoluble, low bioavailability

Formica and Regelson (1995)

α-Tocopherol Antiatherogenic, antidiabetic antioxidation, anticancer

Chemical instability, water insoluble, low bioavailability

Somchue, Sermsri, Shiowatana, and Siripinyanond (2009)

ω-3 fatty acids

Antiinflammatory, anticancer, antioxidation

Strong odor, oxidant instable, water insoluble

Kosaraju, Weerakkody, and Augustin (2009), Richards, Thomas, Bowen, and Heard (2010)

Enhance resistance and treat chronic diseases

Chemical instability, easy to decompose at high temperature

Garcia et al. (2018), Taguchi, Fukusaki, and Bamba (2014)

Maintain the balance of oxidative metabolism in the brain

Chemical instability

Ahn, Kim, and Lee (2005)

Vitamin B2

Promote development and cell regeneration; antioxidation

Decompose by light

Raffy (1939)

Niacin

Prevention and treatment of skin roughness and hyperlipidemia

WaterVitamin C soluble nutrients Vitamin B1

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20.2.2 Oil-soluble nutrients In this section, a few examples of oil-soluble nutrients are given to provide insights into their nutritional properties and potential challenges. 20.2.2.1 Curcumin Curcumin is a polyphenolic compound extracted from the rhizome of turmeric, which has been shown to exhibit various potentially beneficial health effects (Dai et al., 2018). It is a fairly small (Mw 5 368.385 g/mol) and hydrophobic (log D 5 4.12 at pH 3 and 7) molecule that has a functional group that dissociates at higher pH values (pKa 5 8.8) to form a negatively charged molecule under strongly alkaline conditions. Numerous studies have shown that curcumin has antibacterial, antiinflammatory, and antitumor activities (Bordoloi, 2018). It has been shown to be safe for human consumption by the Food and Drug Administration (FDA) in the United States (Cheng, Hsu, Lin, Hsu, & Hsieh, 2001). Nevertheless, there are a number of factors limiting the utilization of curcumin in food products. It has a low water solubility and a poor chemical stability, which means that it is difficult to incorporate into many food products. Moreover, the same factors reduce its bioavailability and bioactivity by decreasing its solubility and by promoting chemical transformations in the gastrointestinal fluids (Dai et al., 2018). 20.2.2.2 β-Carotene β-Carotene (C40H56) is widely found in tomatoes, carrots, spinach and other fruits and vegetables (Durante, Lenucci, D’Amico, Piro, & Mita, 2014). It is a moderately sized (Mw 5 536.888 g/mol) and strongly hydrophobic (log D 5 11.1 at pH 3 and 7) molecule with a very low water solubility that is crystalline at room temperature (Tm 5 180 C). β-Carotene has a strong red-orange color due to its multiple unsaturated bonds. Studies have shown that this carotenoid is an effective precursor of vitamin A and has strong antioxidant activity (Gul et al.,

2015). In addition, it may also have various other health benefits such as reducing the risk of heart disease and other chronic diseases, as well as strengthening the immune system (Tanaka, Shnimizu, & Moriwaki, 2012). However, its use in foods as a functional ingredient is often limited because of its extremely low water solubility and its tendency to chemical degrade when exposed to light, heat and oxygen (Chen, Li et al., 2017; Chen, Liang et al., 2017). 20.2.2.3 Lutein Lutein (C40H56O2) is typically found naturally in plant-based foods such as corn but it may also be present as an additive to provide food color. It is a moderately sized (Mw 5 568.886) and strongly hydrophobic (log D 5 8.55 at pH 3 and 7) molecule with a very low water solubility that is crystalline at room temperature (Tm 5 190 C). Lutein has been reported to have good antioxidant properties, as well as the ability to promote eye health (Yang et al., 2018). In particular, studies have shown that lutein plays an important role in delaying age-related macular degeneration and the development of cataracts (Tan et al., 2008). However, the application of lutein in food is limited because of its low water solubility, poor bioavailability, and poor chemical stability (Steiner, McClements, & Davidov-Pardo, 2018).

20.3 Biological macromolecules used for nutrients delivery The matrix for constructing the nutrient delivery system is generally a food-grade raw material or food additive, and at the same time, it must have good emulsifying properties, viscosity or the characteristics of forming a gel network structure. The materials currently used to construct the delivery system mainly include: protein, polysaccharide, fat etc. Some common substances are shown in Table 20.2.

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20.3 Biological macromolecules used for nutrients delivery

TABLE 20.2 Common substances used in nutrient delivery systems (Gonc¸alves et al., 2018). Category

Name

Source

Main structure type

Protein

Lactoferrin

Whey protein

Spherical

β-Lactoglobulin

Whey protein

Spherical

Gelatin

Pig skin, cowhide, fish skin etc.

Linear

Gliadin

Flour

Spherical

Glycinin

Soybeans

Spherical

Bovine serum albumin

Cow blood/milk

Spherical

Chitosan

Crustaceans/invertebrates

Linear

Pectin

Beet residue/plant cell wall

Branched

Carrageenan

Algae

Linear/Spiral

Xanthan gum

Microorganism

Linear/Spiral

Alginate

Algae

Linear

Agar

Seaweed

Linear

Polysaccharide

20.3.1 Polysaccharides Food-grade nutrient delivery materials are usually assembled from natural polymers such as polysaccharides and proteins. According to the number of monosaccharide units they contain, carbohydrates can be divided into polysaccharides, oligosaccharides, disaccharides, and monosaccharides (Mungure, Roohinejad, Bekhit, Greiner, & Mallikarjunan, 2018). Foodgrade polysaccharides usually come from a wide range of plant or microbial sources. Some natural polysaccharides, such as gum arabic, have emulsifying properties because they have hydrophilic carbohydrate chains attached to hydrophobic proteins chains (Dickinson, 2003). Many polysaccharides can also be used as structure-forming materials due to their ability to be cross-linked by physical or chemical bonds. A large number of studies have shown that polysaccharides can be used to encapsulate bioactive substances such as vitamins and nutraceuticals, which have been reviewed in detail elsewhere (McClements, Bai, & Chung,

2017). For instance, pectin can be combined with calcium ions to form hydrogels, which can be used to contain emulsified oil-soluble bio-actives (An Thi-Binh, Winckler, Loison, Wache, & Chambin, 2014). 20.3.1.1 Pectin Pectin is an acidic polysaccharide that exists in many higher plants, with a molecular weight around 110
150 kDa (Mohnen, 2008). The main chain of pectin molecule is composed of 150
500 α-D-galacturonic acid groups linked through 1-4-glycosidic bonds (An ThiBinh et al., 2014). According to the esterification degree, pectin can be divided into high methoxy pectin (DE . 50%) and low methoxy pectin (DE , 50%) (Wang et al., 2018). Pectin has been claimed to exhibit beneficial biological activities, such as antioxidant, bacteriostasis, antiinflammation, immune regulation, antiglycosylation, and anticoagulation activities (Wang et al., 2018). Pectin is not easily digested by the enzymes secreted in the

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stomach and small intestine, but it is degraded in the colon by pectinases produced by colonic bacteria. Indeed, it has been reported that hydrogels prepared from low methoxy pectin are suitable for colon administration of bioactive substances (Chourasia & Jain, 2003). 20.3.1.2 Alginate Alginic acid is usually extracted from brown algae. Commercially, alginic acid is usually sold in the form of its sodium salt, sodium alginate. Alginic acid is a linear polymer composed of β-1,4-D-mannuronic (M) acid and α-1,4-L-guluronic acid (G), which are often present as M-blocks and G-blocks that may vary in number (Burey, Bhandari, Howes, & Gidley, 2008). Alginate gels can be produced by salt bridge formation between two or more alginate molecules, where the anionic carboxyl groups are bridged by multivalent cations (Ca21). These hydrogels can be used to encapsulate bioactive substances. Hydrophilic substances, like proteins or probiotics, can be directly incorporated in these hydrogels, whereas hydrophobic ones, like oil-soluble vitamins or carotenoids, have to be encapsulated within emulsion droplets first. Hydrogels formed by alginate and Ca21 are thermally irreversible, that is, once formed they are resistant to heating. Calcium alginate hydrogels tend to shrink when the pH is below the pKa value of the carboxyl groups, but expand at higher pH values because of the high charge and electrostatic repulsion (Hari, Chandy, & Sharma, 1996). Calcium alginate hydrogels may dissociate in the presence of calcium chelating agents (such as EDTA) or in concentrated salt solutions that compete with the calcium. Calcium alginate microgels have been used to control the release of encapsulated bioactive substances within the gastrointestinal tract (Zhang, Zhang, Chen, Tong, & McClements, 2015). 20.3.1.3 Carrageenan Carrageenan is a heterogeneous polysaccharide that is typically extracted from red algae by hot alkaline treatments (Morris, 2003). It is

formed by alternately linking sulfated or nonsulfated galactose and 3,6-dehydrated galactose units through α-1,3- and β-1,4-glycosidic bonds (Usov, 1998). There are three main types of carrageenan used in the food industry as functional ingredients: λ-carrageenan, κ-carrageenan and ι-carrageenan. λ-Carrageenan can be used as a thickening agent, but it does not form gels. In contrast, κ-carrageenan and ι-carrageenan can be cross-linked via double helices to form thermally reversible gels. There are differences in the functional properties of these two types of carrageenan due to differences in their linear charge densities. κ-Carrageenan tends to form rigid and brittle gels that dissolve when heated, while ι-carrageenan tends to form soft and elastic gels (Imeson, 2009). The gelling properties of carrageenan mean that it can be used to prepare hydrogels to encapsulate bioactive substances. The functional properties of these hydrogels can be adjusted by changing the type of carrageenan used, changing the cross-linking ions, altering the molecular weight of the carrageenan molecules, or combining carrageenan with other biopolymers (Zhang et al., 2015). 20.3.1.4 Starch Starch is a high abundant polysaccharide that can be economically isolated from corn, wheat, potato, rice, and various other crops (Haaj, Magnin, Petrier, & Boufi, 2013). Starch exists in the form of granules within plants, which consist of alternating layers of crystalline and noncrystalline regions. At the molecular level, starch can be divided into amylose and amylopectin. Amylose is primarily a linear molecule consisting of glucose molecules linked together by α-1,4-glycosidic bonds. Amylopectin is a highly branched molecule consisting of chains of glucose molecules linked together by α-1,4-glycosidic bonds, that are connected at α-1,6 branches (Shalviri, Liu, Abdekhodaie, & Wu, 2010). Starch can be divided into rapidly digestible starch, slowly digestible starch, and resistant starch (RS).

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Previous studies have shown that starch-based hydrogels can be prepared by thermal gelatinization of digestible starch (Firoozmand & Rousseau, 2013). RS cannot be hydrolyzed by enzymes in the mouth, stomach and small intestine but it can be degraded by enzymes released by the gut microbes residing in the colon (Khan et al., 2020). RS has been used to prepare nanoparticles through sonication, and these particles were successfully used as encapsulation materials for delivering bioactive substances to the colon (Sivapragasam et al., 2014). The formation of these nanoparticles can be attributed to the ability of sonication to disrupt the crystalline regions in the starch particles, which leads to their partial dissociation (Gallant, Bouchet, & Baldwin, 1997; Haaj et al., 2013).

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functional characteristics (Gunasekaran, Ko, & Xiao, 2007; Sinha, Radha, Prakash, & Kaul, 2007). Whey protein is composed of a mixture of globular proteins, including α-lactalbumin, β-lactoglobulin, bovine serum albumin, immunoglobulins, and lactoferrin (Chen & Subirade, 2005). β-Lactoglobulin (50%
60%) is present at the highest concentration in whey protein. It contains one free thiol residue and two disulfide bonds, as well as electrically charged and hydrophobic surface groups, which can be utilized to create protein
protein cross-links through covalent or physical interactions (Livney, 2010). Whey protein has commonly been used to create hydrogels, microgels, and nanoparticles to encapsulate and deliver nutrients (Gunasekaran et al., 2007). 20.3.2.2 Casein

20.3.2 Proteins Proteins are widely used as building blocks to assemble delivery systems because of their surface activity and structure-forming properties (Picchio et al., 2018). The surface activity means that it can be used to form emulsions, whereas the gelling properties mean that it can be used to form microgels and hydrogels. Proteins are natural polymers consisting of amino acids linked together through peptide bonds. The molecular weight, conformation, charge and hydrophobicity of proteins are determined by the type, number, and sequence of the amino acids in the polypeptide chains (McClements, 2015). There have been numerous studies on the utilization of proteins to encapsulate and deliver bioactive substances. In this section, we provide a few examples with a focus on milk proteins. 20.3.2.1 Whey protein Whey protein is extracted from the whey produced during cheese production. It has it relatively inexpensive, has a high nutritional value, and exhibits a diverse range of

Casein is usually isolated from milk by acid or enzyme precipitation (Picchio et al., 2018). It consists of a mixture of four main proteins (αS1-, αS2-, β-, and κ-casein) in a weight ratio of about 4:1:4:1 (Holt, Carver, Ecroyd, & Thorn, 2013). Casein contains little secondary and tertiary structure, which means that it tends to have good thermal stability. This is mainly attributed to the fact that casein is rich in proline, which can interrupt the formation of α-helix and β-sheet regions. There have been numerous studies on the utilization of casein to form nanocarriers for the oral delivery of bioactive substances (Chen et al., 2020; Kimura, Imai, & Otagiri, 1991).

20.3.3 Glycoproteins and proteoglycans Glycoproteins consist of protein molecules that are covalently linked to carbohydrates, which are commonly found at the surfaces of biological cells (Tabasum et al., 2017). Typically, these kinds of glycoproteins are comprised of around 2%
30% carbohydrates, which may be assembled from N-acetyl-D-glactosamine,

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N-acetyl-D-glusosamine, D-glucose, D-mannose, rhamnose, L-fucose, xylose, D-galactose, and sialic acid units. The physicochemical properties of glycoproteins are altered by changing the type and content of carbohydrates. For example, the thermal stability of glycoproteins can be changed by glycosylation reactions, and the solubility of glycoproteins can be changed by sulfating the glycans (Kobata, 1993; Sharon & Lis, 1983). In addition, glycoproteins exhibit many beneficial functional properties such as biodegradability, biocompatibility, nontoxicity, antimicrobial activity, and surface activity. Due to these attributes, glycoproteins have been widely used in biomedicine for the delivery of drugs, insulin, and vaccines (Tabasum et al., 2017). Some kinds of glycoproteins may also be used in the food industry for the encapsulation and delivery of edible bioactive substances. Proteoglycans are mainly found in extracellular matrices, at cell surfaces, and within intracellular granules (Sasisekharan & Venkataraman, 2000). Proteoglycans typically have between 1 and 200 highly sulfated glycosaminoglycan polysaccharide chains attached to a central protein molecule (Schaefer & Schaefer, 2010). These polysaccharide chains are unbranded and formed by repeated disaccharide units, which are composed of uronic acid and amino sugars (Ruiz Martinez, Peralta Galisteo, Castan, & Morales Hernandez, 2020). The functional properties of proteoglycans depend on their composition and structure. Consequently, different ones can be used for different applications.

Thakur, 2015; Watkins, Hosur, Tcherbi-Narteh, & Jeelani, 2015). It is widely found in plants, being present at a concentration of around 10%
30%, which means that it is a highly abundant renewable resource (Liu, Wang, Zheng, Luo, & Cen, 2008). More than 90% of industrial lignin is obtained by alkaline extraction, which is usually a by-product of pulping and refining processes (Li, Qiu, Qian, Xiong, & Yang, 2017). Currently, lignin is widely used in nonfood products, such as fillers, adhesives, dispersants, and additives (Figueiredo et al., 2017). Lignin has phenolic and aliphatic hydroxyl groups, which can be chemically modified to prepare and develop new materials (Duval & Lawoko, 2014; Laurichesse & Averous, 2014). Lignin also has good biocompatibility, pH and thermal stability, biodegradability, low toxicity, and several useful structure building properties, which make it suitable for development of delivery systems (Dai, Liu, Hu, Zou, & Si, 2017; Frangville et al., 2012). There have been many studies on the use of lignin to construct delivery systems for drugs and nutraceuticals. For instance, ligninbased nanoparticles have been prepared to encapsulate and release resveratrol, which increased its stability and efficacy (Dai et al., 2017).

20.3.4 Others (lignin as example)

Biopolymer-based delivery systems are kept together by various kinds of molecular interactions, including van der Waals, electrostatic, hydrogen bonding, hydrophobic, and covalent interactions (McClements, 2015) (Fig. 20.1). It is important to understand the origin of these interactions and the factors that affect them, as they have a major impact on the design and fabrication of delivery systems.

There are many other biological macromolecules available for the construction of delivery system for nutrients. For the sake of simplicity, in this chapter, we mainly take lignin as an example. Lignin is an aromatic organic compound that is one of the three main components of lignocellulose biomass (Thakur &

20.4 Molecular interactions that maintain the stability of biopolymer-based delivery systems

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20.4 Molecular interactions that maintain the stability of biopolymer-based delivery systems

FIGURE 20.1

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Common interactions between biopolymers (McClements, 2015).

20.4.1 Electrostatic interactions The electrical properties of proteins and polysaccharides depend on the number and location of the cationic and/or anionic groups. For proteins, the charge distribution on their surfaces is nonuniform, and they typically have both positive and negative patches at most pH values in foods. Generally, electrostatic interactions are affected by pH (proton dissociation/association), ion composition (ionic strength and ion binding effects), and solvent composition (dielectric constant), but temperature has little effect (Pink et al., 2006). Therefore electrostatic interactions can be controlled by manipulating these conditions. The strength of electrostatic interactions is mainly affected by the magnitude of the electrical charges involved, the distance between them, and the ionic strength and dielectric constant of the surrounding medium. Electrostatic interactions are often used to assemble biopolymer microgels and hydrogels, for example, via complexation of anionic polysaccharides and cationic proteins or vice versa.

20.4.2 Hydrogen bonding Hydrogen bonding plays an important role in stabilizing the internal structures of many

biopolymers (such as globular or rod-like molecules), as well as in the formation of intermolecular crosslinks between different biopolymer molecules (such as gelatin and starch). Many factors affect hydrogen bond formation, including temperature, pH, and ionic strength, with temperature being the most important (McClements, 2015). Typically, hydrogen bonds weaken when a food is heated because then conformational entropy effects dominate molecular attractive forces. This is the reason that biopolymers like gelatin or agar melt when they are heated above a critical temperature.

20.4.3 Hydrophobic interactions Hydrophobic interactions play an important role in determining the conformation, interactions, and functional attributes of many food biopolymers. These interactions manifest themselves as strong long-range attractive interactions between nonpolar groups dispersed in water. The strength of the hydrophobic interaction is mainly affected by the oil-water interfacial tension, as well as the surface area of the nonpolar groups exposed to water. The greater the surface area or interfacial tension, the stronger the hydrophobic attraction.

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Hydrophobic interactions tend to get stronger as the temperature is increased. In addition, changes in the pH and ionic strength of the surrounding solution only have a minor impact on their magnitude (unless they cause a big change in water structure) (McClements, 2015).

20.4.4 Covalent interactions Biopolymer molecules may interact with each other through the formation of various kinds of covalent bonds (McClements, 2015). Some of the most important chemical reactions that result in a change in covalent bonds are oxidation, reduction, esterification, amidation, disulfide, and Maillard reactions. These reactions can be triggered by physical, chemical, and/or enzyme treatments. For instance, the Maillard reaction occurs during baking and frying, which involves chemical reactions between reducing sugars (mainly glucose) and free amino groups in proteins. This reaction initially leads to the formation of glycosylamine, which then rearranges into an Amadori product, which then degrades into numerous other reaction products depending on system conditions (especially pH and temperature).

20.5 Retention and release mechanisms Delivery systems are often designed to control the retention and release of bioactive ingredients to achieve specific effects. Therefore a considerable amount of research has focused on identifying and characterizing the release mechanisms for different kinds of delivery system. Some of the most important release mechanisms are diffusion, dissolution, erosion, fragmentation, and swelling (as shown in Fig. 20.2). For the dissolution mechanism, the active ingredients are released from the particles in a colloidal delivery system when it encounters specific solution or environmental conditions and then dissolves (Mogul et al., 1996). The active ingredient may constitute the entire delivery system and is therefore released as the particle dissolves. Alternatively, it may be dispersed within the particle matrix and then be released as the particle matrix dissolves. The release rate depends on the dissolution rate, which depends on the composition and structure of the particles, and the nature of the environmental factors responsible for dissolution.

FIGURE 20.2 Five common release mechanisms encountered for food-grade colloidal delivery systems (McClements, 2015).

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20.6 Nutrient delivery systems based on biological macromolecules

For the diffusion mechanism, the bioactive substance is released from the particles by molecular diffusion through the intact particle matrix. The release rate depends on various factors, including bioactive properties (such as molecular dimensions and partitioning), bioactive-matrix interactions (attraction/repulsion), matrix pore size, particle dimensions (shape and diameter), and fluid flow conditions (Jones & McClements, 2010). For the surface erosion mechanism, the delivery system progressively dissolves from surface-to-interior after it encounters certain solution or environmental conditions, which leads to release of the bioactive substances (Mogul et al., 1996). Erosion can be triggered by physical, chemical, and/or enzyme processes. For instance, a protein particle may be progressive eroded by proteases in the stomach or small intestine, while a starch particle may be eroded by amylases in the mouth or small intestine. The erosion rate is affected by the composition and structure of the particles, as well as the precise nature of the solution and environmental conditions (McClements, 2015). For the swelling mechanism, the bioactive substance is released after the particles absorb water molecules and swell, since this leads to an increase in the pore size, thereby allowing the bioactive materials to escape more easily. Ideally, the particle should remain intact under one set of solution or environmental conditions, but then swell when it encounters another set. Release occurs when the pore size increases to a value similar to the molecular size of the bioactive substance. In this case, the release rate depends on the swelling rate and the time it takes for the bioactive to diffuse through the swollen particle matrix (McClements, 2015). For the fragmentation mechanism, the bioactive substance is released after the colloidal particles become fragmented due to the action of physical, chemical or enzymatic reactions. The bioactive components may then be released through the other three mechanisms

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discussed earlier. In this case, the release rate increases as the degree of fragmentation increases, that is, the particle size decreases, and the surface area increases. The release rate mainly depends on the fracture characteristics of the particles and the shape and size of the fragments formed (McClements, 2015).

20.6 Nutrient delivery systems based on biological macromolecules Various kinds of delivery system have been assembled from biopolymers, including hydrogels, microcapsules, and nanoparticles (Kumari, Yadav, & Yadav, 2010; Narayanaswamy & Torchilin, 2019; Wong, Al-Salami, & Dass, 2018). These delivery systems are usually constructed from polysaccharides (such as starch, alginate, pectin and carrageenan) and/or proteins (such as casein, whey protein, and gelatin) (Zhang et al., 2015). In practice, there is often some overlap between these different kinds of delivery systems in terms of their fabrication, characteristics, and functionality. This section provides a brief overview of some of the most widely used of these delivery systems, including a discussion of their fabrication, properties, and applications. Some common methods used to prepare these kinds of delivery systems are highlighted in Fig. 20.3.

20.6.1 Composition and structure Hydrogels consist of a three-dimensional network of biopolymers linked together by physical and/or chemical bonds, which trap a relatively high amount of water inside. Hydrogel-based delivery systems can be prepared in various physical forms including particles, coatings, and films (Hoare & Kohane, 2008). Microcapsules have a core-shell structure that can encapsulate substances within the core and/or shell (Goodwin & Somerville, 1974). Typically, however, the shell is designed to protect and control the release of the active ingredient (Bah et al., 2020). Microcapsule

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FIGURE 20.3 Some common delivery systems for nutrients. (A) Fabrication of emulsions filled hydrogel particles (Xiao, Sung, Decker, & McClements, 2015); (B) Fabrication of microcapsules (Bah, Bilal, & Wang, 2020); (C) Fabrication of nanoparticles (Idrees et al., 2020); (D) Fabrication of hydrogel (Gautam & Santhiya, 2019).

technology has traditionally been used in the flavor industry to protect volatile substances, control their release, and mask any off-flavors (Lubbers, Landy, & Voilley, 1998). But this technology can also be used to encapsulate many other active substances used in foods (Bah et al., 2020). Biopolymer nanoparticles consist of small particles, typically 10
1000 nm, which are mainly constructed from proteins and/or polysaccharides (DeFrates et al., 2018). They tend to contain less water and be smaller than hydrogel particles, but there are some overlaps.

20.6.2 Fabrication 20.6.2.1 Fabrication of hydrogel Several preparation methods have been developed to create hydrogels, including

coacervation-gelation, denaturation-gelation, emulsification-gelation, injection- gelation, and shearing-gelation methods (Zhang et al., 2015). In addition, molding, microfluidic, and electrospraying methods are also techniques that can be used for this purpose (Burey et al., 2008; Joye & McClements, 2014). Coacervation-gelation methods have been widely used for preparing hydrogels from biopolymers in the food industry (Li & McClements, 2011). This method is based on the formation of electrostatic complexes of two oppositely charged biopolymers, such as an anionic polysaccharide and cationic protein (Weinbreck, Minor, & De Kruif, 2004). Nutrients can be trapped inside the coacervates formed, but they typically have to have some attraction to the biopolymer network. The

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20.6 Nutrient delivery systems based on biological macromolecules

main deficiency of this method is that the coacervates tend to coalesce and sediment over time. In addition, it is usually difficult to control the size of any coacervate particles formed. The most commonly used denaturationgelation method used to form hydrogels involves heating proteins above their thermal denaturation temperature. This process is carried out under conditions that control the attractive and repulsive interactions between the protein molecules, usually by controlling the pH and ionic strength to modulate the electrostatic interactions. After the protein molecules unfold, they tend to aggregate with each other due to an increase in the hydrophobic attraction between them when nonpolar surface groups are exposed (Bromley, Krebs, & Donald, 2006). This approach can be used to form macroscopic gels, coatings, films, or particles. Nutrients can be trapped within the network of aggregated protein molecules by adding them before the thermal denaturation process. Emulsification-gelation methods involve homogenizing an aqueous phase containing the biopolymers with an oil phase containing lipophilic surfactants to form a W/O emulsion, and then taking gelling the internal aqueous phase to form a hydrogel particle (Yu, Jia, Cheng, Zhang, & Zhuo, 2010). These hydrogel particles can then be removed by centrifugation and washing with an organic solvent. Nutrients can be trapped inside the hydrogel particles by adding them to the water phase before homogenization. The injection-gelation method is widely used to form hydrogel particles. A biopolymer solution containing the nutrients is placed in a syringe and then injected, which leads to the formation of a particle. This particle is then gelled by changing the environmental conditions to induce cross-linking of the biopolymers, for example, by heating, cooling, drying, altering pH, increasing ionic strength, or adding crosslinking agents (Wu, Cheng, & Yang, 2019). The

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size of hydrogel particles can be controlled by controlling needle diameter, injection flow rate, and other variables (Zhang et al., 2015). The shearing-gelation method involves continuously stirring an aqueous solution containing the gelling biopolymers and nutrients, and then inducing gelation by changing the temperature, pH or other conditions to promote biopolymer cross-linking (Wolf, Frith, Singleton, Tassieri, & Norton, 2001). In addition to the methods just described, there are numerous other methods that can also be used to prepare hydrogels. For example, molding, microfluidic, electrospray method, and 3D printing methods can also be used (Burey et al., 2008; Joye & McClements, 2014; Martinez, Goyanes, Basit, & Gaisford, 2017). 20.6.2.2 Fabrication of microcapsules Microcapsules with a core-shell structure can be fabricated using physical, chemical, or physiochemical methods (Singh, Hemant, Ram, & Shivakumar, 2010). Chemical methods include interfacial polymerization, in situ polymerization, and layer-by-layer self-assembly; physical methods include spray drying, spray chilling, and fluidized bed granulation, and physiochemical methods include condensation and supercritical CO2 auxiliary methods. In the interfacial polymerization method, two different monomers are dissolved in two different immiscible phases. These two phases are then brought into contact, which causes the monomer molecules to intermingle and react with each other at the interface (Bah et al., 2020). The polymer molecules produced at the interface form a shell around the core material, leading to the creation of microcapsules. In most cases, this method is used to form shells around oil or water droplets in O/W or W/O emulsions, respectively (Ichiura, Morikawa, & Ninomiya, 2006; Raaijmakers & Benes, 2016; Zhang, Liu, Ju, Wang, & Zhao, 2010). The main advantages of interfacial polymerization include simple preparation, fast reaction speeds, and good

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entrapment efficiency (Michel, Aprahamian, Defontaine, Couvreur, & Damge, 1991). In situ polymerization is widely used to produce microcapsules in industry. Two different kinds of monomers are dissolved in a continuous phase and react with each other to form polymers. These polymers then deposit themselves at the surfaces of the core material dispersed in the continuous phase. The nature of the polymer coatings formed can be controlled by changing the temperature, pH, or solvent quality (Nguon, Lagugne-Labarthet, Brandys, Li, & Gillies, 2018). Layer-by-layer (LbL) assembly has also been used to form coatings around core particles (Tong, Song, & Gao, 2012). The principle of LbL assembly is that microcapsules are prepared by sequentially depositing polymers onto the surfaces of colloidal particles, which may then be dissolved if required (Zhang, Chen, Zhang, Deng, & McClements, 2016; Zhang, Wang et al., 2016). The layer-by-layer assembly method can be used to control the composition, thickness, porosity, charge, polarity, and stability of the shells in microcapsules, thereby tuning their functional properties (Caruso, Caruso, & Mohwald, 1998; Decher, 1997). Self-assembly methods can also be used for the preparation of microcapsules, which are based on the tendency for some biopolymer molecules to spontaneously interact with each through van der Waals forces, hydrogen bonds, hydrophobic forces, or electrostatic interactions and form specific structures (Brinker, 2004; Whitesides & Grzybowski, 2002). Spray drying is one of the most common physical methods for preparing microcapsules. It typically produces a powder containing spherical particles that contain the active ingredient trapped within a solid matrix. The composition and structure of the powder particles can be controlled by manipulating the formulation and operating conditions. A spray drier converts a liquid feed into a powder using three main three steps: atomization, solvent evaporation, and

particle collection (Stunda-Zujeva, Irbe, & Berzina-Cimdina, 2017). Depending on the ingredients and operating conditions, spray drying can produce powders with particle diameters ranging from relatively small (10
50 μm) to relatively large (2
3 mm). Despite its versatility, the spray drying method does have some limitations. In particular, some thermosensitive compounds may be damaged due to the relatively high temperatures used during spray drying. Spray drying has been widely used in the food industry to encapsulate bioactive substances (Gharsallaoui, Roudaut, Chambin, Voilley, & Saurel, 2007; Ho, Truong, & Bhandari, 2016). 20.6.2.3 Fabrication of nanoparticles There are many methods available for the preparation of biopolymer nanoparticles, including mechanical crushing, emulsion/ microemulsion templating, and molecular assembly methods. Mechanical crushing method uses physical forces to grind bulk materials into smaller particles. Often, stabilizers also need to be added to protect the small particles from aggregating with each other once formed. The mechanical crushing method has the advantages of simple operation, high efficiency, and low cost. The microemulsion/emulsion-templating methods involve forming a water-in-oil microemulsion or emulsion containing biopolymers in the water phase and then gelling the biopolymers using a suitable cross-linking approach. Typically, cross-linking is achieved by heating, cooling, or adding a cross-linking agent, such as a chemical additive or enzyme. The biopolymer nanoparticles can then be retrieved by centrifugation/filtration combined with washing using an organic solvent. A suitable surfactant must be selected to coat the water droplets in the microemulsions or emulsions. In the case of emulsions, a suitable homogenizer must also be used to prepare them, such as a microfluidizer, high pressure valve homogenizer, or sonicator. The size of the biopolymer nanoparticles

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20.6 Nutrient delivery systems based on biological macromolecules

can be controlled by manipulating the ingredients and processing conditions used to form the microemulsions or emulsions (Vidal-Vidal, Rivas, & Lopez-Quintela, 2006). Molecular assembly methods involve preparing a biopolymer solution and then altering the solution conditions to promote selfassembly of the biopolymer molecules into nanoparticles. Typically, the nanoparticles are held together by intermolecular noncovalent bonds such as hydrogen bonds, electrostatic interactions, hydrophobic forces, or van der Waals forces. The preparation conditions can often be manipulated to create nanoparticles with well-defined sizes and shapes (Nishiyabu, Aime, Gondo, Noguchi, & Kimizuka, 2009).

20.6.3 Properties Biopolymer-based particles, such as hydrogels, microcapsules, or nanoparticles may vary in their compositions, sizes, shapes, internal structures, surface chemistry, and electrical charge characteristics. The functional attributes of a delivery system can therefore be controlled by manipulating the formulation and processing conditions to alter these particle properties. In some cases, the particles should maintain their integrity under one set of conditions (such as a food and upper GIT) but then breakdown under another set of conditions (such as the colon) so they can release the bioactive agents. Therefore it is important to understand the main factors impacting the integrity and mechanical properties of biopolymer particles under different conditions. In general, the integrity and mechanical properties of biopolymer matrices can be changed by controlling the degree of cross-linking. Typically, increasing the number and strength of the crosslinks increases the mechanical properties. The responsiveness of a biopolymer particle to its environment depends on the nature of the crosslinks, such as hydrogen bonding, salt bridges, electrostatic attraction, or hydrophobic forces (McClements, 2015). For example, hydrogen bonds tend to break upon

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heating, as for gelatin, whereas salt bridges and electrostatic attraction may breakdown in the presence of high salt concentrations.

20.6.4 Applications Delivery systems are widely used in the food industry for encapsulating a wide range of bioactive substances, including antimicrobials, flavors, vitamins, minerals, healthy lipids, nutraceuticals, probiotics, and enzymes. These delivery systems may be designed for a variety of purposes, including to improve the storage, handling, and shelf life of the bioactive materials, to mask flavors, to control the release, or to increase the efficacy of the encapsulated components (Gonc¸alves et al., 2018). In this section, a few examples of the use of biopolymer-based delivery systems for food applications are given to highlight their potential. 20.6.4.1 Flavors The aroma of foods is due to the presence of volatile organic molecules, such as vanillin, citral, cinnamaldehyde, and limonene (Benshitrit, Levi, Tal, Shimoni, & Lesmes, 2012). Biopolymer-based delivery systems can be used to inhibit the degradation or evaporation of volatile flavors from foods during storage, as well as to manipulate their release profiles. For instance, biopolymer microgels prepared from calcium alginate have been used to encapsulate allyl methyl disulfide, a volatile lipophilic component from garlic, and control its release during cooking (Wang, Doi, & McClements, 2019). This kind of delivery system may therefore be used to prolong the desirable aroma of foods during food preparation. 20.6.4.2 Enzymes The catalytic activity of enzymes is often lost when they are exposed to certain environmental conditions, such as changes in pH or temperature, or exposure to gastrointestinal fluids. Biopolymer-based delivery systems can be used to improve the stability of enzymes, as well as to control their release. For instance,

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biopolymer microgels fabricated from oppositely charged polyelectrolytes, such as proteins and polysaccharides, are suitable for encapsulating enzymes (Cooper, Dubin, Kayitmazer, & Turksen, 2005). Amphoteric hydrogels have been developed to immobilize glucose oxidase and urease (Ogawa, Ogawa, Wang, & Kokufuta, 2001). Recently, food-grade biopolymer microgels were developed from polysaccharides to encapsulate and protect enzymes, such as lactase and lipase (Zhang et al., 2016; Zhang, Zhang, & McClements, 2017). The enzymes were co-encapsulated with an antacid (magnesium hydroxide), which prevented the interior of the microgels from becoming acidic within the gastric environment, thereby protecting the enzymes from acid-induced degradation. These food-grade microgels may be useful for delivering active forms of these enzymes to the small intestine, which can help treat certain diseases such as pancreatitis or lactose intolerance. 20.6.4.3 Nutraceuticals The development of nutraceutical-enriched functional foods has become a major emphasis of many researchers with the aim of tackling chronic diseases through dietary interventions (Aditya, Espinosa, & Norton, 2017). However, many nutraceuticals have poor water solubility, chemical stability, and bioavailability. Biopolymer-based delivery systems can be designed to overcome these problems. For instance, alginate, gelatin, and starch have been used to encapsulate β-carotene within filled hydrogels, which improve the chemical stability of this hydrophobic carotenoid (Silva, Feltre, Hubinger, & Sato, 2021). Starch nanoparticles have been developed to encapsulate catechins so as to improve their stability and control their release within the intestine (Ahmad et al., 2019). Zein nanoparticles have been used to encapsulate cranberry proanthocyanidins, thereby improving their biological activity (Tao, Zheng, Percival, Bonard, & Gu, 2012).

20.6.4.4 Omega-3 fatty acids Omega-3 polyunsaturated fatty acids (PUFAs) may reduce the risk of heart and brain diseases, thereby playing an important role in maintaining good human health (Comunian & Favaro-Trindade, 2016; Lane & Derbyshire, 2018). However, omega-3 PUFAs are highly prone to oxidation, which leads to undesirable off-flavors and potentially toxic reaction products (Bush, Stevenson, & Lane, 2017; Wang & Shahidi, 2017). Encapsulation of omega-3 PUFAs in biopolymer-based delivery systems can be used to overcome this problem. For instance, encapsulating tuna oil in carrageenan/myofibrillar protein microcapsules prepared by spray drying inhibited the chemical degradation of fish oil during storage (Bakry, Huang, Zhai, & Huang, 2019). Encapsulating flaxseed oil droplets within biopolymer microgels fabricated from calcium alginate and casein has also been shown to be effective at inhibiting the oxidation of omega-3 fatty acids, which was attributed to the antioxidant properties of the casein (Chen, Li et al., 2017; Chen, Liang et al., 2017). 20.6.4.5 Vitamins Vitamins are bioactive molecules necessary for human health, which can prevent diseases and help promote human growth and development. Many vitamins, however, have low water solubility and are chemically unstable, thereby reducing their efficacy (Katouzian and Jafari, 2016). For this reason, researchers have explored the possibility of using biopolymer-based delivery systems to overcome these problems. As an example, whey protein nanoparticles have been used to encapsulate vitamin D3, which reduced their degradation (Abbasi, Emam-Djomeh, Mousavi, & Davoodi, 2014). In another study, soybean protein nanoparticles prepared by sonication have been used to encapsulate and protect vitamin D 3 (Lee et al., 2016).

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20.7 Future trends

20.7 Future trends

20.6.4.6 Minerals Many kinds of minerals are essential for the proper functioning of the human body and must be obtained through the diet. For example, iron is required to maintain the normal function of hemoglobin, myoglobin, and cytochrome. However, incorporating iron into foods may promote undesirable physical changes or chemical reactions, such as precipitation of proteins or oxidation of lipids (Mcclements, Decker, Park, & Weiss, 2009). Moreover, the bioavailability of iron in many foods is often relatively low, which reduces its beneficial nutritional effects. For this reason, researchers are developing encapsulation technologies to reduce the adverse effects of iron in foods, as well as to promote its bioavailability (Lynch et al., 2005). For instance, iron has been encapsulated within biopolymer delivery systems constructed from chitosan (Duffy, O’Sullivan, & Jacquier, 2018), alginate/whey protein (Duran, Churio, Arias, Neira-Carrillo, & Valenzuela, 2020), and alginate/casein (Patwa, Zandraa, Capakova, Saha, & Saha, 2020) with the aim of improving its biological properties. 20.6.4.7 Antimicrobials Bacterial infection is a major cause of infection and death, as well as of food spoilage (Wang, Hu, & Shao, 2017). The application of many kinds of antimicrobial agents is limited because they have poor water solubility, chemical stability, or efficacy. Biopolymer-based delivery systems can be used to overcome these problems. For example, antimicrobial essential oils have been encapsulated in biopolymer matrices assembled from microbial cellulose (Adepu & Khandelwal, 2020), methylcellulose (Serra et al., 2020) and alginate/soy protein (Volic et al., 2018). These systems can be designed to protect the antimicrobials during storage, but then release them at the required site of action.

In this section, we highlight a few areas where we believe that biopolymer-based delivery systems may be particularly useful in the future.

20.7.1 Co-encapsulation of multiple nutrients Most previous researches were focused on the delivery of single nutrients, with a growing interest in the co-delivery of multiple nutrients (McClements, 2020). These kinds of delivery systems are useful for personalized nutrition applications where an individual requires a number of different nutrients to meet their particular nutritional needs. Moreover, synergistic effects can often be achieved by combining two or more nutrients and controlling the time and release rate of each one. These delivery systems may contain one kind of particle that encapsulates all the different nutrients, or they may contain multiple kinds of particle for different nutrients (McClements, 2020). As an example, curcumin and quercetin have been co-encapsulated in biopolymer-based particles comprised of a hydrophobic protein core (zein) and hydrophilic polysaccharide shell (hyaluronic acid) (Chen et al., 2019). A number of other examples are given in a recent review article on this subject (McClements, 2020).

20.7.2 Targeted and controlled release of bioactive molecules Biopolymer-based delivery systems can be designed to release their encapsulated bioactive molecules at a controlled rate (such as rapid or slow) or in response to specific environmental triggers (such as pH, ionic strength, temperature, or enzyme activity) (Chen et al., 2019). These kinds of delivery systems may be useful for many applications in the food and

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other industries, but more research is needed to create this kind of smart delivery system. In particular, biopolymer formulations that produce the required release characteristics under different environmental conditions are still required.

20.7.3 In vivo testing Most previous studies on biopolymer-based delivery systems have used in vitro testing methods to establish their efficacy. In future, it will be important to test them under more realistic conditions in commercial products and within the human body. In summary, biopolymer-based delivery systems have great potential for a wide range of applications, but further research is still needed to meet this potential.

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C H A P T E R

21 Biological macromolecules for nucleic acid delivery Ahmed S. Abo Dena1,2 and Ibrahim M. El-Sherbiny1 1

Nanomedicine Laboratory, Center for Materials Science, Zewail City of Science and Technology, Giza, Egypt 2Department of Pharmaceutical Chemistry, National Organization for Drug Control and Research (NODCAR), Giza, Egypt

21.1 Introduction The emergence of fatal diseases increases every day, and researchers are doing extensive work to find new, safe, economic, and compliant therapeutic agents/techniques for their treatment. Nanotechnology has paved new ways for advanced medications that can be tailored and fine-tuned to achieve the desired properties for treating various diseases. In addition to targeted drug delivery systems (DDSs), controlled and advanced DDSs have been recently explored in order to enhance the therapeutic properties and reduce the adverse side effects of the utilized drugs. Most of the treatments used for chronic diseases face great clinical and therapeutic challenges when conventional therapeutics are used. Sometimes the use of gene therapy is one of the effective ways that can be used for the treatment of these diseases. Drug delivery of genes and nucleic acids is one of the most advanced techniques used for the treatment of chronic diseases.

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00021-X

DDSs can be synthesized from many types of materials because materials science provides the knowledge of materials properties that allows a researcher to select the most suitable material for the desired application. For instance, materials used in DDSs include polymers (natural or synthetic), metals, metal oxides, lipid vesicles, carbonaceous nanomaterials, biological macromolecules, etc. Generally, the selection of a suitable material relies on the properties of the molecule to be delivered and the characteristics of the delivery system to be used. In this chapter, we will focus on the biological macromolecules-based nucleic acid delivery systems. Plants and animals depend on external sources to get nutrient molecules that they cannot synthesize by themselves. These molecules are known as “biological macromolecules,” a term first coined by Herman Staudinger in the 1920s. Animals eat other animals/plants to satisfy their needs of biological macromolecules, while plants absorb them from the soil. Most of these macromolecules are polymers (such as carbohydrates, proteins,

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and nucleic acids) that play crucial roles in the structures and functions of living cells. Lipids constitute the fourth group of biological macromolecules, yet they are not polymers like the former three groups. In biochemistry, the length of short-chain molecules can be specified (e.g., monomer, dimer, trimer, etc.); however, longchain molecules are referred to as “oligomers.” Oligomers are complex macromolecules formed via the noncovalent interaction of few macromolecules (Gardner, Duprez, Stauffer, Ungu, & Clauson-Kaas, 2019).

21.2 Nucleic acids structure and functions Since this chapter is focusing on the delivery of nucleic acids using biological macromoleculesbased systems, the structure and functions of nucleic acids must be summarized herein in order to help the reader understand the chemical basis of material selection for tailored nucleic acids DDSs. Nucleic acids are natural molecules in the bodies of living organisms. They are responsible for all of the inherited traits of the organism because they are the only molecules that carry cellular information. They control the appearance of a certain group of morphological, and even physiological and behavioral, characteristics by directing protein biosynthesis. Nucleic acids include two main classes, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA constitutes the genetic material in all free-living organisms and most of the known viruses, while RNA represents the genetic material of some viruses and is usually found in other living cells functioning in some biological processes such as protein synthesis. Miescher and many other scientists had noticed that the amount of nucleic acid increases before cell division. However, nucleic acids were not believed to act as the genetic material until reporting the work of Oswald Avery, Colin MacLeod, Fredrick Griffith and

Maclyn McCarty. In 1928, Griffith reported that the transformation of living cells could be achieved by extracts obtained from thermallykilled cells. They also proved that this transformation had the ability to permanently alter the genetics of the recipient cell. Griffith group was dealing with two Streptococcus pneumonia strains. The capsulated S strain was found to be virulent, whereas the nonencapsulated R strain is nonvirulent. The mice die after subcutaneous injection of the S strain, whereas the injection of either live R strain or heat-killed S strain does not cause death. On the other hand, injecting mice with a mixture of heat-killed S strain and a live R strain leads to death, and the live S strain can be found in the mouse blood. So, Griffith expected that one of the components of the heat-killed S strain is responsible for transforming the R strain. This component was shown to be DNA in 1944, by MacLeod and McCarty, via isolating a crude DNA extract from the S strain and destroying any other biological macromolecule such as RNA, lipids, carbohydrates and proteins. They found that the isolated DNA was still able to transform the R strain of the bacteria. Whereas when the resultant pure DNA was subjected to DNAse enzyme, it lost the transformation ability (Minchin & Lodge, 2019). DNA was confirmed to function as the genetic material by Alfred Hershey and Martha Chase. They used bacteriophage, a virus that infects bacteria and consists of a protein coat surrounding a central DNA molecule. They demonstrated that when the bacteriophage infects Escherichia coli bacteria, it is the viral DNA, not its protein coat, that enters the bacterial cell (Minchin & Lodge, 2019). Once it had been confirmed that DNA is the genetic material, researchers started the race of determining its 3D structure. Franklin and Wilkins from King’s College London thought that DNA is composed of two strands forming a double helix structure; Franklin further confirmed this thought by X-ray diffraction

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21.2 Nucleic acids structure and functions

(Fig. 21.1). The first three-dimensional structure of DNA was proposed by Watson and Crick in the 1953 (Fig. 21.1), and they shared a Noble Prize for this discovery in 1962. After they had found that the amount of adenine (A) is equal to that of thymine (T), and the amount of cytosine (C) is equal to that of guanine (G), Watson and Crick presumed that a DNA molecule is composed of a double helix whose chains have opposite orientations such that the equal nitrogenous pairs are attached to one another. This postulate requires that the sugar units are oriented to the outside of the helix and the nitrogenous bases lie on the inside. The helix diameter is about 2 nm, opposite bases are away from one another by about 0.34 nm and related by an angle of rotation of

FIGURE 21.1

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36 degrees. As a result, the helical structure repeats every 10 residues. Normally, DNA molecules are very long and there is no restriction to the sequence of bases along the DNA chain. For instance, E. coli genome has a single circular chromosome composed of about 4.6 million base pairs (BP) which is about 1.6-mm long. The single diploid human cell contains 23 pairs of chromosomes that are composed of 2 m of DNA. Therefore, if we assumed that the total number of nucleated cells in the human body is about 3 trillion cells, the whole DNA from one human when put end to end would reach to the sun and back 20 times. The basic structure of nucleic acids is composed of long chainlike molecules of chemically linked units called “nucleotides” making a

DNA structure in the 1950s (left) and Maurice Wilkins (right).

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polynucleotide chain (Fig. 21.2). A nucleotide unit consists of a phosphate group-bearing pentose sugar attached to a nitrogenous aromatic base. The possible organic nitrogenous bases are of five types, namely adenine, guanine, cytosine, thymine and uracil (U). A and G belong to a group of nitrogenous bases called “purines,” while C, T and U are categorized as “pyrimidines.” The nitrogenous bases A, C and G are found in all nucleic acids, but T is only found in DNA and U is only found in RNA. The fivecarbon sugar unit of DNA (20 -deoxyribose) differs from that of RNA (ribose) because the carbon atom at position 20 of the former does not bear a hydroxyl group (aOH); hence the name

“deoxyribonucleic acid.” Successive sugar residues are connected to one another via the phosphodiester bonds between the 50 -hydroxyl group of one sugar and the 30 -hydroxyl group of the subsequent sugar in the nucleic acid chain.

21.3 Biological macromolecules for nucleic acid delivery 21.3.1 Lipid-based drug delivery systems Nowadays, lipid-nanoparticles-based delivery systems have evolved, away from liposomes and lipoplexes, to become effective and

FIGURE 21.2 The molecular structure of the DNA double helix (A) and nucleic acid base pairs (B). Source: Reprinted with permission from Minchin, S. & Lodge, J. (2019). Understanding biochemistry: Structure and function of nucleic acids., Essays in Biochemistry, 63, 433
456. https://doi.org/10.1042/EBC20180038.

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21.3 Biological macromolecules for nucleic acid delivery

efficient delivery vehicles for nucleic acids. Indeed, today’s lipid-nanoparticle-based nucleic acid DDSs are different from the known liposomes. For instance these lipidbased systems do not display an aqueous core surrounded by a lipid bilayer. Moreover, they do not form particles with nucleic acid payloads driven by electrostatic complexation, nor do they need to balance the charges of constituent compounds for effective and efficient delivery into a cell. The first encapsulation of nucleic acids into liposomes was reported in the late 1970s (Dimitriadis, 1978; Hoffman, Margolis, & Bergelson, 1978; Mannino, Allebach, & Strohl, 1979); yet these delivery systems showed poor encapsulation due to using neutral lipids and passive encapsulation. Also lipoplexes have been applied in vitro but have demonstrated tolerability and potency issues; and hence their use in the clinic is precluded. The observed poor tolerability and potency of lipoplexes are in part attributed to the use of cationic lipids that complex with the negatively charged nucleic acids. When these systems are administered via the intravenous route, they suffer from activation of the immune system resulting from their charge, rapid elimination from the blood, tendency to agglomerate and the unencapsulated exposed position of the nucleic acids. Cationic lipid of permanent charges have been used in earlier lipid-based formulations and helped in increasing the efficiency of nucleic acid encapsulation compared to neutral systems. This enhancement in the encapsulation efficiency may be attributed to the charge interactions between the lipid and the nucleic acid. Up until now, these systems experienced poor tolerability because of introducing a highly charged system into the blood. A leap forward in the improvement of pharmacologically adequate lipid systems was the advent of ionizable positively charged lipids (Samaridou, Heyes, & Lutwyche, 2020; Semple et al., 2001). These lipids are cationic at acidic pH and mostly neutral at the physiological pH. The above characteristic gives the lipid nanoparticles

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the merits of being able to bind to nucleic acids and interact with the endosomal membrane so as to release the nucleic acid into the cell cytoplasm. Often, these aminolipids are combined with PEGylated lipids that help in the formulation of nanometer-sized structures, increase stability and help to mask the surface of the lipid nanoparticle from blood components to optimize the product’s biodistribution and pharmacokinetics. However, PEGylation interferes with endocytosis; therefore, a critical design iteration that played an important role in the success of the current generation of lipid nanoparticles is the use of PEGylated lipids having the ability to diffuse out of the lipid nanoparticle upon administration and enable cellular uptake (Semple et al., 2001). The use of solid lipid nanoparticles for the design and synthesis of nucleic acid delivery systems remains a challenge. Researchers have developed solid-lipid nanoparticle-based delivery systems for the delivery of messenger RNA (mRNA) and plasmid DNA (pDNA) (Go´mezAguado et al., 2020). These delivery systems are based on a combination between cationic and ionizable lipids. The study demonstrated that the influence of formulation-related factors on the long-term stability and transfection efficiency of the mRNA vectors is greater compared to the pDNA vectors. Not only the whole DNA/RNA molecules, but also their nucleotide sequences (termed oligonucleotides or genes) can be delivered by lipid-based nanodelivery systems (Moss, Popova, Hadrup, Astakhova, & Taskova, 2019). Interestingly, these lipid-based nanodelivery systems have been approved by FDA, paving the door for developing more nucleic acid-based therapeutics for several kinds of diseases. Improving the structure of the lipid nanoparticle to be suitable for the desired application can be achieved via surface modification with certain functional groups/ligands. This surface decoration allows for tissue or cell-specific delivery of the nucleic acid. Some studies in the literature report that this surface modification has no

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influence on the pharmacokinetics or biodistribution of the lipid nanodelivery system, whereas others report that it has an enhancing effect. The large-scale production of lipid-based delivery systems for nucleic acids remains a great challenge. The reasons for this include the complicated synthetic procedure and the high costs required. In addition, the process needs a large amount of the nucleic acid to be delivered which is difficult to be produced so far. Nowadays, up to 10 mg of RNA can be produced via solid-phase phosphoramidite chemistry. The primary techniques used for producing lipid nanoparticles nowadays are ethanol injection and thin-film hydration methods, although they have issues in scalability and reproducibility (Moss et al., 2019). As a result of these drawbacks, a modified ethanolinjection strategy has been proposed by researches. In 1999, researchers pioneered Tjunction mixing as a technique for the synthesis of DNA-lipoplexes. The method presented a controlled mixing environment and the production showed a good reproducibility. Since this date, techniques based on microfluidic mixing have been utilized in order to load RNA and pDNA into lipid nanoparticles. The most common and advanced strategies used in this technology are microfluidic hydrodynamic focusing and staggered herringbone mixing. The today advances in high throughput screening of lipid nanoparticles have led to more understanding of the nature of macromolecular interactions controlling both the efficacy and the delivery of the lipid nanoparticles. However, further in-vivo investigations are needed to uncover the in-vivo fate of these nucleic acid carriers.

21.3.2 Protein-based drug delivery systems Nowadays, natural proteins represent safe/ biocompatible alternatives for synthetic

polymers in the field of drug delivery. Proteins, natural biomacromolecules in the form of polymers of amino acids, have a wide range of applications in materials and biomedical sciences. Not only being biocompatible, but also these proteins show a high degree of biodegradability, allowing them to act as excellent candidates for the synthesis of nucleic acid delivery systems. In addition, offering a multitude of moieties accessible to modification allows easy tailoring of protein-based systems for drug/nucleic acid binding, as well as imaging and targeting. The different types of natural proteins are considered as excellent materials for the synthesis of nanocarrier systems because of their amphiphilic properties that permit them to interact with the solvent molecules as well as the delivered drug/ nucleic acid. One of the advantages of these materials is that both water soluble (e.g., human and bovine serum albumin, Fig. 21.3) and water insoluble proteins can be used for synthesizing protein nanocarrier systems for nucleic acid delivery. Here, we will give a brief summary about two of the most commonly used proteins in the fabrication of DDSs of nucleic acids, namely albumin and gelatin. Examples of albumin-based nanostructures used for the delivery of nucleic acids are albumin nanoparticles which mainly act to protect the nucleic acid from degradation in the blood stream (Karimi et al., 2016). In addition, albumin nanoparticles loaded with oligonucleotides have a better access to the target cells compared to free oligonucleotides because the latter are normally hydrophilic and charged (Ming, Carver, & Wu, 2013). Ming et al. synthesized 13-nm diameter albumin nanoparticles for the delivery of oligonucleotides for cancer therapy (Ming et al., 2013). They found that the suggested nanoparticle system could improve the half-life of the delivered oligonucleotides in the circulation and their tissue distribution profile compared to the conventional nanocarrier systems.

