Polysaccharide carriers for drug delivery [First edition] 9780081025543, 0081025548, 9780081025536

Table of contents :
Content: Part 1 Fundamentals of polysaccharides for drug delivery 1. Polysaccharides, their modification and characterization 2. Drug delivery & bioactive potentials of polysaccharides and their derivatives Part 2 Biomaterials 3. Polysaccharide-based amorphous solid dispersions (ASD) for improving solubility and bioavailability of drugs 4. Interpenetrating polysaccharide networks as oral drug delivery modalities 5. Polysaccharide nanomicelles as drug carriers 6. Polysaccharide nanoparticles for cancer drug targeting 7. Polysaccharide nanoconjugates for drug solubilization and targeted delivery Part 3 Drug delivery applications of Polysaccharides 8. Biopolymer systems for drug delivery across permeable membranes 9. In situ polysaccharide smart gels and inserts for topical drug delivery applications 10. Polysaccharide hydrogel films/membranes for transdermal delivery of therapeutics 11. Polysaccharide-based orodental delivery systems 12. Stomach-specific buoyant polysaccharide systems for treating localized diseases 13. Advanced colon-specific delivery systems for treating local disorders

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Polysaccharide Carriers for Drug Delivery

Woodhead Publishing Series in Biomaterials

Polysaccharide Carriers for Drug Delivery

Edited by

Sabyasachi Maiti Sougata Jana

An imprint of Elsevier

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2019 Elsevier Ltd. 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-08-102553-6 For information on all Woodhead publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisition Editor: Sabrina Webber Editorial Project Manager: Leticia M. Lima Production Project Manager: Joy Christel Neumarin Honest Thangiah Cover Designer: Greg Harris Typeset by SPi Global, India

Contributors

Shavej Ahmad  Research and Development Centre, Sun Pharmaceutical Industries Ltd., Gurgaon, India Amani Alhibshi  Department of Neuroscience, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Javed Ali  Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Hamdard University), New Delhi, India Iman Almansour Epidemic Diseases Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Sarah Almofty Department of Stem Cell Biology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Dana Almohazey  Department of Stem Cell Biology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Munther Alomari  Department of Stem Cell Biology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Leonard Ionut Atanase  Department of Biomaterials, Faculty of Dental Medicine, “Apollonia” University of Iasi, Iasi, Romania Sanjula Baboota Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Hamdard University), New Delhi, India Waisudin Badri  School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University, Pathum Thani, Thailand Hemant Ramachandra Badwaik  Rungta College of Pharmaceutical Sciences and Research, Bhilai, India

xivContributors

Subham Banerjee Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, India Azam Barzegari  Department of Chemistry, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran Hriday Bera  Faculty of Pharmacy, AIMST University, Bedong, Malaysia; Bengal School of Technology, Sugandha, Hooghly, India Archana S. Bhadauria  Department of Mathematics and Statistics, Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur, India Mallanagouda S. Biradar Shri. B. M. Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapur, India Vasile Burlui Department of Biomaterials, Faculty of Dental Medicine, “Apollonia” University of Iasi, Iasi, Romania Anca Niculina Cadinoiu Department of Biomaterials, Faculty of Dental Medicine, “Apollonia” University of Iasi, Iasi, Romania Kusal K. Das Shri. B. M. Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapur, India Hari Prasanna Deka Boruah  Biological Sciences and Technology Division, Academy of Scientific & Innovative Research, CSIR-North East Institute of Science and Technology, Jorhat, India Abdelhamid Elaissari  School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University, Pathum Thani, Thailand Hatem Fessi  School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University, Pathum Thani, Thailand Animesh Ghosh  Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India Tapan Kumar Giri  NSHM Knowledge Campus, Kolkata Group of Institutions, Kolkata, India Syed Z. Inamdar BLDEA’s SSM College of Pharmacy and Research Centre, BLDE University Campus, Vijayapur, India

Contributorsxv

Sougata Jana  Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol; Department of Health and Family Welfare, Directorate of Health Services, Kolkata, India Subrata Jana  Department of Chemistry, Indira Gandhi National Tribal University, Amarkantak, India Kai Jin School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai, China Chariya Kaewsaneha  School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University, Pathum Thani, Thailand; Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-UMR, Lyon, France Chandrabose Karthikeyan Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Rameshroo Kenwat  Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Raghavendra V. Kulkarni BLDEA’s SSM College of Pharmacy and Research Centre, BLDE University Campus, Vijayapur, India Pranesh Kumar  Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India Awanish Kumar  Department of Biotechnology, National Institute of Technology Raipur, Raipur, India Ashwini Kumar  Department of Biotechnology, National Institute of Technology Raipur, Raipur, India Shobhit Kumar  Department of Pharmaceutical Technology, Meerut Institute of Engineering and Technology (MIET), Meerut, India Dhanabal Kumarasamy NSHM Knowledge Campus, Kolkata Group of Institutions, Kolkata, India Leena Kumari  Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India Balak Das Kurmi Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, India

xviContributors

Yiyang Liu School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai, China Sabyasachi Maiti Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Aanjaneya Mamgain  Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Thingreila Muinao  Biological Sciences and Technology Division, Academy of Scientific & Innovative Research, CSIR-North East Institute of Science and Technology, Jorhat, India Bushra Nabi Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Hamdard University), New Delhi, India Chella Naveen  Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India Amit Kumar Nayak  Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj, India Mintu Pal Biological Sciences and Technology Division, Academy of Scientific & Innovative Research, CSIR-North East Institute of Science and Technology, Jorhat, India Rishi Paliwal  Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Shivani Rai Paliwal Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, India Zhiqing Pang School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai, China M. Prabaharan  Department of Chemistry, Hindustan Institute of Technology and Science, Chennai, India Delia Mihaela Rata  Department of Biomaterials, Faculty of Dental Medicine, “Apollonia” University of Iasi, Iasi, Romania Somasree Ray  Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol, India

Contributorsxvii

Saleha Rehman Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Hamdard University), New Delhi, India Subhadeep Roy Department of Pharmaceutical Sciences, School of Bio-Sciences & Bio-Technology, Babasaheb Bhimrao Ambedkar University, Lucknow, India Sudipta Saha Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India Kalyan Kumar Sen Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol, India Zahra Shariatinia  Department of Chemistry, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran Nalini R. Shastri Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India Ashok K. Singh  Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India P.R. Sivashankari Department of Chemistry, Hindustan Institute of Technology and Science, Chennai, India Saundray Raj Soni Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India Kunjbihari Sulakhiya  Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Kishor Kumar Suryavanshi  Department of Chemistry, Indira Gandhi National Tribal University, Amarkantak, India Harsh Yadav  Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Yuefei Zhu School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai, China

About the Editors

Sabyasachi Maiti is an MPharm, PhD from Jadavpur University, Kolkata, India. He is working as Associate Professor at the Department of Pharmacy, Indira Gandhi National Tribal University (Central University), Amarkantak, Madhya Pradesh, India. He has experience of more than 15  years in pharmaceutical education and research. He is an impressive researcher in the field of drug delivery science and technology. His research works focus on chemical modification of natural polysaccharides, characterization, and their application in the design of novel drug delivery carriers. The outcomes of his research have been appreciated by international peers in this field. He has more than 50 publications to his credit. He also finds a place in the panel of peer reviewers of various international journals of repute. He has written 25 book chapters and is experienced in editing reference books for international publishers. Sougata Jana is a BPharm (Gold Medalist) from West Bengal University of Technology, Kolkata, MPharm (Pharmaceutics) from Biju Patnaik University of Technology, Odisha, India. He was an Assistant Professor of Gupta College of Technological Sciences, Asansol, West Bengal, India. Currently, he is working at the Department of Health and Family Welfare, Directorate of Health Services, Kolkata, India. He is engaged in research for 11 years and that of teaching for 10 years. IPA Bengal branch, Kolkata, India conferred upon him “M.N. Dev Memorial Award” for securing the highest marks in the state of West Bengal in 2005. He bagged “Best Poster Presentation Award” at 21st West Bengal State Science and Technology Congress-2014, and “Outstanding Paper Award” at 1st Regional Science and Technology Congress—2016, organized by Department of Science and Technology, Govt. of West Bengal, India. He published 30 research and review papers in different national and international peer-reviewed journals. He edited books in Springer, Elsevier, and Pharmamedix India Publication Pvt. Ltd. He has more than 25 book chapters to his credit in Elsevier, Springer, Wiley VCH, CRC Press, Taylor & Francis group. His research area of interest includes modification of synthetic and natural biopolymers, microparticles, nanoparticles, semisolids, and interpenetrating network system for controlled drug delivery.

Natural polysaccharides: Structural features and properties

1

Harsh Yadav, Chandrabose Karthikeyan Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India

1.1 Introduction Polysaccharides are polymeric carbohydrates composed of repeating monomeric units of monosaccharides that are covalently linked to each other through glucosidic linkage [1]. The monosaccharides (also known as glycans) usually contain from three to nine carbon atoms and also vary in size and in the stereochemical configuration at one or more carbon centers [2]. Polysaccharides composed of only one kind of monosaccharide are described as homopolysaccharides (homoglycans) [3,4]. Similarly, if two or more different kinds of monomeric unit are present, the class name heteropolysaccharide (heteroglycans) may be used [4,5]. Polysaccharides can exist in the linear form with a straight chain of monosaccharides or a branched form with a monosaccharide chain with arms and turns depending on type of monosaccharide connected and the position of the carbon to which it is connected [6]. Polysaccharides have widespread pharmaceutical applications as excipients, and nowadays they are increasingly used as biomaterials for drug delivery [7–12] for the following reasons. 1. Polysaccharides occur abundantly in nature and the methods for their isolation from natural sources are well characterized and well documented [10,13]. 2. Polysaccharides, compared to synthetic polymers, are biodegradable and biocompatible, and the risk of immunological response associated with polysaccharide use is negligible [10,14]. 3. Polysaccharides can be functionalized through a variety of chemical and enzymatic methods [10,15]. Both natural and functionalized polysaccharides exhibit a wide array of properties that could be exploited for the development of many types of delivery systems [10,16]. For example, ionic polysaccharides show pH and ion-sensitivity; hence, this property could be exploited for the development of stimuli-responsive drug-delivery systems [10,17]. Some polysaccharides have been shown to form gels and interpenetrated polymeric networks (IPN) that exhibit remarkably different physicochemical properties than those of macromolecular constituents [10,17]. 4. The availability of a variety of functional groups permits conjugation or complexation with other biological macromolecules, e.g., proteins, peptides, etc. [10,18]. 5. These characteristics make polysaccharides ideal biomaterials for the formulation of targeted delivery systems that can release the entrapped drug at stipulated time and site of pharmacological response, in response to specific physiological stimuli [10,17]. Polysaccharide Carriers for Drug Delivery. https://doi.org/10.1016/B978-0-08-102553-6.00001-5 Copyright © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1.1  Classification of natural polysaccharides based on their source.

Several polysaccharides are currently in use for development of pharmaceutical delivery systems. They are broadly classified (Fig. 1.1) on the basis of their origin as given here. ●

●

●

●

Polysaccharides of plant origin: starch, cellulose, and exudate gums like arabinogalactan, guar gum, gum arabic (GA), and locust bean gum [19]. Polysaccharides of algal origin: alginates, galactans—carrageenan [19]. Polysaccharides of animal origin: chitin & chitosan, glycosaminoglycans, and hyaluronic acid (HA) [19]. Polysaccharides of microbial origin: dextran, gellan gum, pullulan, and xanthan gum [19].

1.2 Polysaccharides of plant origin 1.2.1 Starch Starch is one of the most abundant polysaccharides found in plants. Starch is composed of repeating α-d-glucose units linked together by α-(1,4)-glycosidic bonds and to some extent by α-(1,6)-glycosidic bonds [20]. Starch consists of two structurally distinct polysaccharides: amylose and amylopectin (Fig.  1.2) [20]. Amylopectin is a highly branched polymer a molecular weight range of 107–109 g mol−1 [20,21]. Amylopectin is made up of hundreds of short chain α-d-glucopyranosyl monomers with α-(1–4) linkages in the linear portion and α-(1–6) linkages at the branching points of the molecule [20,21]. Amylose, on the other hand, has a linear chain with ­α-d-glucopyranosyl units linked to each other through α-(1 → 4) bond with a molecular weight of 105–106 g mol−1 [20,21]. The functional properties of starches, such as crystallinity, viscosity, shear resistance, gelatinization characteristics, solubility, tackiness, paste texture and stability, swelling, and retrogradation are dependent on

Natural polysaccharides: Structural features and properties3

Fig. 1.2  Polysaccharides of plant origin.

the amylose and amylopectin content [22–24]. Starch is a safe, cheap, natural, biocompatible, biodegradable, and multifunctional material with many pharmaceutical applications [23]. Starch is extensively used as an excipient the production of oral solid dosage forms such as bulk granules, tablets, and capsules as well as cosmetic and medicated powders. Native starch is used as binder, disintegrant, diluents, glidant, and lubricants in the manufacture of conventional dosage forms [23]. Although some intrinsic physicochemical properties limit the use of starch as a multifunctional pharmaceutical excipient for formulation of drug delivery carrier system, a large library of modified forms are being currently developed, which may improve its potential as a multifunctional pharmaceutical excipient for drug delivery [23].

1.2.2 Cellulose Cellulose is the most abundant carbohydrate polymer found in nature and it is an important structural component of the cell walls of plants [21,25]. Cellulose is also found in lower organisms like algae, bacteria. Structurally, cellulose is a homopolymer made up of chains of glucose unit ranging from 700 to 25,000 joined together by β1,4 glycosidic linkages(Fig. 1.2) [21]. This linkage in the cellulose is responsible for its linear structure, high crystallinity, and human indigestibility [21,26,27]. Cellulose possesses good thermal and chemical stability, high hydrophilicity, and excellent biocompatibility making it ideal biomaterial for drug delivery systems [28]. However, the pharmaceutical applications of pure cellulose are limited by inherent shortcomings like bad plasticity and dimensional stability, poor solubility in ordinary solvents, and lack of antibacterial activity [28]. Nanocellulose is emerging as a promising biomaterial for pharmaceutical applications owing to its excellent biocompatibility, biodegradability, and low toxicity (ecological and animal) profile [29,30]. Nanocellulose is broadly classified into three categories, namely, bacterial cellulose (BC), cellulose

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nanocrystals (CNCs) (also called as cellulose nanowhiskers or nanocrystalline cellulose), and cellulose nanofibrils (CNFs), depending on their source and methods of production [29,31]. CNCs are obtained from acid or enzyme hydrolysis, whereas cellulose nanofibrils are obtained through mechanical treatments [29]. Bacterial nanocellulose is another highly crystalline form of cellulose, which is obtained mainly from Gluconacetobacter xylinus [29,32]. Nanocellulose has distinct advantage of its nanosize dimension along with free reactive surface hydroxyl groups that are amenable to chemical modification [29]. Nanocelluloses are currently explored for their applications in drug delivery as nanoformulation, microparticles, tablets, aerogels, hydrogels, and transdermal drug-delivery systems [29].

1.2.3 Exudate gums 1.2.3.1 Arabinogalactan Arabinogalactan, a polysaccharide derived from the wood of the larch tree (Larix species primarily Larix occidentalis), is a long, densely branched polysaccharide with varying molecular weights (10,000–120,000) [33]. Arabinogalactan is composed of galactose and arabinose molecules in a 6:1 ratio, with a small amount of glucuronic acid [33,34]. Pharmaceutical-grade larch arabinogalactan is a fine, dry, off-white powder with a slightly sweet taste and mild pine-like odor [33]. Larch arabinogalactan exhibits excellent solubility in water and aqueous alcohol along with water retention properties [35,36]. It is used as thickener, stabilizer, and emulsifier in pharma and food industries [36]. Larch arabinogalactan has also been used as a carrier for hepatic drug delivery [37].

1.2.3.2 Guar gum Guar gum (GG) is a water-soluble polysaccharide derived from the seeds of Cyamopsis tetragonolobus, family Leguminosae [38]. GG is composed of linear chains of (1 → 4)-β-d-mannopyranosyl units with α-d-galactopyranosyl units attached by (1 → 6) linkages (Fig.  1.2) [38,39]. GG contains about 80% galactomannan, 12% water, 5% protein, 2% acidic insoluble ash, 0.7% ash, and 0.7% fat [38]. GG is used as a binder, disintegrant, suspending agent, thickening agent, and stabilizing agent in pharmaceutical preparations. GG can form hydrophilic hydrocolloids on hydration with cold water, which is a viscous pseudoplastic solution [38,40,41]. GG-based hydrogels retard release of the drug from the dosage form and hence can be used for controlled delivery of pharmaceuticals [38]. GG-based hydrogels are also susceptible to degradation in the colonic environment and are useful as vehicles for colon-specific delivery of drugs [38,42–44].

1.2.3.3 Gum arabic GA (also known as arabic gum, gum acacia, and acacia gum) is a gum exudate obtained from the stems and branches of Acacia senegal or of related species of Acacia (Fam. Leguminosae) [45]. GA is a branched-chain multifunctional hydrocolloid with a highly neutral or slightly acidic, arabino-galactan-protein complex containing calcium, magnesium, and potassium [46]. The backbone of GA comprises 1,3-linked ­β-d-galactopyranosyl

Natural polysaccharides: Structural features and properties5

units and the side chains are composed of two to five 1,3-linked β-d-galactopyranosyl units, joined to the main chain by 1,6-linkages [46]. Both the main and the side chains contain units of α-l-arabinofuranosyl, α-l-rhamnopyranosyl, β­ -d-glucopyranosyl, and 4-O-methyl-β-d-glucopyranosyl, the last two mostly as end units [46]. GA exhibits excellent water solubility along with emulsifying, stabilizing, thickening, binding, and film-forming properties [34,46]. This is mainly owing to its highly branched molecular structure and covalent association with a protein moiety [34]. Hence, GA is extensively used in food and pharmaceutical industries as dietary fiber, food additive, shelf-life enhancer, microencapsulator, drug-delivery agent, biomaterial in tissue engineering, as nanoconstruct and functionalizing agent in nanotechnology [47].

1.2.3.4 Locust bean gum Locust bean gum (LBG) is a neutral polysaccharide extracted from the seeds of the carob tree (Ceratonia siliqua) [47]. LBG is composed of a (1–4)-linked β-d-mannose backbone with (1–6)-linked side chains of α-d-galactose; hence, it belongs to the class of galactomannans(Fig. 1.2) [48]. Locust bean gum has the highest average mannose to galactose ratio (3:5) among the commercially available galactomannans (guar gum (1.8) and tara gum (3.0)) [48–50]. The degree of galactose substitution on mannose chain influences the water solubility exhibited by galactomannans, which is why guar gum is soluble in cold water but LBG requires heat treatment for maximum water solubility [51]. LBG is the first galactomannan to be used as an industrial gum in cosmetic, pharmaceutical, food, paper, and textile industries [52]. This is mainly because of its ability to form very viscous aqueous solution at relatively low concentration, to stabilize emulsion, and to replace fat in many food products [52]. Furthermore, LBG solutions are not affected by variations in pH, salt concentration, and heat treatment owing to their nonionic nature [52]. LBG also exhibits synergistic action with other polysaccharides such as carrageenan and xanthan gum to form gels of more elasticity and strength [52]. By virtue of these properties, LBG is mainly used as an additive in the formulation of oral delivery systems based on tablets, hydrogels, and multiparticulate systems [48].

1.3 Polysaccharides of algal origin 1.3.1 Alginates Alginates are an important class of the polysaccharides with both industrial and research applications [53]. Natural alginates are sourced either from algae derived from the sea or as an exopolysaccharide of certain strains of bacteria, e.g., Pseudomonas aeruginosa [54]. Alginates are linear polymers composed of (1 → 4)-α-l-guluronic acid units (GG) and (1 → 4)-β-d-mannuronic acid units (MM), along with some heteropolymeric sequences of M and G (MG blocks) (Fig.  1.3) [53]. Both M/G ratio and the distribution of M and G units along the chain influence the biological and physical properties of alginates in aqueous media [53,55,56]. Monosodium salts of alginates show good aqueous solubility and form a viscous solution in water, which has valuable applications [53], whereas divalent and multivalent cations (except Mg2+)

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Polysaccharide Carriers for Drug Delivery

Fig. 1.3  Polysaccharides of algal origin.

Natural polysaccharides: Structural features and properties7

form gels or precipitates [57,58]. Alginic acid or alginates also form gels in acidic pH through formation of intermolecular hydrogen bonds [58]. The gel-forming properties of alginates led to their extensive use as food additives, emulsifiers, and stabilizers. Alginates are also employed in biomedical field for controlled drug release [59], cells encapsulation [60], tissue engineering [61], and for preparation of dental molds [53,62,63]. Chemical modification of alginates has been actively pursued by several research groups either to enhance their properties or introduce new properties to extend their biomedical and pharmaceutical applications [54,64].

1.3.2 Galactans Galactans represent a large family of polysaccharides found in terrestrial plants and macroalgae [65]. Galactans are generally composed of linear chains of galactoses but for few exceptions in which the backbone is formed by a linear chain of alternate (1 → 3)-β-d-galactopyranose residues (A units) and (1 → 4)-α-d/l-galactopyranose residues (B units) [66]. Galactan linear chains may be ramified by other neutral sugars (Xyl, Glc, Ara, Man) and may possess a methyl ether group or a sulfate hemiester group or a pyruvic acid (cyclic ketal form) [65]. Galactans are mainly represented by agars and carrageenans among which the latter has received increased attention because of their pharmaceutical applications.

1.3.2.1 Carrageenan Carrageenan (CRG) is a natural anionic sulfated linear polysaccharide found in some red seaweeds of the rhodophyceae family (Chondrus, Eucheuma, Gigartina, and Hypnea species) [67]. Carrageenan is a hydrophilic linear sulfated galactan, with molecular weight above 100 kDa [68]. Carrageenan is mainly composed of long linear chains of repeating d-galactose units and d-anhydrogalactose copolymer by alternating α-(1–4) and β-(1–3) linkages with ester sulfates (15%–40%) as substituents (Fig.  1.3) [69]. Traditionally CRG can be classified into six basic forms, namely, Kappa (κ), Iotta (τ), Lambda (λ), Mu (μ), Nu (υ), and Theta (θ)—carrageenan but three (kappa, iota, and lambda—carrageenan shown in Fig. 1.3) of them are the most important commercially with regard to the number and the position of the ester sulfate groups on repeating disaccharide units [70,71]. The number and position of ester sulfate groups and the content of 3,­6-anhydrogalactose (3,6-AG) determine the properties exhibited by CRG [72]. CRG is tasteless, odorless, whitish yellowish to colorless powder with excellent solubility in both cold or hot water [73]. The aqueous solubility of CRG is dependent on its type and the cation linked to the negatively charged sulfate groups [73]. CRG solutions are quite stable at neutral or alkaline pH, whereas the stability of the solutions decreases in acidic pH especially when exposed to higher temperatures owing to the hydrolysis resulting in loss of viscosity and gelation ability [73]. However, performed gels are relatively more stable to acid hydrolysis even at pH 3.5/4 [67,73]. Iotta-carrageenan gels, unlike other types of CRG gels, present a unique thixotropic behavior at low concentrations, which is exploited for suspending insoluble particles [67,73].

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Polysaccharide Carriers for Drug Delivery

CRG has several pharmaceutical applications especially in conventional and s­ ustained-release drug formulations and also for preparation of ­pH-/­temperature-sensitive delivery systems [71,73]. Furthermore, CRG is a biocompatible, biodegradable polymer with water-retention and gel-forming properties promulgating its use in buccal, ophthalmic, and vaginal drug delivery systems, and other biomedical applications, e.g., wound healing and tissue engineering [73,74]. Despite its inherent merits, chemical modifications of CRG and/or CRG-based nanomedicines are not forthcoming owing to their high viscosity [73]. However, modification of CRG by graft polymerization might be a promising strategy to develop CRGs with desirable properties [73].

1.4 Polysaccharides of animal origin 1.4.1 Chitin and chitosan Chitin is a polysaccharide made up of 2-acetamido-2-deoxy-β-d-glucose monomers that are connected via β-(1 → 4) linkages (Fig. 1.4) [21,75]. Chitin naturally occurs in the exoskeleton of anthropods like shrimps, insects, lobsters, crabs, and the cell walls of certain fungi [21,76]. Chitosan—a crystalline, cationic, and hydrophilic polymer with excellent gelation and film-forming property—is obtained by the N-deacetylation of chitin [21,77]. Chitosan is a linear polysaccharide made up of d-glucosamine monomers with randomly located N-acetylglucosamine substituents that are β-(1 → 4) linked (Fig. 1.4) [21]. The level of substitution of the acetamido group with the amine groups is called the degree of deacetylation (DD) of the chitosan and ranges from 30% to 95% [21,78]. The repeating units of chitin and chitosan are shown in Fig. 1.4. The physicochemical properties of chitosan like biodegradability, reactivity, solubility, and cell response depend on the degree of deacetylation [21,79]. Chitosan, despite its crystallinity, is insoluble in water at pH > 7; however, it is soluble in dilute acids owing to the presence of a protonated amino groups in acidic conditions (pH  Zn2+ > Cd2+ > Ni2+ > Co2+ > Ca2+, Eur3+ > Nd3+ > Cr3+ > Pr3+ [44]. Mucoadhesion property of chitosan is particularly important as it provides adhesion when one of the surfaces is mucus. Mucus consists of large amount of water

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Polysaccharide Carriers for Drug Delivery

(>95%) and a glycoprotein mucin [45]. The key sugar residues for mucoadhesive interaction are N-acetyl neuraminic acid or “sialic acid,” few sulfated galactose, and the hydrophobic methyl containing fucose. Chitosan interacts strongly with the negatively charged sialic acid residues [46] through hydrogen bonding and hydrophobic interactions [47]. The chitosan/mucin interaction depends on the zeta potential of the mucin [48], and this change in zeta potential is related to the particle size concentration, molecular weight, charge, and pH [47, 48]. Consequently, the degree of chitosan/ mucin interaction is dependent on the biological source of mucin. Chitosan, therefore, shows a great popularity in drug-delivery systems, particularly in the encapsulation technology involving chitosan nano- or microparticles. In the recent decades, chitosan has been extensively used in biological applications and drug-delivery systems, such as nanoparticle microspheres. It acquired a great deal of interest from the pharmaceutical industry in drug-delivery system and there have been a number of publications on this subject in the last decade. Several aspects including biodegradation, biodistribution, and toxicity [49]; formulations for delivery of nucleic acid-based drugs (NABDs), DNA, siRNA [50], and protein therapeutics [51]; hydrogels for controlled and localized drug delivery [52]; nanostructures for delivery of ocular therapeutics [53]; and the targeted delivery of low-molecular-weight drugs [8] have been reported yet. Application of chitosan has extensively been developed in oral administration for lipid lowering and antihypertensive drugs and in ophthalmic topical preparations for improving the biodistribution and retention of drugs [53]. The nasal insulin delivery is more efficient with chitosan of molar mass greater than 100,000 g/mol because the reversibility of transepithelial chemical resistance (TEER) values decreases with decreased chitosan molar mass [54]. Chitosan has been widely employed in the preparation of nanoparticles for drug delivery [55]. Chitosan nanoparticles can be prepared by at least three different methods: (1) Electrostatic interaction and resultant ionotropic gelation between chitosan and tripolyphosphate (TPP) polyanion [56], (2) Microemulsion for preparation of chitosan-glutaraldehyde complexes [57], and (3) Polyelectrolyte complex (PEC) formation with pectin [58] or hyaluronic acid [59]. This is of particular importance when a constant drug-release profile is not desired [58]. The size of the nanoparticles depends on the molecular weight of the chitosan polymer with higher molecular weight producing larger nanoparticles [56]. The method of crosslinking generally affects the mucoadhesive strength and stability of chitosan nanoparticles [57]. Chitosan is generally used to entrap a variety of drugs to release them in a controlled manner [60]. Spheres of chitosan can be synthesized by crosslinking with glutaraldehyde, formaldehyde, and citric acid, and by the physical agent genipin extracted from the fruit of gardenia [61]. Besides this, chitosan spheres can also be synthesized by techniques, including solvent evaporation, multiple emulsion, successive bark chitosan and coating deposition [62]. Chemical modifications of chitosan to improve its delivery potential are of particular interest, as these modifications do not allow the change in fundamental skeleton and maintain its original physicochemical and biochemical properties. Many changes are reported in the literature, including alkylation, acylation, hydroxy alkylation, carboxy alkylation, phosphorylation, sulfation, oligomerization, enzymatic modifications, and copolymerizations. Chemical modifications

Naturally occurring polysaccharides for drug delivery system

25

provide a wide range of chitosan derivatives with the properties specific for applications in particular areas of drug delivery. Other modifications of chitosan have also emerged as interesting multifunctional macromolecules that include its hybridization with sugar, cyclodextrin, dendrimers, crown ethers, and chitosan itself [63]. Further modifications of chitosan to introduce desired physicochemical and biochemical properties are of great significance for a breakthrough in utilization of their unique features and activities [64]. The possible reaction sites of chitosan mainly include free amino groups, primary and secondary hydroxyl groups, and acetamido groups. Chemical reactions targeted at the hydroxyl groups include etherification, esterification, crosslinking, graft copolymerization, and O-acetylation. Similarly, the chemical reactions targeting amino groups of chitosan mainly include acetylation, quaternization, Schiff base reaction (reactions with aldehydes and ketones), and grafting [65, 66]. Graft copolymerization is an important approach for modification of chitosan toward medical and pharmaceutical applications, such as in wound-dressing materials, orthopedic/ periodontal materials, tissue engineering, and controlled drug/gene delivery. Graft copolymerization of chitosan can be initiated by free radicals initiator systems, by radiation methods, and by enzymatic grafting methods [67]. The resulting macroporous polyacrylamide-grafted chitosan, for example, exhibited superior neuronal cell infiltration owing to the anisotropic porous architecture, high-molar-mass-mediated robustness, and superior hydrophilicity. The plausible chemical modifications of chitin and chitosan are represented in Fig. 2.1.

2.1.4 Pectins Pectins are complex heterogeneous polysaccharides that constitute a large proportion of the primary cell walls of citrus peel and apple pomace [68]. Pectin polysaccharides are made of three main domains: α-(1→4)-linked linear homogalacturonic backbone (HG) with two types of highly branched rhamnogalacturonans regions designated as RG-I and RG-II. The HG region is composed of (1→4) linked α-d-GalpA residues that can be partially methylated at C-6 position and partially acetylated at O-2 and/ or O-3 position. The RG-I region consists of disaccharide-repeating unit [→4)-α-dGalpA-(1→2)-α-LRhap-(1→]n with a variety of side chains consisting of l-arbinose and d-galactose residues (Fig. 2.2). In addition, GalA residues in the RG-I region are partially acetylated, but not methylated. In sugar beet pectin, the neutral side chain sugars are substituted with ferulic acid and the pectin chains are dimerized via diferulic bridges [56]. Moreover, RG-II has a highly conserved structure consisting of eleven different monosaccharide units, including some rare sugars [69]. Although pectins have been extensively used as gelling agent since early 19th century, the use of pectin gels in controlled drug delivery is of recent interest. This is particularly due to their reputation of being nontoxic [70], easy availability [71], and relatively low production costs [72]. It is proposed that pectin could be used to deliver drugs orally, nasally, and vaginally, with good acceptance among patients [73]. The oral delivery using pectin is of particular interest as various approaches can be made to increase drug delivery using enteric coatings [74], micro-/nanosphere formulations, permeation enhancers [75], and protease inhibitors [76]. Apart from this,

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Polysaccharide Carriers for Drug Delivery

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pectins have also been identified as protective agents against enzymatic proteolysis. As pectin is intact in the upper gastrointestinal tract and degraded by colonic microflora, it can provide promising potential for colon-specific drug delivery. It protects polypeptide drugs in the stomach and small intestine prior to pectin digestion by the colonic microflora resulting in the release of drugs [77]. The susceptibility of pectin to enzymatic attack is generally enhanced in the presence of calcium ions and minimized by methyl esterification [78]. The problem associated with pectin formulations is mainly due to its swelling under physiological conditions that leads to premature drug release. This problem can be reduced by preparing pectin combination with other polymers including cellulose and chitosan [56]. Pectin-based systems have emerged as particularly promising for drug absorption through nasal epithelia, which result in very rapid absorption. Low methoxyl pectins are strongly polyanionic polyuronides and can form weak gels in the presence of Ca2+ ions, present naturally in nasal secretions, thereby making them patient friendly in nasal delivery formulations [79]. In addition, this may hold the incorporated drug molecules in the nasal cavity for a prolonged period and thereby improve its rate of systemic absorption. Although pectins generally do not act as an absorption enhancer, they can alter drug-release potential through chelation with calcium ions [80]. They are also highly mucoadhesive, but less than chitosan. Their mucoadhesive power depends on their molecular weight, local pH, viscosity, and their functional groups [73]. Similar to nose, vagina is another possible site for drug delivery owing to its large surface area, rich blood supply, and presence of well-known microflora. Drug-delivery-release rates may vary during the menstrual cycle and this is especially important at the menopause. The vaginal route has been considered to be

28

Polysaccharide Carriers for Drug Delivery

favorable in the delivery of drugs, including propranolol, growth hormones, hormonal contraception, etc. Pectin-based formulations have demonstrated to have mucoadhesive strength, swelling property, and lowest pH reduction, thereby suitable for use in vaginal delivery. Besides these, its several modifications such as gelation of pectin, calcium crosslinked pectinate, composites of pectin and other polymers, including cellulose derivatives, have also been examined and tested for controlled drug delivery [81]. Modification of pectin via grafting with poly(N-vinylpyrrolidone) (PVP) has also been reported to prepare hydrogels that can produce an effective colon-targeted drug delivery [82]. A recent study reported that pectins extracted from spruce bark demonstrated potential immunomodulating activity and can be used as immunostimulant [69].

2.1.5 Heparin Heparin is a polysaccharide consisting of a pentasaccharide sequence. The numerous sulfate groups on glucosamine residues result in a strong negative charge density. This strong negative charge density of heparin molecules is central for their anticoagulant properties. Heparin is widely exploited in acute thrombotic diseases, vascular diseases, and cardiac surgery [83]. It often binds to several naturally occurring neutral and acidic proteins peptides. This binding interaction property, together with anticoagulant activity, is widely used for developing heparin-containing materials. In one study, good anticoagulant action, better cell adhesion properties, lower cell toxicity, and shortened clotting time of the heparin-grafted hybrid materials were observed, compared with heparin itself [84]. A heparin-like structured macromolecule was synthesized for the modification of noncoagulating biomaterials [85]. Another publication showed that a drug-eluting coating, consisting of heparin-loaded microspheres, was effective in suppressing platelet adhesion where the film showed better anticoagulation and biocompatibility with human umbilical veins endothelial cells with improved hemocompatibility [86]. Recently, heparin-coated silica nanoparticles were used for an efficient binding to antithrombin as an anticoagulant drug-delivery system [87]. Synthesis of heparin-based nanocapsules using the inverse miniemulsion process allows to obtain core-shell nanocarriers with defined properties like average morphology, size, and surface functionality [88]. Using miniemulsification, it is also possible to encapsulate efficiently fragile molecules [89]. Till now, heparin has been used as shell material of microcapsules and as modification of nanoparticles. Heparinbased nanocapsules are often highly advantageous due to the reason that (i) hydrophilic active components can easily be encapsulated, (ii) they serve as functional shell material, and (iii) the nanosize makes it feasible to inject it in blood [87]. Heparin coupled with nanomaterials has largely been investigated for chemical and biological properties. It enhances the biocompatibility of nanoparticles and their performance in different biological applications. Heparin has been coupled with several magnetic and metallic nanoparticles and biodegradable synthetic biopolymers. These modifications oriented the role of heparin in novel applications, including improved anticoagulation, cancer therapy, biosensors, tissue engineering, and most importantly in drug-delivery systems [90].

Naturally occurring polysaccharides for drug delivery system

29

2.1.6 Hyaluronic acid Hyaluronic acid consists of the alternate link of glucuronic acid and N-acetylglucosamine and largely occurs in extracellular matrix in mammals. It has an excellent potential to modulate cellular fate by receptor-mediated uptake. For example, it has an ability to bind with cluster determinant 44 (CD44) receptor, hyaluronic acid receptor for endocytosis (HARE), receptor for hyaluronic acid-mediated motility (RHAMM), and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) to induce specific cellular functioning. These properties together with its hydrophilicity, biocompatibility, biodegradability, and nonimmunogenicity make it an excellent drug-delivery candidate [6]. The delivery of anticancer drug doxorubicin by hyaluronic acid has extensively been studied. A homogeneous coating of mesoporous silica (MPS) with hyaluronic acid results in high stability in physiological solution. Additionally, the hyaluronic acid coating of MPS allows the interaction with the CD44 receptor that is often overexpressed in tumors and allows the complex internalization [91, 92]. The hyaluronic acid-modified nanoparticles targeting CD44-overexpressing cancer cells are represented in Fig. 2.2. Hyaluronic acid is digested by some specific enzymes in the cells that lead to the drug release into the cytosol. Chen et al. have found that hyaluronic acid-MPS complex loaded with doxorubicin reduces the viability of human breast cancer more effectively than that of doxorubicin alone [93]. Additionally, when administered to the normal cells, this complex also displayed reduced toxicity compared to doxorubicin alone. Another smart approach to deliver doxorubicin by hyaluronic acid is the development of pH-sensitive modifications. Hyaluronic acid-adipic acid hydrazide (ADH) complex loaded with doxorubicin allowed the hydrolysis of hydrazone bond between hyaluronic acid and doxoubicin faster at acidic pH, allowing the release of doxorubicin in an acidic environment. From the toxicology point of view, unlike free doxorubicin, treatment with hyaluronic acid-doxoubicin complex induced renal and cardiac dysfunction. Moreover, hyaluronic acid-doxoubicin conjugates reduced tumor growth more effectively than doxorubicin alone, with an increased animal survival rate [6]. The delivery of cisplatin by hyaluronic acid-based particles has been investigated by preparing cisplatin-hyaluronic acid conjugate in lung cancer. The idea was to develop a local cisplatin delivery to minimize the side effects of cisplatin. The lung instillation of cisplatin-hyaluronic acid conjugate displayed better pharmacokinetic profile compared to i.v. administration of naked cisplatin. The more targeted localization of cisplatin-hyaluronic acid conjugate resulted in reduced neuro- and nephron-toxic effects compared to naked cisplatin [94]. A further advantage of hyaluronic acid-based carrier is represented by the possibility to be loaded with other drugs.