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FIGURE 21.3 Crystal structures of bovine serum albumin (A) and human serum albumin (B) obtained from the protein databank represented by ribbons.

Cationic human-serum albumin (CHSA)based nucleic acid/gene delivery systems can reduce the drawbacks of other conventional nanocarrier systems including immunogenicity, toxicity and low transfection efficiency. Researchers developed a CHSA-based genedelivery nanosystem based on CHSA/nuclear localization signals/pDNA complex (Guan et al., 2019). In this context, cationic proteins provide an effective family of biological macromolecules for gene and nucleic acid delivery. They can bind to anionic nucleic acids through electrostatic interactions. However, these cationic carriers can cause cytotoxicity when used in the unmodified form due to the excessive positive charges (Lei et al., 2021). Gelatin is a natural polypeptide prepared from collagen (via partial alkaline or acid hydrolysis) and is useful in a lot of food, pharmaceutical and biomedical applications due to its biocompatibility with and biodegradability in biological media. It is an inexpensive, nontoxic, low antigenic and can be easily modified and cross-linked by simple chemical reactions. In addition, it is nonpyrogenic and can be thermally sterilized. For more information about the use of gelatin-based nanocarrier systems for the delivery of genes and nucleic acids, the reader can see the review article puplished in 2013 by Elzoghby (Elzoghby, 2013). The presence of cationic, anionic and hydrophobic

groups in a ratio of 1:1:1 makes gelatin a special polypeptide in nucleic acid delivery applications. Gelatin nanoparticles can be synthesized by various strategies including desolvation, coacervation-phase separation, emulsification-solvent evaporation, reversephase microemulsion, nanoprecipitation, selfassembly and layer-by-layer coating. Gelatin nanoparticles, nonviral nucleic acid delivery vectors, can be bound to moieties that enhance receptor-mediated endocytosis. The to-be-delivered nucleic acids can be conjugated with the gelatin nanocarriers via electrostatic attraction, physical encapsulation or binding with surface-modifying functional groups. The first B type gelatin nanoparticles for the delivery of nucleic acids was synthesized by Kaul and Amiji (2002). Moreover, anionic nucleic acids can be complexed with gelatin via surface modification of gelatin nanoparticles with quaternary ammonium groups such as cholamine (Zwiorek et al., 2008). Another strategy to load nucleic acids onto gelatin nanoparticles is via using avidin-conjugated gelatin nanoparticles (Coester, Kreuter, von Briesen, & Langer, 2000). Truong-Le et al. reported a DNA-gelatin nanospheres system for gene delivery (TruongLe, August, & Leong, 1998). The nanodelivery system was synthesized by salt-induced complex coacervation of gelatin and pDNA. The

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spherical particles size ranged from 200 to 700 nm and the obtained loading efficiency was in the range 25%
30% (w/w) of pDNA. In this study, gelatin was cross-linked by transferrin to provide extra-stabilization of the synthesized nanoparticles. In addition, the proposed system demonstrated a gradual DNA release profile, as 2.2% of the payload was released per day.

21.3.3 Carbohydrate-based drug delivery systems Large carbohydrates or polysaccharides, natural polymers of monosaccharide units joined via glycosidic bonds, have been extensively investigated by researchers in fields related to nanotechnology and drug delivery due to their wide availability, biodegradability, biocompatibility and diverse chemical/physical properties. In addition, carbohydrates can obviously enhance the biocompatibility and/or reduce the cytotoxicity when used as DDSs. The presence of some structural features including chirality, cyclic structures and a large number of hydroxyl groups allows for easy tailoring of the carbohydrate-based nucleic acid DDS. Normally, polysaccharides can be obtained from natural sources, synthesized via enzymatic or ring-opening polymerization. Many natural polysaccharides such as chitosan, pullulan, dextran, hyaluronan and schizophyllan have been investigated as carriers of nucleic acids. In the following sections, we will give an account on two of the most commonly used carbohydrate polymers in nucleic acid delivery, chitosan and pullulan. 21.3.3.1 Chitosan Chitosan is the most widely studied polysaccharide in nucleic acid delivery. It is composed of glucosamine and N-acetyl glucosamine moieties linked via β(1-4) glycosidic linkages (Fig. 21.4). The wide applications

FIGURE 21.4 Chemical structure of chitosan polymer.

of chitosan are attributed to its unique chemical and physical properties, low cost, availability, low toxicity and biodegradability. In addition, chitosan contains a number of hydroxyl groups and primary amine groups that permits easy functionalization of chitosan via certain common chemical reactions. The pKa of the amino groups of chitosan is about 6.5, hence chitosan is positively charged in acidic and neutral solutions. Chemical synthesis of chitosan is achieved via alkaline hydrolysis of chitin, a known carbohydrate in fungi and in the shells of crustaceans/arthropods. Commercial chitosan is usually found with a degree of deacetylation of about 80%, but other methods which produce 100% deacetylated chitosan were also reported in the literature (Mima, Miya, Iwamoto, & Yoshikawa, 1983). Deacetylation degree of chitosan was found to significantly affect the transfection efficiency in nucleic acid drug delivery (Kiang, Wen, Lim, & Leong, 2004). In this study, chitosan with the highest deacetylation degree showed the highest efficiency. Similar results were obtained in the study carried out by Huang et al. where chitosan with the highest degree of deacetylation was found to be more efficient in protecting DNA from degradation (Huang, Fong, Khor, & Lim, 2005). Furthermore, researchers made several attempts to improve the transfection efficiency of chitosan-based nucleic acid delivery systems. One of these attempts relied on grafting chitosan with polyethyleneimine (Kim, Il Kim, Akaike, & Cho, 2005). This was to improve the charge density and buffering capacity of

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chitosan while maintaining its inherent cytotoxicity. Since then researchers developed lots of strategies via which chitosan grafted with polyethyleneimine can be synthesized. Similarly, chitosan can be grafted with imidazole and the obtained properties are better than those of chitosan-g-polyethylemeimine because this strategy does not increase the cationic character of the polymer significantly. Moreover, imidazole can facilitate the endosomal rupture via the proton sponge mechanism due to the presence of protonable nitrogen atoms with pKa values of about 6.15. Chitosan can be functionalized with other groups for gene/nucleic acid delivery. For instance, functionalizing chitosan with thiols (Fig. 21.5) helps in crosslinking via the formation of S
S bonds upon oxidizing thiol groups. This crosslinking protects the payload from degrading enzymes and prevents the premature release of the loaded nucleic acid cargo. In

addition, chitosan also can be functionalized/ grafted with saccharide-based ligands which have the ability to facilitate receptor-mediated endocytosis as they can act as recognition elements. In turn, this allows for specific cell targeting while treating certain diseases (e.g. cancers) with nucleic acids-based therapies. 21.3.3.2 Pullulan Pullulan is a neutral polysaccharide soluble in water and can be obtained by chemical synthesis from starch by the fungus Aureobasidium pullulans. Pullulan is a straight nonbranched polymer consisting of repeating units of maltotriose linked by α-(1-6) glycosidic bonds (Singh & Saini, 2012) (Fig. 21.6). Pullulan is converted into the maltotriose subunits through enzymatic hydrolysis by pullulanase enzyme (Prajapati, Jani, & Khanda, 2013). It has a molecular formula of (C6H10O5)n and a molecular weight varying from 4.5 3 104 to FIGURE 21.5 Chemical structure of urocanic acid-modified chitosan (A) and synthesis of thiolated chitosan (B).

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FIGURE 21.6 Chemical structure of pullulan. O

O

HO

O

HO O

O HO HO

OH

OH

OH HO

O

HO

HO

HO

6 3 105 Da according to the fermentation parameters (Singh, Kaur, Rana, & Kennedy, 2017). It is a white nonhygroscopic powder that has higher solubility in water, alkaline solution, dimethyl sulfoxide and formamide (Kimoto, Shibuya, & Shiobara, 1997). The melting point of pullulan is about 250 C and it starts to decompose at nearly 280 C. It is characterized by being odorless, tasteless, nonmutagenic and having higher safety profile. Due to the presence of hydroxyl groups within the chemical structure which gives it inherent pharmacological actions, the synthesis of new pullulan derivatives through chemical modifications is always possible (Singh et al., 2017). Pullulan has many inherent properties such as significant mechanical strength, adhesiveness, film formation and enzymatic degradation (Prajapati et al., 2013). Also, it is not permeable to oxygen and can escape from the digestive enzymes. As a result, much interest has been paid to the therapeutic applications of pullulan. The aforementioned physicochemical properties of pullulan and its derivatives made it an ideal candidate as a carrier for nucleic acid delivery for the treatment of various diseases such as tumors, hyperlipidemia and immune diseases. The hydrophobic interior part of pullulan can be linked with water-insoluble drugs such as vitamins and cholesterol, and used in the treatment of different diseases. However, pullulan has not been used for the delivery of nucleic-acid-based therapies till 2004. In a study performed by Husseinkhani et al., different pullulan derivatives were investigated as potential candidates for invivo gene delivery (Hosseinkhani, Aoyama,

n

Ogawa, & Tabata, 2002). This work describes interesting strategies for the design of drugdelivery vehicles. Pullulan can also be grafted with spermine resulting in 15 equivalents or 69 equivalents of spermine per primary OH group of pullulan. Spermine-grafted pullulan was proved to enter the cell via clathrin- and caveolaemediated endocytosis. These mechanisms involve sugar-recognition receptors. In addition, similar to chitosan, pullulan molecular weight has a significant impact on the efficiency of transfection. For instance, medium-molecular-weight pullulan (47.3 kDa) was found to have the best transfection efficiency in HepG2 cells ( Jo et al., 2007). Researches on pullulan-based systems applications as nucleic acid delivery vehicles showed that pullulan derivatives act as potential carriers for different types of nucleic acidbased therapies. However, although pullulan itself is not toxic to living cells, some chemical modifications/functionalization or grafting resulted in serious cytotoxicity. These chemical manipulations are unavoidable since pullulan itself is not target-specific, and hence is inefficient in delivery of nucleic acids.

21.4 Conclusions This chapter summarizes the structure and properties of nucleic acids to understand their use in therapeutics. Moreover, the biological macromolecules used for the delivery of nucleic acid-based therapies, including carbohydrates, proteins and lipids, are summarized herein. Reported researches depict that most biological macromolecules and/or their derivatives can be

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References

used for preparing effective nucleic acid delivery systems characterized by safety, biodegradability, and target specificity. However, several studies did not investigate the in-vitro cytotoxicity of the vehicle systems they propose and others did not give any information about the kinds of interactions that may be involved in the encapsulation process or the cell-entry mechanism. It is necessary, by the way, to do a thorough investigation of the targeting mechanisms of these biological macromolecules-based delivery systems. Computational chemistry and computational biology can play a very important role in this area to facilitate the tailoring and designing of new vehicle systems for different nucleic acidsbased therapies.

References Coester, C., Kreuter, J., von Briesen, H., & Langer, K. (2000). Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA). International Journal of Pharmaceutics, 196, 147
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899. Available from https://doi.org/10.1163/156856207781367756. Karimi, M., Bahrami, S., Ravari, S. B., Zangabad, P. S., Mirshekari, H., Bozorgomid, M., . . . Hamblin, M. R. (2016). Albumin nanostructures as advanced drug delivery systems. Expert Opinion on Drug Delivery, 13, 1609
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433. Available from https://doi.org/10.1016/j.jconrel.2021.01.033. Mannino, R. J., Allebach, E. S., & Strohl, W. A. (1979). Encapsulation of high molecular weight DNA in large unilamellar phospholipid vesicles. FEBS Letters, 101, 229
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7949. Available from https://doi.org/10.1016/j.biomaterials.2013.06.066. Moss, K. H., Popova, P., Hadrup, S. R., Astakhova, K., & Taskova, M. (2019). Lipid nanoparticles for delivery of therapeutic RNA oligonucleotides. Molecular Pharmaceutics, 16, 2265
2277. Available from https:// doi.org/10.1021/acs.molpharmaceut.8b01290. Prajapati, V. D., Jani, G. K., & Khanda, S. M. (2013). Pullulan: An exopolysaccharide and its various applications. Carbohydrate Polymers. Available from https:// doi.org/10.1016/j.carbpol.2013.02.082. Samaridou, E., Heyes, J., & Lutwyche, P. (2020). Lipid nanoparticles for nucleic acid delivery: Current

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gelatin nanospheres. Human Gene Therapy, 9, 1709
1717. Available from https://doi.org/10.1089/hum.1998.9.12-1709. Zwiorek, K., Bourquin, C., Battiany, J., Winter, G., Endres, S., Hartmann, G., & Coester, C. (2008). Delivery by cationic gelatin nanoparticles strongly increases the immunostimulatory effects of CpG oligonucleotides. Pharmaceutical Research, 25, 551
562. Available from https://doi.org/10.1007/s11095-007-9410-5.

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C H A P T E R

22 Biological macromolecules in cell encapsulation Milan Milivojevic, Ivana Pajic-Lijakovic and Branko Bugarski Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia

22.1 Introduction In the humans, cells are commonly organized as tissues (except blood cells) and already have some kind of immobilization in extracellular matrix (ECM). The ECM is usually composed of collagen, glycoproteins, and proteoglycans, while cell growth and functions largely depend on anchorage to many different types of ECMs that generally have five main functions in tissues (Chan & Leong, 2008). At first, ECM is structural support for cell attachment, migration, growth and differentiation. It also provides to each tissue type its mechanical properties (rigidity, elasticity, toughness, tensile strength, etc.). In addition, to enable regulation of cell activities, ECM should provide bioactive cues. Fourth function of ECM is to be a reservoir for growth factors and potentiate their activity. Finally, to provide tissue neovascularization and remodeling during morphogenesis, homeostasis, and wound healing, it must be degradable to some extent. Therefore to use the tissue cells for biomedical applications, they mainly have to be somehow immobilized in order to protect/shield them

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00022-1

from unfavorable/adverse environment or/and immunological rejection, while enabling synergistic interactions between adjacent cells. For those reasons it is not surprising that engineers and scientists from different areas are working on new methods, materials and technologies of cell immobilization that may be used to process and handle cellular materials in most appropriate way. Living cells may be immobilized by aggregation (to surface, porous material or self-aggregation), containment, or encapsulation (within coated microparticles, membrane capsules or gel matrix). For the biomedical purposes encapsulation is mainly of primary interest. Cell encapsulation methods may be classified as: 1. chemical, like: interfacial cross-linking, polycondensation, condensation, or polymerization, and in-situ or matrix polymerization; 2. physical, like: solvent evaporation, spray drying, fluid bed or pan coating, droplet forming (by extrusion, vibrating jet or nozzle, rotating disk etc.); and

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3. physicochemical, like: ionotropic gelation, polyelectrolyte complexation, phase separation/coacervation, supercritical fluid extraction. However, some methods should not be applied for biomedical applications, and in many cases not only one method, but even few of them in a combination, may be used to achieve specified requirements. Method limitations and combinations are connected to the properties of the cells that are to be encapsulated, as well as to the application they would be used for. Second, even more important factor for cell immobilization, beside encapsulation technology, is the right choice of encapsulation materials. If it is done properly then it may be expected that therapy effectiveness and safety will be increased, altogether with patient compliance and convenience. The encapsulation material is in the most cases used either as a coating for cell capsule or as a matrix for cell dispersion. In order to be used in biomedical applications polymer must fulfill many different demands like (Neves & Reis, 2016; Onar, 2014): 1. biosafety (should be nontoxic, nonpyrogenic, nonhemolytic, noninflammative, nonallergenic, noncarcinogenic, nonteratogenic, noncytotoxic etc.); 2. biocompatibility (biological, mechanical and interfacial); 3. effectiveness: (should be functional, durable, with desired performances, etc.); and 4. sterilizable (by ethylene oxide, c-Irradiation, electron beams, autoclave, dry heating, etc.). The synthetic polymers are preferably used for many biomedical applications manly because those materials may be produced almost pure, and what is even more important, with highly reproducible characteristics and properties (Neves & Reis, 2016). However, in cell encapsulation natural polymers are

nowadays gaining more and more attention not only due to some disadvantages of the synthetic polymers (high cost, toxicity, nonrenewable sources, side effects, poor patient compliance, etc.), but also because of their biodegradability and inherent biocompatibility, water binding capacity and mucoadhesiveness, abundance and low cost, gelation ability, pseudoplastic behavior and easy reshaping under mild conditions, diversity of individual and quite complex structures, different physiological functions, presence of amenable reactive groups (hydroxyl, carboxylic, and amino) available for different types of chemical (reduction, oxidation, hydrolysis, esterification, etherification, cross-linking, etc.), or enzymatic modifications and/or conjugation with other molecules, etc. (Hamid Akash, Rehman, & Chen, 2015; van Blitterswijk et al., 2008). In addition, all natural polymers that are used in biomedical applications are biocompatible or biodegradable, noncarcinogenic, free from toxicity and harmless before and after degradation (Chen et al., 2018; Liechty, Kryscio, Slaughter, & Peppas, 2010). Since the safety and compliance of the patient are of primary importance for any cell encapsulation system, natural polymers are considered better than synthetic polymers for those applications and they are slowly but steadily taking primary role in that field. However, although those materials mostly good mimic the natural ECM of tissues and are easily processed into various scaffolds without use of harsh chemicals, they also have some drawbacks such as: quality variation (batch-tobatch), high cost, presence of some immunogenic and/or pathogen moieties, crosscontamination susceptibility, suboptimal characteristics, lower properties for electrospinning, etc. (Garg, Bilandi, & Kapoor, 2011; Garg, Rath, & Goyal, 2015). Biopolymers are nowadays among most used materials for producing different biomedical materials and devices for cell encapsulation (or immobilization). Biopolymers may be

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classified as proteins, polysaccharides and polynucleotides. Those biological macromolecules may be produced by some microorganisms (bacteria, fungi, yeast, or algae), plants and animals. The classification of main biological macromolecules used for cell encapsulation is presented in Table 22.1. Special efforts are nowadays taken to research and develop biopolymers that will provide the successful formulations of various novel and smart devices and scaffold with enhanced therapeutic efficacy, better patient compliances, and cost-effectiveness. An appropriate understanding of various potential attributes, such as extraction methodology and sustainable production, chemistry, surface characteristics, rheology, bulk properties, biocompatibility, biodegradability, etc., of biopolymers can help in the designing of various

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biopolymer-based TE scaffolds (Hasnain et al., 2020). Proper design of cell matrix can largely help in optimal cell growth within the scaffold or may limit this growth in some cases (PajicLijakovic et al., 2017; Pajic-Lijakovic et al., 2019). Typically, polymer scaffolds are in the form of fibrous meshes, porous sponges or foams, and hydrogels (Asti & Gioglio, 2014). Proper selection of material for TE applications is not simple task since scaffold must be functional merge of biocompatible and bioactive materials, cells and other important components that will not only induce cell proliferation, growth and differentiation, but also promote tissue growth and regeneration. The most important properties of biopolymers that must be known are its physical, chemical and biological properties. In order to use biopolymer for TE scaffold production its bulk properties (like

TABLE 22.1 Biological macromolecules used in cell encapsulation. Polysaccharides Plant

Cellulose, galactan (galactosan), fructans (levan, inulin), hemicelluloses (arabinogalactan, xylans, glucuronoxylans, arabinoxylans, mannans, galctomannans, glucomannans (i.e., konjac), xylogulcans, β-D-glucans), exudate and mucilage gums (gum arabic, gum tragacanth, gum karaya, gum ghatti, tamarind gum, yellow mustard mucilage, okra mucilage, psyllium gum, flaxseed gum), starch (amylose, amylopectin), pectin (pectinic acid)

Algal

Agar, alginate, carrageenan, cellulose, fucoidan, ulvan

Animal

Chitin and chitosan, glycogen (animal starch), galactogen, glycosaminoglycans (GAGs) or mucopolysaccharides (chondroitin 6-sulfate, heparin, hyarulonic acid, keratan sulfate)

Fungal

Cellulose, chitosan, elsinan, fructans (levan), Galactosaminogalactan, glycogen, lentinan, krestin (or polysaccharide K
PSK), pullulan, scleroglucan (schizophyllan)

Bacterial

Cellulose, chitosan, curdlan, dextran, emulsan, fructans (levan), glycogen, gums (gellan gum, xanthan gum), hyarulonic acid

Proteins

Actin, albumin, collagen, elastin, fibrin (fibrinogen), fibronectin, gelatin (collagen derived polymer), gluten, heparin, keratin, laminin, myosin, proteoglycans, silk (silk fibroin-fibroin, and spyder silkspidroin), spongin

Polynucleotides (e.g., DNA and RNA) Poly(amino acids)

Poly(γ-glutamic acid), Poly(l-lysine)

Polyesters

Polyhydroxyalkanoates

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microstructure, porosity, water content, thermal and mechanical properties, etc.), surface properties (like wettability, topography etc.), molecular weight (MW), physiological degradation, elemental composition, in vitro biological testing (citotoxicity assays, cell morphology and distribution evaluations, cell viability and proliferation assays, biochemical and genotypic expression analysis, histological assessment, and in vivo engineered tissue analyses) need to be obtained. In addition, to be used for cell entrapment, biopolymers should not only have to fulfill many regulatory restrictions (safety, quality, functionality), and rigid guidelines of current good manufacturing practices, but also to allow cell entrapment procedure that will cause minimal loss of cell viability (Milivojevic, Pajic-Lijakovic, Levic, Nedovic, & Bugarski, 2015; Milivojevic, Pajic-Lijakovic, & Bugarski, 2019). Main demands that a material must fulfill in order to be used for the TE scaffolds are complex, numerous and sometimes opposed, and in almost all cases there is not such material that may be used solely, but instead the appropriate blend of several materials must be carefully chosen. The biopolymers have like collagen, gelatin, chitosan, silk fibroin, fibrin, etc., generally have the ability to mimic many properties of ECM, and therefore to induce the cell adhesion, growth, migration, organization and differentiation, but on the other hand they lack mechanical properties (Nosrati, Pourmotabed, & Sharifi, 2018). TE scaffolds usually need to have some, and sometimes all, of the following functions (Dhandayuthapani, Yoshida, Maekawa, & Kumar, 2011; Kohane & Langer, 2008; Nosrati et al., 2018): 1. to promote cell adhesion, interactions between cell and biomaterial, and enable ECM deposition; 2. to be biocompatible at desired biologic environment (it is very important to stress that material which is biocompatible for some tissue may not be for others);

3. to enable appropriate structure and mechanical properties (determined by the target tissue environment and delivered cells); 4. to permit adequate transport of nutrients, gases, and regulatory factors; 5. to biodegrade at a predictable and desirable rate (to have a time-limited architectural and/or other functions that are as close as possible to the rate of the investigated tissue regeneration); 6. to provoke a minimal degree of inflammation and to be neither cytotoxic nor systemically toxic in vivo; and 7. to have some special properties for specific applications (i.e., electrical conductivity, pH or temperature sensitivity etc.). TE scaffold must perform a time-limited function (architectural or other) as it is foreign body within the natural environment and it need to disappear once that function is fulfilled, leaving behind a viable, purely biologic system. Consequently, it must be biodegradable. Further, Scaffold must have mechanical properties that are determined by the target environment and delivered cells (usually to match those of the surrounding tissue). It also need appropriate degree of porosity for in-growth and delivery of nutrients. Finally, in most cases TE scaffold must have biofunctional properties in order to enable cell attachment, growth and proliferation (Kohane & Langer, 2008). Some of the most important issues that need to be known in order to appropriately choose material for TE scaffold are following (Gomes et al., 2008): 1. origin, structure and main properties of biopolymer; 2. biopolymer characteristics that make it particularly interesting for given application; 3. factors that may affect cells/tissue response to biopolymers; 4. processing possibilities of used biopolymer;

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5. areas of TE application for particular biopolymer; and 6. way to improve biopolymer (by blending, modifications, etc.) in order to obtain desired characteristics. On the other hand just having fundamental biomaterial knowledge is not enough to properly design TE scaffold. This also demand to have some basic and/or advanced knowledge in the fields of fundamental and applied biological science, as well as in engineering and TE (Ozdil & Aydin, 2014). Biopolymers have frequently been used in TE applications because they are either components of ECM, or have macromolecular properties similar to the natural ECM (Filippi, Born, Chaaban, & Scherberich, 2020). The good designed scaffold should not only mime the structure but also the biological function of tissue ECM in terms of its chemical composition and physical structure (Asghari, Samiei, Adibkia, Akbarzadeh, & Davaran, 2016). At the beginning, as consequence of their natural presence in the tissues, protein based biopolymers were used in TE applications, while in the recent years there is growing recognition of polysaccharide biopolymers potential for fabrication of solid cellular and hydrogel based cell carriers (Filippi et al., 2020). Except blood cells, almost all other cells in human tissues are anchorage dependent residing in ECM. As there are many different types of ECMs in human tissues, that have multiple components and tissue-specific composition, it is obvious that for each cell and tissue type scaffold composition must be carefully chosen in order to provide residing cells not only with appropriate structural support and physical environment (mechanical properties like rigidity and elasticity), but also to enable them to attach, grow, migrate and respond to signals, to provide bioactive cues, act as reservoir of needed growth factors, and finally to provide a degradable physical environment to allow neovascularization and

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remodeling (Chan & Leong, 2008). This suggests that one biopolymer can’t fulfill all demands and that combination of few must be used for appropriate scaffold fabrication (as is the fact for natural ECMs). The most critical issue for the development of TE scaffolds is immune system as it monitor, recognize, and clear foreign bodies and it activates a response that is not connected with the potential therapeutic effects of implanted biomaterials, but solely based on the information does biomaterial possesses characteristics that “irritate” the immune system or not. So on the one hand, the inflammatory response is the first step in wound and tissue healing, but on the other hand it is also the fundamental reason why many of implanted scaffolds collapsed. Thus the failure or the success of scaffold is mostly controlled by the ability of used biopolymers to “negotiate” with immune system (Mariani, Lisignoli, Borzi, & Pulsatelli, 2019). A deeper understanding of interplay between the scaffold and its biological environment is therefore needed in order to choose right material for its production. And there is the main advantage of biopolymers over synthetic ones despite all their other drawbacks and weakness. However, natural biopolymers induce undesirable immune reactions mainly due to present impurities. In order to overcome problems with innate and adaptive immune responses to insert scaffolds its material need to be tested (or at least its surface). Surface chemistry plays an important role in immune response since biomaterial hydrophobicity (wettability) take part in an intrinsic immunogenicity, different chemical moieties [i.e., amino (
NH2), carboxyl (
COOH), hydroxyl (
OH), and methyl (
CH3) groups] have different levels of the immunological response, while surface charge also modulate immune function either by its activation (particles with positive surface) or by its inhibition (negative particles) In addition scaffold topography (size, shape, and surface texture) also have important role in immune

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response (Mariani et al., 2019). An additional essential aspect that needs to be considered when using biopolymers for TE scaffolds is that even those that normally do no trigger immune responses can induce one if impurities and endotoxins are present due to the source of the material and/or omissions during their isolation and processing (Mano et al., 2007). The main advantages of biopolymers over synthetic ones are high similarity to the host tissue, ability to communicate with the biological systems, metabolic compatibility, nontoxicity and low inflammatory reactions, enzymatic degradation (preferably into the products that may be used in cellular metabolism). They are biologically active and promote cell adhesion and growth, are biodegradable and allow host cells to produce their own ECMs. Common drawbacks of biopolymer use un TE are their sensitivity (which lead to somewhat more troublesome scaffold fabrication), possibility of disease transmission, poor mechanical properties, lack of stability, induction of a strong immunogenic response due to any impurities in the material gained during processing, limited sources, and variable composition and quality depending on batch and source (Akter, 2016; Asghari et al., 2016; Ozdil & Aydin, 2014). This chapter will try to inform readers about the origin, structure and properties of a diversity of biopolymers currently used in TE and to help them to recognize the characteristics that make them particularly interesting for those applications as well as, why some of them are not good choice. It will also be helpful for those who want to find right information about fields of TE that particular biopolymers may be used, about the forms of scaffolds that can be fabricated by them, as well as, about fabrication technologies that may be applied. Although most of the used biopolymers of TE scaffolds is reviewed in this chapter there is still a wide variety of other that are not reviewed but are potentially useful like polynucleotides (DNA, RNA), gluco- and galacto-

mannans, polyhydroxyalkanoates, laminarin, actin, myosin, exudate and mucilage gums, xanthan gum, levan, curdlan, ulvan, fucoidan, mauran, among many others. The reasons that they are rarely used in TE either due to their low availability, difficulties in their isolation and/or purification and processing or lack of information connected to their biocompatibility and toxicity.

22.2 Biopolymers used for cell encapsulation in TE 22.2.1 Agarose Agarose is a polysaccharide derived from red algae (Rhodophyceae Gelidium and Gracilaria spp.) that consists of a galactose-based backbone (from alternating D-galactose and 3,6-anhydro-α-Lgalactopyranose joined by α-(1-3)- and β-(1-4)-linkages with some ionized sulfate groups). The viscoelastic properties of agarose gels decrease with a decrease in the degree of desulfation of its native polysaccharide agar (O’Brien, 2011). Agarose is usually used for cell immobilization in the form of agar (mixture of agarose and agaropectine) (Alaribe, Manoto, & Motaung, 2016). Agarose exhibits a temperature sensitive water solubility that enables it to form strong, stable and inert thermoreversible gels (cold setting gels at approx. 38 C) based on cooperative H-bond interactions between double helices (Mano et al., 2007; Nedovic & Willaert, 2004), but it can also form a gel in the presence of supraphysiological concentrations of calcium ions (Filippi et al., 2020). Since agarose requires a temperature for gelation that is lower than body temperature it needs to be cooled once placed in vivo (Mozafari, Sefat, & Atala, 2019). A standard technique applied to immobilize cells is relatively simple since an agarose/cell suspension is transformed into solid structure of desired form by cooling (i.e., by extrusion of suspension in cold oil or mold) (Filippi et al., 2020). Agarose is

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22.2 Biopolymers used for cell encapsulation in TE

biocompatible and biodegradable biopolymer with high water uptake capability. However, agarose as non ECM polymer has poor abilities (due to low cell adhesiveness) for promotion of cell attachment, growth and differentiation, which results in low cell viability and growth (Chan & Leong, 2008; Mozafari et al., 2019). Therefore it is good for immobilization of cells that grow in suspension and cells that do not have particular surface-attachment requirements, since some anchorage dependent cells can be sustained in a nonproliferating state in agarose (Nedovic & Willaert, 2004). Other drawbacks are slow biodegradation and possibility for cell protrusion through the agarose membrane (Filippi et al., 2020; Mozafari et al., 2019). Due to some biocompatibility-related issues tissue engineers prefer alginate to agarose (Filippi et al., 2020). Agarose and its blends with other polymers and materials have been applied for many different tissue scaffolds. Its scaffolds were used for of bone, cartilage, pancreas, cornea, skin, spinal cord, cardiac, and nerve tissue regeneration (Mano et al., 2007; Mozafari et al., 2019; O’Brien, 2011). They demonstrated good characteristics in promotion of stem cells differentiation (i.e., human MSCs, bovine MSCs, adipose-derived SCs) in chondrocytes, and for differentiation of embryonic stem cells (mouse and primate) into dopaminergic neurons (Alaribe et al., 2016). Agarose gels support chondrocyte proliferation and are reported to sustain the differentiated phenotype (Filippi et al., 2020).

22.2.2 Alginate Alginates is a collective name for many natural, water soluble, linear (unbranched), anionic heteropolysaccharides mainly derived from brown algae species Phaeophyceae (Laminaria spp., Macrocystis spp., Lessonia spp. and other), but they also may be produced as capsular polysaccharides by two Gram-negative bacteria (Azotobacter and Pseudomonas). Bacterial biosynthesis provides

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alginates with more predictable chemical structures and physical properties (Milivojevic et al., 2015). They have high molecular mass (between 32,000 and 400,000 g/mol) and are consisted of two uronate saccharides. Alginates are made up of irregular ratios of β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) linked by 1
4 glycosidic bonds (Milivojevic et al., 2019). This give alginates it specific characteristics that differ from other natural polymers, since variable proportion and distribution of these three types of blocks (homopolymeric MM and GG, and heteropolymeric MG blocks) determine the chemical and physical properties of the alginate molecules (Milivojevic et al., 2015; Mozafari et al., 2019; Nedovic & Willaert, 2004). Some of the main characteristics that allow alginates their wide use in many different biomedical applications are relatively high water solubility, natural abundance, relatively low cost, biocompatibility, bioadhesivity, biodegradability, nontoxicity, relatively nonimmunogenicity, sufficient transparency, relative stability, etc. Milivojevic et al. (2019). But most of all it is their simple, rapid, nontoxic gelation by divalent cations, that may be performed at versatile environmental and physiological conditions (Nedovic & Willaert, 2004; Sethuraman, Krishnan, & Subramanian, 2016), and the ease of their reshaping into number of soft biomaterial forms like particles (nano, micro, beads), fibers, films, sponges, elastic or injectable gels, multilayers, scaffolds etc. (Filippi et al., 2020; Milivojevic et al., 2019). The fast cell entrapment is preferred in order to prevent hypoxic damage to cells (Mozafari et al., 2019). Alginate anionic structure provide polymer with carboxyl end groups making it good mucoadhesive agent with a high degree of swelling, which enable good nutrient and gas transport and provides to immobilized cells an aqueous environment similar to those found in soft tissues (Milivojevic et al., 2019; O’Brien, 2011). Its open structure (due to the high gel porosity) also promotes development of biological tissue

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within alginate gels facilitating vascularization, oxygenation, cell migration, adhesion, and proliferation (Filippi et al., 2020). In vitro studies indicated that alginate scaffolds can support the adhesion, growth, and proliferation of fibroblasts and endothelial cells (Sethuraman et al., 2016). Its hydrophilic character (due to negatively charge) enables maintenance of a moist microenvironment in the scaffold similar to the physiological milieu (Sethuraman et al., 2016), but it also reduces its interactions with the bulk of cells (Singh & Patel Singh, 2016). Nevertheless, this adverse effect on the attachment of immune cells to the scaffold structure may be favorable for the transplantation of nonautologous cells and tissue fragments, where alginate gel may be a selective immune barrier for protecting the transplanted cells from the host immune system by an immunoprotective shell with minimal fibrotic response in vivo (Filippi et al., 2020; Milivojevic et al., 2019; Mozafari et al., 2019). If specific interactions between mammalian cells and alginate in gel are to be improved cell membrane receptor peptide ligands must be incorporated, either by mixing with other materials (chitosan, collagen, gelatin, fibronectin, lecitin, hyaluronan, oligosaccharides, peptides etc.), or by chemically functionalizing them with cell adhesion ligands [i.e., peptides like arginine-glycineaspartic acid (RGD) tripeptide] (Asti & Gioglio, 2014; Milivojevic et al., 2019; Nedovic & Willaert, 2004). It has also been determined that alginates with a high content of MM blocks (with GG portion , 0.10) evoke an inflammatory response by stimulating monocytes to produce proinflammatory cytokines (Nedovic & Willaert, 2004). Another drawback of alginates for some applications may be their relatively low mechanical properties which may be additionally worsened if alginate has high MM blocks content, while on the other hand, those alginate scaffolds with high GG blocks content can inhibit cell metabolic activity if not coated

(Wang & Sakiyama-Elbert, 2018). However, not only the M/G ratio dictate the gelling and mechanical properties of the alginates but also their distribution (Milivojevic et al., 2015; Nedovic & Willaert, 2004). MW of alginate also may have influence on cell survival during scaffold production since for some applications low viscosity alginates are preferred (Kanika, 2018). All this suggests that alginate composition must be carefully considered for tissue regeneration. Alginates are usually combined with different polymers to produce scaffolds with desired properties. In the case that mechanical properties of alginate scaffolds need to be improved for some applications it is usually combined with various polymers among which agarose, gelatin, cellulose and chitosan are mostly used among natural polymers (Mozafari et al., 2019; Sethuraman et al., 2016). Alginate chitosan scaffolds have excellent mechanical and biological properties and usually those scaffolds rarely can be dissolved (Mano et al., 2007; Nedovic & Willaert, 2004), electrospun gelatin nanofibers provide gel reinforcement (Mozafari et al., 2019), while blending alginate with gelatin change solution viscosity (Pina et al., 2019). Alginate is a nonhuman resource so its degradation can’t be done through mammalian cell digestion since they are stable against action of mammalian enzymes (Milivojevic et al., 2019; Mozafari et al., 2019). However, a slow, uncontrollable and unpredictable in vivo dissolution of alginates occurs due to the loss of divalent calcium ions by complexation into surrounding fluids and consequently alginate molecules are eliminated from the body through the kidneys (Asti & Gioglio, 2014; Mano et al., 2007; Milivojevic et al., 2019). Nevertheless, if loss of divalent ions is somehow prevented implanted alginate scaffolds may be stable for years (Sethuraman et al., 2016). Other, less observed but not less important problems that may occur with alginate based scaffolds are connected to the purity of

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22.2 Biopolymers used for cell encapsulation in TE

alginates, since they may contain small amounts of polyphenols that might be harmful to sensitive cells, or presence of pyrogens or immunogenic materials (proteins and complex carbohydrates) may lead to scaffold failure after implantation (Nedovic & Willaert, 2004; Sethuraman et al., 2016). There are many different processing techniques for alginate scaffold production and they have been produced in many different forms. Main application of alginates is in different gels, and in recent years there is promising usage as bioinks for 3D printing. Alginate hydrogels provides permeability for hormonal nutrients and oxygen exchange and reduces the mechanical irritation and shear stress to surrounding tissues and may be also used in the form of injectable hydrogels (Mozafari et al., 2019). Other production methods for alginates like vortexing, homogenization, ultrasonication, air jet spraying, spray drying, a variety of molding and casting techniques (Asti & Gioglio, 2014; Filippi et al., 2020; Mozafari et al., 2019). Alginates are among few biopolymers which have been clinically used for implant fabrication (Filippi et al., 2020; Pina et al., 2019).

22.2.3 Chitin and chitosan Chitin is structural mucopolysaccharide and it is the principal structural component of the exoskeletons of arthropods, invertebrates, such as

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crustaceans and insects, and is also present in the internal parts of body in some animals and cell walls of most fungi and many algae (Asghari et al., 2016). Therefore it is not surprise that chitin is the second most abundant natural polysaccharide after cellulose (Wang & Sakiyama-Elbert, 2018). Chitin is a linear homopolymer of (β-(1
4)N-acetyl-D-glucosamine) residues and it can be converted into soluble derivatives such as chitosan, carboxymethyl chitin, glycochitin, and others (Mano et al., 2007). Among those derivatives particularly interesting is chitosan that represents form of chitin that may be deacetylated fully or partially (usually between 70% and 95%). Chitosan is commercially manufactured by N-deacetylation of chitin from Mollusk shells by boiling chitin in potassium hydroxide. It is a aminopolysaccharide copolymer consisted of repeating units of glucosamine and N-acetyl glucosamine linked through a β-(1
4)-glycosidic linkage [chemical structure similar to HA and GAGs (Mozafari et al., 2019)] with MW that range between 10 and 1000 kDa (Nosrati et al., 2018; O’Brien, 2011). Chitosan is a positively charged polysaccharide, due to the protonation of its free amino groups, and this make it soluble only in acidic pH, when amino groups are protonated, and give it bioadhesive properties (mucoadhesivness) (Filippi et al., 2020; O’Brien, 2011). Generally chitosan is used more than chitin in biomedical applications although chitin promotes growth factor production and acts as a carrier for the release of growth factors (Filippi et al., 2020).

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Chitosan has many advantages that have lead it become one of the most used biopolymers for tissue engineering. It is renewable and inexpensive due to its natural origin and abundance as well as simple production process. In addition it is, as well as its degradation products, biocompatible (bioresorbable, nonimmunogenic, nontoxic and noncarcinogenic) and biodegradable via enzymatic hydrolysis mainly by glycosidases and lysozymes from blood serum and phagocytic cells (Nosrati et al., 2018; O’Brien, 2011). Due to the presence of free amino groups chitosan interact with the negatively charged moieties and this cationic nature provide it bioadhesive (mucoadhesivness) and antimicrobial properties, while also allows chitosan to interact with different water soluble anionic polymers and with GAGs (Mozafari et al., 2019; Sethuraman et al., 2016). Different behavior of chitosan at various pH levels make it pH-responsible and thus suitable for delivery applications. Chitosan hydrophilic nature enable it to form hydrogels without the use of toxic solution by ionic or chemical cross-linking (with glutaraldehyde) and to swells rapidly in acidic pH (Nedovic & Willaert, 2004; Sethuraman et al., 2016). It dissolves in water (become protonated) at pH lower than 6 while at high pH (. 7) chitin is insoluble in most common solvents, but it can be converted by chemical reactions at its amino and hydroxyl groups into soluble derivatives (e.g., carboxymethyl chitin), which extends the domain of chitin applications (Mano et al., 2007; Nosrati et al., 2018). However, while chitosan can be easy chemically modified its MW, as well as its degree of acetylation are very difficult to control, and physicochemical properties like biodegradability, solubility, reactivity, and cell attachment are highly dependent on this subject (Mano et al., 2007; Mozafari et al., 2019). Other important advantages of chitosan are its hemostatic characteristics that accelerate the formation of fibroblasts and inhibit fibroplasia thus promoting wound healing. Its promotion of growth factor production stimulates tissue growth (Sethuraman et al., 2016).

Disadvantages of chitosan are relatively weak mechanical strength and stability and lack of bioactivity. In order to improve those properties it is often combined with other polymers or graft ¨ zkale, polymerized (Nedovic & Willaert, 2004; O Sakar, & Mooney, 2021). Chitosan slow gelling makes it inappropriate for injectable scaffolds and for processing by electrospinning (Sethuraman et al., 2016). Pure chitosan give poor support for neuronal cell types (Wang & Sakiyama-Elbert, 2018), while in bone applications it induce rapid bone regeneration at initial stages followed by very long (several months or even years) period of bone formation after implanting those matrices (O’Brien, 2011). Chitosan have been widely investigated and applied as material for scaffolds for cartilage, bone, vascular and corneal grafts, artificial liver and skin TE, as well as in many other less common applications (Mozafari et al., 2019; Nosrati et al., 2018). This is not surprising since chitosan increases cell adhesion and proliferation of different cell types (i.e., osteoblasts, keratocytes, fibroblasts, and endothelium cells) and many well-documented in vitro and in vivo studies confirmed remarkable integration with the native tissue and reduced inflammation after implantation (Mozafari et al., 2019; Sethuraman et al., 2016). Scaffolds made from chitin and chitosan, and their derivatives and/or blends with other polymers have been prepared by many different techniques (freezing, liophylization, electrospinning, photocroslinking, etc.) in the forms of hydrogels, fibers, sponges, membranes, films, beads, micro and nanospheres, as those polymers are materials with wide capability to be processed in different forms (Mano et al., 2007; Mariani et al., 2019).

22.2.4 Collagen Collagen is the most abundant protein polymer that is readily found in the ECM of animals (particularly in the ECM of connective tissues) and as the main structural protein it make up between 20% and 30% of the total dry weight of

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22.2 Biopolymers used for cell encapsulation in TE

mammals (Mozafari et al., 2019). It have function to interact with cells in connective tissues giving them not only structural and mechanical support but also transducing essential signals for the regulation of cell differentiation, proliferation, anchorage, migration and survival (O’Brien, 2011). Collagen supports tendons, skin, cartilage, bones, ligaments and cornea tissue (Nosrati et al., 2018). Collagen is mostly produced by the fibroblasts and has triple helix structure of polypeptide alpha chains consisted of Gly-X-Y tripeptide sequence (Fig. 22.1) (Asghari et al., 2016; Mozafari et al., 2019). It can be degraded by the enzyme collagenase from the family of matrix metalloproteinases, and serine proteases, which allow locally controlled collagen degradation by cells present in the engineered tissue (Filippi et al., 2020; Sethuraman et al., 2016). Different types of collagen are usually extracted from the skin, tendons, cartilage, and bone of animals (even from exotic sources such as alligator bones or kangaroo) and most commonly from porcine and bovine ligaments or tendons as well as from rat tails, and then simply isolated and purified (Holban & Grumezescu, 2019; Mozafari et al., 2019). However, there is some natural variation of properties and characteristics of collagen materials caused by the specificity of the source. For example, bovine-derived collagen due to its animal origin provokes immune responses, hypersensitivity and

develops dermatomyositis (Sethuraman et al., 2016), while human collagen from cadavers has poor availability and higher risk of disease transmission. This has led to attempts to extract collagen that do not transmit diseases to humans from sources like marine organisms (i.e., jelly fish, sea cucumber and different sea fishes skin), or to create human recombinant collagen by use of transgenic bacteria and plants (i.e., tobacco) (Sethuraman et al., 2016). There have been identified 28 different types of collagens in biological systems divided into eight families (the main families are: network forming, fibril-forming, fibril-associated, anchoring fibrils and transmembrane) (Mozafari et al., 2019). The most investigated and used for scaffolds is collagen I type mainly due its abundance since it comprises almost 90% of the total collagen in the human body. Other collagen types that may be used for fibrous scaffolds fabricating are collagen types II, III, V, and XI, since only fibril-forming collagen and hence can be used for scaffolds (Sethuraman et al., 2016). It is also important to stress that various types of collagen may have dissimilar effects on different cell types (or even different purposes), so using the right combination of collagen and matching cells might significantly affect the regenerative potential of the scaffold (Wang & Sakiyama-Elbert, 2018). Collagen properties that have made it become one of the most frequently used naturally-

FIGURE 22.1

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Structure of collagen.

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derived polymers are connected to its origin. As ECM component it has not only excellent biocompatibility and low stimulation of the immune system since its amino acid sequences and epitope structures are constant among many species (Mozafari et al., 2019). It also has a natural capacity to promote immobilized cell adhesion, proliferation, survival, migration, and differentiation (Holban & Grumezescu, 2019). In addition, collagen high tensile strength, toughness and elasticity, combined with the ability of cross-linking, have made collagen an ideal biomaterials for different tissue engineering applications (Chan & Leong, 2008; Nosrati et al., 2018). Collagen can be easily cross-linked in scaffolds with network-like, highly organized, threedimensional structure with good porosity (Mariani et al., 2019). It can also be modified (by growth factors or other ways) to promote chondrocyte growth and cartilage matrix formation (O’Brien, 2011). If needed it can be easily biodegraded (by enzymes) into nontoxic products that can be absorbed mostly without inflammation (Sethuraman et al., 2016). Collagen-based scaffolds, alone or in combination with other polymers are readily used when biological and/or mechanical properties of scaffolds have to be enhanced (Ozdil & Aydin, 2014). However, collagen also has some drawbacks that are either general or specific. Some of the general disadvantages are possibility for viral and prion contamination, difficulty in controlling rate and extent of degradation, fabrication of scaffolds with homogeneous and reproducible structures, low effectiveness in promoting cell migration, and processing difficulty. Specific drawbacks are connected with high cost of collagen derived by recombinant technologies, poor mechanical strength for some applications (O’Brien, 2011; Ozdil & Aydin, 2014; Sethuraman et al., 2016). Collagen scaffolds have serious problems with conventional sterilization methods as they modify its structure and they are currently sterilized by ethanol, formic acid, or peracetic acid (Sethuraman et al., 2016). One of the biggest

disadvantages of collagen is its rate of degradation that needs to be regulated if it is to be used as scaffold. Usually it is done either physically (by UV irradiation, dehydrothermal treatment), chemically [by adding glutaraldehyde, diisocyanates, carbodiimide, and genipin, or by blending with chitosan, elastin, and glycosaminoglycans (GAGs)], or enzymatically (by transglutaminase enzymes that is most desirable but also most expensive method) (Sethuraman et al., 2016). Having in mind all mentioned advantages it is not surprising that collagen is one of the most applied natural polymers in tissue engineering. Scaffolds can be produced either from a single collagen type or composites of two or more types (O’Brien, 2011). Collagen is also regularly mixed with other natural and/or synthetic polymers in order to improve scaffold properties. This is because collagen enhances growth capabilities of many different cell types and has strong mechanical architecture (Mozafari et al., 2019). It has been widely investigated for applications in wound healing, skin regeneration, cartilage regeneration, genitourinary tract, orthopedic and bone TE, neural and cardiovascular tissue reconstruction, as well as in ligament, dental, renal glomerular tissue, adipose, intervertebral disk, corneal, and many other tissues reconstruction and augmentation (Holban & Grumezescu, 2019; Nosrati et al., 2018; O’Brien, 2011). There are also numerous examples of effective implantation of collagen scaffolds (Mozafari et al., 2019). With the help of the currently available technologies, collagen can be easily reconstituted into many different forms and shapes. Thus by electrospinning it can be produced in the form of nanofibers that mimic the native ECM of the tissues, or it may be easily adapted in desirable shape through a variety of molding and casting techniques and/or freeze-dried and used as solid porous scaffolds (sponges), besides it can be used as a hydrogel, or even crosslinked (Asti & Gioglio, 2014; Mozafari et al., 2019; Sethuraman et al., 2016).

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22.2.5 Gelatin Gelatin is a natural polymer that is obtained by denaturing of collagen using controlled (partial) irreversible hydrolysis at elevated temperature. It represents the mixture of single-stranded proteins and peptides mainly produced from collagen type I taken from animals bones, skin and connective tissue (Asghari et al., 2016; Mozafari et al., 2019). Producing of gelatin may be done either by acid or alkaline treatment which gives two types of gelatin, type A obtained by acid hydrolysis (usually from porcine skin), and type B by alkaline hydrolysis (primarily from bovine hide and bones). The alkaline process hydrolyzes asparagine and glutamine amide groups into carboxyl ones, while acidic treatment has little influence on the amide groups (Nosrati et al., 2018; O’Brien, 2011). Although each process produces gelatin with partially different electrical properties the partial hydrolysis generally results in an unfolding and cleavage of collagen triple helix

that generates polydisperse mixture of proteins in solution, as illustrated in Fig. 22.2. Hydrolysis at elevated temperature may last up to several weeks, and depending on processing time either high MW gelatin is formed (less hydrolyzed) with chemical composition close to collagen, or a low MW fraction of gelatin is obtained if the acid/alkaline treatments are continued until complete hydrolysis. Apart of differences caused by type and duration of treatment the composition and physical properties of gelatin are also affected by collagen origin. The most important advantage of gelatin is its easy, temperature dependent and reversible gelling (hence its name). Gelatin solutions when cooled below 35 C form gel like structures due to hydrogen bonding and van der Waals interactions between interchain gelatin domains. The disordered gel conformation is formed from remaining collagen-like triple helices since unhydrolyzed parts of collagen form junction zones separated by peptide FIGURE 22.2

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Structure of gelatin.

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residues. As junction zones are unstable at temperatures above 35 C formed gel undergoes sol-gel transition and in order to be used as scaffold in human body it need to be chemically crosslinked to avoid dissolution at body temperature. This limitation is overcome by polyion complexation with other polymers (polycationic or polyanionic depending on the type of gelatin) or glutaraldehyde and carbodiimides (O’Brien, 2011). Although hydrolysis reduce to some extent its gelling ability gels formed from gelatin are strong, flexible, transparent, highly hydrophilic, and with good gas permeability, which all make gelatin a valuable biopolymer for TE. It does not induce noticeable antigenicity after implantation (contrary to collagen), has relatively low price with all other properties similar to collagen like good biocompatibility and water solubility, plasticity, adhesiveness, promotion of cell adhesion and growth (Holban & Grumezescu, 2019). In addition to aforementioned gel thermal degradation, its poor mechanical properties like brittleness as well as its rapid degradation profile in some cases reduce gelatin application. Besides, it is soluble only in water and some alcohols. Gelatin is among few biopolymers that have been clinically used and/or investigated in different TE applications such as bone, cartilage, bone, skin, nerve, cardiac and many others (O’Brien, 2011; Pina et al., 2019). Since gelatin is easy to process scaffolds made from it can have many different forms and shapes, while the most common ones are injectable hydrogels, sponges and fibers. A wide plethora of techniques may be used for scaffold production (like melt molding, thermal gelation, freeze drying, layer by layer solvent casting, electrospinning, photocrosslinking, 3D printing, etc.) but it should be mentioned that although gelatin is easily dissolves in water above 35 C aqueous solutions are difficult to electrospun and other solvents must be used (i.e., trifluoroethanol or ethyl acetate
acetic acid
water).

22.2.6 Fibrin Fibrin is a natural ECM fibrous protein formed by the thrombin induced polymerization of the soluble plasma protein fibrinogen during cascade coagulation process (Mozafari et al., 2019). A clot of blood formed at damaged site initiate hemostasis followed by initiation of wound healing. This role of fibrin in hemostasis and tissue repair enable one of its most important advantages, production of the patient’s own scaffold, from fibrinogen and thrombin obtained from autologous blood, thus offering best immune-compatible support for initial cell adhesion, migration, growth, and differentiation (Mariani et al., 2019; O’Brien, 2011). Advantages of fibrin based scaffolds are numerous. This nonimmunogenic carrier for cell delivery contains innate sites for cell binding and has very good mitogenic, hemostatic and chemotactic properties that allow fibrin scaffolds to mimic the native surrounding tissue. Due to its origin (autologous) it may be free from disease transmissions and enhance cell survival. Fibrin is frequently used in tissue engineering applications (even in some clinical) as it is beside mentioned also readily available, low in price material with self-aggregating and gelling abilities (Akter, 2016; Mozafari et al., 2019). If needed rheological properties of fibrin can be easily modified (Mozafari et al., 2019). Naturally, fibrin also has some drawbacks which must be considered and overcome for some applications. As first its fast rate of degradation may be desirable in some cases but for the most of other application it fast and somewhat unpredictable degradation can be a big problem (Sethuraman et al., 2016). In addition, poor mechanical stiffness is one of its biggest weaknesses as fibrin has lowest mechanical strength compared to other protein polymers (Mozafari et al., 2019; Nosrati et al., 2018). Therefore right composition of fibrin scaffolds is an important issue to produce system with applicable mechanical properties.

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22.2 Biopolymers used for cell encapsulation in TE

However, although fibrin poor mechanical properties sometimes limit the level of its application in cartilage and bone TE it still has been widely used for different scaffolds in cartilage, bone, nerve, ocular, cardiovascular, liver and many other TE applications (O’Brien, 2011). The fibrin-based scaffold in many cases has good host-tissue integration but in order to obtain optimal cell adhesion and functions this scaffolds must be further adjusted to accommodate different cell types (Wang & Sakiyama-Elbert, 2018). Many different forms of fibrin scaffolds were produced but most of all gel and glue forms were dominant mainly due to its sealant and adhesive properties (Filippi et al., 2020; Mariani et al., 2019; Mozafari et al., 2019; Sethuraman et al., 2016).

22.2.7 Glycosaminoglycans GAGs or mucopolysaccharides are linear, long chain, negatively charged polymers made of repeated disaccharide units (alternating uronic acid and hexosamine residues) (Mariani et al., 2019). Depending on the sulfation degree six different types of GAGs are synthesized in the Golgi system through complex reaction chains [chondroitin sulfates, dermatan sulfate (DS), keratin sulfate, heparin sulfate, heparin, and hyaluronic acid (HA)], that in all cases except for HA include extensive sulfation of the monosaccharide’s in GAGs (Filippi et al., 2020). In the human body GAG chains are covalently linked to a central protein core (except hyaluronate which is free), and those polysaccharide
protein complex called proteoglycans play a key role in the coordination of various fundamental processes for tissue development and homeostasis. The gel-like matrix of ECM, especially those of connective tissues (like tendon, cartilage, blood vessel walls and skin), is mainly composed of GAGs that surround collagen and other proteins and those GAG chains predominantly determine proteoglycans properties (Mano et al., 2007).

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Only hyaluronate is in the form of free molecule within the ECM and is not covalently attached to the protein. GAGs are group of linear, very long, and highly negatively charged polysaccharides consisted of repeating disaccharide units (uronic acid and glycosamine residue) backbone. Based on backbone structure, five major classes of GAGs are formed with different structural and functional properties that are regulated by many factors such as the sugar composition and degree of sulfation of the GAG, size of the GAG chains, and their ability to form proteoglycans with collagen and other proteins. Based on the difference of repeating disaccharide units comprising GAGs, they can be classified into four main groups: heparin/heparan sulfate, chondroitin sulfate/DS, keratan sulfate (KS), and hyaluronan. Hyaluronan or HA, a very high MW polysaccharide is composed of 250
25,000 b(1/4)-linked disaccharide units, which consists of alternating D-glucuronic acid and N-acetyl-D-glucosamine linked by b(1/3) bonds (Mozafari et al., 2019). Chondroitin-4-sulfate and chondroitin-6-sulfate (KS) differ only in the sulfation of their N-acetylgalactosamine residues, and their derivative DS is obtained by enzymatic epimerization of chondroitin. Among GAGs KS has most heterogeneous composition with variable sulfate content and small amounts of mannose, fucose, GlcNAc and sialic acid, while heparin sulfate (HS) is the most highly charged polymer in mammalian tissues with sulfate residues content that vary around 2.5 per disaccharide unit (Mano et al., 2007). Due to their difference in repeating disaccharide units GAGs may be classified in four main groups: hyaluronan, chondroitin sulfate/DS, heparin/heparan sulfate, and KS. Thus their characteristics are somewhat the same but also differ in some details. Large presence of strongly ionizing sulfate groups make these molecules soluble over a wide pH range, while repeating disaccharide structure give them amorphous structure in the solid state (Filippi et al., 2020). In addition,

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the presence of sulfate groups provide them ability to significantly swell in water which results in the high compressive modulus and excellent resistance to repeated deformation of GAGs rich tissues (i.e., articular cartilage). Most interesting GAG for applications in TE is hyaluronate, the only GAG with significant gel-forming ability. The potential of other GAGs for application in TE as scaffold material mainly came from their ability to form numerous interactions (binding, covalent linking, or complexing) with different proteins and other polymers that have superior mechanical or structural properties. GAGs in turn provide thus formed scaffold with ability to coordinate various fundamental processes for tissue development and homeostasis (cell signaling, its biophysical characteristics, and assembly of the ECM) (Mariani et al., 2019). GAGs as the components of ECM have a major role in cell adhesion, while the magnitude of cell interactions with the ECM is regulated by the degree of sulfation and the MW of GAG (Sethuraman et al., 2016). HA (also called hyaluronate or hyaluronan), is largest GAG and one of the major ECM components in connective tissue, synovial fluid, skin, umbilical cord, and the vitreous humor of the eye. In solution, HA swell significantly occupying a volume up to 1000 times larger than it has in dry state. HA solutions possess viscoelastic properties which make them excellent absorbers and lubricants. HA may be controllably and easily produced by microbial fermentation thus minimizing the risk of animal-derived pathogens (Mano et al., 2007). HA supports cell proliferation, differentiation, and migration, improve cell survival, accelerate tissue repair, and, due to its high-molecular mass, has numerous mechanical functions (in lubrication and shock absorption) (Mariani et al., 2019; Mozafari et al., 2019; Sethuraman et al., 2016). In addition, HA is the most interesting GAG for application in TE as it is the only GAG with significant gel-forming ability. HA gel not only facilitates compressive resistance properties, but also assists diffusion of cell nutrients. The

glycan structure of HA enable easy cross-linking as well as chemical modifications which may help in adhesion and migration of specific cell types. HA has many other attractive features for scaffold use like: easy isolation from abundant natural sources (animals, marine organisms, bacteria), minimal inflammatory response following implantation, and easy formation of gel by covalently cross-linking with hydrazide derivatives which may be enzymatically degraded by hyaluronidase. On the other side, main limitations for the use of HA are the need for complete purification prior to use in order to remove impurities and endotoxins that may transmit disease or trigger an immune response, its long residence time within the body, and limited mechanical properties of its gels (Nedovic & Willaert, 2004). GAGs used in scaffolds and matrixes are usually obtained as salts of sodium, potassium, or ammonia, and in this form they are all water soluble (Filippi et al., 2020). HA is highly preferred for cartilage and dermal TE (Sethuraman et al., 2016), spinal cord injury (Mozafari et al., 2019; Wang & Sakiyama-Elbert, 2018), bone and soft tissue repair (Nedovic & Willaert, 2004), and many others (O’Brien, 2011), and some of them have been clinically used (Pina et al., 2019). Most GAGs are used in TE as scaffolds in the form of hydrogel, while HA has been also used in the form of membranes, sponges and flat sheets that were produced by different techniques like chemical crosslinking, micromolding, photolithography, emulsification, 3D printing, self-assembling, etc.

22.2.8 Silk (fibroion and spidroin) Silks are group of protein biopolymers that are contained in the glands of arthropods, and are mainly obtained from silkworms, spiders, mites, scorpions and flies (Mariani et al., 2019; Nosrati et al., 2018). Generally, there are two main groups of silk: one that is composed of core protein fibroin (70%
80%), which is coated with

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22.2 Biopolymers used for cell encapsulation in TE

small hydrophilic adhesive protein called sericin (20%
30%), and second, spider silk called spidroin, that does not contain sericin. In the first case, silk fibroin is made of a heavy chain and light chain linked by disulfide bridges (between cysteine residues) and by 25 kDa protein called P25 (through noncovalent interactions), while spidroin is composed of major ampullate spidroin proteins I and II. (Mariani et al., 2019; Sethuraman et al., 2016). Main sources for silk extraction are domestic silkworms Bombyx mori, wild silkworms like Antheraea pernyi, worms from the order of Lepidoptera (butterflies and moths), as well as wild several species of Arachnida class and Nephilia clavipes spiders. Source of silk has not only influence on its composition but also on properties. Thus silk fibroin produced from B. mori larvae cocoons lacks cell adhesion RGD (Arg-Gly-Asp) peptide, while those obtained from A. pernyi possesses the RGD peptide and have superior cell adhesion and proliferation. On the other hand proteins from spider silk contain abundance of glycine, alanine, glutamate, proline, and arginine residues which give them excellent mechanical properties (Sethuraman et al., 2016). It also need to be indicated that although sericin also has been explored for TE applications due to its biocompatibility, inherent antioxidant, antimicrobial, and antiapoptotic properties, together with ability to form hydrogels, it must be totally separated from silk fibroin (in prefabrication step called degumming). Namely, sericin in combination with silk fibroin generates toxic effects as it induces a macrophage response (Holban & Grumezescu, 2019; Mariani et al., 2019). Degummed silk fibroin is desirable component of TE scaffolds since it, unlike most other natural polymers, has excellent mechanical properties, combined with availability, easy processing (at ambient and aqueous surrounding) and chemical functionalization, minimal inflammatory response, biocompatibility and slow, but adjustable biodegradation (Akter, 2016; Mozafari et al., 2019; Nosrati et al., 2018).

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While silk fibroin is highly stable to hydrolytic degradation it may be degraded by enzymes such as collagenases, trypsin, and alphachymotrypsin or its degradation rate maybe customized by modifying different mechanical settings of the scaffold synthesis procedure (Holban & Grumezescu, 2019; Sethuraman et al., 2016). Some of the drawbacks exhibited by silk fibroin are as first its brittleness that may be overcome by its blending with other polymer like chitosan (Akter, 2016). Secondly, its poor adsorption of growth factors may be overcome by combining with human keratin, while its lack of cell adhesion RGD peptide is commonly solved either by its chemical functionalization or by blending with polymers like chitosan, PVA, collagen, fibronectin, elastin, laminin, etc. Finally, as a protein, fibroin is very sensitive to the processing conditions such as pH, temperature, and chemical environment (Sethuraman et al., 2016). Spider silk (spidroin) is even more interesting and promising biomaterial for TE applications due its very low weight but very elastic and strong fibers that are comparable to the best synthetized fibers by new technology. It is also biodegradable and environmentally safe material but has very limited production and thus high cost because of the limited production by spiders (Nosrati et al., 2018). The most extensively investigated spidroin is those produced by Nephilia clavipes known as “dragline silk” (Sethuraman et al., 2016). Applications of silk fibroin in TE for scaffolds are numerous and it is among few biopolymers that have been clinically used (Pina et al., 2019). Among those applications most important are in the vascular, bone, skin, nerve, liver, and corneal TE (Holban & Grumezescu, 2019; Mariani et al., 2019; Mozafari et al., 2019; O’Brien, 2011). Scaffolds that have been produced from silk proteins are most extensively used in the form of hydrogels and fibers, as well as porous 3D sponges, nanofibers, films, etc. (Mariani et al., 2019; Mozafari et al., 2019; Sethuraman et al., 2016). Techniques used for fabrication of those

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22. Biological macromolecules in cell encapsulation

scaffolds are also numerous and mainly are based on freeze-drying, electrospinning, solventcasting, salt-leaching, emulsification and so on.

22.2.9 Other natural polymers in TE 22.2.9.1 Cellulose Among other biodegradable and biocompatible natural polymers that have been less extensively used for TE scaffold fabrication one of the main is cellulose and its derivatives. Cellulose as the primary structural component of plant cell walls is the most abundant organic polymer in the world. It is a linear polysaccharide composed of D-glucose units linked by β(1/4) glycosidic bonds. However, other even more important source of cellulose especially for TE applications is bacterial production mainly by Gluconacetobacter Xylinum. Although both celluloses have the same molecular formula their characteristics are considerably different. As first, while cellulose obtained from plan cell walls need to be enzymatically hydrolyzed under mechanical shearing, bacterial cellulose has no impurities like lignin and hemicellulose. In addition, microbial cellulose much more hydrophilic (and therefore has larger water retention), it has larger tensile strength (due higher level of polymerization)and more pronounced crystalline structure. Its structure enables forming of significantly smaller characteristic ribbon-like microfibrils which make bacterial cellulose much more porous. Over all, it is cost-efficient with relatively simple production and can be in situ grown into any shape due to the high moldability (Mano et al., 2007). Scaffolds obtained from cellulose have good mechanical proprieties and high thermal stability, combined with biocompatibility and nonimmunogenicity (Holban & Grumezescu, 2019). Among its drawbacks it has to be noted that cellulose fibers are water insoluble despite its hydrophilicity due to its (1-4)-β-D-glucan chains. If it needs to be dissolved in water it must be modified either by decreasing its MW or by introducing charged groups (i.e., sodium

carboxymethyl cellulose) or branching groups (i.e., methyl cellulose, hydroxylpropyl cellulose, hydroxylpropyl methyl cellulose). Even bigger problem may be its poor degradation in vivo (O’Brien, 2011). Cellulose scaffolds have been investigated mainly for bone, cartilage, cardiac, pancreas and vascular TE applications (Holban & Grumezescu, 2019; Mozafari et al., 2019; O’Brien, 2011). Scaffolds made from cellulose may have different forms but hydrogel, membranes, and different nanostructures produced by physical and chemical crosslinking gelation, radical polymerization, and 3D bioprinting are most common. 22.2.9.2 Starch (starch, amylose, amylopectin) Starch is a storage natural polysaccharide that composed of mixture of glycans that green plants synthesize during the process of photosynthesis as their principal food reserve and then deposits within their seeds, bulbs or tubers. Although starch is shared name for white, tasteless and odorless powder (granule), the granules of starch have specific shape, size, and other characteristic dependent of starch producing plant. Whatever the source of native starch, it is consisted of 70%
85% of amylopectin and 15%
30% of α-amylose. As amylopectin and amylose have different chemical and physical characteristics, overall properties of starch are influenced by their relative proportions. Amylopectin is a branching molecule composed of anhydroglucose chains that consists mainly of α(1/4)-linked glucose residues, with α(1/6) branches at every 24
30 glucose residues. Amylopectin molecules may have up to 106 glucose residues which make them to be among the largest molecules in nature with the MW up to 80,000,000. On the other hand amylose molecules have MW approximately 40,0004340,000, with unbranched glucose chains containing 25042000 anhydroglucose units (Mano et al., 2007). Main properties that have led to starch and its derivatives application for TE scaffold products are its biological renewability, overwhelming abundance and biodegradability.

III. Functional Applications

22.2 Biopolymers used for cell encapsulation in TE

In addition, if necessary it can be easily modified (via crosslinking, acetylation or oxidation) or blended with other polymers in order to improve scaffold characteristics. It is easily biodegraded (amylose is enzymatically degraded by α-amylase from serum) into oligo saccharides that can be readily metabolized to produce energy (O’Brien, 2011). However, due to its semi-crystalline native starch granules are extremely difficult to process as they are very brittle, and either are destroyed, reorganized, or both. Other disadvantage of starch may be its low solubility in cold water and alcohol, while boiling water dissolve starch but also change it slowly into smaller molecules (acid catalyzed hydrolysis). Scaffolds containing starch have been used in Bone and vascular TE, and have been produced by molding, casting, rapid prototyping and 3D printing techniques as they have flexibility to adapt their shape to required forms (Asti & Gioglio, 2014). 22.2.9.3 Carrageenans Carrageenans, like agar belong to the group of galactans, polysaccharides obtained from red algae Rhodophyceae. They are linear polymers consisted of (1/3)-linked β-D-galactose and (1/4)-linked α-D-galactose units that are, depending on the source and extraction conditions, differently substituted and modified into the 3,6-anhydro derivative (Sethuraman et al., 2016). Carrageenans are anionic polysaccharide due to the presence of ester-sulfate that varies between 15%
40% depending on carrageenan type. According to the ester-sulfate content they are classified as κ-carrageenan that has one sulfate group per disaccharide, ι-carrageenan that has 2, and λ-carrageenan that has 3. At higher concentrations all carrageenans twist around each other and form double helical structures however only κ and ι -carrageenan have the ability to form a variety of different gels at room temperature. κ -carrageenan in the presence of potassium ions form strong and rigid thermoreversible gel, while ι -carrageenan in the presence of calcium ions form soft and elastic

509

thermoreversible gel (Mano et al., 2007). Other main advantages that have promoted use of carrageenans in TE applications in recent years were its thixotropic nature, inherent antimicrobial and antioxidant activity and high degree of protein reactivity. However, there are also some problems that may limit their applications like high cost, potential inflammation tendency due to generation of toxic waste, and most of all, gel dissolution in the absence of gelling ions or other gel inducing reagents (Garg et al., 2015; O’Brien, 2011). Main field of carrageenan application is in the bone TE where different forms of pure or combined gels were produced by physical gelation or chemical crosslinking techniques. 22.2.9.4 Dextrans Dextrans are branched microbial exopolysaccharides produced from sucrose by enzyme dextransucrase that is present in different bacterial strains (i.e., Leuconostoc mesenteroides, Lactobacillus brevis, Streptococcus mutans etc.). They consists of repeating glucose units linked in backbone through α 2 1,6 glycosidic links and depending on the branching there are 3 classes of dextrans (Sethuraman et al., 2016). The degree of branching, MW of dextrans, physical and chemical properties of dextrans depend on their source (microbial type and cultivation conditions) and may vary between 0.5% to 60% and 3000 to 2,000,000, respectively (Garg et al., 2015; Mano et al., 2007). Dextrans due to their hydrophilicity are soluble in water and organic solvents and readily form hydrogels. They are stable under mild acidic and basic conditions, biodegradable and biocompatible, while the large number of hydroxyl groups makes them available for modification and/or conjugation with other molecules. In addition, their presence reduces aggregation and may alter cell adhesion in desired direction. However, although dextran is the first microbial product used for pharmaceutical applications and thus has long history of safety, its high cost, tendency for over hydration and risks of coagulation abnormalities and

III. Functional Applications

510

22. Biological macromolecules in cell encapsulation

anaphylaxis have limited its applications in TE. Yet, it is still good material to be used for scaffold production in bone, cartilage, skin, vascular and spinal cord TE, mainly in the form of porous hydrogels (Garg et al., 2015; O’Brien, 2011). Dextrans, together with some of its derivatives (i.e., dextran sulfate), are good candidates for use in 3D printing so it expected that their role in TE may be bigger in the future. 22.2.9.5 Gellan Gellan is anionic, linear, tetra-saccharide consisted of about 50,000 carbohydrate units (MW B 500 kDa). It is consisted of repeating carbohydrate units (1,3-β-D-glucose, 1,4-β
Dglucuronic acid, 1,4-β-D-glucose, and 1,4-α-Lrhamnose). It is produced by bacterium Sphingomonas elodea (earlier Pseudomonas elodea) and commercially present in two forms: native form, or high acyl gellan, and the low acyl gellan (more common and commercially available) (Mano et al., 2007; Milivojevic, PajicLijakovic, Bugarski, Hasnain, & Nayak, 2019; Osmałek, Froelich, & Tasarek, 2014). Native form have the 1-3-linked glucose unit with two acyl substituents: L-glyceryl at O(2) and acetyl at O(6) (with averagely 1/0.5 glyceryl/ acetyl substituents ratio per repeating tetrasaccharide unit). Both substituents are simply removed by hot alkali treatment producing low acyl gellan (Milivojevic et al., 2019). Advantageous properties of gellan are overwhelming [200-]. While at high temperatures gellan molecules exist in the form of random coils at low temperatures have the form of double helices and are present in the form of gel. The rapid gelation is also present in the presence of cations (even at very low gellan concentrations compared to other hydrocolloids) and this gels that may be formed solely or by a combination of thermal gelation and ionic cross-linking is main gellan advantage as it offer wide possibilities for cross-linking densities (Milivojevic et al., 2019; Nedovic & Willaert, 2004; Park et al., 2020). In addition, it

may be blended with many different natural and synthetic polymers with which it may form coupled networks, interpenetrating networks, or complex coacervates, that modify and/or improve gelling characteristics and gel properties in the desired manner (mechanical, diffusivity, etc.) (Milivojevic et al., 2019). Therefore it may be said that possibilities that gellan offer are numerous. Above all that, gellan possesses other advantages as it is biodegradable, biocompatible, mucoadhesiveness, nontoxic, stabile and temperature resistant. The gels produced by high acyl form give transparent, soft, elastic, and flexible gels, with high water holding capacity that is combined with ability to significantly recover after slow mechanical disruption or expulsion of water. On the other hand the low acyl gellan form produces firm, nonelastic, brittle gels (Garg et al., 2015; Mano et al., 2007; O’Brien, 2011). However, despite all mentioned advantages and low number of drawbacks, gellan have been used in TE scarcely for cartilage and bone scaffold fabrications in the form of thermally or ionotropically produced gels. Main reason that gellan full potential in biomedical applications is still not realized may be its relatively short history in the field (compared to other gelling agents) and there is a hope that in the future it will become a useful component for different TE scaffolds. 22.2.9.6 Pullulans Pullulans are neutral, hydrophilic polysaccharides predominantly produced by the fungus Aureobasidium pullulans from starch. They are composed of repeating maltotriose, a(1/4)linked units joined by a(1/6) linkages with the inclusion of occasional maltotetraose units (Mano et al., 2007). Pullulans have a unique linkage in their structure, which provides them a high adhesion (that promote chondrogenesis) and excellent mechanical properties compared to other hydrogel forming systems (Park et al., 2020). Pullulans easily dissolve in water in the form of random coils and form stable, viscous

III. Functional Applications

22.3 Advantages, drawbacks, applications, forms and manufacturing methods

511

and nongelling solutions. Their properties such as nonimmunogenicity and biodegradability, in vitro and in vivo biocompatibility and hemocompatibility, as well as high hydration capacity, offer promising role of pullulans for TE applications (Garg et al., 2015). However, there is a mayor limitation for their wider usage as they lack a surface that can support cell adhesion and spreading which limits their ability for cell proliferation and osteogenesis. In order to overcome this problem pullulans may be easily derivatized to provide incorporation of RGD ligands or can be mixed with cell recognizable molecules like gelatin. Other, less important disadvantages are its relatively high cost and instability. Those materials have been mainly used in bone and cartilage TE to produce hydrogels, fibers and oxygenimpermeable films.

aggregate above critical temperature (O’Brien, 2011). It applications are mostly for different vascular, epidermal, cervical and bone TE scaffolds, with nanofiber electrospinning as most important technique for their fabrication.

22.2.9.7 Elastin

Fibronectin is a glycoprotein of the ECM where it binds to collagen, fibrin, integrins, and other proteoglycans. Although fibronectin has better cell attachment properties and improved nerve regeneration qualities than fibrin application of fibronectin as hydrogel scaffold is not desirable as it gel smoothly and form particulates which make tissue repair more difficult (Mozafari et al., 2019). However, due to its important role in peripheral nerve regeneration its use in scaffolds for peripheral nerve TE scaffolds is almost inevitable while it has been also used for liver and pancreatic TE application (Mozafari et al., 2019; Wang & Sakiyama-Elbert, 2018). Most useful technique for fibronectin containing scaffold fabrication is photolithography.

Elastin is fibrous ECM protein synthesized by vascular smooth muscle cells and it is a major component in connective tissue and different tissues that need high elasticity for their physiological function (i.e., uterus). It is made of few simple amino acids (glycine, alanine, valine and praline), and within ECM it forms elastic matrices (fibers and sheets) in combination with several unbranched microfibrils (fibrillin, vitronectin, fibulin, emilin) and microfibril-associated glycoprotein-1 (Mozafari et al., 2019). Its main function in the ECM of tissues is to provide it with elasticity, tensile strength and flexibility and enable recover of shape after deformations. It is one of the most stable components of the ECM and as such a highly popular for different TE applications. Elastin has low polydispersity (Precise MW), it is biocompatible with easy controllable degradation and it also promote cell adhesion, proliferation, and infiltration (Garg et al., 2015; Sethuraman et al., 2016). However, although it is soluble and nonglycosylated due to its hydrophobic nature, it becomes insoluble and

22.2.9.8 Laminin Laminin is another ECM protein component that is used for TE scaffold fabrication since it plays an important role in peripheral nerve regeneration as it promotes neurite growth, and it promotes cell adhesion, proliferation, and infiltration, as well as stem cell differentiation. Except of its wide use in nerve TE it has been also used for cardiac and liver TE scaffolds, mainly in the form of hydrogels (Mozafari et al., 2019; Sethuraman et al., 2016; Wang & Sakiyama-Elbert, 2018). 22.2.9.9 Fibronectin

22.3 Advantages, drawbacks, applications, forms and manufacturing methods Main characteristics (advantages and drawbacks) and applications of different biopolymers used it TE are highlighted in Table 22.2 (Akter, 2016; Asti & Gioglio, 2014; Dhandayuthapani

III. Functional Applications

TABLE 22.2

Main characteristics and applications of biopolymers used it TE.

Advantages

Drawbacks

Applications (cells)

Manufacturing methods of material or composites

(1) Chitosan • • • • • • • • • • • • • • • • •

• • •

Biologically renewable; Biodegradable; Biocompatible; Nonantigenic; Nontoxic; Biofunctional; Bioadhesive; Inexpensive; Positively charged; Antibacterial; Tissue compatible; High blood compatibility and macrophages are less active; Injectable form prepared without the use of toxic solution; pH-sensitive behavior; Additional control over chitosan’s final property; Bioresorbable (its degradation products are nontoxic, nonimmunogenic, noncarcinogenic); Hemostatic agent which accelerates the formation of fibroblasts and increases early-phase reactions related to healing; Allow osteoconduction due to its porous structure; Promotes growth factor production and acts as a carrier for the release of growth factors; Inhibits fibroplasia in wound healing and promotes tissue growth.

• Rapid bone regeneration at • Bone [Human bone marrow cells, initial stages but long delay in Human articular chondrocytes, Mouse bone formation (after several BMSCs, Rat calvarial osteoblasts, MSCs, months or years); Osteoblastic MC3T3-E1 cells, Human bone MSCs, Osteoblast-like human cell • Processing take long time; line (MG63), Primary human • Limited supply; osteoblasts, Human dermal • Difficult processing by microvascular endothelial cells, Human electrospinning; dermal neonatal fibroblasts]; • Immunogenicity; • Relatively weak mechanical • Cartilage (Articular chondrocytes, strength and stability; Human articular chondrocytes, Porcine articular chondrocytes, Human • Insolubility in most common adipose-derived adult stem cells, Cattle solvents (can be converted into articular chondrocytes, Rat soluble derivatives by chemical chondrocytes, Bovine articular reactions); chondrocytes, Rabbit articular • Chitin derivatives are chondrocytes, Rabbit rib chondroenzymatically degraded by progenitor cells, Mouse BMSCs, lysozyme, but degradation rate hBMSC); depends on the degree of acetylation. • Vascularization/angiogenesis [micro and macrovascular endothelial cells, human umbilical vein endothelial Cells (HUVEC)]; • Dental (human periodontal ligament cells, fetal rat calvarial osteoblastic cells); • Osteochondral (bone marrow mesenchymal cells); • Peripheral nerve (mice neural stem cells, olfactory ensheathing cells); • Blood vessel (human dermal fibroblasts); • Cardiac tissue (cardiomyocyte); • Skin (human oral/epidermal keratinocytes and fibroblasts);

• Thermally induced phase separation, • Freeze drying, • Microsphere fusing, • Co-precipitation, • In-situ precipitation, • Freezing and lyophilization, • Particle aggregation/ agglomerization, • Photolithography, • Wet spinning, • Microfiber wetspinning, • Robocasting, • Sol-gel method combined with 3D plotting, • Electrospuning, • Chemically crosslinked injectable hydrogels, • Photopolymerization of hydrogels, • Injectable Hydrogels, • Ionotrophic or covalent gelation.

• Trachea (fibroblasts and chondrocytes); • Meniscus (Rat BMSCs); • Liver (porcine pancreatic islets, primary hepatocyte/endothelial cells, HepG2 cells, primary mouse hepatocytes, hepatocytes/NIH3T3 fibroblasts). (2) Fibrin (fibrinogen) • • • • • • • • • • • •

• •

Highly biocompatible; Biodegradable; Low price; Readily available; Transparent; Easy to handle; Free from disease transmission; Hemostatic, chemotactic and mitogenic properties; Tissue sealant; Induce improved cellular interaction, and has good tolerance to cells; Allows production of patient’s own scaffold—can be used as an autologous transplant; Naturally contain sites for cell binding, providing the starting support for cell adhesion, migration, growth, and differentiation; Supports adipogenesis in vivo; Cytocompatible.

• Instable; • Rapid degradation in vivo; • Low mechanical stiffness— difficult to maintain structural integrity; • Hard processing; • Requires chemical cross linking; • Has not been formulated as 3D porous scaffold; • Insoluble in water and low stability in aqueous environment.

• Bone (human osteoblasts, hUCMSCs); • Proteolytic cleavage gelation, • Cardiac (endothelial cells, skeletal myoblasts, cardiac myocytes, bone • Emulsification. marrow mononuclear cells, carotid artery-derived cells, cardiac-derived stem cells, adipose-derived stem cells, human mesenchymal progenitor cells,); • Cardiovascular (human foreskin fibroblasts cell line, human venous myofibroblasts); • Cartilage (human articular chondrocytes, rabbit articular chondrocytes, porcine articular chondrocytes, bovine articular chondrocytes, human adipose-derived adult stem cells, embryonic chondrogenic cells, porcine chondrocytes, infrapatellar fat padderived stem cells); • Vascularization/angiogenesis (human umbilical vein endothelial cells, outgrowth endothelial cells, rat aortic smooth muscle cells); • Osteochondral (human bone marrow derived mesenchymal stem cell, rabbit bone marrow derived mesenchymal cells, rabbit BMSCs); • Spinal cord (chick dorsal root ganglia cell culture, murine embryonic stem cells); • Peripheral nerve [Chick dorsal root ganglia cell culture, mESC-derived NPCs, mESC-derived progenitor Motor neurons (pMN), Schwann cells]; • Nerve (human mesenchymal progenitor cells); (Continued)

TABLE 22.2

(Continued)

Advantages

Drawbacks

Applications (cells)

Manufacturing methods of material or composites

• Cornea (epithelial, stromal and endothelial cells); • Skin (human fibroblasts cell line, NIH 3T3 fibroblast, human keratinocytes (HaCaT) cell lines, septal chondrocytes, tracheal epithelial cells, murine embryonic stem cells, mesenchymal progenitor cells, keratinocytes, urothelium cells); • Uterus (telomerase-immortalized human endometrial stromal cells, Ishikawa adenocarcinoma epithelial cells). (3) Silk, silk fibroin, spidroin • Biocompatible; • Biodegradable; • Excellent mechanical properties (comparable to the best synthetic fibers); • Light weight; • Extremely strong and elastic; • Slow degradation; • High thermal stability; • High mechanical strength; • Transparent; • Reducing aggregation and adhesiveness; • Adjustable degradation; • Easy processing; • Environmentally safe; • Offer excellent environment for cell growth; • Induces slight inflammation reaction in vivo; • Lack of osteogenic activity in vivo; • Free from disease transmissions.

• High cost; • Over hydration; • Reduced availability (e.g., low production of spidroin by spiders); • High brittleness (may need to be combined with another polymer i.e., chitosan); • Residue contaminants.

• Bone (bone marrow-derived mesenchymal stem cells, osteoblasts, adipose-derived stem cells and human osteoblasts, human adipose-derived stem cells, human adipose stem cells (hASCs), MC3T3-E1 cells); • Cartilage (pig chondrocytes, MSCs); • Vascularization/angiogenesis (endothelial cells, endothelial cells and smooth muscle cells); • Skin (keratinocytes and fibroblasts, human oral/epidermal keratinocytes and fibroblasts, dorsal fibroblasts of newborn littermates of C57 BL/6 mice); • Liver (hepatocytes, rat hepatocytes, FLC-4, HepG2 cell lines); • Tendon/ligament (bone marrow-derived mesenchymal stem cells, mesenchymal progenitor cells); • Spinal cord (rat olfactory ensheathing cells); • Trachea (fibroblasts and chondrocytes);

• Freeze drying, • Freezing and lyophilization, • Emulsification/ freeze-drying, • Salt-leaching/freezedrying, • Electrospinning, • Solvent casting, • Injectable Hydrogel, • Simple calcium phosphate coating, • 3D bioprinting.

• Muscle (human smooth muscle cells and myoblasts); • Eardrum (human tympanic membrane keratinocytes); • Cardiac (HL-1 atrial cardiomyocytes, human embryonic stem cell-derived cardiomyocytes); • Dental (dental pulp stem cells); • Sciatic nerve (bone marrow MSCs); • Peripheral nerve (olfactory ensheathing cells, neural stem cell (NSC). (4) Gelatin • Biocompatible and biodegradable in physiological environment; • Low antigenicity; • Easy processing into different shapes; • Cheap derivatives; • Promotes cell adhesion and helps in exudates removal; • Supports adipogenesis in vivo; • Antithrombogenic; • Good cell recognition properties; • Clinically approved; • Provide minimal inflammation; • Controllable biodegradation; • Amenable for increasing degrees of cross-linking and decreasing biodegradability.

• • • • •

Poor mechanical properties; Brittleness; Low stability; Chemical cross-linking needed; Primary use as material for microspheres.

• Bone (osteoblast-like MC3T3-E1 cell line, Rat marrow stromal osteoblasts, bone marrow stromal cells (transfected), rat bone marrow stromal osteoblasts, osteoblast); • Cartilage (bovine chondrocytes, human adipose-derived adult stem cells, fibroblast-like NIH/3T3 cell line, human nasal chondrocytes, adult human mesenchymal stem cells, mesenchymal stem cells, embryonic chondrogenic cells, bovine articular chondrocytes, human articular chondrocytes, rabbit articular chondrocytes, adult rabbit chondrocytes, chondrocytes, human adipose mesenchymal stem cells); • Adipose (human preadipocytes, human intervertebral disk cells, mature adipocytes); • Dental (human periodontal ligament cells); • Osteochondral (rabbit bone marrow derived mesenchymal stem cells); • Blood vessel (human dermal fibroblasts); • Skin (keratinocyte, NIH 3T3 fibroblast, human keratinocytes (HaCaT) cell lines); • Tendon (adipose stem cells);

• • • • • • • • • • •

Melt molding, Thermal gelation, Freeze drying, Layer by layer solvent casting, Electrospinning, Photocrosslinking gelation, Emulsification,3D printing, Freezing and lyophilization, Electrospinning, Chemically crosslinked Injectable hydrogels.

(Continued)

TABLE 22.2

(Continued)

Advantages

Drawbacks

Applications (cells)

Manufacturing methods of material or composites

• Meniscus (New Zealand white rabbits meniscal cells, rat BMSCs); • Nerve (PC12 cells); • Peripheral nerve (HUVECs and Schwann cell line (RT4-D6P2T)); • Liver (rat hepatocytes,). (5) Collagen • Biocompatible; • Good cell recognition and high cell binding; • Low antigenicity; • High mechanical strength; • Abundant, • Biodegradable (easily destroyed by enzymes in the body); • Low stimulation of the immune system’ • Cross-linking ability; • Promotes favorable adipose outcomes; • Well characterized; • Fibrous morphology • Nontoxic • Nonantigenic; • Mimic native bone ECM topography; • Mimic native ECM of skin in structure and topography; • Biologically renewable; • Bioadhesive; • Biofunctional; • Supports neural cell attachment and growth; • Support axonal regeneration; • Collagen fibrils provide physical support to tissues;

• Poor mechanical properties; • High cost (when derived by recombinant technologies); • Difficult to process; • Chances of viral and prion contamination; • Extent and rate of degradation is difficult to control; • All sterilization methods incur some degree of alteration; • Poor mechanical properties; • Low stability; • Fusion of nanofibers in aqueous environment; • Low melting point;

• Bone (alveolar osteoblasts, osteoblasts, mouse BMSCs, bone marrow-derived mscs, adipose-derived stem cells and human osteoblasts, rMSCs, osteogenic cells, MC3T3-E1 preosteoblast cells, umbilical cord stem cells, MC3T3-E1 cells); • Cardiovascular (bone marrow-derived mesenchymal stem cells); • Cartilage (chondrocytes, autologous chondrocytes, bone marrow stromal cells, human articular chondrocytes, rabbit articular chondrocytes, rabbit chondrocytes, primary chondrocytes); • Adipose (human intervertebral disk cells, human preadipocytes); • Dental (porcine third molar cells, human periodontal ligament cells, human dental pulp stem cell); • Urogenital tract (human smooth muscle cells); • Renal glomerular tissue (glomerular mesangial cells, epithelial cells); • Liver (hepatocytes,); • Heart valve (porcine mitral valve interstitial cells an endothelial cells); • Kidney (glomerular mesangial and epithelial cells);

• Electrospinning, • Nanofiber electrospinning, • Phase separation, • Thermally induced phase separation, • Solvent casting/ particulate leaching, • Solvent casting/salt leaching, • Layer by layer solvent casting, • Fiber Bonding, • Gelation by neutralization, • Photopolymerization of hydrogels, • Injectable Hydrogel, • Compression, • Molding, • Freeze drying, • Immersion method, • 3D printing, • 3D bioprinting, • Organ printing (Melt-based rapid prototyping), • Freezing and lyophilization,

• Promote cell adhesion and proliferation better than synthetic polymers; • Provides structural support and controls many cellular functions; • Good substrate for culturing keratinocytes and other cell types.

• Meniscus (meniscus cells, MSCs and • Emulsification, • Simple calcium MCs, adipose tissue-derived phosphate coating. mesenchymal stem cells); • Trachea (autologous tracheal epithelial cells and chondrocytes); • Osteochondral (chondrocytes, BMSCs); • Tendon (tendon cells); • Skin (human oral/epidermal keratinocytes and fibroblasts, adipose derived mesenchymal stem cell, human endometrial stem cells, epidermal keratinocytes and dermal fibroblasts, human foreskin-derived neonatal epidermal keratinocytes); • Peripheral nerve (Schwann cells, hESCderived neural progenitor cells (NPCs), rat cortical neurons); • Uterine (human stromal 1 epithelial primary cultured endometrial cells, rabbit stromal 1 epithelialmprimary cultured mendometrial cells, human telomerase-immortalized stromal cells, human BM-MSCs, Telomeraseimmortalized human endometrial stromal cells and ishikawa adenocarcinoma epithelial cells, human endometrium-like cells derived from hESC, human umbilical cord-derived mesenchymal stem cells); • Pancreas (pancreatic islet).

(6) Hyaluronic acid • • • • • • • • • •

Highly biocompatible; Biodegradable; Excellent viscoelasticity; Excellent water solubility; Natural component of ECM and structurally similar to GAGs; Easily functionalized; Favorable mechanical properties; Easy manipulation; Negatively charged; Good cell recognition;

• Poor mechanical properties; • Expensive; • Expense of preservation and storage in a cryo-freezer; • Difficult processing by electrospinning (due to high viscosity and surface tension).

• Skin (melanocytes, keratinocytes); • Cartilage (bovine articular chondrocytes, periostal explants); • Vascularization/angiogenesis (human umbilical vein endothelial cells); • Adipose (human intervertebral disk cells); • Osteochondral (rabbit bone marrow derived mesenchymal stem cells, human bone marrow derived

• Chemically crosslinked hydrogels, • Photocrosslinked hydrogels, • Injectable hydrogel, • Micromolding, • Photolithography, • Emulsification, • 3D printing, (Continued)

TABLE 22.2

(Continued)

Advantages

Drawbacks

• Easily and controllably produced in a large scale by microbial fermentation; • Healing mediator (regulating tissue injury, accelerating tissue repair); • Supports adipose tissue formation; • Advantageous in bone TE applications; • Promote skin cell migration and proliferation.

Applications (cells)

Manufacturing methods of material or composites

mesenchymal stem cells); • Bone (hASCs); • Peripheral nerve (neural stem cell (NSC), NSC with rPDGF, ESC- derived V2a interneuron); • Liver (primary hepatocyte/endothelial cells).

(7) Alginate • • • • • • • • • •

Biocompatible; Biodegradable; Simple gelation method; Resistance to acidic conditions; Good mucoadhesiveness; High degree of swelling; Negatively charged; Crossilinkable and injectable; Easy functionalization; Adjustable properties based on the two monomer content; • Promote angiogenesis; • Increase axonal elongation; • Hemostatic properties;

• Poor mechanical properties; • Uncontrolled degradation kinetics; • Difficult sterilization and handling; • Limited ability to support cell attachment, growth and differentiation.

• Bone (bone marrow stromal cells, human bone marrow cells, |human articular chondrocytes, osteoblasts, MC3T3-E1 cells/BMP-2); • Cartilage (human bone marrow cells, human articular chondrocytes, auricular chondrocytes, rabbit articular chondrocytes, rabbit mesenchymal stem cells, porcine articular chondrocytes, bovine articular chondrocytes, periostal explants, bovine and human chondrocytes, chondrocytes, primary chondrocytes, hBMSC); • Vascularization/Angiogenesis (human dermal microvascular endothelial cells, human umbilical vein SMCs); • Liver (rat hepatocytes, adipose tissuederived stem cells, HepG2 cells, human hepatocytes from metabolic-disordered children, primary mouse hepatocytes, hepatocytes/NIH3T3 fibroblasts); • Pancreas (murine insulinoma βTC3 cells, Langerhans, porcine pancreatic islets, Pancreatic islets); • Osteochondral (chondrocytes); • Skin (human oral/epidermal keratinocytes and fibroblasts); • Peripheral nerve (Schwann cells);

• Ionic crosslinking gelation, • Chemically crosslinked injectable hydrogels, • Injectable hydrogels, • In situ coprecipitation, • Freeze drying, • Micromolding, • Microfluidics, • Inkjet printing, • Freezing and lyophilization, • 3D bioprinting, • Co-axial printing.

(8) Pullulan • • • •

Good water solubility; Biocompatibility; Nontoxicity; Easy derivatization.

• Instable; • High cost.

• Cartilage (Bone marrow stem cells).

• Thermal gelation.

(9) Polylysine • Good thermal stability; • Safe; • Improved cell attachment.

• High internal colloid osmotic pressure.

(10) Pectin • Exhibit stable viscosity; • Itself gelling property without use of harmful solvents; • Superior mechanical properties.

• Readily degradable in aqueous solution; • Acid sensitive.

(11) Elastin • • • • • • • •

Biocompatible; Easily functionalized; Easily controllable degradation; Confers elasticity; Precise molecular weight; Low polydispersity; Resistance to fatigue; Potent autocrine regulator of vascular smooth muscle cells activity.

• Poor mechanical properties; • Expensive; • Becomes insoluble and aggregate at a critical temperature.

• Nanofiber electrospinning.

(12) Dextran • • • •

Biodegradable; • High cost; Biocompatible; • Over hydration; Reduces aggregation and adhesiveness; • Risk of anaphylaxis. Readily available in a wide range of molecular weights along with several derivatives; • Enable cell adhesion and promote vascular phenotypes;

• Bone (rat calvarial cells, bone marrow stromal cells (transfected)); • Dental (human periodontal ligament cells) • Guided cell and axonal regeneration (primary embryonic chick dorsal root ganglia cells).

• 3D printing.

(Continued)

TABLE 22.2

(Continued)

Advantages

Drawbacks

Applications (cells)

Manufacturing methods of material or composites

• Soluble in both water and organic solvents; • Stable under mild acidic and basic conditions; • Contains a large number of hydroxyl groups available for modification/ conjugation; • with other molecules; • Promote neovascularization and skin regeneration; (13) Dextran sulfate • Increased durability; • Reversible nature; • Stable.

• High cost; • Chances of contamination.

• Gelation by physical/chemical crosslinking, or radical polymerization.

(14) Chondroitin sulfate (GAGs) • Nonimmunogenic; • Readily water-soluble nature; • Degrades to nontoxic oligosaccharides; • Instable [usually crosslinked • Shock absorber, provides elasticity and with other polymers, such as tensile strength to the scaffold; chitosan, gelatin, collagen, hyaluronan, poly(vinyl • Involved in cell adhesion, migration, alcohol)]. proliferation, and differentiation, binding and modulation of growth factors and cytokines.

• Bone (rat calvarial cells); • Cartilage (rabbit articular chondrocytes, articular chondrocytes); • Vascularization/angiogenesis (human saphenous vein endothelial cells, porcine vascular endothelial cells, NIH 3T3 fibroblasts); • Heart valve (porcine mitral valve interstitial cells and endothelial cells)

(15) Carrageenan • • • •

Thixotropic nature; Highly flexible molecules; No surfactant needed; Form a thermoreversible gel at room temperature;

• • • •

High cost; Generation of toxic waste; High melting temperature; Gel dissolution in the absence of a gel-inducing reagent.

• Gelation by physical/chemical crosslinking, or radical polymerization.

• • • •

High degree of protein reactivity; High molecular flexibility; Thixotropic nature Possess an inherent antimicrobial and antioxidant activity.

(16) Carboxymethyl chitin • Easy processing; • Good efficacy.

• Stability problem; • Multistep procedure.

(17) Agarose • • • • • • •

Biocompatible; Biodegradable; Low antigenicity; Easy to use; High water uptake capability; Forms thermoreversible gels; Forms a stable and inert gel at room temperature; • Support chondrocyte proliferation.

• • • •

Hard to process; Difficult to obtain; Slow biodegradation; Low cell attachment growth and differentiation, results in low cell viability and growth adhesiveness; • Possibility of cellular protrusion through the agarose membrane.

• Bone (human marrow stromal cell); • Pancreas (pancreatic islets and insulinoma cells); • Cornea (epithelial, stromal and endothelial cells); • Cartilage (primary chondrocytes); • Spinal cord (rat cortical neurons); • Uterus (telomerase-immortalized human endometrial stromal cells and Ishikawa adenocarcinoma epithelial cells).

• Thermal gelation, • 3D bioprinting/

(18) Glycosaminoglycans (GAGs) • Responsible for the coordination of manifold fundamental processes for tissue development and homeostasis, such as biophysical characteristics, cell signaling, and assembly of the extracellular matrix.

• Thermally induced phase separation.

(19) Chitin • High strength; • Biodegradability; • Nontoxicity.

• Peripheral nerve regeneration (mice neural stem cells)/

(20) Starch • Inherent biodegradability into oligosaccharides;

• Extremely difficult to process; • Brittle;

• Bone (osteoblasts, bone marrow stromal • 3D printing, cells); (Continued)

TABLE 22.2

(Continued)

Advantages

Drawbacks

Applications (cells)

Manufacturing methods of material or composites

• Overwhelming abundance; • Renewability; • Easy modification, conversion into thermoplastic or blending with other polymers.

• Structure of semicrystalline • Vascularization/Angiogenesis [micro native starch granules is either (HPMEC-ST1.6 R) and macrovascular destroyed, reorganized, or both. (HUVEC) endothelial cells)].

• Melt-based prototyping, • Rapid prototyping.

• Low acyl (LA) gellan gum • Cartilage (Bone marrow stem cells). produces firm, nonelastic brittle gels.

• Thermal or ionotropic gelation.

(21) Gellan gum • Resistant to heat and acid; • High acyl form produces transparent, soft, elastic, and flexible gels at polymer concentrations higher than 0.2%. (22) Cellulose • • • • • • • •

Most abundant polymer Low cost; Easily converted into derivatives; Stabilizes the structure; Porous; Biocompatible; Exceptional strength; Water insoluble.

• Poor degradation in vivo; • Long regeneration time.

• Bone (osteoblast-like MC3T3-E1 cell line, • Gelation by physical osteogenic cells); or chemical crosslinking, and by • Pancreas (Langerhans); radical • Vascularization (endothelial cells, polymerization, smooth cells, fibroblasts). • 3D bioprinting,

(23) Galactose • Improved cell attachment, viability, and metabolic functions.

• Low stability.

(24) Heparin (GAGs) • Preserve growth factor stability and bioactivity.

• Reduced cell growth rate/

(25) Peptidesd • Biocompatible • Biodegradable

• Poor mechanical properties

• Vascularization/angiogenesis (human umbilical vein endothelial cells); • Liver (rat hepatocytes,).

(26) Keratin (GAG) • Biocompatible; • Biodegradable.

• Poor mechanical properties;

• Skin (wound dressing) [NIH 3T3 fibroblast, Human keratinocytes (HaCaT) cell lines].

• Self-assembled process.

• Peripheral nerve (olfactory ensheathing cells, embryonic stem cells, Schwann cells, hESC- derived neural progenitor cells (NPCs), induced pluripotent stem cells, adipose-derived stem cells, mesenchymal stem cells).

• Photolithography

(27) Spongin • Biocompatible; • Inexpensive; • Low risk of transmission of infectioncausing agents; • Widely available; • Well-established farming techniques; • Appropriate porosity and surface chemistry; • Stable in vitro;

• Need for determining aquaculture systems or farming • (when ex situ cultivation is difficult) • Species-dependent variability of characteristics and composition.

(28) Hemoglobin and myoglobin • Biocompatible; • Excellent oxygen permeation; • Alleviate wound hypoxia.

• Less stable; • Difficult to produce nanofibers.

(29) Fibronectin • Plays an important role in peripheral nerve regeneration; • Supports cell migration and • growth and remodeling and resynthesis of connective tissue matrix.

(30) Laminin • Plays an important role in peripheral nerve regeneration. (31) Arabinogalactan • Highly branched polysaccharide with high water solubility.

• Peripheral nerve (Schwann cells).

524

22. Biological macromolecules in cell encapsulation

et al., 2011; Filippi et al., 2020; Garg et al., 2011; Garg et al., 2015; Mano et al., 2007; Mozafari et al., 2019; Nosrati et al., 2018; O’Brien, 2011; Pina et al., 2019; Sethuraman et al., 2016; Singh et al., 2016). Main forms and manufacturing methods for different biopolymers used in TE are

given in Table 22.3 (Akter, 2016; Asti & Gioglio, 2014; Dhandayuthapani et al., 2011; Filippi et al., 2020; Garg et al., 2011; Garg et al., 2015; Mano et al., 2007; Mozafari et al., 2019; Nosrati et al., 2018; O’Brien, 2011; Pina et al., 2019; Sethuraman et al., 2016; Singh et al., 2016).

TABLE 22.3 Main forms and manufacturing methods for biopolymers used it TE. Scaffold material

Forms

Manufacturing methods of material or composites

(1) Chitosan

• • • • • • • • • •

Hydrogels, Injectable hydrogels, Sponges, Microspheres, Scaffolds, Nanofibers, Membranes, Fiber mesh, Tubes, Particles.

• • • • • • • • • • • • • • • • •

(2) Fibrin (fibrinogen)

• • • •

Hydrogels, Beads, Scaffolds, Glue.

• Proteolytic cleavage gelation, • Emulsification.

(3) Silk, silk fibroin, spidroin

• • • •

Scaffolds, Hydrogels, Fibers, Nanofibers.

• • • • • • • • •

Freeze drying, Freezing and lyophilization, Emulsification/freeze-drying, Salt-leaching/freeze-drying, Electrospinning, Solvent casting, Injectable Hydrogel, Simple calcium phosphate coating, 3D bioprinting.

(4) Gelatin

• • • • • • •

Scaffolds, Microspheres, Hydrogels, Sponges, Nanofibers, Disks, Beads.

• • • • • • • • • • •

Melt molding, Thermal gelation, Freeze drying, Layer by layer solvent casting, Electrospinning, Photocrosslinking gelation, Emulsification, 3D printing, Freezing and lyophilization, Electrospinning, Chemically crosslinked Injectable hydrogels.

Thermally induced phase separation, Freeze drying, Microsphere fusing, Coprecipitation, In-situ precipitation, Freezing and lyophilization, Particle aggregation/agglomerization, Photolithography, Wet spinning, Microfiber wet-spinning, Robocasting, Sol-gel method combined with 3D plotting, Electrospuning, Chemically crosslinked injectable hydrogels, Photopolymerization of hydrogels, Injectable hydrogels, Ionotrophic or covalent gelation.

(Continued) III. Functional Applications

22.3 Advantages, drawbacks, applications, forms and manufacturing methods

525

TABLE 22.3 (Continued) Scaffold material

Forms

Manufacturing methods of material or composites

(5) Collagen

• • • • • • •

Hydrogels, Sponges Microspheres, Scaffolds, Nanofibers, Membranes, Films.

• • • • • • • • • • • • • • • • • • • • •

Electrospinning, Nanofiber electrospinning, Phase separation, Thermally induced phase separation, Solvent casting/particulate leaching, Solvent casting/salt leaching, Layer by layer solvent casting, Fiber bonding, Gelation by neutralization, Photopolymerization of hydrogels, Injectable hydrogel, Compression, Molding, Freeze drying, Immersion method, 3D printing, 3D bioprinting, Organ printing (melt-based rapid prototyping), Freezing and lyophilization, Emulsification, Simple calcium phosphate coating.

(6) Hyaluronic acid

• • • • •

Hydrogels, Membranes, Scaffolds, Sponges, Flat sheets.

• • • • • • •

Chemically crosslinked hydrogels, Photocrosslinked hydrogels, Injectable hydrogel, Micromolding, Photolithography, Emulsification, 3D printing.

(7) Alginate

• • • • • •

Hydrogels, Microparticles, Membranes, Matrix, Injectable vehicles, Scaffolds.

• • • • • • • • • • •

Ionic crosslinking gelation, Chemically crosslinked injectable hydrogels, Injectable hydrogels, In situ coprecipitation, Freeze drying, Micromolding, Microfluidics, Inkjet printing, Freezing and lyophilization, 3D bioprinting, Coaxial printing.

(8) Pullulan

• • • •

Double network hydrogels, Matrix, Fibers, Films.

• Thermal gelation.

• Nanofiber electrospinning.

(9) Elastin (10) Dextran

• Hydrogels.

• 3D printing. (Continued)

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22. Biological macromolecules in cell encapsulation

TABLE 22.3 (Continued) Scaffold material

Forms

Manufacturing methods of material or composites

• Gelation by physical/chemical crosslinking, or radical polymerization.

(11) Dextran sulfate

(12) Chondroitin sulfate (GAGs)

• Hydrogels, • Microspheres.

(13) Carrageenan

• Polyelectrolyte for cell encapsulation.

• Gelation by physical/chemical crosslinking, or radical polymerization.

(14) Agarose

• • • • •

• Thermal gelation, • 3D bioprinting.

Gels, Sponges, Hydrogels, Matrix, Microcapsule.

• Thermally induced phase separation.

(15) Glycosaminoglycans (GAGs) (16) Starch

• Scaffolds.

• 3D printing, • Melt-based prototyping, • Rapid prototyping.

(17) Gellan gum

• Double network hydrogels, • Scaffolds.

• Thermal or ionotropic gelation.

(18) Cellulose

• Hydrogels, • Scaffolds, • Membranes.

• Gelation by physical or chemical crosslinking, and by radical polymerization, • 3D bioprinting.

(19) Heparin (GAGs)

• Hydrogels.

(20) Keratin (GAG)

• Sponges.

(21) Heparan sulfate

• Hydrogel.

• Self-assembled process.

(22) Fibronectin

• Photolithography.

(23) Polyesters

• Melt-blown nonwoven fiber.

(24) Laminin

• Hydrogel.

22.4 Conclusions Biopolymers are nowadays among the most investigated materials in many different fields because of their preferable characteristics. Their exclusive biochemical and biophysical features like biocompatibility, biodegradability, gel forming ability, nontoxicity, nonimmunogenicity, and fluid adsorption capacity, along with antibacterial,

antifungal, and/or antitumor properties have made them promising candidates for TE applications. Many different biopolymers like alginate, chitosan, collagen, elastin, gelatin, HA, keratin, etc., are already most effectively used for different hard and soft TE scaffolds in bone tissue regeneration and repair, cartilage repair, wound healing, etc. This chapter provides an attempt to highlight the main properties, applications, and

III. Functional Applications

References

fabrication technologies of most widely used biopolymers in order to promote and improve the further development of scaffold design and development.

Acknowledgment This work was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (Contract No. 451
03
9/2021
14/200135).