2.1.7 Dextran Dextran constitutes a group of microbial polysaccharides often produced by Leuconostoc spp. and Lactobacillus spp. It is chemically composed of 1,6-linked α-d-glucopyranose units with varying proportions of other bond types, including α1,3, α-1,2, and α-1,6 as branch linkages. Dextran is considered an appropriate delivery material owing to its hydrophilic character, biodegradability, biocompatibility,

30

Polysaccharide Carriers for Drug Delivery

and possibility of easier chemical modifications [6]. A dextran-mediated delivery of doxorubicin has been developed to produce microcapsules fabricated via host-guest interaction between polyaldehyde dextran-graft-adamantane and carboxymethyl dextran-graft-β-cyclodextrin [95]. This kind of interaction has strong stability but is pH dependent due to the presence of acid-sensitive hydrazone bonds in polyaldehyde dextran-graft-adamantane. Adamantane was used as doxorubicin-linking site and the presence of pH responsive bonds is introduced to confer the ability to release drug in acidic cancer tissues. In particular, at pH 5.5 about 80% of the loaded drug was released, while at physiological pH only 18% of the drug was delivered. The pH-responsive property together with the excellent biocompatibility makes this a very promising conjugated delivery system [95]. Another interesting and innovative dextran-mediated delivery has been reported to deliver two different drugs from the same particle. To achieve this, dextran nanovesicles were constructed with a dextran backbone incorporated with a hydrophobic pentadecyl phenol (DEX-PDP) group through an aliphatic ester linkage. The amphiphilic nature of designed nanovesicles allows the simultaneous loading with the water-soluble doxorubicin and the hydrophobic irinotecan, an inhibitor of topoisomerase I. The combined administration of doxorubicin and irinotecan in a 4:1 ratio showed the best cytotoxicity. Notably, the activity of the particles loaded with the two drugs was always superior to that of the same particles loaded with the two drugs separately [96]. Additionally, it could be interesting to investigate the effectiveness of nanovesicles loaded with different combinations of the drugs.

2.1.8 Pullulan The exocellular Pullulan, produced by the yeast-like fungus Aureobasidium pullulans, is a linear polymer composed of glucose residues polymerized into repeating maltotriose units. The maltotriose units are linked via α-1,6 bonds; within each unit the glucose residues are linked α-1,4. Nevertheless, some 1,6-α-d-maltotetraose-repeating units and 1,3-linked glucose residues have also been found as components of pullulan primary structure [6]. Pullulan structure has a unique characteristic, i.e., coexistence of α-(1→4) and α-(1→6) linkages. Due to this unique structure of pullulan, its nanoparticles have both hydrophobic and hydrophilic features. It can be functionalized using different chemical reactions to enhance its utility in the field of controlled drug delivery. Pullulan-drug conjugates support safe and targeted delivery of various drugs to a specific site and exhibit high bioactivity with the release of cytotoxic molecules. Thus, pullulan drug conjugates can be used for targeted drug delivery and targeted gene delivery for the treatment of several diseases in liver, lungs, brain, spleen, etc. [97]. Furthermore, pullulan is considered a potential interesting polysaccharide due to its excellent water solubility, biocompatibility, lack of immunogenicity, and presence of several hydroxyl groups that can be functionalized. Notably, pullulan has unique ability to target hepatic tissue as it can interact with the asialoglycoprotein receptor present on hepatocytes. A notable example of drug-delivery potential of pullulan is demonstrated by Lee et al., where paclitaxel-incorporated nanoparticles have been prepared using pullulan acetate. It was demonstrated that paclitaxel was released with an initial burst that lasted after 2 days, followed by a reduced release over one week [98].

Naturally occurring polysaccharides for drug delivery system

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Pullulan nanoparticle-paclitaxel conjugate demonstrated an activity superior to naked paclitaxel in reducing tumor growth. Importantly, pullulan nanoparticle-paclitaxel conjugate induces a reduced body weight loss and a reduced systemic toxicity when compared to the naked paclitaxel [98]. Further in addition to the slow-release kinetics, the effectiveness of pullulan nanoparticle-paclitaxel conjugate with reduced side effect also depends on the enhanced permeability and retention (EPR) effect.

2.1.9 Other modified polysaccharides The purpose of chemical modification of polysaccharides is to improve chemical and mechanical properties, biocompatibility, biodegradability, solubility, and toxicity. These modifications to polysaccharides mainly involve following approaches: (1) Combination or chemical ligation with other biopolymers, (2) Surface coating of micro- or nanosphere polysaccharides with other biocompatible polymers, (3) Crosslinking with specific types of reagents, (4) Enhancement of hydrophobicity through alkylation reactions [99]. Using these techniques, numerous types of modified polysaccharides to establish optimal drug-delivery systems have been prepared. Glucose-based polysaccharides, including glycogen, cellulose, amylase, and amylopectin, possess multiple free reactive hydroxyl groups. Several other polysaccharides offer both hydroxyl and carboxylic acid moieties that can be easily modified. Alginate is an interesting polysaccharide composed of α-l-guluronic acid and β-d-mannuronic acid with 1,4 linkages, modifications of which induce various physiological changes depending on the requirement. For example, oxidation of hydroxyl groups increases their biodegradability, while sulfonation supports enhanced blood flow by producing a heparin-like polysaccharide [99]. Modification of chitosan is an interesting approach to be discussed herewith. Interestingly, the modification of primary amino groups with various alkyl groups can be used to improve solubility and bioactivity [100]. A variety of modified polymers are prepared by chemical conjugation between insoluble drugs and water-soluble polymers through a biodegradable spacer. The selected spacers for this purpose should be stable in the bloodstream but cleavable after approaching the target site by hydrolysis to improve local drug release and to avoid toxicity [100]. In particular, for tumor treatment, such drug conjugates are selectively accumulated at the tumor-specific site by the enhanced permeability and retention (EPR) effects. A promising example is N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-doxorubicin conjugate (PK1) and HPMA copolymer-doxorubicin conjugate containing galactosamine as a targeting moiety (PK2), which have been developed for treating liver cancer. Based on similar concept, numerous chitosan-conjugated drugs have been investigated; one of them is doxorubicin-conjugated glycol-chitosan complex using carbodiimide chemistry [101]. Few small-sized ligand molecules can be easily fitted on the surface of nanoparticles that allow the attachment of several molecules on a nanoparticle. Further connection of nanoparticles with linkers allows the introduction of thousands of such small molecules. Following this concept, chitosan has been modified with oleoyl to prepare self-assembled nanoparticles as carriers for delivery of hydrophobic antitumor agents,

32

Polysaccharide Carriers for Drug Delivery

such as doxorubicin [102]. The amine group of chitosan has been modified with phthalic anhydride and primary hydroxyl with polyethylene glycol and carbolytic acid to synthesize amphiphilic-chitosan-monodispersed nanospheres with pH sensitivity [103]. Thiolated chitosan was prepared by formation of amide bonds between amine groups of chitosan and carboxylic acid of glutathione (GSH). Further, the insoluble anticancer drug paclitaxel (PTX) was incorporated with poly(methyl methacrylate) nanoparticles-coated chitosan-glutathione system [104]. In this strategy, folic acid [105] and various sugars have been extensively used due to essential role of folic acid in cell survival, and folate receptors are overexpressed in many types of cancer cells. Mansouri et al. synthesized and characterized nanoparticles folic acid-chitosan-DNA and evaluated their cytotoxicity. Folic acid-chitosan nanoparticles have proved to be promising candidates as nonviral gene vectors [106]. Besides, several long-chain fatty acids, including decanoic acid, hexanoic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, and oleic acid, have also been extensively used for modifying polysaccharides [107]. Recently, the grafting of synthetic polymers to polysaccharides has attracted huge attention. Graft copolymerization reactions are employed to introduce side chains that lead to the formation of novel hybrid molecules composed of natural and synthetic origin. Polysaccharides and their derivatives can be covalently crosslinked to synthesize nanoparticles as drug-delivery carriers. This crosslinking phenomenon involves formation of covalent bonds between functional groups of crosslinking agents. For example, the free amino groups in chitosan contribute to the reactivity with various crosslinking agents. The microspheres prepared from chitosan crosslinked with glutaraldehyde are very promising for controlled drug delivery [108]; however, its toxicity on cell viability limits its use in pharmaceutics [107]. Various crosslinking agents, including genipin, glyoxal, and other polymers, have been investigated for their possible biomedical applications. Microspheres crosslinked with glyoxal were more hydrophobic, nonspherical, and smaller in size due to the high degree of crosslinking. These microspheres enhance the controlled-release period for ormeloxifene when compared with chitosan and glutaraldehyde crosslinked microspheres [109]. Chitosan has been successfully modified by condensation reaction with naturally occurring di- and tricarboxylic acids to form pH-sensitive nanoparticles [110]. A novel biocompatible chitosan derivative, N-succinyl-chitosan (NSCS), with a well-designed structure, has been successfully synthesized that is nontoxic and has regular nanosphere morphology in water with cell-compatible fashion [111].

2.2 Polysaccharides for the delivery of nucleic acid-based drugs Besides clinically approved drugs, other experimental molecules with potential therapeutic value can be potentially benefited with the polysaccharide-based drugdelivery approach. One category of these experimental molecules represents nucleic acid-based drugs (NABDs). These compounds constitute short sequences of either DNA or RNA and include antisense oligonucleotides, decoy oligonucleotides, triple helix-forming oligonucleotides, aptamers, DNAzymes, ribozymes, small interfering

Naturally occurring polysaccharides for drug delivery system

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RNAs (siRNAs), and microinterfering RNAs (miRNAs) [111]. As all NABDs are made up of short nucleic acid sequences, they have comparable physical-chemical properties and therefore required similar type of the delivery materials. Thus, the polysaccharide-based delivery systems used for siRNAs/miRNAs can also be fundamentally effective for NABDs. Although the therapeutic potential of miRNAs/siRNAs (collectively NABDs) has been recognized in various types of cancer therapy, their practical use is limited. This is mainly due to the absence of optimal delivery systems that can protect and direct them to the desired target tissue. If administered systemically in the naked form, NABDs have to face the obstacle of blood nucleases that rapidly trigger their degradation. Moreover, naked NABDs can be eliminated by the reticuloendothelial system by kidney filtration [112], and, in some cases, tend to activate the innate immunity [113]. The crossing of the vascular wall before reaching the targeted tissue is another obstacle to NABDs action. To approach the desired tissue, NABDs have to cross the cell membrane that is a difficult step due to the repulsion caused by negatively charged surface of cell membranes as well as phosphate groups present in NABDs. Secondly, the hydrophilic nature of NABDs disfavors the crossing of the hydrophobic layer of the cellular membrane. Within the cells, NABDs are also susceptible to further degradation by cellular nucleases and subjected to the cellular trafficking issue. Due to these aspects, NABDs may be sequestered into cytoplasmic vesicles (endosomes) that make the target mRNA unable to reach into the cell cytoplasm [114]. Altogether, these considerations clearly suggest that naked NABDs are unable to provide their therapeutic action. The problem associated with NABDs degradation can be overcome through the chemical modifications of their structure to make them resistant to nucleases-mediated degradation. A second possibility is the complexation or binding to other synthetic polymer materials. Among the several available materials, the use of several polysaccharides such as chitosan and pectin is considered better delivery material due to the wide spectrum use and versatility [115].

2.3 Pharmacological significance of bioactive polysaccharides The bioactive polysaccharides refer to the polysaccharides that can be produced by living organisms or functionalized from sugar-based materials and exert biological effects on living organisms. Additionally, the biological effects produced by polysaccharides are limited to therapeutic activities for curing diseases as well as toxic activities for causing diseases [116]. The pharmacological activities of polysaccharides are greatly affected by their chemical structure and chain conformations. The polysaccharides in algae and lichens have gained increasing attention particularly due to their beneficial biological properties, such as antithrombotic, anticoagulant, antiviral, antioxidant, antiinflammation, immunomodulating, and antitumor activity [117]. Polysaccharides in the dietary fibers including cellulose, hemicelluloses, pectins, and gums may be biologically active in their original form or after chemical/enzymatic modifications. For example, the cellulose and hemicellulose can directly activate the bowel movement, while the inulin requires to be fermented into short-chain fatty acids

34

Polysaccharide Carriers for Drug Delivery

by microflora in order to prevent various gastrointestinal (GI) disorders [118]. On the other hand, pectins, inulin, and gums help to minimize the movement of food in the GI tract, to reduce the blood cholesterol level, and to minimize the rate of sugar absorption that leads to avoid sudden hyperglycemia after food intake. Epidemiological and clinical studies suggested that the higher intakes of dietary fiber can effectively control the risk of diabetes [119], cardiovascular diseases including hypertension and coronary heart disease [120], hyperlipidemia [121], obesity, as well as colorectal cancer [122]. Polysaccharides have been known as major active compounds responsible for numerous pharmacological activities, including antitumor, immunomodulatory, antioxidant, antiviral, hepatoprotective, radioprotective, and antifatigue activity [123]. Various herbal polysaccharides are believed to be active in their natural form to activate human immune systems, to scavenge free radicals, to inhibit viral replication, and to inhibit lipid peroxidation [124]. The polysaccharides in algae and lichens have attracted an increasing attention owing to their excellent beneficial biological activities, such as antioxidative, anticoagulant, antithrombotic, antiinflammatory, antiviral, antitumor, and immunomodulatory activity. [125]. The sulfated polysaccharides are considered as the most interesting and attractive ingredients in the marine algae, including fucoidans and laminarans in brown algae (Phaeophyceae), ulvans in green algae (Chlorophyceae), and carrageenans in red algae (Rhodophyceae) [126]. Anticoagulant activity could be the most attractive property of the sulfated polysaccharides. For example, the sulfated fucoidans from brown algae and the sulfated galactans (carrageenan) from red algae have been known for high anticoagulant properties. These polysaccharides have been reported to possess stronger activity than that of heparin [127]. Other beneficial pharmacological significance of sulfated polysaccharides has been identified from algae. For example, (1) antitumor activity of algae was found to originate from its free-radicals scavenging and antioxidant properties [128]; (2) the highest antioxidant activity responsible for protection against damage from reactive oxygen species (ROS) was found in fucoidan followed by alginate and laminaran; (3)  antiviral activity of fucoidans, carrageenans, and rhamnogalactans was established by exerting inhibitory potential on the entry of herpes and HIV viruses into cells; (4) immunomodulatory activity was identified by enhancing the phagocytic activity of macrophages and by inducing the production of nitric oxide, ROS, and cytokines [129]. Polysaccharides isolated from lichens have been reported to possess a wide range of pharmacological activities, such as antitumor, immunomodulatory, and antiviral activity [130]. The β-glucans from lichens and lichenan manifested the immunomodulatory activity by activating a wide range of immune responses including cytokine release, ROS generation, nitric oxide production, and release of arachidonic acid. Lichenan has also been reported for showing anticoagulant and antithrombotic activity [131]. These activities of the sulfated polysaccharides from algae and lichens greatly depend on their structural features, including sulfate content and its distribution on the main chain, molar mass, and stereochemistry. Therefore, it is an urgent need for rational modification of the natural sulfate polysaccharides to obtain the polysaccharides of particular interest. Polysaccharides from wood mainly include cellulose and a few primary groups of hemicelluloses including glucomannans, arabinans, xylans,

Naturally occurring polysaccharides for drug delivery system

35

g­ alactans, and glucans [132]. Galactoglucomannans and pectins from wood have been identified to show immunomodulating activities and free radical-scavenging activities [133]. Xylans from wood and fibers have also been identified to possess excellent prebiotic potential for various medical applications [134]. Cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose, and hydroxylpropylmethylcellulose, have reported to show promising applications in numerous medical, pharmaceutical, and cosmetics fields. The sulfated glycosaminoglycans, including heparansulfate (HS), chondroitin sulfate (CS), dermatansulfate (DS), and keratin sulfate (KS), represent another important animal-derived group of bioactive polysaccharides [135]. Heparin, the most highly sulfated heparin sulfate, has largely been used as an effective clinical anticoagulant. Anticoagulant activity of the heparin highly depends on its specific structure sequence, where the binding site of a protein antithrombin III is crucial to prevent the generation of a fibrin clot formed by the action of thrombin [136]. CS/ DS chains are composed of variably sulfated N-acetylgalactosamine and glucuronic acid disaccharides repeating units [137]. Growing evidence reveals that these are the key molecules of the brain extracellular matrix, regulating proliferation, cell adhesion and migration during wound healing, and signaling of growth factor in skeleton [138]. Animal-derived hyaluronic acid (HA) is a linear nonsulfated glycosaminoglycan with repeating disaccharide units of β-(1-4)-d-glucuronic acid and β-(1-3)-N-­acetyld-glucosamine. HA is essentially found in extracellular matrix, controlling cellular signaling, morphogenesis, wound healing, and matrix organization. HA is, therefore, clinically used for various medical applications, including viscosupplementation and eye surgery [139]. Chitin, the second most abundant polymer after cellulose, is a copolymer of β-(1-4)-linked N-acetyl-glucosamine (acetylated unit) and N-glucosamine (deacetylatedunit). Partial deacetylation of chitin under alkaline conditions or by chitin deacetylase yields the most important derivative of chitin, i.e., chitosan [140]. Oligomers obtained from partial acid hydrolysis of chitin and chitosan have been recognized for their strong bioactivity, including antiinflammatory, antitumoral, hemostatic, antibacterial, and fungicidal activities [141]. The biological effects of important bioactive polysaccharides are represented in Table 2.1.

2.4 Conclusion Today polysaccharides have become a major focus of attention in many areas of research as they possess strong bioactive and drug-delivery potential. Particularly, polysaccharides of marine origin, including chitosan, alginate, carrageenan, agar, and collagen, are playing a key role with remarkable drug-release capacity. The discovery of “magic bullet” concept by Paul Ehrlich attracted many researchers to develop the drugs that selectively target disease cells without harming healthy cells. Consequently, numerous polysaccharides underwent cytotoxicity screening toward healthy cells prior to development of any drug-delivery system. Among such targeted delivery systems, chitosan and its derivatives possessed various advantages, including biocompatibility, biodegradability, low toxicity, mucoadhesivity, and other unique biological properties that made them fit for optimal drug delivery. However, a limitation of using these

36

Table 2.1  A brief summary of bioactive polysaccharides from different sources Type

Name

Composition

Sources

Biological effects

References

Vegetal

Cellulose

β-(1-4) Linked-d glucopyranose Xylans, mannans, β-glucans with mixed linkages and xyloglucans

Grains, fruit, vegetables, nuts Annual and perennial plants, fruit, legumes, and nuts

[120]

α-(1-4)-d-galacturonic acid and rhamnose backbone, arabinose, galactose, xylose side chains, partially O-methyl/acetylated β-(1-4)-d-Glucose and β-(1-3)-d-glucose

Plant primary cell wall, soft tissues of fruit and vegetable

Increase stool bulk and help to regulate bowel movement Immunomodulating, antithrombotic, free radicals eliminating, bowel movement regulating and cholesterollowering effects Intestinal immunomodulating and cholesterol-lowering effect, decrease gastric emptying and small intestine transit time

[147–149]

Starch

α-(1-4) and/ or (1-6)-Linked d-glucopyranosyl

Gums

Galactan, xylan, xyloglucan, glucuronic mannan, galacturonic rhamnosan type β-(1-2)-d-Fructofuranosyl

Potatoes, rice, green bananas, legumes, and food containing modified starch Locust bean gum, gum arabic, and guar gum

Cholesterol lowering, blood glucose regulating, antihypertensive, and immune system-stimulating effects Prevention of colonic cancer, hypoglycemic and hypocholesterolemic effects, prebiotic, mineral absorption enhancer Hypocholesterolemic and hypotriglyceridemic effects, gel forming to increase satiety, decrease gastric emptying Hypolipidemic and prebiotic effects, which influence gut microbiota, mineral absorption enhancer

Hemicellulose

Pectins

β-Glucans

Chicory root, wheat, onion, garlic

[145, 146]

[150]

[151, 152]

[153–155]

Polysaccharide Carriers for Drug Delivery

Inulin

Oats, barley grains

[142–144]

Amorpho-phallus konjac plant

Cholesterol lowering, reduces the risk of constipation

[156]

Animal

Shell of marine invertebrates

[56]

2-O-sulfated iduronic acid and 6-O-sulfated, Nsulfated glucosamine Alternate link of glucuronic acid and N-acetylglucosamine

Liver and mucosal tissue of animals such as pigs and cattle Rooster comb, human umbilical cord, and bovine vitreous

Deacetylated chitin is used in medicine as tablet component and absorption enhancing agent Anticoagulation, cancer treatment, tissue engineering, and biosensors

[158]

Dextran

μ-1,6 d-Glucose

Pullulan

α-1,4- and α-1,6-Glucan or maltotriose

Anticoagulant and plasma expander

[97]

Ginseng polysaccharides

(1-4)-Linked homogalacturonan backbone, (1-2)-linked rhamnose on position 4 as a part of backbone or ramified regions, (1-5)-linked arabinose with branch points at position 3, (1-3)-linked terminal galactose

Bacterial (Leuconostoc and Streptococcus) Yeast-like fungus Aureobasidium pullulans Ginseng, root of Panax ginseng

Useful in cancer, wound repair, inflammation, granulation, cell migration, skin healing, fetal wound healing Used in medicine as plasma expander

Antirotavirus activity

[160]

Chitin and chitosan Heparin

Hyaluronan

Microbial/ fungal

[157]

[159]

Naturally occurring polysaccharides for drug delivery system

β-(1-4)-Linked d-glucose and β-(1-4)-linked d-mannose Deacetylated P-1,4 N-acetyl-d-glucosamine

Glucomannan

Continued 37

38

Table 2.1  Continued Type

Name

Composition

Sources

Biological effects

References

Astragalus

α-(1-4)-d-Glucan with α-(1-6)-branches Seven monosaccharides including rhamnose, xylose, glucose, mannose, arabinose, galactose, and glucuronic acid in a molar ratio of 7.45:18.63: 25.15:0.93:8.35:2.79:5.69 (1-3)-Linked galactose, (1-3) linked arabinose, (1-4)-linked glucose and terminal (1-4)-linked xylose residues. Sulfations occur on O6 of galactose and O3 of arabinose Backbone of alternating β-(1-3)- linked dgalactosyl units and α-(1-4)-linked lgalactosyl, (1-6)-sulfate or 3,6-anhydro-α-l galactosyl units

Astragalus roots

[161, 162]

Acanthopanax senticosus leaves

Immunomodulating and antiviral activity Antioxidant and immune-biological activity

Green algae, Caulerpa racemosa

Antiviral activity (herpes simplex virus types 1 and 2)

[164]

Red algae, Porphyra haitanensis

Antioxidant and anticoagulant activity

[165]

Acanthopanax senticosu

Algae and lichens

Green algae sulfated polysaccharides

Polysaccharide Carriers for Drug Delivery

Red algae sulfated polysaccharides (porphyran)

[163]

Naturally occurring polysaccharides for drug delivery system

39

polysaccharides for oral drug delivery is its fast dissolution rate in the stomach. Since chitosan is positively charged at low pH values (below its pKa value), it generally attracts negatively charged polyatomic ions in solution to furnish polyelectrolyte complexes. Using polysaccharide-based materials, other numerous drug-delivery systems have been designed for anticancer drugs that deliver drugs for prolong periods at controlled rates. However, little effort has been given to developing systems for the controlled release of nucleic acid. Recently, a novel gene transfer method that allows prolonged release of plasmid DNA in animals was established. In this system, a natural polymer collagen was used as carrier for the controlled delivery of plasmid DNA. Once introduced, the collagen derived biomaterial carrying plasmid DNA to animals and gradually released plasmid DNA to animals with prolonged biological effects. Despite numerous evidence of potential anticancer value of NABDs, their practical use is often limited. This is mainly due to their delivery problems that include fast degradation in biological fluids, difficulties to cross cell membrane, and sequestration into cellular lysosomes. Thus, for conventional anticancer drugs as well as NABDs, the development of optimal delivery systems can radically improve their therapeutic efficacy. Among the several delivery materials studied so far, polysaccharides represent very attractive candidates as they can be obtained easily from natural sources, can undergo a wide range of chemical modifications, are biocompatible, biodegradable, and have low immunogenic properties. Altogether, these features make polysaccharides excellent molecules for the development of smart delivery systems capable to release at appropriate time and site of action. However, the development of such smart approaches needs to consider several aspects. First, it is required to optimize the polysaccharide-drug interaction that depends on the physicochemical properties of the polysaccharide materials and drugs. Second, it needs to optimize pharmacokinetics and pharmacodynamics through a careful evaluation of the ADMET profilings. Third, it is required to confer the targeting ability of polysaccharides to limit their effects toward healthy tissue. Finally, for the applications considering drug delivery from a gel matrix, the study of release mechanisms of the drug from the gels is also important. Although an ideal delivery system is still unexplored, the approaches discussed in the present chapter indicate that many interesting options, based on the use of polysaccharides, are emerging nowadays. In the last decade, there has been a great deal of interest particularly in chitosan and pectin in drug-delivery systems. However, several important issues still remain to be resolved fully to enhance their potential in drug delivery. With chitosans, these include: (i) their stability, without any limitation of being soluble at pH 2.5) but when it comes in contact with water, it absorbs water, swells, and produces a clear to opalescent viscous solution (DS =1.3–2.5) [61]. Their viscosity does not change with change in pH and they remain stable over a wide range of pH 2–12. However, with increase in temperature above 29±2°C, the viscosity of MC increases remarkably resulting in the formation of a thermoreversible gel [62]. MC therefore is classified as a lower critical solution temperature (LCST) polymer [61]. MC is used for treating constipation, hemorrhoids, diverticulosis, irritable bowel syndrome, and dry eyes. They serve as an efficient water retention agent and are administered in powder form which is not digestible in the body. They do not cause any allergic reaction since they consist of an interesting dietary fiber. The pharmaceutical uses of MC comprises of an emulsifying agent, suspending agent, capsule disintegrants, binders, controlled release agents and viscosity enhancer in oral and topical formulations [63, 64]. Hirasawa et  al. exhibited potential of MC for improvement in dissolution and bioavailability by preparing tablets of Nilvadipine based on SD technology using MC as the carrier. The tablets were reported to exhibit higher solubility and dissolution rate and indicated good physical stability during storage [65]. However, much less research has been carried out on MC pertaining to solubility enhancement as newer forms have been developed giving better outcomes.

10.5.1.2 Ethylcellulose Ethylcellulose (EC) is one of the few cellulose ethers which are hydrophobic in nature i.e. insoluble in water but soluble in many polar organic solvents. The replacement of hydroxyl groups of AGU by ethyl ether (OCH2CH3) groups result in the formation of EC. It is a nonionic polymer, which is odorless, tasteless and not

284

Polysaccharide Carriers for Drug Delivery

affected by the change in pH [60, 63]. EC is often used in the pharmaceutical industry as a binding, coating, granulating, flavoring, thickening, extended, and sustained release agent [51]. The extended release property of EC is attributed to the formation of a viscose gel around the tablet which hinders the free release of the drug from the formulation [66]. EC has several applications in modified release dosage forms, however, it is used in conjunction with water soluble polymers like MC and HPMC (hydroxypropylmethylcellulose) in aqueous coating of liquids [67]. EC has also been used as a carrier for preparing SD of Dimenhydrinate and Indomethacin for its solubility and dissolution enhancement, although it is not widely utilized for this technology [68, 69].

10.5.1.3 Hydroxyalkyl cellulose Hydroxyalkyl celluloses such as hydroxyethyl and hydroxypropyl cellulose are cellulose ethers synthesized by replacing deprotonated hydroxyl groups in AGU with hydroxyethyl/hydroxypropyl groups by reacting it with ethylene oxide/propylene o­ xide [70]. The ring opening reaction of epoxides generates a new hydroxyl group at the terminal end, distant from the main cellulose chain which is more reactive and can be further modified. However, so far, the hydroxyalkyl cellulose derivatives available in the commercial market are very few owing to the harsh conditions used in the alkaline etherification procedures [71]. Therefore there is a need of developing new and more efficient methodologies for their synthesis taking into consideration the high urge of sustainable biomaterials. Hydroxypropyl cellulose (HPC) is a nonionic water-soluble polymer which is freely soluble in water below 38°C but precipitate in hot water (40–45°C). It is a pH insensitive cellulose derivative with DS of 3.0. HPC is widely employed as a food additive, thickening, stabilizing, binding, and disintegrating agent in the pharmaceutical industry. It is also known to be used as a carrier in ASDs and the drug release from these dispersions are primarily dependent on the molecular weight of HPC which ranges from 37,000 (Type SSL) to 150,000 (Type H) [72]. The release rate of flurbiprofen was found to improve with low molecular weight HPCs and with increased proportion of HPC in SD [73]. The high Tg of the polymer is another factor responsible for stabilizing the prepared formulations by restricting the drug recrystallization. In addition, both the hydrophobic and hydrophilic moieties of HPC are required for maintaining the drug in a supersaturated state which provides optimum dissolution rate to ASDs [74]. The improvement in solubility using HPC has been investigated on nifedipine which showed promising results [75]. In a study involving preparation of dispersions of fenofibrate using spray-drying technique, HPC was compared with Eudragit E-100 and Solutol HS15 for improvement in dissolution. However, Eudragit E-100 was found to exhibit faster dissolution efficiency than the other two polymers but HPC also showed favorable results in augmenting the dissolution profile of fenofibrate [76]. The polymer HPC has also been used in combination with EC to control the release of the water-soluble drug oxprenolol hydrochloride. The mechanism involving this is that HPC swells in water and is then trapped in water-insoluble EC which is responsible for the slow release of drug [51].

ASDs for improving solubility and bioavailability of drugs

285

Percent drug dissolved

8 HPC-SSL 6

PVP-VA Pure drug

4 2

0 0

60

120

180

240

300

Time (min)

Fig. 10.3  Dissolution profile of felodipine and its solid dispersions. Reproduced with permission from Sarode AL, Malekar SA, Cote C, Worthen DR. Hydroxypropyl cellulose stabilizes amorphous solid dispersions of the poorly water soluble drug felodipine. Carbohydr Polym 2014;12:518.

The low viscosity grade of HPC (HPC-SSL) has also been effectively utilized as a carrier in ASD. Research was done using HPC-SSL for investigating its potential in stabilizing the ASDs of felodipine, a poorly water-soluble drug. The effect was compared with vinyl acetate-substituted, polyvidone-vinylacetate (PVP-VA) and the results demonstrated that HPC-SSL was successful in providing stability to the felodipine ASDs when they are stored at lower or room temperature with elevated humidity. The dissolution profile of felodipine as shown in Fig. 10.3 exhibited that HPC-SSL displayed higher dissolution rate than PVP-VA [77]. Hydroxyethyl cellulose (HEC) is partially substituted polyhydroxylethyl ether of cellulose. Like EC, it is also nonionic water-soluble cellulose ether which readily disperses in water but is insoluble in organic solvents. HPC is available in many grades differing in their viscosity which depends on DS and molecular weight [63]. The polymer finds its use as a matrix for modified release tablet, as a film former, stabilizer, thickener, and suspending agent for oral and topical drug delivery. HEC is primarily used as a gel in topical formulations owing to its nonionic and water-soluble property [60]. Although the research pertaining to its use in ASD is limited, the recent research involved preparation of ASDs of etoricoxib using three water-soluble polymers—polyvinyl alcohol, PVP, and HEC—and further evaluating the kinetic solubility advantage of these prepared ASDs. The study confirmed the potential of HEC to maintain supersaturation state and imparting stability to the prepared formulation [78]. Therefore the hydroxyalkyl cellulose demands further research in the area of solubility and bioavailability enhancement of poorly water-soluble drugs.

10.5.1.4 Sodium carboxymethylcellulose Sodium carboxymethylcellulose (NaCMC) is a white, odorless, tasteless, and granular powder which is polyanionic in nature [79]. It is a product formed by substituting the

286

Polysaccharide Carriers for Drug Delivery

H atoms of the hydroxyl groups of AGU by CH2CO2Na with the DS ranging from 0 to 3 [80]. NaCMC is the most important industrial derivative as well as the most exploited commercial salt of carboxymethylcellulose in comparison to its other salts (potassium, calcium, ammonium etc.). Due to its ability to easily dissolve/disperse in water, it forms highly viscous solutions which are used as a thickening or suspending agent. Moreover, their swelling behavior is observed at high pH which finds its applicability in formulating pH sensitive drug delivery of aspirin, diclofenac etc. The polymer retards the drug release at low pH values in the stomach thereby releasing the drug at high pH values in the intestine [80, 81]. NaCMC has found wide applications in the food, textile, paper, and cosmetic industry. In the pharmaceutical industry, NaCMC has been employed as a binding, emulsifying, film forming, coating, and stabilizing agent. It has been extensively used as a constituent of vehicles used for formulating oral suspension. The viscosity increasing properties have also been employed for topical formulations including gel, emulsions etc. [82]. Further, the use of NaCMC as a carrier in SD for solubility enhancement of poorly soluble drugs is well known. The latest research signifying the potential of NaCMC in augmenting dissolution and bioavailability involved the preparation of SD of Cefdinir using spray-drying method. The in vivo bioavailability was found to be increased 6.77-fold using this method [83]. The SD of Tacrolimus prepared using NaCMC and sodium lauryl sulfate reported a 2000-fold increase in the solubility and a 10-fold increase in dissolution in comparison to the tacrolimus powder [84]. In another study, the effect of NaCMC on aqueous solubility of a nonsteroidal antiinflammatory agent, flurbiprofen was investigated, where it was reported to deliver the poorly water-soluble drug flurbiprofen with enhanced solubility and bioavailability [85]. Similarly, numerous other research studies demonstrated NaCMC to be a promising vehicle in providing better dissolution and bioavailability profile for poorly ­water-soluble drugs [86–88].

10.5.1.5 Hydroxypropylmethylcellulose The most common mixed ether cellulose derivative employed in the pharmaceutical industry is Hydroxypropylmethylcellulose (HPMC). It is a water soluble nonionic polymer with molecular weight ranging from 10,000 to 150,000. It is prepared by derivatizing 16.5%–30% of the hydroxyl groups by methyl groups and 4%–32% with hydroxypropyl groups [89, 90]. HPMC acts as a binding, thickening, emulsifying, ­viscosity-controlling agent and has the ability of water retention in pharmaceuticals and personal care products. It has been used in the preparation of controlled and sustained release dosage forms where it forms a gel on coming in contact with water, following the two mechanisms for the drug release: drug diffusion through the gel layer and drug release by membrane erosion [51]. Furthermore it has been extensively employed in ASDs for improvement in solubility and is the sole pH-independent cellulose derivative utilized for the same. HPMC is reported to form a solid solution with poorly water-soluble crystalline drugs. Earlier research involving HPMC in augmenting the solubility of nifedipine [91], ER-34122 (5lipoxygenase/cyclooxygenase inhibitor) [92], and Itraconazole [93] has been r­eported.

ASDs for improving solubility and bioavailability of drugs

287

The polymer exhibited highest supersaturation level in the dissolution studies in comparison to the other polymers involved in the research. Since then, numerous studies have been done which unfolded the potential of HPMC in solubility enhancement, inhibition of crystallization, as well as maintaining the supersaturated state. Xie and Taylor investigated the crystallization inhibitory properties of HPMC when incorporated in the SD of Celecoxib in order to attain an improved drug release [94]. Further, Vora and his associates investigated the maintenance of supersaturation state by HPMC as the drug traverses from acidic to neutral pH. They prepared SD of dipyridamole using three molecular weight grades of HPMC (HPMC E5, E15 and E50) and found that HPMC E50 was the best polymer in inhibiting the precipitation and extending the supersaturation. However, the solubility and dissolution improvement was reported with all three grades [95]. Adibkia and coworkers prepared and characterized solid dispersion of naproxen using HPMC E4M. The maximum drug release owing to the hydrophilicity and gel forming capacity of HPMC was obtained at drug/carrier ratio of 1:2 as shown in Fig. 10.4. The dissolution was also greatly affected by the pH of the dissolution media being higher at pH 7.4 than at pH 3.0 due to increased ionization at high pH leading to improved solubility [96]. The increase in solubility and drug release was also obtained when HPMC was used as a carrier to prepare the SDs of Itroconazole [97] and Cefdinir [83].