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C H A P T E R

23 Biological macromolecules for enzyme immobilization Hamza Rafeeq1, Sarmad Ahmad Qamar1, Hira Munir2, Muhammad Bilal3 and Hafiz M.N. Iqbal4 1

Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan 2Department of Biochemistry and Biotechnology, University of Gujrat, Gujrat, Pakistan 3School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai’an, China 4Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico

23.1 Introduction Enzymes, being the natural catalysts, have many applications in synthetic chemistry and these biocatalysts can be utilized in different novel applications. Bio-catalysis involves the exploitation of microbial species and enzymes and defines some new limitations of their abilities (Bilal et al., 2018; Qamar, Asgher, & Bilal, 2020). Almost a hundred years ago, the use of biocatalysts in the development of many valuable and important biochemicals has been documented such as the use of plant extract to make (R)-mandelonitrile, glucose isomerase to convert glucose into fructose, and to produce antibiotics from penicillin G acylase (Bilal & Iqbal, 2019). Depending on the technologies and applications, biocatalysis was divided into three categories or three waves that came with the new inventions in scientific world. First wave involves the

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00023-3

immobilization of enzymes to enhance the restricted stability of enzymes. In the second wave between 1980 and 1990, diverse substrate ranges were achieved by protein engineering approaches like structure-guided technologies, which allowed the industrial production of many synthetic products like for pharmaceuticals. At the end, in the current era third wave of biocatalysis was started by a method of direct evolution invented by Frances Arnold and PimStemmer. This method has shown the ability to quickly modify enzymes and biocatalysts on higher extent in relatively short time period. Additionally, random changes of amino acids became possible in third wave of biocatalysis, which provided enhanced stability to biocatalysts and allowed them to specifically select the substrate (Bilal et al., 2018). At the starting of 1916, scientific world has been introduced with a new idea, in which the

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23. Biological macromolecules for enzyme immobilization

enzyme present in a solvent with free mobility will be transformed into the movement restricted enzymes having active size movements only. As the enzyme portion other than active site maintain the orientation and shape of the active site directly involved in the catalysis. This maintenance helps in accurate fitting of enzyme with the substrate for catalysis (Powers et al., 2006). The first enzyme that has been immobilized based on the idea of movement restriction was invertase. At the end of 1916, invertase was immobilized on aluminum hydroxide and charcoal-based matrices and resulted in the same catalytic activity as before immobilization (Nelson & Griffin, 1916). This study became the foundation of immobilization techniques currently being used. After the testing in laboratories different immobilizing techniques were used by industries to design the immobilized catalysts for commercial scale production. For example, proteases, invertases, penicillin G acylase and lipases were being used in many industrial scale production processes (Cherry & Fidantsef, 2003). Since the first enzyme was immobilized, enzyme immobilization became a

hot research topic as many patents and approximately 10,000 papers have been published based on enzyme immobilization techniques, matrices used for immobilization, and their applications. The industrial value of enzymes as catalysts has drastically boosted up by the invention of immobilization technique as it provides useful properties to enzymes like, longer storage time, broad catalytic activity in different chemical and physical conditions, higher stability, easier product recovery, and high reusability potential (Fig. 23.1). Any inert, inorganic, organic and insoluble material can be used to immobilize enzyme. After 100 years of study and research, still enzyme immobilization is considered as trial and error-based technique. As the only hurdle in this technique is to build a suitable and effective method to be used at the industrial scale (Sheldon, 2007; Torres-Salas et al., 2011). Applications of immobilized enzymes has drastically spread out in almost every field of life. Recently nanotechnology enhances the revolution in enzyme immobilization. Still, enzyme immobilization is a research topic that attracts FIGURE 23.1 Main characteristics of enzyme immobilization platforms.

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531

23.1 Introduction

biophysicist, biochemist molecular biologist, and now even for immunologist and cell biologist for their research work (Dwevedi, 2016). Heterogeneous biocatalysts present high reusability potential, and own vast field of applications, however they provide a solid foundation for the development of nanosized biodevices which are being utilized in biomedical, analytical, energetic and analytical fields. These nanosized devices also have applications in solid-phase protein chemistry (Mugo & Zhang, 2019). Three types of immobilization protocols have been reported in the literature: (1) prefabrication of binding support for

enzyme, (2) enzyme entrapment or encapsulation, and (3) crosslinking with bifunctional reagents without any carrier (Sheldon, 2007). Another study has reported three strategies, that is, (1) ionic interaction (2) interaction with covalent bonding, and (3) immobilization based on enzyme adsorption (Cardoso, Miranda, de Paula, de Paula Carmo, & Eller, 2020). Empirical-bases can be taken to draw a rough sketch of immobilization protocol, in addition to the observation about the range of enzymes with which the immobilizing material can be used efficiently (Basso et al., 2007; Vasconcelos et al., 2020). Table 23.1 illustrates

TABLE 23.1 Studies reporting immobilization of different enzymes using various biopolymers for potential applications in various sectors. Enzyme

Support material

Immobilization type

Purpose of immobilization

Reference

Pectinase

Na-alginate/ graphene oxide beads

Covalent immobilization

Food sector

Dai et al. (2018)

Pectinase

Ca-alginate beads

Entrapment

Fruit juices

de Oliveira, Dias, da Silva, and Porto (2018)

Protease

Ca-alginate beads

Entrapment

Leather and textile sector

Qamar, Asgher et al. (2020)

Laccase

Ca-alginate

Encapsulation

Biodegradation of sediment-bound hydrocarbons

Kucharzyk, Benotti, Darlington, and Lalgudi (2018)

Peroxidase

Guar gum- alginateagarose

Entrapment

Textile industry effluents

Ali and Husain (2018)

Tannase

Ca-alginate

Entrapment

Food sector

de Lima et al. (2018)

Lysozyme

Alginate-graphene oxide

Adsorption

Food and pharmaceutical sector Li, Ma, Chen, Huang, and He (2018)

Glucose oxidase

Ca-alginate beads

Encapsulation

Production of reduced alcohol wines

Ruiz et al. (2018)

Laccase

Cu-alginate

Entrapment

Bioremediation of textile effluents

Sondhi, Kaur, Kaur, and Kaur (2018)

Inulinase

Ca-alginate-gelatin

Entrapment

Fructose synthesis for inulin bioconversion

Hang, Wang, Cheng, Li, and Song (2018)

Peroxidase

Ca-alginate

Entrapment

Oxidation of environment matters

Torres, Villanueva, La´zaro-Martı´nez, Copello, and Dall’Orto (2018)

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532

23. Biological macromolecules for enzyme immobilization

studies reporting immobilization of different enzymes using various biopolymers for potential applications in various sectors.

23.2 Biological macromolecules for enzyme immobilization 23.2.1 Chitin and chitosan Chitin and chitosan are naturally derived polymers and have wide range of applications in field of enzyme immobilization (Kapoor & Kuhad, 2007). It is the second most abundant biopolymer which also presents broad range of applications in enzyme immobilization field because of the beneficial characteristics desired for the immobilization like biocompatibility, biodegradability, hydrophilicity, low-cost, ease in availability, metal ions chelation, adsorption, and film formation can be resulted from exoskeleton ¨ zogul, & Regenstein, of crustaceans (Hamed, O 2016; Rinaudo, 2006). Enzymes can directly bind to chitosan without any linker due to the presence of amine and hydroxyl groups present in the molecular structure of chitosan. Chitosan molecule has ß-(1,4)-linked 2-amino-2-deoxy-Dglucose (D-glucosamine) and 2-acetamido-2deoxy-D-glucose (N-acetyl-D-glucosamine) units combined with each other and has a linear formation (Qamar, Ashiq, Jahangeer, Riasat, & Bilal, 2020). Chitosan has varied ranges of molecular weight from 10 to 1000 kDa and degree of deacetylation from 70%
95%. Ration of N-acetyl-Dglucosamine and D-glucosamine determines the biocompatibility, biodegradability, physiochemical properties and catalytic activity (Younes & Rinaudo, 2015). Chitin and chitosan are very important in the field of catalytic immobilization because they possess some specific and beneficial characteristics as mentioned above. Because of that chitin and chitosan are the hot research topics since last few years for the researchers doing work on enzyme immobilization (Jahangeer,

Qamar, Mahmood, & Asgher, 2019). Many discoveries of industrial application of chitin and chitosan have been made since past few years and these biological macromolecules resulted in many bioproducts production that is, paper and pulp industry, agriculture, wastewater treatment, cosmetics, toiletries and in food industry (Hamed et al., 2016; Krajewska, 2004). However, chitosan has the ability to attain different forms such as microcapsules, microspheres/ beads, fibers, sponges, membranes, gels, and coatings. This structural diversity enhances the usefulness of chitosan as immobilizer or carrier support for wide range of biomolecules and industrial enzymes to enhance their stability (Bilal & Iqbal, 2019). Singh, Singh, and Kennedy (2017) reported the immobilization of inulinase, an extracellular enzyme extracted from Kluyveromyces marxianus and purified by ethanol precipitation and gel filtration chromatographic techniques. Immobilization of inulinase was studied on chitosan beads to be used in batch system for inulin hydrolysis. The results of study have shown that the immobilization of enzyme resulted in hydrolysis 87% inulin just within 4 h of reaction time. Immobilized inulinase has the ability to be reused up to 14 cycles and after 5th cycle inulinase exhibit 78% of its initial activity. Furthermore, a study on eugenyl benzoate to attain good yields in minimum time was conducted (Abd Manan, Attan, Zakaria, Mahat, & Abdul Wahab, 2018). Researchers reported hybrid composites-based on biodegradable, and ecofriendlier biopolymer in which chitin and chitosan nanowhiskers plays the role of supporter and carrier, then lipase enzyme from Rhizomucor miehei was crosslinked on these nanowhiskers. Results indicated that the enzyme immobilized on chitin and chitosan hybrid composite has maximum of 56.3% catalytic activity, while lipase in free form has only 47.3% maximum activity under optimized conditions. Additionally, alginates-chitosan blends have been used to

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23.2 Biological macromolecules for enzyme immobilization

serve biocatalytic immobilization purpose. Enzyme immobilization with chitosan resulted in the ionic and physical interactions between them which drastically reduces the leaching effect of enzyme (Datta, Christena, & Rajaram, 2013). Chitosan-clay-based biocomposite have been shown to have efficient enzyme entrapment efficacy due to the presence of amino and hydroxyl groups in their structure. These groups make enzyme binding easy and provide high permeability and hydrophilicity to the enzyme. Chitosan beads can immobilize double the concentration of enzyme (Chang & Juang, 2007; Salehi, Daraei, & Shamsabadi, 2016). After heating the composites at 60 C for 3 h, the noncrosslinked and crosslinked immobilized CTs retained 51.6% and 70.7% of the initial activity, respectively, while the free CTs retained only 29.6% of the initial activity. In addition, noncross-linked and cross-linked immobilized CTs retained 85.7% and 84.9% of the initial activity, respectively, whereas the free CTs retained only 18.8% of the initial activity after 20 days. When the MCNCs were used to immobilize the CT molecules, the enzyme loading capacity was enhanced up to 6.threefold upon cross-linking. Moreover, the immobilized CTs could be easily separated and recycled from the reaction system by a magnetic force. Huang, Wang, Xue, and Mao (2018) immobilized chymotrypsin enzyme on magnetic chitin nanofiber composites (MCNCs) and observed that cross linkage of enzyme with magnetic nanoparticle enhances the stability of enzyme and its activity was also improved. After keeping the cross-linked composite in 60 C for 3 h, it shows 70.7% initial activity as compared to noncrosslinked composite which shows 51.6% activity and free enzyme only retains 29.6% initial activity. The immobilized enzyme exhibits enhanced storage and thermal stability. It possesses loading capacity up to 6.threefold upon crosslinking and can be reused by separating

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it from the reaction system. The steps taken for the immobilization have been represented in Fig. 23.2.

23.2.2 Agarose Agarose is a natural biomacromolecule, has linear structure and is a hetero polysaccharide obtained from red seaweed. Structure of cellulose contains ß-D-galactose and 3,6-anhydro-α-Lgalactose units, combined by ß-1,4 glycosidic and α-1,3 glycosidic linkages (Zucca, FernandezLafuente, & Sanjust, 2016). Agarose has inert nature and being a carrier, it also possesses high level of biocompatibility as well as specific mechanical properties that increases its importance (Nawaz et al., 2016). Agarose has porous structure, void-like spaces in its structure, which provide a favorable and free-space for enzyme and substrate interaction and after catalytic activity of enzyme product can easily exit out of the construct (Fatin-Rouge, Starchev, & Buffle, 2004). By reducing the temperature below 35 C, agarose can attain stable and rigid structure with highly ordered arrangements because of the gelation property of agarose (Delattre, Andrea, & Michaud, 2011). Agarose also possess different forms like microcapsules, beads and fibers. Ability to exhibit different forms increase the use of agarose on industrial scale. Agarose has the ability of multipoint covalent immobilization which surely increases the stability of the enzyme immobilization. Multiple bonds strengthen the bonding between enzyme and support carrier makes enzyme strong and reduces the chance of enzyme deactivation due to the conformational changes of constructs. However, multiple covalent bonding increases the functional group density, surface structure of enzyme and support becomes indistinguishable and recycling becomes impractical, which are some disadvantages of multiple covalent bonding (Zucca et al., 2016). To address these advantages,

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FIGURE 23.2 Chymotrypsin immobilized onto chitin-nanofibers with magnetic nanoparticles, increases the crosslinking to give favored characteristics to the enzyme.

agarose surface is functionalized by epoxy or amino groups (Urrutia, Mateo, Guisan, Wilson, & Illanes, 2013). For example, Guerrero, Vera, and Illanes (2017) reported immobilization of α-galactosidase purified from Aspergillus oryzae on glyoxal-agarose, amino-glyoxal-agarose and chelate-glyoxal-agarose. These constructs were utilized for the production of lactulose in continuous batch process. Results reported enzyme immobilized on these constructs gave highest lactulose yields as compared to the free enzyme. In other studies by Bilal and coworkers (Bilal et al., 2017; Bilal, Adeel, Rasheed, Zhao, & Iqbal, 2019) immobilized manganese peroxidase (MnP) on fabricated agarose beads, and used these construct for treatment of dyes containing textile waste effluent. Immobilized MnP exhibit high thermal stability with enhanced storage time, as well as its tolerance towards pH and temperature deviations

increases as compared to the free/soluble enzyme. After ten repetition immobilized MnP was still able to recycle, after 3rd and 5th cycle enzyme retains 81.3% and 58.1% catalytic activity, respectively.

23.2.3 Alginate Alginate is alginic acid of different mineral salts like sodium, calcium and magnesium which is extracted from cell wall of brown algae. Alginate has been widely used for the purpose of immobilization in the form of alginate-polyacrylamide gels, xanthan-alginate beads and calcium alginate beads which increase the reusability of enzyme with increased enzymatic activity. Additionally, by combining alginate with glutaraldehyde and divalent ions by cross-linking significantly

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improves the enzyme stability (Bhushan, Pal, & Jain, 2015; Datta et al., 2013; Qamar, Asgher, et al., 2020). In calcium alginates, divalent Ca21 ions give some beneficial properties to the constructs like reduced production cost, nontoxicity and biocompatibility with wide range of enzymes in the presence of mild environment. All these properties are crucial in developing alginate-based supports materials for immobilization. Molecular weight of these constructs ranges between 32,000 g/mol to 4,00,000 g/mol and are available in markets for research purposes. These ranges allow the use of alginatebased immobilizers to give specific properties to the constructs, like physiochemical properties can be modified by choosing specific molecular weight. For example, high molecular weight can increase the viscosity of the construct. However, these high viscosity solutions are not considered favorable during the encapsulation process of proteins and cells in alginate-based immobilizers (Freeman & Kelly, 2017; Kong, Smith, & Mooney, 2003). Another advantage of wide range of molecular weight is that it allows the regulation of pre and post gelling limitations by precisely distribute the biopolymer in the working environment (Hazur et al., 2020). Alginates beads, hydrogels and capsules are commonly used forms of alginate-based immobilizer supports for enzyme entrapment and encapsulation (Qamar, Asgher, et al., 2020). Calcium and sodium ions are commonly used with alginates. For example, de Oliveira et al. (2018) reported the encapsulation of pectinase from Aspergillus aculeatus in microcapsules of alginate and applied it to clear apple and umbu juice on industrial scale in a continuous process. They optimized the concentration of calcium chloride and sodium alginate while doing immobilization by using central composite rotational design (CCRD) for optimization purpose. This optimization resulted in more than 80% of its original catalytic activity even after reusing it in the third cycle. In another

535

study Ca-alginate microspheres activated by glutaraldehyde increases the stability of xylanase enzyme purified from Bacillus licheniformis Alk1. It was also observed that the efficiency of enzyme after recycling increases, as well as its storage stability. They reported that the enzyme retained 80% and 37% of its starting activity after 30 days at 4 C, after 5 consecutive reaction cycles (Kumar, Haq, Prakash, & Raj, 2017). Alginate-based immobilization of enzyme has several benefits, but it also has a few drawbacks like leaching of enzyme during application and low mechanical strength of alginate, force the users to use this polysaccharide in association with other biopolymers such as pectin, starch, gelatin and chitosan, which are compatible with alginate and enzyme immobilization (Bilal & Iqbal, 2019). Recently, entrapment of an in-house extracted horseradish peroxidase from Armoracia rusticana was reported. Researcher used a blended hydrogel microsphere made up of fabricated polyvinyl alcohol-alginate and to entrap enzyme in this microsphere, sodium nitrate is to cross-link the enzyme with the hydrogel. The entrapped enzyme showed 60% more activity after ten repetition cycles and exhibit more storage and thermal stability then the enzyme in free state (Bilal, Rasheed et al., 2017).

23.2.4 Cellulose and its derivatives Cellulose is the most abundant biomacromolecule which is most common and is widely used in enzyme immobilization industry and is compatible with huge variety of enzyme for example tyrosinase (Labus, Turek, Liesiene, & Bryjak, 2011), lipase (Huang et al., 2011), and many other enzymes (Klein et al., 2011). Two natural sources for cellulose are plant and bacteria. From plant pure plant cellulose can be attained but commonly cellulose is attained in the form of lignocellulose. However, bacterial cellulose can be extracted in pure form without any hemicellulose or any other biopolymers

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(Asgher, Nasir, Khalid, & Qamar, 2020; Heinze, 2015). Bacterial cellulose possesses some useful properties which makes it more valuable than plant cellulose, as bacterial cellulose is highly polymerizable and are more crystalline than plant cellulose. Bacterial cellulose can be extracted with high purity while plant cellulose has other plant materials like pectin and lignin associated with it. Bacterial cellulose is ultra-fine and is nanosized as well as possess high capacity to hold water and has high tensile strength as compared to plant cellulose (KhosraviDarani, Koller, Akramzadeh, & Mortazavian, 2016). The properties of cellulose that makes it highly demanded in for immobilization purposes is high degree of sorption, diversity based on chemical modification and high extent of hydroxyl groups improves the effective binding with enzyme and also allows surface modification of cellulose by glutaraldehyde, diethylaminoethanol or introduction of epoxy groups as described by Feng et al. (2014). Cellulose and its derivatives have recently gained huge interest in the field of enzyme immobilization (Nikolic et al., 2017; Talingtaisong, Vongsetskul, Panatdasirisuk, & Tangboriboonrat, 2017). Cellulose also have huge number of derivatives like cellulose supports associated with Diethyl aminoethyl (DEAE) provide huge capability of storage for long time durations (Al´ Adhami, Bryjak, Greb-Markiewicz, & PeczynskaCzoch, 2002; Heinze, El Seoud, & Koschella, 2018). Another derivative of cellulose is used for starch degradation in which cellulose is coated on magnetite nanoparticles. Then, α-amylase enzyme was attached to cellulose dialdehyde resulted in starch degradation system. And for better flexibility and formability, enzyme is immobilized on ionic liquid-cellulose film which is activated by addition of glutaraldehyde (Akhond, Pashangeh, Karbalaei-Heidari, & Absalan, 2016; Namdeo & Bajpai, 2009). Being cost-effective, durable, and stable support materials for enzyme immobilization, cellulose and its derivatives are being used in construction of many cellulose-based constructs

with desired catalytic properties having high stability and makes this biological macromolecule highly demanded for attachments of biocatalysts for biomedical applications as well. Recently, Lv, Ma, Anderson, and Chang (2018) reported the use cellulose spheres crosslinked with citric acid for the immobilization of urease enzyme. Cellulose sphere with citric acid gets oxidized by sodium periodate to introduce dialdehyde groups which enhances the efficiency of enzyme immobilization. Immobilized enzymes observed to possess high thermal stability and capacity of urea adsorption also increases. Bacterial nanocellulose has ultra-fine network structure which makes it suitable to be used in wound dressing, it provides the wound area with biocompatible and moist environment, thus also protects the open wound from microbial growth and infection (Sampaio et al., 2016).

23.2.5 Gelatin for enzyme immobilization Gelatin have high amino acid concentration and is being used as hydrocolloid to promote wound healing. Long shelf-life is one of the prominent properties that increases its demand in enzyme immobilization field. Gelatin can be used in polyacrylamide system in combination with other carrier systems like potassium chromium, chromium-acetate, and chromium-sulfate, but the cross-linking of gelatin with chromiumacetate was reported to be more beneficial then cross-linking of gelatin with potassium chromium, and chromium-sulfate (Datta et al., 2013; Emregul, Sungur, & Akbulut, 2006). Calcium phosphate used for enzyme immobilization can be deposited on a composite of calcium alginate with gelatin. Furthermore, gelatin associated with polyester films resulted in 75% efficiency for enzyme loading (Ate¸s & Dogan, 2010; Shen et al., 2011). In edible or degradable films, gelatin is the most prevalent one which have proteins or peptides that are produced from the collagen

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23.2 Biological macromolecules for enzyme immobilization

degrading enzymes. It contains diverse type of applications in pharmaceutical and food industries which owns its physical properties like as closeness of the melting point to physiological temperature levels (Joly-Duhamel, Hellio, & Djabourov, 2002). For the entrapment of enzyme and whole microbial cells the immobilization can be done by using the gelatin as support material. The crosslinkers usage is recommended for the facilitated joining of the irreversible gelatin process which is happened due to lack of effective immobilization and temperature provided to it (Tanriseven & Do˘gan, 2002). Enzyme laccase has been immobilized on alginate-gelatin supports for decolorizing the synthetic dye pollutants. Enzymes obtained from following practice gives the greater stability and optimum performance at the temperature of 50 C for removing crystal violets that remain active up to 85% after consecutive five cycles. Glutaraldehyde decreased the 42.7% color of apple juice by functionalizing the gelatin-hydrogel which is encapsulated in the MnP that also reduced the 36% of its turbidity. It gave more than 60% of activity after repeatedly used six cycles (Bilal, Asgher, Iqbal, Hu, & Zhang, 2016). A biosensor was reported for the detection of hydrogen peroxide, a composite made of chitosan-gelatin biopolymers, was used as support material to immobilize horseradish peroxidase. The constructed biosensor observed to be more stable, high reactivity and response rate and higher extent of reusability, as well as reduced limit of detection and high sensitivity (Teepoo, Dawan, & Barnthip, 2017).

23.2.6 Dextran for enzyme immobilization Dextran is a natural polysaccharide with biodegradable and compatible nature towards biological environment. Dextran is produced by Leuconostoc mesenteroides bacterium by using the D-glucose subunits linked together via 1, 6 glycosidic linkage

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mostly (95% bonds) while contain very few numbers of 1, 3-linkage (nearly about 5%) (Kara, Demirel, & Tu¨mtu¨rk, 2006). Degradability of Dextran is performed by the dextranase enzyme which is known to be its prominent feature. Aldehyde-dextran obtained by dextran oxidation by periodate is considered as appropriate crosslinking poly aldehyde polymer for enzyme molecule with the linkage of its amino-group. Previously, has been used for the crosslinking of monomers of multimeric catalysts (Pessela et al., 2008; Rocha-Martin, Acosta, Berenguer, Guisan, & Lopez-Gallego, 2014), reducing the enzyme desorption from support, and to give the shield on the surface modification on the enzymes (LopezGallego et al., 2007). Structural form of oxidized dextran is polyaldehyde-dextran. Unstable Schiff’s bases are produced as a result of chemical reaction taking place in amino acid and aldehyde groups. If this polyaldehyde is incubated at different pH ranges, different vicinal aldehydes are produced, and result in few cyclic structures formation which can act as precursors of stable linkages with other amino acids (Mateo et al., 2006). Hence, dextran can perform excellently in enzyme surface coating, due to this stable cross linking (Pizarro et al., 2012; Rocha-Martin et al., 2014). For example, the use of polyaldehyde dextran as spacer arm for enzyme immobilization and the mechanism reported in the study has been shown in Fig. 23.3. Orrego et al. (2018) used hydrophobic octyl-Sepharose for immobilization of lipase extracted from three different species of fungi. As compared to the lipase in free state, immobilized lipase exhibit 70% catalytic activity. Greater density of aldehyde groups in polyaldehyde dextran increase the bonding of amino groups of lipases with support, hence, enhanced the stability of immobilized enzyme. 23.2.6.1 Carrageenan for enzyme immobilization Carrageenan is a name given to group of sulfate polysaccharides having high molecular wight.

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FIGURE 23.3

23. Biological macromolecules for enzyme immobilization

Polyaldehyde dextran as spacer arm for enzyme immobilization.

These are extracted from Rhodophyceae class of edible red sea weeds. Chemical structure of carrageenan contains two types of subunits 3, 6anhydro-galactopyranose and D-galactopyranose, interlinked via α-1,3 and ß-1,4-glycosidic bond (Jiao, Yu, Zhang, & Ewart, 2011; Necas & Bartosikova, 2013). There are six different form of carrageenan, and all of them contain 22%
35% sulfate groups but three of them are most commercial because of their industrial applications. Position and number of 3, 6-anhydrogalactopyranose as well as the ester sulfate groups are the main reasons of properties difference in these forms of carrageenan. Each form of carrageenan is hydrophilic in nature and are soluble in water. Amount of sulfate and cations like Na1 and K1 determines the viscosity, water solubility and carrageenan ability of gel formation. To specifically improve the stability of enzymes a sulfated linear polysaccharide (carrageenan) which is compatible with variety of different enzymes.

Carrageenan has pseudoplastic nature, due to which it can bare shear stress and after the stress removed it can recover back to its original state and regain its original viscosity (Tu¨mtu¨rk, Karaca, Demirel, & Sahin, 2007). A few years ago, a study reported the incorporation of carrageenan with agar to improve the storage, operational, and thermal stabilities of immobilized enzymes. Immobilization of enzyme on agar was realized by a two-step methodology. In the first step, agar was treated with polyethyleneimine, and in the second step, glutaraldehyde treatment was given as shown in Fig. 23.4 (Wahba & Hassan, 2017). Jegannathan, Jun-Yee, Chan, and Ravindra (2010) reported 42.6% efficacy achieved in enzyme encapsulation by carrageenan for production of biodiesel. The clinical studies of carrageenan revealed that it has very less or no toxicity. The blending of two crosslinked polymers leads to synthesis of interpenetrating networks of polymer. The potential of interpenetrating polymer

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23.2 Biological macromolecules for enzyme immobilization

FIGURE 23.4

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Preparation of agar-CAR gel disks for enzyme immobilization.

networks (k-carrageenan-based) was depicted by researchers as supporting matrix for immobilizing enzymes (Makas, Kalkan, Aksoy, Altinok, & Hasirci, 2010). Laccase, a multicopper oxidase was entrapped into semi-interpenetrating polymeric networks fabricated by mergingcarrageenan either with poly (acryl amide-itaconic acid) or poly (acrylamide-acrylic acid). In contrast to free enzymes, immobilized enzymes on both systems were observed to have 80% initial catalytic activity after storage duration of 42 days (Makas et al., 2010).

23.2.7 Pectin for enzyme immobilization Pectin is a structural heteropolysaccharide (Servais et al., 2018), used to improve the strength and durability by reducing the brittleness of support material. Blending pectin with

0.2%
0.7% glycerol increases the plasticity of the support and reduces its brittleness. Plasticized pectin has been used in papain immobilization, moreover, it has also been reported in the formation of materials applied for treatment of skin injury (Gouveia, Biernacki, Castro, Gonc¸alves, & Souza, 2019). Pectin derivatives like pectin
calcium alginate and pectin
chitin supports have been used to make the entrapped enzymes thermally stable, resistant against denaturants, and enhance their catalysis efficiency, because pectin-based support materials exhibit strong bonding with enzymes due to the formation of highly stable polyelectrolyte complexes (Meka et al., 2017; Satar, Matto, & Husain, 2008). In food industry pectin lyase is utilized to remove and degrade pectin from fruit juices and to achieve the properties that is, viscosity decrease, upgrading in liquefication, and clarification,

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higher yield and improved filterability. In apple pectin, hydrolysis is performed by beta elimination (breaking glycosidic bonds) because of active nature of pectin lyase toward hydrolysis of highly esterified pectin (Wang, Yeats, Uluisik, Rose, & Seymour, 2018). The structural limitations or potential contact diffusion of immobilized enzyme with the substrate causes the hydrolysis issues for immobilized enzyme on macromolecular substrate. The pectin lyase is found active for hydrolysis even after the immobilization by variety of different supports. The catalyst is recycled through immobilization on solid support as well as pectin decomposition using fruit juice synthesis by continuous process (DiCosimo, McAuliffe, Poulose, & Bohlmann, 2013). Several supports are used to immobilized pectin lyase such as porous glass DEAE cellulose activated with titanium salt, Eupergits C, chitin, nylon, g-alumina, bentonite and alginate gel (DiCosimo et al., 2013; Sheldon & Woodley, 2018). Several commercial enzyme mixtures forms of alginate immobilized pectin lyase were analyzed to determine the catalytic capability. These enzymatic industrial mixtures include Pectinex, Danisco (GrindamylTM 3PA), (Rapidases C80 (Gist Brocades) and CCM (Quest International) (Kohli & Gupta, 2019). The thermal and operational stability of enzyme preparation was also studied along with the determination of pH, impacts of immobilization, kinetic factors and temperature profile. The 7.2 pH and temperature 55 C were considered the conditions to attain maximum solubility and enzyme immobilization. The 35% decrease in viscosity of apple pectin was observed at 401 C after 30 min of treatment. To attain this purpose, Immobilized Rapidase C80 was recycled in four consecutive batch reactions. The lower level of pH and efficient operational stability was observed after the immobilization of pectin lyase from Penicillium italicum on nylon 6. The acid cleavage of amide groups of nylon took place and

resulted into primary amino groups. Then these primary amino groups were coupled with the enzyme acquired through the immobilization by covalent binding to Nylon 6 through glutaraldehyde cross linkage (Kohli & Gupta, 2019; Wong, Senecal, & Goddard, 2017). Different fruit juices of (melon, apricot, and peach) were utilized to analyzee the impact of immobilized enzyme on the viscosity. After 60 min of incubation for each fruit juice, 25% decrease in viscosity was analyzeed. whenever the 12 consecutive batch reactions were used to recycle enzyme and no activity was determined. The soluble enzyme demonstrated the higher Michaelis
Menten constants as compare to the immobilized enzyme activity for hydrolysis of citrus pectin (DE 70%) at pH 6.0 (DiCosimo et al., 2013; Sheldon & Woodley, 2018).

23.2.8 Xanthan for enzyme immobilization Firstly, Allene Rosalind Jeanes at the United States Department of Agriculture USA, introduced the xanthan in 1950s. The utilization of xanthan as thickening agent in many food items and stabilizers was approved by FDA in 1969. In 1960s, Kelco Company started its industrial synthesis which is recently known as CP Kelco. The xanthan gum was synthesized at industrial scale in the Europe Jungbunzlauer Austria AG and Solvay, under the trade name Rhodopol. The largest xanthan producer has been china since 2005 (Tao et al., 2012; Wibberg et al., 2015). The different strains named as Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas citri, Xanthomonas fragaria, Xanthomonas gummisudans, Xanthomonas juglandis, Xanthomonas phaseoli, Xanthomonas vasculorium are known to have gram negative bacteria of genus Xanthomonas, known to produce xanthan gum (Saddler, 2007; Tao et al., 2012; Verma, Sakure, & Badwaik, 2017). The most generally used xanthan gum for

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Conflicts of interest

industrial level synthesis is known as X. campestris (Palaniraj & Jayaraman, 2011). The excellent reviews are available in literature on synthesis factors or parameters such as optimum pH and temperature of growth medium and oxygen transfer rate, on the fermentation yield, sort and amount of nutrients in the growth medium, types of bioreactors, continuous or batch mode of fermentation (Alle et al., 2020; Palaniraj & Jayaraman, 2011; Rosalam & England, 2006). Different conditions like temperature, pH, xanthan amounts, molar ratio of biopolymers, shearing conditions and ionic strength of medium are used to control interactions between proteins, polypeptides and polysaccharides (Gharanjig, Gharanjig, Hosseinnezhad, & Jafari, 2020; Wiederschain, 2007). The immobilization of enzyme and shielded confirmation are maintained through hydrophilicity of polysaccharides that give them protective surroundings. For example, the immobilization of different enzymes like xylanases, proteases and lipases are immobilized through complexes of chitosan and xanthan. The 0.65 wt.% xanthan solution and the desired enzyme was added drop wise in a 0.65 wt.% chitosan solution to prepare the complexes of xanthan and chitosan (Kim et al., 2017). The higher thermal stability was noticed by immobilized enzyme, achieved through increased enzyme concentration in the xanthan solution in order to enhance the enzymatic activity of immobilized enzymes (Kim et al., 2017). An enzyme with biocidal characteristics and isoelectric point at 10.7 (Muszanska, Busscher, Herrmann, van der Mei, & Norde, 2011) has proved to have been used for xanthan hydrogels carriers (Bueno & Petri, 2014). The attraction between negatively charged xanthan and positive charged residues of Lyz cause the low release of Lyz (Bengani, Leclerc, & Chauhan, 2012; Dario, de Paula, Paula, Feitosa, & Petri, 2010; Kumar, Rao, & Han, 2018). The bactericidal activity was observed by Lyz loaded xanthanbased hydrogels films. CD spectra demonstrated

the bactericidal activity because of conserving the native structure of Lyz loaded xanthan-based hydrogel films (Bueno & Petri, 2014; Patel, Maji, Moorthy, & Maiti, 2020).

23.3 Conclusions and future outlook The utilization of biopolymers in various domains as a support material is a very promising field in which exploration of advanced and novel materials are attained from natural origins, which can be manipulated to meet the world’s increasing demands. These materials, either polysaccharides or proteins, are found to have extensive usability in different industries due to their outstanding properties, that is, biocompatibility, biodegradability, and high responsiveness to chemical functionalization. Different multifunctional biocatalysts are developed by using support carriers of a large number of biopolymers and their derivatives such as alginate, chitosan, pectin, carrageenan, gelatin, cellulose, dextran, agarose, agar-agar, and carrageenan. The immense biotechnological interest has made possible to extensively utilize these biopolymers-based composite materials having incredible properties and novel structures for enzyme immobilization. Moreover, the biocatalytic potentialities can also be increased by the utilization of advanced materials. The new biopolymers materials along with novel enzyme immobilization technique can be used to increase the catalytic performance of the insolubilized enzymes at high level.

Acknowledgment The listed author(s) are thankful to their representative universities for providing the literature services.

Conflicts of interest The listed author(s) declare that no competing, conflicting, and financial interests exist in this work.

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Further reading Bilal, M., Wang, Z., Cui, J., Ferreira, L. F. R., Bharagava, R. N., & Iqbal, H. M. N. (2020). Environmental impact of lignocellulosic wastes and their effective exploitation as smart carriers—A drive towards greener and ecofriendlier biocatalytic systems. Science of the Total Environment, 722, 137903.

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C H A P T E R

24 Carbohydrates mimetics: enzyme inhibitors and target molecules in several diseases Vero´nica E. Manzano1,2, Custodiana A. Colmenarez Lobo2 and Evangelina Repetto1,2 1

Faculty of Exact and Natural Sciences, Department of Organic Chemistry, University of Buenos Aires, Buenos Aires, Argentina 2Research Center in Carbohydrate Chemistry (CIHIDECAR), National Scientific and Technical Research Council (CONICET)-UBA, Buenos Aires, Argentina

24.1 Introduction 24.1.1 Biomass and biobased materials Biomass offers a richness and molecular diversity with interesting properties and great potential for new uses and applications. Materials from renewable resources are invaluable because they give vital elements of sustainability (Gandini & Belgacem, 2008). For the last few decades, there has been a growing interest in the use of compounds from the biomass as sustainable sources of new materials and chemicals, in replacement of the fossilderived ones. Considering that the petroleumbased materials are generally nonbiodegradables, which leads to environmental problems. In this sense, many researchers from different areas, ranging from chemistry, food,

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00024-5

medicine to material science and agriculture, focused their studies on the use of biobased derivatives from renewable biomass in the search of new compounds and materials (Gandini, Lacerda, Carvalho, & Trovatti, 2016; Gandini, 2010; Mohamed, El-Sakhawy, & ElSakhawy, 2020). It is expected that products derived from the biomass present enhanced biodegradability, low toxicity and better biocompatibility in contrast to the petroleumbased counterparts. Furthermore, its biodegradation is expected to generate environmentally friendly products. Also, these compounds should present easy access and low cost in contrast to petroleum derivatives. Among the wide range of compounds from the biomass, carbohydrates stand out as promising scaffolds for this issue.

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© 2022 Elsevier Inc. All rights reserved.

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24. Carbohydrates mimetics: enzyme inhibitors

Carbohydrates are the main source of energy and stored (i.e., starch, glycogen) in nature. These are vital molecules in many biological processes (Mishra, Tiwari, & Schmidt, 2020) and sometimes they have structural functions as in the case of chitin and cellulose. Carbohydrates are the most easily accessible and abundant biomolecules of the biomass. They present a unique structural diversity combined with the large number of stereocenters and the multiple hydroxy groups which make them versatile building blocks in asymmetric synthesis (Boysen, 2007; Seeberger, 2008) and interesting scaffolds in the design of bioactive compounds (Ernst & Magnani, 2009; Werz & Seeberger, 2005), drug discovery (Galan, Benito-Alifonso, & Watt, 2011; Valverde, Arda´, Reichardt, Jime´nez-Barbero, & Gimeno, 2019), biomedical applications (Gopinath, Saravanan, Al-Maleki, Ramesh, & Vadivelu, 2018) and new materials (Asgher, Qamar, Bilal, & Iqbal, 2020; Herniou-Julien, Mendieta, & Gutie´rrez, 2019). In addition, they present an easily modifiable architecture, which enables the tailoring of different properties turning carbohydrates in interesting molecules for varied applications. In this sense, carbohydrates offer a source of enantiomerically pure starting materials. These compounds are used for the synthesis of a variety of molecules and have also been used as chiral auxiliaries.

24.1.2 Carbohydrates As it is well known, carbohydrates are polyhydroxylated compounds containing an aldehyde or ketone group. Carbohydrates can be found in nature as monosaccharides, as cyclic structures formed by a hemiacetal linkage, or they can be also linked to another monosaccharide or even other kind of molecules by an acetal bridge (glycosidic linkage), to give more complex structures.

Structurally, monosaccharides can be found in pyranosic or furanosic configurations and the linkages may occur in different positions, producing many possible structural arrangements. The ability to build complex structures and diverse types of connections is a unique structural characteristic of carbohydrates, compared to peptides or nucleic acids, which results in their great functional diversity in biological systems. There are many examples of carbohydrate-based drugs, with many and varied function, as shown in Fig. 24.1. The large number of hydroxyl groups confer carbohydrates their polar characteristics, high hydrophilicity, and the possibility of interacting through hydrogen bonds with other molecules. Also, as result of the presence of many hydroxyl groups they can form supramolecular networks like gels, which turn them interesting molecules for the biomedical area, drug delivery and tissue engineering (Gim, Zhu, Seeberger, & Delbianco, 2019). As the interaction between two different backbones of carbohydrates is weak, this hydrogen bond can be broken and this gives the materials based on carbohydrate the property of being reversible (responsive materials). This response can be tuned upon certain stimulus, making interesting materials for different applications. Conformation is also an aspect that influences biological activity and the properties of the final product. Glycoside conformation is governed mostly by the stereochemistry of glycosidic linkage, configuration of substituent groups and the possibility of pseudorotation in the pyranosic or furanosic rings. The wide variety of carbohydrates properties is related to their structures, which include linear or branched polymers (polysaccharides) or complex structures conjugated with other types of molecules. In this last case, they are usually found in conjunction with proteins (glycoproteins), lipids (glycolipids) and other glycoconjugates, which can be found in all living systems.

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24.1 Introduction

FIGURE 24.1

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Molecular diversity of carbohydrates in biological applications.

For all the reasons stated before, carbohydrates have attracted the attention of many researcher and its chemistry and uses have become an interesting field of study due to all the possibilities they give rise to, as summarized in Fig. 24.2. They have been used in supramolecular chemistry (Evenou et al., 2018), in biomedicine as drugs (Mishra et al., 2020; Valverde et al., 2019), glycomimetics (Compain, 2018; Hevey, 2019) or in delivery systems (Pacho, Manzano, & D’Accorso, 2019; Prusty & Swain, 2019), in food packaging (Asgher et al., 2020; Herniou
Julien et al., 2019) and even in material science for the development of different polymers based on

carbohydrates (Galbis & Garcı´a-Martı´n, 2008), just to mention some relevant examples. In recent years, new compounds based on carbohydrate derivatives have been studied, observing an improvement in enzyme inhibition and different properties. Carbohydratebased macromolecular systems which can potentially inhibit glycosidases, have also been on the rise. Among these, the multivalent inhibitors and polysaccharides from biomass can be mentioned. In this chapter we will focus on the use of carbohydrates as glycomimetics, especially as enzyme inhibitors for the treatment of different diseases. We will discuss the different chemical

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FIGURE 24.2

24. Carbohydrates mimetics: enzyme inhibitors

Examples of biomedical applications of carbohydrates.

modification present in each glycomimetic and response caused by its variation. This chapter will encounter the recently advances in this field, but it is important to make clear that we will discuss the lately researches but there much more work done on the field.

24.1.3 Biological and medicinal interest of carbohydrates Carbohydrates play an important role in biological functions (Shade, Conroy, & Anthony, 2019) and therefore they are strongly connected to the biochemical, pharmaceutical and medicinal areas. They are essential biomolecules for life, whose role is not only limited to energy storage as it was mentioned before, but they are also key elements in a variety of biological processes (Pohl, 2010) as signaling and cell adhesion, cellular recognition (Glavey et al., 2015; Szabo & Skropeta, 2017), growth and differentiation, fertilization, angiogenesis, intercellular communication, and also they can be target molecules (van Kooyk & Rabinovich, 2008). Besides, it is well established that when certain disease is present, this modifies the expression of the different components in the cell, which leds into changes in the composition of cell membrane

glycans and in the enzymes related to the carbohydrates, that is glycosyl hydrolases or glycosyl transferases (Hevey, 2019). Thus, understanding the function or interaction between carbohydrates and cells can lead to the development of new synthetic mimics which can act as therapeutic agents interfering with the enzymes that play an essential role in the progress of a disease (Bertozzi & Kiessling, 2001). The study of the function of carbohydrates (mono or oligosaccharides and lectines) is key to design new drugs, vaccines and biomedical devices. Diseases, such as diabetes, arthritis, Alzheimer’s disease, infections (either viral or bacterial), thrombosis, tumor cell metathesis and cancer are related or mediated by carbohydrates. This relationship between carbohydrates and enzymes is explained by the fact that a large group of enzymes is required for their synthesis, modification, and degradation of the vast diversity of carbohydrate-based structures occurring in nature (Henrissat & Davies, 1997). For this reason, it is known that a wide variety of metabolic disorders and diseases are mediated by glycosidases (Davies & Henrissat, 1995; Zechel & Withers, 2000).

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24.1 Introduction

Currently, multivalent compounds are widely studied as they are a promising alternative to obtain powerful glycosidase inhibitors for the treatment of some diseases. If thermodynamic modifications in the binding are considered when going from a monovalent to a multivalent system, the affinity and selectivity are improved. Therefore an inhibitor can become more effective and selective in a group than being alone. It is important to remark that when a multivalent effect occurs, the entropic cost to be paid in the second interaction of the ligand with receptor is lesser than when there are monomeric ligands, since a preorganization of the entire system occurs. Generally, if the enzyme has several active sites, it can interact efficiently with such multivalent inhibitors. However, if the enzymes have only one active site or deep active sites, this potential chelate effect is ruled out. These latter cases could be argued with aggregation processes, additional unspecific interactions with a noncatalytic subsite or statistical regrouping mechanisms, depending on the case. Furthermore, it should be considered that the union of multiple inhibitors does not always lead to an efficient multivalent system, since there are several influencing parameters, such as the length and stiffness of the system (Carta, Dumy, Supuran, & Winum, 2019; Martı´nez-Baile´n et al., 2020).

24.1.4 Glycosidases Carbohydrates as glycoproteins, glycolipids or polysaccharides play essential roles in development of microbes, plants or animals and even in cell physiology. On the other hand, glycosidases are a widespread class of enzymes, which act on a wide variety of substrates in nature, as carbohydrates. They are able to cleave the glycosidic bond between two sugar (saccharide) molecules, which is one of the most stable bonds in nature, stimulating different physiological responses or

551

transferring saccharides to other molecules such as sugar moieties, lipids or proteins (Davies, Gloster, & Henrissat, 2005). Recently, the study of glycosidases have attracted many researches in view of their predominant role in several biological processes (the enzymatic processes that are catalyzed by these enzymes are essential for the normal functioning of mainly all organisms) and consequently, their inhibition might constitute an interesting strategy for the development of new therapeutics to treat diverse diseases (Borges de Melo, da Silveira Gomes, & Carvalho, 2006; Wadood et al., 2018). The “carbohydrate-active enzymes” (CAZymes) have been grouped into several families which include glycosidases (GHs), also known as glycosyl hydrolases; glycosyltransferases (GTs); polysaccharide lyases and carbohydrate esterases as it is shown in Fig. 24.3. In this opportunity, we will focus on glycoside hydrolases because, as it was mentioned before, they are related to the biosynthesis of glycoconjugates which are associated to several disorders. GHs and GTs are enzyme that catalyze the transfer of an activated donor sugar to a suitable acceptor, usually another sugar, lipid, protein or small molecule. They are classified into over 150 different families in the CAZymes database based the resemblance or similarity on the amino acid sequence (Davies & Williams, 2016). Glycoside hydrolases are the most known and characterized enzymes which act on disaccharides, oligosaccharides and polysaccharides. They have been classified and divided in groups using different indices. Just to mention, based on substrate specificity, the EC 3.2. class includes cleaving O-, S-or N-glycosides. They can be classified also into GHs families according to their amino acid sequence resemblances because of advancements in genomic science (Lombard, Golaconda Ramulu, Drula, Coutinho, & Henrissat, 2014). This system further separates GHs families into groups, given the improved conservation of protein fold than the sequence (Henrissat et al., 1995).

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24. Carbohydrates mimetics: enzyme inhibitors

(A)

(B)

(C)

(D)

FIGURE 24.3 Different classes of enzymes: (A) GHs, responsible for the hydrolysis of the glycosidic bond; (B) GTs, which use the energy derived from activated sugar donors to drive glycosidic bond synthesis; (C) polysaccharide lyases which catalyze the β-elimination reaction on uronic acid glycosides and (D) carbohydrate esterases, responsible of the de-O and de-N acetylation of acetylated sugars.

The carbohydrate degradation mechanism by these proteins is based in the recognition of some specific carbohydrate unit and then the hydrolysis of the glycosidic bond. Glycosidases are capable of distorting sugars conformation to accommodate them at catalytic sites during the process that leads to the glycosidic bond cleavage (French, 2012; Johnson, Petersen, French, & Reilly, 2009). It is possible to distinguish between an exoand endo-glycosidase; the first one acts at the non-reducing end of a glycan chain, but the second one cleaves any point along an oligosaccharide chain. This can be in a random way or “chemical environment”-specific manner. GHs are essentially catabolic, degrading enzymes. Several glycosidases are specialized

in the cleavage of glycosidic bonds based on the position, number or configuration of the hydroxyl groups presents in the sugar molecule. The common catalytic mechanisms for GHs were studied and suggested some decades ago (Koshland, 1953). Based on this, the enzymes may be classified in two most significant groups, based on the mechanisms involved in the hydrolysis of the glycoside linkage of substrates (Lai, Morris, Street, & Withers, 1996; Vasella & Davies, 2002; Zechel & Withers, 2000), the inverting enzymes and the retaining enzymes (Fig. 24.4). The mechanism for the first group (inverting glycosidases) starts by generating an oxocarbenium ion and then this transition state goes under inversion of configuration at the anomeric center. On the

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24.1 Introduction

553

(A)

(B)

FIGURE 24.4

(A) Mechanism of inverting glycosidases; (B) Mechanism of retaining glycosidases.

other hand, retaining glycosidases act via initial formation under inversion of configuration of a covalent enzyme
glycon bond which is then hydrolyzed in a second inversion step. In this case, the configuration at the anomeric carbon is retained as shown in Fig. 24.4B. It is well known that GHs are crucial in numerous biological processes. For this reason, in the last years many attempts have been made to project and produce new inhibitors of glycosidases (Simone et al., 2017). So, inhibition of glycosidation pathways is a novel way for designing new drugs. In this sense, the study of their inhibition mechanisms is key to the development of new and powerful inhibitors. In recent decades, an increase in the search for new potent and selective glycosidase inhibitors has been observed (Colomer et al., 2016; Driguez, 2001; Garcı´a-Herrero et al., 2002), as these enzymes are found in almost all living organisms.

Continuous advances in this area allowed to discover a large number of compounds representing the structures of monosaccharide and oligosaccharides from natural sources or even synthesized in the laboratory (Asano, Nash, Molyneux, & Fleet, 2000; Asano, 2009; Butters, Dwek, & Platt, 2005). Carbohydrates unique biological activities have led the search for new analogs with improved biological properties, because they are able to act as analogs of the natural substrates of these enzymes and might interact with their active site, inhibiting their activity. They are “carbohydrate mimetic compounds,” in which their molecular structure shows reminiscent of a natural sugar, but with modifications that can affect their biological activity and metabolism, making them very useful to investigate processes mentioned above (Marcaurelle & Bertozzi, 1999; Sears & Wong, 1999).

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24. Carbohydrates mimetics: enzyme inhibitors

24.2 Glycomimetics In the last decades, an interesting approach that emerges and remains as a very challenging task for the development of new drugs, is the design and synthesis of glycomimetics. A glycomimetic is defined as chemical entities that mimic the structure or function of a native carbohydrate. This new molecule present structural modifications in comparison to the natural counterpart, comprising from replacement of the oxygen of the cycle to substitution of the glycosidic or a different oxygen with other atoms such as nitrogen (iminosugar), sulfur (thiosugar), carbon (carbasugar), selenium or phosphorus (phosphasugars), to mention some. On the other hand, the glycosidic or pseudoglycosidic molecules have been used to develop new therapeutic and diagnostic tools, however they showed poor permeation properties and deficient metabolic stability, but a rapid approval of carbohydrate-based drugs. Besides, these mimetics are helpful molecules for understanding biological processes in which carbohydrates play a significant role. Then, it is important to note that this modification avoids degradation by specific enzymes, enhances the stability and affinities, increases the bioavailability, and/or mimics the transition state of the glycosyl hydrolase. Inhibitors of this enzymes are relevant because of their potential as antiviral, anticancer and antidiabetic agents or even as therapeutic agents for certain genetic diseases (Asano, 2003; Gruner, Locardi, Lohof, & Kessler, 2002; Leiria Campo, Araga˜o-Leoneti, & Carvalho, 2013). Consequently, the inhibition of these GHs can produce considerable effects on the intestinal digestion, maturation, transport and secretion of glycoproteins, catabolism of glycolipids in lysosome and also can alter the recognition of cellcell or cell-virus processes providing a basis for their potential use in diabetes, genetic lysosomal disorders, cancer, and viral infection (Asano,

2009; Horne, Wilson, Tinsley, Williams, & Storer, 2011; Nash, Kato, Yu, & Fleet, 2011). Numerous compounds, of natural or synthetic origin, that mimic the structures of monosaccharides or oligosaccharides were discovered, as it was mentioned. Most of the designs were based on mimicry of the transition state formed during the hydrolysis of the glycoside, which has significant oxocarbenium character. It is believed that their inhibitory function depends on the structural and electronic resemblance at physiological pH to the oxocarbenium transition state of the natural counterpart of these enzymes. Examples concerning compounds where the oxygen atom was replaced by carbon, sulfur or nitrogen will be discussed.

24.2.1 Iminosugars Iminoalditols, or iminosugars are polyhydroxylated alkaloids which are structurally related to common carbohydrates but in these compounds the endocyclic oxygen atom, which is O-4 when the compound is furanosic or O-5 when its structure is pyranosic, was replaced by a nitrogen atom (Horne et al., 2011). Iminoalditols, which are extensively distributed in nature, are found in bacteria, fungi and plants (Asano et al., 2000). Interest in this research area, started around 1962
63, when carbohydrate researchers studied the possibilities and limitations of its (sugars) synthesis and of related compounds. This was a consequence of the growing concern in the influence and impact of antibiotics containing amino sugar on science and in health-care systems (Hanesslan & Haskell, 1963; Paulsen, 1962). Many iminosugars have been discovered from natural sources. Just to mention the first important discoveries, 5-amino-5-deoxy-Dglucose which is known as the first natural iminosugar, was isolated from Streptomyces

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24.2 Glycomimetics

nojiriensis (Nishikawa & Ishida, 1965), characterized and termed as Nojirimycin (Inouye, Tsuruoka, & Niida, 1966; Ishida, Kumagai, Niida, & Tsuruoka, 1967) and then its structure and synthesis were described (Inouye, Tsuruoka, Ito, & Niida, 1968). The first iminosugar synthetically obtained was 1,5-dideoxy-1,5-imino-Dglucitol, also known as 1-deoxynojirimycin (1DNJ) whose synthesis was reported in 1966 (Paulsen, Sangster, & Heyns, 1967; Paulsen, 1966). Some years after a new type of alkaloid was isolated from the plant called Derris elliptica, identified as 2,5-dideoxy-2,5-imino-D-mannitol or as 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine or DMDP (Welter, Jadot, Dardenne, Marlier, & Casimir, 1976). (Fig. 24.5) More discoveries like these, supported the idea that iminosugars and their structural relatives could be a common family of natural products, highly distributed in plants, in different climate zones and with varied biological effects related. So, an entire interdisciplinary scientific research field emerged to initiate the development of this new class of glycomimetics (Martin & Compain, 2007; Stu¨tz & Wrodnigg, 2016; Stu¨tz, 1999). It was from 1984 that this promising iminosugar area received reports from research groups which made a strong impact on this

field. [Fig. 24.6 (1) (Fleet, Smith, Evans, & Fellows, 1984), (2) (Fleet, Shaw, Evans, & Fellows, 1985), (3) (Fleet et al., 1985), (4) (Fleet et al., 1986), (5) (Legler & Julich, 1984)] These class of glycomimetics constitutes the best developed and major class of monosaccharide mimetics described to date, showing that their basic properties, due to the presence of the nitrogen atom, are responsible for their significant biological activity. Structurally, iminosugars can be classified in two groups if they were obtained from natural resources (plant or microbes) or from synthetic origin, such as monocyclic systems such as piperidines, pyrrolidines and azepanes or bicyclic scaffolds, such as indolizidines pyrrolizidines or nortropanes (Fig. 24.7). Both classes were recently extensively reviewed (Stu¨tz & Wrodnigg, 2011). Iminoalditols obtained from natural products or even through many excellent synthetic approaches, proved to be of great value as glycosidases inhibitors displaying high pharmacological potential in numerous therapeutic areas. As it was mentioned, iminosugars inhibit carbohydrate processing enzymes, such as GHs, GTs and even other classes of enzymes (Lahiri, Ansari, & Vankar, 2013; Martin & Compain, FIGURE 24.5 First important iminosugars discovered from natural sources.

N

FIGURE 24.6

D

Relevant examples that contributed to first developments in the field of iminosugars.

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24. Carbohydrates mimetics: enzyme inhibitors

2007; Stu¨tz & Wrodnigg, 2011). They behave as potent inhibitors, which usually bind to the active sites of GHs in a competitive and reversible mode. Generally, these mimetics possess simple and small structures firmly related to their natural counterpart although most of them are stable, chemically and biologically, during the main processes carried out by carbohydratemodifying enzymes. The nitrogen atom in the iminosugars is protonated, allowing the carbohydrate analog to compete and inhibit the process of hydrolysis. For this reason, iminosugars turn out to be one of the most popular class of carbohydrate processing enzyme modulators over the last years. The inhibition specificity of some iminosugars such as piperidines, may be easily predicted from the configurations of hydroxy groups, since they

are identical to those in the corresponding pyranose. For instance, 1-DNJ which is the pyranic analog of glucose has high inhibition specificity against α-glucosidases (Fig. 24.8). In the same way, the polyhydroxylated piperidine analogs of mannose (1,5-dideoxy-1,5-imino-D-mannitol (deoxymannojirimycin (DMJ)) or galactose (1,5-dideoxy1,5-imino-D-galactitol (deoxygalactonojirimycin (DGJ)), exhibit the expected inhibition for α-mannosidase and α-galactosidase, respectively (Kato et al., 2005). However, it worth to mention the difficulty of identifying subtle differences in the isoenzyme located in tissues or multiple organs. For this reason, the synthesis of selective inhibitors is relevant to avoid side effects resulting from undesired interactions with isoforms not involved in the targeted pathology. In fact, this FIGURE 24.7 Iminosugar structural motifs. (A) monocyclic systems (B) bicyclic scaffolds.

(A)

(B)

(A)

(B)

(C)

FIGURE 24.8 Piperidine iminosugars inhibitors. IC50 values for: (A) lysosomal acid α
glucosidase (B) Jack bean α
mannosidase (C) lysosomal α
galactosidase A.

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24.2 Glycomimetics

could be solved with glycomimetics because they are considered to be more selective than their close sugars in inhibiting enzymes but sometimes this inhibition selectivity which is based on their high similarity with the substrate might produce side effects. This group of glycomimetics demonstrated a widespread variety of biological activities related with an excellent drug profile. For these reasons, these compounds result attractive therapeutic candidates for numerous medical interventions. Actually, this family of compounds proved to be interesting for treatment of various diseases, such as diabetes (Ferhati et al., 2019), cancer (Gueder et al., 2017), bacterial infections (Hsu et al., 2014), HIV (Yan et al., 2018), HPV (Wetherill et al., 2018), influenza (Tyrrell, Sayce, Warfield, Miller, & Zitzmann, 2017), hepatitis (Jacob, Mansfield, You, Tennant, & Kim, 2007), the dengue virus (Miller, Tyrrell, & Zitzmann, 2018), malaria (Evans, Tyler, & Schramm, 2018) and fungal infections (Chavan et al., 2017). Even showed promising results as pharmacological chaperones of defective glycosidases, in the treatment of lysosomal storage diseases such as Gaucher and Fabry diseases. In these disorders, the role of iminosugars is to bind to the enzyme, thus stabilizing its conformation in order to prevent premature degradation and to save its catalytic activity. Therefore extensively studied iminosugars, constant efforts in their chemical synthesis and the increasing knowledge of their affinity and

target selectivity (Sa´nchez-Ferna´ndez et al., 2016) remarkably improved their progress as possible drug candidates. Actually, some products are currently commercially marketed as drugs (Fig. 24.9). It worth to mention that polyhydroxylated alkaloids of the iminosugar family based on modifications of 1-DNJ are intensely studied compounds. For example, the N-substituted 1-DNJ derivative miglitol (Glyset, Pfizer, New York, NY, United States), is an inhibitor of the intestinal α-glucosidases which constitutes an effective and harmless treatment option in patients with diabetes mellitus (type 2) who are not able to be adequately controlled with diet or oral sulfonylurea therapy (Campbell, Baker, & Campbell, 2000). N-butyl-DNJ (miglustat; Zavesca, Actelion Pharmaceuticals Ltd., Allschwil, Switzerland), inhibits glucosylceramide synthase and is indicated in the treatment of Gaucher disease, caused by a malfunction of lysosomal β-glucocerebrosidase (GCase) (Stirnemann et al., 2017). It worth to mention that Parkinson’s and Alzheimer’s diseases have been related to GCase deficiency too (Gatto, Da Prat, Etcheverry, Drelichman, & Cesarini, 2019). Recently, an inhibitor of α-galactosidase, the DNJ epimer DGJ, as the corresponding hydrochloride salt (migalastat; Galafold, Amicus Therapeutics, Cranbury, NJ, United States) was successfully developed for the treatment of Fabry disease. For adults and adolescents aged $ 16 years in the United States, having a FIGURE 24.9 Structure of the iminosugars 1-DNJ and 1-DNJ-related drugs: miglitol, miglustat, and migalastat.

D

M

M

M

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confirmed diagnosis of Fabry disease with amenable mutations, it was approved as oral monotherapy for their long-term treatment. Besides, phase III development of the drug is being carried out in the United States, Canada and other countries (Markham, 2016). A wide number of glycomimetics based on iminosugars have been investigated and that this research area is in constant development. Some examples of the latest reports will be mentioned. Wolfsgruber et al. (2020) were especially interested in iminoalditol structures, with modifications at the ring nitrogen. So, they synthesized N-alkylated D-gluco and D-xylo iminosugar which were evaluated as inhibitors of human Gcase, related to Gaucher disease. In this opportunity, their synthetic pathway is based on the coupling of three main building blocks (Fig. 24.10): The iminosugar scaffold

which acts as an active site ligand; the interface moiety that enables variation in length and therefore properties of the final product and the terminal building block (tag) which provides various functional groups that can be modified for further applications such as nitrile, azide or alkyne, nonafluoro-tert-butyl and N-dansyl functionalities. The biological evaluation with different GHs proved that all synthesized compounds bind tightly to GCase with Ki values ranging the micro and nanomolar scale. Most of them display good selectivities, highlighting their potential to be used as enzyme inhibitors, pharmacological chaperons or active site directed ligands for enzyme labeling. Zamoner, Araga˜o-Leoneti, and Carvalho (2019) also reported a small library iminosugars, including novel compounds (Fig. 24.11) with diverse stereochemistry, different ring FIGURE 24.10

Three main building blocks to get N-alkylated iminosugar based inhibitors in the D-gluco or D-xylo configuration.

S S

I

I

T

FIGURE 24.11 Synthesis of iminosugars from D-mannitol, via bis-epoxides.

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24.2 Glycomimetics

size, and N-substitutions synthesized from a common precursor, D-mannitol. The key reaction was an SN2 aminocyclization via two isomeric bis-epoxides. Inhibition toward α- or β-glucosidases was showed for some of these piperideines and azepanes. Thus they observed that glucosidase inhibition promoted by some polyhydroxypiperidines was accomplished by proportional inhibition of the corresponding polyhydroxyazepane isomers bearing the same N-chain regardless of the ring stereochemistry. The glycosidase inhibition profiles of the sixteen piperidine analogs of the same amount of aldohexoses are quite well correlated, as it was mentioned before, with the structures of their parent sugars; but, the 10 pyrrolidine analogs of the eight ketohexoses might show many interesting results (Ayers et al., 2012). In this sense, Wibowo et al. (2020) synthesized pyrrolidine-based iminosugar derivatives to evaluate them as inhibitors of α-glucosidase and for molecular docking (Fig. 24.12). The compounds studied were hydrazinyl, hydroxyamino, and hydroxypyrrolidine derivatives. Docking studies revealed that all the new analogs synthesized occupied the same region as the DNJ inhibitor on the enzyme binding site, and also that the most potent compounds established interactions with key residues. Sometimes the progress in the search of new inhibitors can also contribute to understand enzymatic processes specially when the structure and interactions in the catalytic site of the (A)

559

enzyme stay unknown. That is the case of relevant reports from Oliveira Udry and coworkers (Oliveira Udry, Repetto, Vega, & Varela, 2016; Udry, Repetto, & Varela, 2014) which include stereospecific synthesis of enantiomeric polyhydroxyalkylpyrrolidines via 1,3-dipolar cycloadditions from stabilized azomethine ylides and sugar enones (dihydropyranones) derived from pentoses, to be evaluated as inhibitors of a β-Galactofuranosidase from Penicillium fellutanum. These results interesting because this enzyme is only found in some pathogenic microorganisms, but it has not been found in higher eukaryotes, so, its inhibition could prevent the proliferation of some pathogens such as Mycobacterium tuberculosis or Trypanosoma cruzi, the agents of tuberculosis or Chagas disease, respectively. New pyrido-pyrrolidine hybrid compounds were designed, developed and screened against a series of α-glycosidases (Fig. 24.13) by the group of Sen (Luthra, Banothu, Adepally, Kumar, & Chakrabarti, 2020). Recently diabetes researchers started to look for other potential therapies including these inhibitors to treat type I diabetes in addition to insulin therapy (DeGeeter & Williamson, 2014). The purpose of this novel class of molecules based on pyrido-pyrrolidine hybrids is to help to control high blood glucose level in diabetes mellitus in general. On the other hand, even with their therapeutic potential, most iminosugars present a low specificity maybe as a consequence of the

(B)

FIGURE 24.12 (A) Potent glycosidase inhibitors pyrrolidine-based iminosugars (B) Relevant examples of new pyrrolidine-based iminosugar derivatives evaluated for α-glucosidase inhibitory activity.

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flexible conformations. For this reason, bicyclic iminosugars such as pyrrolidizines, indolizidines and nortropanes have drawn great interest. This bicyclic compounds with rigid structures, could induce a higher and more specific glycosyl hydrolase inhibitory activity, making them promising drug candidates (Chen, Huang, Li, Gao, & Zhou, 2018; Massicot et al., 2019; Tamariz, Burguen˜o-Tapia, Va´zquez, & Delgado, 2018). It worth to mention that the synthesis of some conformationally locked iminosugars has also been explored. For instance, imidazopiperidine derivatives, triazole and tetrazole fused iminosugars were also studied. Alkaloids of pyrrolizidine and indolizidine constitute an important part of the alkaloids family, often similar to each other only by the presence of one single nitrogen atom. Many different approaches for their synthesis have been developed, in all the cases the key step is forming azabicyclic skeleton. Chen et al. (2019)

described recently, a divergent and concise route to synthesize bicyclic iminosugars, particularly polyhydroxylated pyrrolizidine and indolizidine-based molecules. This group showed before that their synthetic polyhydroxylated pyrrolizidines were more potent α-glucosidase inhibitors than the corresponding polyhydroxylated pyrrolidines (Cheng, Guo, Lin, & Jiang, 2015). Based on these results, they developed novel bicyclic iminosugars against mannosidases, other kind of glycosidases. So, six natural product-like bicyclic alkaloids including (-)-Swainsonine (a potent mannosidase inhibitor) were conveniently prepared and tested for inhibition study of α-mannosidases. In this opportunity, they reported that the C-8 hydroxyl group with appropriate orientation plays a crucial role for inhibitory activity, as shown in Fig. 24.14. The favorable properties of this kind of sp2iminosugars as glycosidase inhibitors constitutes an interesting strategy for the discovery of FIGURE 24.13 Design of novel hybrids α-glucosidase inhibitors.

FIGURE 24.14 Bicyclic iminosugars prepared for inhibition study against α-mannosidases.

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24.2 Glycomimetics

carbohydrate-based drugs. Although multicyclic iminosugars (with three or more fused rings) having sp2-character on the anomeric carbon have attracted much less attention on their biological activities. Just to mention some examples, this group (Yan et al., 2019) reported recently the synthesis and glycosidase inhibitory activity of novel tricyclic sp2-iminosugar-fused benzimidazoles (Fig. 24.15) based on the fact that those synthetic compounds which have benzimidazole moieties displayed a variety of biological activities, such as antidiabetes, anticancer, antibacterial, antivirus, and antiglycosidase activities (Adegboye et al., 2018; Akhtar et al., 2017; Bansal ¨ zil, Parlak, & Balta¸s, 2018). & Silakari, 2012; O Also, the synthesis of new iminosugar with fused bicyclic oxazolidin-2-one was described by Domingues et al. (2020). 1,2-fused oxazolidinone iminosugars were obtained in good yields and studied as a new class of glycosidase inhibitors, extending the family of known 1-DNJ bicyclic analogs (Fig. 24.16). Compounds were evaluated

561

against diverse glycosyl hydrolases such as α,β-D-glucosidase, α,β-D-galactosidase, α,β-Dmannosidase, and β-D-glucuronidase. D- and L-arabino based iminosugars showed moderate inhibition properties with remarkable selectivity on β-D-glucosidase and α-D-galactosidase respectively, inspiring the synthesis of novel GHs inhibitors containing this unusual scaffold.

24.2.2 Carbasugars Carbasugar, or pseudosugars as firstly known, is the type of glycomimetic where the oxygen of the ring was replaced with a methylene group. This bioisosteric replace confers a greater chemical stability as well as resistance to endogenous enzymatic degradation (Kiefel, 2010). Lately, Somsa´k and coworkers (Somsa´k, Bokor, Juha´sz, Kun, & La´za´r, 2019) reported the synthesis of C-glycopyranosyl heterocycles of five and six membered rings as well as

FIGURE 24.15

Synthetic tricyclic benzimidazole-iminosugars.

FIGURE 24.16

Examples of fused oxazolidinone iminosugars, evaluated as a new class of glycosidase inhibitors.

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anhydro-aldimine derivatives and exo-glycals. It is very interesting to note that, most of them, were efficient glucose analog inhibitors of glycogen phosphorylase turning them into potential therapeutics of these enzymes. As result of this modification, any interaction or stereo-electronic effect that may be due to the presence of the oxygen of the cycle is now detached and directly affects the biological response. It is expected that due the structural similarity to the natural counterpart, carbasugars may be recognized by enzymes or biological systems modifying their response.

FIGURE 24.17

Carbasugars are rarely found in nature; there are only few examples of carbafuranoses and carbapyranoses isolated from natural sources (Roscales & Plumet, 2016). Five members cyclitols are encountered in the first group while in the second one it is found carba-α-Dgalactopyranose and cyclohexanes highly substituted by hydroxyl groups or epoxides cyclohexanes and cyclohexenes. There are also examples of carbasugar with amine group also isolated from microorganism. These aminocarbasugars are often second metabolites and are minor components in the fermentation process. Finally, there are some carbaoligosaccharides

Examples of natural carbasugars.

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24.2 Glycomimetics

such as the family of validamycins. All these compounds are compilated in Fig. 24.17. One of the first attempts of using compounds containing carbasugar in the biomedical field, was the ascarbose (see Fig. 24.1 in introduction), which is an effective oral antidiabetic agent, used for the treatment of type II diabetes. Its role is to inhibit the glycosyl hydrolase (Junge et al., 1984; Schmidt et al., 1977), inhibiting the release of glucose. The authors postulated that the carbasugar unit (the unsaturated cyclitol) is presented in a boat conformation and the similarity of this unit to the glucosyl analog may explained the high activity based in the transition state analog theory. In this sense and with the objective to elucidate the mechanism underlying the interaction and inhibition of glycosyl hydrolases, (Ren et al., 2018). designed various glycomimetics of α-galactopyranoside using cyclohexene as starting material, which showed potent inhibition of α-galactosidase from Thermotoga maritima. These compounds inhibit temporally the hydrolase since the activity was restored when the covalent intermediate was hydrolyzed, similar mechanism of retaining glycosidases (Fig. 24.4B). They also synthesized the isotopologues of three carbasugars (Ren et al., 2020) in order to analyze the transition state involved in the interaction with the enzyme. As model enzyme they used the α-galactosidase from T. maritima. They demonstrated that the intact inhibitor as well as the product, presented in the cyclohexene fragment a half-chair conformation (2H3) while it flattens when it links covalently to the enzyme. All the results were

FIGURE 24.18

563

contrasted and confirmed by hybrid QM/MM computational analysis. Danby and Withers (2017) also prepared carbon glycomimetics and studied the transition state stability and the structures involved in the interaction. They concluded that the transition states that lead to the allylic and oxocarbenium ions are similar, almost identical. They postulated that the isoprenyl structure is the key intermediate when carbasugars are used, in contrast to the oxocarbenium which is the well-established intermediate for glycosyl derivatives. The authors claimed that stability observed for the carbasugar intermediates is not a consequence of the stability of the allylic cation. They argued that the instability of the oxocarbenium intermediate is the reason for this difference and that it is due to the electronegative hydroxyl substituent of the glycoside summed to the planar geometry that destabilize the positive charge. Recently, the group of Baran (Karakılıc¸, Durmu¸s, Sevmezler, S¸ ahin, & Baran, 2018) reported the regio- and stereoselective synthesis of pyranose carbasugars from cis-furan, as shown in Fig. 24.18. Inhibition studies over α-glucosidase of Saccharomyces cerevisiae and β- glucosidases from almonds were performed showing that all the products were inhibitors of the enzyme in different degrees but less than ascarbose. This constitutes an interesting approach for the development of new drugs based in glycomimetics for the treatment of varied disorders such as diabetes, viral infections, influenza to mention some. Also (Narayana et al., 2018) designed the synthesis of carbasugars analogs of N-acetyl

Compounds synthesized and IC50 (μM) inhibition of the enzyme activity.

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glucosamine in both families L- and D- using as key steps a Ferrier carbocyclization, a Peterson olefination and an asymmetric epoxidation. The choice of the synthesis of these analogs was based on the biopotential of similar active molecules, such as Validamine (see Fig. 24.17) or Voglibose, which are used as therapeutics that inhibit glycosyl hydrolases. From all the compounds synthesized only three of them showed inhibition of certain glycosidases, their structures are shown in Fig. 24.19. All the products obtained were assayed as potential inhibitors of various glycosyl hydrolases. The author found certain selectivity of one of the compounds for the inhibition of bovine liver β-galactosidase.

24.2.3 Thiosugars Another interesting group of glycomimetics comprises the thiosugars which are characterized by the replacement of an endocyclic or glycosidic oxygen with a sulfur atom. These modifications may be present in

monosaccharides, in the furanose or pyranose form, disaccharides or even oligosaccharides. It is well-established the important role of sulfur compounds in biological systems. In this same sense, there was an increased development and use of sulfur analogs for the design of new drugs and therapeutics for the treatment of many diseases (Das & Banik, 2020), such as cancer disease (Sarnik et al., 2020). This approximation is based on the resemblance between sulfur and oxygen, which gives this compounds unique electronic, conformational and physicochemical properties as well as pronounced biological activities in contrast to the natural counterpart. It is also worthy to mention that the introduction of a sulfur atom confers new and pronounced properties such as the stability to degradation and the ability to resist and inhibit glycosyl hydrolases, which are useful for biomedical applications (Witczak & Culhane, 2005). The first report of a natural thiosugar used for the inhibition of glycosyl hydrolases was the well-known Salacinol (Fig. 24.20) (Yoshikawa et al., 1997). This potent inhibitor of FIGURE 24.19 Carbasugar analogs to N-Acetyl glucosamine synthesized by Narayana et al.

FIGURE 24.20

Examples of thiosugars isolated from Salacia species.

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24.2 Glycomimetics

the α-glucosidase is a sulfonium sulfate compound which was isolated from Salacia reticulata, an antidiabetic Ayurvedic traditional medicine. Also, other sulfonium ions were isolated from the same source and proved their inhibitor activity against the α-glucosidase. This was the case of salaprinol, ponkoranol and kotalanol, which were promising therapeutics for many diseases, especially for type II diabetes. This inspired many groups to develop an efficient synthetic route to get these compounds and diverse derivatives of them. The group of Pinto (Eskandari, Kuntz, Rose, & Mario Pinto, 2010) reported the synthesis of neoponkoranol, the de-O-sulfonated derivative and its 5’-epimer, in a high yield. These compounds proved to be potent inhibitors of the human maltase glucoamylase. Along these years, there were many synthetic approaches to the synthesis of thiosugars, specially related to the construction of the thioglycosidic linkages. Some of these involved substitution of a good leaving groups with an appropriate thiol or thioaldose (Kern & Pohl, 2020), substitution mediated by metals (Ibrahim, Alami, & Messaoudi, 2018), conjugate addition of a thioaldose to an α,β-unsaturated system (Cagnoni, Uhrig, & Varela, 2009; Cristo´falo, Nieto, & Uhrig, 2020; Dada, Manzano, & Varela, 2018; Witczak, Lorchak, & Nguyen, 2007), nucleophilic opening of three membered rings of epoxides and thiiranes (Manzano, Uhrig, & Varela, 2008; Repetto, Manzano, Uhrig, & Varela, 2012; Tamburrini et al., 2017), metal catalyzed reactions (Benmahdjoub, Ibrahim, Benmerad, Alami, & Messaoudi, 2018) or phtotoinducedreactions as the thiol-ene reactions (Borba´s, 2020), to mention some. An interesting approach was reported by (Dada et al., 2018) that synthesized stereoselectively 2-acetamido-2,3-dideoxythiodisaccharides by a conjugate addition (Michael Addition) of 1thioaldoses to a sugar enone system of an OAcetyl oximes. The reduction and later

565

acetylation of the oxime led to the formation of amine analog in C-2. All the products were studied as inhibitor of the β-galactosidase of Escherichia coli and they were a competitive and a mixed inhibitor of the enzyme. Their structures are shown in Fig. 24.21. Another interesting approach is the one proposed by Ueda, Pi, Makura, Tanaka, and Uenishi (2020) that designed the sulfur analog of sucrose, the ( 1 )-5-thiosucrose, stereoselectively. The anomeric configuration was controlled in a single step. Inhibitory activities against α-glucosidase were also examined but no inhibition was observed. In the search of new drugs for the treatment of influenza, Yamabe, Fujita, Kaihatsu, and Ebara (2019) synthesized a sulfur-glycoside containing sialoside. This compound was able to occupy three sites in which the sialic acid bind and was resistant to neuraminidase (NA) that cleaves the O-glycosidic bond on the surface of virus. Although this compound showed certain resistance to NA, it still maintained high binding affinity for various influenza viruses. In order to design new pesticides for Ostrinia furnacalis (a destructive agricultural pest), Shen et al. (2020) designed and synthesized ureido thioglycosides that inhibit a the β-N-acetylhexosaminidase OfHex1, a key enzyme involved in the development of the exoskeleton composed mostly by polysaccharides (mainly chitin). Thus interference of this enzyme results in the death of the insect. The thiosugar were synthesized from glucosamine,

FIGURE 24.21 Thiosugars and inhibition constant for α-galactosidase from Escherichia coli synthesized by Dada et al.

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24. Carbohydrates mimetics: enzyme inhibitors

in a procedure that involved the formation of the thioglucosamine, which was then coupled with a naphthalimide or benzoyl derivative. Evaluation of enzyme inhibition showed that two of the compounds synthesized presented high inhibitory activities against OfHex1.These structures are shown in Fig. 24.22.

24.3 Hybrid carbohydrates It has been shown that binding of carbohydrates to other inhibitors compounds can increase bioavailability and permeability in the biological barrier, decrease hepatotoxicity and improve inhibitory activity. Among these compounds we can mention tacrine, which was the first drug for the treatment of Alzheimer’s disease, but it was withdrawn from the market due to its hepatotoxicity levels. Lopes et al. (2018) synthesized tacrine hybrids with derivatives of natural carbohydrates, D-xylose, Dribose and D-galactose. The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was studied, and all the compounds showed a high inhibitory activity on a nanomolar concentration scale. A D-ribose derivative (7a) and a D-xylose derivative (6b) were more selective for BuChE. In addition, almost all D-xylose (6a-e) compounds exhibited selectivity for AChE. Meanwhile, D-galactose (8a) was the derivative with greatest preference for this enzyme. Finally, the most active compound for both enzymes turned out to be a derivative of D-xylose (6e). Fig. 24.23 summarized all the structures.

FIGURE 24.22

On the other hand, molecular coupling was studied, which showed that inhibitors could provide optimal interactions with the junction cavity and thus efficient blocking. In these systems the main contribution of the linker was to allow a correct positioning of the two moieties. Also, it was noted that there is no linear correlation between linker size and binding affinity. As the synthesized hybrids did not show predicted hepatotoxicity, performance a high inhibitory potential and obey the Lipinski rules, they are promising compounds for Alzheimer’s treatment. Besides, there are tannins that have several promising biological properties, such as their antidiabetic character. However, they have the disadvantage of being difficult to obtain in good purity individually. Cardullo et al. (2020) studied the inhibition of α-glucosidase and α-amylase with fourteen C-glucosidic ellagitannins and three gallolated glucoses. It was observed that most of the derivatives could potently inhibit α-glucosidase with IC50 values less than 10 μM, lower than those of acarbose (260.5 μM), an antidiabetic drug. However, these showed a moderate inhibition against α-amylase, where only four compounds inhibited it considerably. The selectivity shown by these derivatives can be a great advantage since the strong inhibition of α-amylase generates unwanted side effects in most cases. Then, fluorescence quenching studies of the best α-glucosidase inhibitors were carried out. It was noted the same trend as the values obtained

Ureido thioglycosides inhibitors of β-N-acetylhexosaminidase OfHex1.

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24.4 Macromolecules

567 FIGURE 24.23 Tacrine-(carbohydrate-derived) hybrids.

from IC50, being possible to confirm the high affinity of these derivatives for such enzyme, through a static quenching mechanism. This interaction with α-glucosidase was also confirmed by the circular dichroism study. Finally, by evaluating the kinetics of inhibition of α-glucosidase, it was determined that the more active derivative C-glucosidic ellagitannin roburin D (RobD) acts as a competitive inhibitor and the derivative α-pentagalloylglucose (α-PGG) is an inhibitor of mixed type. These compounds may be encouraging for their use as food ingredients with antidiabetic properties, playing a fundamental role in the prevention of diabetes mellitus (Fig. 24.24).

24.4 Macromolecules 24.4.1 Multivalents In the area of macromolecules that inhibit this type of enzymes, carbohydrates also play a fundamental role. Among them it is possible to describe the so-called multivalent inhibitors,

wherein multiple replicas of a sugar are incorporated into a platform. This provides a substantial improvement in the affinity for the enzyme, compared to the monosaccharide. Recently, (Martı´nez-Baile´n et al., 2020). developed a chemical library of iminosugars based on mutimeric pyrrolidines (Fig. 24.25). They evaluated the potential of these compounds to inhibit human α-galactosidase A (α-Gal A) and GCase. The aromatic moiety of some derivatives was found to be important in the inhibition of both enzymes. On the other hand, it was observed that two differently configured iminosugar epitopes were discriminated only by α-Gal A. In addition, the structure-activity relationship was also studied. One of the nonavalent inhibitors showed a potent multivalent effect in the inhibition of α-Gal A (0.20 μM, competitive inhibition), more potent than the monovalent reference. Furthermore, these inhibitors can be used as pharmacological companions, that are capable of binding to the deficient enzyme, and form an enzyme-inhibitor complex. In this way, they help the correct folding of the enzyme, and

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24. Carbohydrates mimetics: enzyme inhibitors

FIGURE 24.24

C-glucosidic ellagitannins and galloylated glucoses.

FIGURE 24.25

Multivalent pyrrolidine iminosugars.

improve traffic to lysosomes, thus avoiding their early degradation. After the mutant enzyme stabilizes and reaches the lysosomes, the complex dissociates and the enzyme can hydrolyze the substrate, preventing accumulation in the lysosomes. Therefore the best α-Gal A inhibitors were evaluated as potential

pharmacological companions. For this, its ability to increase the activity of this enzyme in R301G fibroblasts was studied in patients with Fabry disease. The nonavalent inhibitor presented a higher enzymatic activity, increasing the activity of the misfolded enzyme to 2.5 mM. Results places this study as a pioneer

III. Functional Applications

24.4 Macromolecules

of multivalent inhibition for the human α-Gal A enzyme, also being the first evidence that multivalent iminosugars have to act as potential pharmacological companions in the treatment of this disease. Another way to build multivalent compounds is through supramolecular self-assembly. Li et al. (2019) synthesized an amphiphilic derivative of deoxynojirimycin FA-DNJ with fatty chain conjugates. This derivative was able to efficiently selfassemble in aqueous medium at different pH values, in spherical micelles, as shown in Fig. 24.26. The inhibition of FA-DNJ against α-mannosidase (Jack bean), α-galactosidase (Green coffee beans), β-mannosidase (Helix pomatia) and β-galactosidase (E. coli) was studied in vitro. Micelles showed a powerful inhibitory activity of α-galactosidase and α-mannosidase, achieving for the latter an improvement with respect to known miglitol. After in vivo and cell viability studies, FA-DNJ was found to have a hypoglycemic effect similar to that of miglitol and nontoxic behavior, making it a promising multivalent glycosidase inhibitor for clinical applications.

24.4.2 Polysaccharides Within this group of macromolecules are polysaccharides, obtained from natural products. These compounds have been studied extensively for their various biological activities, for not presenting toxicity, for not causing significant side

FIGURE 24.26

569

effects, and because they provide inhibition against these enzymes. Thus multiple studies have focused on the analysis of natural polysaccharides as inhibitors of α-glycosidases. Deng et al. (2020) analyzed a new polysaccharide fraction obtained from seeds of Chaenomeles speciosa. The polysaccharide was extracted, purified and physical and chemical characterized. It was observed that its structure was composed of D-rhamnose, D-galactose, Dglucuronic acid and L-arabinose. Then it was performed hypoglycemic studies and a good inhibition of α-amylase and α-glucosidase was observed. Therefore this new natural polysaccharide has potential for use in postprandial hypoglycemia, delaying its effects. Another research focused on the characterization and study of inhibition of polysaccharides, is the work of Gu et al. (2020). They obtained an alkali-extracted acidic polysaccharide from residues of Annona squamosa, a fruit specie. A new polysaccharide called AWPA was characterized, finding that it is composed of L-arabinose, D-galactose, D-glucose, D-mannose and D-galacturonic acid. Its inhibitory activity in vitro against α-glucosidase and α-amylase was studied, showing that the polysaccharide inhibited both enzymes. Besides, enzymatic kinetics were evaluated, and it was noted that inhibitory effects of α-glucosidase were reversible and mixed-type. However, the inhibitory effects were reversible and competitive on α-amylase. Finally, results

FA-DNJ structure and schematic diagram of self-assembling into micelles.

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show that AWPA is a potential alternative in the development of inhibitors, helping in avoiding the waste of Annona squamosa residues. These results prompted the author to postulate that natural polysaccharide are a promising source of inhibitors.

24.5 Conclusions Carbohydrate constitutes an attractive source of compounds that are being studied for application in varied of areas, ranging from the biomedicine to the materials areas. Its great structural diversity, accessibility and possibility for tuning their properties by physical or chemical modification, turns them into promising compounds for the development of new drugs and materials. On the other hand, the study of this widespread group of enzymes, the glycosidases, have attracted the attention over the last years because of their predominant role in many biological processes. This growing importance suggests that the inhibition of these enzymes constitutes an interesting and novel strategy to approach new therapies against numerous diseases. In this point glycomimetics are crucial. The present overview highlights this strong interest and some of the latest and more relevant reports showing all their potential as result of their excellent ability to interact with mentioned enzymes. Thus the results reported here try to demonstrate that diverse families of glycomimetics have the possibility to act as main players in different therapeutic treatments.

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1933. Available from https://doi.org/10.1016/j. carres.2007.06.005.

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References

Wolfsgruber, A., Thonhofer, M., Weber, P., Nasseri, S. A., Fischer, R., Schalli, M., et al. (2020). N-alkylated iminosugar based ligands: Synthesis and inhibition of human lysosomal-glucocerebrosidase. Molecules (Basel, Switzerland), 25, 4618. Available from https://doi.org/ 10.3390/molecules25204618. Yamabe, M., Fujita, A., Kaihatsu, K., & Ebara, Y. (2019). Synthesis of neuraminidase-resistant sialoside-modified three-way junction DNA and its binding ability to various influenza viruses. Carbohydrate Research, 474, 43
50. Available from https://doi.org/10.1016/j.carres.2019.01.008. Yan, L., Lui, H., Sun, J., Gao, L., Lu, X., Li, X., et al. (2019). Synthesis of tricyclic benzimidazole-iminosugars as potential glycosidase inhibitors via a Mitsunobu reaction. Carbohydrate Research, 485, 107807. Available from https://doi.org/10.1016/j.carres.2019.107807. Yan, L., Yin, Z., Niu, L., Shao, J., Chen, H., & Li, X. (2018). Synthesis of pentacyclic iminosugars with constrained butterfly-like conformation and their HIV-RT inhibitory

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activity. Bioorganic & Medicinal Chemistry Letters, 28, 425
428. Available from https://doi.org/10.1016/j. bmcl.2017.12.025. Yoshikawa, M., Murakami, T., Shimada, H., Matsuda, H., Yamahara, J., Tanabe, G., et al. (1997). Salacinol, potent antidiabetic principle with unique thiosugar sulfonium sulfate structure from the ayurvedic traditional medicine Salacia reticulata in Sri Lanka and India. Tetrahedron Letters, 38, 8367
8370. Available from https://doi.org/ 10.1016/S0040-4039(97)10270-2. Zamoner, L. O. B., Araga˜o-Leoneti, V., & Carvalho, I. (2019). Iminosugars: Effects of stereochemistry, ring size, and n-substituents on glucosidase activities. Pharmaceuticals, 12. Available from https://doi.org/ 10.3390/ph12030108. Zechel, D. L., & Withers, S. G. (2000). Glycosidase mechanisms: Anatomy of a finely tuned catalyst. Accounts of Chemical Research, 33, 11
18. Available from https:// doi.org/10.1021/ar970172 1 .

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C H A P T E R

25 Current challenging issues of biological macromolecules in biomedicine Y. De Anda-Flores1, E. Carvajal-Millan1, A.C. Campa-Mada1, K.G. Martı´nez-Robinson1, J. Lizardi-Mendoza1, A. Rasco´n-Chu2, A.L. Martı´nez-Lo´pez3 and J. Tanori-Cordova4 1

Biopolymers-CTAOA, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico 2Biotechnology-CTAOV, Research Center for Food and Development (CIAD, A.C.), Hermosillo, Mexico 3NANO-VAC Research Group, Department of Chemistry and Pharmaceutical Technology, University of Navarra, Pamplona, Spain 4Department of Polymers and Materials Research, University of Sonora, Hermosillo, Mexico

25.1 Introduction As their name indicates, biomacromolecules are macromolecules of biological origin essential for all life forms (animals, plants, and microorganisms). According to their structure and function, biomacromolecules are classified into four major classes: carbohydrates, proteins, lipids, and nucleic acids. These are formed from smaller molecules (monomers) and repeated sequence, connected by covalent bonds (Krishnamurthy, 2017). The study of these biomolecules has led to the development of biomaterials of biomedical importance as they can mimic the human body’s in vivo environment. Biomaterials based on biomolecules can be designed to adapt to various forms and complement synthetic materials. These systems are on a molecular scale that ranges

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00025-7

from macro, micro, and nano. Due to their biological nature, these biomaterials offer great advantages such as biocompatibility, molecular recognition, response to biological stimuli, and ability to adapt in biochemical environments (Datta, Manchineella, & Govindaraju, 2020). A biomaterial must present biological characteristics such as nontoxicity, noncarcinogenicity, nonallergenicity, noninflammatory, biocompatibility, and biodegradability. Therefore, the study of polymeric biomaterials of natural origin has revealed that they can present these biological characteristics and specific binding sites for proteins and other biochemical signals that make them suitable for use in biomedicine (Samadian et al., 2020). Currently, advances in biomedicine have made possible the design and manufacture of

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25. Current challenging issues of biological macromolecules in biomedicine

biomaterials from macromolecules to be used in different applications as peptide, protein, and drug delivery systems (Agrahari, Agrahari, & Mitra, 2016). Proteins and peptides are being used as therapeutic biomolecules due to their important role in medicine; these therapies were established using insulin. This protein was the first to be approved for commercialization and served as a treatment for diabetes mellitus (Muheem et al., 2016). Polysaccharide-based biomaterials for biomedical applications have been on the rise. Some of the polysaccharide polymers commonly used are chitosan, alginate, starch, and cellulose, among others. These can be easily modified to improve their mechanical, optical, and biocompatibility properties. In biomedicine, they are used as vehicles for drug delivery, wound healing, and tissue engineering (Harling et al., 2020). Oral drug administration is a method that provides many benefits to patients because it is painless, noninvasive, and comfortable to handle. The drugs and other molecules that can be delivered orally consist of tablets, powders, drops, and liquids. However, this technique can have disadvantages, as it must overcome multiple barriers in the human body (Reinholz, Landfester, & Maila¨nder, 2018). For this reason, the use of coatings can prevent the degradation of the drug and increase its absorption rate when it is delivered orally. These techniques are highly supportive of protein therapies, including enzymes, peptides, and cytokines to treat diseases such as cancer, autoimmune diseases, and metabolic disorders (Wang, Liu, & Mo, 2020). Other techniques consist of the use of biomaterials for wound healing and tissue engineering. Damage and failure in tissues and organs’ function is usually the result of injuries, diseases, and other types of damage that lead to health problems. Currently, treatment methods include surgery, drug therapy, transplants, or prostheses. However, on occasions when these interventions do not help all these

conditions, tissue engineering has proven to be a complementary alternative in biomedicine (Abbasian, Massoumi, Mohammad-Rezaei, Samadian, & Jaymand, 2019). Likewise, this method combines engineering and chemistry, molecular biology, and materials science, which replace or repair damaged tissues or organs using living cells, scaffolds, and signal molecules. It is important to consider the proper use of natural biological macromolecules and synthetic polymers to obtain successful biomaterials (Ullah & Chen, 2020). This chapter focuses on the biomedical challenges and applications of biological macromolecules in developing biomaterials for targeted drug delivery, tissue engineering, and wound management. This contribution aims to present important information from the literature that shows various biomaterials derived from two or more types of biomolecules and their applications in biomedicine.

25.2 Biological macromolecules Biological macromolecules are molecules necessary for life and are built from smaller ones. Carbohydrates, lipids, proteins, and nucleic acids are the main biological macromolecules with an important cellular function, constituting all living beings’ basic structures and functions (Bovey, 1979). Carbohydrates are macromolecules with complex chemical structures that have physiological functions in the living system. The vast majority are obtained from plants, animals, microbes, fungi, and algae. They can build structural polysaccharides such as pectins, hemicelluloses, cellulose, alpha hand, and alpha/beta galactan, and reserve polysaccharides such as glycosaminoglycans, glycogen, and starch (Kaur & Singh, 2020; Roger, Westerlund, & Aman, 2006; Song, Fan, Hu, Cheng, & Xu, 2020). Proteins are monodisperse macromolecules that contain a sequence of 20 amino acids that correspond to

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25.3 Macromolecules in biomedical applications

20 different sides of the R chain. Therefore, they can adopt many complex but defined secondary structures, and the conformation of the chains shows a high degree of variability from protein to protein. They participate in a wide variety of structural functions in cell walls, skin, bones, the transport and storage of oxygen, such as hormones, immune protection, and the control of gene function (Bovey, 1979; Borchmann, Carberry, & Weck, 2014). Lipids have multiple essential functions, e.g., as cell membranes, energy storage, and their participation in signaling processes (Di Mascio et al., 2019). Nucleic acids are composed of sugars, deoxyribose for deoxyribonucleic acid (DNA) and ribose for ribonucleic acid (RNA), interconnected by a phosphodiester bridge between the 30 and 50 carbons. A nitrogenous base is linked to each sugar using an N-glycosidic bond, which gives the nucleoside name; when this nucleoside is linked to more phosphates, it is called a nucleotide. In nucleic acids, nitrogenous bases are classified into two categories: pyrimidine bases (cytosine, thymine [DNA] and uracil [RNA]), and purine bases (adenine and guanine) as described in Table 25.1 (Geinguenaud, Guenin, Lalatonne, & Motte, 2016). Due to their structures and functions, macromolecules have received great interest in biomedicine. The use of these and their derivatives have been increasing due to the immense demand for functional and smart biomaterials in both the biomedical and

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pharmaceutical industries (Chakhalian, Shultz, Miles, & Kohn, 2020).

25.3 Macromolecules in biomedical applications Innovations in the biomedical industry have had a great impact and, therefore, it is divided into four sectors: pharmaceutical industry, biotechnology industry, medical technology, and health information technology. The materials used in these sectors have driven biodegradable, biocompatible, bioabsorbable, and bioactive materials (Lee, 2018). Macromolecules have been studied due to their numerous applications in tissue engineering, drug delivery, immunological engineering, and medical device manufacturing (Borchmann et al., 2014; Wang et al., 2020; Xue et al., 2019). Biomaterials are defined as a natural or synthetic material intended to interact with a biological system. These biomaterials are currently used in many biomedical and pharmaceutical preparations, playing a central role in repairing, reinforcing, or replacing functional parts of the human body (Trimukhe, Pandiyaraj, Tripathi, Melo, & Deshmukh, 2017). Biomaterials have their challenges based on its intended application at the biological site. Despite the promising results, there is an urgent need to develop new types of biomaterials with adequate mechanical properties, durability, and

TABLE 25.1 Chemical properties and functions of biological macromolecules. Macromolecule Basic formula

Monomer

Functions

Carbohydrates

CH2O

Monosaccharides Energy storage and structure

Proteins

NH2 1 -COOH 1 R group

Amino acids

Storage, signals, structural, contractile, defensive, enzyme, transport, and receptors

Lipids

R-COOH

Fatty acid and glycerol

Energy, storage, protection, chemical messengers, repel water

Nucleic acids

Sugar, a nitrogenous base, Nucleotides phosphate

Genetic information

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25. Current challenging issues of biological macromolecules in biomedicine

functionality (Abbasian et al., 2019). The interactions of a biomaterial with biological components (tissues, blood, and body fluids) are closely related to the material’s surface and the biological stimuli. The biological stimuli are triggered in living tissues by being in contact with these extrinsic biomaterials; these interactions at the molecular level determine the biocompatibility of the biomaterial. Likewise, other important characteristics are their stability, tissue compatibility, zero toxicity, and reproducibility. Materials used in vivo studies must be approved by the FDA (Food and Drug Administration); otherwise, the material must undergo a series of biocompatibility tests: Cytotoxicity, hemolysis, intravenous toxicity, mutagenicity, oral toxicity pyrogenicity, and sensitization (Park & Lakes, 2007; Ratner, Hoffman, Schoen, & Lemons, 2004).

25.4 Macromolecules in targeted drug delivery Targeted drug delivery seeks delivery to receptors or organs in a specific part of the body, improving their efficacy, and reducing side effects (Manish & Vimukta, 2011). These help to model the pharmacokinetics of the drug and its therapeutic effect. Due to their nature, biomaterial systems derived from macromolecules (proteins, polysaccharides, lipids, and nucleic acids) offer many advantages as biocompatibility, biomolecular recognition, and responsiveness to biological stimuli for a wide variety of application routes (Table 25.2) (Bovey, 1979; Kowalski, Bhattacharya, Afewerki, & Langer, 2018). Currently, the main classes of approved drugs are small molecules (,100 atoms) and therapeutic proteins. Small molecules can adapt, which allows them a rapid passive diffusion through cell membranes, unlike protein therapies that cannot cross this cell barrier, limiting protein therapeutics to extracellular targets (Verdine & Walensky, 2007). Proteins are biomolecules with several applications in

biomedicine. These are beneficial due to their biogenic capacity, which makes them no toxic. Likewise, this characteristic leads to bioavailability problems because they can degrade rapidly under environmental conditions and in the absence of proteases. Also, proteins can trigger negative immune responses (Borchmann et al., 2014). Protein-based drugs can have high specificity to various targets or be used to replace missing or mutated proteins. Still, their size and stability can limit their use for specific targets or diseases. This limitation prevents them from being targeted at all genes or certain proteins, so protein messenger RNA (mRNA) and DNA precursors are promising macromolecules for therapeutic use. They can be targeted in a specific way through the base-pairing of WatsonCrick in gene editing treatments (Kaczmarek, Kowalski, & Anderson, 2017). The nucleic acid delivery has focused on designing methods and materials for drug administration based on DNA and RNA, nucleobases, and their derivatives. The specific assembly of the sequence helps the biomaterial manufacturing process. Oligonucleotides, mRNA technologies, as well as RNA modification therapies [RNA interference (RNAi), antisense oligonucleotides] that silence transcribed mRNA from disease genes, are advancing in clinical trials for drug development (Morrison, 2018; Yin, Kauffman, & Anderson, 2017). RNA drugs have emerged to treat diseases at the gene and RNA level, but their delivery remains one of the main challenges. They are prone to degradation by nucleases and can activate the immune system. Therefore, they are often designed in conjunction with pH-sensitive biomaterials, such as lipids or polymers that contain ionizable functional groups (Kaczmarek et al., 2017; Kowalski, Bhattacharya et al., 2018). Various biomolecules such as lipids, polymers, peptides, proteins, and extracellular vesicles have been designed and explored to deliver mRNA in vitro and in vivo. Polymers have been used to develop delivery systems capable

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25.5 Biomaterials as targeted drug delivery

TABLE 25.2 Macromolecules as drug delivery system and their applications. Active Macromolecule molecule Biomaterial

Delivery route

Study Comments

References

CalciumAlginate

OVA

Microsphere

Oral delivery

In vivo

The uncoated microparticle had a macroporous structure and rapid dissolution at intestinal pH.

Suksamran et al. (2013)

INA

IDM

Microsphere

Oral delivery

In vitro

IDM was released only in a simulated colonic fluid.

Jain, Sood, Bora, Vasita, and Katti (2014)

PEG

BCA

NCL

Oral delivery

In vitro

The PEG-NCL offered a possible solution to improve the oral bioavailability of a poorly water-soluble drug.

Wang et al. (2015)

SWNT-CHIHA

DOX

Nanotubes

Intravenous injection

In vivo

The SWNT-CHI-HA-DOX showed high drug loading efficiency, controlled and sustained release, and targeting to cancer cells.

Mo et al. (2015)

Dextran

5-FU

Microspheres

Oral delivery

In vivo

The uncoated microspheres released the Rai, Yadav, 5-FU before they reached the colon. Jain, and Agrawal (2015)

ApS

DOX

DNA Intravenous nanostructures injection

In vivo

The ApS recognized their targets and showed affinity and selectively transported the drug into cancer cells.

Liu et al. (2016)

PPD

DOX

MNRs

In vivo

The MNRs can achieve intracellularlevel drug delivery

Chen et al. (2019)

Intravenous injection

ApS, Aptamer-dsDNA; BCA, Biochanin A; DOX, Doxorubicin; 5-FU, 5-Fluorouracil; IDM, Indomethacin; INA, Inulin acetate; MNRs, Multifunction nanorods; NCL, Nanostructured lipid carriers; OVA, Ovoalbumin; PEG, Polyethylene glycol; PPD, Polypeptide-polyethylene glycol (PEG) derivative; SWNT-CHI-HA, Carboxyl single-walled carbon nanotubes-Chitosan-Hyaluronan.

of carrying small molecules, such as proteins and nucleic acids. These polymeric carriers have been designed for the delivery of short RNAs (B7
14 kDa) (antisense oligonucleotides and interference RNA) and the delivery of mRNA molecules (B600
1000 kDa) (Kowalski, Capasso Palmiero et al., 2018). Lipids and materials derived from them have been used to administer DNA, RNAi, and mRNA through the preparation of liposomes, lipid nanoparticles, lipid emulsions, among others (Weng et al., 2020). According to the global report on medicine (IMS, 2019), it is expected that by 2023 the world pharmaceutical market will exceed

1.5 trillion dollars, and its annual growth rates will increase from 3% to 6%. The main drivers of growth will continue to be the United States with 4%
7% and pharmerging markets with 5%
8% annual growth.

25.5 Biomaterials as targeted drug delivery 25.5.1 Hydrogels for drug delivery Hydrogels are three-dimensional polymeric networks capable of retaining water or

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25. Current challenging issues of biological macromolecules in biomedicine

biological fluids, giving them the possibility of swelling and making them insoluble due to their covalent, ionic, or physical crosslinks hydrophilic residues (carboxyl, hydroxyl, ether, and amino groups) (Peppas, 2000). These hydrogels can be made from various polysaccharides such as chitosan, alginate, dextran, arabinoxylans (AX), and starch derivatives (Carvajal-Millan, Guilbert, Morel, & Micard, 2005; Coviello, Matricardi, Marianecci, & Alhaique, 2007). The physicochemical properties of hydrogels are given by their chemical composition, functional groups, manufacturing method, and the polymer chain’s molecular weight, among others. Their water absorption capacity, flexible nature similar to natural tissue, and biocompatibility give them various potential applications. An example of these is their use as food additives, enzyme immobilizers, controlled release devices, and tissue engineering scaffolds, having multiple applications, mainly in sectors biomedical and pharmaceutical (Berlanga-Reyes et al., 2009; Herna´ndez-Espinoza, Pin˜o´n-Mun˜iz, Rasco´nChu, Santana-Rodrı´guez, & Carvajal-Millan, 2012). The first hydrogel manufactured for biomedical use was originated in 1960, and over the years, hydrogels have been improved and modified according to advances in biomedicine (De France, Cranston, & Hoare, 2020; Wichterle & Lı´m, 1960). Hydrogels are found in many sizes and shapes with characteristic properties; they are classified as macrogels, microgels, and nanogels. Macrogels are materials with pores bigger than 50 nm, unlike microgels are particles with sizes ranging from 0.1 to 100 μm. On the other hand, nanogels are defined as structures with lattice sizes between 1 and 100 nm (Kudaibergenov, Nuraje, & Khutoryanskiy, 2012). Macrogels can be previously formed and implanted in the body for local delivery; they can also include microparticles and nanoparticles inside a composite hydrogel (MarquezEscalante & Carvajal-Millan, 2019; Morales-

Ortega et al., 2014; Mu, Li, Tong, & Guo, 2019). Due to their size, microgels can be used for oral or pulmonary delivery; they can encapsulate peptides, probiotics, and other classes of microorganisms and proteins (McClements, 2017; Morales-Burgos et al., 2019). Nanogels have properties similar to nanoparticles and can be functionalized for targeted delivery into cells or specific body areas (Kim, Moon, Kim, Heo, & Jeong, 2018; Maciel, Yoshida, Pereira, Goycoolea, & Franco, 2017; Mohammed, Syeda, Wasan, & Wasan, 2017). Hydrogels can be divided into ordinary and smart. Ordinary hydrogels are not sensitive to environmental changes, unlike smart ones affected by pH, temperature, and photoelectricity (Aeridou, Dı´az Dı´az, Alema´n, & Pe´rez-Madrigal, 2020). 25.5.1.1 Injectable hydrogels as drug delivery systems Hydrogels, as a sustained release injectable drug delivery system, have presented various challenges because they need to be biocompatible, biodegradable, nontoxic, and capable of providing sustained drug release. These characteristics are given according to the type of material used to form the gel and the time it takes to form. Recently, injectable hydrogels have attracted attention because they can be targeted at the site of interest using minimally invasive approaches. They can be cross-linked in situ using ultraviolet (UV) light, enzymes, ions, or temperature (Fan, Tian, & Liu, 2019; Mathew, Uthaman, Cho, Cho, & Park, 2018; Shrestha, Regmi, & Jeong, 2020). Generally, in situ crosslinking of hydrogels can present problems as there may be leakage of the initial precursor solution into adjacent tissue or the bloodstream and catheter blockage as a result of premature polymerization. For this reason, shear-thinning hydrogels have been developed, allowing them to lose their shape once in contact with needles and catheters and to revert to their original shape

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25.5 Biomaterials as targeted drug delivery

once the mechanical force is removed (De France et al., 2020; Yu & Ding, 2008). Ye et al. (2016) designed a self-healing hydrogel of polyglycerol sebacate-polyethylene glycol methyl ether methacrylate (PGSPEGMEMA)/α-cyclodextrin (αCD). This hydrogel could be sheared into a liquid during injection and has the potential to reverse into gel postinjection. The gel was biocompatible and biodegradable. It presented cell viability with human fibroblast cells of at least 80%. It was degraded under accelerated degradation conditions (acidic and basic environment), which allows being a hydrogel suitable for its use in vivo studies. The hydrogel exhibited the ability to recover into gel after being diluted by shear into a liquid, indicating a thixotropic property (Fig. 25.1). In in vitro studies of drug

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release, the gel presented release in two phases; the first phase took place during diffusion and the second phase occurred after the erosion of the gel. The development of injectable hydrogels, with properties such as tissue adhesiveness and pH sensitivity, is desirable for the localized delivery of drugs to treat diseases. In this type of materials, their design has been a challenge. Samimi Gharaie, Dabiri, and Akbari (2018) designed an injectable biomaterial composed of Chi microgels (pH-sensitive), chitosan microgels, and poly N-isopropyl acrylamideco-acrylic acid (PNIPAM). They were placed in laponite and gelatin, as seen in Fig. 25.2. This biomaterial was developed to use pH as an external stimulus for the localized delivery of drugs.

FIGURE 25.1

Shear-thinning of the PGS-PEGMEMA/αCD hydrogel in a liquid and its conversion to a gel at rest. Source: Adapted from Ye, H., Owh, C., Jiang, S., Ng, C., Wirawan, D., & Loh, X. (2016). A thixotropic polyglycerol sebacate-based supramolecular hydrogel as an injectable drug delivery matrix. Polymers (Basel), 8, 130. https://doi.org/10.3390/polym8040130.

FIGURE 25.2 Diagram of manufacturing a gel with pH-sensitive microgels. Source: Adapted from Samimi Gharaie, S., Dabiri, S., & Akbari, M. (2018). Smart shear-thinning hydrogels as injectable drug delivery systems. Polymers (Basel), 10, 1317. https://doi.org/10.3390/polym10121317.

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With the development of hydrogels as treatment systems for drug delivery in cancer therapies, drugs are retained and injected directly into or alongside the tumor. Thermosensitive, sensitive to pH changes, photosensitive, and dual sensitive biomaterials, have been designed for tumor treatments (Fan et al., 2019). pHsensitive materials are the most widely used since tumors exhibit extracellular pH lower than normal tissues and bloodstream. Liang et al. (2019) designed injectable hydrogels with sensitivity to pH 5 and mucosal adhesion as localized drug carriers. These were modeled using dihydrocaffeic acid solution grafted with chitosan, oxidized pullulan, and doxorubicin as the anticancer drug. These hydrogels exhibited great advantages such as fast gelation time, good injectability, pH-dependent, swelling, and good rheological properties. It turned out to be a self-adhesive drug delivery system that maintained a sustained release system for colon cancer therapies. Temperature-sensitive hydrogels can alter their volume according to the ambient temperature changes. These gels tend to be manufactured with hydrophobic and hydrophilic groups, causing this temperature variation to affect the hydrophobic interaction of their groups (Cirillo, Spizzirri, Curcio, Nicoletta, & Iemma, 2019). Resistance to drugs in chemotherapy treatments is usually a problem; an example is the use of doxorubicin as a chemotherapeutic agent. This drug has limitations concerning its side effects and resistance to therapy, so the use of hydrogel-assisted chemotherapy can reduce these effects, which is still under study. Chung et al. (2020) developed poloxamer 407 hydrogels with doxorubicin and studied its pharmaceutical, biological, and physicochemical parameters. The P407 hydrogels were kept as fluids at a temperature of 4 C and solidified at 37 C (Fig. 25.3). These materials managed to retain the drug at the injection site, and the antitumor effects were observed.

FIGURE 25.3 Doxorubicin loaded P407 hydrogels at 4 C (liquid-state) and 37 C (solid-state). Source: Adapted from Chung, C. K., Garcı´a-Couce, J., Campos, Y., Kralisch, D., Bierau, K., Chan, A., et al. (2020). Doxorubicin loaded poloxamer thermosensitive hydrogels: Chemical, pharmacological and biological evaluation. Molecules (Basel, Switzerland), 25, 2219. https://doi.org/10.3390/molecules25092219.