10.5.1.6 Cellulose acetate phthalate Cellulose acetate phthalate (CAP) is a cellulose ester containing acetyl (21.5%–26%) and phthalyl or o-carboxybenzoyl (30%–36%) groups. It is synthesized by reacting phthalic anhydride and partial acetate ester of cellulose. CAP is mainly employed as an

100

80

Cumulative release (%)

Cumulative release (%)

100

60 40 20

80 60 40 20

0

0 0

20

Naproxen

40

60

80

100

0

120

10

20

PM4 (NPX:HPMC, 1:0.5)

SD4 (NPX:HPMC, 1:0.5)

PM5 (NPX:HPMC, 1:1)

SD5 (NPX:HPMC, 1:1)

PM6 (NPX:HPMC, 1:2)

30

40

50

60

Time (min)

Time (min)

Naproxen

PM6(NPX:HPMC, 1:2)

SD6(NPX:HPMC, 1:2)

SD6 (NPX:HPMC, 1:2)

(A)

(B)

Fig. 10.4  Dissolution profile of pure naproxen, physical mixtures, and solid dispersions in (A) pH 3.0 and (B) pH 7.4. Reproduced with permission from Adibkia K, Barzegar-Jalali M, Maheri-Esfanjani H, Ghanbarzadeh S, Shokri J, Sabzevari A, Javadzadeh Y. Physicochemical characterization of naproxen solid dispersions prepared via spray drying technology. Powder Technol 2013;246:452–453.

288

Polysaccharide Carriers for Drug Delivery

enteric coating agent in delayed-release dosage forms which confers gastro-­resistance and it readily dissolves in a neutral or mildly acidic intestinal environment [98]. The polymer is insoluble at low pH and dissolves/swells at neutral to high pH. Due to its distinctive properties such as high Tg value, ability to ionize at low pH, and as a concentration enhancer, it was evaluated for its potential as a carrier in ASDs. A study involving CAP/Itraconazole matrix revealed twofold improvement in its oral bioavailability in comparison to the marketed formulation of Itraconazole (SPORANOX) in rat models. The improvement in bioavailability was due to the enhanced intestinal targeting and increased extent of supersaturation exhibited by CAP [99]. Recently, CAP displayed significant results when evaluated in comparison to other cellulosics for increasing the solubility of Lopinavir [100]. However, the polymer lacks sufficient research pertaining to its use in ASDs for solubility enhancement.

10.5.1.7 Carboxymethyl cellulose acetate butyrate Carboxymethyl cellulose acetate butyrate (CMCAB) is a hydrophobic ester derivative of water-soluble, anionic cellulose ether, carboxymethylcellulose (CMC) [101]. The DS of CMCAB polymers varies depending on the feed ratios and reaction conditions with different DS values for each of carboxymethyl (0.29–0.35), butyryl (1.37–1.64), and acetyl (0.30–0.55) groups. The synthesis of CMCAB is done by the esterification of NaCMC with acetic and butyric anhydrides. It is a thermoplastic polymer with high molecular weight and high Tg [102]. Being a nonpolar compound, it is insoluble in water but soluble in common organic solvents such as ketones, alcohols, esters, and ethers. However, CMCAB swells in water when it is partially ionized [103]. The use of CMCAB in oral drug delivery employs two very effective methods as described by Posey-Dowty and associates. The first, being the easiest one, involved preparing physical blends of ibuprofen with CMCAB by direct compression which resulted in extended drug release at zero-order at pH 6.8 [103]. The second method, being more effective, involved preparation of ASD of a poorly water-soluble drug (Fexofenadine HCl) which resulted in enhancement of its solubility [104]. The ASDs of poorly water-soluble drugs prepared using CMCAB produce stable supersaturated solution with zero-order release profile [105]. The potential of CMCAB for increasing solubility in ASDs were also evaluated in quercetin [106], curcumin [107], naringenin [105], ellagic acid [108], and resveratrol [109]. CMCAB has also recently been employed in novel techniques involving preparation of nanoparticles of cellulose acetate-based SD matrices. The ASD of p­ olymer-drug nanoparticles of ritonavir (RTV) and efavirenz (EFV) was prepared using flash ­nano-precipitation method. CMCAB showed best results among other polymers employed in this research (cellulose acetate propionate 504-0.2 adipate 0.33, cellulose acetate propionate adipate 0.85, and cellulose acetate 320S sebacate). There was a 10- to 20-fold improvement in the solubility of RTV and EFV using these cellulose derivatives [110]. An earlier similar study has been reported involving preparation of CMCAB nanoparticles containing acyclovir [111]. Thus CMCAB is a carboxylated cellulose derivative strongly recommended for use in ASD due to its unique properties

ASDs for improving solubility and bioavailability of drugs

289

of high Tg, low toxicity, impeding crystallization, maintaining supersaturation, and imparting pH controlled drug release [112].

10.5.1.8 Hydroxypropylmethylcellulose acetate succinate HPMCAS is known to be a premier polymer excipient available in the industry due to its high Tg value (133°C), benign toxicity profile, maintaining supersaturation state, and hampering crystal growth. The synthesis of HPMCAS is very complicated and requires critical control of the five substituents, i.e. methoxyl, hydroxypropyl, acetate, and succinate [113]. Due to these hydrophobic (methoxyl and acetate) and hydrophilic (hydroxypropyl and succinate) groups attached on the AGU chain, HPMCAS behaves as an amphiphilic polymer which facilitates strong interaction with hydrophobic drugs, thus leading to the formation of stable dispersions in water. The high Tg of the HPMCAS-based dispersions is also one of the reasons for the formation of physically stable dispersions having shelf lives of more than 2 years under standard storage conditions [114]. In addition, HPMCAS being less hygroscopic does not allow moisture uptake thus contributing stability to the formulation in which it is incorporated. HPMCAS is an enteric polymer and its dissolution behavior can be controlled by controlling the number of acetyl and succinoyl groups attached at the AGU chain. Thus the amorphous dispersion of HPMCAS with poorly soluble drugs was found to release the drug at the pH of the small intestine and showed no release at the pH of the stomach [16]. Hydroxypropylmethylcellulose acetate succinate or Hypromellose acetate succinate (HPMCAS) was initially developed as a cellulosic enteric coating agent in 1984. In addition, it also proved its efficacy as a carrier in the preparation of SDs [115]. HPMCAS was first patented by Shin-Etsu in 1987 and its use as a carrier in ASD was developed and patented by Pfizer Inc. in alliance with Bend Research. The mere admixture of HPMCAS with the basic or zwitterionic drugs was reported to improve the solubility ~1.5-fold [116]. In a study, the effect of polymer type on the solubility and dissolution enhancement of ASDs containing felodipine was studied, where HPMCAS outperformed other polymers (HPMC & PVP) in maintaining the supersaturated state subsequent to the dissolution of amorphous solids. In addition, it was found to impede crystallization thus reducing the growth of felodipine crystals and contributing to its enhanced dissolution [117]. Li et  al. demonstrated the solubilizing potential of HPMCAS in several studies and compared it with other carriers. In one such study, fast and complete drug release was obtained for ASDs containing curcumin:HPMCAS at 1:9 as shown in Fig.  10.5. The dispersions were also found to inhibit crystallization and protect curcumin against chemical degradation, thereby yielding stable dispersions [107]. HPMCAS was able to achieve high solubility and bioavailability in quercetin [106], ellagic acid [108], naringenin [105], and resveratrol [109] and was found more effective than other polymers in inhibiting crystallization and maintaining a supersaturated state. Recently, ASD of quercetin was prepared using cellulose ester, HPMCAS to improve its aqueous solubility, provide stability against crystallization, and impart pH-triggered release [118]. Similar effects were observed in progesterone [35], nifedipine [119], celecoxib [94], and danazol [120].

Polysaccharide Carriers for Drug Delivery

Curcumin dissolved (%)

290 50 45 40 35 30 25 20 15 10 5 0

1/9 1/3 1/1 3/1 9/1

0

5

10

15

20

2

Time (h)

Fig. 10.5  Dissolution profile of curcumin/HPMCAS solid dispersions in pH 6.8 buffer. Reproduced with permission from Li B, Konecke S, Wegiel LA, Taylor LS, Edgar KJ. Both solubility and chemical stability of curcumin are enhanced by solid dispersion in cellulose derivative matrices. Carbohydr Polym 2013;98(1):1114.

10.5.2 Natural gums The natural carriers have always evoked tremendous interest of the researchers due to their being nontoxic, biocompatible, biodegradable, cost-effective, chemically inert, and available in abundance. Moreover, they can be tailored easily to obtain the material of desired properties thus giving tough competition to the available synthetic excipients [121]. The orientation of the pharmaceutical industry as well as the increase in patients’ interest towards these natural agents has led to the improvement in their methods of extraction and purification to give higher yield. The investigations on the natural carriers majorly center around polysaccharides. Natural gums (polysaccharides) have gained interest in recent years and have been investigated for drug delivery applications and in the biomedical field. Moreover, they are devoid of any toxicity and thus have been categorized as GRAS by USFDA [122]. India has been a rich source of these gums among the Asian countries because of its environmental and geographical position [123]. The gums have diverse application in the pharmaceutical industry as a tablet binder, disintegrant, emulsifying agent, suspending agent, targeting, and for immediate and sustained-release preparation [124, 125]. Moreover, many natural gums have found their use in formulation of solid dispersion for solubility enhancement of poorly soluble drugs [126]. They have been investigated by many researchers and have shown promising results in modifying drug release from the formulations [127]. The natural gums are also associated with disadvantages such as microbial contamination, batch to batch variation, uncontrolled rate of hydration, and reduced viscosity on storage which demands modification of existing natural agents for their successful incorporation in novel drug delivery systems (NDDS) [124]. In addition, they readily dissolve in water and swell forming a viscous gel layer around the dosage form leading to its sustained release. Due to these inherent limitations and undesired properties, the gums are modified by heating at a specific temperature for a definite period and then sieved

ASDs for improving solubility and bioavailability of drugs

291

and stored at 25° C. This step reduces the viscosity and increases the water retention capacity of the gums causing minimal changes in their swelling behavior [128]. Thus there is huge scope for research on these gums for exploiting them for largescale manufacturing of different drug delivery systems in the pharma industry.

10.5.2.1 Neem gum The use of neem (Azadirachta indica Family: Meliaceae) in Ayurvedic medicine dates back 4000  years and it is considered as a wonder tree in India, offering treatments for almost every ailment [129]. Azadirachtin and nimbin are the main constituents of neem gum. Neem gum is composed of mannose, galactose, arabinose, glucosamine, glucose, fucose, and xylose [123]. Several compounds have been isolated from different parts of the tree, used for antifungal, antimalarial, antibacterial, antiviral, antitumor, antiinflammatory, antipyretic, analgesic, antiulcer, antiglycemic, and antifertility activity [130]. Besides its medicinal uses, neem gum has certain pharmaceutical applications. Previous studies have shown the use of neem gum as a directly compressible excipient [131], tablet binder [132], suspending agent [133], thickening agent [134], sustained release agent, and film coating agent [135]. Recently, the applicability of neem gum as a solubility-enhancing agent has been discovered; however, very little research has been done in this area. Neem gum has a very low viscosity which makes it suitable as a solubilizer. Rodde and coworkers have prepared solid dispersion of Atorvastatin using neem gum as a hydrophilic carrier. The results exhibited an increase in solubility (~85.8%) of the drug with increase in gum concentration (drug: polymer=1:9). The in vitro, ex vivo and in vivo studies further confirmed the potential of neem gum in enhancing the solubility and bioavailability of Atorvastatin [136]. A similar study utilized neem gum as a prospective carrier for bioavailability enhancement of the poorly soluble drug Aceclofenac, achieving enhanced dissolution profile. The in vivo pharmacodynamic studies exhibited improved analgesic potential of the solid dispersion-based tablet formulation when compared to that of the pure drug and the marketed formulation [137]. Hence, more exhaustive research and thorough investigations are a prerequisite for neem gum to be established as a solubility enhancing agent.

10.5.2.2 Locust bean gum Locust bean gum (LBG) is also known as carob gum or carubin, is a galactomannan vegetable gum obtained by removing and processing of endosperm of the pods of the tree Ceratonia silique (Family: Leguminosae/Fabaceae) [122]. It is a high molecular weight polysaccharide consisting of 80% d-galacto-d-mannoglycan (mannose:galactose=4:1) and the rest 20% consisting of proteins cellulose and impurities [138, 139]. USFDA has categorized LBG as GRAS and has defined 74.25 mg as the approved limit for its use. LBG is partially soluble in water at room temperature; therefore in order to attain maximum viscosity, hydration, and solubilization, modified LBG (MLBG) is prepared by heating at above 85° C for 10 min [140, 141]. LBG has been widely used as an excipient in the pharmaceutical industry owing to its gelling, thickening, and stabilizing property [142]. It is also employed as a

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­superdisintegrant [141], emulsifying, binding, granulating agent [125], mucoadhesive, coating agent [143], and for the controlled and sustained delivery of the drugs [144]. The extensive use of LBG has been credited to its high swelling behavior, high water retention capability, chemical compatibility, binding, and digestible nature [128]. MLBG has been used as a carrier for the solubility enhancement of lovastatin using solid dispersion method. The results revealed enhanced solubility and improved dissolution which was found to be dependent on LBG concentration and method of preparation. Further the in vivo study confirmed a significant reduction in HMG Co-A (3-hydroxy-3-methyl-glutaryl-coenzyme A) reductase activity [128]. In another study involving preparation of solid dispersion of loratadine using MLBG as a carrier, enhanced solubility and dissolution was achieved owing to the synergistic effect of reduced crystallinity, reduced drug particle size and improved wettability [145]. MLBG also enhanced the solubility of glibenclamide (from 26 to 97 μg/mL) on the same principle and the in vivo studies exhibited better activity in alloxan induced diabetic rat model. Similar studies were conducted which demonstrated the superior potential of MLBG in the biopharmaceutical field for solubility enhancement [146–149].

10.5.2.3 Karaya gum Karaya gum is a dried gummy exudate which is obtained from a large bushy tree (30 ft.) Sterculia urens (Family: Sterculiaceae) which is native to India. The gum is an acid polysaccharide which includes d-galactose, d-glucouronic acid, l-rhamnose, xylose residues, and acetyl groups as its main constituents [122]. Karaya gum is categorized as GRAS by USFDA and has been widely used as a food additive [150, 151]. In addition, the use of this gum as a laxative is evident from the literature [152]. It has been employed as a mucoadhesive, emulsifying agent, suspending agent, and sustained release agent [153]. The prior investigations have also indicated its use as a disintegrant and hence it can be used as an alternative to synthetic superdisintegrants owing to its abundant availability, biocompatibility, and low cost. The varied uses of this gum are due to its high water retention capacity, viscosity, and swelling behavior, antimicrobial property, and abundant availability [140]. The use of karaya gum as a tablet binder and as a disintegrant has been limited by its high viscosity which led to modifications in the gum. Modified karaya gum (MGK) has been prepared by heating at 120°C for 2 h to modify its viscosity and for its better processing and handling during the preparation of solid mixtures. However, the swelling behavior of MGK has not been compromised and is found comparable to unmodified karaya gum. Some researchers have also indicated the potential of MGK in dissolution enhancement of poorly soluble drugs. In a research study involving preparation of solid dispersion of Nimodipine using MGK as the carrier, the superiority of MGK over unmodified gum was demonstrated. MGK was found to be a potential carrier in dissolution rate enhancement of nimodipine as shown in Fig. 10.6 [154]. They later conducted in vivo study which also confirmed the efficiency of MGK as a solubility and bioavailability enhancing agent [155]. In another study, which involved preparation of solid mixtures of Nimesulide with MGK by physical mixing, kneading, and solid dispersion techniques, solid dispersion exhibited the most promising results

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Fig. 10.6  Dissolution profile of nimodipine and its solid dispersions. Reproduced with permission from Murali Mohan Babu GV, Prasad CDS, Ramana Murthy KV. Evaluation of modified gum karaya as carrier for the dissolution enhancement of poorly watersoluble drug nimodipine. Int J Pharm 2002;234(1–2):12.

in solubility enhancement of nimesulide [156]. MGK also enhanced the dissolution rate of glimepiride and was found to overcome the high viscosity issues observed with karaya gum [157]. Several other studies have been done in the past involving preparation of solid dispersion with MGK confirming its potential as a carrier for solubility and bioavailability enhancement [150, 158].

10.5.2.4 Guar gum Guar gum, also known as cluster bean or guaran, is a galactomannan extracted from the endosperm of the seeds of Cyamompsis tetragaonolobus (Family: Leguminoseae). It is high molecular weight polysaccharide consisting of a linear chain of galactose and mannose residues (mannose:galactose=2:1) [159, 160]. Therapeutically guar gum has been utilized as a bulk-forming laxative, in peptic-ulcers, as an adjunct in treatment of Type II diabetics and as an appetite depressant [125]. The pharmaceutical uses of guar gum encompass tablet binding, disintegrating, stabilizing, thickening, emulsifying, as a sustained release agent, and for colon targeted drug delivery [161–163]. For their use in pharmaceutical industry they are modified by heating at 125–130°C for 2–3 h. The viscosity of the modified guar gum (MGG) was found to be reduced by ~3 times without any change in the swelling and water retention capacity, thus paving the way for their use in solubility enhancement. Solid dispersion of licofelone was prepared by using guar gum and MGG at a ratio of 1:6 (drug:gum). The results clearly

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indicated the efficacy of MGG in enhancing the solubility of licofelone. The reason was the swelling property of the gum which increased the surface of carrier coming into contact with the dissolution medium resulting in enhanced dissolution rate [164]. Another study involved the preparation of solid dispersion of gliclazide using guar gum as the carrier which resulted in 96.79% release within 60 min [165]. Maximum solubility and in vitro dissolution were attained when solid dispersion of cefixime was prepared using MGG as the carrier by using solvent evaporation method [166]. The ability of guar gum to enhance the dissolution rate of poorly soluble drugs has been widely studied thus establishing it as a prospective solubilizer [167–169].

10.5.2.5 Hupu gum Hupu gum or kondagogu gum is a dried gummy exudate obtained from Cochlospermum gossypium (Family: Cochlospermaceae/Bixaceae). It is an anionic polysaccharide belonging to the class of substituted rhamnogalacturonans which consist of rhamnose, glucuronic acid, galacturonic acid, galactopyranose, glucose, galactose, fructose, mannose, and arabinose with sugar linkages [170]. The gum is modified to reduce the viscosity, increase water-holding capacity and improve mucoadhesiveness by the process of carboxymethylation or heating at 140°C for 2 h [171]. The utilization of hupu gum in the field of pharmaceutics include solubility enhancement, colon targeting, as an emulsifying agent, and for controlled and sustained drug release [172–175]. The ability of the gum to swell to a considerable size proves to be favorable for improving the solubility of a poorly water-soluble drug. A study was conducted to improve the dissolution profile of an antidiabetic drug, pioglitazone HCl, using hupu gum and modified hupu gum (MHG) as carriers using solid dispersion methodology. The unmodified gum was found to form lumps with the drug thereby decreasing dissolution whereas the solid dispersion formed using MHG as the carrier resulted in easily dispersed particles. Thus MHG proved its efficacy in enhancing the solubility of pioglitazone HCl over that of unmodified hupu gum [176]. Thereafter, studies were conducted involving preparation of solid dispersion of the drugs using different carriers including hupu gum and comparing their potencies in solubility enhancement [165, 169, 177]. Hupu gum showed promising results but needs further exploration for it to be established as a potential solubilizer.

10.5.2.6 Xanthan gum Xanthan gum is an anionic, high molecular weight polysaccharide produced by the bacterium Xanthomonas campestris. It consists of a backbone made of glucose with mannose and glucose units [122, 172]. The pharmaceutical uses of xanthan gum include sustained and controlled drug release [178], as a suspending agent [179], and for colon-specific drug delivery [180]. Xanthan gum is modified to reduce its viscosity by heating at a temperature of 120°C. Modified xanthan gum (MXG) was found to possess less viscosity, comparable swelling index, and increased water retention capacity which proves to be advantageous for enhancement in dissolution [172]. The enhancement of dissolution profile of valsartan was achieved when it was formulated as solid dispersion using MXG as

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carrier. The solubility studies demonstrated that drug solubility increased with the increase in concentration of polymer [169]. In a study, MXG increased the percent drug release from 40% to 70% owing to the enhanced wettability and dispersibility of gliclazide in the surrounding dissolution medium [165]. Similar results were obtained when simvastatin [181] and pioglitazone HCl [182] was formulated as solid dispersion using MXG. Xanthan gum requires further studies to confirm its applicability in the formulation technology.

10.5.2.7 Tamarind gum Tamarind gum is a biodegradable polysaccharide obtained from the endosperm of the kernels of Tamarindus indica (Family: Leguminosae). The gum constitutes 65% of the total seed components [183]. It is a galactoxyloglucan consisting of a backbone of d-(1-4)-galactopyranosyl unit which is substituted with side chains of xylose and galactose linked to glucose. This polysaccharide is a monomer of glucosyl:xylosyl:galactosyl in the ratio of 3:2:1 [123]. Being under the GRAS category, it has been widely utilized as a tablet binder, disintegrant [184], emulsifier [185], suspending agent [186], gelling agent [183], and for colon targeting [187, 188]. The gum possesses mucoadhesive properties due to which it has been used in the past as a controlled release excipient for drug delivery through nasal mucosa [189]. Owing to its viscosity and swelling behavior it has been employed for increasing the solubility of Aceclofenac, Atorvastatin and Irbesartan [190]. The combination of tamarind gum with xanthan gum has yielded promising results for solubility enhancement of BCS Class II drugs [125]. However, there is scope for more research for exploration of solubility enhancement potential of tamarind gum.

10.5.2.8 Mango gum Mango gum is isolated from the barks of Mangifera indica (Family: Anacardiaceae). It is a dried gummy exudate polysaccharide obtained from the incised trunk which is rich in mangiferin, protocatechuic acid, γ-aminobutyric acid, catechin, indicoside A and B, manghopanal, mangocoumarin, manglupenone, mangsterol, and mangiferolic acid methyl ester. Mango gum has inherent antidiabetic, antiviral, antiparasitic, antidiarrhoeal, antitumour, antispasmodial, antifungal, antibacterial, and immunomodulatory activity [121]. However, the pharmaceutical applications of mango gum include its use as a tablet binder [191], disintegrant [192], and for sustained release [193]. Although mango peel pectin has been utilized as a carrier for enhancing the dissolution rate of a poorly water-soluble drug, Aceclofenac, the exploitation of mango gum for solubility enhancement is still pending [194].

10.5.2.9 Aegel marmelos gum Aegel marmelos gum (AMG) is obtained from the fruits of Aegle marmelos belonging to family Rutaceae. The gum is rich in d-galactose, l-Rhamnose, l-arabinose and d-galacturonic acid. The medicinal uses of AMG include reducing glutathione concentration in the liver, stomach, intestine, and kidney, decreasing lipid peroxidation,

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plasma insulin, and liver glycogen level [140]. In addition to augmenting the solubility of poorly soluble drugs, AMG has been utilized as a tablet binder and as a mucoadhesive agent [140, 195]. The solubility enhancing property of AMG has been studied in two separate research studies, which involved preparation of solid dispersion of aceclofenac and atorvastatin. The solubility of aceclofenac was found to markedly increase from 0.0816 to 15.65 mg/mL with the increase in concentration of AMG [196]. Similarly, solid dispersion of atorvastatin with AMG resulted in a twofold increase in its solubility [197]. Therefore AMG can be utilized as a prospective natural carrier for solubility enhancement.

10.5.3 Sugar and urea Sugars are the first generation carriers which were earlier used in the preparation of SD but now have very limited use. Although they possess advantages such as very high water-solubility, very little to no toxicity, and easy availability, which makes them a suitable carrier, their disadvantages such as high melting point, low solubility in organic solvents, and hygroscopic nature outweigh these advantages thus leading to rare use in SD [89, 198]. Moreover, the SDs which resulted using these carriers were found to be crystalline in nature exhibiting a slow drug release as opposed to the rapid drug release by ASDs. Mannitol, having a high melting point of 165–168°C and decomposition at above 250°C can be used to prepare SD by hot melt method in some cases. A research study involved preparation of SD of celecoxib using mannitol by physical trituration, solvent evaporation, and melt methods. The results reported highest increment in solubility in SD prepared by melt/fusion method at 1:5 drug:mannitol ratio. However, the results were insufficient to indicate whether the prepared SD was amorphous or crystalline [14]. A recent study utilized various sugar carriers like d-mannitol, d-fructose, d-­dextrose and d-maltose for the solubility enhancement of clotrimazole by the SD approach. The results indicated improvement in solubility, dissolution, and antifungal activity of clotrimazole. The solubility was increased 806-fold as compared with the plain drug using saturated solution of mannitol. This study inferred that the sugar alcohols like mannitol are suitable carriers for SD in comparison to the monosaccharide and disaccharide having aldehyde and ketone group [199]. Another study involved the preparation of SD of griseofulvin by roll mixing method using saccharides such as corn starch, maltose, and lactose as carriers. The dissolution was enhanced ~170-fold and griseofulvin in the roll mixture was found to be present in the amorphous state as detected by absence of drug peaks in XRD [200]. Some of the past research involving use of other sugar carriers such as xylitol [201], sorbitol [202], dextrose, sucrose [203], galactose [204], mannitol [205], trihalose [27], and isomalt [206] have been reported by researchers. All these carriers were found to report increase in solubility when employed as carriers in solid dispersions but do not find much application currently due to the availability of more effective synthetic polymers. Urea is the end product of human protein metabolism and is the first generation agent employed in SD. It is reported to exhibit no toxicity. Urea is found to have sufficient solubility in water and to exhibit good solubility in organic solvents. The earlier

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and perhaps the first study incorporating urea as the carrier in SD was that by Sekiguchi and Obi who reported better absorption in rabbits when sulphathiazile was given as a eutectic with urea [24]. Similarly, better dissolution was observed with solid dispersion of chloramphenicol [207] and ursodeoxycholic acid [208] using urea as the carrier. However, since then, the use of urea in SDs has become more or less obsolete due to the introduction of second and third generation carriers which were found more effective. Though there are few research studies done lately to support the use of urea as a carrier in SD, the most recent research was based on preparation of clarithromycin-urea solid dispersion by solvent evaporation, electrospraying, and freeze-drying method. Among all these, the SDs prepared by freeze-drying displayed best results in terms of improvement in solubility and bioavailability as shown in Fig. 10.7 [209]. Urea exhibited improvement in dissolution of cefuroxime axetil [210] and rofecoxib [211] as well when incorporated in their SDs.

10.5.4 Inutec SP1

Plasma Concentration (ng/ml)

Inutec SP1 is a polydisperse polysaccharide which is extracted from the roots of Cichorium intybus (Family: Asteraceae) consisting mainly of fructosyl fructose units and not necessarily one glucopyranose unit. It is obtained by reacting isocyanates and the polyfructose backbone using tertiary amine or a Lewis acid as the catalyst which introduces alkyl groups on its backbone [212]. The polymer depicts excellent surfactant properties (with both hydrophilic and lipophilic parts on the chain) which present an interesting approach for it to be utilized as a carrier in SDs [213]. Mooter and his coworkers introduced the new use of Inutec SP1 as a suitable carrier and evaluated its potential by incorporating it in SD of itroconazole, an antifungal

100

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Clarithromycin

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Freeze drying (Drug:urea=1:3) Drug powder

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2

4

6

8

10

12

Time (h)

Fig. 10.7  (A) Dissolution profiles of clarithromycin and its solid dispersions prepared by freeze drying method and (B) in vivo blood concentration of clarithromycin powder and solid dispersions prepared by freeze drying method. Reproduced with permission from Mohammadi G, Hemati V, Nikbakht M, Mirzaee S, Fattahi A, Ghanbari K, Adibkia K. In vitro and in vivo evaluation of clarithromycin–urea solid dispersions prepared by solvent evaporation, electrospraying and freeze drying methods. Powder Technol 2014;257:173.

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Fig. 10.8  Dissolution profile of itroconazole and its solid dispersions using (A) Inutec SP1, (B) Inutec SP1, and PVPVA 64. Reproduced with permission from (A) Van den Mooter G, Weuts I, De Ridder T, Blaton N. Evaluation of Inutec SP1 as a new carrier in the formulation of solid dispersion for poorly soluble drugs. Int J Pharm 2006;316(1–2):3. (B) Janssens S, Humbeeck JV, Van den Mooter G. Evaluation of the formulation of solid dispersions by co-spray drying itraconazole with Inutec SP1, a polymeric surfactant, in combination with PVPVA 64. Eur J Pharm Biopharm 2008;70(2):502.

BCS Class II drug. However, the SDs prepared were found to be crystalline in nature as revealed by DSC and XRD studies and despite this the dissolution was improved as compared to the pure drug [3]. However, in another research by Janssens and associates, the XRD studies of the solid dispersion of itroconazole reported amorphous content of the drug. In addition to inutec SP1, they incorporated PVP-VA 64 which kept the drug molecularly dispersed and led to an immense improvement in the degree of dissolution in comparison to earlier research as shown in Fig. 10.8 [214]. Later, a research study which involved evaluation and comparison of the solubilizing potential of inutec SP1 with PVP was done for the poorly soluble drugs such as diazepam, fenofibrate, ritonavir, and efavirenz. The results depicted the outstanding performance of inutec SP1 in increasing the solubility of the respective drugs. The prepared SDs were also found to possess good physical stability when tested for three months [215]. Moreover, besides being used as a carrier in SDs, inutec SP1 is employed successfully as a surfactant, emulsifying, stabilizing, and suspending agent because of its excellent surfactant properties, nontoxicity, and biodegradability [213].

10.5.5 Pectin Pectins are heterogeneous polysaccharides with a linear homo-galacturonic backbone alternating with two types of highly branched rhamno-galacturonans regions (RG-I & RG-II) [216, 217]. It is the methylated ester of polygalacturonic acid which makes up about 33% of the cell wall dry substance of higher plants. They find their main application in the food industry as gelling or thickening agents, particularly pectins

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derived from citrus peel and apple pomace under mildly acidic conditions. In the pharmaceutical industry, it is mainly employed as an excipient due to its nontoxic and nonirritating nature, abundant availability, and low production costs. Pectins are also used for colonic targeting drug delivery, controlled drug delivery, and as an immunostimulant [218, 219]. However, despite the several advantages offered by pectins, only one study reports the use of pectin in SDs. The pectin used in the study was extracted from mango peel and was used in the preparation of SDs of Aceclofenac. The findings revealed that with the increase in pectin content, the dissolution of the drug was significantly improved [194]. Therefore more research needs to be conducted on pectins pertaining to their use in SDs.

10.5.6 Corn starch Starch is synthesized by the plants in granular form and consists of linear amylose, branched amylopectin, and minor constituents including proteins, lipids, and minerals [220]. It is a nontoxic, biodegradable and renewable material and therefore has tremendous potential in the industry. On the other hand, the inherent properties of starch limit their direct industrial applicability, for which several physical, chemical, and enzymatic modifications are needed [221]. In addition, the use of corn starch as a carrier in SD is also very limited. In one study the solubility enhancement of Griseofulvin [200] was observed and another study used a mixture of corn starch and lactose to achieve improved dissolution for aceclofenac [222]. Recently, corn starch with varying ratios of amylose to amylopectin was used to study the thermodynamic interactions with the model drugs (acetaminophen and phenazone). Holt-melt extrusion method was used to prepare the SDs but the findings revealed that the drug in SD showed crystalline behavior [223].

10.5.7 Chitosan Chitin, a nitrogenous polysaccharide, is isolated from the exoskeleton of marine organisms (largely crabs and shrimps) which are crushed, washed, and treated with sodium hydroxide to obtain crude chitin [224]. Chitin has been widely used for lowering serum cholesterol and in hypertension and ophthalmic formulations. However, the practical use of chitin has declined owing to its semicrystalline structure with extensive hydrogen bonds and it being insoluble in most of the solvents [218]. Therefore its derivative chitosan was developed, which is a deacetylated product obtained from chitin and is a copolymer of β-(1→4)-linked 2-acetamido-2-deoxy-d-glucopyranose and 2-amino-2-deoxy-dglucopyranose [224]. The different grades of chitosan contain varying amounts of these two monosaccharides with molecular weight ranging from 50 to 2000 kDa, each differing in their viscosity and pKa values [225]. Chitosan is reported to be insoluble in water and organic solvents. However, it exhibits pH-dependent solubility, being soluble at pH 6.0 and insoluble above pH 7.0, therefore it cannot be used for biological applications which require a neutral environment. Hence, modifications of chitin/chitosan in the form of esterification, etherification, O-acetylation, cross-linking, and graft copolymerization can be done to introduce desired properties in the polymer [226].

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Chitosan is the second most abundant natural polysaccharide after cellulose and has attracted researchers’ interest owing to its nontoxic, biocompatible, biodegradable, bioadhesion, bioabsorbable, and renewable nature supplemented with its wide availability, flexibility in usage, and low cost. It has consequently emerged as one of the most promising polymers achieving the status of most desired polymer for use in therapeutic interventions [227, 228]. As reported firstly by Allan & Hardwiger in 1979, chitosan is believed to have an excellent antimicrobial activity. They demonstrated that chitosan possesses a wide spectrum of activity and a high killing rate against both gram-positive and gram-negative bacteria [229, 230]. A recent study investigated the antimicrobial and antioxidant properties of abietic acid-chitosan SDs. It suggested that abietic acid and chitosan displayed a synergistic effect at 1:1 ratio. In addition, increased antimicrobial and antioxidant activity was observed with the drug in its amorphous state [231]. Some authors have also reported the use of chitosan in enhancing solubility and consequently bioavailability of poorly soluble drugs. The solubilizing potential of chitosan was investigated by preparing ASD of telmisartan using chitosan as the carrier by different methods. The results advocated use of cogrinding method to achieve markedly improved dissolution, reduced particle size, and drug amorphization which increased with the increase in concentration of chitosan [232]. Mura et al. prepared binary solid dispersions of naproxen with chitosan and studied the effect of varying chitosan molecular weight, drug/chitosan (w/w) ratio, and preparation method on the dissolution rate of naproxen. The results revealed that the dispersions prepared by cogrinding method using low molecular weight chitosan (CS-Lw) and with drug:chitosan of 1:9 increased the dissolution rate 10-fold. The relative increase in dissolution efficiency for solid dispersions prepared by different methods using chitosan at low (CS-Lw) and medium (CS-Mw) molecular weight is depicted in Fig. 10.9 [233]. Earlier studies validated the potential of chitosan in enhancing the solubility and bioavailability of poorly soluble drugs [234–236].

10.5.8 Carrageenan Carrageenan is a natural heteropolysaccharide which is extracted from a species of red seaweed (Class: Rhodophyceae). The main sources are Eucheuma spinosum, Eucheuma cottonii, and Chondrus crispus. Chemically, carrageenan is a high molecular weight linear polysaccharide comprising of repeating galactose units. It is soluble in water and insoluble in most of the organic solvents. Carrageenan at very low concentrations in water forms a weak gel and is unstable in highly acidic conditions. The pharmaceutical uses of carrageenan comprise of suspending, stabilizing, thickening, and gelling agents. It is most commonly used for oral and topical drug delivery, dentrifices, wound cleaning, cosmetics, suppository bases, and in controlling humidity in industrial plants [237]. Carrageenan has been shown to possess solubilizing potential, however, only one study has been reported which depicted solubility enhancement of efavirenz by formulating its SD using carrageenan as a carrier [238, 239]. Therefore the polymer needs further exploration in this area to establish its solubilizing potential.

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rel. incr. DE 6 5 4 3 2 1 0

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COE

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P.M. = Physical mixture COE = Co evaporation KN = Kneading GR = Cogrounded

Fig. 10.9  Relative increase in dissolution efficiency for 3:7 (w/w) products of naproxen with chitosan. Reproduced with permission from Mura P, Zerrouk N, Mennini N, Maestrelli F, Chemtob C. Development and characterization of naproxen–chitosan solid systems with improved drug dissolution properties. Eur J Pharm Sci 2003;19(1):73.

10.5.9 Alginate Sodium alginate (SA), the most available form of alginate, is a hydrophilic polysaccharide which is mainly isolated from the cell walls of brown seaweeds. Chemically it consists of guluronic acid and mannuronic acid arranged in an irregular pattern with reactive sites like hydroxyl and carbonyl groups present along the backbone [218, 240]. Alginate is nontoxic, biocompatible, and biodegradable and has been categorized as GRAS by FDA since 1982 [241]. Alginate has been invariably used in forming hydrogels, microparticles, and in drug delivery [218]. It has been employed in solid dispersion as an ester derivative for enhancing the solubility of poorly soluble drugs [242]. In another recent study, SA showed enhanced solubility and dissolution rate of telmisartan when employed as a carrier in its SD prepared by ball milling method as displayed in Fig. 10.10. The polymer has also been reported to inhibit the recrystallization of the drug [241]. The most recent research explored the potential of SA as a diphase SD carrier in enhancing solubility and solubility of two model drugs: indomethacin and lovastatin. SA was successful in providing stability and improving dissolution in comparison to HPMCAS-based systems [243]. As not much research has been done using SA as a carrier in SD due to limited molecular interaction between alginate and drugs, the area needs further exploration.

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Dissolution efficiency (%)

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M

ic ar

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Fig. 10.10  Dissolution profiles of telmisartan and its solid dispersions. Reproduced with permission from Borba PAA, Pinotti M, de Campos CEM, Pezzini BR, Stulzer HK. Sodium alginate as a potential carrier in solid dispersion formulations to enhance dissolution rate and apparent water solubility of BCS II drugs. Carbohydr Polym 2016;137.

10.6 Marketed amorphous solid dispersions Solid dispersion has managed to attract the attention of researchers in the area of solubility enhancement but still the number of products reaching the stage of commercialization is relatively very few. However, most of the SDs available on the market contain polysaccharides as the carriers as shown in Table 10.4. The reason for the lesser availability of marketed formulations employing an SD approach can be primarily attributed to the scale-up problems, physicochemical instability in the manufacturing process, or during storage leading to crystallization and phase separation [5, 17, 244].