25.5.1.2 Oral hydrogels as drug delivery systems Oral administration of drugs continues to be the route most studied by scientists and preferred by patients since it does not need to be administered by health professionals. The disadvantage presented is the early release due to the variation in pH and the biological environment in the gastrointestinal (GI) tract (Muheem et al., 2016; Tyagi, Pechenov, & Anand Subramony, 2018). The GI tract is capable of digesting carbohydrates, proteins, and other nutrients and breaking them down to their monomers (amino acids and simple sugars, respectively). Likewise, the GI tract can avoid pathogens’ passage; therefore, orally administered drugs must overcome various biological obstacles before absorption (Brown, Whitehead, & Mitragotri, 2020). The manufacture of drug-bearing biomaterials has presented several challenges. These biomaterials tend to degrade due to changes in pH throughout the GI tract, the presence of proteolytic enzymes, the epithelial barrier, composition and thickness of the mucosa, and their rapid excretion, which leads to drug loss and makes

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it difficult to access the systemic bloodstream (Reinholz et al., 2018; Soleymani-Goloujeh & Mahmoodpour, 2016). This disadvantage leads to lower systemic bioavailability of drugs administered orally than those administered intravenously (Mathew et al., 2018). Therefore, the development of materials that improve its administration and extend the drug’s release time throughout the GI tract has been of great importance (Vertzoni et al., 2019). Due to the challenges involved in this drug delivery route, various action mechanisms have been studied for biomaterials used for this purpose. One of them is the mucoadhesion and mucopenetration of biomaterials; these being mucoadhesives increase the drug’s residence time in the desired site and reduce the dilution effects. Mucous membranes are found in various body regions such as the eyes, the respiratory tract, the GI tract, and the reproductive organs. In many cases, this mucus layer becomes an obstacle to drug delivery to the underlying epithelium (Netsomboon & Bernkop-Schnu¨rch, 2016). One of the polysaccharides most studied and with mucoadhesive capacity is chitosan because it is a hydrophilic, pH-sensitive, and cationic polysaccharide. These chitosan-based mucoadhesive particles can extend the residence time in mucosal areas such as the small intestine, which helps prolong the drug’s absorption (Ways, Lau, & Khutoryanskiy, 2018). Mucopenetrating biomaterials can penetrate the mucus layer, which facilitates the transit of proteins and peptides reaching the epithelium. In these mucopenetrant biomaterials, two systems are studied: passive and active. The passive system does not have the ability to interact with the underlying mucus or epithelium actively. The challenge of this system is to maintain minimal surface interactions with the micro and nanocarrier systems. On the other hand, the active system can change the mucosa’s properties or properties through chemical reactions (Netsomboon & Bernkop-Schnu¨rch, 2016).

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Another mechanism is enzyme inhibition. The use of enzyme inhibitors is a strategy used to prevent the drug’s breakdown in the intestine. An example is the use of proteases capable of inactivating proteolytic enzymes present in the digestive tract and avoiding protein degradation (Banerjee & Mitragotri, 2017). The mechanisms that enhance paracellular permeation are divided into first and second generation enhancers. The first generation can open tight junctions through intracellular signaling mechanisms, and the second generation act through the direct interruption of homophilic interactions in the cell adhesion recognition sequences (Maher, Brayden, Casettari, & Illum, 2019). On the other hand, transcellular permeation enhancers allow the protein load’s translocation, facilitating its diffusion through the cell (Dahlgren, Sjo¨blom, Hedeland, & Lennerna¨s, 2020). 25.5.1.2.1 Hydrogel-based microparticles as drug delivery systems

Micro and nanomaterials biomaterials for oral delivery systems are the most studied systems (Yu et al., 2016; Zhang, Sang, Feng, Li, & Zhao, 2016). Microparticles or microgels have a three-dimensional network, which allows them to contain drugs, proteins, nucleic acids, and even cells. Their type of structure confers stability to the particle. This network can protect the molecule loaded, delivered to the targeted place, and respond to external stimuli by volume contraction and size reduction (Burchard, 2007; McClements, 2017). For example, biomaterials based on polysaccharides such as alginate, chitosan, carboxymethylcellulose, carrageenan, gelatin, AX, and pectins are used to form these biomaterials by different technologies such as coacervation, electrospray, spraydrying, emulsion, among others (Anal & Singh, 2007; Ghayempour & Mortazavi, 2013; Wu & Zhang, 2018). Moreno et al. (2018) report the manufacture of chitosan microparticles by electrospray. These microparticles showed diameters of 1 μm and self-adhesive properties.

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Galdioli Pella´ et al. (2020) synthesized hybrid microgels based on modified chitosan and silicon oxide nanoparticles (NP SiO2-VTS) to deliver them in treatments for gastric wounds. The NP SiO2-VTS was added to coat the microgels’ structure and improve the matrix to avoid degradation. The results indicated that the matrix at pH 1.2 suffered from degradation caused by the weakening of the microgel’s chemical bonds, so the NP SiO2-VTS was not an efficient coating to functionalize the microgel. The encapsulation of insulin for oral administration in patients with Type 1 diabetes has presented a great challenge due to the easy degradation of insulin by the small intestine’s proteolytic enzymes (McClements, 2018; Muheem et al., 2016). Type 1 diabetes is an autoimmune disease that destroys most insulin-producing islets’ beta cells, while the remaining cells are unable to produce enough insulin to maintain glucose homeostasis (Atkinson & Eisenbarth, 2001). There is no treatment to stop this disease, but insulin treatment is necessary to control hyperglycemia. This treatment consists of three to four subcutaneous injections of recombinant human insulin per day, with long-acting or fast-acting doses. This type of treatment is uncomfortable for patients, as it can have side effects such as peripheral hyperinsulinemia or portal hypoinsulinemia (Panchal, Shah, & Upadhyay, 2011; Salsali & Nathan, 2006). The innovation and improvement of biomaterials for the supply of insulin by different routes such as oral, transdermal, ocular, buccal, nasal routes, among others, have been promoted (Grassin-Delyle et al., 2012; Morales-Burgos et al., 2019; Paz-Samaniego et al., 2018; Souto et al., 2019; Zaman, Hanif, & Khan, 2018). Oral insulin delivery has gained interest because of its cost benefits, lower risk of infection, and practicality for patients. However, the development of biomaterials for the oral delivery proteins or drugs capable of improving their

absorption has been a great challenge (Mohammed et al., 2017; Patel, 2013). Colorectal cancer (CRC) represents one of the leading causes of mortality and morbidity in the world (Issa & NouredDine, 2017). Recently, research has focused on developing bioactive materials for oral delivery treatments of CRC to the colon (Zhang et al., 2016). Among the challenges for delivering oral insulin and drugs for CRC treatments, directing their arrival and release in the colonic region has allowed the design of biomaterials capable of carrying them intact through the stomach and small intestine. This strategy allows preserving their integrity so they can be absorbed by the systemic route (circulation). The epithelial cells of the intestine do not transport macromolecules such as insulin or drugs that are directed to cancerous tumors (Amidon, Brown, & Dave, 2015; Moroz, Matoori, & Leroux, 2016; Tyagi et al., 2018). Microencapsulation can protect insulin and drugs against degradation in the upper GI tract and targeted release in the circulatory system. The colon has become a preferred site for delivering peptides, therapeutic proteins, and drugs because digestive enzymes and metabolic enzymes are less in this part of the GI tract compared to the small intestine. The colon is the place with the most abundant microbiota in the entire GI tract, predominantly anaerobic bacteria such as Bacteroides, Bifidobacteria, Eubacteria, Enterobacteria, among others. These bacteria produce a large amount of reducing and hydrolytic enzymes such as β-glucuronidase, β-xylosidase, α-arabinoside, β-galactosidase, hydroxylase, among others. The mechanism of action to release at the colon level has led to the development of systems activated by microbiota enzymes and sensitive to pH (Lee et al., 2020). Studies have shown the development of biomaterials for the controlled release of drugs activated by the colonic microbiota. Likewise, these biomaterials must maintain the efficiency of the proteins or drugs within their system. This release system occurs through the use of

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25.5 Biomaterials as targeted drug delivery

its microbiota, which promotes the release of drugs embedded in matrices capable of being degraded by the enzymes of colonic bacteria. Some authors have reported the use of polysaccharides such as pectin, guar gum, inulin, chitosan, and AX as metabolized systems by the colonic microbiota, facilitating drug release (Wong, Al-Salami, & Dass, 2018). Martı´nezLo´pez et al. (2019) fabricated microspheres from AX/insulin by enzymatic crosslinking (insulin trapped in situ), with an average diameter of 320 μm. In the in vitro release study, the AX microspheres minimized insulin loss in the upper GI tract, retaining B75% insulin in the matrix. Besides, encapsulated peptides in polysaccharide matrices can modify the gel’s rheological properties (elasticity and gelation time) due to thermodynamic incompatibilities between insulin/AX. Zhu et al. (2019) developed a porous starch, chitosan, and pectin system to encapsulate doxorubicin. In vitro results showed a drug release rate of 13.80% in the upper GI tract. pH-dependent polymers, such as those derived from methyl methacrylate copolymers, cellulose acetate phthalates, among others, have been used to coat polysaccharides and improve their mucoadhesiveness and drugdependent release. These polymers must resist low pHs present in the stomach and small intestine and be dissolved by the ileum and colon’s pH. These systems help patients who come to present pH alterations due to disease or other causes (Lee et al., 2020). Ansari, Sadarani, and Majumdar (2019) fabricated pectin beads to encapsulate pterostilbene and were coated with Eudragit S100. The in vitro release profile showed the coating managed to protect the pH 1.2 and 6.8 beads, releasing the drug in the presence of pH 7.4. 25.5.1.2.2 Hydrogel-based nanoparticles as drug delivery systems

Pharmaceutical nanotechnology has developed biomaterials such as liposomes,

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dendrimers, nanogels, nanoparticles, and polymeric micelles (Bhatia, 2016). Nanogels have benefits over micro and macro size systems due to their higher loading capacity and high stability. Nanomaterials are capable of being integrated into biomedical devices because most biological systems are nano-sized. These nanomaterials have been used in the oral delivery of proteins and drugs. They can improve the material’s stability against enzymatic and hydrolytic degradation in the GI tract, facilitating its intestinal absorption (McClements, 2017; Rajput, Narkhede, & Naik, 2020). The size of nanomaterials used for drug delivery contributes to their oral absorption and biodistribution. The 100 nm nanomaterials have shown increased uptake in the GI tract and a higher level of translocation to the lymphatic organs than 1000 nm nanomaterials, contributing to better absorption (Carvalho, Felı´cio, Santos, Gonc¸alves, & Domingues, 2018). Likewise, other factors contribute to nanomaterials’ mechanism; these factors are mucoadhesion, release in vivo studies, cell uptake, and transport through the intestinal epithelium (Netsomboon & Bernkop-Schnu¨rch, 2016). Nanomaterials can deliver therapeutic agents to the target site in necessary concentrations. Depending on the particles’ design, they can efficiently contain various charges on the core and the outer surface by conjugation or adsorption. Nanomaterials passively pass through sites such as capillaries and penetrate cells depending on their size, shape, and charge; these must not be toxic and must degrade in the body without producing harmful byproducts (Su et al., 2019). Allergen immunotherapy has presented clinical disadvantages, and the use of nanoparticle-based allergen delivery systems has become an alternative treatment. Pereira et al. (2018) evaluated poly (anhydride) nanoparticles for cashew nuts’ oral immunization. The nanoparticles were loaded with cashew allergens, and their immunomodulation was

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evaluated by in vivo tests. The results showed that these nanoparticles led to a pro-Th1 and Treg immune response to be used as a treatment for cashew allergy. Due to the degradation of polysaccharide matrices in the GI tract, ˘ and Erel, Kotmakc¸ı, Akbaba, So¨zer Karadaglı, Kantarcı (2016) developed a double-layer nanoparticles system based on chitosan loaded with insulin encapsulated as a microemulsion (w/o) to protect insulin, taking advantage of the electrostatic properties of chitosan. The results showed that a double layer of protection by the emulsion decreased insulin release at pH 2.5. The stability of the nanoparticles to avoid their aggregation in the GI tract remains a challenge. Chang, Wang, Hu, and Luo (2017) developed protein-polysaccharide-based nanoparticles using covalently cross-linked caseinate, zein, and three polysaccharides (pectin, carboxymethylcellulose, and gum arabic). Curcumin was encapsulated and delivery model for this system. The results showed that the polysaccharides provided different physicochemical properties to the nanoparticles, such as colloidal stability, encapsulation efficiency, kinetic release, and drying. Rodriguez, Hu, and Luo (2019) prepared nanoparticles based on zein and sodium caseinate with an oxidized dextran coating for curcumin encapsulation. The cross-linked proteins with the polysaccharide were carried out to aid the stability of these particles. The results showed that this system’s polysaccharide presence improved its colloidal stability and the controlled release rate of curcumin in gastric and intestinal conditions significantly. The oxidized dextran has the potential to improve the dispersibility of nanoparticles fabricated by spray-drying. Liposomes are small spherical vesicles with a lipid bilayer and an internal aqueous cavity. They can be classified according to their diameter, small unilamellar vesicles from 20 to 100 nm and large ones vary from 200 to 1000 nm. Multilamellar are formed from multiple phospholipid bilayers, and their sizes vary between 400 and 3500 nm. These biomaterials

have been used as a drug delivery system for cancer, gene therapy, vaccines, and biomimetic models (Ramos, Cruz, Tovani, & Ciancaglini, 2017). De Leo et al. (2018) developed liposomes for colonic delivery with Eudragit S100 pHsensitive polymer coating to encapsulate curcumin. The results showed curcumin encapsulation efficiency of 98%, and the Eudragit 100 layer helped protect the liposome against changes in pH. Dendrimers have three drug entrapment sites, site 1: voids (molecular entrapment); 2: branch points (hydrogen bonds); and 3: outer surface groups (load-load interaction). Commercial dendrimers have amino, hydroxyl, carboxyl, and pyrrolidinone groups on their surface. A dendrimer can improve the solubility and targeting of a drug (Chauhan, 2018). Zhang et al. (2018) developed folic acidmodified and DOX-conjugated poly (amidoamine) (PAMAM) dendrimers for use in cancer cell-targeted chemotherapy. The results showed that these dendrimers presented a high release rate under acidic pH conditions. Likewise, the folic acid conjugate in the dendrimers allowed a specific orientation toward cancer cells, showing DOX’s therapeutic activity.

25.5.2 Gene delivery Gene therapy consists of the transfer of DNA, RNA, and exogenous genes to patients’ somatic cells to treat or prevent diseases. Gene therapy consists of correcting genetic defects through gene expression, replacing defective genes, and introducing new genetic material. Still, it depends on the systemic delivery of genetic material to the affected site and its insertion into a specific cell. Viral vectors (retroviruses) and adeno-associated viruses have been used for this therapy to supply genetic material. Vectors have the function of evading the host’s defense and introducing the genome of the target cells. Likewise, these vectors have limitations due to

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their immunogenicity, and another type of therapy includes direct injection of genetic material, nonviral vectors, and liposomes (Dunbar et al., 2018; Tyagi & Santos, 2018). These therapies are used to treat cancers, neuronal pathologies, infectious diseases, among others. Vector therapies can present high specificity, transduction, and gene expression. These can also present several disadvantages, such as high toxicity, immunogenicity, mutagenicity, low specificity, and high costs. Therefore, the use of nonviral vehicles, such as nanocarriers, presents flexibility and safety. The functionalization of these nanoparticles with peptides, proteins, polysaccharides, and antibodies decreased their toxicity and increased their stability and cellular specificity (Xu, Li, & Si, 2014). Chitosan is a polysaccharide used as a polycationic nonviral vector because it is biodegradable and biocompatible. It presents a low capacity to transfect under physiological conditions efficiently. Baghdan et al. (2018) manufactured nanoparticles from chitosan (CsNP) encapsulated in liposomes using ionic gelation. The CsNPs were incubated together with the anionic liposomes forming lipochitoplexes (LCPs) to encapsulate plasmid DNA (pDNA). The results showed that the LCP matrix improved the protection of pDNA. In the in vivo studies, it was observed that the LCPs were able to transfect the chorioallantoic membrane without damaging the surrounding blood vessels, making it an optimal vehicle for gene delivery.

Cystic fibrosis is a lethal genetic disease due to mutations in the transmembrane conductance regulator gene of cystic fibrosis. Genetic therapies to correct this defective gene have been limited due to increased immune responses to vectors. Ferna´ndez Ferna´ndez et al. (2018) formulated polymeric nanoparticles of poly lactic-co-glycolic acid (PLGA) and chitosan to encapsulate RNA oligonucleotides modified with locked nucleic acid (LNA) Fig. 25.4. The results showed that nanoparticles had high loading capacity, making them candidates for use as nonviral vectors. In gene therapy, studies to silence genes at the mRNA level in the group of nanocarriers capable of gene delivery are the amylose. Wu et al. (2017) developed amylose nanoparticles loaded with superparamagnetic iron oxide and functionalized with folate (Fig. 25.5) for the delivery of survivin intertransfer RNA (siRNA) to hepatocellular carcinoma cells (HCC). The results showed that these nanoparticles had high transfection efficiency, exerting a silencing effect on the survivin gene in HCC cells. It is considered an efficient and safe gene delivery vector.

25.6 Macromolecules on tissue engineering Tissue engineering turns out to be a multidisciplinary field as branches such as medicine, biology, chemistry, engineering, and materials FIGURE 25.4 Graphic representation of nanoparticles based on PLGA/chitosan-LNA. Source: Adapted from Ferna´ndez Ferna´ndez, E., Santos-Carballal, B., de Santi, C., Ramsey, J., MacLoughlin, R., Cryan, S.-A., et al. (2018). Biopolymer-based nanoparticles for cystic fibrosis lung gene therapy studies. Materials (Basel), 11, 122. https://doi.org/ 10.3390/ma11010122.

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FIGURE 25.5 Diagram of the synthesis of amylose nanoparticles loaded with folate-functionalized superparamagnetic iron oxide. Source: Adapted from Wu, Z., Xu, X.-L., Zhang, J.-Z., Mao, X.-H., Xie, M.-W., Cheng, Z.-L., et al. (2017). Magnetic cationic amylose nanoparticles used to deliver survivin-small interfering RNA for gene therapy of hepatocellular carcinoma in vitro. Nanomaterials, 7, 110. https://doi.org/10.3390/nano7050110.

science converge to develop solutions that help restore and improve tissue functions. Although this field benefits from smart biomaterials, significant challenges remain for these to be scaled up to the clinic. One of the limitations of the design of biomaterials in tissue engineering is the poor understanding of the interactions between the material and cells or tissues. For them, tissue engineering has focused on manufacturing smart biomaterials capable of

incorporating specific functional groups, providing control of their physical, chemical, and biological properties (Kowalski, Bhattacharya et al., 2018). These systems effectively mimic the structure and functions of the extracellular environment that is being replaced. Biomaterials such as hydrogels are used in tissue engineering as scaffolds and can be modified to mimic the extracellular matrix of native tissue. Scaffolds must provide adequate

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25.6 Macromolecules on tissue engineering

mechanical and chemical signals to direct appropriate cellular behavior to achieve regeneration and replace necessary biological functions. They also serve as a temporary and transport three-dimensional (3D) mechanical support to stimulate their adherence, proliferation, and differentiation (Abbasian et al., 2019; Tchobanian, Van Oosterwyck, & Fardim, 2019). Tissue engineering can incorporate nanoparticles as delivery elements of drugs, genes, therapeutic agents, among others, in the scaffold to improve the interaction between cells and the extracellular environment. The delivery of genes present in the scaffolds can induce signals in cells, significantly and temporarily, to maintain tissues. Therefore, these genes can enhance the incorporation of tissue construction, growth, and assimilation with nearby tissues, serving as a treatment in the field of regenerative medicine (Nitta & Numata, 2013). Degeneration or tissue damage from trauma or disease is a severe problem since tissue engineering’s current clinical approach does not achieve complete regeneration. These biomaterials can be manufactured from various natural sources (proteins and polysaccharides) and synthetic materials using two-dimensional (2D) and three-dimensional (3D) biomaterial architectures. To prepare these biomaterials (2D and 3D) it is important to consider the pore size, distribution, geometry (surface area), and the material’s mechanical properties. Membranes

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are other biomaterials that can be created for tissue engineering that can be manufactured through electrospinning, decellularization, or compaction of materials (Carvalho, Ina´cio, Sousa, Reis, & Silva, 2020). Proteins such as collagen I have been used as scaffolds to treat wounds on the skin or organs. One of the challenges before using collagen is studying their osteogenic potential before using them as a scaffold. Elango et al. (2018) studied the osteogenic potential of collagen I isolated from shark skin. Acid-soluble type I collagen (ASC) and pepsin (PSC) were studied. In this study, it was observed that PSC presented greater thermal stability. Both types of collagen promoted osteoblast cells’ growth and increased collagen synthesis in bone cells, a desirable characteristic for this type of biomaterials for biomedical application. Fig. 25.6 shows that both collagens (ASC and PSC) presented a fibrillar network with many squamous orientation layers; these characteristics influence growth, mitigation, and proliferation, among others. These factors are of great importance for their use in tissue engineering. The production of scaffolds by 3D technology has been of great relevance; Kankala, Xu, Liu, Chen, and Wang (2018) manufactured 3D porous scaffolds based on inks composed of gelatin (GEL), nanohydroxyapatite (n-HA), and poly (lactide-co-glycolide) (PLGA) using hybrid technology based on 3D printing and

FIGURE 25.6 Morphology of the fibrillar network of the collagen ASC and PSC from blue shark skin. Source: Adapted from Elango, J., Lee, J. W., Wang, S., Henrotin, Y., De Val, J. E. M. S, Regenstein, J. M., et al. (2018). Evaluation of differentiated bone cells proliferation by blue shark skin collagen via biochemical for bone tissue engineering. Marine Drugs, 16, 350. https://doi.org/10.3390/ md16100350.

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lyophilization technology used for bone tissue engineering. These were made by printing the PLGA scaffolds and then coated with the Gel/ n-HA complex to obtain a Gel/n-HA/PLGA scaffold (Fig. 25.7). These scaffolds allowed osteoblasts (MC3T3-E1) to adhere as a layer and promoted their growth and differentiation, and bone growth was demonstrated by measurements of key biomarkers in the ossification process. Therefore, these can be of use in applications in regenerative medicine. Bone remodeling consists of replacing old bone with new tissue, which requires interaction between cellular phenotypes and regulated by biochemical and mechanical factors. Osteoclasts (reabsorb bone tissues) and osteoblasts (deposit calcium in the bone matrix) participate in this process, following a sequence of activation-reabsorption events. Bone is a porous mineralized structure made up of vessel cells and crystals of calcium components (hydroxyapatite), continuously remodeled throughout life (Hadjidakis & Androulakis, 2006). The bone structure helps locomotion and protects vital organs such as the bone marrow and the brain and functions as a reservoir for calcium and phosphate. Osteoblasts can create osteoid at the site of bone regeneration, which helps to mineralize new bone. Osteoclasts are bone resorption cells and originate from hematopoietic stem cells (Kim, Lin, Stavre, Greenblatt, & Shim, 2020). (The challenges in the manufacture of biomaterials for

bone regeneration involve the disadvantages that they can present at the time of cell proliferation, biocompatibility, and their mechanical properties. Li, Zhang, and Zhang (2018) fabricated chitosan (CS)/nanohydroxyapatite (HA) scaffolds with PLGA microspheres, and these in turn loaded with simvastatin (SIM). The results showed that the encapsulated SIM was released in a sustained manner for 30 days. In vitro studies on mesenchymal stem cells showed that SIM-loaded scaffolds had a great capacity to accelerate cell proliferation and were capable of inducing osteogenic differentiation. Also, in vivo studies showed that SIMloaded scaffolds affected bone regeneration. Bi et al. (2018) used a PLGA/HA scaffold, coated it with collagen, and incorporated the amino acids Asp-Gly-Glu-Ala (DGEA) to be used for the repair of defects of the skull in rats. The results showed that the collagen coating improved mechanical resistance and hydrophilic properties, which helped cell adhesion and proliferation. In in vivo studies, this scaffold released DGEA, which helped promote osteogenesis, and new bone tissue was observed 12 weeks after implantation. The bone tissue engineering strategy also involves using three-dimensional (3D) porous scaffolds used as temporary support for bone regeneration and development. Polley et al. (2020) designed by 3D printing scaffolds of piezoelectric and porous barium titanium (BaTiO3)/hydroxyapatite (HA) (Fig. 25.8). The FIGURE 25.7 Images of the Gel/n-HA/PLGA scaffolds. (A) PLGA 3D printed scaffolds. (B) Scaffolds coated with the Gel/nHA/PLGA complex. Source: Adapted from Kankala, R., Xu, X.-M., Liu, C.-G., Chen, A.-Z., Wang, S.-B. (2018). 3D-printing of microfibrous porous scaffolds based on hybrid approaches for bone tissue engineering. Polymers (Basel), 10, 807. https://doi. org/10.3390/polym10070807.

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(nHAC) scaffolds. The results showed the insulin could release itself on the scaffold and maintain controlled release kinetics. In vitro tests showed good biological function, which conferred adhesion and proliferation of mesenchymal stem cells, and in vivo tests showed bone regeneration capacity at eight weeks of treatment.

25.6.1 Wound management

FIGURE 25.8 Cylindrical scaffolds based on BaTiO3/ HA printed in three dimensions (3D) (A) Before synthesizing, (B) after synthesizing at 1320 C. Source: Adapted from Polley, C., Distler, T., Detsch, R., Lund, H., Springer, A., Boccaccini, A. R., et al. (2020). 3D printing of piezoelectric barium titanate-hydroxyapatite scaffiolds with interconnected porosity for bone tissue engineering. Materials (Basel), 13, 1773. https://doi.org/10.3390/MA13071773.

in vitro studies showed that the preosteoblastic cells (MC-3T3) were able to bind to the surface of the material and presented high cytocompatibility, as well as piezoelectric properties that mimic dry bone. It was observed a material with high porosity, which leads to weak mechanical properties. However, this makes it a promising biomaterial for bone regeneration studies. In bone regeneration, it has been observed that the use of proteins such as insulin helps osteogenesis and bone turnover, and their short periods of activity and controlled release make it less promising. Wang et al. (2018) incorporated insulin-loaded PLGA nanospheres into nanohydroxyapatite/collagen

Wounds are injuries caused by accidents or trauma and often damage the skin integrity and soft parts, where there is neuronal tissue that does not contain stem cells, and they cannot self-regenerate. These usually have different characteristics according to different factors such as the patient’s condition, location, and whether they have an infection. The use of dressings and bandages has been useful for the treatment of these wounds. Their adaptability depends a lot on the type of wound and the characteristics of the material. Among the desired characteristics for the use of dressings is their ability to maintain moisture in the wound, permeability (water and oxygen), ability to absorb exudates, protect the skin, mechanical protection, adaptability, protection against bacterial and infectious agents, biocompatibility, biodegradability, temperature balance, absence of toxicity, inflammatory stimulation, ease of use and accessibility (Bianchera, Catanzano, Boateng, & Elviri, 2020).

25.6.2 Development of skin substitutes The skin is the outer layer of the human body and serves as a defense against external mechanical, biochemical, and environmental factors. It is divided into the epidermis, dermis, and hypodermis. The epidermis corresponds to the outer and thinnest layer, does not have blood vessels, and has multiple layers of keratinocytes antigens. The dermis is the second

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layer, and this one does have blood vessels, nerve endings, and glands, and it is composed of fibroblasts capable of synthesizing collagen. As the last layer is the hypodermis, this is a fat-storage system and works as a lipid barrier rich in stem cells, hormones, and growth factors. This last layer is responsible for reepithelialization, wound healing, and the angiogenesis process (Byrd, Belkaid, & Segre, 2018). In tissue engineering, skin tissue replacement or modeling is a construction that attempts to imitate its shape and physiological function. A skin substitute has not been shown to restore normal structure and physiological function completely. A great variety of strategies have been developed for its functionality; these involve delivering cells or signals capable of stimulating or participating in tissue repair. These designs have been used in research to develop skin constructs for application as wound dressings or skin substitutes for severe skin injuries. This field remains in development since clinical studies have presented various challenges to achieve successful results, which has been sought to continue studying new methods (Yu et al., 2019). Synthetic materials were widely used to maintain the stability of these materials. It was observed that the use of biomolecules and skin cells to functionalize synthetic materials would help to form a more stable scaffold. They can facilitate cell adhesion, have greater compatibility, and be easily degraded; the biomolecules used are collagen, gelatin, keratin, chitosan, and dextrans. Likewise, these constructions also incorporate growth factors, and cells such as fibroblasts, keratinotics, and others to help facilitate the growth of native cells (Tchobanian et al., 2019). One of the challenges in making biomaterials is to mimic tissues. Nanofibrous structures can imitate extracellular matrices and have been used as materials for wound healing due to their high surface-volume ratio, high porosity, and permeability. Zahedi, Esmaeili, Eslahi, Shokrgozar, and Simchi (2019) fabricated

fibrous scaffolds with a central layer of chitosan, polycaprolactone, and keratin to encapsulate aloe vera using the coaxial electrospinning technique. In vitro studies showed increased cell growth and adhesion in materials with the presence of aloe vera and, at the same time, did not show cytotoxic effects. Hydrogels are biomaterials widely used in tissue engineering. Because they are threedimensional networks capable of hydrating and increasing their volume, they can absorb exudates from wounds, allow oxygen diffusion, and maintain moisture. These characteristics allow them to be used as dressings to cover skin wounds (Tavakoli & Klar, 2020). Certain polymers are capable of mimicking the dermal extracellular matrix when used to make wound dressings. For example, in a study by Ren, Gan, Zhou, and Chen (2020), a hydrogel-based on two polymers [polyvinyl alcohol (PVA) and fish gelatin (FG)] was developed, and salicylic acid was incorporated as an antimicrobial. The FG was added to the polymer to improve the mechanical resistance and the hydrogel’s swelling response, so it was used in different concentrations (0.00%, 0.75%, 1.50%, 2%
25%, 3.00%, and 3.75%) (Fig. 25.9). The results showed while FG concentration increased (0%
3.75%), the swelling of the hydrogel also increased from 54% to 83% in a period of 8 h, making them an ideal biomaterial to absorb liquids exudates. In in vitro studies of salicylic acid, the release showed that these hydrogels presented sustained release; also, in the study of antimicrobial activity, the hydrogels demonstrated good antimicrobial effect. Alves et al. (2020) fabricated a thermoreversible hydrogel based on xanthan gum-konjac glucomannan (XG/KGM). These hydrogels were formed at temperatures of 37 C; XG’s presence gave their thermosensitive character. These hydrogels also presented a transparent and hydrated appearance, which helps monitor the wound healing process without needing to

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FIGURE 25.9 PVA/FG hydrogels with different FG content. Source: Adapted from Ren, T., Gan, J., Zhou, L., & Chen, H. Physically crosslinked hydrogels based on poly (vinyl alcohol) and fish gelatin for wound dressing application: Fabrication and characterization. Polymers (Basel), 12, 1729. https://doi.org/10.3390/POLYM12081729.

FIGURE 25.10 (A) The liquid-solid gel transition at 50 C and 37 C. (B) XG/KGM macrogels at 1 and 2% concentration. Source: Adapted from Alves, A., Miguel, S. P., Araujo, A. R. T. S., de Jesu´s Valle, M. J., Sa´nchez Navarro, A., Correia, I. J., et al. (2020). Xanthan gum
konjac glucomannan blend hydrogel for wound healing. Polymers (Basel), 12, 99. https://doi.org/10.3390/polym12010099.

remove the dressing (Fig. 25.10). In addition, these materials presented biological properties that help cell adhesion, migration, and proliferation. Autografts have been used to treat chronic wounds, but these techniques usually have

limitations such as immune rejection. The use of scaffolds to treat these wounds has become an alternative for their treatment; even so, these scaffolds present their limitations since they do not cover the affected area. Chanda et al. (2018) manufactured a scaffold based on chitosan (CS)/polycaprolactone (PCL) and hyaluronic acid (HA), using the electrospinning technique. In vitro tests showed increased proliferation and migration of cells in the scaffold. The electrospinning technique allowed scaffold fibers sizes of 362.2 nm, which are in the range of collagen fibers present in extracellular matrices. Tonda-Turo (Tonda-Turo et al., 2018) made gelatin-based scaffolds with gentamicin and silver nanoparticles as antibacterial agents. The results showed that the nanofibers’ formation was not affected by antimicrobials, and these were efficient against Gram-negative bacteria. In the in vitro tests, it was also observed that gelatin nanofibers favor cell adhesion due to the material’s rough surface, which allowed supporting its proliferation and cell regeneration. Miguel, Ribeiro, Coutinho, and Correia (2017) manufactured a scaffold based on polycaprolactone and coated with a chitosan/aloe vera compound. The results revealed that the scaffolds had mechanical properties similar to native skin. The adhesion and proliferation capacity of fibroblast cells in their lower layer was also observed, thanks to the fact that aloe vera can maintain a suitable humid environment.

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25.7 Conclusion Macromolecules in biomaterials play an increasingly important role as therapeutic agents for use in biomedicine. The advantages that macromolecules present in the manufacture of these biomaterials provide solutions to the limitations of traditional techniques in development and application. However, these macromolecule-based biomaterials can present challenges, which can be solved by functionalizing them using two or more macromolecules. An example of this occurs in drugs’ targeted delivery because these biomaterials must ensure drug loading, controlled release, and prevent its degradation. These biomaterials must also be biocompatible, biodegradable, nontoxic, mutagenic, and stable. Therefore, the use of macromolecules will continue to provide a broad development of the biomedical area and new challenges.

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694. Available from https://doi.org/10.1016/ j.ijbiomac.2019.04.197. Aeridou, E., Dı´az Dı´az, D., Alema´n, C., & Pe´rez-Madrigal, M. M. (2020). Advanced functional hydrogel biomaterials based on dynamic B
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konjac glucomannan blend hydrogel for wound healing . Polymers (Basel) (12, p. 99). Available from https://doi.org/10.3390/polym12010099.

Amidon, S., Brown, J. E., & Dave, V. S. (2015). Colontargeted oral drug delivery systems: Design trends and approaches. AAPS PharmSciTech, 16, 731
741. Available from https://doi.org/10.1208/s12249-015-0350-9. Anal, A. K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends in Food Science and Technology, 18, 240
251. Available from https://doi. org/10.1016/j.tifs.2007.01.004. Ansari, M., Sadarani, B., & Majumdar, A. (2019). Colon targeted beads loaded with pterostilbene: Formulation, optimization, characterization and in vivo evaluation. Saudi Pharmaceutical Journal, 27, 71
81. Available from https://doi.org/10.1016/j.jsps.2018.07.021. Atkinson, M. A., & Eisenbarth, G. S. (2001). Type 1 diabetes: New perspectives on disease pathogenesis and treatment. Lancet, 358, 221
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matrix . Polymers (Basel) (8, p. 130). . Available from https://doi.org/10.3390/polym8040130. Yin, H., Kauffman, K. J., & Anderson, D. G. (2017). Delivery technologies for genome editing. Nature Reviews Drug Discovery, 16, 387
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589. Available from https://doi.org/10.3109/ 1061186X.2015.1128941. Zhang, M., Zhu, J., Zheng, Y., Guo, R., Wang, S., Mignani, S., et al. (2018). Doxorubicin-conjugated PAMAM Dendrimers for pH-responsive drug release and folic acid-targeted cancer therapy. Pharmaceutics, 10, 162. Available from https://doi.org/10.3390/pharmaceutics 10030162. Zhu, J., Zhong, L., Chen, W., Song, Y., Qian, Z., Cao, X., et al. (2019). Preparation and characterization of pectin/ chitosan beads containing porous starch embedded with doxorubicin hydrochloride: A novel and simple colon targeted drug delivery system. Food Hydrocolloids, 95, 562
570. Available from https://doi.org/10.1016/j. foodhyd.2018.04.042.

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26 Future perspectives of biological macromolecules in biomedicine Ana R. Neves, Ru´ben Faria, Taˆnia Albuquerque, Telma Quintela, ˆ ngela Sousa and Diana Costa A CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal

26.1 Bio-nanotechnology Nanotechnology arises as a powerful tool in many domains, ranging from industry, agriculture, informatics, energy, catalysis to pharmaceutical and medicine applications (Jeevanandam, Barhoum, Chan, Dufresne, & Danquah, 2018; Wu et al., 2020). In the last decade, the development of nanomaterials for this wide range of applications conquered special interest among researchers (Faria, Sousa, Neves, Queiroz, & Costa, 2019; Jeevanandam et al., 2018; Shafiq, Anjum, Hano, Anjum, & Abbasi, 2020). The formation of nanomaterials with small size to large surface, commonly 10
300 nm, constitutes a relevant tool in various fields due to their tunable physicochemical and biological properties, and their performance is aimed at a specific function and in vitro/in vivo biocompatibility (Faria et al., 2019; Sztandera, Gorzkiewicz, & KlajnertMaculewicz, 2020). The continuous progress in the bio-nanotechnology area led to considerable advances in the design and conception of new drug or gene delivery systems presenting low

Biological Macromolecules DOI: https://doi.org/10.1016/B978-0-323-85759-8.00026-9

cytotoxicity, able of cellular uptake internalization and displaying targeting capacity. This concerted effort contributes for accomplishments of therapies based on drug or gene release and, in turn, to progresses on the treatment of diseases, such as cancer (Faria et al., 2019; GonzalezFernandez et al., 2017; Neves et al., 2020). Additionally, the simultaneous delivery of different pharmaceutics, namely the release of anticancer drugs and tumor suppressor genes, has led to considerable success on cancer therapy, due to synergistic effect (Li, Thambi, & Lee, 2018; Paris & Vallet-Regi, 2020). For this has contributed the capacity of a drug or gene-based carrier to surpass the cellular membrane, resist to degradation and, ultimately, reach the cellular organelle where it should exert its therapeutic function. For this contributes the fact that biological macromolecules and nanoparticles can be targeted to specific cellular compartments or accumulate in tumor environment opening the route for particle surface decoration, with tumor targeting sequences, for enhanced results (Costa et al., 2017; Faria et al., 2019). The delivery of

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nucleic acids to specific cells promote the expression or inhibition of certain proteins, a main strategy in restoring normal cell function. Since nucleic acids and most drugs are not able to easily cross the cellular membrane of target cells and due to nucleic acids degradation, by the action of nucleases, the formulation of a promising delivery system to ensure high payload loading and protection is crucial for the success of intracellular delivery of the cargo and, consequently, for the desired therapeutic effect (Faria et al., 2019; Gonzalez-Fernandez et al., 2017; Li et al., 2018; Neves et al., 2020). Therefore, the development of a convenient nanoparticle delivery system is a relevant step for the implementation of drug and/or gene release protocols toward biomedicine applications.

26.1.1 Delivery systems In the last decades, researchers have made considerable efforts in the search of the optimal delivery system to overcome the major drawbacks of payload delivery, namely, cellular uptake/internalization, endosomal escape, targeting to a specific subcellular compartment, and ultimately, induction of therapeutic action (Costa et al., 2017; Faria et al., 2019; GonzalezFernandez et al., 2017; Li et al., 2018; Lundstrom, 2018; Neves et al., 2020). Viral vectors have been largely employed for the treatment of metabolic, cardiovascular, hematologic, infectious diseases and cancer (Lundstrom, 2018). Although the transfection mediated by these vectors can be a very efficient process, their use can also represent great disadvantages such as their immunogenicity, toxicity, the need to design a system for each target molecule, possible random mutagenesis and the insufficient information concerning the dose dependency of cellular expression limit their application as carriers (Hinderer et al., 2018). In contrast, nonviral vectors show many advantages over viral ones and are a convenient tool

for drug and gene therapy. These carriers are easy to fabricate and manipulate, can accommodate large quantities of genetic payloads and exhibit lack of immune response. Due to their tailored characteristics, nonviral delivery systems have been intensely investigated for drug and/or gene delivery with great accomplishments on clinical context (Chen et al., 2019; Gonzalez-Fernandez et al., 2017; Li et al., 2018; Morrison, 2018; Neves et al., 2020). 26.1.1.1 Nonviral vectors Vectors produced by synthetic procedures can be easily fabricated and adequately tailored, display no immune responses and have a great capacity to encapsulate and transport large genetic materials, being considered useful platforms for the intracellular delivery of therapeutics (Chen et al., 2019; Costa, Albuquerque, Queiroz, & Valente, 2019; Morrison, 2018; ReyRico & Cucchiarini, 2019). Synthetic vectors can be chemical or physically produced and optimized and also functionalized in their surface to promote their recognition by the cells, ensure specific targeting and to potentiate the gene transfection process (Coutinho, Batista, Sousa, Queiroz, & Costa, 2017; Du et al., 2017). In this sense, one tool that has been significantly explored was the chemical modification of vectors with specific ligands with the ability to bind to the receptors overexpressed in cancer cells; this skill can improve treatment outcomes (Faria et al., 2019; Lee et al., 2016). For example, methotrexate (MTX), an anticancer drug used in cancer care has the capacity to bind to the folate receptors and this drug is able to inactivate the enzyme dihydrofolate reductase that plays an important role in both cell survival and division (Wong & Choi, 2015). The functionalization of vectors with MTX revealed to be an adequate approach to potentiate targeting and therapeutic efficacy leading to significant progresses on cancer care (Lee et al., 2016; Zhang et al., 2017). The conjugation of chemotherapy, gene therapy and the cell

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targeting strategy represent a significant advance in the development of novel therapeutic protocols in cancer treatment (Faria et al., 2019; Lee et al., 2016; Lin et al., 2019). Among the diverse synthetic carriers commonly used for therapeutic cargo release, complexes formulated by the electrostatic binding between a cationic agent (lipids, dendrimers or polyethylenimine) and an anionic molecule attracted special interest. In this sense, cationic liposomes have been deeply investigated as a delivery platform able to ensure stability of drug and/or nucleic acid therapeutics in the blood stream while promoting their intracellular release (Das & Huang, 2019; Yonezawa, Koide, & Asai, 2020). Cationic lipids commonly exhibit a positively charged head group and one or two hydrophobic tails composed of hydrocarbon chains, sometimes steroid structures can be present. Due to its cationic charge, the lipids can efficiently load and encapsulate both drugs and genes, while favorably interacting with the negative cellular membrane (Das & Huang, 2019; Radchatawedchakoon et al., 2020; Yonezawa et al., 2020). Moreover, the incorporation of pegylated lipids or functional lipids on liposomes allows for their surface modification with direct repercussion on their ability for cellular internalization and transfection efficiency (Hattori, Shimizu, Ozaki, & Onishi, 2019). Also, currently it is known that the liposomes delivery performance can be strongly affected by the types of helper lipids and their compositions (Rangasami et al., 2019). Additionally, liposomes revealed to be biodegradable after administration in vivo, instigating effective clinical translation (Ashrafzadeh, Akbarzadeh, Heydarinasab, & Ardjamand, 2020). Dendrimers are nano-sized polymers with a defined spherical architecture containing multiple positive charges on the surface. In particular, Poly(amidoamine) dendrimers (PAMAM) gained attention due to a set advantages for the release of both anticancer drugs and genes,

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considering physical or chemical ways of conjugation (Bhatt, Ghosh, & Biswas, 2020; Rompicharla, Kumari, Ghosh, & Biswas, 2018). Moreover, they possess functional groups on the surface that allows them to be anchored by various peptides/ligands/antibodies with the aim of targeting cancer cells (Bhatt et al., 2020). The PAMAM dendrimers are highly accessed dendrimers for payload delivery, which present ethylenediamine core, amine and repetitive amide bonds in the periphery. The number of primary amine groups (theoretically, 64 amines for PAMAM dendrimer, generation 4 [G4] or 128 for PAMAM dendrimer generation 5 [G5]) on the surface of these spherical polymers could efficiently condense nucleic acids (Bahadoran, Moeini, Bejo, Hussein, & Omar, 2016; Zarebkohan et al., 2016). Along with acting as a gene delivery vector, the well-defined core in the PAMAM dendrimer could encapsulate hydrophobic anticancer drugs, or conjugate the drug on the surface (Li, Liang, Liu, & Wang, 2018). Moreover, several examples concerning the recent employed strategies for the production of biomacromolecules loaded/ functionalized dendrimers, for therapeutic applications, can be found in the literature (Carvalho, Reis, & Oliveira, 2020; Maysinger et al., 2019; Sharma et al., 2020). Polyethylenimine (PEI) has been immensely used for the formation of drug and DNA or RNA based nanoparticles in therapeutic protocols (Appelbe et al., 2018; Costa, Briscoe, & Queiroz, 2015; Faria et al., 2019; Sousa et al., 2020). This polymer presents a high cationic charge density and due to its ionization degree with pH, PEI exhibits endosomolytic activity. In addition, the positive amine groups of PEI electrostatically interact with the negatively phosphate groups of nucleic acids leading to their condensation into particles at nanoscale (Faria et al., 2019; Sousa et al., 2020). The properties of PEI namely the molecular weight, the architecture of the chains or the degree of branching are important parameters, as they

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can define the physicochemical characteristics of DNA-PEI complexes, as well as, the cytotoxic profile and determine the efficiency of transfection (Costa, Valente, Queiroz, & Sousa, 2018; Sousa et al., 2020; Utsuno et al., 2016). Generally, the higher molecular weight polymers are more cytotoxic but in turn these PEIs can promote higher transfection efficiency rates. Furthermore, the lower molecular weight systems can be altered to potentiate the transfection process (Zhao et al., 2016). The architecture of the chains also influences this process. Branched PEI (bPEI) compacts nucleic acids in a more effective way than the linear polymer, fact that can be due to the higher density of primary amines. Also, the capacity of PEInucleic acid systems for gene release is also dependent on the PEI nitrogen to nucleic acids phosphate groups (N/P) ratio, as this property is crucial for the size and surface charges presented by the carriers (Costa et al., 2015, 2018; Faria et al., 2019; Sousa et al., 2020). Following this, the drug MTX has been encapsulated into complexes based on PEI and a plasmid DNA (pDNA) containing a tumor suppressor gene (p53) and the cancer-cell targeting specificity of the formed systems has been studied (Faria et al., 2019). An evaluation on fibroblast and HeLa cells revealed that the anticancer drug is able of cell targeting by recognition and binding to folate receptors. The efficiency of transfection can be modulated by the amount of MTX, what demonstrated to have influence on the expression of p53 gene. The therapeutic effect monitored on HeLa cells showed that the codelivery vector induced a great decrease on their viability (Faria et al., 2019). Moreover, PEI polyplexes functionalized with the mitochondrial affinity compound tryphenylphoshonium (TPP) have been explored to target mitochondria in view of promoting gene and mitochondrial protein expression (Sousa et al., 2020). Cell-penetrating peptides are considered as interesting molecules, in the biomaterials field, mainly due to their characteristics of structure

and sequence (Konate et al., 2019; Silva, Almeida, & Vale, 2019). These peptides are short, up to 30 amino acids, and can be separated into arginine-rich and amphipathic peptides (Patel, Zaro, & Shen, 2007). The amphipathic class have hydrophilic and hydrophobic regions that promote the interaction with nucleic acids and cell uptake. The RALA peptide exhibits 7 arginine residues and an alpha helical structure with hydrophobic and hydrophilic amino acids. RALA can strongly condense nucleic acids leading to the formation of nanometric vectors with adequate properties for in vitro release (Bennett et al., 2015; Jain et al., 2015; Neves et al., 2020). In a recent study, nanoparticles based on RALA and pDNA were conceived at different N/P ratios and characterized, in terms of shape, size, surface charges and cytotoxic profile for gene delivery purposes (Neves et al., 2020). The properties of the formed peptide/pDNA carriers can be tailored and by varying the N/P ratio. A microscopy evaluation showed the cellular uptake and internalization into cancer cells. Moreover, studies on gene expression revealed effective transfection, p53 production and apoptosis in HeLa cells (Neves et al., 2020).

26.2 Mitochondrial gene therapy Mitochondrion is a cellular organelle constituted by enzymatic complexes with the main function of chemical energy production (ATP). This organelle has important functions in various processes occurring in the cell and it also has influence on apoptosis (Hajno´czky et al., 2006; Tavassoly et al., 2015). Similar to nucleus, mitochondria contain their own genome, the mitochondrial DNA (mtDNA). Mutations in mtDNA lead to a variety of diseases mainly affecting vital organs such as the heart and the brain (Ciccone, Maiani, Bellusci, Diederich, & Gonfloni, 2013; Spangenberg et al., 2016; Zsurka & Kunz, 2015). The treatment of

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mtDNA based disorders is, in most of the cases, ineffective. Mitochondrial gene therapy can be a promising tool to correct mtDNA mutations. In order to turn this therapy feasible, the fabrication of an effective mitochondrial gene-based system is crucial.

26.2.1 Mitochondrion Human mitochondrion is a dynamic cytoplasmic organelle in virtue of fusion and fission cycle in response to cell needs and environment. Mitochondria are involved in a variety of cellular processes such as, signaling, ion homeostasis and in the metabolism of lipids, steroids, nucleotides and other molecules. This organelle also exerts control over cell cycle and cell growth (Hajno´czky et al., 2006; Tavassoly et al., 2015). This cellular organelle produces ATP through the mitochondrial respiratory chain, composed by four complexes. Complexes I, III and IV are energy coupling centers. These complexes, the complex V and the electron carriers ubiquinone and cytochrome c, constitutes the system of oxidative phosphorylation. This system is responsible to give ATP to the cell, when needed. Mitochondria also has a role on apoptosis process (Tavassoly et al., 2015). This mechanism of cell death includes the activation of caspase proteases. Two distinct pathways for apoptosis are well identified and studied, revealing the significant action of mitochondria: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway (Dai, Meng, & Kaufmann, 2016). As the nucleus, mitochondrion has its own genome, the mtDNA. This molecule is a circular double stranded DNA with approximately 17 kb pairs. This genome is present in hundreds to thousands of copies per cell and contains 37 genes, 2 rRNAs and 22 tRNAs, all exclusive to the mitochondria. Seven proteins are integrated in subunits of complex I, one is

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involved in complex III, three are included in the complex IV and two are part of ATP synthase. The other genes left contain information related with the translational machinery of the mitochondrial genome. Along with this, in the nucleus approximately one thousand mitochondrial proteins are encoded, translated in the cytoplasm and translocated to mitochondrion by a complex mechanism of protein import. Despite the fact that mtDNA is quite small, mutations in this genome gave raise to a large variety of syndromes/diseases. Therefore, mitochondrial diseases are estimated to have an incidence of 1.6 in 5000.

26.2.2 Mitochondrial mutations Although being smaller when compared to nuclear genome, mDNA is around ten times more prone to mutations than nuclear genes. Contrary to nuclear DNA, mtDNA do not possess an effective protection mechanism as the chromatine to pack and protect DNA. Besides, and due to few noncoding regions in mtDNA, mutations in this genome are much more likely to be pathogenic. Furthermore, packaged as nucleoids, mtDNA localizes in the vicinity of the inner mitochondrial membrane and the oxidative phosphorylation complexes. Therefore, this genome is exposed to high levels of reactive oxygen species which greatly contribute for its high rate of mutation. To keep the functionality of mitochondrial genome, different DNA repair pathways have emerged. Over the last two decades it was confirmed that mitochondrion displays DNA repair mechanisms (Pinz & Bogenhagen, 1998) and the understanding of their function has increased. The most studied DNA repair pathway is the base excision repair pathway. It repairs small DNA modifications in mtDNA caused by alkylation, deamination, or oxidation (Prakash & Doublie´, 2015). This mechanism recognizes the damage, and proceed with various enzymatic steps to

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remove the lesion keeping the integrity of mtDNA. Nowadays, researchers believe that the mismatch repair pathway, thought to occur solely in the nucleus, take place in mammalian mitochondrion. The genes encoded in the genome of mitochondria are all critical for their normal way of function. A variety of diseases are associated to mutations in specific genes and mitochondrial tRNA. Somatic mutations, mutations that were not present in the germ cell line, may also take place in mitochondrion; point mutations along with deletions are the most frequent. It is well documented that mutations in the mitochondrial genome can cause a wide range of metabolic and neuromuscular degenerative syndromes and usually involve tissues with high energy requirements. Mutations and/or polymorphisms in mitochondrial genome are a frequent cause of cytopathy giving raise to diverse clinical phenotypes associated with severe metabolic dysfunctions, including progressive cardiomyopathy, encephalopathy, leukodystrophy, Leigh’s syndrome or ragged red fibbers syndrome and premature age-related symptoms (Hadzsiev et al., 2010; Mustafa, Fakurazi, Abdullah, & Maniam, 2020; Zaragoza, Fass, Diegoli, Lin, & Arbustini, 2010). Moreover, deletions in mtDNA genome are linked to pathologies such as chronic progressive external ophtalmoplegia, Pearson syndrome or Kearns-Sayre syndrome, (Farruggia, Di Marco, & Dufour, 2018; Mustafa et al., 2020) while, a single point mutation in mtDNA seems to be correlated with diabetes (Wei & Chinnery, 2020). Michael Lin et al. identified, for the first time, significant levels of mtDNA mutations in substantia nigra neurons in both early stage Parkinson disease (PD) and incidental Lewi body disease (Lin et al., 2012). Later, other authors also conclude that mtDNA mutations accumulate within dopaminergic substantia nigra neurons and contribute to the progress of PD (Simon, Matott, Espinosa, & Abraham, 2017). Alzheimer disease (AD), one of the most common neurodegenerative disorders, is believed to

strongly depends on the mitochondrion function. It is known that in AD brains, the activity of the proteins responsible for the creation of energy such as complex IV cytochrome c oxidase or ATP synthase complex, among others, were found decreased. Additionally, somatic mtDNA mutations in AD brains are higher when compared to healthy brains (Cenini & Voos, 2019). Furthermore, the link between mitochondrial genome mutations and the higher susceptibility to develop cancer has been intensively studied. Large-scale sequencing and clinical studies have highlighted the prevalence of mtDNA mutations in human tumors and their roles in cancer progression (Gammage, & Frezza, 2019). A review article from Hertweck and Dasgupta deeply identifies the actual information related to the quantity and distribution of somatic mitochondrial mutations and elucidates the mechanisms affecting the fate of mutations in cancer cells (Hertweck & Dasgupta, 2017). Furthermore, the accumulation of mtDNA mutations was found to be related with premature aging (Kujoth et al., 2005).