10.7 Concluding remarks The pharmaceutical development pipeline is comprised of a large number of drugs which are poorly water-soluble and present significant challenges to formulation scientists. Over the past few decades, ASD technology has emerged as a powerful solubility enhancing technology which stabilizes the drug in the amorphous form both in the dosage form as well as during the supersaturation state. This technique has been considered as a major advancement in the area where poor water-solubility is a concern. ASDs are accompanied by various benefits such as drug stabilization, enhanced drug release, and ease of administration by the patient compliant oral route. However, the physical instability and the processing difficulties observed with ASDs are the

Marketed product

Drug

Carrier

Manufacturer (year of approval)

Indication

Orkambi Noxafil

Lumacaftor/Ivacaftor Posaconazole

HPMCAS/SLS HPMCAS

Vertex Pharmaceuticals Inc. (2015) Merch Sharp & Dohme Ltd. (2014)

Kalydeco Zelboraf Incivek

Ivacaftor Vemurafenib Telaprevir

HPMCAS HPMCAS HPMCAS-M

Abbott Laboratories (2012) Roche (2011) Vertex Pharmaceuticals (2011)

Certican Onmel Zotress

Everolimus Itraconazole Everolimus

HPMC HPMC HPMC

Novartis (2010) Merz Pharma. Inc. (2010) Novartis (2010)

Intelence Crestor Afeditab CR Rezulin

Etravirine Rosuvastatin Nifedipin Troglitazone

HPMC HPMC HPMC/PEG HPMC

Tibotec (2008) Astra Zeneca (2003) Watson lab. (2002) Pfizer (1997)

Prograf Sporanox Nivadil Nimotop Isoptin SRE

Tacrolims Itraconazole Nivaldipine Nimodipine Verapamil

HPMC HPMC/PEG HPMC – HPC/HPMC

Astellas (1994) Janssen-Cilag (1992) Astellas (1989) Bayer (1988) Abbott (1987)

Cystic fibrosis Aspergillosis coccidioidomycosis Candidiasis mycoses Cystic fibrosis Certain types of melanoma Chronic hepatitis C genotype 1 infection Antineoplastic agent Onychomycosis Prophylaxis of organ rejection in adult patients HIV Hypolipidemic agent Hypertension Antihyperglycemic agent (withdrawn from US market) Immunosuppressant Onychomycosis Hypertension Hypertension Mild to moderate hypertension and coronary heart disease

ASDs for improving solubility and bioavailability of drugs

Table 10.4  Polysaccharide-based amorphous solid dispersions available in market

303

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Polysaccharide Carriers for Drug Delivery

r­easons behind its limited product progression and exposure into the commercial market. Owing to present needs, significant upfront development is a prerequisite to produce stable ASDs. The noteworthy improvements in this field by researchers have finally paved the way for the use of amorphous products for solubility enhancement which were once avoided owing to their high-energy amorphous state. Consequently, ASDs enjoy superiority over other solubility enhancing formulation approaches because of their added advantages. ASDs have exhibited remarkable results using both synthetic and natural polymers. However, synthetic polymers do have a number of limitations, therefore natural polymers such as polysaccharides prove beneficial in ASD preparation. Some of these polymers are modified for better chemical stability and for minimizing processing issues to exploit their potential to the fullest. It is anticipated that these carriers with easy-to-tune structures will result in remarkably improved oral bioavailability. This chapter attempted to discuss the various polysaccharide carriers which are employed in the preparation of amorphous solid dispersions.

10.8 Future perspectives The enhancement of solubility and bioavailability of lipophilic drugs by formulating them into ASDs is challenging and a mostly unsettled frontier. Future development in this field demands use of novel polymers or their combinations coupled with thorough understanding at molecular level which can drive the use of this technology towards increase in solubility and bioavailability and most importantly for further modulation of pharmacokinetic profiles. The current need demands an exhaustive study of the interactions occurring at molecular level for the rational design of products based on amorphous solid dispersion. The physicochemical properties governed by the molecular and thermodynamic factors need to be properly addressed. The high energy amorphous state of the incorporated drug limits their commercial applicability by reverting them to crystalline forms. Therefore, better knowledge about the thermodynamics, glass transition temperature, crystallization, molecular mobility, and drug-polymer interactions is imperative for the rational design of amorphous solid dispersion products. Moreover, the solid-state stability and maintenance of a supersaturation state should also be critically focused upon in order to produce efficient ASDs with desirable properties. Irrespective of the underlying challenges, the area warrants collaboration between formulators, chemists, and biopharmaceutics scientists for fruitful implementation of the strategy in the pharmaceutical discovery and development phases.

Acknowledgment The authors acknowledge Jamia Hamdard, New Delhi, India for providing the “Jamia HamdardSilver Jubilee Research Fellowship-2017” (AS/Fellow/JH-5/2018).

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Sougata Jana*,†, Sabyasachi Maiti‡, Kalyan Kumar Sen*, Subrata Jana§ ⁎ Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol, India, † Department of Health and Family Welfare, Directorate of Health Services, Kolkata, India, ‡ Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India, § Department of Chemistry, Indira Gandhi National Tribal University, Amarkantak, India

11.1 Introduction Polysaccharides represent an important category of polymers that is widely being explored as building blocks of interpenetrating network (IPN) structures in due consideration of their biodegradable, biocompatible, and nontoxic properties as well as low cost of production, abundance, and the ability to form metallic or polyelectrolyte complexes. Additionally, the reactive functional groups present along in the backbone of polysaccharides can be used for chemical modification for the development of novel materials for drug delivery [1–5]. Recent studies show that the single network of natural polysaccharides offered weak mechanical strength and response to swelling by the external stimulus (pH and temperature). To enhance the mechanical properties and swelling characteristics, multicomponent network systems as IPNs have been fabricated [6]. IPNs are crosslinked polymer networks, where at least one of the polymers is synthesized and/or cross-linked in the immediate presence of the other, which cannot be separated unless the chemical bonds are broken [7, 8]. A combination of the polymers can effectively produce a multicomponent polymeric system, with newer physicochemical characteristics [9]. Natural polysaccharide IPNs are innovative biomaterials in that they can control the delivery of the entrapped drug for a prolonged period of time [10]. In particular, alginate, chitosan, carragenan, gellan gum, locust bean gum, cellulose, and xanthan gum hyaluronic acid polysaccharides have received increasing attention for the development of IPN or (semi)-IPN for controlled drug delivery and tissue engineering applications [11–18]. This chapter gives an overview of the IPN/semi-IPN based on natural polysaccharides, especially alginate, chitosan, carrageenan, xanthangum, and locust bean gum towards their applications in oral drug delivery.

Polysaccharide Carriers for Drug Delivery. https://doi.org/10.1016/B978-0-08-102553-6.00011-8 Copyright © 2019 Elsevier Ltd. All rights reserved.

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11.2 Polysaccharide-based IPN carriers 11.2.1 Alginate-based IPNs Alginate (Alg), a linear polysaccharide obtained from sea algae, is composed of 1-4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G), arranged block-wise fashion as homopolymer blocks (MM, GG) or alternating blocks of M and G with different M/G ratios [19, 20]. It can be easily cross-linked by divalent ions, which mainly bind to the guluronic residues for the synthesis of hydrogel systems. Due to this ability, it has been widely used as one of the biomaterials in fabricating IPN systems in combination with other natural/synthetic polymers for drug delivery [21]. The thermo-sensitive hydrogels, which respond to temperature and swell accordingly, have also been tested as drug delivery carriers. Consequently, the drug release rate from the encapsulated hydrogel can be controlled by changing the temperature of the local environment [22, 23]. Zhao et al. [24] synthesized thermo-sensitive semiinterpenetrating polymer network (semi-IPN) hydrogels via in situ copolymerization of Nisopropylacrylamide (NIPAAm) with poly(ethylene glycol)-co-poly(ɛ-caprolactone) (PEG-co-PCL) macromer in the presence of hydrophilic polysaccharide sodium alginate (Na-Alg) by UV irradiation method. The swelling behavior of the hydrogels was dependent on the Alg content, salt, and temperature. The swelling studies in distilled water showed that an increase in Alg content in IPN increased the swelling propensity perhaps due to more ingress of water molecules within the hydrophilic polymeric networks. The hydrogels swelled less as the temperature was increased as a consequence of greater physical interlocking with copolymerized networks of the macromer with NIPAAm and Na-Alg chains. Therefore, the phase transition was mainly regulated by the thermo-sensitive poly (N-isopropylacrylamide) (PNIPAAm) component. The incorporation of Na-Alg into the network increased the mechanical strength of the hydrogel. Bovine serum albumin (BSA) as a model protein was incorporated into the hydrogel. An in vitro burst release of BSA in 1 h, followed by a sustained release (1–11 h), and a plateau region (11–95 h) was evident. In another study, Prajapati et al. [25] prepared oxcarbazepine-loaded Alg-egg albumin IPN beads by ionotropic gelation method (Fig.  11.1). Oxcarbazepine is a novel anticonvulsant, antiepileptic and mood stabilizing drug, used primarily in the treatment of epilepsy. Alg-egg albumin IPNs were optimized by 32 factorial design, the average size, drug entrapment efficiency, and cumulative drug release at 8 h (Q8h, %) was found to be in the range of 976–1084 μm, 65%–91%, and 73%–94%, respectively. The in vitro drug release from IPN beads showed sustained release profiles with anomalous non-Fickian diffusion mechanism in pH 1.2 and pH 7.4. Boppana et  al. [26] fabricated pH-sensitive polyacrylamide-grafted-gum ghatti (PAAm-g-GG) and Alg IPN microbeads for gastro-protective release of ketoprofen. The IPN microbeads were developed by dual cross-linker Ca2+ions and glutaraldehyde (GA). The drug release from IPN microbeads was relatively fast in pH 7.4 as compared to drug release in medium of pH 1.2. The antiinflammatory activity, in vivo pharmacokinetic data, and stomach histopathology observations in wistar rats were promising for possible application in drug delivery. Currently, Jana et al. [27]

Interpenetrating polysaccharide networks as oral drug delivery modalities321 COONa O OH

O

OH

OH

OH O

O COONa

Sodium alginate

NH2 Egg Albumin

Ca+2

Sodium alginate-Egg albumin IPN matrix Calcium chloride crosslinked sodium alginate chain Calcium chloride crosslinked egg albumin chain

Fig. 11.1  Schematic representation of sodium alginate-egg albumin containing oxcarbazepine IPN matrix. Reprint with permission from Prajapati VD, Gandhi AK, Patel KK, Patel BN, Chaudhari AM, Jani GK. Development and optimization of modified release IPN macromolecules of oxcarbazepine using natural polymers. Int J Biol Macromol 2015;73:160–9. Copyright (2015), Elsevier.

d­ eveloped and characterized an O-carboxymethyl Tamarind gum (CTG)-Alg IPN hydrogel (Fig. 11.2) system for an antiviral drug, acyclovir. The maximum drug entrapment efficiency of IPN hydrogel was ∼70%. In vitro drug release was slow in acidic environment (pH 1.2). The non-Fickian drug release behavior was observed after fitting the drug release data into the Korsmeyer-Peppas kinetic model. The drug release kinetic data revealed release of drug control by diffusion and swelling kinetics. Upadhyay et al. [28] developed locust bean gum-Alg IPN microbeads containing capecitabine by QbD (Quality by Design) approach. The % drug entrapment of about 74% was obtained for the optimized microspheres, which controlled the drug release

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Fig. 11.2  Field emission scanning electron microscopy (FE-SEM) image of IPN hydrogel. (A) Single hydrogel IPN microsphere, (B) Surface morphology of hydrogel IPN microsphere. Reprint with permission from Jana S, Sharma R, Maiti S, Sen KK. Interpenetrating hydrogels of O-carboxymethyl Tamarind gum and alginate for monitoring delivery of acyclovir. Int J Biol Macromol 2016;92:1034–9. Copyright (2016), Elsevier.

over 12 h in pH 6.8 buffer solution. The oral administration of the optimized formulation in Albino Wistar rats exhibited better pharmacokinetic parameters than free drug. Further, a significant reduction in the growth of HT-29 cells was noticed, indicating their potential for treating colon cancer. Alginate-PVA IPN hydrogels were synthesized by free radical polymerization using 2-Acylamido-2-methylpropane-sulfonic acid as monomer [29]. The hydrogels demonstrated pH independent swelling and drug release behaviors. Recently, Ganguly et al. [30] reported nanoclay embedded poly(methacrylic acid) grafted alginate semi-IPNs with enhanced mechanical strength and biocompatibility. These material features supported their application in the field of tissue engineering. Matricardi et al. [31] stated that the tablets of semi-IPNs based on Alg-scleroglucan (Sclg)-borax could exhibit a strong pH dependent release of the protein myoglobin. Considering their pH-dependent mechanical properties, they suggested that such a new matrix could protect protein molecules from an adverse gastric environment, and allow its subsequent sustained release in the intestine. Later on, the authors [32] presented the effect of Alg on semi-IPN systems in terms of drug release and mechanical properties. Alg strongly influenced the intestinal release of myoglobin from the tablets which completed drug release after 24 h; whereas only 60% of loaded myoglobin was released from Scgl/borax tablets. However, the effect of Alg was relatively weaker in the case of drug release from theophylline tablets. The presence of Alg improved the gel strength by an order of magnitude and imparted a thermo irreversible character. The homogenous IPNs were fabricated by the water-in-oil emulsification method using GA as Alg-cross-linker [33]. The release of 5-flurouracil (5-FU) from Alg-Nisopropylacrylamide (NIPAAm) semi-IPN microspheres at 25°C and 37°C confirmed their thermosensitive nature and continued drug release up to 12 h in vitro. IPN hydrogel tablets of tamarind seed polysaccharide and Alg were investigated for controlled release of an antihypertensive drug, propranolol HCl [34]. The IPN tablets loaded with drug or drug-resin complex (resinate) exhibited a distinct drug release pattern.

Interpenetrating polysaccharide networks as oral drug delivery modalities323

The resinate-loading caused prolonged drug release up to 24 h from the IPN matrices. Higher cross-linking led to a non-Fickian mechanism in the event of a subsequent slow drug release process. The effect of size and hydrophobicity of alginate microspheres on cellular uptake and their toxicity to phagocytic cells was examined by Kidane et al. [35]. Cellular uptake studies revealed that alginate-methylcellulose-Pluronic L61 microspheres coated with poly-l-lysine (3.7 μm) were most efficiently phagocytosed by mouse macrophages. However, the difference in cellular uptake among the microspheres without coating was not statistically significant. Toxicity to macrophages was dependent on the ratio of microspheres to cells. Because the M-cells in the peyers patches are prevalent in the upper intestine, this system could be useful for oral delivery of delivery of vaccine or antigen. IPN hydrogels composed of Alg and hydrophobically modified ethyl hydroxyl ethyl cellulose were developed by Choudhary et al. [36]. The mechanical property of this type of IPN system was determined by the ratio of two polymers and high molecular weight of cellulose fraction. The use of hydrophobic cellulose materials in the hydrogel matrix caused an improvement in the entrapment of sulindac. Alginate seemed to dominate the gelation kinetics of the IPNs but the effect of the cellulosic component was also evident at its low concentration. Raz et al. [37] reported Ca + 2 cross-linked Alg and N,N′-methylenebis (acrylamide) cross-linked poly (N-isopropylacrylamide) IPN microgels using microfluidic synthesis for cell flow study. Increase in alginate content made the microgels stiffer, but the relaxation was faster. The concentration of PNIPAm in the microgels and its degree of cross-linking had a negligible impact on the mechanical properties of microgels. Shi et al. [38] fabricated pH- and temperatureresponsive semi-IPN hydrogel beads, composed of calcium alginate (Ca-alginate) and PNIPAAM for a nonsteroidal antiinflammatory drug, indomethacin. At pH 2.1, only about 10% drug was released in 400 min, while this value approached 95% at pH 7.4 in 3 h. The drug release rate was faster at 37°C than at 25°C and increased slightly with increasing PNIPAAM content. Almeida and Almeida [39] developed GA-crosslinked Alg-gelatine IPN beads containing pindolol, a poorly soluble drug used for the treatment of angina pectoris, hypertension, and other cardiovascular diseases. The swelling of cross-linked beads did not lead to complete erosion as observed only with the alginate matrix. Ultimately, the cross-linking improved pindolol retention within the matrix and allowed controlled release of pindolol. Shikanov et al. [40] investigated fibrin-alginate IPN for testing in vitro growth of ovarian follicles. The rate of meiotically competent oocytes produced by culture in the IPN was 82%, significantly greater than in alginate alone.

11.2.2 Chitosan-based IPNs Chitosan (CS) is a linear, cationic polysaccharide, composed of β-(1 →  4)-2-­amino2-deoxy-d-glucopyranose and β-(1 → 4)-2-acetamido-2-deoxy-d-glucopyranose units, randomly distributed along the polymer chain. It attracted the attention of a large pool of pharmaceutical scientists due to its outstanding biological characteristics such as biocompatibility, biodegradability, nontoxic, and antibacterial activity. It contains amino and hydroxyl functional groups, which can easily be modified for the

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intended purpose, in particular as controlled drug release carriers [41–43]. CS has received GRAS (Generally Recognized as Safe) status by FDA and thus has found wide application in many biomedical applications including drug delivery, food technology, and tissue engineering [44]. Methoxy poly (ethylene glycol) grafted carboxymethyl chitosan and Alg were employed for the encapsulation of Bovine serum albumin as a model protein drug [45]. The formation of IPN hydrogel improved protein loading capacity as compared to the formation of hydrogel after physical blending of these constituent polymers. The IPN suppressed burst release of the protein at pH 1.2, while releasing the protein at pH 7.4, suggesting their potential for site-specific protein drug delivery in the intestine. Guo and Gao [46] synthesized pH and thermo-responsive carboxymethyl CSPNIPAm semi-IPN polyampholyte hydrogels using N,N′-methylenebisacrylamide as a cross-linker for oral delivery of coenzyme A. PNIPAm is a widely used temperature-­ sensitive polymer because it has a LCST in the range of 30–32°C, close to human body temperature (37°C). Dual responsive system is more important because the pH- and temperature can be easily controlled for both in vitro and in vivo conditions. The accumulative release of the enzyme was ~22% in acidic medium (pH 2.1) and ~89% in alkaline medium (pH 7.4) at 37°C up to 24 h. The extent of enzyme release was higher at 37°C than 25°C in an alkaline medium. In another study [47], they used poly(dimethylaminoethyl methacrylate) instead of PNIPAm for the fabrication of semi-IPN polyampholyte hydrogels. Semi-IPN hydrogel swelled outside its isoelectric pH and exhibited low degree of swelling between 30°C and 50°C at pH 6.8 buffer solution. The release rate of coenzyme A was relatively higher at 50°C than that observed at 37°C. Further, the content of carboxymethyl chitosan was found to control the release rate of the enzyme. Another report on stimuli-sensitive biopolymeric systems for controlled drug delivery carriers was disclosed by Bashir et al. [48]. The pH responsive N-succinyl CS-g-poly (methacrylic acid) hydrogels were tested for the oral delivery of theophylline. The swelling and drug release characteristics of the copolymer hydrogel was affected by factors such as the amount of monomers, cross-linking agent (N,N′methylenebisacrylamide), initiator (ammonium persulfate), ionic strength of salts, and pH. The drug entrapment efficiency of the formulation varied widely and lay in the range 58%–94%. A maximum of ~90% theophylline released in simulated intestinal fluid. Angadi et  al. [49] reported covalently cross-linked CS and hydroxyethyl cellulose (HEC) blend IPN microspheres of 66–82 μm size for controlled delivery of an antitubercular drug isoniazid. HEC is a water-soluble, nonionic, and nontoxic carbohydrate used in the pharmaceutical field as a polymer for pH-independent drug release. IPNs had drug encapsulation efficiency of 50%–66%. The drug release was influenced by the degree of cross-linking as well as blend composition of the IPN matrix. The ionotropic pregelation of insulin-entrapped alginate (ALG) core, followed by polyelectrolyte complexation with CS, produced nanoparticles of 100–200 nm size [50]. The nanoparticles showed ∼85% of insulin encapsulation and pH sensitivity in the course of in vitro drug release. The nanoparticles retained almost the entire amount of encapsulated insulin in acidic media followed by its sustained release in simulated intestinal fluid. The in  vivo hypoglycemic effects with enhanced relative bioavailability (∼8.11%) and lack of systemic toxicity proved the efficacy of these core-shell

Interpenetrating polysaccharide networks as oral drug delivery modalities325

nanoparticles as an oral insulin carrier. Bulut [51] used water-in-oil emulsification method for the preparation of IPN blend microspheres of CS with poly(vinyl alcohol). Ibuprofen is a poorly soluble, antiinflammatory drug used to treat osteoarthritis, rheumatoid arthritis, and moderate pain [52–54]. To avoid the ulcerogenic effect in the gastrointestinal tract and other side effects, ibuprofen was incorporated into polymeric microspheres to control its release in gastric fluid. The release of an antiinflammatory drug, ibuprofen, in simulated gastrointestinal pH from the microspheres decreased with increasing drug/polymer ratio and extent of GA cross-linking and increased with increase in CS/PVA ratio. About 62% release was accounted at the end of 6 h. Further, they altered the polymer system for the design of IPNs and delivery of ibuprofen [55]. They used CS-graft-polyacrylamide and methyl cellulose for the preparation of IPNs by the same water-in-oil (w/o) emulsion-GA cross-linking method. The release of ibuprofen increased with the increase of the drug/polymer ratio in the microspheres. Cationic polysaccharide-based hydrogels with favorable pH-sensitive swelling characteristics have been used for stomach-specific targeted drug delivery systems. Such systems provide adequate drug release in gastric pH (acidic) condition. Vaghani and Patel [56] studied pH-sensitive CS-polyvinyl pyrrolidone (PVP) semi-IPN hydrogel for controlled oral delivery of clarithromycin. Clarithromycin belongs to the class of macrolide antibiotics, widely used in the treatment of peptic ulcers caused by Helicobacter pylori and upper respiratory tract infections, with an adult oral dosage of 500 mg twice daily [57, 58]. GA cross-linked CS-PVP semi-IPN demonstrated considerable swelling and mucoadhesion under acidic conditions. In acidic conditions, ionized chitosan interacted with the negatively charged mucus and contributed to the mucoadhesive property. Semi-IPN of chitosan (2% w/v) and PVP (4% w/v) in the ratio of 21:4 showed complete drug release after 12 h in lower pH and the drug release kinetic followed non-Fickian diffusion mechanism. The analysis of surface morphology of semi-IPN after dissolution in simulated gastric fluid (pH 1.2) revealed the existence of pores in the membrane. Novel semiinterpenetrating polymer network (IPN) hydrogel microspheres of chitosan (CS) and hydroxypropyl cellulose (HPC) were prepared by the emulsioncross-linking method using glutaraldehyde (GA) as a cross-linker. Chlorothiazide (CT), a diuretic and antihypertensive drug with limited water solubility, was successfully encapsulated into IPN microspheres. Various formulations were prepared by varying the ratio of CS and HPC, the percentage drug loading and amount of GA. Microspheres were characterized by Fourier transform infrared (FTIR) spectroscopy to investigate the formation of IPN structure and to confirm the absence of chemical interactions between drug, polymer and cross-linking agent. Scanning electron microscopy (SEM) was performed to study the surface morphology of the microspheres. SEM showed that microspheres have smooth surfaces. Particle size, as measured by the laser light scattering technique, gave an average size ranging from 199 to 359 μm. Differential scanning calorimetry (DSC) was performed to discover the formation of IPN structure. X-ray diffraction (X-RD) studies were performed to understand the crystalline nature of the drug after encapsulation into IPN microspheres. Encapsulation of drug up to 76% was achieved as measured by UV spectroscopy. Both equilibrium and dynamic swelling experiments were performed in 0.1 N HCl. Diffusion c­ oefficients (D)

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for water transport through the microspheres were estimated using an empirical equation. In vitro release studies indicated the dependence of release rate on the extent of cross-linking, drug loading, and the amount of HPC used to produce the microspheres; slow release was extended up to 12 h. The release data were also fitted to an empirical equation to compute the diffusional exponent (n), which indicated that the release followed the non-Fickian trend. Chlorothiazide (CT) is a diuretic and antihypertensive drug. It has less water solubility, so it is used for development of IPN microspheres for prolong drug delivery. Rokhade et al. [59] investigated semi-IPNs hydrogels MPs of CS-hydroxypropyl cellulose (HPC) containing CT, for controlled drug delivery. MPs were fabricated by the GA cross-linked emulsification method. Particle size range of MPs was 199–359 μm which was measured by laser light scattering technique. In vitro drug release experiments were evaluated in 0.1 N HCl and pH 7.4 medium and the results suggested they were sustained up to 12 h. In vitro release kinetic data showed the drug release from the matrix followed the non-Fickian type. Genipin, a naturally occurring cross-­ linking agent, is significantly less cytotoxic than glutaraldehyde. Chen et al. [60] used genipin for the cross-linking of water-soluble chitosan derivative (N,O-carboxymethyl chitosan, NOCC) with alginate. The semi-IPN of the genipin-cross-linked NOCC/ alginate hydrogel restricted the release of bovine serum albumin at pH 1.2 to 20%, while that at pH 7.4 increased significantly to 80%. In vitro release characteristics of NOCC/alginate hydrogel are depicted in Fig. 11.3.

100

pH 7.4 pH 1.2

90

(n = 5)

Cumulative release (%)

80 70 60 50 40 30 20 10 0 0

50

100

150

200

250

300

350

Time (min)

Fig. 11.3  BSA release profiles from a genipin cross-linked NOCC/Alg IPN hydrogel at pH 1.2 and 7.4. Reprint with permission from Chen S, Wu Y, Mi F, Lin Y, Yu L, Sung H. A novel pH-sensitive hydrogel composed of N, O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. J Control Release 2004;96:285–300. Copyright (2004), Elsevier.

Interpenetrating polysaccharide networks as oral drug delivery modalities327

The formation of hydrogen bonds between NOCC and alginate restricted swelling of these hydrogels at low pH values; while a large swelling force prevailed as a consequence of electrostatic repulsion of ionized caroboxyl groups at high pH and, thus, correlating the pH-dependent event of drug release. The results supported that the genipin-cross-linked NOCC/alginate hydrogel could be a potent system for intestinal protein drug delivery. In the fabrication of IPN, Jana et al. [61] used tamarind seed polysaccharide (TSP) as one of the natural polymers in addition to chitosan for the development of IPNs for aceclofenac. The microparticles showed drug entrapment efficiency of 86% to 92%. IPN microparticles showed sustained release of aceclofenac over 8 h and obeyed the anomalous (non-Fickian) diffusion mechanism for drug release. These data in conjunction with amicable antiinflammatory activity in carrageenan-induced rats over a prolonged period after oral administration highlighted their potential application in drug delivery. Jana et al. [62] further compressed these microparticles in the form of matrix tablets, which sustained the release of aceclofenac up to 8 h.

11.2.3 Carrageenan-based IPNs Carrageenan (CG) is a marine polysaccharide belonging to the red algae family and has a distinctive structure and functional properties. CG is an anionic, sulphated polygalacton composed of alternating long linear chains of α-1,3-d-galactose and β-1,4 glycosidic linkages in 3,6-anhydro-galactose with ester sulphates (15%–40%) similar to naturally occurring glycosaminoglycans [63]. Depending on the source of extraction, sulphate content, and solubility, CG is classified into different types such as Kappa (κ)-, Iota (ɩ)-, Lambda (λ)-, Mu (μ)-, Nu (ν)- and Theta (θ). Among them κ-CG is widely used as a drug delivery carrier [64, 65]. Recently, κ-CG has gained significant importance for oral drug delivery carriers in the pharmaceutical field [66–68]. Polysaccharide-based pH-responsive multi-particulate systems have emerged as smart drug delivery carriers owing to their responsiveness to external stimulus (pH, temperature), which can alter their properties such as network structure, strength, swelling pattern, and permeability. Kulkarni et  al. [69] synthesized pH-sensitive PAAm-g-κ-CG-NaAlg IPN beads for the intestinal delivery of ketoprofen. PAAmg-CG was synthesized by free radical polymerization followed by alkaline hydrolysis in the N2 environment. The drug-loaded IPN beads were prepared by ionotropic gelation and covalent cross-linking. The pulsatile swelling and drug release behaviors were evident for the IPNs. The outcomes of stomach histopathology of albino rats were interesting in that the release of ketoprofen in the stomach did not show any evidence of erosion of gastric mucosa, ulceration, and hemorrhage. The magnetic nanocomposite hydrogels of polyvinyl alcohol, kappa-carrageenan, and magnetite Fe3O4 were obtained in situ [70]. The produced magnetite-polymers were cross-linked with freezing-thawing technique and subsequently with K(+) solution. The dynamic swelling kinetics of hydrogels followed the second-order model. The hydrogel was efficient in releasing diclofenac sodium under physiological simulated pHs and external magnetic fields. The drug-loaded hydrogels demonstrated

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Polysaccharide Carriers for Drug Delivery

NH3·H2O

FeCl3·6H2O

win g

FeSO4·4H2O

K+

Fre e

zing

Carrageenan

Release

Magnetic nanocomposite

-tha

PVA

.4

in PH=7

Sodium diclofenac .2

=1

se

in

PH

ea

l Re

Fig. 11.4  A simple scheme for synthesizing of magnetic kappa-carrageenan/PVA nanocomposite hydrogel. Reprint with permission from Mahdavinia GR, Etemadi H. In situ synthesis of magnetic Cara PVA IPN nanocomposite hydrogels and controlled drug release. Mater Sci Eng C 2014;45:250–60. Copyright (2014), Elsevier.

significant antibacterial activity by inactivating Gram-positive Staphylococcus aureus bacteria. The hydrogels containing kappa-carrageenan showed good mucoadhesiveness in both simulated gastric and intestinal conditions (Fig. 11.4). To minimize the gastric side effect of nonsteroidal antiinflammatory drugs, Lohani et al. [71] developed κ-CG-Na CMC IPN beads for controlled delivery of ibuprofen. They studied the effect of polymer ratio, AlCl3 concentration and gelation time on in vitro drug release in different pH (1.2 and 7.4). Metronidazole is an antibacterial drug used against infections in the mouth, intestine, vagina, genital tract, fumigating tumors, bones, and skin ulcers. Microwave irradiated IPN of carageenan and guar gum was reported by Swain and Bal [72] for metronidazole. IPNs were micro-porous and possessed mucoadhesive property. Honeycomb-like internal architecture could provide high mechanical strength for the IPN for controlled release of metronidazole. In vitro release of optimized formulation showed that 72% of MTZ release at 12 h.

Interpenetrating polysaccharide networks as oral drug delivery modalities329

11.2.4 Gellan gum-based IPNs Gellan gum (GG) is a negatively charged, linear, exopolysaccharide. GG is commercially developed by microbial fermentation from bacterium Sphingomonas elodea or Pseudomonas elodea [73–75]. It is composed of tetrasaccharide repeat unit of glucose, glucuronic acid, and rhamnose in a molar ratio of 2:1:1 [76]. The pure drug diltiazem showed rapid and complete dissolution within 60 min, while drug release from drug-resinate was extended for 3 h and that from IPN microcapsules was still slower. The ionically cross-linked microcapsules were capable of releasing drug up to 9 h, and that from dual cross-linked microcapsules was extended up to 15 h. The microcapsules which were prepared with higher concentration of glutaraldehyde released the drug more slowly. Kulkarni et al. [77] prepared gellan gum-egg albumin (ALB) IPN microcapsules and diltiazem-resin complex was entrapped. Diltiazem HCl is an antihypertensive drug, widely used for the treatment of hypertension and angina pectoris. It has short biological half-life (3–4 h) and is administered orally 3–4 times daily [78]. Hence, this necessitates development of controlled release formulation for diltiazem. By means of ionotropic gelation by Ca2+ and covalent cross-linking by GA, IPN systems were produced as depicted in Fig. 11.5. The drug-resinate complex extended drug release only up to 3 h, however, encapsulation in IPN microcapsules further slowed the drug release process. The dual cross-linking of the microcapsules extended drug release up to 15 h, compared to that observed with ionically crosslinked microcapsules, which controlled drug release up to 9 h. Another antihypertensive drug—carvedilol—was successfully loaded into IPN microspheres of gellan gum and poly(vinyl alcohol) by the emulsion cross-linking method [79]. The chemical stability and crystalline dispersion of carvedilol in the microspheres were noted. The particle size measured by laser light scattering technique ranged between 230 and 346 μm. Carvedilol encapsulation efficiency up to 87% was achieved in the polymeric matrices. In  vitro release studies were performed in the simulated gastric fluid or simulated intestinal fluid. The release of carvedilol was continued up to 12 h in simulated gastrointestinal fluids.

11.2.5 Locust bean gum-based IPN Locust bean gum (LBG) is a high molecular weight branch polysaccharide, obtained from the seeds of Ceratonia siliqua. The molecular weight of LBG ranges between 300,000 and 1,200,000 Da. Chemically it is composed of α (1,4)-linked βd-­mannopyranose backbone with linked to (1,6) α-d-galactose. LBG is a nonstarch galactomannan polysaccharide containing galactose and mannose in the ratio of 1:4. The nontoxic LBG can be used as drug delivery retardant biomaterials [80–82]. The bio-safety and in  vivo pharmacokinetic study of LBG-poly(vinyl alcohol) (PVA), CMLBG-PVA and polyacrylamide-grafted-LBG-PVA IPN microspheres for controlled oral delivery of buflomedil hydrochloride [83]. CMLBG-PVA IPN microspheres showed a similar Tmax value with drug oral suspension in the rabbit model. However, compared to LBG-PVA, the acrylamide-g-LBG-PVA IPN microspheres showed controlled release property under in vivo condition.

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Polysaccharide Carriers for Drug Delivery COO–M+

CH2OH

CH2OH

O O

HO OH

OH

O O

OH Gellan gum

O O

OH OH

CH3

OH

OH

n

+ NH2 Egg albumin

Ca2+

Lonically crosslinked gellan gum chains Uncrosslinked egg albumin chains

OHC–(CH2)3–CHO glutaraldehyde

GA crosslinked gellan gum chains GA crosslinked egg albumin chains

GG-ALBIPN matrix

Fig. 11.5  Schematic representation of GG-ALB IPN matrix. Reprint with permission from Kulkarni RV, Mangond BS, Mutalik S, Sa B. Interpenetrating polymer network microcapsules of gellan gum and egg albumin entrapped with diltiazemresin complex for controlled release application. Carbohydr Polym 2011;83:1001–7. Copyright (2014), Elsevier.

Dey et al. [84] focused on the importance of the hydrogelation period in the development glipizide-loaded carboxymethyl LBG-Alg IPN beads by Al+3 ions-mediated cross-linking methods in an aqueous environment. The drug release in phosphate buffer solution could be extended for at least 8 h.

Interpenetrating polysaccharide networks as oral drug delivery modalities331

To minimize the gastro-ulcerative side effects of the nonsteroidal antiinflammatory drug (NSAID), LBG-based polysaccharide-based IPN systems have been sought. To overcome stability and burst drug release of Alg microspheres at higher pH values, Jana et  al. [85] prepared Alg-LBG IPN microspheres by calcium ion Ca+2 induced ionotropic gelation technique for prolonged release of aceclofenac, an antiinflammatory drug [86]. The drug entrapment efficiency of the IPN microspheres of 406–684 μm size varied widely from 59% to 93%. The microspheres released aceclofenac in phosphate buffer solution (pH 6.8) over a period of 8 h and exhibited prolonged antiinflammatory activity in the carrageenan-induced rat paw model after oral administration. GA cross-linked CS-LBG IPN nanocomposites (Fig 11.6) of aceclofenac were developed using glutaraldehyde [87]. Increasing the amount of LBG in the IPN decreased the drug entrapment from 72% to 40% and produced larger particles. LBG: CS mass ratio of 1:5 provided maximum drug entrapment efficiency of 78.92%, smallest composites of 318 nm size and slowest drug release profiles in phosphate buffer solution (pH 6.8) up to 8 h. The composite systems efficiently suppressed the burst release of aceclofenac in acidic medium (pH 1.2). The data suggested that the IPN system could minimize gastrointestinal side effects of the drug. Box-Behnken design was used to optimize capecitabine-loaded Ca2+-LBG-Alg IPN microbeads [88]. The % drug entrapment and particle size of the beads was ~81% and ~494 μm, respectively. The optimized formulation released 92% drug in 12 h and followed an anomalous mechanism for drug discharge. The formulation with improved pharmacokinetics than pure drug clearly suggested their potential as a carrier for anticancer drug delivery.

OH

OH H

H OH

O H

H

O

O

H

O H

N

OH

n

OH H O H

OH H

N

O

O

n

n

OH

H O OH

OH

H

Locust bean gum

N H

OH

H

Chitosan

OH Glutaraldehyde

O H

H OH

H

OH H

N H

O OH

OH

n

Fig. 11.6  The reaction mechanism of GA cross-link CS-LBG IPN. Reprint with permission from Jana S, Sen KK. Chitosan-locust bean gum interpenetrating polymeric network nanocomposites for delivery of aceclofenac. Int J Biol Macromol 2017;102:878–84. Copyright (2014), Elsevier.

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11.2.6 Xanthan gum-based IPNs Xanthan gum (XG) is a high-molecular-weight, extracellular anionic polysaccharide, obtained from the gram-negative bacterium Xanthamonas campestris. It has been widely utilized in the pharmaceutical field [89, 90]. The natural polysaccharides showed some limitations, such as uncontrolled swelling and hydration, uncontrolled release of drug, viscosity changes on storage, and microbial contamination. These types of problems can be reduced following modification by blending, cross-linking, functional group modification etc. Kulkarni and Sa [91] evaluated hydrogel beads of polyacrylamide-grafted-XG and sodium carboxymethyl cellulose (NaCMC) for pH sensitive release of ketoprofen. Microscopic examination revealed porous matrix structure of IPNs in alkaline pH whereas nonporous matrix structure was observed in acidic pH. The swelling and drug release was a function of pH of the medium. As pH of the medium was changed to alkaline, a considerable increase in swelling was noticed for the IPN beads. At higher pH, the polymeric carboxyl groups underwent ionization and consequently built an osmotic pressure inside the beads and caused higher swelling. Ray et  al. [92] reported pH-sensitive XG-poly vinyl alcohol (PVA) IPN microspheres, prepared by emulsion cross-linking method using GA as a cross-linker and examined their potential to deliver an antiinflammatory drug—diclofenac sodium—to the intestine. The drug was chemically stable and molecularly dispersed after incorporation into 310–477 μm size microspheres. Drug encapsulation of up to 82.94% was achieved and the drug release was dependent on the extent of cross-linking and the ratio of XG:PVA present in the microsphere. In vivo pharmacokinetic results of the drugloaded IPNs in rabbits were encouraging compared to the drug solution. IPN hydrogel beads of Al+3 and GA cross-linked pectin‑sodium carboxymethyl XG were prepared by Giri et  al. [93] for sustained delivery of diltiazem hydrochloride. The hydrogel beads swelled less and released a small amount of drug in pH 1.2 buffer solution and prolonged the same in pH 6.8 buffer solution depending on polymer concentration, strength, and exposure period of cross-linker and drug content in the IPN matrices. IPNs of sodium carboxymethyl cellulose and sodium carboxymethyl XG containing diclofenac sodium (DS), a nonsteroidal antiinflammatory drug, were developed by an emulsion gelation process using AlCl3 as the cross-linking agent [94]. The drug encapsulation efficiency of IPN beads was reasonable with a good control over intestinal drug release. Further, Bhattacharya et al. [95] reported IPNs based on XG and poly(vinyl alcohol) for sustained release of ciprofloxacin hydrochloride. Regardless of polymer ratios and cross-linking density, all the microspheres exhibited satisfactory in vitro release characteristics and followed the non-Fickian trend in drug release. IPN beads of sodium carboxymethyl XG and Alg were prepared by the ionotropic gelation process using AlCl3 as a cross-linking agent [96]. The authors extensively examined the influence of concentration of total polymer, cross-linking agent, gelation time, and drug load on the release of ibuprofen. Increase in the concentration of total polymer, gelation time, and drug load decreased the drug release in both the acidic and alkaline media. However, the effect reversed as the concentration of cross-linking agent was increased. A maximum 14% of the loaded drug was released in 2 h in the acidic medium.

Interpenetrating polysaccharide networks as oral drug delivery modalities333

IPNs were found to complete their drug release in phosphate buffer solution within a maximum of 5.5 h. The drug release occurred in accordance with the swelling kinetics of IPN beads. Giri et  al. [97] fabricated theophylline-loaded NaCMXG-casein IPN beads. The formulations exhibited slow drug release in pH 1.2 KCl/HCl buffer with a maximum drug release of about 20% in 2 h. Increasing casein concentration, glutaraldehyde (GA) concentration, and drug loading suppressed the release of theophyline from the hydrogels in phosphate buffer solutions (pH 6.8).

11.3 Conclusion In this chapter, the reports on polysaccharide-based IPNs were extensively and critically analyzed in an attempt to control the delivery of a variety of drugs. Their versatile properties, such as cost effectiveness, nontoxicity, biodegradability, desirable drug release profile at target site, and broad regulatory acceptance were the key to develop IPNs based on natural polysaccharides. The different studies revealed that the fabricated IPN systems had significant mechanical strength and could control swelling and drug release properties in a pH-dependent manner. Much emphasis was placed on a number of polysaccharides such as alginate, chitosan, carrageenan, locust bean gum, gellan gum, and xanthan gum, and their development as IPN microspheres, beads, nanoparticles, and hydrogels for oral drug delivery. Numerous combinations of polymers were tested for their potential application in drug delivery. However, the reports on in vivo performance of the IPN carriers and safety issues must be critically examined before their appearance in clinics.

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Chella Naveen, Nalini R. Shastri Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India

12.1 Introduction The advancement of biotechnology has led to the development of many new chemical entities that can target the diseases more effectively and precisely. However, the success of these molecules depends on efficiency of delivery system to place the drug in sufficient concentration at correct location (spatial or temporal placement of drug) at an optimized rate. Novel delivery systems based on nanotechnology like liposomes, nanoparticles, and micelles offer similar advantages by direct targeting an organ or specific tissues. These systems can also improve the therapeutic efficiency with reduced side effects of many drugs (either small molecules or proteins) by regulating their pharmacokinetics (ADME) and biodistribution process [1]. Hence, nanotechnologybased products have emerged as superior alternatives with better patient quality as compared to conventional therapy. Polymeric micelles are spontaneously formed colloidal carriers in nanosize range (20–80 nm) composed of hydrophobic core and hydrophilic shell. These are made up of amphiphilic molecules with the unique self-assembling ability and capacity to load hydrophobic and hydrophilic molecules for a wide variety of therapeutic applications in drug delivery and diagnostics [2]. Polymeric micelles possess several advantages of the nanocarriers: (i) Passive targeting or accumulation at the desired site by Enhanced Permeation and Retention (EPR) effect due to small size (nanosize range) with narrow size distribution of the particles. (ii) Possibility of active targeting by attaching variety of ligand on to different functional groups available on the surface. (iii) Higher payloads for hydrophobic drugs by incorporation into inner hydrophobic core. (iv) Increased circulation half-life by decreased renal clearance and prevention of opsonization due to outer hydrophilic portion. (v) Ability to incorporate both hydrophilic and hydrophobic drugs due to hydrophobic inner core and outer hydrophilic surface. (vi) The structural stability rendering them more suitable for in vivo application [3]. Tailoring micelles from block copolymers was initiated by Ringsdorf et al., in 1980, and thereafter extensive studies were performed on the micelle properties and modified accordingly to meet the requirement of pharmaceutical applications. Recently, naturally occurring biodegradable materials such as polysaccharides in place of synthetic polymers have received much attention from drug-delivery scientists due to their wide range of availability, biodegradability, biocompatibility, nontoxicity, ­nonimmunogenicity, and occurrence of abundant surface functional groups for modification and targeting delivery [4]. Polysaccharide Carriers for Drug Delivery. https://doi.org/10.1016/B978-0-08-102553-6.00012-X Copyright © 2019 Elsevier Ltd. All rights reserved.

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Polysaccharides are polymeric carbohydrate molecules isolated from renewable resources including plants, animals, microbials, and algae (marine). They are most abundant in nature, easy to isolate, and undergo enzymatic or hydrolytic degradation to nontoxic products. The major function of polysaccharides in an organism is to provide structural support and act as an energy store. Due to biochemical similarity with human extracellular matrix, these are more readily accepted by the body. The hydrophilicity of most of polysaccharides facilitates longer stability with increased circulation half-life [5]. The interaction between epithelial and mucous membrane of biological tissues and functional groups (carboxyl, amine, and hydroxyl) present on the surface of polysaccharides provides bioadhesion or mucoadhession. This bio/ muco adhesiveness nature further helps to increase the residence time and also provides site-specific or targeted drug delivery of loaded active moieties. These properties make them biocompatible, biodegradable, nontoxic, highly stable, thus making them amenable for use in drug-delivery systems over synthetic biopolymers [6]. Polysaccharides are diversified structurally and functionally with varying chemical composition (repeating monosaccharide units), chain length and degree of branching, wider molecular weight distribution, and large number of reactive groups on their surface. The general formula of polysaccharides is Cx(H2O)y, where “y” has a value between 200 to 2,500. These are produced by condensation of monosaccharide predominantly, d-glucose, and others like d-fructose, d- and l-galactose, d-arabinose and d-xylose. The glycosidic linkages between the glycosyl residue of the hemiacetal and the hydroxyl residue of another monosaccharide result in linear to branched structure of polysaccharide. Depending on the repeating monosaccharide units, these are classified into homopolysaccharides (single monosaccharide unit) and heteropolysaccharides (two or more different monosaccharide units). Based on the presence of charge, polysaccharides are further classified as positively charged (chitosan), negatively charged (hyaluronic acid (HA), alginate, gellan gum, and pectin), and neutral (dextran, amylose, cellulose). They can exist as linear (amylose, cellulose, pectin, alginates) and branched (guar gum, locust bean gum, Xanthan gum) polysaccharides (Fig. 12.1).

12.1.1 Graft copolymerization Polysaccharides, despite playing an important role in drug delivery due to their unique properties, also suffer from drawbacks like microbial contamination, uncontrolled hydration, poor mechanical strength, and alteration in viscosity on storage, which limits their application in drug delivery and biomedical field [7]. Chemical modification of these natural polysaccharides brings further changes in their physicochemical properties (solubility, permeability, melting point, glass transition temperature, and chemical reactivity) while retaining their original characteristics, and viscoelastic behavior making the backbone of natural polymers more suitable for pharmaceutical applications. Hence, chemical modification of natural materials with synthetic monomers or polymers through grafting has gained much interest in last few years due to combined advantages of both natural and synthetic polymers. The availability of diverse functional groups such as hydroxyl, amino, aldehyde, and carboxylic acid groups on the surface of most of the polysaccharides acts as

Polysaccharide nanomicelles as drug carriers341

Fig. 12.1  Schematic representation of polysaccharide classification based on their structural units.

r­ eactive sites and makes it easy for chemical modification. The attachment of different ligands on to these functional groups makes them more suitable for biomedical applications such as drug delivery systems, tissue engineering, and regenerative medicine [8]. There are different chemical modification techniques such as conjugation of small molecules, synthetic polymers, surface modification, or covalent attachment of different functional groups and graft polymerization. The selection of modification method depends on the nature of the polysaccharide, their properties, required characteristic properties needs to be incorporated, and its final application. Graft copolymerization offers desirable properties into the polymer backbone by hybridizing synthetic and natural polymers. Grafting procedure involves covalent attachment of side chains onto the polymer backbone to form a copolymer with branched structure. The grafting procedure brings backbone and polymer side chain together by chemical bonding rather than leaving them as simple physical mixtures. The backbone and the attached side-chain polymers can be homopolymers or copolymers [9]. The first step in graft copolymerization is generation of active sites on the backbone followed by attaching monomers on to the active sites produced. The graft copolymerization occurs through chemical methods, radiation induction, enzymatic methods, or plasma polymerization with either covalent or noncovalent bonds between

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Polysaccharide Carriers for Drug Delivery

p­ olysaccharide backbone and the graft chain. The detailed procedure for synthesis along with their advantages and limitations were discussed by Atanase et al. [10]. The polymers used for grafting procedure should be inert without any biological activity, able to protect the natural polymers in the biological milieu, and able to modify distribution profile in the body. The final properties of graft polymer depend upon the molecular structure, length, and number of grafted polymer chains. Desirable properties can be achieved by controlling grafting frequency and grafted chain length. The modified polysaccharide derivatives (amphiphiles) obtained by grafting hydrophobic chains on to the natural hydrophilic polysaccharides are able to self-assemble in the aqueous media leading to formation of nanocarriers (micelles). These nanomicelles are used to deliver hydrophobic drugs, proteins, and genes. The natural polysaccharides that are modified in full or partial using hydrophobic moieties such as cholesterol, PLGA, PLA, PCL, Pluronics, bile acids, and long-chain fatty acids (hexanoic acid, linoleic acid, linolenic acid, palmitic acid, or stearic acid) include chitosan, HA, sodium alginate, and dextran. These grafted copolymers are purified to remove the unreacted monomers and other impurities with the help of solvent precipitation and characterized for their molecular weight, average composition, grafting density, and the corresponding molar distributions using suitable analytical techniques [10].

12.2 Polysaccharides in drug delivery Natural polysaccharides and their derivatives are widely used in pharmaceutical, cosmetic, and biomedical industry for drug delivery, cosmetic, and diagnostic applications. Few examples include chitosan, HA, dextran, sodium alginate, starch, gaur gum, gellan gum, pectin, gum kondagogu, pullulan, cellulose, heparin, and polysialic acid. In the present chapter, we have limited our review to three major polysaccharides and their derivatives that are mostly used to produce nanomicelles in drug-delivery applications.

12.2.1 Chitosan Chitosan is a cationic, hydrophilic, linear heteropolymer containing d-glucosamine and N-acetylglucosamine units linked by β(1 → 4) glycosidic bonds. It is the second most abundant polysaccharide after cellulose. Chitosan is obtained by partial deacetylation of chitin, which is the main component in the exoskeleton of crustaceans (e.g., shells of crab, shrimp, and lobster) under alkaline conditions. Generally, the production of chitosan from crustacean shell comprises four basic steps: demineralization, deproteinization, decoloration, and deacetylation [11]. The degree of deacetylation (DA) conveys about fraction of N-acetyl-d-glucosamine relative to the total number of units and decides the existence of either chitin (DA > 50%) or chitosan (DA < 50%) [12]. The physicochemical properties of chitosan differ with respect to its molecular weight, degree of deacetylation, and surrounding pH conditions. It is degraded into oligomers and finally to N-glucosamine by enzymes such as lysozyme and c­ hitosanase in vivo. Two products, namely, Tegasorb by 3M (wound dressings) and Hem-con by

Polysaccharide nanomicelles as drug carriers343

HemCon (hemostatic patch) containing chitosan are already approved by US Food and Drug Administration (FDA). It is used as dietary additive in Japan, Italy, and Finland, and LD50 is reported to be 16 mg/kg in mice after oral dose [13]. Chitosan is preferred in drug delivery due to its biocompatible, biodegradable, nontoxic, antiinflammatory, mucoadhesive, antimicrobial properties, and neuroprotective behavior that ensure effective and safe therapy [14]. Chitosan also has the ability to open the tight junctions of biological membranes and thus aid in paracelluar transport of the loaded drug molecules. Despite these advantages, the use of chitosan is limited due to its insolubility above pH 6 and in organic solvents. Grafting of chitosan can modulate the physicochemical properties without altering its biocompatibility and biodegradability. Two reactive sites available on the chitosan backbone for grafting include hydroxyl groups at C3 and C6 carbons on acetylated or deacetylated units and free amino group produced as a result of hydrolysis of the deacetylated units at C2. These reactive sites are produced either by physical methods or chemical methods. Amphiphilic chitosan can be produced by grafting directly hydrophobic drug molecules or hydrophobic groups such as alkyl, acyl, cholesterol, cholic acid, deoxycholic acid, 5β-cholanic acid on to the amine groups by N-acylation reactions. These hydrophobically modified chitosan derivatives can self-associate in aqueous environment spontaneously or after sonication to form micellar nanocarriers that are useful in drug-delivery applications [15]. The micellar carriers produced by chitosan and its derivatives improve loading of hydrophobic drugs, control drug release, improve stability of sensitive drugs, increase retention time in plasma either by improved mucoadhesiveness or increased residence time, tumor targeting (passive or active targeting), reduce adverse effects and bioavailability enhancement (Table 12.1). Other derivatives of chitosan reported for drug delivery include carboxymethyl chitosan, N-methylene phosphonic chitosan, carbohydrate-branched chitosan, and alkylated chitosan. Chitosan, being cationic in nature, adheres to the negative mucosal surface. This makes chitosan versatile among other polysaccharides, as chitosan micelles can be used in oral delivery to improve the permeability and absorption of drugs. Chitosan micelles can also deliver DNA and RNA molecules by conjugation due to electrostatic attractions.

12.2.2 Hyaluronic acid/hyaluronan The journey of HA began in 1934, when it was extracted from bovine vitreous humor by Karl Meyer and John Palmer [27]. Later in 1942, a patent was applied on the use of HA in bakery products as a substitute for egg white by Endre Balazs. The first medical application in humans was reported in 1950s as a vitreous substitution/replacement during eye surgery. To encompass its existence in both polyanionic and acid form and to conform to the international nomenclature, the term “hyaluronan” was introduced in 1986. HA is a naturally occurring hydrophilic, anionic, mucopolysacharide, composed of repeating disaccharides, d-glucuronic acid, and N-acetyl glucosamine linked by β(1 → 3) and β(1 →4) glucosidic bonds. It is a major component of cellular matrix, connective tissues, and synovial fluid in different joints. It is abundant in the vitreous of the eye, skin, heart valves, umbilical cord, and skeletal tissues [28]. Previously, HA

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Table 12.1  List of conjugates attached to Chitosan, drug loaded along with their application in drug delivery Polymer grafted

Drug

Application

References

Poly-(N-3carbobenzyloxylysine) with transactivator of transcription (TAT) β-Cyclodextrin and benzimidazoleterminated poly(ε-caprolactone) Palmitic acid

Doxorubicin and p53 plasmids

Higher gene transfection efficiency, higher apoptosis, and low viability in HeLa cells

[16]

Doxorubicin

pH-dependent drug release at desired site

[17]

Tamoxifen

[18]

N-octyl-O, N-carboxymethyl

Silybin (SLB)

A glycyrrhetinic acid/cystaminepoly(ε-caprolactone) copolymer N-arylsuccinyl chitosan TPGS and stearic acid

Doxorubicin and curcumin

Significant enhancement in the cytotoxicity of tamoxifen on MCF-7 cancer cells Enhanced oral absorption of SLB by increasing the permeability and inhibition of efflux mechanism Improved efficacy due to synergism by combination therapy

[21]

Folic acid-conjugated stearic acid vitamin E succinate

pDNA

Stearic acid grafted to depolymerized chitosan Polyoxyethylated (Pluronic F127)

Itraconazole

Improved solubility makes it suitable for oral delivery Enhancement in docetaxel oral bioavailability by 2.52fold compared to docetaxel solution Increased transfection efficiency up to 2.3-fold Improved intestinal absorption (4.5-fold) and improved drug accumulation in intestinal villi Stable micelles for inhalation delivery of itraconazole Improved intraocular absorption and enhanced corneal permeability of dexamethasone

[26]

Meloxicam Docetaxel

Paclitaxel

Dexamethasone

[19]

[20]

[22]

[23] [24]

[25]

was obtained from bovine vitreous humor. Nowadays, commercially, it is produced from bacteria (Bacillus sp.) by fermentation. HA has wider molecular weight distribution ranging from 4000 Da to 10 MDa depending on the origin and exhibits high degree of polymerization [29]. The biological activity of HA depends on its molecular weight. High-molecular-weight HA

Polysaccharide nanomicelles as drug carriers345

possess antiangiogenic, antiinflammatory, immunosuppressive properties and inhibits cell proliferation, whereas low-molecular-weight HA promotes angiogenesis and enables cell migration [7]. HA is degraded by hyaluronidase. Native HA can be used as a drug molecule and based on its properties, two products of HA, such as Hylan GF-20 (Genzyme) and Healon (Abbott), were approved for osteoarthritis and eye surgery, respectively [30]. The readers are suggested to refer cited literature for further details on structure, synthesis, degradation, physicochemical, pharmacological, and pharmacokinetic properties [31–34]. HA is one of the widely used polysaccharides in medical, cosmetics, and pharmaceutical industry due to its biocompatible, biodegradable, nonimmunogenic, nontoxic nature, and specific binding affinity toward CD44 and RHAMM (receptors for HAmediated motility) receptors, which are overexpressed on tumor surface [35]. Apart from these mentioned reasons, presence of multiple functional groups like hydroxyl and carboxyl groups makes them attractive for chemical modification either with drug molecules or other block polymers/lipids and stealth characteristics that stabilizes drug molecules in plasma, rendering them amenable to drug delivery. HA is modified either by chemical or biochemical means to obtain amphiphilic molecule that can spontaneously produce micelles in aqueous environment for drug delivery and targeting (Table 12.2). Crosslinking or conjugations are two methods by which HA can be chemically modified. The first report of HA prodrug resulted from conjugation of drug with HA was published in 1991 [49]. Primary hydroxyl group on the N-acetylglucosamine and carboxyl group on the d-glucuronic acid are the two most exploited groups on HA for graft copolymerization. The possible reactions on to hydroxyl group include ester and ether linkages and onto the carboxyl group are amidation and esterification [29]. The major agents used in grafting are either synthetic polymers (PLA, PLGA, and PCL) without any specific biological activity or bioactive polypeptides. The resulting copolymer can improve the pharmacokinetic behavior of HA and can be used for targeted drug delivery. The first HA grafting copolymer obtained was HA-g-PLA derivative synthesized by Giammona research group. This group employed low-molecular-weight HA (200 kDa) and short-molecular-weight PLA (8 kDa) to obtain colloidal aggregates or viscous physical hydrogels in the concentration range of 0.125 to 1% w/v by changing degree of derivatization from 1.4 to 8 mol% [50]. HA derivatives developed by grafting PLA alone or in combination with PEG (HA-g-PLA and PEG-g-HA-g-PLA) with 1.8 mol% degree of derivatization were employed to produce micelles for the successful delivery of doxorubicin. Critical aggregation concentration was between 0.04 and 0.06 g/L and particle diameter of the prepared micelles was found to be 41 and 37 nm for Ha-g-PLA and PEG-g-Ha-g-PLA, respectively. Micelles prepared with PEGg-Ha-g-PLA conjugate showed larger particle size, narrow size distribution, and improved hydrophilicity. The presence of PEG chains further increased charge from −61 to −46.7 mV due to shielding effect and reduced number of free carboxylic groups on HA chains after PEGylation. Drug loading was found to be 4.8% in non-PEGylated micelles, whereas PEGylated micelles showed improvement in drug loading (10.2%). In vitro cytotoxicity results revealed no significant difference in cytotoxicity between PEGylated and non-PEGylated micelles, indicating no interference of PEG chains in receptors recognition. Further, in vitro study using J774 A.1 mouse macrophage cell

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Table 12.2  List of different conjugates and drugs loaded in respective micelles of HA Conjugate moiety

Drug loaded

Activity evaluated

Reference

PLGA

Doxorubicin

[36]

1,2-Distearoyl-sn-glycero3-phosphoethanolamine (DSPE); 1,2-dimiristoylsn-glycerol-3phosphatidylethanolamine (DMPE) Vitamin E succinate

Triamcinolone

In vitro cytotoxicity using HCT-116 cells Biological fate of micelles after intraarticular injection to joints

[39]

Polycaprolactone

Radioactive iodine-131labeled lipiodol Doxorubicin

Antitumor efficacy in 4T1 tumor-bearing mice Cytotoxicity and the cell uptake of micelles Tumor growth inhibition in H22 cell-bearing mice Antitumor efficacy in a MCF-7 breast tumor model Cell viability assay for MCF-7/MDR cancer cells Tumor growth inhibition in H22 tumor-bearing mice Theranostic applications such as chemotherapy, hyperthermia, and bioimaging For the treatment of rheumatoid arthritis Intracellular delivery carrier for efficient tumor therapy

[41]

Glycyrrhetinic acid and l-histidine

Doxorubicin

Deoxycholic acid-histidine

Paclitaxel

Pluronic L61 and poly(propylene glycol)

Doxorubicin

Quercetin

–

Doxorubicin

–

Methotrexate

–

Paclitaxel

[37, 38]

[40]

[42]

[43]

[44]

[45]

[46] [47, 48]

line showed a very low cell uptake from the PEGylated micelles, which confirms that PEGylated micelles are less sensitive to macrophage uptake [51]. Uthaman et al. showed enhancement in tumor targeting and photothermal capability of IR-780 dye, using CD44-targeted HA-based micelles. The researchers synthesized two types of HA and octadecyl amine (C18) conjugates and later R-780 dye was loaded in to the micelles by dialysis. Various studies, including cell viability, photothermic effect studies (both in vitro and in vivo), CD44-specific competitive ­inhibition

Polysaccharide nanomicelles as drug carriers347

studies, in vivo biodistribution studies, tumor-targeting efficiency, ex vivo organ analysis, were performed on the prepared micelles and the activity was compared to that of IR 780 alone. The IR 780-loaded HAC18 micelles showed significant toxicity upon laser irradiation with higher accumulation (due to EPR and CD44 targeting) in tumor tissue without causing any toxicity to other organs [52]. HA or conjugated HA micelles can be used in diagnostic imaging, drug delivery, gene delivery, and combination of both diagnosis and therapy (theranostic) with single delivery system [53, 54]. In case of drug delivery, they are delivered mainly through oral [55], parenteral [37], topical [56] routes.

12.2.3 Dextran Dextrans are hydrophilic and water-soluble polysaccharides used as plasma expanders for more than 50 years. Recently, the application of these agents in drug delivery and imaging is being explored. Dextrans are produced extracellularly through dextransucrase from bacteria of the family Lactobacillaceae and mainly from cultures of Leuconostoc mesenteroides NRRL B-512F by chemical synthesis from sucrose. Chemically, it is synthesized from levoglucosan (1,6-anhydro-β-d-glucose) via a cationic ring-opening polymerization. Structurally, the dextran consists of a linear polymer backbone with mainly 1,6-α-d-glucopyranosidic linkages with some α-1,2, α-1,3, and α-1,4 modifications (branching). The degree of branching (0.5%–60%) and molecular weight (4–200 kDa) determines the physicochemical properties of the polymer and may vary depending on the polymer source [57]. The aqueous solubility of dextran decreases as the percentage of branching increases and vice versa. Although natural dextran has large degree of poly dispersity, the commercial grades are monodisperse with less percentage of branching. Dextrans from microbial origin are purified by partial depolymerization by acid hydrolysis and fractionation for clinical application. Chemically, these dextrans are stable under acidic and basic conditions. Dextran can be used as carrier either in parenteral delivery or for local delivery (to colon) due to its inability to absorb through epithelial cells and its ability to degrade in GI tract when given orally. In the body, these dextrans are metabolized or degraded by different α-1-glucosidases (dextranases) present in various organs, including liver, spleen, kidney, and lower parts of GI tract [58]. Grafting of hydrophilic dextran with hydrophobic moieties can help in designing self-assembling carriers for drug delivery, including proteins/peptides and in imaging applications. Conjugation of dextran improves in vivo stability of the polysaccharide by preventing degradation by dextranases and alters the pharmacokinetics of the drug molecule. The conjugates also increase drug circulatory life by impeding their renal filtration due to high molecular weight. Retention of dextran drug conjugates in the reticuloendothelial cells leads to accumulation of these carriers in liver that can be used to target liver diseases. The presence of large number of hydroxyl groups on the surface facilitates modification either directly with the drugs or through a linker [59]. Various chemical methods such as direct esterification, carbonyldiimidazole activation method, carbonate or carbamate ester method, periodate oxidation method,

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cyanogens bromide activation method, and etherification of dextran are reported in the literature for synthesizing dextran conjugates [58–61]. Streptodekase, a conjugate of dextran and streptokinase, is the first protein conjugate to be tested in humans [62]. Conjugated dextran derivatives are utilized for various applications in drug delivery as shown in Fig. 12.2. Especially, amphiphilic copolymers produced after conjugation of dextran with different hydrophobic polymers are able to form micelles that can be used for tumor targeting, site-specific delivery, stimuli-based delivery systems, solubility enhancement of poorly soluble drugs, to overcome the multidrug resistance (MDR) and P-gp efflux (Table 12.3).

12.3 Preparation of micelles Polysaccharide micelles are prepared by different techniques depending on the properties of polysaccharide and grafted copolymer. Generally used techniques include ultrasonication, dialysis, emulsification, nanoprecipitation (Table 12.4).

12.3.1 Ultrasonication method Polysaccharide or modified copolymer is dispersed in aqueous media along with hydrophilic drugs. Hydrophobic drugs are dissolved in organic solvent and added to the dispersion. The organic solvent used is removed using rotary evaporator and the

Fig. 12.2  Applications of dextran conjugate in drug delivery.

Polysaccharide nanomicelles as drug carriers349

Table 12.3  Dextran micelles in drug delivery Delivery system

Application

Reference

Dextran-doxorubicin prodrug micelles Curcumin-dextran micelles

Solid tumor therapy

[63]

pH-dependent drug delivery for enhanced therapeutic efficacy in cancer cells Enhanced cytotoxicity and better pharmacokinetic profile pH-responsive delivery of doxorubicin Targeted doxorubicin delivery in acute leukemia For the oral delivery of poorly watersoluble drugs (Cyclosporin A) For reducing drug resistance of colorectal cancer by boosting etoposide cellular uptake Improved in vitro antitumor efficacy of doxorubicin in human nasopharyngeal epidermoid carcinoma cancer cells As an excipient for dry powder inhalations to deliver hydrophilic and hydrophobic drugs Solubilization of the poorly watersoluble drug, Cyclosporin A

[64]

Near-infrared light-sensitive micelles for enhanced intracellular drug delivery To deliver the paclitaxel for the treatment of Multidrug resistance tumors To overcome multidrug resistance in cancer therapy with doxorubicin

[73]

Dextran-PLGA-loaded docetaxel micelles Histidine-modified dextran-gcholesterol micelle Folate-targeted dextran/ retinoic acid micelles Hydrophobically modified dextran micelles Dextran stearate polymeric micelles α-Tocopherol succinatemodified dextran micelles

Stearylamine dextran micelles

Dextran-g-polyethylene glycolalkyl ether polymeric micelles Dextran-graft-(2-diazo-1,2naphthoquinone) Indomethacin-conjugated dextran-based micelles Dextran (DEX)-SSindomethacin micelles

[65] [66] [67] [68] [69]

[70]

[71]

[72]

[74]

[75]

Table 12.4  List of various preparation methods of micelles and incorporated drugs Method

Drugs used

Reference

Dialysis

Methotrexate; Doxorubicin Cyclosporin A Doxorubicin; Paclitaxel; Quercetin Doxorubicin; Losartan Nile Red (NR), Coenzyme Q10; Paclitaxel

[57, 68, 76]

Ultrasonication method Emulsification method Solvent evaporation

[24, 41, 77] [78, 79] [56, 80]

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resulting solution is sonicated to get the required size range micelles [81]. This method is recommended for hydrophobic polysaccharides with good swelling properties.

12.3.2 Dialysis Polymer/copolymer and drug are dissolved in water-miscible organic solvents like ethanol, methanol, tetrahydrofuran, dimethyl sulfoxide, acetonitrile, acetone, and N,N-dimethylformamide and dialyzed against water. Micelles are formed as the organic solvent is evaporated [4]. The advantage with this method is evasion of stabilizer or emulsifier in formulation.

12.3.3 Emulsification method Water-immiscible solvents like chloroform, dichloromethane, or ethyl acetate is used to dissolve the drug and added to aqueous solution containing polymer and homogenized to get O/W emulsion. Slow evaporation of organic solvent results in core shelltype polymeric micelles [4].

12.3.4 Solvent evaporation Polysaccharides are dissolved in selected solvents like methanol and slowly added to aqueous phase under stirring to remove the organic solvent. Evaporation of the organic solvent results in formation of nanomicelles [56].

12.4 Evaluation and characterization The detailed descriptions of various analytical techniques were discussed in literature. Here, the parameters needs to be measured, suitable analytical procedures and instruments preferred for the same are listed in Table 12.5.

12.5 Applications in drug delivery 12.5.1 Polysaccharide micelles as smart delivery systems Smart polymers are those that change their physical properties such as size, shape, or hydrophobicity with corresponding changes in the surrounding environment. The major environment factors include pH, temperature, enzymes, or chemicals present at the site of action. Human body presents various pH conditions in different parts of the body like endosomes, lysosomes, and tumor tissue environment, which has lower pH. The smart delivery systems can carry the drug molecules specifically to the required site avoiding unnecessary exposure to healthy tissues. These types of delivery systems are more useful in tumor targeting. These micelles are stable at blood/plasma pH 7.4, but start to release the drugs under acidic conditions of tumor. Under acidic

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Table 12.5  List of various characterization tools along with principle and equipment required Characterization parameter

Principle

Equipment used

Reference

Critical micellar concentration (CMC) Critical association concentration (CAC) Size and size distribution Morphology

Solubilization method using fluorescent dyes

Microplate reader

[52, 82, 83]

Dynamic light scattering Electron microscopy

Zeta sizer

Surface charge

Electrophoretic mobility Differential scanning calorimetry X-ray diffraction Fourier transforminfrared spectroscopy 1 H NMR spectrum Extraction of drug in suitable solvent

Physical/solid-state characterization or interaction studies

Entrapment efficiency, drug loading, and in vitro drug release In vitro permeation studies

Transepithelial electrical resistance

Scanning electron microscopy Transmission electron microscopy Atomic force microscopy Zeta sizer IR spectrophotometer Differential scanning calorimeter X-ray diffractometer NMR spectrometer UV spectroscopy HPLC analysis LC-MS analysis Electrical resistance System

pH ­conditions, the attached sensitive groups protonate, resulting in repulsion between polymer chains and leading to destabilization of micelle and release of entrapped drug. Cong et al. prepared a pH-sensitive drug delivery system composed of crosslinked chitosan micelles loaded into alginate hydrogel matrix for oral delivery of emodin. Chitosan micelles were prepared by crosslinking chitosan using calcium chloride solution. These micelles were dispersed in trisodium citrate dehydrate and mixed with sodium alginate solution under mixing. The stability of hydrogel was further increased by allowing sodium alginate to crosslink with calcium chloride solution. The hydrogel micelle composites showed lower swelling and degradation in simulated gastric fluid (less than 10% drug release) and complete swelling and release in simulated colon fluid confirming the potential of these systems for colon-specific controlled drug delivery [84]. Qiu et al. reported that conjugation of 2-(Octadecyloxy)-1,3-dioxan-5-amine with HA resulted in pH-responsive graft copolymer. Micelles prepared by this conjugate were able to load doxorubicin that demonstrated efficient internalization via CD44 receptor-mediated endocytosis. In vivo studies indicated higher accumulation of drug in tumor sites [85].

352

Polysaccharide Carriers for Drug Delivery

Benjamin et  al. synthesized pH-responsive dextran block copolymer using c­ opper-mediated click chemistry. The micelles were prepared using dextran blocks modified by combined microwave irradiation and aniline-mediated reductive amination methods. The resulting micelles were stable at neutral pH that degraded under slightly acidic conditions and enhanced the stability of hydrophobic drugs like curcumin in aqueous solutions for extended periods. The authors finally concluded that modified polysaccharides resulted in production of stable and biocompatible micelles in nanosize range that are sensitive to pH and produced sustained drug release with enhanced stability of loaded drugs in aqueous environment [86]. The micelles prepared by Blanco-Fernandez et  al. using dextran in combination with poly(2-aminoethylmethacrylate) and poly(N-isopropylacrylamide) showed dual sensitivity to both pH and temperature. Dextran grafting was performed via controlled radical polymerization and methotrexate was loaded into the prepared micelles. The micelles showed complete drug release, high internalization in tumor cells with enhanced cytotoxicity when compared to the free drug [57]. Literature also cites various examples on the use of pH responsive micelles [87, 88], pH-/light-responsive [89], and tumor microenvironment-responsive micelles [55] in drug delivery.

12.5.2 Polysaccharide micelles as targeted delivery systems Polysaccharide polymeric micelles are useful in delivering water-insoluble anticancer drugs due to their smaller particle size, higher solubilization capacity, core shell structure, and good in vitro stability. The smaller size of the micelles combined with PEGylation generally aids in escaping RES uptake and passive targeting by EPR effect. Further, the linkage of specific ligand to their surface makes them more amicable for targeted therapy along with passive targeting. Nowadays, the use of HA in targeted delivery systems has increased due to its tumor-targeting ability, especially stem cells, due to binding of HA to CD44 receptor, improved drug distribution in tumor tissues, and possibility of overcoming the MDR effect as discussed by Dosio et al. [35]. Uthaman et al. showed the targeting ability of HA-based micelles loaded with IR 780 iodide to enhance the photothermal therapy effects in tumors by CD44-targeting and EPR effect using fluorescence. Upon irradiation with an 808-nm laser, a significant enhancement in toxicity to tumor tissue was observed without causing any toxicity to other organs [90]. Lee et al. synthesized HA paclitaxel conjugates using a novel solubilization methodology and assessed antitumor activity of the resulting micelles in cell lines. These conjugates formed nanomicelles in aqueous solution under acidic condition and resulting micelles exhibited higher cytotoxicity in HA receptor overexpressing cancer cells than in HA receptor-deficient cells. Based on these results, the authors suggested that these HA-paclitaxel conjugate micelles can be potentially utilized as tumor-specific nanoparticulate therapeutic agents [48]. Hu et al. prepared stearic acid-grafted chitosan micelles using mitomycin C as a model drug and studied effect of PEGylation on the cellular uptake of respective micelles in normal and tumor cells. The results did not show any significant differences in cellular uptake of PEGylated micelles and non-PEGylated micelles. However, a significant reduction in internalization of micelles into macrophage was observed after

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PEGylation. In vitro antitumor studies showed significant increase in cytotoxicity of the drug from stearic acid-grafted chitosan micelles [91]. Polysaccharide or modified derivatives were used to prepare micellar nanocarriers for site-specific delivery of many anticancer drugs using various targeting approaches such as intracellular targeting [92, 93], transferrin receptors [94], folate receptors [67], CD44- and EPR-based tumor accumulation [52], asialoglycoprotein receptor [95].