26.2.3 Targeting Mitochondria Mitochondrial gene therapy can be explored through allotopic expression involving the expression of genes in the nucleus, followed by their synthesis in the cytosol and subsequent targeting and protein imported into mitochondrion. Some authors have conveniently investigated this concept and, by using viral vectors, reported significant progresses in the treatment of Leber neuropathy (Wan et al., 2016) and in the conception of adequate animal models for mtATP6 mutations (Dunn & Pinkert, 2012). Despite being a valuable method in various circumstances, the allotopic expression presents some disadvantages namely, the fact hydrophobic proteins are difficult to import to mitochondrion and the apparent complementation attributed to revertants of the original mutations in the mitochondrial genome. The direct

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transfection of mitochondria emerges as a powerful alternative approach for mitochondrial gene therapy. The direct delivery of genes of therapeutic interest to mitochondrion requires the formation of a suitable DNA based delivery system exhibiting mitochondrial targeting ability. In line with this, the development of a mitochondrial gene system has emerged as imperative but also as a challenging task. A group of outstanding researchers devoted their attention to this issue and relevant achievements were attained. For instance, advances were made in the cloning of mouse mtDNA in Escherichia coli and by using in vitro transposition reaction followed by in organello analysis (Bigger et al., 2001; Yoon & Koob, 2003). Bigger et al. made use of yeast to clone the human mtDNA in a single copy plasmid (Bigger, Liao, Sergijenko, & Coutelle, 2011). More recently, Costa et al. cloned, for the first time, the mitochondrial gene ND1 (mitochondrially encoded NADH dehydrogenase 1 protein) in E. coli, that was maintained in pCAG-GFP plasmid (Coutinho et al., 2017). To assure the intracellular access and bioavailability of the fabricated mitochondrial vector, its loading/complexation into a nanosystem is imperative. The design of mtDNA based delivery systems also brought significant difficulties. The development of delivery systems based on mitochondriotropic compounds have been a strategy followed by many researchers who demonstrated the feasibility of mitochondrial gene therapy and the possible clinical translation (Biswas, Dodwadkar, Piroyan, & Torchilin, 2012; Coutinho et al., 2017; Santos, Sousa, Queiroz, & Costa, 2014; Sousa et al., 2020; Weissig, DSouza, & Torchilin, 2001). These compounds display great lipophilicity and possess delocalized charges accumulating in the mitochondrion due to the existence of mitochondrial membrane potential. Rhodamine 123, an amphiphile molecules with delocalized cationic charge, accumulates in the mitochondrion without cytotoxic effects. This compound

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exhibits green fluorescence and has been explored as a mitochondria probe (Chazotte, 2011). A study, using model plasmid DNAs, reported the formation of rhodamine plasmid DNA nanoparticles with appropriate size and charge on the surface for cellular internalization (Santos et al., 2014). Moreover, these nanovectors were internalized into fibroblasts and HeLa cells and were found to target mitochondria, as revealed by fluorescence confocal assays (Santos et al., 2014). Pursuing the goal of targeting mitochondria, Weissig and his team gave a great contribution to the field of mitochondrial gene therapy with the creation of dequalinium-based liposomes aiming the delivery of pDNA and drugs to the site of mitochondria (Weissig, 2015; Weissig et al., 2001). Mitochondriotropic compounds were also employed by other authors in the formulation of mitochondria-targeted systems for innovative therapeutic approaches, namely, drug delivery in cancer therapy, address the problem of drug resistance, or gene release (Ahn et al., 2018; Cohen-Erez, Harduf, & Rapaport, 2019). In some situations, fluorimetric analysis on isolated mitochondria were also performed to illustrate the selective and efficient mitochondria targeting capacity of the developed delivery systems (Ahn et al., 2018). TPP, a lipophilic cation, has affinity for mitochondrion and its combination of with therapeutic payloads has been studied to promote specific mitochondria targeting (Ozsvari, Sotgia, & Lisanti, 2018; Zhang & Zhang, 2016). Recently, Costa et al. synthesized a polyethyleneimine (PEI)-based nanosystem, by conjugating poly (ethylene glycol) (PEG) and TPP moiety to PEI of various molecular weights (Sousa et al., 2020). In this study, to reduce the cytotoxicity of PEI, this polymer was linked to PEG and the resultant polycation (PEG-PEI) at different molecular weights was conjugated with TPP. PEG-PEI-TPP was employed to complex ND1 and green fluorescent protein (GFP) based plasmids and design of experiments tool

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26. Future perspectives of biological macromolecules in biomedicine

has been used to optimize the polyplexes formation revealing the vectors possessing the most promising characteristics for mitochondrial gene therapy purposes (Sousa et al., 2020). The properties of these pDNA/TPPpolyplexes have been studied by means of several techniques revealing the size and surface charge of the formed nanoparticles (Faria et al., 2020). Spherical particles were formulated for both pGFP- and pND1-PEG-PEI-TPP vectors, and their size varied with PEI molecular weight and N/P ratio. Similarly, the zeta potential is also affected by these parameters. As the N/P ratio increases, more positive charges from the polymer are present and neutralize the negative pDNA charges. Therefore, the surface charges tend to increase. Although at a less extent, PEI molecular weight also affects the zeta potential exhibited by the vectors (Faria et al., 2020). Moreover, in vitro studies using revealed the capacity of the conceived complexes for cell uptake. A microscopy study performed on cancer cells, after transfection mediated by PEG-PEI25 kDa-TPPpND1 nanoparticles, showed the pDNA colocalization with mitochondrion (Fig. 26.1) (Sousa et al., 2020).

2011). These scientists created an artificial mitochondrial genome pmtGFP that has been conveniently encapsulated into a DQAsome delivery vector that can fuse with the mitochondrial membrane. Taking into account the unique genetic code of mitochondria, they designed a pDNA specifically expressed in these organelles by incorporating a codon which can only be read by mitochondrial ribosomes. The mitochondrial genome differs from the nucleus genetic code by four codons. The codon “uga” which codes for a tryptophan if read by the mitochondrial ribosomes has been inserted in pmtGFP (Lyrawati et al., 2011). If read by the cytosolic ribosomes, the same codon is identified as a stop codon. By using techniques such as immunofluorescence, immunohistochemical and molecular assays they demonstrated the release of the pmtGFP to the mitochondria of a mousse macrophage cell line.

26.2.3.1 Gene and protein expression Despite the intense research on the mitochondria gene therapy field, to date few reports have presented effective transgene delivery and expression into this cellular organelle (Jang & Lim, 2018). Contrary to nuclear gene therapy, a subject hardly researched in the last decades contributing to great achievements as the various ongoing clinical trials worldwide can proof, gene therapy toward mitochondrial disorders has still considerable space for evolution. Pursuing the aim of expressing a mitochondrial gene into mitochondria, several authors have developed suitable approaches allowing significant advances in this area. Lyrawati et al. presented innovation with a novel approach for expression of GFP in mitochondrion (Lyrawati, Trounson, & Cram,

FIGURE 26.1 Evaluation of transfection ability and intracellular colocalization of PEG-PEI25 kDa-TPP-pND1 delivery vectors. Nucleus is stained blue by DAPI and green represents the pND1 labeled with FITC dye. (A) Mitochondria stained by MitoTracker Orange; (B) pND1 stained with FITC; representative images of HeLa cells 24 h after transfection mediated by PEG-PEI25 kDa-TPP-pND1FITC nanoparticles formed at N/P ratios of (C) 5 and (D) 10. Scale bar 5 10 μM. Source: Adapted from Sousa, A., Faria, R., Albuquerque, T., Bhatt, H., Biswas, S., Queiroz, J.A., & Costa, D. (2020). Design of experiments to select triphenylphosphoniumpolyplexes with suitable physicochemical properties for mitochondrial gene therapy. Journal of Molecular Liquids, 302, 112488.

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26.2 Mitochondrial gene therapy

Despite the low efficiency, this method led to the expression of mRNA and protein. Transfection studies in other cells also revealed feasibility (Lyrawati et al., 2011). Exploring the assets of this pDNA specifically expressed in the mitochondria, another research team formulated geminisurfactant-based DNA complexes to target mitochondria and promote gene delivery (Cardoso et al., 2015). These gemini-surfactantpDNA systems were taken up by cells by endocytic pathways and exhibited skills to promote the destabilization of the membranes. Additionally, the complexes strongly interact with lipid membrane models resembling the composition of the mitochondrial membrane evidencing the favored interaction with mitochondrion (Cardoso et al., 2015). Costa et al. analyzed the GFP gene expression after transfection of HeLa cells mediated by PEG-PEI25 kDa-TPPpDNA delivery systems. Significant levels of GFP mRNA were detected in HeLa when compared to the mRNA levels found in control cells, as presented in Fig. 26.2 (Faria et al., 2020). The mRNA

615

content varied with the N/P ratio; carriers developed at N/P ratio of 10 led to more intense bands, in the agarose gels, from the GFP transcripts (Faria et al., 2020). Another work evaluated the mutant levels originated from the transfection of wild-type mitochondrial pre-tRNAPhe by using amplification refractory mutation systemquantitative PCR. A strong decrease in the mutant rate of tRNAPhe was achieved and the induced therapeutic effect was sustained (Kawamura et al., 2020). Park et al. published an interesting review highlighting the challenges and techniques for the investigation of gene expression into mitochondria (Park, Lee, & Min, 2020). The protein expression into mitochondria has also been evaluated by Costa et al. ND1 and recoded GFP proteins production in HeLa cells were quantified, by ELISA immunoassay, after 48 h transfection with PEG-PEI25 kDa-TPPpDNA vectors. GFP and ND1 protein levels are dependent on the N/P ratio used in the vector’s formation step; as this parameter increases more efficient is the transfection FIGURE 26.2 PCR analysis of GFP mRNA extracted from mitochondria isolated from HeLa cells after 24 h of transfection mediated by PEGPEI25 kDa-TPP-pGFP vectors conceived at different N/P ratios (2, 5 and 10; R2, R5 and R10, respectively). (A) negative control, (B) mRNA samples from mitochondria of untreated cells, (C) mRNA from mitochondria of cells transfected with pGFP. Source: Adapted from Faria, R., Albuquerque, T., Neves, A. R., Bhatt, H., Biswas, S., Cardoso, A.M., Lima, M. C. P., Jurado, A., & Costa, D. (2020). Physicochemical characterization and targeting performance of triphenylphosphonium nanopolyplexes. Journal of Molecular Liquids, 316, 113873.

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26. Future perspectives of biological macromolecules in biomedicine

process and a higher mitochondrial protein content can be produced. A comparison of protein levels found in the cytosol and mitochondria of HeLa cells revealed that when the developed nanoparticles are directed to the cytosol, GFP and ND1 proteins cannot be expressed due to differences in the genetic code of mitochondria. Table 26.1 presents the ND1 protein levels in the cytosol and mitochondria of HeLa cells, after transfection with the mentioned polyplexes, and illustrates this phenomenon. This study represents a great advance in gene release into the site of mitochondria and transgene expression aiming restore the normal functionality of this organelle. Additionally to the topic of conception of mitochondria-targeted vectors, for effective and sustained gene expression, the fabrication of mitochondrial disease models toward common mutations will deeply contribute for novel outcomes on mitochondrial gene therapy. TABLE 26.1 Quantification of ND1 protein levels (ng/mL) in the cytosol and mitochondria of HeLa cells after 48 h of transfection mediated by PEG-PEI-pND1 and PEG-PEI-TPP-pND1 carriers, at different N/P ratios. Cytosol

Mitochondria

System

Control cells

0

98 6 4.2

PEG-PEI-pND1 N/P 2

0

98 6 3.9

PEG-PEI-pND1 N/P 5

0

100 6 5.1

PEG-PEI-pND1 N/P 10

0

100 6 3.8

PEG-PEI-TPP-pND1 N/P 2

0

266 6 4.6

PEG-PEI-TPP-pND1 N/P 5

0

420 6 5.0

PEG-PEI-TPP-pND1 N/P 10

0

485 6 6.9

The values were calculated with the data obtained from three independent measurements (mean 6 SD, n 5 3). Data were analyzed by one way or two-way ANOVA (GraphPad Software version 6.01, Inc., CA, USA). Statistical significance was accepted at a level of p , 0.05. Adapted from Faria, R., Albuquerque, T., Neves, A. R., Bhatt, H., Biswas, S., Cardoso, A.M., Lima, M. C. P., Jurado, A., & Costa, D. (2020). Physicochemical characterization and targeting performance of triphenylphosphonium nano-polyplexes. Journal of Molecular Liquids, 316, 113873.

26.3 Crosstalk between chronobiology and cancer The physiology of several organisms is adapted to a 24 h day-night cycle, anticipating environmental changes controlled by the rotation of earth (Paschos, Baggs, Hogenesch, & Fitzgerald, 2010). As a consequence, daily rhythms in various physiological and metabolic processes are subjected to circadian regulation that can result from cyclic neuronal and humoral signals coming directly or indirectly from the suprachiasmatic nucleus (Levi & Schibler, 2007). Chronobiology is the quantitative study of the biological daily rhythms and mechanisms in living systems. The human sleep-wake cycle is originated from a circadian mechanism. The sleep-wake cycle normally occurs at a specific phase in relation to both the internal circadian rhythms and the external light-dark cycle. Parameters such as sleep homeostasis, ocular photoreception or interindividual variations in the 24 h intrinsic period of endogenous circadian pacemaker can influence the sleep-wake cycle. Molecular chronobiology studies, sometimes recurring to animal studies, help providing new insights into circadian rhythm sleep disorders and highlighted the relevance of chronobiology on the emergent topic of sleep disorders medicine. The importance of the role of chronobiology on both the toxicity and the antitumor activity of cancer drugs and consequently on cancer treatment has gone through a significant development in the last years (Fu & Kettner, 2013). In fact, several studies have shown not only a circadian variation on the pharmacokinetics of anticancer drugs, but also an increase in tissue sensibility determined by the time of the day of drug administration (Dallmann, Okyar, & Levi, 2016; Kiessling & Cermakian, 2017). Thus, the impact of clock system on the success of cancer therapy through the administration of anticancer drugs in specific phases of the circadian rhythm is likely to improve their therapeutic effects.

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26.3 Crosstalk between chronobiology and cancer

In the following sections, the main topics related with cancer chronobiology, namely the consequences of circadian disruption and the commitment of circadian clock to improve cancer chronotherapy, also recurring to delivery vectors based on biological macromolecules, will be addressed in detail.

26.3.1 Circadian clock and cancer development In mammals, the clock system is constituted by the central circadian clock which is located in the suprachiasmatic nucleus of the hypothalamus and peripheral oscillators. The process of timekeeping involves a set of circadian clock regulated genes, which include circadian locomotor output cycles kaput (Clock), brain and muscle ARNTL-1 (Bmal1), Period (Per1 and Per2) and Cryptochrome that interact between them, forming transcriptional autoregulatory feedback loops. The positive feedback loop is powered by the heterodimerization of CLOCK protein with BMAL1. CLOCK/BMAL1 promotes the transcription of clock-controlled genes (CCGs) which include the ones encoding for PER and CRY proteins. The negative feedback loop closes at the point where PER/ CRY after shuttling to the nucleus binds with CLOCK/BMAL1, inhibiting their own gene transcription (Hastings, Maywood, & Brancaccio, 2018; Takahashi, 2017; Welsh, Takahashi, & Kay, 2010). Nuclear retinoic acid receptor-related orphan receptor (ROR) and REV-ERB, a nuclear receptor that acts as a ligand-dependent suppressor of gene transcription (Burris, 2008) are involved in the first auxiliary loop, with CLOCK/BMAL1 mediating the transcriptions of REV-ERB through E-boxes in their promoters (Crumbley & Burris, 2011). REVERB represses while ROR positively regulates the expression of many genes including BMAL1 (Logan & McClung, 2019; Preitner et al., 2002). CLOCK/BMAL1 complex directly promotes the expression of the D-box binding protein (DBP) (Ripperger, Shearman, & Reppert, 2000) while RORs and REV-ERB, drive the transcription of the

617

nuclear factor, interleukin 3 regulated (NFIL3) (Takeda, Jodhi, Birault, & Jetten, 2012). These two PAR bZIP transcription factors form the second auxiliary loop, where DBP positively and NFIL3 negatively regulate the transcription of various CCGs by linking to D-box response elements in its promotors (Mitsui, Yamaguchi, Matsuo, Ishida, & Okamura, 2001; Takahashi, 2017). The circadian clock is controlled by a set of transcriptional regulatory factors (Shafi & Knudsen, 2019) and can regulate many intracellular pathways related to cancer processes. Indeed, up to 50% of the genome is thought to be under circadian control. The prevalence of mutations and aberrant expression of clock genes in several cancer types is closely associated with abnormalities in cell cycle events and in the activation of intracellular inflammatory and oncogenic signaling pathways, therefore, supporting the link between circadian dysregulation and cancer development (Fu & Kettner, 2013; Morgan et al., 2019; Sulli, Lam, & Panda, 2019). The connection between circadian disruption and cancer appears to be bidirectional, due to the fact that tumors in one organ can influence the homeostatic levels of systemic factors, and this situation may lead to the disruption of the circadian rhythms other organs (Pariollaud & Lamia, 2020; Sulli et al., 2019). Furthermore, circadian disruption is associated with low outcomes, for instance fatigue, sleep disorders, and survival (Pariollaud & Lamia, 2020). Epidemiological studies and experiments in animal models have linked circadian clock disruption to night shift work or the use of intense light at night (LAN) exposure with cancers that are hormone-dependent (Verlande & Masri, 2019). Additionally, clinical evidence associated methylation of clock gene promoters with various cancers, such as breast, (Cordina-Duverger et al., 2018; Hojo et al., 2017; Lesicka et al., 2018; Van Dycke et al., 2015; Wegrzyn et al., 2017) pancreatic (Jiang et al., 2016) lung (Xiang et al., 2018) prostate (Papantoniou et al., 2015) rectal (Papantoniou et al., 2018) ovarian (Khan et al., 2018) and colon (Bishehsari et al., 2020).

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To follow the modification in core clock genes or CCGs taking place worldwide, novel informatic strategies have analyzed patient data from The Cancer Genome Atlas, Gene Expression Omnibus, among others. This effort has allowed to verify that around 90% of “clock genes” were expressed, in a different way, in at least one tumor type. Moreover, the circadian repressors were linked to the inhibition of gene expression regulating apoptosis and other cellular processes (Ye et al., 2018). Some external environmental factors have been proved to disturb circadian system and influence risk of cancer. Irregular light exposure patterns such as LAN or artificial light exposure can potentially disrupt circadian regulation (Blume, Garbazza, & Spitschan, 2019; Chang, Aeschbach, Duffy, & Czeisler, 2015; Figueiro, 2017; Haim & Zubidat, 2015). Other possible disruptors include night shift work (Li, 2019; Lunn

et al., 2017; Pahwa, Labre`che, & Demers, 2018; Pham et al., 2019) geographic factors like the length of daylight and darkness in different seasons (Adamsson, Laike, & Morita, 2016; ˚ kerstedt et al., 2015; Papantoniou et al., 2015; A Wang et al., 2015; Weissova, Skrabalova, Ska´lova´, Bendova´, & Koprivova, 2019) misaligned feeding and excessive intake of fats (Srour et al., 2018; Sulli et al., 2019) jet lag (Kelly et al., 2020; Kettner et al., 2016; Koopman et al., 2017) and disturbance sleep (Lu et al., 2017; Phipps et al., 2016). A 2017 study confirmed the association between long-term rotating night shift work and higher risk of breast cancer, and found that this connection was particularly significant among women who were shift workers during young adulthood, suggesting that age at exposure could be a factor in this association (Pariollaud & Lamia, 2020; Wegrzyn et al., 2017). Fig. 26.3 presents and summarizes the external

FIGURE 26.3 Several external environment and genetic factors that can cause disruption of the circadian rhythms and its cancer-related targets.

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26.3 Crosstalk between chronobiology and cancer

environmental factors linked to circadian cycle disruption and related with cancer occurrence and/or development. Circadian clock dysregulation has also been associated to inner causes such as clock gene mutations, abnormalities in cell cycle events and the activation of intracellular inflammatory and oncogenic signaling pathways. Studies indicated that Clock and Bmal1 may play tumor-suppressive roles. In humans, polymorphisms and/or upregulation in Clock, Bmal1, Cry1,2 and Per1, 2, or 3 genes are connected with great susceptibility to some cancer types and tumor suppression (Dong et al., 2019; Shafi & Knudsen, 2019). Two population studies have demonstrated a link between polymorphisms in the Clock gene and breast cancer (Morgan et al., 2019). Other studies have revealed the phosphorylation and the modification of histones in the promoters of clock genes in cancer cells that are associated with the deregulation of clock gene expression (Masri, Kinouchi, & Sassone-Corsi, 2015; Reszka & Zienolddiny, 2018; Yuan et al., 2019). Recent studies have found that Per1 knockdown significantly decreases the transcript expression levels of p53 (Fig. 26.3) (Fu, Li, Yang, Chen, & Tang, 2016; Li et al., 2016; Yuan et al., 2019). Dysregulation of Per2 is also related with lower levels of p53 (Yuan et al., 2019). This may be explained due to a role of Per2 in the stabilization and nuclear translocation of p53 (Gotoh, Vila-Caballer, Liu, Schiffhauer, & Finkielstein, 2015; Sulli et al., 2019; Zhanfeng et al., 2016). Bmal1 has been found to play a role on γ-radiation (γ-IR) induced p53 and p53-dependent p21 activation to arrest the cell cycle upon DNA damage (Yuan et al., 2019). At the early G1 phase, the Bmal1/Clock heterodimer downregulates the transcription of c-Myc oncogene to prevent its overexpression (Li, 2019). Another example is the expression of the Wee1 gene, an important G2/M checkpoint kinase, which is controlled by the Bmal1/Clock complex (Schmidt et al., 2017).

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These new knowledge and understandings has instigated the research that links circadian clock and cancer therapy (Jacob, Curtis, & Kearney, 2020).

26.3.2 Chronobiology and cancer treatment Nowadays, the complex connection between biological rhythms and cancer occurrence is beyond question. There is a growing body of evidence concerning circadian variation in cytotoxic drug metabolism and tissue sensitivity of anticancer agents (Baraldo, 2008; Dallmann, Brown, & Gachon, 2014). Consequently, the inclusion of circadian timing into cancer therapy may potentially offer a more effective approach and can be considered an emergent concept in the field of therapeutics. Conventional chronotherapy refers to the administration of a treatment into a specific circadian time window under the assumption that the time, at which anticancer drugs are provided, can minimize the inevitable side effects and enhance their efficacy (Ohdo, 2010). Over the last years, substantial evidence has been indicating that not only circadian clocks can alter cancer biology, but they can also influence the pharmacokinetics of several drugs and the targeted responses (Ozturk, Ozturk, Kavakli, & Okyar, 2017). Daily oscillations in drug absorption, distribution, metabolism and excretion can profoundly impact parameters, such as, the abundance, activity, blood concentrations, bioavailability and the effectiveness of molecules relevant to cancer chemotherapy (Baraldo, 2008). The tolerability of more than 40 anticancer drugs has been assessed, and in turn has revealed the importance of the inclusion of the circadian rhythms as a variable in both drug pharmacokinetics and pharmacodynamics (Levi, Okyar, Dulong, & Innominato, 2010; Ozturk et al., 2017). Several studies performed on animals and also

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26. Future perspectives of biological macromolecules in biomedicine

valuable information from human trials have shown promising results, instigating the implementation of cancer chronotherapy on the clinics. In a recent study, researchers have subjected cells to circadian desynchrony. Chronic circadian desynchronization promoted phosphorylation of the retinoblastoma (RB) protein (a tumor suppressor protein) by cyclindependent kinase (CDK) 4/6, favoring G1/S phase cell cycle progression and increasing cell proliferation. Additionally, they did also report that the antitumor effect of one drug named PD0332991 (Palbociclib), an CDK4/6 activity inhibitor, varies in a time-of-day
specific manner. The treatment based on the drug administration in the morning is more effective than the drug treatment at night. Also, the efficacy of PD-0332991 was decreased in cells and in mice when their circadian rhythms were disrupted (Lee et al., 2019). A deep exploitation of this subject must include a better understanding of the chronic disruption effect at a molecular level, as this can contribute to the development of more efficient treatment strategies. Such approaches may include the timing delivery of cancer therapies for a maximum benefit and/or the correction of the circadian disruption. More scientific evidence on this topic is presented in a set of clinical trials clarifying the potential benefits of chronotherapy for cancer patients. For instance, the application of chronochemotherapy to head and neck squamous cell carcinoma, in comparison to conventional chemotherapy, showed higher and longer tumor objective response rate and overall survival in chronomodulated chemotherapy group with lower incidence of adverse events (Chen, Cheng, Yang, Ma, & Yang, 2013). Interestingly, concerning radiotherapy application, time of radiation administration also affects treatment outcomes in cancer patients (Akgun et al., 2014; Shuboni-Mulligan, Breton, Smart, Gilbert, & Armstrong, 2019). Overall survival improvement and further tumor

regression has also been observed for chronomodulated drugs administration in other clinical trials (Li, Chen, Ji, Zou, & Zhu, 2015; Pilancı et al., 2016). Despite these findings, some results have been inconsistent. Findings from meta-analysis studies indicate overall survival improvement only in males under the chronomodulated schedule (Giacchetti et al., 2012). The complexity of cancer biology, as well as, the individual variability differences in age, sex, lifestyle, and genetics variations are altogether relevant factors for the determination of individual chronotypes, which can modify circadian clocks and the metabolism pathways dynamics, thus affecting the optimal timing of drug administration. To overcome these concerns, noninvasive and clinically validated techniques for determining the circadian timing system status have been developed for more accurate and personalized cancer therapies. Another question that needs to be addressed is whether cancer development is a result of circadian disruption, if circadian disruption is an outcome of cancer itself, or both. The relevance of this matter to cancer chronotherapy lies in novel opportunities for cancer treatment that includes the manipulation of the circadian clock and the regulatory factors as possible molecular targets. Table 26.2 summarizes some of the cancer chronotherapy studies currently available in the literature. 26.3.2.1 Cancer chronodrug/gene delivery Despite the great accomplishments on cancer research, cancer remains one of top global causes of death. In fact, unsuccessful chemotherapeutic treatments are often a result of multifactorial issues dependent on pharmacokinetics, tumor microenvironment, ineffectiveness targeting of cancer cells or drug resistance (Sabnis & Bivona, 2019). Besides, conventional chemotherapies lead to considerable toxicity, resulting in lack of wellbeing and quality of life of patients (Wang et al., 2019). This

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26.3 Crosstalk between chronobiology and cancer

621

TABLE 26.2 List of cancer chronotherapy studies in cancer patients. Cancer type

Administered drug

Methods

Main findings

References

Nonsmall cell lung cancer (NSCLC)

Cisplatin

• 41 patients • Patients were divided into two groups based on minimized randomization by receiving cisplatin at different time: 18:00 (Group A) and 6:00 (Group B)

Cisplatin-based chronotherapy has advantage in relieving side effects of chemotherapy, and circadian could influence the metabolism of cisplatin

Li et al. (2015)

High grade glioma

Temozolomide • Randomized with parallel (TMZ) assignment (ongoing) • 27 patients • TMZ administration in the morning (before 10 am) or in the evening (after 8 pm)

Am dosing of temozolomide was associated with higher incidence of adverse events

Campian et al. (2018)

Metastatic Oxaliplatin colorectal cancer and capecitabine

• Single-arm phase II • 30 patients • Oral administration of 50% dose at 8 am and 50% dose at noon

Absence of grade 3
4 gastrointestinal toxicities and no patients with grade 3
4 hand-foot syndrome in comparison with previous studies without chronomodulated administration

Pilancı et al. (2016)

Oral squamous cell carcinoma

Docetaxel, cisplatin and 5-Fluorouracil (FU)

• Crossover • 9 patients • Morning-dosing (10:30) versus evening-dosing (18:30)

Undesirable effects were smaller during evening-dosing than during morning-dosing.

Tsuchiya et al. (2018)

Nasopharyngeal carcinoma

Docetaxel, cisplatin and 5-FU

• Phase II trial • 148 patients • 1-day dose of cisplatin by sinusoidal chronomodulated infusion from 10:00 am to 22:00 pm

Chemotherapy followed by concurrent chronochemotherapy is a promising treatment due to its curative effect while reducing adverse effects and improving immune function

Zhang et al. (2018)

demands for the quest of accurate technologies for drug administration. Over the past decades, research on drug release has addressed protocols toward constant drug release rates. However, new findings on cancer chronotherapy have motivated the development of novel chronodrug delivery systems (ChrDD) according to the time of greatest need. The major challenge in the development of drug delivery systems that match the circadian rhythm might be relying in the appropriate technology (Sunil et al., 2011). There have been some efforts to develop drug products intended for

chronotherapy for a variety of diseases. Some of them include biodegradable polymers and stimuli-sensitive hydrogels for oral administration (Peppas & Leobandung, 2004; Sunil et al., 2011). External programmable pumps have also contributed for advances on cancer chronotherapeutics. IntelliJectTM and MelodieTM are two electronically engineered programmable multichannel pumps, that enable combination schedules for more than one chemical given according to a circadian modulated delivery program (Ballesta, Innominato, Dallmann, Rand, & Levi, 2017; Levi, Karaboue´,

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26. Future perspectives of biological macromolecules in biomedicine

Etienne-Grimaldi, Paintaud, & Focan, 2017) and require minimal nursing intervention. Although used in several clinical trials, the accuracy and safety of infusion pumps revealed significant inconsistencies, remain a critical issue in the clinics (Koopman et al., 2017). In this regard, mathematical models are being developed to optimize delivery, since it can for instance predict the intended drug profile and actual plasma concentrations of a given drug, minimize error and inconsistencies. The optimal cancer chronotherapy should depend upon delivering a drug at right time, right target and right amount (Hill, Innominato, Levi, & Ballesta, 2020). Gene therapy has emerged over the decades as a promising tool for the development of innovative treatments of several types of cancers. Gene therapy as an adjuvant to chemotherapy, as various genes can sensitize tumor cells to drugs promoting a synergistic effect (Liu et al., 2018). Most of the progress that has been done in this broad filed are focused on strategies that include the delivery of tumor suppressor genes, oncogene inhibition, suicide gene therapy, antiangiogenic gene therapy, multi drug resistance associated genes and oncolytic vir´ yen, Martı´nez, Marchal, & Boulaiz, otherapy (A 2018). Although numerous genes might be responsible for triggering oncogenic processes, tumor suppressor genes are usually more associated with the disease. Among these, p53 tumor suppressor gene is one of most studied and applied in gene therapy. The p53 gene keeps the genome integrity by regulating several cellular pathways. Therefore, not surprisingly, mutations in p53 are found in 50% to 70% of all human tumors (Cheah & Looi, 2001). Several studies have shown that the wilt-type p53 gene transfection results in the re-establishment of the normal p53 expression levels and function inducing cancer cells apoptosis (Valente, Queiroz, & Sousa, 2018). The codelivery of p53 gene and anticancer drugs such as doxorubicin has proved to potentiate

the synergistic effect causing a high apoptotic rate of tumor cells both in vitro and in vivo (Li, Xu, Bai, & Liu, 2015). The combination of chronobiology with nanotechnological formulations aiming targeted drug/gene delivery should be able to overcome the limitations of current methods applied to cancer therapy. Nanoformulations can be effective carriers of anticancer drugs and/or therapeutic genes by improving the circulation time and enhancing their bioavailability (Chenthamara et al., 2019) without secondary effects, costs and risks for the patients (Levi & Okyar, 2011). Although all the evidence supporting the application of gene therapy or chronotherapy, studies fail to consider the involvement of circadian clocks on a variety of processes concerning the application of drug loaded nanocarriers systems to cancer gene therapy, such as, the cell uptake and consequent internalization of the nanocarrier, its mechanism of action and, ultimately, the induced therapeutic effect. To the best of our knowledge, only one study addressed yet the dimension of time into nanoparticle formulations. In a recent study, the effect of paclitaxel (PTX)-loaded polymeric nanoparticles (PTX-NPs) combined with chronomodulated administration on lung cancer A549 cells was tested. The results exhibited PTX-NPs greatest antitumor activity and showed a circadian dependence, suggesting a potential treatment for lung cancer (Hu et al., 2017). Recently, other nanocarriers have also been investigated for chronotherapeutics. Costa et al. developed PEI polyplexes able of MTX and p53 loading and encapsulation. The PEI/MTX/p53 polyplexes have appropriate properties concerning size, morphology, surface charge and cytotoxicity for drug/gene release into cancer cells (Costa et al., 2018; Faria et al., 2019). In addition, Fouriertransform infrared spectroscopy were applied to further characterize the developed complexes and confirm the encapsulation of both

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26.3 Crosstalk between chronobiology and cancer

MTX and p53 based plasmid into the nanoparticles. To investigate the influence of the circadian clock in the performance of drug/gene delivery vectors and, therefore, their efficacy in cancer therapy, Costa et al. used the PEI/ MTX/p53 complexes to transfect cancer HeLa cells, following a chronotherapy protocol (unpublished data). To fulfill this propose, HeLa cells have been transfected with these delivery systems at six time points (2, 6, 10, 14, 18 and 22 h) after cells were synchronized and different phenomena, such as, cell uptake/ internalization, transport, and efficacy of transfection were compared during the 24 h. Fig. 26.4 illustrates the hypothesis that are on the basis of the chronotherapy approach under investigation. In this preliminary study, the cell-associated pDNA and MTX fluorescence have been monitored and quantified. High levels of both anticancer drug and pDNA were found in cancer cells for some time points, probably indicating a circadian clock effect on

FIGURE 26.4

623

the cellular uptake and internalization of the drug/gene nanoparticles. Moreover, the p53 protein levels were quantified by an ELISA kit and the obtained data revealed high protein production for the same time points at which MTX and p53 gene were strongly identified in the cancer cells (data not shown). Although preliminary, these results seem to show a direct influence of circadian timing into the cellular transfection mediated by PEI/MTX/p53 delivery systems. A deep investigation of the circadian role on the delivery performance of these drug/gene carriers is ongoing and includes the evaluation of p53 gene expression, the monitorization of the cellular internalization and nucleus colocalization by fluorescence microscopy and, ultimately, the analysis of the induced apoptotic effect on cancer cells. Altogether, these studies will certainly unravel the mechanism underlying the circadian control of transfection efficiency and therapeutic effect.

The role for the circadian clock in the therapeutic effect mediated by drug/gene codelivery.

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The lack of more studies reinforces the necessity of investigating the inclusion of the circadian timing variable into research. Hopefully, more results can encourage the incorporation of the circadian rhythms alongside with other therapeutic strategies. Another point that is worth exploring refers to receptors expressed on cell surface. For instance, transferrin receptor 1 (TfR1) is abundantly expressed on cancerous cells, correlating with the rate of cell proliferation. It was found that TfR1 in colon cancerbearing mice exhibited a 24-h rhythm in mRNA and protein levels (Okazaki et al., 2010). Thus, the efficiency of transferrin conjugated delivery systems could be optimized accordingly. Determining the moment of higher TfR1 expression could be used not only to improve the target capability of the delivery system but also to ensure an increased of chemotherapeutic agents into malignant cells. In addition to anticancer drug and gene delivery, the pharmacological modulation of circadian rhythms can also be an interesting approach to find out novel creative outcomes in cancer therapy. In recent years, significant efforts have been employed in order to develop novel small-molecule modulators of circadian clocks-related proteins that can adjust clock functions (Kettner et al., 2016).

26.4 Concluding remarks In the last decade, the growing research interest in the nanotechnology area has led to great progresses in a broad range of fields from industry, agriculture to biomedical/clinical applications. Concerning the last topic, particular focus has been given to the potentialities of biological macromolecules as therapeutic agents as well as to the design and development of suitable delivery systems to load/encapsulate those macromolecules in view of novel and advanced protocols in biomedicine. Among the wide variety of questions

to address, the treatment of severe and serious diseases affecting humankind, to which current medicine strategies are far from being effective, such as cancer and mtDNA based disorders, conquered special attention. Mutations in mtDNA genome are related to several neurological and metabolic diseases, Parkinson and Alzheimer diseases, diabetes and, even, cancer. Current treatments of these diseases include the administration of a set of drugs to minimize/alleviate the symptoms, therefore, not providing a definite cure. Mitochondrial gene therapy arises as a powerful alternative as it acts in the actual cause of the disorder, being promising in the restoration of normal mitochondrial function. To turn gene therapy toward mitochondria feasible and viable, the conception of a mitochondrial gene-based targeted carrier is imperative. In this context, compounds exhibiting mitochondrial affinity reveal great potential to be incorporated into the delivery systems employed to target mitochondria. Targeting mitochondria appears essential for further gene expression and ultimate protein expression into this organelle. The use of a recoded GFP encoding gene or the cloning of the mitochondrial gene ND1 are both well succeeded approaches in the quest for an efficient mitochondrial vector to mediate the transfection protocol directed to mitochondria. The discussed strategies are both considered a significant contribution for the implementation of mitochondrial gene therapy, instigating other research teams to pay attention into therapies centered into mitochondrion. These works demonstrate mitochondria targeting, effective cellular transfection, gene expression into this organelle, and protein production. Together, the studies reported pave the way for the intensification of the research focused on the development of mutant cells along with adequate animal models of mitochondrial disease, shortening the distance to the desired achievement of clinically viable gene therapy toward mitochondria.

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Chronobiology is a quite new and emerging research field. This is an interdisciplinary area focused on the study of the circadian timing at the organismal and cellular levels. The circadian clock exists in simple unicellular organisms and in more complex ones such as in mammals. In mammals, the circadian clock is organized into the main pacemaker, input signaling pathways and output signaling pathways. Changes and/or perturbations in the circadian clock system lead to various molecular dysfunctions and can originate severe pathologies, such as, cancer. Several studies reported the link between the disruption of the circadian timing system and the occurrence and/or development of several types of cancer, elucidating the relevance of a functional clock system. In fact, dysregulation of the circadian clock might affect many cancer-related cellular processes. Furthermore, it is well documented that tumor suppressors like p53, are controlled by the circadian clock and the disruption of circadian oscillations might affect p53 expression. In this sense, the clock system may also impact on the efficacy of anticancer medications, and the timely delivery of drugs in specific phases of the circadian rhythm revealed to enhance the therapeutic outcomes. Besides the development of suitable drug delivery vectors for controlled delivery, the study of the effect of these systems on circadian clock components of cancer cells and the investigation of the influence of the circadian system on the therapeutic effect of the drug delivery formulations is mandatory to acquire more solid data that will, for sure, bring more understanding and knowledge into this promising field. This effort in consolidating the bridge between nanotechnology and chronobiology may have a remarkable impact in personalized cancer treatment, where matching the drug administration with the individual circadian cycle could be rationally implemented as a standard practice in the clinics.

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IV. Others

Index

Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Aceclofenac, 349 Acetaminophen, 342 Acetohexamide, 229
230 Acetylcholinesterase (AChE), 566 Acetylglucosamine, 72
73 Acotinum coreanum, 254
255 Acylated homoserine lactones (AHLs), 32
33 Adalimumab, 60, 274
275 Adenine (A), 61, 481
482 Adenosine diphosphate (ADP), 15 Adenosine triphosphate (ATP), 15 Adipose-derived stem cells (ADSCs), 383 Ag nanoparticles (AgNP), 187 Agar, 9 agar-based injectable hydrogels, 410
411 Agaricus bisporus, 36
37 Agarose, 496
497, 533
534 Agarwood, 266 Age-related macular degeneration (AMD), 106 Agrobacterium tumefaciens, 32
33 Albuminoids, 13 Albumins, 12 albumin-based nanostructures, 484 Algae, 203 bioactive macromolecules, 203
213 alkaloids, 208 peptides, 209
210 phenolics, 207
208 polyketide, 210 polysaccharides, 208
209 steroids, 206
207 terpenoids, 203
206 polyunsaturated fatty acids, 210
213 Algal-derived proteins, 151
152

Alginate(s), 3
4, 7
8, 8f, 128, 173, 183
184, 382
383, 408, 460, 497
499, 534
535, 585
586 alginate-based hydrogels, 407
408 alginate-based nanocomposite, 187 Alginic acid, 460 Alicyclobacillus acidoterrestris, 32
33 Alkaline phosphatase (ALP), 383
384 Alkaloids, 99
102, 208 caffeine, 99
100 capsaicin, 101 chemical structure of, 101f theobromine, 101
102 Alkyl spacer, 313 All-trans retinoic acid (atRA), 396 Allergen immunotherapy, 591
592 Allograft techniques, 381 Allophycocyanin, 151 Alpha-linolenic acid (ALA), 210
211 α-amylase, 230 α-bisobolol (BIS), 401 α-cyclodextrin (αCD), 587 α-galactosidase, 569 α-glucosidase inhibitors, 230, 560 α-linolenic acid, 120 α-mannosidase, 569 α-pentagalloylglucose (α-PGG), 566
567 Alzheimer’s disease (AD), 3
4, 219, 612 Amantadine, 317 Amino acids, 24 deficiency, 297
298 5-amino-5-deoxy-D-glucose, 554
555 Aminoglycosides, 307
308 Aminolipids, 483 Amoxicillin, 395 AMP-activated protein kinase (AMPK), 256 Amphenicols, 307
308

633

Amyloid-beta and tau protein, 219
220 Amylopectin, 289
290, 460
461, 508
509 Amylose, 289
290, 460
461, 508
509 Andrographis paniculata, 146 Angelica sinensis, 141 Animal derived carbohydrates with nutraceutical activity, 126 Animal-derived polysaccharides, 146
147 Animal-derived proteins, 150
151 Annona squamosa, 569
570 Antheraea pernyi, 506
507 Anthocyanins, 103 Anti-IL-23 antibody, 274
275 Antibacterial activity, synthetic macromolecules with, 313
317, 314t Antibiotic(s), 99, 307
308 drugs, 165 resistant tuberculosis, 306 Anticancer agents biological macromolecules for cancer therapy, 244
269 carbohydrates, 244
258 lipids, 262
269 natural compounds or biological macromolecules, 245t proteins and nucleic acid, 258
262 Antidiabetic agents advantages, limitations, and future perspectives, 238 biological macromolecules, 232
238 types, 230
232 Antifungal activity, synthetic macromolecules with, 318
319 Antifungal polymers, 306 Antigen presenting cells (APCs), 275

634 Antimicrobial activity, 306 antibacterial activity, synthetic macromolecules with, 313
317 antifungal activity, synthetic macromolecules with, 318
319 antiparasitic activity, synthetic macromolecules with, 319
320 antiviral activity, synthetic macromolecules with, 317
318 classification of antimicrobial polymers, 308
309 factors affecting antimicrobial activity, 311
313 alkyl spacer, 313 charge density, 312
313 counter ion effect, 312 hydrophilic/hydrophobic balance, 311
312 molecular weight, 312 polymeric architecture, 313 history of antimicrobial agents and antimicrobial polymers, 307
308 preparation routes for antimicrobial polymers, 309
311 synthetic macromolecules with, 306
320 Antimicrobial agents, 306
308 antimicrobial activity of biological macromolecules, 172
180 fatty acids, 177
180 polysaccharides, 172
174 proteins, 175
177 antimicrobial activity of macromolecule composites, 180
185 chitosan-alginate, 180
181 chitosan-cellulose, 184 collagen-alginate, 183
184 gelatin-chitosan, 181
182 keratin-chitosan, 182
183 lactoferrin-oleic acid, 185 applications, 190
193 drug delivery, 191
192 food packaging, 190
191 wound dressing, 192
193 classification of biological macromolecule, 167
172 carbohydrate, 167
169 lipid, 170
171 protein, 169 nanotechnology based antimicrobial macromolecule, 185
190

Index

Antimicrobial peptides (AMPs), 183
184, 203, 307
308 Antimicrobial polymers, 307
308 classification of, 308
309 preparation routes for, 309
311 Antimicrobial surfaces, 308 Antimicrobials, 471 Antioxidants, 140
141, 320 activity methods evaluation of antioxidant activity, 325t synthetic macromolecules with, 320
324 food-based applications, 156
158 future trends, 159 limitations of biological macromolecules, 158
159 macromolecules as, 152
156 polymers, 306 types and sources of biological macromolecules, 141
152 Antioxidative macromolecules, 152 nonextractable polyphenols, 152 Antiparasitic activity, synthetic macromolecules with, 319
320 Antisense oligonucleotides, 62, 584
585 Antiviral activity, synthetic macromolecules with, 317
318 Antiviral polymers, 306 Antrodia cinnamomea, 257 Apolar lipids, 120 Aptamer, 63 Arabinose (Ara), 254 Arabinoxylans (AX), 585
586 Arachidonic acid (AA), 180, 278
279 Arctigenin (Arc), 253 Arctium lappa, 253 Armoracia rusticana, 535 Asamycines, 307
308 Ascophyllum nodosum, 232 Ascorbic acid (C6H6O6), 456 Aspergillosis, 318 Aspergillus A. aculeatus, 535 A. flavus, 41 A. fumigatus, 318 A. niger, 172 Astragalus A. membranaceous, 254 A. membranaceus, 141
146 Astragalus polysaccharide (APS), 254

Atom transfer radical polymerization (ATRP), 309
310 Aureobasidium pullulans, 510
511 Auricularia polytricha, 292 Autografts, 599 techniques, 381 Avena sativa, 232 Avian influenza A, 306 Avrainvillea, 204

B B-glucans, 276 Bacillus B. cereus, 31 B. subtilis, 42, 204
206 B. thuringiensis, 32
33 Bacillus striatum polysaccharide (BSP), 128
129 Bacterial cellulose (BC), 173 Bacterial polysaccharides, 149 Bacteroides, 590 Barium titanium (BaTiO3), 596
597 Base pairs (BP), 481 Beads-free fibers, 401
402 Beetle (Massonia pustulata), 261
262 1,4-benzoquinone moiety, 205 Bergamot essential oil (BEO), 265 Bergapten-free BEO (BEO-BF), 265 β-carotene, 105
106, 458 β-galactosidase, 569 β-glucan, 56 β-glucocerebrosidase (GCase), 557 β-lactoglobulin, 461 β-(1,4)-linked D-glucosamine, 72
73 β-mannosidase, 569 β-N-acetylhexosaminidase OfHex1, 565
566 ureido thioglycosides inhibitors of, 566f β-tricalciumphosphate (β-TCP), 395 Bicyclic iminosugars, 559
560, 560f Bifidobacteria, 590 Bio-catalysis, 529 Bio-nanotechnology, 607
610 delivery systems, 608
610 Bioactive compounds, 305 Bioactive lipids, 120 in cancer stem cells, 123
124 Bioactive macromolecules, 305 Bioactive materials, 315 Bioactive molecules, 471
472 Bioactive peptides, 109
110, 308

635

Index

Bioactive polysaccharide, chitosan as, 36
42 Bioactive proteins, 14 enzymes as, 24
36 Biobased materials, 547
548 Biocatalysts, 529 Biocidal polymers, 308 Biocide releasing polymers, 308 Biocompatibility, 492 Biodegradable chitosan, 349 Biodegradable polymers, 393 Biological interest of carbohydrates, 550
551 Biological macromolecules, 3, 23, 53, 69, 70f, 141, 166, 339
340, 381, 479
480. See also Synthetic macromolecules as antidiabetic agents, 232
238 carbohydrates, 232
233 lipids, 233
234 nucleic acids, 237
238 proteins, 235
237 bio-nanotechnology, 607
610 in BTE, 382
387 for cancer therapy, 244
269 carbohydrates, 4
9 crosstalk between chronobiology and cancer, 616
624 chronobiology and cancer treatment, 619
624 circadian clock and cancer development, 617
619 drug delivery using, 340
367 for enzyme immobilization, 532
541 lipids, 9
11 mitochondrial gene therapy, 610
616 nucleic acids, 14
17 for nucleic acids delivery, 482
488 for nutrients delivery, 458
462 nutrients delivery systems based on, 465
471 proteins, 11
14 sources and bioactivities of macromolecules, 142t types and sources of, 141
152 antioxidative macromolecules, 152 polysaccharides, 141
149 proteins, 149
152 Biomacromolecules, 191
192, 581 Biomass, 547
548

Biomaterials of biomedical, 581 as targeted drug delivery, 585
593 gene delivery, 592
593 hydrogels for drug delivery, 585
592 Biomedicine, 64 biological macromolecules in, 53
54, 582
583 carbohydrates, 54
56 chemical properties and functions of, 583t lipids, 60
61 nucleic acids and oligonucleotides, 61
63 peptides, 56
58 proteins, 58
60 synthesis of macromolecules, 63
64 biomaterials as targeted drug delivery, 585
593 macromolecules in biomedical applications, 583
584 targeted drug delivery, 584
585 macromolecules on tissue engineering, 593
599 nucleic acids and nutraceutical properties used in, 112
119 Biomolecules, 274
275 biomolecules-based therapeutics, 53 Biopassive materials, 313
315 Biopolymers, 403, 440
441, 492
493, 495 biopolymer-based delivery systems, 462
464 covalent interactions, 464 electrostatic interactions, 463 hydrogen bonding, 463 hydrophobic interactions, 463
464 other natural polymers in TE, 508
511 used for cell encapsulation in TE, 496
511 agarose, 496
497 alginate, 497
499 chitin and chitosan, 499
500 collagen, 500
502 fibrin, 504
505 gelatin, 503
504 glycosaminoglycans, 505
506 silk, 506
508 Biosafety, 492

Biosensing, 113
114 Biotransformation of food, 289 Bixalomer, 327
328 Bletilla striata, 292
294 Blowfly (Chrysomya megacephala), 146 Bombyx mori, 506
507 Bone morphogenic proteins (BMPs), 445 Bone regeneration, 439
441 growth factors in, 441
443 scaffolds and fibers, 446
447 Bone remodeling, 596 Bone tissue engineering (BTE), 381
382 biological macromolecules in, 382
387 alginate, 382
383 carrageenan, 384
385 chitosan, 383
384 fucoidan, 385 gelatin, 386
387 ulvan, 385
386 strategy, 596
597 Bordetella bronchiseptica, 149 Bovine lactoferrin, 176
177 Bovine serum albumin (BSA), 399, 448 nanoparticles, 352
353 Brain cancer, lipids in, 265
266 Branched PEI (bPEI), 609
610 Breast cancer carbohydrates in, 252
255 lipids in, 264
265 proteins and nucleic acid in, 259
261 Brome mosaic virus (BMV), 117 Brown alga (Sargassum henslouianum), 148 Buccal patches, 349 Butanetetracarboxylic acid, 397 Butylated hydroxyanisole (BHA), 321 Butylated hydroxytoluene (BHT), 321 Butyrylcholinesterase (BuChE), 566

C C-type lectins, 222 Caffeine, 99
100 Calcium phosphate cement (CPC), 433 Calcium Resonium, 327 Callophyllis concepcionensis, 156 Campylobacter jejuni, 176
177 Cancer Genome Atlas, 618 Cancer(s), 3
4, 243, 306 bioactive lipids in, 123

636 Cancer(s) (Continued) cancer stem cells, 123
124 cancer chronodrug/gene delivery, 620
624 chronobiology and, 616
624 circadian clock and cancer development, 617
619 list of cancer chronotherapy studies in cancer patients, 621t of GS, 289 Candida albicans, 42, 318 Candidiasis, 318 Cantharidin, 261
262 Capsaicin, 101 Capsicum C. annuum, 101 C. baccatum, 101 C. chinense, 101 C. frutescens, 101 C. pubescens, 101 Capsosiphon fulvescens (CF), 251
252 Carbasugars, 561
564 analogs to N-Acetyl glucosamine, 564f natural, 562f Carbazole-loaded gliadin nanoparticles, 354 Carbohydrate macromolecules. See also Synthetic macromolecules biological macromolecules, 70f cellulose, 69
71 chitin and chitosan, 72
73 hemicelluloses, 71
72 lignin, 72 modification of carbohydrate biological macromolecules, 73
86 chemical modification of cellulose, 75t chemical modification of chitin/ chitosan, 80t chemical modification of lignin, 76t classification of modification, 73f enzymatic modification of cellulose, 76t enzymatic modification of chitin/ chitosan, 84t enzymatic modification of lignin, 77t modification techniques for hemicellulose, 74f

Index

physical modification of cellulose, 74t physical treatments for chitin/ chitosan, 80t principle groups of carbohydrates macromolecule, 70f Carbohydrate-active enzymes (CAZymes), 551 Carbohydrate(s), 3
9, 23, 53
56, 124, 222
223, 230, 305, 548
550, 582
583 as antidiabetic agents, 232
233 as antimicrobial agents, 167
169 disaccharide, 167
168 monosaccharide, 167 oligosaccharide, 168 polysaccharide, 168
169 biological and medicinal interest of carbohydrates, 550
551 biomedical applications of, 550f for cancer therapy, 244
258 breast cancer, 252
255 cervical cancer, 244
250 colon cancer, 250
252 lung cancer, 255
257 others, 258 pancreatic cancer, 257
258 carbohydrate-based drug delivery systems, 486
488 carbohydrate-based macromolecular systems, 549 drug delivery using, 341
349, 350t esterases, 551 glycomimetics, 554
566 glycosidases, 551
553 classes of enzymes, 552f hybrid, 566
567 molecular diversity in biological applications, 549f monosaccharides, 5 as nutraceuticals, 124
129 alginate, 128 animal derived carbohydrates with nutraceutical activity, 126 BSP, 128
129 carbohydrates from plants with nutraceutical activity, 125 carbohydrates with nutraceutical activity from microorganisms, 128 cellulose and hemicellulose, 125
126

chitosan and chitin, 127
128 dextran, 128 heparin, 126
127 hyaluronic acid, 127 immunostimmulatory effect of carbohydrates, 125 polysaccharides role in extracellular membrane, 124
125 oligosaccharides, 5 polysaccharides, 5
9 role in gastrointestinal system, 289
295 role on nervous system, 223 in retino-tectal system, 223 therapeutics based on carbohydrates, 55
56 Carbon, 4 Carboxylated cellulose nanocrystals (CCNCs), 397
398 Carboxylic acids, 177 Carboxymethyl-hexanoyl derivative, 232
233 Carboxymethylcellulose (CMC), 42, 397, 429 Cardiovascular diseases, 419
421 Carrageenan, 3
4, 384
385, 460, 509 for enzyme immobilization, 537
539 Casein, 461 Casein-coated diltiazem HCl, 351 Cassia obtusifolia L., 146 Cassia tora L., 146 Castleman’s disease, 274
275 Catalase, 140
141 Catechol-modified methacryloyl chitosan, 407 Cationic human-serum albumin (CHSA), 485 Cationic lipids, 609 Cationic polymerization, 309
310 CCK-8 assay kit, 256 Cedrelopsis grevei, 265 Cell biology studies, 401 cell-penetrating peptides, 610 cell-signaling, 441 cycle proteins in CNS injury, 220, 221f proliferation, 441
442 replication, 16 targeting strategy, 608
609 Cell encapsulation

Index

advantages, drawbacks, applications, forms and manufacturing methods, 511
525 characteristics and applications of biopolymers used TE, 512t main forms and manufacturing methods for biopolymers used TE, 524t biological macromolecules used in, 493t biopolymers used for cell encapsulation in TE, 496
511 methods, 491
492 Cell-wall targeting (CWT), 28
30 Cellular damages, 27 Cellular processes, 441 Cellulose, 6, 7f, 69
71, 125
126, 166, 168
169, 169f, 173, 289
290, 508 based nanocomposite, 187
189 chemical modification of, 75t and derivatives for enzyme immobilization, 535
536 enzymatic modification of, 76t molecular structure of cellulose molecule-numbering system, 71f physical modification of, 74t Cellulose acetate (CA), 397
398 Cellulose nanocrystals (CNC), 6, 173 Central composite rotational design (CCRD), 535 Central fatty hypothesis, 222 Central nervous system (CNS), 3
4, 219 carbohydrates, 222
223 role on nervous system, 223 in case of neurodegenerative diseases, 225 cell cycle proteins, 220 central fatty hypothesis, 222 cPLA2 role in cerebral ischemia, 225 glycans, 223
224 role in neural development, 224 homer/vesl proteins, 220
222 lipids, 224
225 peroxidation, 225
226 proteins, 219
220 in sensory organs, 223 Cephadrine, 409 Cephalins, 10