12.5.3 Polysaccharide micelles for topical delivery Enhanced permeability of drugs through the stratum corneum by nanocarriers has gained attention of pharmaceutical industry to focus on the development of nanocarriers for topical/transdermal delivery of various therapeutic agents for local or systemic effect. However, the clinical application of nanosystems for topical delivery is still under debate due to insufficient understanding of interaction between nanocarriers and the skin. Use of biodegradable and biocompatible materials like HA, a constituent of extracellular matrix, has shown promising results. Smejkalova et al. developed micelles with two acylated derivatives of HA, loaded with Coenzyme Q10 and their in vitro skin penetration from excised porcine skin was compared with that of nonpolymeric micelles. The fluorescence and confocal image studies indicated enhancement in the cutaneous availability due to increased permeation through stratum corneum into deeper epidermal and dermal layers of loaded drugs using HA polymeric micelles. The authors also studied the mechanism of transport and effect of molecular size of loaded molecules on the skin permeation. The results revealed that both HA and drug enters into the skin layers through transcellular pathway and permeation decreased with increasing molecular weight of the drug molecule [56]. Dellera et al. developed chitosan oleate ionic micelles encapsulating silver sulfadiazine for wound healing. Encapsulation of drug into the micelles markedly improved solubility and also accumulation of drug at the site of action in higher concentration. Further, encapsulation of drug inside the micelles protected the human cells against drug cytotoxicity without affecting its antimicrobial properties. The micelles also showed good compatibility with platelet lysate allowing its association with platelet lysate. Overall, chitosan oleate micelles showed improved delivery of silver sulfadiazine to wounds [96]. Cerchiara et  al. reported increased permeability of hydrophilic molecule (propranolol HCl) from the chitosan hydrogels compared to pure drug solution. The authors have synthesized different conjugates using high-molecular-weight chitosan (MW 600,000) with degree of deacetylation 85% and crosslinkers such as lauric, myristic, palmitic, and stearic acid in presence of N-hydroxysuccinimide. Conjugation was confirmed by NMR and FTIR studies. Hydrogels were prepared by dispersion of resulting conjugates in water glycerol mixture (5:1) and adding lyophilized propranolol HCl. In vitro permeation of drug through porcine skin showed enhanced permeation of drug from hydrogels compared to commercial propranolol hydrochloride. Among the conjugates, the permeability of the drug was higher from the lowest length of the crosslinker acyl chain conjugates (chitosan laurate and

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chitosan myristate) hydrogels compared to conjugates containing longer acyl chain (chachitosan palmitate and chitosan stearate) hydrogels [97].

12.5.4 Polysaccharide micelles in oral delivery Intravenous administration of drugs may initially cause a sudden rise in levels of toxic molecules and minimum levels at the end of dosing interval due to faster clearance. Further, parenteral dosage forms require stringent sterility conditions that increase the cost. In contrast, the oral route has been the major route of drug delivery for chronic treatment of number of diseases and is the simplest and easiest way of administering drugs. However, factors like low aqueous solubility, slower dissolution, and poor permeability of the drug molecules hinder the oral bioavailability leading to therapeutic failure. Recently, micellar delivery systems made of grafted polysaccharide copolymers showed promising results in the delivery of poorly soluble molecules by oral route. The hydrophobic portion of these amphiphiles solubilizes the hydrophobic drug molecule; mucoadhesiveness offered by few polymers increases the residence time and promotes drug permeation leading to enhanced bioavailability. Micelles produced by modified polysaccharide containing Pluronic block copolymers further improve the bioavailability by inhibiting the activity of P-glycoprotein (P-gp)-mediated drug efflux and Cytochrome P450 metabolism [98]. Francis et al. investigated the suitability of dextran-g-polyethyleneglycolalkyl ether (DEX-g-PEG-C16) micelles for the oral delivery of a poorly soluble drug cyclosporine A, whose oral bioavailability is limited by its hepatic metabolism in presence of CYP3A4 and P-gp efflux in the small intestine. Attachments of small number of polyethylene glycol n-alkyl ether chains onto the dextran backbone impart ability to self-assemble in presence of aqueous environment and also ability to load hydrophobic drugs like cyclosporine A. Solubility of cyclosporine A in micelles was enhanced with increasing number of polyethyleneglycolalkyl ether units grafted per dextran chain and decrease in dextran molecular weight. The cytotoxicity of DEX-g-PEG-C16 polymeric micelles was significantly lower than that of free PEG-C16 molecules toward Caco-2 cells, as tested by MTT cytotoxicity [72]. Zhang et al. evaluated the permeability and bioavailability of insulin loaded in micelles prepared from chitosan modified with N-octyl-N-Arginine, a permeation enhancing peptide. Chitosan was modified to N-octyl chitosan and then allowed to react with activated l-arginine solution for 48 h under stirring. This results in N-arginine chitosan, a positively charged conjugate. The micelles were produced through self-­ assembly of positively charged N-arginine chitosan and negatively charged insulin under magnetic stirring. The obtained micelles were of size in the range of 257.5– 327.3 nm, with zeta potential +4.61 to +6.31 mV, 39.4%–75% entrapment efficiency and 8.1%–13.4% drug loading. Incubation of formulation in simulated intestinal fluid showed 50% of insulin recovery after 4 h incubation, compared to only 6.4% recovery from plain insulin solution, indicating protective effect of micelles. Apparent permeability coefficient was found to be increased approximately 22-fold for insulin from micelles compared to plain solution. In vivo hypoglycemic studies resulted in 25.9%

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reduction in blood glucose levels at 2 h, with 16.8% relative pharmacological availability compared to subcutaneous injection of 2 IU insulin/kg [99]. Literature also cites use of various polysaccharides and their derivatives in oral delivery of different drug molecules including paclitaxel [24, 100, 101], docetaxel [22], gambogic acid [102], meloxicam [87], doxorubicin [103], and curcumin [88].

12.5.5 Polysaccharide micelles for ophthalmic delivery Topical administration is the most preferred route in ocular delivery due to its ease of administration and noninvasive nature. However, the natural protective mechanisms of the eye like lacrimal drainage, low capacity of human cul-de-sac, dilution by tears, and metabolizing enzymes in lachrymal fluid combined with poor permeation through cornea limits the availability of drug applied by topical administration. Polymeric micelles produced from either natural or grafted polysaccharides provide a promising approach in ocular drug delivery due to its ability to load hydrophobic drugs, ­permeation-enhancing activity, and mucoadhesion properties, which improve drug permeability across ocular epithelia. Di Prima et  al. developed inulin-based mucoadhesive micelles loaded with different corticosteroids (dexamethasone, triamcinolone, and triamcinolone acetonide) and assessed their ability to penetrate across the corneal barrier. Grafting of ethylendiamine and retinoic acid on to the backbone of inulin improved the hydrophobicity and mucoadhessive properties. The resulting copolymer produced stable micelles that demonstrated augmentation in drug permeability coefficient and flux across corneal epithelial cells, thus reducing drug loss due to retention inside the cells, with a great potential for clinical use [104]. Pepic et al. prepared micelles composed of Pluronic F127 and cationic polysaccharide chitosan and loaded dexamethasone as a hydrophobic model drug. The resulting micelles were in the size range of 25–30 nm with a zeta potential around 10–20 mV and 0.4%–0.58% drug loading. Micelles containing chitosan showed enhancement in dexamethasone release rate and its transportation across Caco-2 cell monolayers, as compared to the chitosan-free F127 micelles. In  vivo studies in rabbits showed 2.4-fold significant increase in ocular bioavailability of dexamethasone from chitosan F127 micelles compared to commercial dexamethasone eye drops [105]. Various other studies were also reported in the literature on the potential of polysaccharide micelles in improving ocular delivery of metipranolol [106], cyclosporin A [107], dexamethasone [26], indomethacin [108].

12.5.6 Polysaccharide micelles for parenteral delivery Delivery of hydrophobic drugs by parenteral route is more problematic due to insolubility. The methods used to deliver hydrophobic drugs currently use cosolvents or surfactants. However, these may precipitate upon dilution with physiological fluids or the excipients used may be toxic and produce adverse reactions [109]. For example, Cremophor EL, which is used to administer paclitaxel, causes hypersensitivity reactions at the injection site along with neuro- and nephrotoxicity [110].

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Kumar et al. developed novel chitosan-grafted-Pluronic F-127 copolymer-based nanomicelle for the sustained delivery of amphotericin B with improved activity. Chitosan g-Pluronic F127 copolymer was synthesized by carbodiimide crosslinking method. The prepared polymer was purified by dialysis and amphotericin B was loaded into micelles by ultrasonication at different drug to polymer ratio. Resulting micelles were of 150-nm particle size range, and surface charge was found between −21.2 to +31.1 mV by changing drug to polymer ratio from 1:1 to 1:4. Higher concentrations of polymer induced positive charge to the micelles. Drug loading was found to be 33.3% and in vitro drug release showed sustained drug release (80 % after 48 h). In vivo toxicity studies showed reduction in adverse effects of amphotericin B compared to Fungizone (marketed product) treated mice indicating parenteral administration of amphotericin B in chitosan-g-Pluronic f127 micelles as safer delivery system [111]. Pang et  al. developed a prodrug of quercetin for targeted delivery using HA as polymeric carrier. Quercetin was conjugated via hydroxyl group to adipic dihydrazide-­ modified HA. The conjugate on addition to water resulted in micellar carriers with the diameter of 172.1 nm, potential of −20.3 mV and showed pH-sensitive sustained release with initial burst effect. In vitro cell line studies showed higher cytotoxicity toward CD44-expressing cells. Pharmacokinetics after single intravenous administration in rats showed significantly higher plasma concentrations and delayed mean residence time of quercetin from micelles compared to solution. Significant tumor inhibition with micellar carriers was observed in in vivo tumor models compared to free quercetin [44].

12.6 Marketed products/patents/technologies available To date (based on clinical trial.gov.in), there were no reported clinical trials on polysaccharide micelles for drug delivery. However, two trials (phase I) were published in journals on dextran conjugates with anticancer agents like dextran—DOX (AD-70) and dextran-exatecan (DE-310). These conjugates form micelles in aqueous environment due to their amphiphilic nature. In one of the studies conducted using dextran-conjugated doxorubicin (AD-70, DOX-OXD) in 13 patients at 3 dose ranges ranging from 12.5 to 40 mg/m2 doxorubicin equivalent showed profound but reversible thrombocytopenia and hepatotoxicity. Doselimiting toxicities were observed in two patients at 40 mg/m2 and significant toxicity in two patients at a dose of 20 mg/m2. Based on the results, they have recommended 12.5 mg/m2 doxorubicin equivalent will be the suitable dose for phase II study [112]. In another study, 27 patients received a total of 86 administrations of dextran-­exatecan (DE-310). Neutropenia and grade 3 thrombocytopenia, and grade 3 hepatotoxicity with veno-occlusive disease, were dose-limiting toxicities observed. Overall, there was enhancement in efficacy and reduction in toxicity of topoisomerase I inhibitors [113].

12.7 Conclusions and outlook The unique self-assembling feature along with biocompatibility and biodegradability offered by polysaccharide micelles has gained interest on their use as potential carrier

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to improve the delivery of hydrophobic drugs. The targeting ability of HA to CD44 cells and the mucoadhesive nature of chitosan have rendered them as superior carriers in targeted delivery, especially for anticancer agents. Even though animal studies have demonstrated success in reducing tumor burden, preventing metastasis, and overcoming the drug resistance, few trials in humans has revealed dose-limited toxicities due to uptake of these particles by reticuloendothelial system (RES). This calls upon requirement of extensive work in area of safety and improved understanding of these molecules before achieving clinical success.

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Polysaccharide nanoparticles for cancer drug targeting

13

Yuefei Zhu, Yiyang Liu, Kai Jin, Zhiqing Pang School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai, China

Abbreviations ALG ApoG2 BBB CG CGH-NPs Chol CI COX-2 CPT CS CSaSt CS-SA-nps Cy5.5 DCT Dex-PLGA DHA-NPs DOX EphB4 EPR ETB FA FA-CS-UA-NPs FTIR GA GAGs GC GCNP-ES GTAC HA HACE HAS HAS1 HAS2 HAS3 HA-SPIONs

alginate apogossypolone blood brain barrier cationically modified gelatin CS copolymer‑gold hybrid nanoparticles cholesterol chitosan oligosaccharide-indomethacin conjugate cyclooxygenase 2 camptothecin chitosan cationic stearic acid-grafted starch chitosan/sodium alginate nanocomplexes cyanine 5.5 docetaxel amphiphilic copolymer poly(lactic-co-glycolic acid)-g-dextran DOX-loaded HA nanoparticles doxorubicin Ephrin receptor B4 enhanced permeation and retention erlotinib folic acid folate modified-chitosan nanoparticles loaded with ursolic acid Fourier transform infrared spectroscopy gum arabic glycosaminoglycan particle nanoclusters galactosylated chitosan estrone-modified glycol chitosan nanoparticles glycidyl trimethyl ammonium chloride hyaluronic acid hyaluronic acid-ceramide hyaluronan synthases hyaluronan synthases 1 hyaluronan synthases 2 hyaluronan synthases 3 hyaluronan-coated superparamagnetic iron oxide nanoparticles

Polysaccharide Carriers for Drug Delivery. https://doi.org/10.1016/B978-0-08-102553-6.00013-1 Copyright © 2019 Elsevier Ltd. All rights reserved.

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HES IR LCST LHRH LNCAP MCF-7 MLDC NCs MRP MSRS-CS-MCs MTD MW NIR PDI PDP PEC PEG pegfp PEI PEMs PLL PMAA-PS 80-g-St PS 80 PTX RITC ROS SA SA siRNA STEMI T-CS TEM TMC TPP

Polysaccharide Carriers for Drug Delivery

hydroxyethyl starch inhibition rate lower critical solution temperature luteinizing hormone-releasing hormone prostate cancer cell line human breast adenocarcinoma cell line mulberry-like dual-drug nanoparticles multidrug resistance-associated protein multistimuli responsive smart chitosan-based microcapsules maximum tolerated dose molecular weights near infrared polymer dispersity index pentadecyl phenol polyelectrolyte complexation poly(ethylene glycol) plasmid encoding for green fluorescent protein polyethyleneimine proton exchange membranes poly-l-lysine poly(methacrylic acid)-polysorbate 80-grafted-starch polysorbate 80 paclitaxel Rhodamine B isothiocyanate reactive oxygen species sodium alginate stearic acid small interfering RNA ST-segment elevation myocardial infarction a homing peptide modified chitosan-g-stearate polymer micelle transmission electron microscopy trimethylated chitosan tripolyphosphate

13.1 Introduction As cancer is one of the major causes of mortality worldwide today, cancer therapy is no longer just a focal point of researchers but also an involved problem that needs to be solved urgently in clinical practice. Over previous decades, the design of new treatments for cancer has been facilitated by the understanding of the tumor microen‑ vironment [1]. Hence, the current treatments for cancer may go far beyond the conven‑ tional therapeutic options (i.e., radiation therapy and chemotherapy). Recent studies have brought nanoparticle drug delivery systems into the mainstream, developed to surmount the obstacles in conventional drug delivery for cancer. The unprecedented expansion in the field of nanomedicine arises and brings about innovation in thera‑ peutic strategies, with the development of new nanoparticles for the diagnosis and treatment of various cancers.

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Greater targeting selectivity and better delivery efficiency are the major advantages of nanomedicines in cancer therapy. Nanoparticles possess unique biological prop‑ erties given their mesoscopic size range of 5–200 nm, permitting them to overcome the poor biopharmaceutical characteristics of chemotherapeutic agents including non‑ specific biodistribution, poor water solubility, endothelial association/extravasation and targeting, as well as low therapeutic indices. Moreover, their ultra-small volume, tunable size, shapes, and surface characteristics endow them high stability, high drug loading capacity, high compatibility, and the ability to incorporate both hydropho‑ bic and hydrophilic substances, thereby making them highly appealing in many as‑ pects of medicine. Once used as carriers, these nanoparticles will either entrap and encapsulate drugs or bind drugs on their surface. They can protect drugs from dena‑ turation and degradation during the drug delivery process. Furthermore, the large sur‑ face area-to-volume ratio capacitates them to absorb, bind, and carry large-­molecule compounds like DNA, RNA, and proteins. The innovative design and chemical func‑ tionalization of nanoparticles have boosted and brought about new nanoparticle drug delivery systems. Polymeric materials play a pivotal role in both the material world and modern in‑ dustrial economics. They are solid, nonmetallic compounds of high molecular weight [2]. Polymeric materials used for preparing nanoparticles are not only manifold, but also involute with varied stimuli-sensitive linkers such as disulfide, hydrozone, etc. Furthermore, drug delivery systems can be prepared with server materials such as viruses (viral-like nanoparticles), lipids (liposomes), and polymers (macromolecules, micelles, or dendrimers) with distinct characteristics. Among them, as the biodegrad‑ able polymers found in all living organisms, polysaccharides turn out to be the most recognized biomaterials to prepare nanoparticles for targeting drug delivery systems. Polysaccharides are natural carbohydrate polymers containing from approxi‑ mately 35 (usually >100) to as many as 60,000 monosaccharide units. These poly‑ mers were perfected with many characteristics from nature, which include energy storage (starch and glycogen), structural support for plants (cellulose), interphase adhesion in the cell walls of plants (hemicellulose), components in plant cell walls and middle lamellae (pectin), structural roles for aggrecan assemblies and extra‑ cellular components (hyaluronic acid), structural support in arthropods (chitin), cell wall constituents (alginate), anionic extracellular cell wall constituents (gellan gum), and components in the seed husk (psyllium husk). Thus polysaccharides are ideal candidates for drug delivery and biomedical applications, which can be eas‑ ily obtained from natural sources. On account of the composition of monosaccha‑ ride units, polysaccharides can be classified as homopolymers (i.e., formed from the same monosaccharide repeats, like glycogen, starch, cellulose, pullulan, pectin, etc.) or heteropolymers (i.e., formed from different monosaccharide units, like chitosan (CS), heparin, hyaluronic acid (HA), chondroitin sulfate, keratan sulfate, heparan sulfate, and dermatan sulfate) [3]. With a wide range of general structures (from linear to various branched structure) and shapes, polysaccharides can be subjected to an extensive range of chemical and enzymatic reactions. Recent studies have con‑ sidered polysaccharides as amorphous polymers, which may provide a safe, biode‑ gradable, and tunable option for making newer and smarter materials for biomedical

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applications. Particularly, with hydrophilic groups such as carboxyl, hydroxyl and amino groups, most natural polysaccharides could form noncovalent bonds with bio‑ logical tissues (mainly epithelia and mucous membranes), forming bioadhesion [4]. Those polysaccharides—such as CS, starch, and alginate—are all benign bioadhesive materials, which once engineered as nanoparticles for drug delivery could prolong the residence time and therefore increase the absorbance of loaded drugs. Moreover, those functional groups empower them for the ideal preparation of a wide array of nanoparticles. Miscellaneous polysaccharide derivatives can be synthesized by easy chemical or biochemical modifications due to the presence of other derivable groups. Hence, a polysaccharide-based drug delivery system may have a promising future for cancer-targeting therapy. As for polysaccharide-based nanoparticles, recent studies have revealed great advantages of those nanocarriers in drug delivery systems. Firstly, polysaccharidebased nanoparticles possess high drug loading capacity resulting from the elonga‑ tion and branching of their chains. Moreover, polysaccharide-based nanoparticles can encapsulate or immobilize a high amount of anticancer drugs through covalent or noncovalent interaction, which results from their relatively large (functional) surface area. Secondly, polysaccharides have biological functions including energy storage, protection of cell organelles, and easy modification with peptides or pro‑ teins, which might endow nanocarriers with additional advanced properties. Thirdly, ­polysaccharide-based nanoparticles may possess high hydrophilicity. In particular, the majority of natural polysaccharides have hydrophilic groups such as carboxyl, hydroxyl, and amino groups, which could form noncovalent bonds with biological tissues (mainly epithelia and mucous membranes), forming bioadhesion. As a conse‑ quence, polysaccharide nanoparticles could prolong the residence time and increase the absorbance of loaded drugs, which ensures they will be a promising delivery system in cancer treatment [5]. Fourthly, the protein-repellent property of these nanoparticles renders their interaction with certain proteins/cell surfaces, making carbohydrates very promising elements for the construction of future therapeutics [6]. For instance, it has been reported that hydroxyethyl starch (HES), a synthetically modified starch derivative, dextrin, or other saccharides can cut down the protein adsorption on nanocarriers and prolong their circulation time in the bloodstream. Fifthly, polysaccharide-based nanoparticles are biocompatible, biodegradable, and nonimmunogenic owing to their natural origin. For this reason, polysaccharides are also often employed in hybrid nanostructures to reduce the toxicity of synthetic ma‑ terials (e.g., poly(ethyleneimine) or PEI) [7]. This review article will focus on polysaccharide-based nanoparticle drug delivery systems for cancer therapy. Firstly, it will elucidate and address the types and char‑ acteristics of polysaccharides, specifically highlighting the nature of the polymer em‑ ployed for the preparation of targeted nanoparticles and the fabrication method for the architecture. Secondly, it will present the design philosophy and application of poly‑ saccharide nanoparticles in cancer drug delivery including small chemotherapeutics, genes, and proteins. Finally, perspectives on design and translation of polysaccharide nanoparticles for cancer targeting will be provided.

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13.2 Polysaccharides 13.2.1 Chitosan Composed of randomly distributed β-(1,4)-linked d-glucosamine and N-acetyl-dglucosamine units, CS is a linear and positively charged polysaccharides derived from natural sources of chitin, one of the main components of marine crustacean shells and the cell wall of fungi [8]. As a cationic polysaccharide, CS is a very popular candidate among natural polysaccharides for nucleic acid complexation and drug delivery [8]. It is also correspondingly known as amino polysaccharide [9]. Being part of the poly‑ saccharide family, the major properties of CS are biocompatibility, biodegradability, antibacterial, low toxicity, and low immunogenicity, which are in line with other poly‑ saccharides [10–12]. For instance, its biodegradability and the similarity of its chemical structure to extracellular matrix glycosaminoglycans render it an attractive biopolymer for tissue repair [13, 14]. In addition, CS possesses other biological properties such as mucoadhesion, transfection, controlled drug delivery, colon targeting, permeation enhancement, in situ gelation, a remarkable affinity to protein, and efflux pump inhibi‑ tion, all of which are credited to its primary amine functional group [15, 16]. Besides, more salutary features of CS and CS derivatives are their permeation-enhancing and mucoadhesive properties, explaining their frequent use in tissue engineering or mu‑ cosal drug delivery [17]. The versatility and multifunctional nature of CS is due to its active amino groups which may act as the reactive site for a variety of new group attachment under mild reaction conditions. Structurally, CS has one amino (NH2) group and two hydroxyl (OH) groups in each repeating glycosidic unit [18]. It can remain as a polycationic species, owing to protonation of amino group with increased soluble prop‑ erty at low pH [19, 20]. On account of its surficial positive charges, CS can interact with negatively charged molecules such as polyanions, nucleic acids, polysaccharides, and negatively charged proteins [21, 22]. Moreover, CS’s cationic property can also be re‑ versed via sulfonation to introduce an anionic character, with better paste fluidity, high water reducing ratio, water soluble, and anticoagulant property [23]. Its preparation procedure and original sources vary its molecular weight (MW), which ranges from 300 to over 1000 kDa with a degree of deacetylation from 30% to 95% [24]. Initially, CS was used universally in medical applications such as slimming, wound dressing, and tissue engineering. As research progressed, CS has gradually been developed as a prominent candidate for drug delivery systems [25]. In addition, CS has already been used intensively for drug delivery systems in virtue of its low-cost and well-established safety [26]. It has also been slated for dietary applications in Italy, Finland, and Japan and has been approved for wound dressing by the FDA [27, 28]. Despite these unique and positive characteristics, CS has some disadvantages that impose restrictions on its application. Firstly, CS has poor solubility in neutral and al‑ kaline conditions, resulting from the linear aggregation of chain molecules. Likewise, the intermolecular hydrogen bonding of CS also causes the rigid crystalline structure [29]. Secondly, CS has finite biological activity compared with commercial drugs, lagging behind the standards of commercial development [22]. Thirdly, CS has low

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transfection efficiency on account of its relatively low cationic density which causes less compact of CS/gene complexes [30]. Fourthly, natural CS possesses high crystal‑ linity and lacks mobile protons; it has low proton conductivity and thus it is difficult to meet the requirement of proton exchange membranes (PEMs). To overcome these shortcomings, many modification strategies, including chemical cross-linking [31], chemical modifications [32], and organic-inorganic composite [33], have been used. Moreover, the modification of the structure of pristine CS to improve its solubility and other properties has been used frequently in drug delivery systems. It appears that various CS derivatives are obtained by conjugating different molecules to CS’s back‑ bone. For example, trimethylated chitosan (TMC) [34], galactosylated chitosan (GC) [35], N-alkylated chitosan [36], N-acetylated chitosan [37], N-dodecylated chitosan [38], PEGylated chitosan [39], TAT peptide (cell-penetrating peptide RKKRRQRRR) chitosan [40], chitosan quaternary ammonium salt, thiolated methylated dimethylam‑ inobenzyl chitosan [37], and so on.

13.2.2 Hyaluronic acid As a promising hydrophilic material in the area of nanoparticle system design, HA is one of the most common glycosaminoglycans found in vertebrate tissues and is responsible for carrying out certain vital activities [41,42]. The name of this com‑ pound stems from the Greek word hyalos meaning “glass” which refers to its physical properties [43]. It is a linear polysaccharide composed of alternating d-glucuronic acid and N-acetyl-d-glucosamine units, which are connected repeatedly by β-1,3 and β-1,4 glycosidic bonds. The chemical structure of HA is fairly regular, with the exception of occasional deacetylated glucosamine residues [44]. Moreover, it mostly indwells in the extracellular matrix, synovial fluid of articular joints, and the vitreous humor of the eye [45]. HA is synthesized by three transmembrane hyaluronan synthases (HAS1, HAS2, and HAS3) on the inner surface of the cell membrane, and secreted in the ex‑ tracellular matrix [46]. Primarily occurring in vivo as sodium hyaluronate, HA plays a significant role in the maintenance of moisturization of tissues and joint lubrication in the hydration, the transition of matter from tissues, the movement, differentiation, and division of cells [47]. Compared with other natural and synthesized biopolymers, HA enjoys some pronounced properties such as high water holding capacity, high vis‑ coelasticity, nonimmunogenicity, degradability, plasticity, and good biocompatibility. HA is hydrophilic, and its high water holding capacity renders it to uptake water and expand its solid volume by up to 1000 times. Thus it can further form a very elastic and viscous gel with a large hydrodynamic volume [48, 49]. With the capacity of bind‑ ing a large number of water molecules, HA has been widely applied to the fields of surgery, arthritis treatment, drug development [50]. It has been shown that hyaluronic acid plays a role in important biological functions such as water retention, provision of nutrients for cells, and removal of wastes from cells, especially for cells without direct blood supply, such as cartilage cells [51]. In oncotherapy, HA can specifically bind to cancerous cells, where HA receptors are overexpressed. Exploiting this ligand-­ receptor interaction, HA-based nanoparticle is now a rapidly growing platform for targeting CD44-overexpressing tumor cells to improve anticancer therapy.

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13.2.3 Alginate Alginate is an anionic and hydrophilic polysaccharide. Commonly, alginate is one of the most affluent biosynthesized natural materials and is derived primarily from two sources: marine plants, i.e., brown seaweed (40% of dry matter) and bacteria [52, 53]. As a water-soluble linear polysaccharide, alginate comprises linear blocks of (1→4)-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) monomers in structure presentation [52]. These blocks consist of three different forms of poly‑ mer segments: consecutive G residues, consecutive M residues, and alternating MG residues. Thus the copolymer composition, sequence, and molecular weights may vary with the source and species that produce the copolymer [54]. This unbranched polysaccharide is a natural copolymer and is an important component of algae such as kelp; it is also an exopolysaccharide of bacteria including Pseudomonas aeruginosa [55]. Owing to the abundance of algae in water bodies, there is a large amount of alginate material present in nature. Alginate can be obtained from both algal and bacterial sources. Commercially, available alginates currently derive only from algae [56]. Alginate has been extensively reviewed with respect to its physical and chem‑ ical features [57]. Generally referred to the natural biopolymer family, alginate also enjoys excellent biodegradability, biocompatibility, nontoxicity, chelating ability, and relatively low cost [58]. Moreover, it demonstrates strong gelling properties and re‑ markable convenience for processing and modification [59]. Alginate possesses strong ability to cross-link and form network in aqueous solutions. It is ionotropic upon interand intramolecular cross-linking with divalent ions to form hydrogels [60]. Under this occasion, alginate is of growing importance in the pharmaceutical industry. Alginate is included in tablets to improve the bioadhesive property of buccal adhesive tablets [61]. Although alginate has so many inspiring features useful for drug delivery and cell immobilization, the high hydrophilicity of alginate may be disadvantageous as it can cause low selectivity and poor membrane stability. Hence, chemical modification via covalent cross-linking can be performed for membrane property enhancement [55]. Recently, alginate derivatives have been applied to improve drug delivery efficiency. Most alginate derivatives are synthesized via acetylation of alginates, phosphorylation of alginates, sulfation of alginates, and hydrophobic modification [55]. Consequently, modification of alginates to obtain unique and ideal characteristics leads to prolonged and desired drug release behavior compared with unmodified alginate gels.

13.2.4 Heparin Heparin is a sulfated polysaccharide belonging to the class of glycosaminoglycans which consists of 1,4-linked disaccharide repeating units of uronic acid and glucos‑ amine residues [62]. Widely used as an anticoagulant and antithrombotic drug, heparin is merely produced in mast cells, where it is cleaved from the core protein (serglycin) at the end of synthesis [63]. In clinical application, heparin can be explored to mod‑ ulate numerous biological processes. Moreover, heparin sulfate is the derivative of heparin which is also a linear polysaccharide. Both biopolymers are crucially involved in various biological processes, including regulation of anticoagulation, ­angiogenesis,

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suppression of inflammation, and cancer progression. As an anticoagulant drug, hep‑ arin not only has anticoagulant properties but also exerts a broad range of cytoprotec‑ tive actions, including prevention of apoptosis and modulation of vascular adherence [64]. It has already been demonstrated that heparin may reduce the risk of ischemic events among patients with ST-segment elevation myocardial infarction (STEMI) a decade ago [65]. In addition to the broad properties of heparin, it can interact with a variety of functional proteins such as extracellular matrix components, growth fac‑ tors, and adhesion molecules via electrostatic interactions. Heparin has capacity to bind with the serine protease inhibitor, antithrombin, which is responsible for some selectivity and specificity toward cancer cells [63, 66]. It is also noteworthy that low-­ molecular-weight species of heparin appear to prolong the survival rate of patients with cancer [67].

13.2.5 Starch As the second largest biomass on earth, starch is a natural, cheap, available, renew‑ able, and biodegradable polymer [68]. Typically, it is isolated from plants in the form of microscale granules. Normal starch in granular form is generally composed of two types of molecules: amylose and amylopectin [69]. Amylose is a linear polysaccharide obtained from plant extracts and an unbranched constituent of starch (15%–30%) con‑ sisting of several thousands of glucopyranose units (α-1,4-d-glucose) linked through α (1→4) bonds [53]. Amylopectin is a branched structure containing both (1,4)-αd linkages between d-glucose residues and (1,6)-α-d branch points [70]. Therefore, starch turns out to be a flexible polysaccharide in application. It is not only treated as a wholesome food, but is also used in industrial and clinical practice. Some properties such as low cost, worldwide availability, nontoxicity, and modifiability enable starch to meet the requirements of drug delivery systems. However, most native starches present limitations such as high viscosity, limited digestibility for some people, sus‑ ceptibility to retrogradation, and limited solubility [71]. Besides, most starches cannot always withstand extreme processing conditions, i.e., freeze thaw cycles, high tem‑ perature, alkali treatments, strong acid, and high shear rates [72]. Thus native starches are modified via different techniques to enhance or inhibit their inherent properties and obtain ideal candidates for novel drug delivery systems.

13.2.6 Dextran Similarly, dextran is an abundantly available natural polymer, which possesses renew‑ able, cheap, and nontoxic features. Dextran is defined as a homopolysaccharide of glu‑ cose with a linear backbone of α-linked d-glucopyranosyl repeating units. It belongs to α-d-glucans containing α-(1→6) linkage in the main chain and variable amounts of α-(1→2)-, α-(1→3)-, or α-(1→4)-branched linkages [73]. Dextran occurs naturally as a tiny proportion in foods such as refined crystalline sugar, sauerkraut juice, maple syrup, and honey, and also as a component of dental plaque [74]. Dextran has some admirable specialties including its colloidal nature, water solubility, hydrophilic, and is inert in biological systems, which has been a boon to researchers exploring dextran

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as a polymeric carrier in novel drug delivery systems [75]. Moreover, dextran enjoys a remarkable diversity in chain length and in physicochemical properties because of the variation in the degree of branching in their glucose backbone. Dextran also offers other useful features such as stability to alkali and acids at room temperature. These features enable dextran to be a commercially available product with applications in medicine [76]. Thus it has been extensively applied in multifarious drug delivery sys‑ tems such as micelles, hydrogels, core-shell type nanoparticles, and other morpholo‑ gies for targeted drug delivery to tumors [53].

13.2.7 Other polysaccharides As natural polymers, most polysaccharides are highly safe, nontoxic, stable biode‑ gradable, and biocompatible. They have abundant and various classifications and there are other polysaccharides like xanthan gum, pullulan, chondroitin, pectin, chondroitin sulfate, cyclodextrin, and so on [53]. These other polysaccharides also serve as a fan‑ tastic field for both drug delivery systems and industrial applications and extensive reviews on these polysaccharides can be found elsewhere [77].

13.3 Fabrication of polysaccharide-based nanoparticles 13.3.1 Covalently cross-linked polysaccharide nanoparticles Cross-linking of biodegradable polymers is momentous to control swelling and deg‑ radation rates. It is also an early preparation approach of polysaccharide nanopar‑ ticles. Among various polysaccharides, CS was an early one to be used to prepare nanoparticles [78]. During the fabrication of CS nanoparticles, glutaraldehyde was ever used as a cross-linking agent. As reported by Zhi et  al. [79], glutaraldehyde could solidify the chitosan polymer once it was added into the nanoemulsion system. Afterwards, CS nanoparticles with an average diameter of 22 nm were obtained. The effect of cross-linking on the adsorption capacity of CS nanoparticles was investi‑ gated via varying the glutaraldehyde concentration in its nanoemulsion [80]. For the CS molecules with two different molecular weights (MW), 10% glutaraldehyde gave higher adsorption capacity than 50% glutaraldehyde. Furthermore, the measurement on —NH2 content indicated that lower glutaraldehyde concentration generated higher content of —NH2. However, because of the toxicity of glutaraldehyde on cell viability, its utility in the field of drug delivery as a covalent cross-linker is limited. Along with the use of biocompatible cross-linkers, biocompatible covalent cross-linking is promising. With the aid of a water-soluble condensation agent of car‑ bodiimide, natural di- and tricarboxylic acids, including succinic acid, malic acid, tartaric acid, and citric acid, are employed for intermolecular cross-linking of CS nanoparticles. The condensation reaction is performed between the carboxylic groups of natural acids and the pendant amino groups of CS, through which biodegradable chitosan nanoparticles are obtained. This method allows the formation of polycations, polyanions, and polyampholyte nanoparticles. The prepared nanoparticles are stable

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in aqueous media at low pH, neutral, and mild alkaline conditions. Pujana et al. [81] reported the synthesis of CS nanoparticles via cross-linking with two biocompati‑ ble dicarboxylic acids: polyethylene glycol dicarboxylic acid and tartaric acid. The water-in-oil (W/O) microemulsion method yielded particle size around 10–15 nm in the dried state (TEM) and 200–700 nm in the swollen state from commercial chitosan. Moreover, polyethylene glycol dicarboxylic acid was shown to be more efficient for CS cross-linking than tartaric acid. All the synthesized nanogels showed improved water solubility and most of them were stable at physiological pH.

13.3.1.1 Ionically cross-linked polysaccharide nanoparticles Over the past two decades, nanoparticles prepared through ionic cross-linking of poly‑ electrolytes have been explored in a wide range of applications, including drug deliv‑ ery, medical imaging, and food science [82]. Compared with covalently cross-linking, ionic cross-linking enjoys more advantages, such as mild preparation conditions and simple procedures. For charged polysaccharides, low MW polyanions and polycations could act as ionic cross-linkers for polycationic and polyanionic polysaccharides, re‑ spectively [78]. For instance, the amidogens existing in the molecular chain of CS can protonize so that it can dissolve in dilute acid solutions. In the same way, the carbox‑ ylic acid of alginates’ residues can ionize to make a polysaccharide with a negative charge. Hence, these polyelectrolytes can aggregate into nanoparticles by means of cross-linking with micromolecular substances with opposite charge via electrostatic interaction [83]. To date, the most widely used polyanion cross-linker for the cat‑ ionic polysaccharide is tripolyphosphate (TPP) which is nontoxic and has multivalent anions [84]. Bhat et  al. [85] reported chitosan nanoparticles cross-linked with TPP. CS nanoparticles were developed by ionic gelation method using chitosan and TPP anion with some modifications according to their previous studies. The constructed nanoparticles were successfully conjugated with eurycomanone with high entrapment efficiency. Cross-linking of TPP-chitosan is based on the intermolecular interaction. The hydrogen boding and electrostatic interaction occurs between the hydrogen and amine groups of CS and TPP. The greater the concentration of TPP, the higher the den‑ sity of ionic cross-linking, which results in more compactness and smaller structure of the nanoparticles. Recently, calcium-cross-linked negatively charged polysaccharide nanoparticles have been explored as drug carriers. Some polysaccharides bearing carboxylic groups on molecular chains can be cross-linked by bivalent calcium ion to form nanoparti‑ cles. Angelescu et al. [86] prepared calcium alginate nanoparticles by using the waterin-oil microemulsion method. Sodium alginate (SA) and CaCl2 were solubilized in the aqueous phase of two microemulsions, and their mixing induced cross-linking of the alginate polymer by calcium ions. To examine the potential of nanoparticles for drug delivery, they tested the radial distribution of the chain. It was shown that lower densities were obtained near the center of the cavity and at the cavity wall, whereas local maxima denoting chain crossing were found at intermediate distances. The ra‑ dial extension was not strongly affected when both mono- and divalent ions were present, suggesting that the gel formation did not shrink further. Analogously, Daemi

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and Barikani [87] synthesized calcium alginate nanoparticles by ion-induced gelation method and characterized them with Fourier transform infrared spectroscopy (FTIR). Their results revealed significant improvement of size and distribution of calcium algi‑ nate nanoparticles with decrease of SA and increase of calcium cation concentrations, respectively. Owing to the random distribution of diequatorial and diaxial linkages, al‑ ginate chains possess different conformations that afford multivalent cations different interactions with each block. In addition, Wu et al. [88] showed nanoscale complexes currently being developed via ionic cross-linking can also play a significant role in biomedical applications. They examined in detail the effect of calcium ions on the binding of lysozyme to chitosan (CS)/SA nanocomplexes (CS-SA-NPs) to develop a complex system for lysozyme de‑ livery. In this research, the external gelation technique was used to modulate the prop‑ erties of lysozyme-loaded CS-SA-NPs by controlling calcium ions concentration. The results showed that with the increase of calcium ions (Ca2+) concentration, the value of zeta potential and loading efficiency increased and the addition of 5.0 mM Ca2+ contributed to uniform distribution of the nanocomplexes. Moreover, the release rate of lysozyme from CS-SA-NPs could be slowed by the increase of Ca2+ concentration.

13.3.1.2 Polyelectrolyte complexation (PEC) of polysaccharide nanoparticles Polyelectrolyte polysaccharides can form PEC with oppositely charged polymers through intermolecular electrostatic interaction. Apart from covalent cross-linking of polysaccharide biopolymers, polysaccharide-based PEC nanoparticles can be ob‑ tained by dint of adjusting the MW of component polymers in a certain range [78]. Theoretically, polyelectrolyte could interact with polysaccharides to fabricate PEC nanoparticles. However, in many practical cases, some polyelectrolytes are restricted to those water-soluble and biocompatible polymers in view of safety. Based on such a situation, CS has been found to be an ideal polysaccharide that can form nanoparticles via PEC. Moreover, the amino groups of CS are ionized at acid pH, thus making it hydrosoluble and positively charged. These properties enable it to interact with oppo‑ sitely charged polymers by intermolecular electrostatic interaction, forming PEC [89]. At present, there are many negative polymers complexed with chitosan to form PEC nanoparticles, which can be divided into polysaccharides, peptides, the polyacrylic acid family, and so on [78]. Most of these negatively biopolymers belong to the poly‑ saccharide family, such as dextran sulfate [90], alginate [91], and heparin [92]. Tan et al. [93] developed polysaccharide-based nanoparticles by the polyelectrolyte complexation between chitosan (CS) and gum arabic (GA) as novel drug delivery systems for curcumin. During the fabrication of nanoparticles, they found that the positively charged amino groups of CS could strongly interact with the negatively charged GA at pH 4.0 and mixing ratio of 1:1, forming hydrophilic, stable, and homo‑ geneous nanoparticles. Alginates are another potential experimental subject other than CS; Sarika and James [94] reported hybrid PEC nanoparticles, which were prepared from cationically modified gelatin (CG) and SA by electrostatic complexation be‑ tween the polymers. Moreover, just simple mixing of protonated CG and anionic SA

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formed these nanoparticles with spherical morphology. These PEC nanoparticles were found to be suitable matrixes for encapsulation and delivery of curcumin to carcinoma cells. They not only showed considerable encapsulation efficiency of curcumin, but also exhibited sustained release of curcumin in  vitro. Coincidently, Kim et  al. [95] also generated alginate nanoparticles by microfluidic-aided polyelectrolyte complex‑ ation. In the microfluidic mixing device, aqueous Ca-alginate pregels and cationic poly-l-lysine (PLL) solutions were mixed, and the polyelectrolyte complexation that subsequently occurred between the pregel and PLL resulted in precipitates, yielding alginate nanoparticles. Owing to the faster microfluidic mixing compared with the conventional bulk mixing method, the alginate nanoparticles exhibited enhanced sta‑ bility. In this way, the resulting alginate nanoparticles may have great potential in various biomedical applications, such as drug delivery, food processing, and enzyme immobilization.

13.3.1.3 Self-assembly of hydrophobically modified polysaccharides Self-assembly is an important mechanism to acquire new materials with different properties by exploiting only/mainly intermolecular interactions. It brings about far-ranging possibilities without involving breaking or forming the covalent bond. Self-assembly also allows more control of the properties due to the reversibility and response to external stimuli [96]. During the assembly process, biomacromolecules with well-defined monomer units are usually introduced to direct and control the as‑ sembly of particles to gain the desired structure [97]. Moreover, self-assembly is typi‑ cally driven by simple interactions such as electrostatic interaction, hydrogen bonding, hydrophobic interaction, and van der Waals forces [98]. In recent years, the selfassembly of nanoparticles into high-order and stable structures has become an attrac‑ tive strategy. Thus numerous studies have been carried out to investigate the synthesis and the application of self-assembled polysaccharide nanoparticles as drug delivery systems. Widely studied, self-assembled CS nanomaterials have been explored to meet varying applications, ranging from wound-healing to gene delivery. Owing to the natural properties and structure of CS, the intra- and intermolecular hydrogen bonds due to the presence of the ―OH and ―NH2 groups along the chitosan backbone contribute to the self-assembly of CS nanoparticles [98]. For the monocomponent system, the self-assembly of CS and its derivatives is typically driven by hydropho‑ bic interactions or hydrogen bonds; for the multicomponent system, self-assembly is typically actuated by electrostatic interactions or hydrogen bonds between CS and other molecules. In both cases, van der Waals interaction with the cross-linker may be part of the self-assembly process, but will not take the major role [98]. For prob‑ ing deeply into the mechanism of self-assembled CS, Dey et al. [99] investigated the self-assembly of chitosan, based on the proton balance between its protonated and deprotonated residues, to promote facile nanoparticle synthesis. The proton balance was observed to be dependent on pH modulation, which governed the degrees of pro‑ tonation of chitosan and helped to obtain the desired self-assembly during formation of CS nanoparticles, in the presence of a suitable cross-linker. It was noteworthy that

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the criticality of p­ rotonation was also dependent on the degree of deacetylation of chi‑ tosan. Furthermore, high protonation did not influence the self-assembly, in virtue of high repulsive forces between the residues and chains of chitosan. Likewise, complete deprotonation of CS led to agglomeration of the polymer, making it unsuitable for any further application. Therefore, neither complete protonation nor complete depro‑ tonation aided the self-assembly, and parity among the protonated and deprotonated groups led to equilibration of various driving forces like electrostatic force, hydro‑ phobic force, hydrogen bonding. Apart from CS nanoparticles, Pistone et al. prepared stable alginate nanoparticles by self-assembly using zinc as the cross-linker without the need for additional polycations [100]. They utilized multivariate evaluation to opti‑ mize the formulation factors for the preparation of alginate‑zinc nanoparticles by ion‑ otropic gelation. The researchers found that after using a high alginate concentration (0.09%), they obtained a reduction in the PDI by simultaneously increasing the ionic strength of the solvent and the zinc concentration.

13.4 Design and application of polysaccharide nanoparticles for cancer drug targeting 13.4.1 Chitosan-based nanoparticle delivery systems for cancer therapy Due to the favorable properties of CS, chitosan-based nanoparticle delivery sys‑ tems have achieved wide appreciation in cancer therapy. Recently, Lee et  al. [101] developed CS oligosaccharide-indomethacin conjugate (CI) nanoparticles as passive tumor-­targeted drug delivery carriers. They served as chemosensitizing nanoparticles to enhance antitumor activity and prolong blood circulation. The design concept was to obtain amphiphilic CS compound by grafting indomethacin to the chitosan oligo‑ saccharide backbone as a hydrophobic residue. The chemosensitizing mechanism of CI/DOX NPs for cancer therapy was indicated in Fig. 13.1. Moreover, this ingenious conjugate would form self-assembled nanoparticles in the aqueous environment where doxorubicin (DOX) can be loaded into their hydrophobic cavity. The size of these nanoparticles was found to be 99% of antibacterial activity against E. coli and S. aureus. Annur et al. [62] fabricated the chitosan/PEO composite electrospun nanofibers loaded with Ag nanoparticles. In this study, initially, chitosan/PEO electrospun nanofibers were prepared and loaded with the silver precursor. Then, these fibers were treated with argon plasma to form the Ag nanoparticles within the fibers’ matrix. The average size of Ag nanoparticles obtained by this approach was found to be ~1.5 nm. The developed electrospun nanofibers presented an improved antibacterial activity against E. coli with the increase in plasma treatment time. Similarly, Bajpai et al. [58] found that polyelectrolyte complex based on chitosan and poly(acrylamide-co-itaconic acid) films loaded with Ag nanoparticles had an antibacterial activity against E. coli with a zone of inhibition of 2.6 cm. Bone implants should exhibit good antimicrobial property besides possessing osteoinductivity and biocompatibility as implant-associated infections can arise within two weeks after surgery. Hence, different types of chitosan-based scaffolds loaded with Ag nanoparticles were developed for bone-tissue-engineering applications. Cao et al. [54] fabricated Ag-loaded MgSrFe-layered double hydroxide/chitosan composite scaffolds. These scaffolds exhibited good biocompatibility against human bone marrow-derived mesenchymal stem cells (hBMSCs) and ~97% of bacterial inhibition against S. aureus. The scaffolds based on chitosan/carboxymethyl cellulose nanocomposite incorporated with cellulose whisker modified Ag nanoparticles were fabricated by the freeze-drying method and their function in bone regeneration has been studied [63]. Apart from improving the stability of the scaffolds, Ag nanoparticles increased the mechanical strength matching that of the cancellous bone. Moreover, the Ag nanoparticles incorporated scaffolds showed good antimicrobial activity, decreased swelling, and degradation rate. They also supported the growth and proliferation of MG-63 cell lines in vitro. PVA-capped Ag nanoparticles were prepared by microwave-assisted route and their ability as a nanofiller in improving the mechanical strength of the chitosan matrix and antibacterial activity when deposited on titanium substrates were evaluated [53]. The Ag nanoparticles increased the elastic modulus of the titanium implants coated with chitosan/PVA-capped Ag nanoparticles in both dry and hydrated conditions. Loading of Ag nanoparticles in chitosan matrix also resulted in the sustained release of Ag nanoparticles for up to 120 h, which is essential for the prolonged antibacterial activity of the implants.

19.4.2 Chitosan/  TiO2 composites TiO2is one of the most elaborately studied metal oxides due to its remarkable photocatalytic activity. TiO2 nanoparticles are widely used in pharmaceutical, food industry, and cosmetics. TiO2 nanotubes have been found to support bone regeneration, prevent bacterial infection, and enhance blood clotting in cases of hemorrhage. Moreover, TiO2 nanoparticles were found to exhibit the slow release of titanium ions, which exert good antimicrobial and wound healing property [64]. The drawback of using TiO2 is its lack of stability in suspensions, which makes it difficult to use as such.

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Therefore, TiO2 nanoparticles have been blended with polymers and fabricated into films, nanofibers, and porous scaffolds to widen their applications in various domains ranging from photocatalysis to biomedical fields [65]. It was found that titanium ions can interact with the electron rich oxygen atoms present in the glucosidic and hydroxyl groups of chitosan through electrostatic interactions to form the stable complexes [66]. Haldorai and Shim prepared the chitosan/TiO2 composites and studied their antimicrobial activity against E. coli [67]. The developed composites showed 98% reduction in microbial growth when compared to pure chitosan that exhibited only a 7% reduction in 24 h. This observation might be due to the reaction of oxygen species generated during the photocatalytic activity of the TiO2 nanoparticles with the bacterial cells and the interaction between the positively charged chitosan/TiO2 composites and negatively charged lipid-rich cell membrane of E. coli. Behera et al. [68] fabricated the chitosan/TiO2 nanoparticles composite membranes by varying the concentrations of TiO2 (0.25%, 0.5%, 0.75%). Due to the presence of TiO2 nanoparticles, these composite membranes showed an increased surface roughness and thereby enhanced the cell adhesion and proliferation. The composite membrane with 0.25% of TiO2 was found to decrease reactive oxygen species (ROS) formation when compared to pristine TiO2 nanoparticles. This membrane favored cell-cycle progression of L929 fibroblast cells with the increase in cells at synthesis phase, whereas cell grown on pure chitosan membranes did not proceed to synthesis phase of the cell cycle but remained in the G0/ G1 phase. In this work, the reverse transcription polymerase chain reaction (RT-PCR) analysis was carried out for fibroblast marker genes such as TGF-β, FGF-2, and proliferating cell nuclear antigen (PCNA). It was found that the highest expression of the genes was observed by the cells cultured over the composite membrane with 0.25% of TiO2 and the least expression was observed from the cells cultured on pure chitosan membranes. Cells cultured in pure TiO2 nanoparticles significantly downregulated the expression of the marker genes, suggesting that pure TiO2 nanoparticles do not support the cell proliferation [68]. A bilayer wound dressing consisting of human adipose tissue-derived ECM as the lower layer and electrospun chitosan nanofibers loaded with TiO2 nanoparticles as upper layer was fabricated [66]. The upper chitosan/TiO2 nanofibrous layer used to prevent the microbial growth in the wound, while the lower ECM layer supported the tissue regeneration. In this study, the chitosan/TiO2 nanofibrous layer was fabricated by electrospinning of chitosan nanofibers followed by immersion in 1 wt.% TiO2 solution. The developed bilayer dressing inhibited E. coli and S. aureus up to 33.9% and 69.58%, respectively. In rat full-thickness cutaneous wound models, the bilayer wound dressings adhered to the wound area and were able to absorb exudates. After 28 days, wounds treated with both control and bilayer composite healed efficiently with a remaining wound area of 23.1% and 17.4%, respectively. Similarly, chitosan/ pectin polyelectrolyte complex incorporated with TiO2 nanoparticles was evaluated as wound dressing material [69]. This polyelectrolyte complex exhibited good antibacterial activity, biocompatibility against L929 and NIH3T3 cells lines, and wound closure in the in vivo rat subcutaneous wound models. After 16 days of wound treatment with the samples, chitosan/pectin/TiO2 nanoparticles -based composite exhibited the maximum wound closure rate of 99.01%. This superior wound-healing property of

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the composite is due to the combined action of both chitosan and TiO2. Histological observation revealed that the wounds treated with chitosan alone were infiltrated with inflammatory cells with loosely packed dermis and epidermis. Whereas in the chitosan/pectin/TiO2 nanoparticles-treated groups, well-developed dermis and epidermis with the absence of any inflammation was observed [69]. A dressing material based on chitosan/PVP/TiO2 nanoparticles was examined for its wound healing property [64]. Compared to the control, the chitosan/PVP/TiO2 composite exhibited good antibacterial activity against S. aureus and E. coli, and biocompatibility against L929 and NIH3T3 cells. On application in rat models, the chitosan/PVP/TiO2 exhibited the highest wound closure rate (99.09%) within 16 days. In situ fabrication of TiO2 nanoparticles in chitosan matrix is more advantageous than the ex situ method, as in situ synthesis results in the formation of homogeneous dispersion and stable interaction between the TiO2 nanoparticles and the polymer matrix. It was found that with the increase in chitosan concentration, the size of the TiO2 nanoparticles reduced from 10.58 nm (without chitosan) to 4.5 nm (2 g/L of chitosan). Immersion of the nanocomposite in 1.5% SBF for 21 days resulted in the formation of the apatite layer, illustrating the osteoinductive nature of the nanocomposites [70]. Chitin/chitosan-based porous scaffolds incorporated with TiO2 nanoneedles were employed for bone regeneration [71]. These scaffolds displayed a reduced degradation rate when compared to pure chitin/chitosan scaffolds due to the presence of TiO2 nanoparticles. In addition, the developed chitin/chitosan/TiO2 scaffolds exhibited an adequate biocompatibility against MG-63, hMSCs, and L929 cells. In the chitin/chitosan/TiO2 scaffolds, the cells were spread out well within the pores due to the increased surface roughness of the scaffolds. It was noted that incubation of the developed scaffolds in SBF for 7 and 14 days resulted in HA formation. These results indicated that the incorporation of TiO2 nanoparticles within the chitosan-based scaffolds is highly favorable for bone-tissue-engineering applications.

19.4.3 Chitosan/ZnO composites ZnO nanoparticles have been explored for a wide range of applications in cosmetics, environmental, and biomedical fields due to their UV light absorption, optical, and antibacterial properties [72]. These nanoparticles have been generally recognized as safe by FDA [73, 74]. Hence, ZnO nanoparticles have been mixed with chitosan-based materials to develop the wound dressing and scaffolds materials with the desired properties in recent years. The chitosan/ZnO/gentamicin composites have been proved to be an excellent wound dressing material [75]. These composites showed an increased zone of inhibition (17 mm) than gentamicin controls (11 mm) against S. aureus. This result proved that the combination of ZnO nanoparticles with chitosan and the antibiotic system would be highly effective in killing the microorganism than pure drug. Sudheesh Kumar et al. [76] developed the porous chitosan bandage loaded with ZnO nanoparticles. This hybrid material displayed superior tensile strength, antibacterial activity, and blood-clotting effect. Furthermore, in the in vivo wound-healing models, the ZnO-loaded chitosan bandage exhibited good wound healing when compared to bare wound and Kaltostat-treated wounds. Around 90% of wound closure was seen

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in wounds treated with ZnO-loaded chitosan bandage, whereas only 70% of wound closure was seen in control and kaltostat-treated wounds after 2 weeks of treatment. Ullah et  al. [74] fabricated the composite scaffolds based on chitosan/collagen/ ZnO nanoparticles and studied their properties. It was observed that the incorporation of ZnO nanoparticles to the polymer matrix increased the tensile strength of the resulting scaffolds. With the increase in ZnO nanoparticles, the average pore size of the scaffolds decreased but the porosity of the scaffolds increased. Moreover, the cell adhesion and proliferation onto the chitosan/collagen matrix were increased with the increase in ZnO concentrations. Similarly, the wound-dressing materials based on chitosan/silk sericin/ZnO nanoparticles also showed good biocompatibility, compressive strength, and antimicrobial activity against E. coli and S. aureus [77]. The similar type of observation was also reported for the wound-healing agents based on castor oil/chitosan/ZnO nanoparticles [78]. Zhai et al. [73] developed ZnO-loaded chitosan/ keratin-based porous hydrogel scaffolds as bandages for burn wound-healing applications. These ZnO-loaded scaffolds exhibited an improved tensile strength (0.31 MPa) when compared to the pristine chitosan/keratin scaffolds (0.15 MPa). Moreover, they showed about 95% cell viability against NIH3T3 fibroblasts after 3 and 7  days of incubation. In vivo wound-healing studies in Sprague Dawley rats showed that the developed scaffolds displayed about 95% of wound closure after 14 days. Baghaie et al. [79] evaluated the antibacterial and in  vivo wound-healing property of ZnO-loaded PVA/starch/chitosan hydrogel membranes. These membranes showed an increased tensile strength and zone of inhibition due to the presence of ZnO nanoparticles. The increased antibacterial activity of membranes is due to the release of Zn2+ ions from the polymer matrix, which damages the proteins and lipids present in the cell wall of the bacteria. The hydrogel-treated wounds exhibited more fibrin deposition than the control. The control wounds exhibited hemorrhage, which was not seen in the case of hydrogel-treated ones. After 14 days of treatment, the percentage of wound healing was about 96%, 95%, and 79% for the wounds treated with ZnO-loaded hydrogel, hydrogel without ZnO, and control (wound without any treatments), respectively. A combination of genipin crosslinked chitosan/PEG films loaded with ZnO/Ag nanoparticles has proved to be a highly efficient wound-dressing material [80]. Since chitosan films are highly rigid in nature, PEG has been added as a plasticizing agent for the formation of films that can be easily handled. These composite films exhibited higher tensile strength when compared to films without ZnO/Ag nanoparticles. Moreover, they displayed the highest zone of inhibition (20–21 mm in diameter) than the pure chitosan/PEG films (8–10 mm) and films with only ZnO nanoparticles (15–16 mm) or Ag nanoparticles (9–11 mm) against both gram-positive and gram-negative microbes. Recently, graphene oxide decorated chitosan were loaded with ZnO nanoparticles by in situ method, and their antibacterial activity was studied [81]. This material showed an improved antibacterial activity due to the presence of ZnO nanoparticles by the production of ROS leading to the oxidative damage of the microbes. The minimal inhibitory concentration of the nanocomposite against E. coli and S. aureus was 2.5 μg/ml and 5 μg/mL, respectively. Iman et al. [82] evaluated the regeneration of transected sciatic nerve with ZnO-loaded chitosan conduits in rat models. In this study, the developed conduits were placed in the sciatic nerves by surgical

Bioactive nanomaterials/chitosan composites as scaffolds for tissue regeneration573

procedures and the regeneration was studied after 12 weeks of surgery. A significant difference was observed in behavioral and functional tests in the rat models implanted with ZnO-loaded chitosan conduits and pure chitosan conduits. The amounts of regenerated fibers were found to be higher in the ZnO-loaded chitosan conduits–treated groups than the chitosan-treated groups.

19.5 Chitosan/carbon nanomaterial composites In recent years, carbon-based nanomaterials like carbon nanotubes (CNTs), graphene, and graphene oxide (GO) have received more attention because of their superior mechanical, optical, electrical, and biological properties (Fig.  19.4) [83, 84]. Due to these properties, carbon-based nanomaterials have been utilized as a nanofiller for the fabrication of several tissue-engineering scaffolds. The scaffolds incorporated with ­carbon-based nanomaterials have found to be highly beneficial in improving the stability and biological property [85, 86]. Moreover, these scaffolds possess good electrical conductivity that is required for nerve and cardiac regeneration. The carbon-based nanomaterials were found to increase the protein adsorption ability of the scaffolds

(A)

(B) COOH COOH COOH OH COOH O O

O

HOOC

O

HO

COOH HO

O O O

COOH

O

OH

(C)

COOH

COOH

Fig. 19.4  Structure of (A) CNT, (B) graphene, and (C) GO.

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due to their large surface area and ability to interact with protein through van der Waal’s interaction, ionic bonding, hydrophobic, and π-π interactions [87, 88]. Since the scaffolds loaded with carbon-based nanomaterials had an improved surface roughness, they showed a better cell adhesion and proliferation [89, 90].

19.5.1 Chitosan/CNT composites CNTs possessing high aspect ratio are stiff and strong and can be synthesized by various methods such as arc discharge, chemical vapor deposition, etc. [91, 92]. Singlewalled CNTs have been utilized as a filler in chitosan-based scaffolds, especially for nerve regeneration [93, 94]. Several methods like electrospinning, thermally induced phase separation, freeze drying, etc. have been employed for the fabrication of chitosan/CNT composite scaffolds [95]. These scaffolds displayed an increased tensile strength up to 97%, porosity, and pore diameter ranging from 100 to 300 μm at the optimum composition of chitosan/CNT [96, 97]. Huang et al. [93] prepared the scaffolds based on chitosan/HA/multiwalled CNTs. These scaffolds supported the proliferation of preosteoblast MC3T3-E1 cells and filopodial extensions were seen on the composite scaffolds. Moreover, they exhibited good electrical conductivity, mechanical strength, and favored the proliferation of rat pheochromocytoma cells (PC12) cells. Due to the electrical conductivity of CNTs, the developed scaffolds were found to be suitable for nerve tissue engineering. Similarly, chitosan/PVA nanofibers loaded with single-walled CNTs were developed, and their suitability as tissue-engineering scaffolds was assessed [94]. The prepared composite nanofiber was found to possess superior mechanical strength compared to the control. In addition, the human glioblastoma-astrocytoma (U373-MG) cell line exhibited good viability over the nanofibers incorporated with single-walled CNTs. The composite films based on chitosan/multiwalled CNTs prepared by simple solution evaporation method were found to be useful for cardiovascular tissue engineering [97]. Ovine carotid myofibroblast cells cultured over the composite films exhibited good viability and proliferated rapidly. The composite films were found to be highly biocompatible and did not induce any adverse reaction even after 8 days of culture in vitro. The nerve conduits based on polypropylene functionalized chitosan/ carboxylic acid functionalized multiwalled CNTs were prepared for regeneration of peripheral nerve injury [98]. These nerve conduits were made up of two layers in the form of a tube consisting of inner aligned nanofibers and outer random nanofibrous mats. It was found that the growth, proliferation, and differentiation of Schwann and PC12 cells were higher in aligned nanofibrous mats than in random nanofibers. The carboxylic acid functionalized multiwalled CNTs increased the porosity and surface area of the mats, which resulted in an improved adhesion and proliferation of cells.

19.5.2 Chitosan/graphene composites Graphene is made up of a single sheet of graphite, which possesses superior electrical, optical, thermal, and mechanical properties [99]. Although several methods such as chemical exfoliation, mechanical exfoliation, arc discharge methods are available

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for the synthesis of graphene, chemical vapor deposition method is commonly used for the large-scale production of graphene [100]. The aromatic structure of graphene, strong carbon-carbon interaction, and the presence of free π electrons make graphene as a desirable material for various applications ranging from sensors to tissue engineering [101]. Some of the favorable characteristics of graphene are its biocompatibility, mechanical strength, and ability to undergo chemical modification. Incorporation of hydrophilic groups on the graphene surface increases its capability for cell adhesion and proliferation [100]. Due to these desirable properties, recently, graphene has been considered as a potential filler to improve the performance of chitosan-based scaffolds. Chitosan/graphene composites showed an increased tensile strength and thermal stability [99]. Moreover, these composites presented the superior mechanical strength even at lower concentrations of graphene (0.1–0.3 wt.%) within the polymer matrix [102]. The electrical conductivity of graphene makes it as one of the desirable materials for the nerve regeneration. Hence, chitosan/gelatin composites loaded with polypyrrole/graphene and polyaniline/graphene were developed recently and their properties were studied [103, 104]. These materials showed the higher electrical conductivity and cell adhesion than pure polymer scaffolds. Lu et al. [105] analyzed the antibacterial activity of grapheme-loaded chitosan/PVA nanofibers against E. coli and yeast. These materials were found to exhibit antibacterial activity against E. coli. However, they did not inhibit the growth of yeast. The mechanism behind the specific cytotoxic activity of graphene is due to the presence of free electrons in the graphene sheets, which, on meeting the prokaryotic cells, enters the nucleus and damages the DNA of the microbes. On the other hand, due to the presence of a nuclear membrane in eukaryotic cells (yeast), the electrons present in the graphene sheets could not enter the nucleus, thus preventing cell death. Recently, Yu et al. [106] prepared the hydrogels based on chitosan/HA/graphene by crosslinking chitosan with genipin and reducing GO using sodium ascorbate. The resulting hydrogels exhibited high porosity and increased deposition of HA, which is desirable for bone tissue engineering. Rat bone marrow-derived stem cells (rBMSCs) exhibited the improved cell viability on the developed composite hydrogels up to 14 days.

19.5.3 Chitosan/GO composites GO is commonly prepared from graphene by modified Hummer’s method. In recent years, GO is considered as an ideal material for biomedical applications due to its hydrophilic nature and the presence of carboxyl, hydroxyl, and epoxy functional groups. Due to the presence of carboxyl groups, GO has been conjugated with chitosan by the carbodiimide reactions [107, 108]. The GO-conjugated chitosan scaffolds exhibited superior mechanical strength, hydrophilicity, reduced degradation rate, and water retention capacity when compared to scaffolds prepared by simple blending chitosan and GO [109]. Due to the presence of GO, these composite scaffolds showed less swelling degree, biodegradation, and improved resistance toward the lysozyme-­ induced degradation [110, 111]. GO also favored the formation of apatite crystals onto the surface of the composite scaffolds [111].

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Chitosan/GO-based two-dimensional films containing ˂1.5 wt.% of GO fabricated by solution casting method exhibited an increase in tensile strength due to the interaction between the primary amine group of chitosan and the carboxyl group of GO [112–114]. However, when increasing the GO content from 1.5 to 6 wt.%, the scaffolds produced adverse effects such as decreased mechanical strength and cell growth [115, 116]. These results suggest that the concentration of GO in the composite plays a vital role in achieving the favorable characteristics required for tissue regeneration. Li et al. [110] developed the chitosan/GO composite scaffolds for bone regeneration. These scaffolds improved the formation of HA in presence of SBF through biomineralization due to the presence of GO. Due to the increased surface roughness, the scaffolds based on chitosan/hyaluronic acid/GO exhibited the improved cell adhesion when compared to the control chitosan/hyaluronic acid matrix [117]. When loaded with simvastatin, the chitosan/hyaluronic acid/GO scaffolds exhibited an adequate cell adhesion and proliferation. MC3T3 osteoblast cells exhibited good adherence and proliferation on the scaffolds with simvastatin after 24 and 48h of culture. Addition of simvastatin also induced the biomineralization after 14 days of incubation in SBF [118]. Saravanan et al. [119] fabricated the chitosan/gelatin/GO composite scaffolds for bone tissue engineering. It was observed that the pore size of the scaffolds was decreased from 60% to 48% when increasing the GO content of the scaffolds. In this work, Rat tibial bone defect was used as the model system to evaluate the suitability of chitosan/gelatin/GO composite scaffolds for bone regeneration. It was found that the scaffolds with GO exhibited good wound closure and degradation with the regeneration of bone. Histological studies of the tissue section revealed that the animals treated with scaffolds containing 0.25% of GO showed more amount of collagen deposition when compared to the controls. Chitosan/PVA nanofibrous mats loaded with GO were found to exhibit good biocompatibility toward mouse chondrogenic cells (ATDC5) [120]. Cell viability analysis using MTT assay was done after 4, 7, and 14 days of culture, which inferred that chitosan/ PVA fibers with 6 wt.% of GO displayed an increase in ATDC5 cells proliferation than the nanofibrous mats possessing 4 wt.% of GO. This result suggested that the increase in GO content in the nanofibrous mats favors the proliferation of chondrocytes. Azarniya et  al. [121] developed the chitosan/bacterial cellulose/GO nanofibrous mats for wound dressing and skin-tissue-engineering applications. It was found that incorporation of GO into the polymer matrix decreased the average fiber diameter, the hydrophilicity of the nanofibers, and water vapor permeability. Similarly, electrospun nanofibers composed of chitosan/PEO/PVP/GO were prepared as a wound-dressing material [122]. In  vivo studies in rat wound models suggested that nanofibers with 1.5 wt.% of GO had a wound closure rate of about 33% without the formation of scar or inflammation. Recently, hydrogels based on oxidized konjac glucomannan/ carboxymethyl chitosan/GO were prepared for tissue engineering [123]. The gelling time of these hydrogels was decreased with the increase in GO content due to the hydrophilic nature of GO and its hydrogen bonding with the polymer networks. The compressive modulus of the hydrogels was found to be increased from 144% to 296% when increasing the GO concentration in the composites. The results of these studies indicated that chitosan/GO composites would be ideal wound-dressing and tissue-­ engineering materials.

Bioactive nanomaterials/chitosan composites as scaffolds for tissue regeneration577

19.6 Conclusions In the present chapter, preparation, properties, and application of various bioactive nanomaterials/chitosan composites have been discussed. These composites showed an improved biocompatibility, cell adhesion, and proliferation due to the presence of bioactive nanomaterials. Chitosan, when combined with HA or β-TCP, was found to improve the biomineralization, which is an important process for successful bone regeneration. Bioactive glass combined with chitosan-based scaffolds has proved to be a desirable bioactive nanomaterial for bone tissue engineering as it can influence the genes that are responsible for osteogenesis. On the other hand, metal/metal oxide nanoparticles such as Ag, TiO2, and ZnO nanoparticles can impart antibacterial activity and antimicrobial drug resistance to chitosan-based wound dressings. Moreover, the cells cultured on the chitosan/metal or metal oxide composites were found to increase the expression of fibroblast marker genes such as FGF-2 and TGF-β. Carbon-based nanomaterials such as CNTs, graphene, and GO are relatively new to the tissue-­engineering field, but they have proved to be the versatile nanofillers for the development of chitosan-based scaffolds for soft- and bone-tissue regeneration. Since the chitosan/carbon-based nanomaterials showed an improved biomineralization during the bone regeneration, these materials were widely considered as the scaffolds for bone tissue engineering. Also, the chitosan/graphene composite scaffolds were found to be suitable for the fabrication of nerve conduits and cardiac regeneration due to their electrical conductivity. Based on this extensive review given in this chapter, bioactive nanomaterials/chitosan composites could be ideal biomaterials for the development of potential scaffolds for various tissue regenerations.

Acknowledgment The authors greatly acknowledge DST-Nano Mission (SR/NM/NS-1260/2013), Department of Science and Technology, Government of India, for financial assistance.

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Biopolysaccharide-based hydrogel materials for drug delivery

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Dhanabal Kumarasamy, Tapan Kumar Giri NSHM Knowledge Campus, Kolkata Group of Institutions, Kolkata, India

20.1 Introduction Hydrogels are crosslinked polymer networks comprising hydrophilic group bearing monomers in large number. These polymer networks have great affinity for water; however, they do not dissolve owing to the physical or chemical bonds that interlink the polymer chains as an integrated network. Water accesses the networks through hydrophilic groups in the polymer, which results in swelling of the hydrogel. Completely swollen hydrogels have a number of physical properties familiar to living tissues. These are soft, elastic consistency, compatibility with biological fluids owing to low interfacial tension [1]. The soft and elastic nature of the swollen hydrogels leads to reduce irritation to the neighboring tissues following the implantation of hydrogel. The reduced interfacial tension between body fluid and hydrogel minimizes the protein adsorption and cell adhesion, resulting in diminished negative immune reactions. Additionally, hydrogels have numerous additional characteristics that render them as outstanding drug-delivery agent. Various polymers were used in the preparation of hydrogel, having a bioadhesive and mucoadhesive property that improves the tissue permeability and drug residence time [2]. The bioadhesive property is arising due to interchain connection between the functional groups containing hydrogel polymers and the mucus glycoproteins [3], that improves site-specific binding to colon, nose, and vagina [4]. Hydrogels are prepared using natural, semisynthetic, and synthetic polymers [5–7]. The polymeric networks produced by chemical crosslinking have the most stable connections, whereas physical networks result in temporary connections. Physical networks arise from either physical interactions or polymer chain entanglements usually involving more than one of the following, hydrogen bonds, ionic interactions, and hydrophobic interactions [8]. Physically hydrogels appear as a film, matrix, or microspheres due to varying techniques of polymerization used to prepare them. Interpolymer complex, ionic, hydrophobic associations and polyelectrolytes are the four major physical interactions that lead to the chitosan solution gelation (Fig. 20.1). Gel creation can be reversed since the network formation by all of these interactions is purely physical. Natural polymers like polysaccharides and proteins are known to form hydrogels. Hydrogels prepared from natural polysaccharides are frequently used to deliver drug molecules as these are biocompatible and biodegradable. Various naturally occurring polysaccharides are used in the preparation of hydrogel. Distinctively, these Polysaccharide Carriers for Drug Delivery. https://doi.org/10.1016/B978-0-08-102553-6.00020-9 Copyright © 2019 Elsevier Ltd. All rights reserved.

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Polysaccharide Carriers for Drug Delivery Ionic interactions

Thermoreversible complex

Anionic molecules

Hydrophobic interactions

Chitosan Hydrophilic groups Ionic interactions

37° Hydrogel formation

Chitosan

Polyelectrolytes Hydrophobichydrophilic repulsion

Crystallite & secondary bonding

20°

(A)

Added polymer

Polymer dissolution

(B)

Fig. 20.1  Schematic representation of chitosan-based hydrogel networks derived from different physical associations: (A) networks of chitosan formed with ionic molecules, polyelectrolyte polymer and neutral polymers; (B) thermoreversible networks of chitosan graft copolymer resulting in semisolid gel at body temperature and liquid below room temperature. (Reprinted from Bhattarai N, Gunn J, Zhang M. Chitosan-based hydrogels for controlled, localized drug delivery. Adv Drug Deliv Rev 2010;62:83–99, Copyright (2010), with permission from Elsevier.)

p­ olysaccharides of natural origin in combination with other polymers present desirable biological and chemical advantages. Recently, numerous studies have significantly contributed to our current perceptive of polysaccharide-based hydrogel networks. Besides their vast application in modulating small molecule drug delivery, as a matrix hydrogels are much exploited for controlled release of biopharmaceuticals like protein molecules and encapsulate living cells for in situ delivery. There are extremely large number of polysaccharides that were investigated for hydrogel preparations as suitable delivery systems. This chapter presents a collection of most important polysaccharides that are widely used in the fabrication of hydrogels and the techniques employed in the construction of hydrogel network for the purpose of regulated drug delivery.