Cerebral ischemia, cPLA2 role in, 225 Cerium (Ce), 384
385 Cervical cancer, carbohydrates in, 244
250 Chaga mushroom (Inonotus obliquus), 256 Charge density, 312
313 Chelating agent soluble solids (CHSS), 249 Chemically modified polymers, 309 Chemotherapy, 608
609 Chenopodium quinoa, 236
237 Chitin, 72
73, 127
128, 499
500 chemical modification of, 80t enzymatic modification of, 84t for enzyme immobilization, 532
533 physical treatments for, 80t Chitosan (CS), 3
4, 8
9, 72
73, 127
128, 166, 172, 191, 232
233, 252
253, 276
277, 383
384, 395, 486
487, 499
500, 585
586, 593, 596 based nanocomposite, 185
187 as bioactive polysaccharide, 36
42 bioactivity of chitosan with modified functional group, 41
42 relationship of chitosan physicochemical property and bioactivity, 37
40 chemical modification of, 80t chitosan-alginate, 180
181, 429
430 chitosan-cellulose, 184 chitosan-clay-based biocomposite, 533 enzymatic modification of, 84t for enzyme immobilization, 532
533 physical treatments for, 80t Chitosan oligosaccharide (CSO-OA), 179, 292 Chlorella pyrenoidosa, 252 Chlorococcum sp., 252 Chlorpropamide, 229
230 Cholesterol, 11, 170
171 conjugated 5-fluorouracil, 361 Cholestyramine, 326 Chondrococcus hornemanni, 205 Chondroitin, 166, 173
174 Chondroitin sulfate (CS), 173
174, 429, 505 Chromoproteins, 13

637 Chronobiology and cancer treatment, 616
624 Chronodrug delivery systems (ChrDD), 616
624 Chrysomya megacephala. See Blowfly (Chrysomya megacephala) Cigarette smoke, 139 Ciprofloxacin (CP), 173, 264, 352 HCl, 398 Circadian clock, 617 and cancer development, 617
619 dysregulation, 619 Citrullus lanatus, 236
237 Clarithromycin, 354 Click chemistry, 309
310 Clock-controlled genes (CCGs), 617 Clove buds (Syzygium aromaticum), 268
269 Coacervation-gelation methods, 466
467 Coagulated proteins, 14 Coaxial emulsion electrospinning, 400 Cocculas hirsutus, 347
349 Codium iyengarii, 206 Cohesive system, 64 Colesevelam, 326
327 Colestimide, 326
327 Colestipol, 326 Collagen, 166, 175, 402, 426, 432, 500
502, 501f based nanocomposite, 190 collagen-alginate, 183
184 Collagen-binding VEGF (CB-VEGF), 426 Colon cancer, 250
252 carbohydrates in, 250
252 lipids in, 263
264 proteins and nucleic acid in, 259 Colony forming unit (CFU), 183
184 Colorectal cancer (CRC), 590 proteins and nucleic acid in, 259
261 Combretum caffreum, 244 Communicable diseases (CDs), 98 Composites materials for GFs delivery, 430 Compound lipids, 10 Concentrated alkali soluble solids (CASS), 249 Conjugate lipids, 10 Conjugated proteins, 13 Constipation, 289 Convulsive disorders, 3
4

638 Cordyceps sinensis, 256 Corn silk polysaccharide (CSP), 244 Coronary diseases, 419
421 Coronavirus (CoVs), 111
112 Coronavirus disease (COVID-19), 123 bioactive lipids in, 123
124 Corynebacterium diptheriae, 206 Counter ion effect, 312 Covalent bonding, interaction with, 531
532 Covalent interactions, 464 cPLA2 role in cerebral ischemia, 225 CRISPR, 117
119 Crohn’s disease, 281
282 Croton flavens L., 263 Cryptococcus neoformans, 318 CsNP. See Nanoparticles from chitosan (CsNP) Cucurbita C. moschata, 232 C. pepo, 236
237 Curcuma longa. See Turmeric (Curcuma longa) Curcumin, 98, 102
103, 354
355, 458, 592 Cutaneous Leishmaniasis, 319 Cyanobacteria, 259
260 Cyclic guanosine monophosphateadenosine monophosphate synthase (cGAS), 276
277 Cyclin-dependent kinase (CDK), 219, 619
620 Cyclodextrin, 445
446 Cyclopentano perhydro phenantherene, 11, 11f Cyclophosphamide, 354 Cysteine, 396 Cystic fibrosis, 593 Cytidine diphosphate (CDP), 15 Cytidine triphosphate (CTP), 15 Cytochrome c, 256 Cytosine (C), 61, 481
482 Cytotoxicity, 583
584 test, 250
251

D D-galactose, 566 D-ribose, 566 D-xylose, 566 DC-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), 282 Deacetylation degree (DD), 276
277

Index

Dectin-1, 276 Degummed silk fibroin, 507 Delivery systems, 469 bio-nanotechnology in, 608
610 nonviral vectors, 608
610 for GFs, 421
423 biological macromolecules for, 424
433 composites materials for, 430 materials for, 423
424 polysaccharide combinations for, 429
430 polysaccharide-based materials for, 427
429 polysaccharide-polysaccharide composites for, 433 protein-based composite for, 430
432 protein-based materials for, 425
427 protein-polysaccharide composites for, 432
433 Denaturation-gelation method, 467 Dendrimers, 318, 592, 609 Dendritic cells (DCs), 280 Deoxycytidine (dCyd), 264 Deoxygalactonojirimycin (DGJ), 556 Deoxymannojirimycin (DMJ), 556 1-deoxynojirimycin (1-DNJ). See 1,5dideoxy-1,5-imino-D-glucitol Deoxynojirimycin FA-DNJ, 569 Deoxyribonucleic acid (DNA), 3, 15
16, 61, 171
172, 230, 480, 481f, 582
583 fragmentation, 27 functional sequence, 116
119 CRISPR, 117
119 gene therapy, 117 non-mRNA sequences, 116
117 nanostructures, 113
116 biosensing, 113
114 drug delivery, 114
115 immunomodulatory, 115
116 nanotechnology, 358 Deoxyribose, 61 Derived lipids, 10
11 Derived proteins, 13
14 Dermatan sulfate (DS), 505 Dermis, 597
598 Derris elliptica, 554
555 Dexamethasone (DXM), 401
402 Dextran (Dex), 7, 128, 399, 509
510, 585
586

for enzyme immobilization, 537
539 carrageenan for enzyme immobilization, 537
539 Dextran sulfate sodium (DSS), 289
290 DHA. See Docosahexaenoic acid (DHA) Diabetes, 229 Diarrhea, 289 1,5-dideoxy-1,5-imino-D-glucitol, 554
555 2,5-dideoxy-2,5-imino-D-mannitol, 554
555 Diethyl aminoethyl (DEAE), 536 Diffusion mechanism, 465 2,5-dihydroxymethyl-3, 4dihydroxypyrrolidine (DMDP), 554
555 Diluted alkali soluble solids (DASS), 249 Dimethyl (DM), 319 Dioscorea species, 342 Dipeptidyl-peptidase-IV (DPP-IV), 229
230 2,2-diphenyl-1-picrylhydrazyl (DPPH), 40, 244 Dipotassium hydrogen orthophosphate, 406 Disaccharides (DS), 5, 167
168, 289
290 Dissolution mechanism, 464 Diterpenoids, 203
204 Docosahexaenoic acid (DHA), 11, 121
123, 210
211, 278
279 Dopamine, 222 Doxorubicin, 352 doxorubicin-loaded zein nanoparticles, 353
354 Doxycycline monohydrate (DCMH), 401
402 Dragline silk. See Nephilia clavipes Drug delivery, 114
115 antimicrobial agents on, 191
192 using biological macromolecules, 340
367 using carbohydrates, 341
349, 350t using lipids, 359
367, 366t using nucleic acids, 355
359 using peptides, 349
355, 356t using proteins, 349
355, 356t

639

Index

drug delivery-based on lipids, 60
61 Drug delivery systems (DDSs), 393, 479 “Drug-free” macromolecular therapeutics, 273
274 Drug-loaded electrospun fibers in tissue engineering, 394
403 drug-loaded polysaccharidesbased ESF, 395
400 drug-loaded protein-based ESF, 401
403 Drug-loaded injectable hydrogels in tissue engineering, 403
411 Drug-loaded polysaccharides-based ESF, 395
400 Drug-loaded protein-based ESF, 401
403 Durvillaea antarctica, 148

E Ebola, 306 Echinacea purpurea, 292
294 Eicosapentaenoic acid (EPA), 11, 121
123, 210
211, 278
279 Elaidic acid (EA), 263
264 Elastin, 426, 511 Elav-like protein 1, 235
236 Electrospinning process, 393
394, 399 Electrospun fibers (ESF), 394
395 drug-loaded polysaccharides-based, 395
400 Electrostatic interactions, 463 Elsholtzia ciliata, 265
266 Emulsification-gelation methods, 467 Emulsion electrospinning, 402 Endo-glycosidase, 552
553 Endogenous antioxidants, 140
141 Endogenous digestive enzymes, 230 Engeletin (ENG), 255
256 Enhanced permeability and retention effect (EPR effect), 358 Enterobacteria, 590 Enterococcus, 173
174 Enteromorpha E. compressa, 156 E. linza, 148 Enzyme immobilization biological macromolecules for, 532
541 agarose, 533
534 alginate, 534
535

cellulose and derivatives, 535
536 chitin and chitosan, 532
533 dextran, 537
539 gelatin, 536
537 pectin, 539
540 xanthan, 540
541 characteristics of enzyme immobilization platforms, 530f studies reporting immobilization of different enzymes using biopolymers, 531t Enzyme(s), 469
470, 529 as bioactive proteins, 24
36 LAOs, 25
28 lysostaphin, 28
32 metallo-β-lactamase-like lactonase, 32
36 carbohydrates for enzyme inhibition biomass and biobased materials, 547
548 glycomimetics, 554
566 hybrid carbohydrates, 566
567 macromolecules, 567
570 enzyme-assisted extraction method, 230
232 Epidermal growth factor receptor (EGFR), 281
282 Epidermis, 597
598 EPL. See Poly(ε-lysine) (EPL) Erythropoietin (EPO), 63
64 Escherichia, 173
174 E. coli, 6, 27, 42, 149, 166, 206 Especialized metabolites with nutraceutical activity, 107, 108t Essential fatty acids (EFA), 120, 298
299 Essential oil from fresh leaves of O. kilimandscharicum (EOOK), 266
267 Essential oils (EO), 262
263, 267 Ethnomedicines, 64 Ethosomal formulation, 362
364 Ethyl cellulose (EC), 396 Eubacteria, 590 Eucheuma E. cottoni, 206, 207f E. serra, 209
210 Eupatorium adenophorum (EA), 266 Eurysterols A, 206 Eurysterols B, 206 Exo-glycosidase, 552
553

Extracellular matrix (ECM), 124
125, 393, 419, 491 Extracellular membrane, polysaccharides role in, 124
125 Extracellular polysaccharides (EPSS), 252 Extraction method, 230
232

F Fabrication, 466
469 of hydrogel, 466
467 of microcapsules, 467
468 of nanoparticles, 468
469 “Facially amphiphilic” approach, 311
312 Fascioquinols A, 205
206 Fascioquinols B, 205
206 Fascioquinols C, 205
206 Fascioquinols D, 205
206 Fats, 10 Fatty acid methyl ester (FAME), 177
179, 212 Fatty acids, 177 antimicrobial activity of, 177
180 arachidonic acid, 180 FAME, 177
179 linoleic acid, 179
180 oleic acid, 179 role in gastrointestinal system, 298
300 Ferrier carbocyclization, 563
564 Fibers, 446
447 Fibrin, 166, 176, 504
505 Fibrinogen, 426
427 functionalization synthesis of, 431f Fibroblast growth factor (FGF), 442 Fibroion, 506
508 Fibronectin, 511 Ficus pandurata, 244
249 First generation monoclonal antibodies, 59 First-line defense antioxidants, 140
141 Fish gelatin (FG), 598 Flavin adenine dinucleotide (FAD), 25
26 Flavonoids, 243 Flavors, 469 5-fluorouracil (5-FU), 263
264, 361, 403 5-fluorouracil-loaded zein nanoparticles, 353
354

640 Food additives, 158 Food and Drug Administration (FDA), 57
58, 274
275, 280, 458, 582
583 Food packaging, 157
158 antimicrobial agents on, 190
191 Food-based applications antioxidants in, 156
158 food additives, 158 food packaging, 157
158 functional foods, 156
157 other applications, 158 Fourier transform infrared spectroscopy (FTIR), 250
251, 622
623 Fourth generation monoclonal antibodies, 60 Fragmentation mechanism, 465 Free fatty acid receptor 1 (FFA-1), 233
234 Free radicals, 139 polymerization technique, 309
310, 322
323 Fructose, 167, 167f, 289
290 Fucoidan (FCD), 55
56, 257, 385, 411 Fucosterol, 206, 206f Functional foods, 156
157 Functional peptides, 109
110 Fungal infections, 318 Fungal polysaccharides, 149, 258

G G protein-coupled receptor (GPR), 277 Gabapentin, 397
398 Galactomannan, 429 Galactose (Gal), 254, 289
290 Galacturonic acid (GalTA), 254 Galaxaura marginata, 209
210 Gallic esters, 320
321 γ-linoleic acid, 278
279 Ganglioseries, 223 Gas chromatography (GC), 254
255 Gastrointestinal systems (GS), 289 carbohydrates role in, 289
295 fatty acids role in, 298
300 nucleic acids role in, 300 proteins role in, 295
298 types of biological macromolecules, 290f Gastrointestinal tract (GI tract), 289, 588
589

Index

Gelatin (Gel), 3
4, 166, 175, 386
387, 397
398, 426, 485, 503
504, 595
596 based nanocomposite, 189
190 for enzyme immobilization, 536
537 gelatin-chitosan, 181
182 hydrogels, 446 microspheres, 448 Gellan, 510 Gemcitabine, 264 gemcitabine-conjugated albumin nanoparticles, 352
353 Gene delivery, 592
593. See also Drug delivery cancer chronodrug/gene delivery, 620
624 Gene expression, 614
616 Gene Expression Omnibus, 618 Gene therapy, 117, 608
609 Ginseng polysaccharides (GP), 56 Glibenclamide, 229
230 Globoseries, 223 Globulins, 12 Gloiopeltis furcate. See Red alga (Gloiopeltis furcate) Glucagon-like peptide-1 (GLP-1), 237
238 receptors, 229
230 Glucose (Glc), 167, 167f, 254, 289
290 Glutathione (GSH), 255 Glutathione reductase, 140
141 Glutelins, 12 Glycans, 223
224 role in neural development, 224 Glycine max, 236
237 Glycinin peptide, 176 Glycogen, 9, 146, 289
290 Glycolipids, 10, 548 Glycomimetics, 549
550, 554
566 carbasugars, 561
564 iminosugars, 554
561 thiosugars, 564
566 Glycopeptides, 307
308 Glycoproteins, 13, 461
462, 548 Glycosaminoglycans (GAGs), 126, 449, 505
506 Glycosidases (GHs), 551
553 Glycoside conformation, 548 Glycoside hydrolases, 551 Glycosyl hydrolases, 551 Glycosyltransferases (GTs), 551 Gracilaria fisheri OS (GFOS), 292

Gracilaria spp., 496
497 G. chilensis, 156 G. lemaneiformis, 148 Graft polymerization, 323 Grafting techniques, 381 Granulation tissue, 440 Graphene oxide (GO), 395 Green alga (Ulva fasciata), 148 Green fluorescent protein (GFP), 612
614 Green tea, 258 Growth factors (GFs), 419 biological macromolecules for delivery systems of, 424
433 composites materials for, 430 polysaccharide combinations for, 429
430 polysaccharide-based materials for, 427
429 polysaccharide-polysaccharide composites for, 433 protein-based composite for, 430
432 protein-based materials for, 425
427 protein-polysaccharide composites for, 432
433 biomacromolecules as carriers of, 443
445 classification of families of, 420f delivery systems for, 421
423 materials for delivery systems of, 423
424 in tissue and bone regeneration, 441
443 Guanine (G), 61, 481
482 Guanosine diphosphate (GDP), 15, 405
406 Guanosine triphosphate (GTP), 15 Gum acacia, 7 Gum Arabic, 7 Gut microbiota modulation, 277

H Halimeda, 204 Halloysite nanotubes (HNT), 399 Halogen polymers, 308
309 Health care, nutraceutical compounds in, 97
99 Heamophillus influenza, 274 Heat shock protein, 297
298 Heavy metals, 139 Helicobactor pylori, 173

Index

Hemagglutinin esterase (HE), 111 Hemicelluloses, 71
72, 125
126 modification techniques for, 74f Hemocyanins, 282 Hemolysis, 583
584 Heparin, 54, 54f, 126
127, 166, 173, 505 Heparin sulfate (HS), 505 Heparin/heparan sulfate, 505 Hepatic cancer, proteins and nucleic acid in, 261
262 Hepatocellular carcinoma cells (HCC), 593 Heptoses, 5 Hericium erinaceus, 251 Herpes viruses, 56 Heterogeneous biocatalysts, 531
532 Heterogeneous nuclear ribonucleoprotein (hnRNP F), 235
236 Heterogeneous nuclear ribonucleoprotein K (hnRNP K), 235
236 Heteropolysaccharides, 5, 168
169 Hexoses, 5 Hialuronic acid (HA), 409 Hibiscus rosa-sinensis, 342 High branched isoterpenoid (HBI), 204 High Performance Gel Permeation Chromatography (HPGPC), 244
249 High performance liquid chromatography (HPLC), 254 High-performance anion-exchange chromatography (HPAECPAD), 256 Hippophaerhamnoides L. See Seabuckthorn (Hippophaerhamnoides L.) Hirustella sinensis, 256 Histone deacetylases (HDACs), 277 Histones, 13 Homer/vesl proteins in CNS injury, 220
222 Homopolysaccharides, 5 Hordeum vulgare, 236
237 Host defense peptides, 307
308 Hot buffer soluble solids (HBSS), 249 Human adipose-derived stem cells (hADSCs), 385 Human immunodeficiency virus (HIV), 56, 361

Humira. See Adalimumab HUS. See Scoville Heat Units (HUS) Hyaluronan, 505
506 Hyaluronate, 506 Hyaluronic acid (HA), 126
127, 166, 168
169, 169f, 174, 406, 430, 505 injectable hydrogels on, 410t Hybrid carbohydrates, 566
567 tacrine-(carbohydrate-derived) hybrids, 567f Hydralazine, 342
343 Hydro distillation (HD), 265
266 Hydrogels, 394, 445
446, 465
466, 586, 598 for drug delivery, 585
592 injectable hydrogels as drug delivery systems, 586
588 oral hydrogels as drug delivery systems, 588
592 fabrication of, 466
467 hydrogel-based microparticles as drug delivery systems, 589
591 hydrogel-based nanoparticles as drug delivery systems, 591
592 Hydrogen, 4 bonding, 463 Hydrogen peroxide (H2O2), 25
26 Hydrolyzable polyphenols, 152 Hydrophilic L-amino acids, 26
27 Hydrophilic/hydrophobic balance, 311
312 Hydrophobic interactions, 463
464 2-hydroxy-3-trialkylammonium propyl, 42 Hydroxyapatite (HA), 381
382, 395, 596
597 Hydroxypropyl cellulose (HPC), 396
397 Hydroxypropyl methylcellulose (HPMC), 342 Hyperkalemia, 327 Hyperlipoproteinemia, 10 Hyperphosphatemia, 327
328 Hypnea pannosa, 205 Hypodermis, 597
598 Hyriopsis cumingii, 146 Hyrtios erectus, 204
205

I Iminoalditols, 554 Iminosugars, 554
561, 555f, 556f DNJ and 1-DNJ-related drugs, 557f

641 fused oxazolidinone iminosugars, 561f piperidine iminosugars inhibitors, 556f potent glycosidase inhibitors pyrrolidine-based iminosugars, 559f synthetic tricyclic benzimidazoleiminosugars, 561f Immature GFs, 419 Immobilization based on enzyme adsorption, 531
532 Immobilized Rapidase C80, 540 Immunoglobulins (Ig), 273
274 Immunohistochemical assay, 254 Immunomodulation, 273
274 biomolecules, and applications, 274
275 Immunomodulatory approach, 115
116, 273 for different health conditions, 274f immunomodulation, 274 lipids, 277
280 polysaccharides, 275
277 proteins, 280
282 Immunostimmulatory effect of carbohydrates, 125 In situ polymerization, 468 In vitro biological testing, 493
494 In vivo testing, 472 Indolizidines, 559
560 alkaloids of, 560 Infectious diseases, 306 Inflammation, 273, 289 Inflammatory bowel diseases (IBD), 274
275 Inflammatory cell activation sites, 139 Inflammatory cytokines, 281
282 Infliximab, 274
275 Influenza A virus, 317 Infra-red analysis (IR analysis), 244
249 Injectable hydrogels, 404 as drug delivery systems, 586
588 drug-loaded, 403
411 Injection-gelation method, 467 Inonotus obliquus. See Chaga mushroom (Inonotus obliquus) Insulin-like growth factor 1 (IGF-1), 442 Insulin-like growth factor 2, 235
236 Insulin-loaded transferosomes, 364
367

642 Intelligent packaging, 322 IntelliJectTM, 620
622 Interfering RNA (RNAi), 61 Interferon (IFN-γ), 254 Interleukin (IL) IL1, 102
103 IL-1β, 123
124 IL-2, 254 IL6, 102
103, 123
124 IL-12, 274
275 Interpenetrating polymeric network (IPN), 345 Intestinal epithelial cells (IECs), 277 Intravenous toxicity, 583
584 Invertases, 529
530 Inverting enzymes, 552
553 Inverting glycosidases, 552
553 Ionic gelation technique, 343
344 Ionic interaction, 531
532 Ionic polymerization, 309
310 Irgasan (IRG), 402 Irradiation of microwave, 230
232 Irritable bowel syndrome (IBS), 289 Isoascorbic acid, 321 Itraconazole-loaded pectin-based nanoparticles, 345
347

J Juglans mandshurica, 236
237 Juvenile Rheumatoid arthritis, 281
282

K k-carrageenan, 384
385, 409 Kaempferia rotunda, 259 Kalimate, 327 Kayexalate, 327 Keratan sulfate (KS), 505 Keratins, 166, 175, 425
426, 444, 505 biomacromolecules, 444 extraction processes of, 427f keratin-based hydrogels, 352 keratin-based nanoparticle, 190 keratin-chitosan, 182
183 Klebsiela pneumonia, 206 Korsmeyer-Peppas kinetic model, 396
397

L L-amino

acid oxidases (LAOs), 25
28, 29t Lablab purpureus, 149
150 Lactobacillus, 289
290

Index

L. brevis, 509
510 Lactoferricin (LFcin), 176
177 lactoferrin-oleic acid, 185 Lactonases, 32
33 Lactose, 289
290 Lactoseries, 223 Lactosylceramide, 276 Laminaria L. digitata, 183
184 L. hyperborea, 183
184 L. japonica, 147
148, 183
184 Laminin, 511 Lapacho tree (Tabebuia avellanedae), 244 Layer-by-layer assembly (LbL assembly), 468 Leber neuropathy, 612
614 Lecithins, 10 Leishmaniasis, 319 Leuconostoc mesenteroides, 509
510, 537 Leukocytes, 58
59 Leukotrienes, 278
279 Levofloxacin (LEVO), 402 Lewis lung carcinoma (LLC), 255 Lichenan. See Lichenin Lichenin, 56 Light at night (LAN), 617 Lignin, 71
72, 72f, 462 chemical modification of, 76t enzymatic modification of, 77t LIN28 gene, 235
236, 300 Lincosamides, 307
308 Lindra thalassiae, 205 Linoleic acid (LA), 120, 179
180, 263
264 Linum usitatissimum, 236
237 Lipases, 529
530, 541 Lipid nanoparticles (LNPs), 60, 234, 279
280 Lipid(s), 3, 9
11, 23, 53, 60
61, 230, 262
269, 277
280, 305, 582
583 as antidiabetic agents, 233
234 as antimicrobial agents, 170
171 nucleic acid, 171
172 phospholipid, 170 sterol, 170
171 triglyceride, 170 basic functions of, 12t brain cancer, 265
266 breast cancer, 264
265 colon cancer, 263
264 compound or conjugate, 10 derived, 10
11

drug delivery using, 359
367, 366t drug delivery-based on lipids, 60
61 function, 339
340 immunomodulatory effect of lipids, 278
280 lipid-based drug delivery systems, 482
484 liver cancer, 266 as nutraceuticals, 119
123 omega-3 PUFAs role in some disorders, 121
123 polyunsaturated fatty acids, 120
121 psychiatric and neurological disorders, 122t others, 267
269 ovarian cancer, 266
267 peroxidation, 27, 225
226 role on nervous system, 224
225 phospholipids, 224
225 simple, 10 skin cancer, 267 Lipopeptides, 307
308 Lipoproteins, 10, 13 Liposomes, 401
402, 592 Listeria monocytogenes, 166, 401
402 Liver cancer, lipids in, 266 Living cells, 491
492 Local fungal infections, 318 Locked nucleic acid (LNA), 593 Long chain fatty acid, 298 Low molecular weight (LMW), 385 Low-density lipoprotein cholesterol (LDL), 324
326 Lung cancer, carbohydrates in, 255
257 Lutein (C40H56O2), 106, 458 Luteolin, 104 Lycopene, 105 Lymphocytes, 274 Lyngabya confervoides MK012409, 259
260 Lysostaphin, 25, 28
32, 29t

M Macrocystis pyrifera, 156 Macrogels, 586 Macrolides, 307
308 Macromolecules, 53
54, 567
570. See also Biological macromolecules as antioxidants, 152
156

643

Index

nonextractable polyphenols as antioxidants, 156 polysaccharides as antioxidants, 152
155 proteins as antioxidants, 155
156 in biomedical applications, 583
584 multivalents, 567
569 polysaccharides, 569
570 synthesis of, 63
64 in targeted drug delivery, 584
585 on tissue engineering, 593
599 Macrophages, 274 Macular pigment (MP), 106 Magnetic chitin nanofiber composites (MCNCs), 533 Magnetite nanoparticles, 179 Maillard reaction, 464 Maltodextrin, 289
290 Maltose, 289
290 Malus micromalus, 146 Mannitol, 54, 55f, 289
290 Mannose receptors (Mr), 282 Marine fungi, 148 Marine-derived polysaccharides, 147
149 Massonia pustulata. See Beetle (Massonia pustulata) Mechanical crushing method, 468 Medical therapy, 98 Medicinal interest of carbohydrates, 550
551 Medium chain fatty acid, 298 MelodieTM, 620
622 Meroterpenoids, 205
206 Mesenchymal stem cells (MSCs), 401
402, 444
445 Messenger RNA (mRNA), 16
17, 62, 483, 584 Metallo-β-lactamaselike lactonase (MLL), 25 Metalloproteins, 13 Metaproteins, 13 Metformin, 229
230 Methacryloyl chitosan (MC), 407 Methotrexate (MTX), 608
609 Microalgae, 203 antimicrobial peptides, 203 Microalgal polysaccharides, 148 Microcapsules, fabrication of, 467
468 Micrococcus luteus, 31 Microemulsion/emulsion-templating methods, 468
469 Microencapsulation, 590

Microgels, 586 Microorganisms, carbohydrates with nutraceutical activity from, 128 MicroRNAs (miRNAs), 62, 116, 260
261 Migalastat, 557
558, 557f Miglitol, 557
558, 557f Miglustat, 557
558, 557f Millettia pulchra, 253
254 Minerals, 140
141, 471 Minimum inhibitory concentration (MIC), 316 miR-22, 261 Misgurnusanguilli caudatus, 146 Mitochondria peroxisomes, 139 Mitochondrial DNA (mtDNA), 610
611 Mitochondrial gene therapy, 610
616 mitochondrial mutations, 611
612 mitochondrion, 611 targeting mitochondria, 612
616 Mitochondrial membrane potential (MMP), 251
252 Mitochondrial mutations, 611
612 Mitochondrion, 610
611 Mitomycin-C, 351 Modification of carbohydrate biological macromolecules, 73
86 cellulose chemical modification of, 75t enzymatic modification of, 76t physical modification of, 74t chitin/chitosan chemical modification of, 80t enzymatic modification of, 84t physical treatments for, 80t classification of modification, 73f hemicellulose, modification techniques for, 74f lignin chemical modification of, 76t enzymatic modification of, 77t Molecular assembly methods, 469 Molecular interactions, 462
464 covalent interactions, 464 electrostatic interactions, 463 hydrogen bonding, 463 hydrophobic interactions, 463
464 Molecular weight (MW), 36
37, 273
274, 312, 493
494 Momordica M. charantia, 236
237

M. cymbalaria, 236
237 M. dioica, 236
237 Monoclonal antibodies (mAb), 58
59, 280, 281t first generation, 59 fourth generation, 60 second generation, 59 third generation, 59
60 Monocutaneous Leishmaniasis, 319 Monomethyl (MM), 319 Monophosphoryl Lipid A, 279
280 Monosaccharides (Ms), 4
5, 167, 289
290, 548 Monoterpenes, 243 Monoterpenoids, 203
204 Moringa oleifera, 236
237 MP optical densities (MPOD), 106 mRNA, 255 technologies, 584
585 Mucoadhesive nonwoven fibers, 396 Mucopolysaccharides, 505 Multidrug resistant microbes (MDR microbes), 165 Multifunctional natural antioxidants, 159 Multilamellar vesicles, 592 Multivalents, 567
569 pyrrolidine iminosugars, 568f Muricauda olearia, 32
33 Mutagenicity, 583
584 Mycobacterium tuberculosis, 559 Myrica gale L., 263

N N,N,N-trimethyl chitosan (TMC), 42 N-(2-hydroxypropyl)methacrylamide copolymer, 306 N-butyl-DNJ, 557 N-vinylpyrrolidone, 305 N. sativa essential oil nano emulsion (NSEO-NE), 264
265 Nanoassemblies, 448
449 Nanofiber scaffolds, 447 Nanogels, 586 Nanohydroxyapatite (n-HA), 595
596 Nanomaterials, 591, 607
608 Nanoparticles (NPs), 395, 439, 448
449 fabrication of, 468
469 Nanoparticles from chitosan (CsNP), 593 Nanotechnology, 479, 607
608

644 Nanotechnology (Continued) based antimicrobial macromolecule, 185
190 alginate-based nanocomposite, 187 cellulose based nanocomposite, 187
189 chitosan based nanocomposite, 185
187 collagen based nanocomposite, 190 gelatin based nanocomposite, 189
190 keratin-based nanoparticle, 190 oleic acid based nanoparticle, 190 based macromolecule composites, 166 Naringenin, 104 National Program for Science and Technology Development Plan, 53
54 Natural antioxidants, 320 Natural bioactive micromolecular compounds, 141 Natural bone mineral (NBM), 426 Natural fatty acids, 262
263 Natural immunomodulatory proteins, 282 Natural killer cell (NK cell), 274 Natural polymers, 305 in TE, 508
511 carrageenans, 509 cellulose, 508 dextrans, 509
510 elastin, 511 fibronectin, 511 gellan, 510 laminin, 511 pullulans, 510
511 starch, 508
509 Natural surfaces, 308 Naturally derived biological macromolecules, 3
4 Nelumbo nucifera, 141 Nephilia clavipes, 506
507 Nervous system, carbohydrates role on, 223 Neural development, glycan role in, 224 Neuraminidase (NA), 565 Neurodegenerative diseases, 225 Neurological disorders, 219 Neutrophils, 274

Index

Nigella sativa L. (NS), 264
265 No methyl (NM), 319 Non-mRNA sequences, 116
117 Non-small cell lung cancer/carcinoma (NSCLC), 262, 268 Noncoding DNA (ncDNA), 116 Noncoding ribonucleic acid, 300 Noncommunicable diseases (NCDs), 98 Nonextractable polyphenols, 152 as antioxidants, 156 Nonspecific-action immunomodulators, 274 Nonviral vectors, 608
610 Norepinephrine, 222 Nortropanes, 559
560 Nuclear factor, interleukin 3 regulated (NFIL3), 617 Nuclear factor-kB (NF-kB), 102
103 Nuclear magnetic resonance (NMR), 244 Nucleic acid(s), 3, 14
17, 23, 61
63, 230, 289, 305, 582
583 as antidiabetic agents, 237
238 as antimicrobial agents, 171
172 biological macromolecules for delivery, 482
488 carbohydrate-based drug delivery systems, 486
488 lipid-based drug delivery systems, 482
484 protein-based drug delivery systems, 484
486 for cancer therapy, 258
262 colon cancer, 259 colorectal and breast cancer, 259
261 hepatic cancer, 261
262 NSCLC, 262 DNA, 15
16 drug delivery using, 355
359 nucleosides, 15 nucleotides, 15 as nutraceuticals, 112
119 in biomedicine, 112
119 DNA/RNA functional sequence, 116
119 DNA/RNA nanostructures, 113
116 perspectives, 119 RNA, 16
17 role in gastrointestinal system, 300

structure and functions, 480
482 Nucleoproteins, 13 Nucleosides, 15 Nucleotides, 14
15, 481
482 Nutraceuticals, 97
98, 470 alkaloids, 99
102 carbohydrates as, 124
129 future views, 107 history of applications of nutraceutical compounds in health care, 97
99 lipids, 119
123 nucleic acids and nutraceutical properties used in biomedicine, 112
119 phenolic compounds, 102
104 potential use of bioactive lipids in cancer stem cells and coronavirus disease, 123
124 proteins and peptides with biological activity of medical interest, 107
112 terpenes, 105
107 Nutrients, 455
458 bioactive molecules, 471
472 biological macromolecules for delivery, 458
462 glycoproteins, 461
462 polysaccharides, 459
461 proteins, 461 proteoglycans, 461
462 co-encapsulation of, 471 delivery systems based on biological macromolecules, 465
471 applications, 469
471 composition and structure, 465
466 fabrication, 466
469 properties, 469 health benefits and limitations of, 457t molecular interactions, 462
464 oil-soluble nutrients, 458 retention and release mechanisms, 464
465 substances in nutrient delivery systems, 459t in vivo testing, 472 water-soluble, 456
457 Nutrition, 97, 120
121 Nylon-3 polymers, 319

Index

O

P

Ocimum kilimandscharicum, 266
267 Odontella aurita, 148 Oils, 10 oil-soluble nutrients, 456, 458 Oleic acid, 185 Oleic acid based nanoparticle, 190 Oleoylethanolamide (OEA), 123 Oligomers, 479
480 Oligonucleotides, 53, 61
63, 584
585 therapeutics based on, 61
63 antisense oligonucleotides, 62 aptamer, 63 siRNA, 62
63 Oligosaccharides (OS), 4
5, 168, 289
290. See also Polysaccharides (PS) Omega-3 fatty acid (O3FA), 210
211, 298 Omega-3 polyunsaturated fatty acids, 470 in disorders, 121
123 Omega-6 fatty acids, 278
279, 298 Ophiopogon japonicus, 257
258 Optimal cancer chronotherapy, 622 Oral drug administration, 582 Oral hydrogels as drug delivery systems, 588
592. See also Injectable hydrogels hydrogel-based microparticles as drug delivery systems, 589
591 hydrogel-based nanoparticles as drug delivery systems, 591
592 Oral toxicity pyrogenicity, 583
584 Oregano (Origanum vulgare), 243 Organocatalytic living ring-opening polymerization, 306 Organotin polymers, 317
318 Origanum vulgare. See Oregano (Origanum vulgare) Oseltamivir, 317 Osteoarthritis, 439
440 Osteoblasts, 596 Osteocalcin (OCN), 385 Osteoclasts, 596 Osteopontin (OPN), 385 Osteoporosis, 439
440 Ostrinia furnacalis, 565
566 Ovarian cancer, lipids in, 266
267 Overhydroxylation, 439
440 Oxidative stress, 139 Oxygen, 4

P407 hydrogels, 588 Paclitaxel (PTX), 359, 622
623 Paclitaxel-loaded polymeric nanoparticles (PTX-NPs), 622
623 Padinasanctaecrucis, 147
148 Panax ginseng, 292
294 Pancreatic cancer, carbohydrates in, 257
258 Paracetamol suspensions, 341
342 Parageobacillus caldoxylosilyticus, 35 Parasites, 319 Parkinson’s disease (PD), 3
4, 219, 612 Passive polymers, 315 Pasteur, L. (father of microbiology), 307 Pathogen-associated molecular patterns (PAMPs), 276 Pathogenic microbes, 165 Pathogenic microorganisms, 305
306 Patiromer, 327 Pavlova viridis, 148 Pectins, 7, 8f, 459
460 for enzyme immobilization, 539
540 Penicillin G acylase, 529
530 Penicillins, 99 Penicillium P. fellutanum, 559 P. italicum, 540 Penicillus, 204 Pentoses, 5 Peptides, 14, 53, 56
58, 203, 209
210, 581
582 alkaloids, 99 with biological activity of medical interest, 107
112 drug delivery using, 349
355, 356t therapeutics based on peptides, 57
58 Peptones, 14 Periploca laevigata, 146 Permeability of glycoprotein (p-gp), 234 pH-dependent polymers, 591 Phaeodactylum tricornutum, 212 Phalaris canariensis, 236
237 Phallaidin, 402 Pharmaceutical nanotechnology, 591 Phaseolus vulgaris, 236
237 Phenolic compounds, 102
104

645 anthocyanins, 103 catechins, 104 curcumin, 102
103 luteolin, 104 naringenin, 104 quercetin, 103 resveratrol, 103 Phenolics, 207
208 Pholidota chinensis, 250
251 Phosphate buffered saline (PBS), 447 Phosphobetaine, 315 Phospholipids, 10, 170, 171f, 224
225 polymers, 315 Phosphoproteins, 13 Phosphotidic acids, 10 Phycobiliproteins, 151 Phycocyanin, 151 Phycoerythrin, 151 Phycoerythrocyanin, 151 Phytophthorainfestans, 172 Pituranthos tortuosus, 267 Plants carbohydrates from, 125 plant-derived polysaccharides, 141
146 plant-derived proteins, 149
150 Plasmalogens, 10 Plasmid DNA (pDNA), 483, 593, 609
610 Plasmodium P. falciparum, 205
206, 319 P. knowlesi, 319 P. malariae, 319 P. ovale, 319 P. vivax, 319 Platelet-derived growth factor (PDGF), 384 Pleurotus sajor-caju, 36
37 Plocamium cornutum, 205 Plumula nelumbinis, 141 Pluronic F-127, 398 Polar lipids, 120 Polo-like kinase 1 (PLK1), 117 Poloxamines, 445
446 Poly (amidoamine) (PAMAM), 592, 609 Poly (caprolactone) (PCL), 395 Poly (lactide-co-glycolide) (PLGA), 595
596 Poly (vinyl alcohol) (PVA), 395 Poly N-isopropyl acrylamide-coacrylic acid (PNIPAM), 396, 587

646 Poly(1-naphthylamine-azobenzene) (PNA-AB), 319
320 Poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV), 446
447 Poly(aniline-azobenzene) (PANI-AB), 319
320 Poly(dimethylacrylamide), 315 Poly(dimethylsiloxane), 315 Poly(epichlorohydrin-co-ethylene oxide) (PECO), 401
402 Poly(ethylene glycol) dimethacrylate (PEGDMA), 431 Poly(ethylene oxide) (PEO), 396 Poly(ethyleneimine), 316 Poly(lactic acid) (PLA), 402 Poly(lactic-co-glycolic acid) (PLGA), 399, 431, 446
447, 593 Poly(luminol-azobenzene) (PLu-AB), 319
320 Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA), 312
313 Poly(N-vinylpyrrolidone), 315 Poly(o-phenylenediamineazobenzene) (PPd-AB), 319
320 Poly(vinylpyrrolidone) (PVP), 396 Poly(ε-lysine) (EPL), 401 Polyampholytes, 315 Polydatin (PD), 255 Polyethylene glycol (PEG), 232
233, 315, 421, 612
614 Polyetyhylenimine (PEI), 308
309, 609
610 PEI-based nanosystem, 612
614 Polyglycerol sebacate-polyethylene glycol methyl ether methacrylate (PGSPEGMEMA), 587 Polygonatum cyrtonema Hua (PCH), 249 Polygonatum sibiricum, 146, 255 Polygonatum sibiricum polysaccharides (PSPs), 255 Polygonum cuspidatum, 255 Polyguanidines, 316
317 Polyketide, 210 Polymer sequestrants, 324
328 Polymeric architecture, 313 Polymeric bile acid sequestrants, 324
326 Polymeric biocides, 308 Polymeric composites, 309, 430

Index

Polymeric polyphenols, 152 Polynucleotides, 496 Polyols, 289
290 Polysaccharide derivative from wild R. griseocarnosa (PRG1
1), 249
250 Polysaccharides (PS), 3
9, 55, 141
149, 208
209, 275
277, 289
290, 459
461, 548, 569
570, 585
586 alginate, 460 animal-derived, 146
147 antimicrobial activity of, 172
174 alginate, 173 cellulose, 173 chitosan, 172 chondroitin, 173
174 heparin, 173 hyaluronic acid, 174 as antioxidants, 152
155 bacterial and fungal, 149 carrageenan, 460 combinations for GFs delivery, 429
430 derived from herbs in nature, 126t drug-loaded polysaccharides-based ESF, 395
400 gut microbiota modulation, 277 immunomodulatory, 275
277 lyases, 551 marine-derived, 147
149 pectin, 459
460 plant-derived, 141
146 polysaccharide-based biomaterials, 581
582 polysaccharide-based materials for GFs delivery, 427
429 polysaccharide-polysaccharide composites, 433 role in extracellular membrane, 124
125 sources and functions of, 9t starch, 460
461 Polysaccharides extracted from Inonotus obliquus (IOP), 256 Polyunsaturated fatty acids (PUFAs), 120
121, 121t, 210
211 from algae, 210
213 precursors and types of, 121f Polyurethane (PU), 315
316, 395 Polyvinyl alcohol (PVA), 423, 598 Polyzwitterions, 308
309 Portieria hornemannii, 205

Procambarus clarkia, 252
253 Proinflammatory cytokines, 276
277 Proinflammatory mediators, 292
294 Prolamins, 13 Propidium iodide (PI), 249 Protaetia brevitarsis L (PBL), 263 Protamines, 13 Proteans, 13 Proteases, 529
530, 541 Protein Data Bank (PDB), 24 Protein(s), 3
4, 11
14, 12f, 23, 53, 58
60, 149
152, 230, 280
282, 305, 461, 581
583 algal-derived proteins, 151
152 animal-derived proteins, 150
151 as antidiabetic agents, 235
237 antimicrobial activity of, 175
177 collagen, 175 fibrin, 176 gelatin, 175 keratin, 175 lactoferrin, 176
177 soy protein, 176 as antimicrobial agents, 169 types and functions, 170t as antioxidants, 155
156 with biological activity of medical interest, 107
112 for cancer therapy, 258
262 colon cancer, 259 colorectal and breast cancer, 259
261 hepatic cancer, 261
262 NSCLC, 262 in CNS injury, 219
220 amyloid-beta and tau protein, 219
220 conjugated, 13 derived, 13
14 drug delivery using, 349
355, 356t drug-loaded protein-based ESF, 401
403 engineering approaches, 529 expression, 614
616 immunomodulatory, 280
282 plant-derived proteins, 149
150 protein-based composite for GFs delivery, 430
432 protein-based drug delivery systems, 484
486 protein-based materials for GFs delivery, 425
427

Index

protein-polysaccharide composites for GFs delivery, 432
433 protein-polysaccharide-based nanoparticles, 592 role in gastrointestinal system, 295
298 simple, 12
13 therapeutics based on proteins, 58
60 mAb, 58
59 protein-based therapeutics, 58 Proteoglycans, 13, 461
462 Proteoses, 14 Pseudoalteromonas bacteriolytica, 205 Pseudomonas, 173
174 P. aeruginosa, 313 P. elodea, 510 Pseudosugars, 561
562 Pullulans, 487
488, 510
511 Punica granatum, 244 Pure zein fibers, 401 Purines, 481
482 bases, 582
583 Pyrimidines, 481
482 bases, 582
583 Pyrrolidizines, 559
560 alkaloids of, 560

Q Quaternary phosphonium salts (QPS), 315 Quercetin, 103 Quercus brantii. See Zagros oak (Quercus brantii) Quorum quenching (QQ), 32
33 Quorum sensing (QS), 32
33

R Radix astragali, 141
146 Raffinose, 168, 168f, 289
290 Reactive oxygen species (ROS), 27, 55
56, 105, 139, 187, 249
250, 276 Reactive oxygen/nitrogen species (ROS/RNS), 139 Real-time quantitative reverse transcription PCR (qRT-PCR), 255, 258 Receptor-related orphan receptor (ROR), 617 Recombinant human bone morphogenetic protein-2 (rhBMP-2), 443

Red alga (Gloiopeltis furcate), 148 Regenerative medicine, 393 Resistant starch (RS), 460
461 Resveratrol, 103 Retaining enzymes, 552
553 Retaining glycosidases, 552
553 Retention and release mechanisms, 464
465 Retino-tectal system, 223 Retinoblastoma (RB), 619
620 Reversible addition-fragmentation chain transfer polymerization (RAFT), 309
310 Rhamnose (Rha), 254 rhBMP2, 401
402 Rheumatoid arthritis, 281
282 Rhodophyceae gelidium, 496
497 Riboflavin (C17H20N4O6), 456
457 Ribonucleic acid (RNA), 3, 16
17, 61, 171
172, 230, 480, 582
583 functional sequence, 116
119 CRISPR, 117
119 gene therapy, 117 non-mRNA sequences, 116
117 nanostructures, 113
116 biosensing, 113
114 drug delivery, 114
115 immunomodulatory, 115
116 nanotechnology, 358 Ribose, 61 Ribosomal RNA (rRNA), 16
17 Rifampicin, 355 Rimantadine, 317 Ring-opening metathesis polymerization (ROMP), 306, 309
310, 323
324 RNA interference (RNAi), 584
585 RNA-induced silencing complex (RISC), 62 Roburin D (RobD), 566
567 Rosmarinus officinalis L., 266 Runt-related transcription factor 2 (run-2), 385 Russula griseocarnosa, 249
250

S S-lac type lectins, 222
223 Saccharomyces, 289
290 S. cerevisiae, 258, 563 Salacinol, 564
565 Salmonella, 176
177 S. typhimurium, 166 Salvia officinalis, 267

647 “Same centered” approach, 311
312 Sarcinochrysis marina, 148 Sarcodia ceylonensis, 148 Sargassum S. fluitans, 147
148 S. macrocarpum, 205
206 S. wightii, 232 Sargassum henslouianum. See Brown alga (Sargassum henslouianum) Saturated fatty acid, 298 SBPW3, 258 Scaffolds, 446
447, 507
508 Scanning electron microscope (SEM), 251 Scenedesmus sp., 252 Schinus molle L., 264 Schinus terebinthifolius, 264 Scoville Heat Units (HUS), 101 Scutellaria barbata, 258 Scytosyphonlomentaria, 156 Seabuckthorn (Hippophaerhamnoides L.), 146 Seaweeds, 210 Second generation monoclonal antibodies, 59 Second-line defense antioxidants, 140
141 Secondary derived proteins, 14 Secondary metabolites (SM), 97
99, 107 with nutraceutical activity, 108t “Segregated monomer” approach, 311
312 Selectivity index (SI), 264 Self-assembly methods, 468 Self-healing polymers, 315 Sensitization, 583
584 Sensory organs, 223 Serotonin (5-HT), 222 Sesquiterpenoids, 203
204 Sesterterpenoids, 203
204 Severe acute respiratory syndrome (SARS-CoV-2), 123, 306 Shear’s polysaccharide, 275 Shearing-gelation method, 467 Short chain fatty acids (SCFA), 177, 277, 298 Sickle cell disease (SCD), 119 Silk, 506
508 Silk fibroin-based patches (SF-based patches), 401
402 Silver nanoparticles (Ag NPs), 173
174, 179

648 Simvastatin (SIM), 596 Single nozzle electrospinning technique, 395 Skin cancer, lipids in, 267 Skin regeneration, 442 Skin substitutes development, 597
599 Skin tissue replacement, 598 Small interfering RNA (siRNA), 62
63, 116
117 Snake venoms (SV), 25 Snigella dysentri, 206 Sodium alginate (SA), 398 Sodium bicarbonate (NaHCO3), 405
406 Sodium erythorbate, 321 Solid lipid nanoparticles, 362 Solid-liquid phase separation, 443 Sorbitol, 289
290 Soy protein, 166, 176 Soy protein isolated (SPI), 399 Sphingomyelins, 10 Spider silk, 507 Spidroin, 506
508 Sponges, 445
446 Spray drying, 468 Stage specific embryonic antigen 1 (SSEA1), 224 Staphylococcus, 173
174 S. aureus, 6, 27, 149, 166, 205
206, 313 S. epidermidis, 149, 176
177 S. simulans, 28
30 Starch, 5
6, 6f, 460
461, 508
509 Steroids, 11, 206
207 Sterols, 11, 170
171, 171f Stigma maydis, 244 Stimulator of IFN genes (STING), 276
277 Streptococcus, 289
290 S. mutans, 509
510 S. pneumoniae, 42 Streptogramins, 307
308 Streptomyces nojiriensis, 554
555 Stromal cell-derived factor (SDF), 430
431 Strontium (Sr), 383
385 Structure-guided technologies, 529 Structure
activity relationship (SAR), 23
24 chitosan as bioactive polysaccharide, 36
42

Index

enzymes as bioactive proteins, 24
36 Sucrose, 167
168, 168f, 289
290 Sulfate, 505 Sulfobetaine, 315 Sulfur nanoparticles, 191 Sulphamethoxazole suspensions, 341
342 Sulphated polysaccharide of A. cinnamomea (SPS), 257 Sulpholipids, 10 Supercritical fluid extraction approach, 230
232 Superoxide dismutase (SOD), 140
141 Superworm (Zopho basmorio), 146 Supramolecular chemistry, 549 Surface erosion mechanism, 465 Survivin intertransfer RNA (siRNA), 593 Sweet potato glycoproteins (SPG), 259 Swelling mechanism, 465 Synthesis of macromolecules, 63
64 Synthetic antioxidants, 320 Synthetic drugs, 229
230, 243 Synthetic macromolecules with antibacterial activity, 313
317 with antifungal activity, 318
319 with antimicrobial activity, 306
320 with antiparasitic activity, 319
320 with antiviral activity, 317
318 with biological activity, 305 polymer sequestrants, 324
328 synthetic macromolecules with antioxidant activity, 320
324 Synthetic polymers, 305, 319, 492 Synthetic surfaces, 308 Systemic fungal infections, 318 Syzygium aromaticum. See Clove buds (Syzygium aromaticum)

T T helper cells (Th cells), 280 T regulatory cells (Tr cells), 280 T-cell receptors (TCR), 275
276 Tabebuia avellanedae. See Lapacho tree (Tabebuia avellanedae) Tannins, 566 Targeted drug delivery biomaterials as, 585
593 macromolecules in, 584
585, 585t Targeting mitochondria, 612
616 gene and protein expression, 614
616

Tau protein, 219
220 Temperature-sensitive hydrogels, 588 Terfaira occidentalis, 236
237 Terpenes, 105
107, 106f β-Carotene, 105
106 lutein, 106 lycopene, 105 zeaxanthin, 107 Terpenoids, 203
206 isomers of, 205f Terpyridine, 173 2-tert-butylhydroquinone (TBHQ), 321 Tetracycline, 307
308 HCl, 397
398 Tetraterpenoids, 203
204 Tetroses, 5 Theobroma cacao, 236
237 Theobromine, 101
102 Therapeutic(s) based on carbohydrates, 55
56 based on oligonucleotides, 61
63 based on peptides, 57
58 based on proteins, 58
60 macromolecules, 273
274 Therapies, biological macromolecules in, 53
54 Thermotoga maritima, 563 Thiosugars, 564
566 and inhibition constant for α-galactosidase from Escherichia coli, 565f isolated from Salacia species, 564f Third generation monoclonal antibodies, 59
60 Third-line defense antioxidants, 140
141 Three-dimensional porous scaffolds (3D porous scaffolds), 596
597 Thyme essential oil (TEO), 401
402 Thymine (T), 61, 481
482 Tissue engineering (TE), 381, 393, 494 BTE, 381
382 drug-loaded electrospun fibers in, 394
403 drug-loaded injectable hydrogels in, 403
411 interaction and application in, 405f macromolecules on, 593
599 skin substitutes development, 597
599 wound management, 597 scaffolds, 494

649

Index

effects of alkali soluble polysaccharide, 291f Ultraviolet (UV), 14, 586
587 Ulva fasciata. See Green alga (Ulva fasciata) Ulva sp., 156 U. intestinalis, 148 U. lactuca, 148 Ulvan, 3
4, 148, 385
386 Uncharged polymers, 315 Undaria pinnatifida, 253 Unsaturated fatty acids, 123 Uracil (U), 61, 481
482, 582
583 Uridine triphosphate (UTP), 15 Ustekinumab, 274
275

Tissue regeneration, growth factors in, 441
443 TMC. See N,N,N-trimethyl chitosan (TMC) Tolbutamide, 229
230 Toll-like receptor (TLR), 276 Toll-like receptor 4-mitogen-activated protein kinase/nuclear factor (TLR4-MAPK/NF-κb), 255 Transfer RNA (tRNA), 16
17 Transferrin receptor 1 (TfR1), 624 Transforming Growth Factor (TGF), 276
277 Transmission electron microscope (TEM), 250
251 Transport proteins, 339
340 Tricalcium phosphate, 444
445 Trichosanthes kirilowii, 141
146 Triglycerides, 120, 170, 171f Trimethylxanthine. See Caffeine Trioses, 5 Tripolyphosphate, 448 Trisaccharides, 5 Trisodium trimetaphosphate, 399 Triterpenoids, 203
204 Triticum aestivum, 236
237 Trypanosoma cruzi, 559 Tryphenylphoshonium (TPP), 609
610 Tumor necrosis factor (TNF), 274
275 TNF-α, 102
103, 254 Tumor rejection antigens (TRA1), 224 TUNEL assay, 256 Turmeric (Curcuma longa), 149
150 Type 1 diabetes, 229 Type 2 diabetes, 229 Type 3 diabetes, 229

Vaccination, 115
116, 274 Vaccines, 274 Vascular endothelial growth factor (VEGF), 399, 441
442 Very long chain fatty acid, 298 Vigna V. angularis, 236
237 V. unguiculata, 236
237 2-vinylfuran, 305 2-vinylpyrrole, 305 2-vinyltiophene, 305 Vipera ammodytes ammodytes, 26
27 Viruses, 317 Visceral Leishmaniasis, 319 Vitamin B2. See Riboflavin (C17H20N4O6) Vitamin C. See Ascorbic acid (C6H6O6) Vitamins, 470 Volumetric Muscle Loss treatment (VML treatment), 425
426

U

W

Udotea, 204 Ulcer, 289 Ulcerative colitis (UC), 289
290

Water-soluble nutrients, 456
457 ascorbic acid, 456 riboflavin, 456
457 Water-soluble polysaccharides, 146

V

Waxes, 10 Western blot, 255 Whey protein, 461 Wound dressing, antimicrobial agents on, 192
193 Wound management, 597

X Xanthan for enzyme immobilization, 540
541 Xanthan gum-konjac glucomannan (XG/KGM), 598
599 Xanthomonas X. arboricola, 540
541 X. axonopodis, 540
541 X. campestris, 540
541 X. citri, 540
541 X. fragaria, 540
541 X. gummisudans, 540
541 X. juglandis, 540
541 X. phaseoli, 540
541 X. vasculorium, 540
541 Xenograft models of mice, 251
252 Xylanases, 541

Y Y-box binding protein1 (YBX1), 235
236 Yulangsan polysaccharide (YLSPS), 253
254

Z Zagros oak (Quercus brantii), 141 Zanamivir, 317 Zeaxanthin, 107 Zidovudine, 361 Zika, 306 ZnO nanoparticles (ZnO NP), 185
186 Zopho basmorio. See Superworm (Zopho basmorio) Zwitterionic polymers, 315 Zwitterionic polysaccharides (ZPs), 275
276