20.2 Hydrogels Hydrogels are hydrophilic polymer networks capable of absorbing large quantity of water or fluids of biological origin [9]. The networks consist of copolymers or homopolymers and are insoluble owing to the existence of either physical or chemical crosslink or combination of them. There exist numerous reported techniques for the preparation of hydrogels. The first method involves crosslinking of comonomers using multifunctional comonomers acting as crosslinking agent. The crosslinking reaction is affected by an initiator. The second approach involves crosslinking of linear polymers by chemical compounds or by irradiation. The monomers used in the preparation of hydrogel contain an ionizable group or a functional group that can be substituted with an ionizable group through a chemical reaction, subsequent to the completion of

Biopolysaccharide-based hydrogel materials for drug delivery587

polymerization. Thus, the prepared hydrogels contain weakly basic groups like substituted amines, or weakly acidic groups like carboxylic acid, or a strong basic and acidic group like quaternary ammonium compounds and sulfonic acids. Commonly used crosslinking agents include ethylene glycol, divinylbenzene, and dimethacrylate. Hydrogels are used as new materials with diverse range of applications in sanitary products, biomedical engineering, bioseparation, agriculture, food processing, and oil recovery, to mention a few. They were effectively employed as superabsorbent materials, drug-delivery systems, tissue repairing, and in the encapsulation of living cells and maintenance of their in vitro viability owing to their inherent capability to hold a high amount of water or other biological fluids coupled with excellent biocompatibility. The water-holding and retention property of the hydrogels facilitates an appropriate drug diffusion pathway and, consequently, as carriers of drugs, many hydrogel polymer networks were developed. The release patterns of drugs are directly correlated with the rate and extent of hydrogel swelling; these essential parameters can be manipulated to bring out a desirable pattern in drug release. The property of hydrogel swelling when placed in contact with water is favorable in many drug-delivery circumstances. Surface of hydrogel in its early stage contact with water molecules, the latter access the surface rapidly to solvate and start gaining access toward the deep polymeric network. The unsolvated glassy phase is alienated from rubbery region with a moving boundary. The meshes of polymer network at the rubbery phase will start growing, recurrent events allow other water molecules to enter deep in the network. Achilleos et al. have devised a method for profiling and visualizing dynamic deformations during the swelling process of hydrogels [10]. There exists a converse elasticity force, in resistance to the positive osmotic force, which prevents its deformation by balancing the stretching of the network. There is no additional swelling at equilibrium, because the osmotic and elasticity forces are balanced. The rate of swelling of a hydrogel is an important and key attribute that determines its right application. Though there exist a number of parameters, the degree of porosity and the nature of porous structure are essential parameters that influence the rate of swelling (Table 20.1). One of the essential properties of hydrogels is its equilibrium water content. Higher the water content of a hydrogel, better and more beneficial it is for biomedical applications [11]. Generally, a large swelling leads to poor mechanical properties of the hydrogel network. The desirable mechanical properties can be augmented by increasing the density of crosslinking; this affects unfavorably on the swelling. Hydrogels with desirable swelling and improved mechanical properties are accessed by copolymerizing hydrophilic monomer with a lesser hydrophilic monomer [12]. Some hydrogels are known as responsive hydrogels, as they undergo reversible swelling and shrinking cycles by numerous hundred times in response to minute changes in temperature, pH, solvent composition, electric field, ionic strength, and light. Designing of sustained released systems for pharmaceuticals, drugs, and agriculture pesticides requires the knowledge of swelling kinetics and is vital to bring out a desirable release pattern [13]. Recently nanotechnology was applied in the preparation hydrogels that results in nanogel systems. Nanotechnology improves the materials physical, chemical, and biological properties [14–16].

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Table 20.1  Hydrogel swelling with respect to the porosity Type Nonporous Microporous

Macroporous Superporous

Major swelling mechanism

Swelling rate

Application

Diffusion through free volumes Diffusion and convection in the water-filled pores

Very slow

Diffusion in the water-filled pores Capillary forces

Fast

Contact lenses to artificial muscles Biomedical applications and controlled release technology Superabsorbent in baby diapers Drug delivery system

Slow

Very fast

20.3 Preparation of polysaccharide hydrogel Polysaccharides have been widely used as structural materials in the preparation of hydrogels mainly due to their ready availability, low toxicity, biocompatibility, its susceptibility to enzymatic degradation, and biodegradability [17–19]. Polysaccharides have advantage over other naturally derived materials, characterized by low immunogenicity and source-originated pathogen-transmitting potential. Various methods of hydrogel preparation using polysaccharides are given here.

20.3.1 Ionic complexes Ionic interactions can occur between polysaccharides that contain anionic or cationic groups and oppositely charged molecules or ions. Ionic complexation of mixed charge systems can be produced between polysaccharide (chitosan) and anionic molecules like carboxylic acids, i.e., acetate, citrate, or inorganic anions, i.e., sulfate, phosphate, or anions of transition metals, i.e., Mo (VI), Pd (II), and Pt (II). The resulting hydrogels have variable material properties; the main controlling factors are the charge density of the counter ions, molar ratio, size of the anion/cation, extent of deacetylation, and the concentration of the chitosan polymer used. Chitosan nanoparticles were prepared by ionic gelation method (Fig. 20.2). An aqueous solution of chitosan was prepared by dissolving it in a weakly acidic solution, protonation of amino groups of chitosan, making it soluble. Then it was added drop wise to the polyanionic tripolyphosphate solution under constant stirring. Cationic chitosan forms ionic complexation with polyanionic triphosphate; the net result is ionic gelation and chitosan precipitates as spherical particles. Hydrogels of carboxymethyl guar gum-sodium alginates have been prepared using an ionic complexation with barium ions [20]. In simulated gastric and intestinal fluids, swelling capacity of these hydrogels was found to be about 15 and 310%, respectively. This hydrogel loaded with vitamin B12 displayed a release of 20% and 70% in simulated gastric and intestinal fluids, respectively. Hydrogels that showed 50% entrapment efficiency were crosslinked with 5 or 6%(w/v) barium chloride solution.

Biopolysaccharide-based hydrogel materials for drug delivery589

Fig. 20.2  Preparation of chitosan nanoparticles by ionic gelation method.

20.3.2 Polyelectrolyte complexes The links between the polyelectrolytes and polysaccharide through ionic interactions are stronger than other common binding interactions, i.e., hydrogen bonding and van der Waals interactions. The advantages of this type of complex are that the preparation of hydrogels does not require any catalyst, reactive organic precursors, or any other reactive crosslinking agents, thus relieving the anxiety about their safety concerns in in vivo application or crossreactivity toward a therapeutic consignment. Additionally, since polyelectrolyte complexes consist of polyelectrolyte and polysaccharide, their complexation is reversible and simple. Chitosan-based polyelectrolyte complex networks have been formed by water-soluble, anionic macromolecules such as DNA, anionic polysaccharides (e.g., alginate, heparin, hyaluronic acid, chondroitin sulfate, dextran sulfate, pectin, carboxymethyl cellulose, xanthan, etc.), proteins (e.g., collagen, gelatin, keratin, albumin, fibroin, etc.), and anionic polymers of synthetic origin polyacrylic acid. The stability of these complexes is largely reliant on the ionic strength, solvent, charge density, temperature, and pH [21].

20.3.3 Physical mixtures and secondary bonding Polymer blends are yet another method of making hydrogels with desired properties. They are prepared by blending polysaccharides with other water-soluble nonionic polymers. Such mixtures create confluence points in the form of crystallites and through interpolymer complexation, which forms during the process of lyophilizing and remains thereafter or requires a series of freeze-thaw cycles to achieve the desirable interpolymer network [22,23]. The crosslinking sites of such ­hydrogels of ­physical

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mixtures of polymers are their chain-chain interactions. Hydrogel consisting of a polymer blend of polyethylenimine and chitosan was prepared [24]. Polyethylenimine is a polycationic material that has a wide application in the area of gene transfection [25]. A hydrogel was formed within 5min by mixing the chitosan with the polymer and is stable under cell culture environment. Thus, the hydrogel scaffold could sustain the growth of primary human fetal skeletal cells. Chitosan alone also used to prepared hydrogel without the addition of any other complexing molecule or polymer. Such hydrogels are created by using hydroalcoholic solvation method that relied on the chitosan's amino groups charge neutralization by using a solution of sodium hydroxide [26]. This leads to forbidden ionic repulsions among the polymer chains, thus allowing the formation of hydrogen bonds, hydrophobic interactions, and chitosan crystallites. Hydrogels in the order of cubic centimeters could be achieved using this technique.

20.3.4 Thermoreversible hydrogels Researchers have prepared a novel category of thermosensitive hydrogel system known as thermoreversible gels. They assume the state of short-lived gel or liquid depending on the temperature of their environment. The polysaccharides form a semirigid gel from flowable liquid solution through hydrophobic interactions. This interaction leads to the formation of intersections between the chains. Particularly, when system temperature surpasses the lower critical solution temperature, the material experiences a reversible hydrophilic to hydrophobic transition. The inherent property of the solution of a polymer with low viscosity at room temperature and formation of a gel beyond their lower critical solution temperature that is in the range of body temperature has potential use in biomedical applications. The solutions of these materials are injectable and assume the state of gel in situ where the body temperature is past lower critical solution temperature, offering the prospective as carrier of vast range of small drug molecules as well as biopharmaceuticals besides having other potential biomedical applications [27]. These injectable thermoreversible hydrogel systems can be administered into the body without the need of invasive surgeries and can transport the drug molecules to the targeted defective body site without significant side effects (local heating and formation of toxic byproducts). A chitosan thermoreversible gel was developed as injectable that employed the chain interactions for gelation. The hydrogel was made by a chitosan polyethylene glycol copolymer that was formed by chemically grafting monohydroxy polyethylene glycol into the chitosan backbone. The injectable solution (room temperature) of copolymer undergoes a thermoreversible transition to form a gel at body temperature. The optimization of the desired properties at varied temperature is achieved by changing the polymer's polyethylene glycol content and the molecular weight of polyethylene glycol used. Therefore, the solution maintained below its transition temperature could be injected and converted into a transparent gel above the transition temperature. In solution state (low temperatures), the hydrogen bonding between water molecules and polyethylene glycol dominates, whereas at high temperatures the hydrophobic interactions were overwhelming among the polymer chains [28,29]. This ­temperature-dependent hydrophobic hydrophilic transition results in reversible gel

Biopolysaccharide-based hydrogel materials for drug delivery591

formation. This type of gel formation has been observed in other cellulose derivatives grafted with hydrophilic moieties also [30]. In recent times, hydrogels using combinations of chitosan and poly (N-isopropyl acrylamide) and poloxamers were developed. These materials are designed in such a manner that the hydrophobic group interactions dominate at elevated temperatures. These grafted polymers are established as materials of choice for in situ reversible hydrogel formation [31].

20.3.5 Chemical crosslinking Physically linked hydrogels endowed with the benefit of gel formation without the use of any crosslinking agent are desirable for biomedical applications. However, they suffer from the following limitations, challenge in precise control of the physical gel pore size, degradation or dissolution, and chemical functionalization. These lead to poor in vivo performance of the material. On the other hand, the properties of chemically crosslinked hydrogels principally depend on the density of crosslinking, i.e., the molar ratio of crosslinker molecules and polymer repeating units [32]. Several bifunctional molecules like diglycidyl ether, glutaraldehyde, diacrylate, diisocyanate, and others were employed to crosslink the chitosan polymer. Hydrogels formed by using these molecules can offer advantageous properties; however, the ­application-specific biocompatibilities of large number of crosslinkers are unknown. In addition to that, the fate of several of these crosslinking agents in the body is largely unknown. In the light of these facts, chemically crosslinked hydrogels are subjected to rigorous purifications to avoid even trace amounts of unreacted crosslinking agents, prior to in vivo administration. Genipin is an outstanding naturally occurring crosslinker, obtained from the fruits of Gardenia jasminoides. It has been found to be a biocompatible crosslinking agent [33]. Genipin has been reported to covalently bind with biopolymers and biological tissues [34]. Genipin is a useful crosslinking agent for polymers containing amino groups like proteins, collagen, gelatin, and chitosan and is much less cytotoxic than glutaraldehyde [35]. Genipin crosslinked chitosan hydrogel showed extended drug release in situ [36,37]. Moreover, genipin crosslinked chitosan membranes display a slower rate of degradation than their counterpart chitosan membranes crosslinked with glutaraldehyde [38]. Substantial research has been dedicated to develop polymer mixtures bearing photosensitive functional groups that can form hydrogels in situ through crosslinking upon exposure to light of specific wavelength. Chitosan is functionalized with these photosensitive/reactive groups; the modified chitosan polymer forms crosslinkages upon irradiation with UV light. This method offers significant advantages (Safe, ease of formation, low cost, etc.) over conventional chemical methods, which usually necessitate the use of various initiators, catalysts, or reactive species. A chitosan hydrogel prepared by photocrosslinking method having desired in situ properties has been achieved by functionalizing the polymer with azide groups [39]. The azide is converted into a reactive nitrene intermediate, which further reacts with chitosan's free amino groups to form crosslinks causing gelation within 60 seconds of UV irradiation. This chitosan hydrogel loaded with variety of growth factors displayed

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sustained release of its cargo, thus serving as a novel carrier for the induction of neovascularization in vivo [40,41]. An injectable hydrogel based on chitosan was produced from chitosan via grafting of glycolic acid, through water-soluble chitosan derivatives and phloretic acid by an enzymatic crosslinking with horseradish peroxidase and hydrogen peroxide [42]. Gelation time varying from 4 min to 10s can be achieved by escalating concentration of the polymer from 1% to 3% (w/w). The desirable mechanical properties, gel content, degradation by lysozyme, hydration, and water content can be achieved by altering the initial polymer concentration. Tyrosinase is a copper-containing oxidase enzyme of monophenol monooxygenase class present in plant and animal tissues. It has been used to crosslink chitosan with gelatin to form a hydrogel in situ. The enzyme specifically oxidizes the tyrosyl residues of gelatin, forming reactive quinone residues, and chitosan's electron-rich amino groups are covalently added to the electron-­ deficient quinone moiety, thus forming intermolecular crosslinkages [43,44]. Hyaluronic acid-based hydrogels has been prepared by first converting the hyaluronic acid into adipic dihydrazide derivative, then crosslinking it with poly propiondialdehyde-(ethylene glycol). These hydrogels could swell sevenfold in buffer solutions in the time period of less than 2 min. Morphological characterization and enzymatic degradation (hyaluronidase) were studied using scanning electron microscopy and spectrophotometric assay, respectively. This hydrogel has been used for sustained release of biomolecules at wound sites [45].

20.3.6 Complex coacervation Complex coacervate hydrogels were produced by mixing polycation with a polyanion. The underlying principle of this method is that polymer strands with opposite charges align together in a coordinated manner due to ionic interaction. These mixtures of polymers can form soluble and insoluble complexes depending on the pH and concentration of the relevant solutions. An example of this class of hydrogel is made by coacervating polycationic chitosan with polyanionic xanthan [46]. Proteins below the isoelectric point are positively charged and are likely to associate with oppositely charged macroions such as anionic hydrocolloids and form poly ionic complex hydrogel [47].

20.3.7 Hydrogen bonding Extensively hydrogen-bonded hydrogel systems can be obtained from acidic polymers with carboxylate functional groups. The pH of aqueous solution of polymers was lowered to keep the carboxylic acid group unionized, thereby the solubility is greatly reduced and it favors the gel formation due to extensive hydrogen bond network of polar functional groups. Carboxymethylated chitosan hydrogels are fabricated by crosslinking in the presence of acids or polyfunctional monomers that favor extensive hydrogen bonding. Hydrogels of mixed system are also prepared in a variety of combinations, one such hydrogel system is obtained by mixing xanthan andalginate. The molecular interaction of xanthan and alginate by means of hydrogen bond f­ ormation causes

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the change in matrix structure due to intermolecular hydrogen bonding between the two different strands of polymer. The net result is formation of insoluble hydrogel networks.

20.3.8 Grafting The process of polymerizing a monomer on the preformed polymers backbone is known as grafting and the resulting material is grafted polymer. The preformed polymers that were chosen as backbone chains are activated by applying chemical reagents or treating them with high-energy radiation. The progress of functional monomers’ growth on activated polymer chain macroradicals results in branching and further crosslinking of the chains of backbone polymer. In chemical grafting, numerous reagents are used to activate polymer backbones. Starch grafted with acrylic acid with the use of N-vinyl-2-pyrrolidone as activating reagent is an example of chemical grafting [48]. Such carboxylate group-grafted hydrogels show distinguished characteristic of pH-dependent swelling and are ideal performance materials with application in the delivery of drug and vitamins to the small intestine. Grafting without the use of chemical reagents is also possible by irradiating the mixture with high-energy radiation like gamma rays and electron beam. Said et  al. reported acrylic acid–grafted carboxymethyl cellulose hydrogels by irradiating the aqueous solution of the mixture with high-energy electron beam, which leads to free radical-induced polymerization of acrylic acid on the backbone of carboxymethyl cellulose [49]. The use of aqueous reaction medium also produces radiolytic products of water, which abstracts protons of macromolecular backbones, thereby facilitating the polymerization of acrylic acid on the polymer. Propagation and termination of free radical reaction result in grafting of polymer backbone and the net result is hydrogel formation. This hydrogel has been proposed for recovery of multivalent metal ions like nickel, copper, lead, and cobalt. Also, they found application as temporary skin cover dressing. Zhai et al. reported the preparation of a series of polyvinyl alcohol-starch blend hydrogels at room temperature and grafting affected by the applying gamma and electron beam radiation [50]. The influence of radiation dose and the content of starch in the blend systems were studied. Increased proportion of starch in the blend increased the gel strength; at the same time, it decreased the swelling index. Through the design of experiments using different blends of amylose and amylopectin with polyvinyl alcohol, it was found that the amylose component of starch as key reactive component of starch and its concentration has direct correlation with the properties of the resulting hydrogels. Cai et al. reported a hydrogel preparation that is endowed with dual sensitivity for temperature and pH changes [51]. The hydrogel was prepared by graft copolymerization of N-isopropyl acrylamide and chitosan. It was found that increased concentration of monomer along with increased irradiation dose increased the percentage and efficiency of grafting. The chitosan graft N-isopropylacrylamide is a responsive hydrogel, sensitive to the change in temperature and pH while having desirable swelling property.

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Guar gum-grafted acrylamide hydrogels were prepared using glutaraldehyde as a crosslinker. The hydrogel was designed as a carrier for calcium channel blockers, nifedipine and verapamil hydrochloride. These drugs may be loaded following crosslinking or incorporated while the grafting is carried out [52].

20.3.9 Interpenetrating polymer network Interpenetrating polymer networks are usually defined as close combination of two polymers, in which one of the polymer components is produced or crosslinked in the immediate presence of other. The interlocked structure of the crosslinked interpenetrating polymer network components is supposed to assure bulk stability and surface morphology of the material [53,54]. The major advantages of interpenetrating polymer network are comparatively film dense hydrogel matrices. They feature tougher and stiffer mechanical properties, physical properties that are controllable. Moreover, this type of hydrogels is more proficient in drug loading in comparison to conventional hydrogels. Guar gum-polyvinyl alcohol interpenetrating polymer network hydrogels are prepared using glutaraldehyde as a crosslinker [55]. It was observed that an augmented crosslink density modified the release of nifedipine from Fickian to non-­Fickian diffusion. The pattern of drug release was shown to be reliant on the load of drug, crosslink density, and the method of loading. The semi-interpenetrating polymer network was attained through dispersion of dextran methacrylate derivative chains into calcium alginate hydrogel. The rheological property of the resultant hydrogel is different from that of calcium alginate hydrogel [56]. This modification allows the semi-interpenetrating polymer network injection in a hypodermic needle without difficulty. Curing of the semi-interpenetrating polymer network by UV light leads to crosslinking of methacrylate moieties, resulting in an interpenetrating polymer network hydrogel that is hard sufficiently for orderly delivery of bioactive molecules. Acrylamide-grafted dextran (AAm-g-Dex) semi-interpenetrating polymer network hydrogel microspheres were prepared for the oral delivery of acyclovir [57]. The synthesis of hydrogels was represented in Fig. 20.3. The release of drug from the synthesis hydrogel was extended up to 12 h.

20.4 Drug-delivery applications 20.4.1 Oral drug delivery The administration of bioactive agents orally leads to absorption and internalization at different sites of the enteric system, i.e., at the oral cavity, stomach, intestine, or colon. Aqueous solubility of the molecules plays a vital role to achieve the required concentration of drug to reach the systemic circulation through oral route [58,59]. A variety of methods are available to enhance the water solubility of drugs, thereby enhancing their bioavailability [60,61]. Using drug-delivery system, clinicians can particularly target various tissue systems within the gastrointestinal tract for local drug action or

Biopolysaccharide-based hydrogel materials for drug delivery595 OCH2 O OH

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Fig. 20.3  Schematic representation of the synthesis of AAm-g-Dex. (Reprinted from Rokhade AP, Patil SA, Aminabhavi TM. Synthesis and characterization of semi-interpenetrating polymer network microspheres of acrylamide grafted dextran and chitosan for controlled release of acyclovir. Carbohydr Polym 2007;67:605–13, Copyright (2007), with permission from Elsevier.)

delivery of drug to the vasculature can be achieved through the extensive capillary beds of small intestine.

20.4.1.1 Drug delivery in the oral cavity The local delivery of therapeutic molecules to the mouth itself can be exploited to treat various types of oral cavity diseases such as stomatitis, periodontal disease, viral and fungal infections, and oral cavity cancers. Desirable treatment outcome can be achieved with negligible systemic exposure of drugs. Additionally, achieving the therapeutic concentration of drugs by administering the drug through oral mucosa provides some distinctive advantages, including evasion of the hepatic first-pass metabolism and the high acidity of stomach, in which drug may be degraded, and proteolytic activity of the rest of the gastrointestinal tract does not spare the protein biopharmaceuticals.

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Drug uptake through oral mucosa is usually low unless a suitable excipient is employed. Chitosan-based hydrogels were accepted as an outstanding excipient for oral mucosal delivery of number of therapeutics due to their mucoadhesive properties. The studies indicating that these hydrogels have improved drug penetration within the mouth, enhancing therapeutic efficacy in treating periodontal disease by maintaining high levels of antimicrobial agents in the gingival crevicular fluid with minimal systemic uptake [62]. In a porcine oral mucosa in  vitro system, chitosan hydrogels have demonstrated an outstanding capability to enhance the paracellular permeability of the mucosal epithelia, which results in effectual transport of the peptide drug-­ transforming growth factor beta (TGF-β). Chitosan hydrogel systems were designed for the local release of numerous drugs in the oral cavity, majority of them being antimicrobials. In addition, chitosan polymers themselves have shown antifungal activity. Chitosan hydrogels were capable of limiting the adhesion of Candida albicans to human buccal cells. Sustained release of antimicrobial agent chlorhexidine gluconate was achieved by these hydrogel formulations [63]. Chitosan hydrogels were also designed to offer sustained delivery of bone building agent ipriflavone at periodontal pockets. For the purpose, layered (mono and multi) composite systems comprising chitosan along with another biodegradable copolymer poly (lactic-co-glycolic acid) were designed for a prolong drug release. Studies in vitro demonstrated a sustained release of ipriflavone for 20 days [64].

20.4.1.2 Drug delivery in the GI tract Successful, localized delivery of drug molecules within the gastrointestinal tract faces a number of challenging barriers, i.e., extremely acidic environment of stomach, harsh cocktail of enzymes, and limited residence time of the drug. The combined effect results in reduced therapeutic efficacy of drug. In spite of these, targeting stomach and colon for drug delivery is exceedingly essential for the local treatment in certain pathological conditions such as inflammation, Crohn's disease, infection, ulcerative colitis, and malignant tumors [65]. Drug release to the particular tissue greatly reduces the dosage that is required in case of systemic treatment. Also, nontargeted tissues are not exposed to the drug thereby minimizing the side effects. The extensively ranging pH microenvironments within the gastrointestinal tract permits selective delivery by a drug-delivery system that shows pH-responsive swelling and drug release. Chitosan hydrogels can be easily modified to have the pH-dependent response or enzyme-specific triggering of drug release, thus making them suitable for use in oral drug-delivery system. Delivery of drug molecules to the acidic environment of stomach has been demonstrated by chitosan-based hydrogel that swells rapidly under acidic environments. Such quick response is an important property that is ideal owing to the rapid gastric emptying time; consequently, the residency time of drug molecules in the stomach is short. Patel et al. explored pH-sensitive and selective release mechanism in vivo using a chitosan polyethylene oxide semi-interpenetrating polymer network hydrogel [66]. These hydrogels achieved the local delivery of the metronidazole and amoxicillin to the stomach. Enzymatically degradable semi-interpenetrating polymer network

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c­ omprising gelatin-polyethylene oxide was achieved with the pH-sensitive character and also having desirable swelling properties, intended for oral drug delivery [67]. The integration of gelatin in the interpenetrating polymer network made it feasible for quick swelling at the acidic pH of the gastric fluid. The swelling is owing to the protonation of basic amino acid residues present in gelatin. This proteinaceous interpenetrating polymer network is degraded by proteolytic enzymes present in the gut pancreatin and pepsin. Alginate/hydroxypropyl-methylcellulose gel beads were developed to investigate the release of bovine serum albumin [68]. Gel beads exhibited high bovine serum albumin loading efficiency for all formulations. Incorporation of hydroxypropyl-­ methylcellulose increased the swelling ability of the beads without affecting particle size. The presence of hydroxypropyl-methylcellulose resulted in enhanced bovine serum albumin release in physiological saline solution (Fig. 20.4).

20.4.1.3 Drug delivery to the colon Enzymatic activity in the colon is less than that of small intestinal. Thus, large intestine is considered as a protected site for absorption of orally delivered proteins and peptides. Chitosan-based hydrogel drug-delivery system loaded with 5-amino salicylic acid, acetaminophen, insulin, and sodium diclofenac showed acceptable uptake within the colon [69–72]. The microflora of the colon present a degradation mechanism to the chitosan polymer itself; thus, the progress of degradation leads to controlled release of the loaded drug. An interpenetrating polymer network hydrogel of sodium alginate/carragenan is grafted with polyacrylamide and is crosslinked with glutaraldehyde released through the NSAID drug ketoprofen to intestine. In acidic environment only 10% drug was released, whereas when there was a shift in pH 7.4, the remaining load of the drug was released in a controlled way. The effectiveness of these hydrogels in protecting the ulcers induced by ketoprofen is tested in vivo. Entrapment of ketoprofen into hydrogel beads reduced the side effects common to NSAIDs such as gastric bleeding, gastric mucosal erosion, and ulceration [73]. Calcium-pectinated hydrogel microspheres and beads have been developed in the past decade with a focus on sustained release of different drugs, mainly for diminishing the release in gastric environment and prolonging it in the intestine [74–77]. Calcium pectinate hollow beads released a negligible amount of bioactives in acidic environment and then progress to complete the release at basic environment in a pulsed manner owing to swelling of the hydrogel beads at basic pH [78]. A separate in vivo study also confirmed that calcium pectinate gel beads having adequate degree of crosslinking precisely delivered the antimetabolite anticancer drug 5-fluorouracil to the colon [79]. Numerous polysaccharide-based hydrogels are presently being exploited as a prospective approach for colon-specific drug delivery. They comprise physically or chemically crosslinked polysaccharides, e.g., amidated pectin [80], dextran [81], ­inulin [82–84], and guar gum [85]. These hydrogels are designed in a manner that facilitates

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their degradation or are highly swollen in the incidence of colonic microflora or enzymes that dictate the colon specificity in drug delivery. pH-sensitive nanospheres were prepared for colon-specific delivery of 5-­aminosalicylic acid [86]. Sodium alginate was oxidized with sodium periodate and then modified by incorporating 4-aminothiophenol to the backbone (Fig. 20.5). This modified derivative could form core crosslinked nanospheres by self-assembly in deionized water. It was observed that the 5-aminosalicylic acid released over 15 h from the nanospheres in the glutathione-free media was around 25%, while the release of 5-aminosalicylic acid was increased markedly in pH 6.0 medium with micromolar glutathione (Fig. 20.6).

20.4.2 Ophthalmic delivery In ophthalmic delivery of drug, several physiological barriers, as a defensive mechanism in the eye, prevent successful drug delivery, for instance, effectual tear drainage, low permeability, and blinking of the cornea. Therefore, the drugs administered in the form of conventional eye drops tend to be removed quickly from the eye, resulting in inadequate absorption of the administered drug and reduced ocular bioavailability. In consequence of the short retention of drug, frequent administration of the eye drops is necessary to achieve the desired therapeutic concentration of the drug.

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(B) Fig. 20.5  (A) Synthetic scheme of TSA and (B) assembly process of disulfide crosslinked nanospheres. (Reprinted from Chang D, Lei J, Cui H, Lu N, Sun Y, Zhang X, et al. Disulfide cross-linked nanospheres from sodium alginate derivative for inflammatory bowel disease: preparation, characterization, and in vitro drug release behavior. Carbohydr Polym 2012;88:663–9, Copyright (2012), with permission from Elsevier.)

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Fig. 20.6  (A) Release profiles of 5-ASA from NS-2: in control media (■) and in simulated gastrointestinal media (●) and (B) Release profiles of 5-ASA from NS-1 (□), NS-2 (●) and NS-3 (∆) in simulated gastrointestinal media. (Reprinted from Chang D, Lei J, Cui H, Lu N, Sun Y, Zhang X, et al. Disulfide cross-linked nanospheres from sodium alginate derivative for inflammatory bowel disease: preparation, characterization, and in vitro drug release behavior. Carbohydr Polym, 2012;88:663–9, Copyright (2012), with permission from Elsevier.)

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Therefore, there is a great scope in developing novel liquid-based formulations of gels, colloidal systems, and nanoparticles [87]. Exploring the bioadhesives and penetration enhancers is a major avenue that was envisaged in search of future components in ocular therapeutics. Ocular mucoadhesion of the drug formulation prolongs the residence time of drug and is based on the affinity of some polymers toward the mucin coat covering the corneal surface and conjunctiva. The other strategy is to increase the rate and amount of drug transport with the help of penetration enhancers like surfactants [88]. Hydrogels prepared using chitosan hold great potential in ocular drug delivery owing to their inherent property of penetration-enhancing and mucoadhesive properties [89,90]. Chitosan hydrogels were evaluated for their ocular tolerability and precorneal residence time and have demonstrated to elevate corneal residence time of antimicrobial drug tobramycinin in comparison to commercial formulation of tobramycin [91]. Cohen et al. prepared an in situ gelling system of pilocarpine-loaded alginate [92]. The natural polymer having high guluronic acid content is chosen as a material of choice for the ocular delivery purpose. The duration of the pressure-diminishing effect of pilocarpine-loaded alginate gel system was 10 h and was 3 h for the conventional formulation of pilocarpine nitrate solution. Carlfors et  al. studied the rheological properties of deacetylated gellan gum (Gelrite) for their potential application as ocular drug delivery vehicle [93]. The solution of gelrite undergoes gel transition upon instilling into the eye. The observed long precorneal contact times (20 h in humans) of in situ gelrite gel are owing to their high rate of the sol/gel transition.

20.4.3 Transdermal delivery The dermal route of administering drug is very different from the enteric route, in which drug is exposed to the harsh surroundings of the gastrointestinal tract. The therapeutic intervention through transdermal route draws benefit from controlled drug release, and drug withdrawal is straightforward with the removal of the drug delivery system. Transdermal hydrogel drug delivery system offers attractive advantages due to their high water content, relaxed sensation on the patient's skin, and easy removal. Thus, they show the way to enhanced patient compliance with the course of the therapy [94,95]. Glimepride, an antidiabetic sulfonylurea drug with poor bioavailability due to poor water solubility, has shown potential for an effective delivery by transdermal chitosan hydrogel system. Therapeutic efficacy of this transdermal gel system was evaluated in mice and showed consistent antidiabetic effect over 48 h, thus signifying its promising application in clinical settings [96]. Chitosan hydrogels, in combination with penetration enhancer, were proficient in the delivery of benzyl isoquinoline alkaloidberberine, which is a quaternary ammonium salt having poor oral bioavailability [97] and is also successfully developed for an enantioselective delivery of S-enantiomer from the racemic propranolol using a reservoir-type transdermal patch [98]. The latter study employed molecularly imprinted polymer thin-layer composited cellulose membrane with S-propranolol selectivity for constructing the enantioselective controlled release system.

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Ketoprofen-loaded transdermal reservoir system, produced by a matrix of sodium alginate-grafted polyacrylamide, was crosslinked with glutaraldehyde. Using rat abdominal skin in vitro, ketoprofen release studies were carried out in modified diffusion cells. The cathode was placed in the donor compartment containing formulation, whereas phosphate buffer of pH 7.4 and anode were placed in the receptor cell. The deswelling of hydrogels due to the electric stimulus led to faster drug release. However, control hydrogels of crosslinked sodium alginate did not show any electric sensitivity. Pulsated release of ketoprofen was observed by repeated cycle of switching on and off electric stimulus [99]. As vehicles, numerous hydrogel-based preparations have been investigated for transdermal iontophoresis to achieve improved permeation of sodium nonivamide acetate, a capsaicinoid [100], luteinizing hormone releasing hormone [101], enoxacin [102], and nicotine [103].

20.4.4 Rectal delivery The rectal route of administering is preferable for various types of drugs, though patient satisfaction is inconsistent owing to the uneasiness from the administered dosage forms. The main application is to achieve local treatment of diseases connected with the rectum like hemorrhoids. Conventional suppositories are still now employed as a dosage form for rectal route of administration. They are solids at room temperature and melt or soften at the body temperature. There are some problems associated with rectal route of administration of suppositories such as uncontrolled drugs diffusion from suppositories, inability to retain for specific time and at specific position in the rectum, rare migration of suppositories upwards to the colon. This leads to inconsistencies in the bioavailability of a certain group of drugs that undergo extensive firstpass metabolism at liver. In this context, hydrogel designed with sufficient bioadhesive property after rectal administration offers some means to overcome the problems and limitations associated with conventional suppository formulations. Miyazaki et al. investigated the possible utility of xyloglucan gels endowed with thermal gelling property as vehicles for rectal drug delivery [104]. Xyloglucan hydrogel sol-gel transition temperature of the processed polysaccharide is around 22–27°C, and therefore, it can be a gel at body temperature. Alternatively, it can be simply administered as enema because it is a liquid at room temperature. Indomethacin-loaded xyloglucan gel administered rectally in rabbits showed well-controlled plasma concentration of the drug, showing increased bioavailability when compared with commercial indomethacin suppositories.

20.4.5 Nasal delivery Insulin-loaded thiolated chitosan nanoparticulate hydrogel considerably enhanced the absorption of insulin at nasal mucosa in comparison to the nonthiolated chitosan nanoparticulate hydrogel in addition to soluble chitosan [105]. Thiolated chitosan nanoparticulate hydrogel exerted enhanced mucoadhesion properties that lead to longer residence time at nasal mucosa. In addition, thiolated chitosan nanoparticulate hydrogel showed a quicker swelling and release in comparison to nonthiolated chitosan

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nanoparticles, while diffusion of the entrapped drug is assisted by swelling. Intranasal administration of thiolated nanoparticles loaded with insulin in rats showed the swift decrease in blood glucose levels. The decrease in glucose levels was similar to the group that received insulin subcutaneously. Nanoparticulate hydrogels comprising chitosan and negatively charged cyclodextrin derivative (sulfobutylether-β-cyclodextrinor carboxymethyl-β-cyclodextrin) were made and characterized [106]. A cell culture model showed that nanoparticulate hydrogel of chitosan-sulfobutylether-β-cyclodextrin induced lower Trans-epithelial electrical resistance (TEER) of Calu-3 cells than chitosan-carboxymethyl-β-cyclodextrin nanoparticulate hydrogel. However, intranasal administration of these two nanoparticulate hydrogels loaded with insulin demonstrated comparable reduction effects on the plasma glucose levels in rates. However, the direct measurement of plasma insulin concentrations of the treated animals may offer a direct indication of absorption enhancement of insulin administered through nasal route, which is not studied. Hydrophilic nonionic surfactant poloxamer 407 (Pluronic F127) was used as a penetration enhancement adjuvant in chitosan hydrogels loaded with Bordetella bronchiseptica antigens [107]. The intranasal administration of this hydrogel formulation showed an outstandingly enhanced systemic IgG and particularly S-IgA antibodies in comparison to free antigen used as control. They have shown only lower serum IgG titers when compared to intramuscularly administered vaccine. However, the animals vaccinated with the chitosan hydrogel added with ­permeability-enhancing adjuvant showed better protection to the nasal spray of B. bronchiseptica bacteria than the intramuscularly vaccinated group. Hagenaars et al. reported analogous results; intranasal vaccination of trimethyl chitosan-coated whole inactivated influenza virus (WIV) vaccine provided protection against the viral challenge [108]. They have reported that intranasal immunization with WIV strains induced stronger HI, IgG, IgG1, and IgG2a/c titers than WIV alone. The observed results indicate that quarternization of amino group of chitosan decreased the transepithelial resistance of epithelial cell monolayer, thus facilitating the absorption of WIV.

20.4.6 Subcutaneous delivery The capability of polysaccharide-based hydrogel that gel inside the body offers controlled delivery of the drugs. This distinct advantage made hydrogels a popular and versatile material in the area of subcutaneous delivery and implantable therapeutics. Polysaccharides are also a favored material owing to lack of inflammation and immunogenicity; these are common side effects of other materials, which are subcutaneously implanted. Treatments with certain growth factors having short biological half-life necessitate recurrent dosing to maintain an effective therapeutic concentration. Subcutaneous implantation of chitosan-albumin hydrogel microspheres in rates have shown a stable release pattern for over 3 weeks, signifying possible achievement of high degree of neovascularization in vivo [109]. A chitosan-based hydrogel crosslinked with glutaraldehyde [110,111] and loaded with (131) I-norcholesterol was prepared and implanted in 4T1 cell-induced tumors in

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xenograft mice models. This hydrogel demonstrated a lessening in the succession rate of the tumor growth and prevented 69% of tumor reappearance and metastatic spread. Significantly, there was little or no systemic circulation of the radioisotope from the hydrogel implantation. In recent times, a new in situ gelling hydrogel system of chitosan/dipotassium orthophosphate was developed for targeted delivery of doxorubicin to the site of malignancy [112]. The doxorubicin-incorporated hydrogel not only considerably repressed the growth of primary and secondary osteosarcoma, and lung metastasis at the same time, but also decreased the adverse effects of doxorubicin in mice models in comparison to conventional administration of the chemotherapeutic drug.

20.4.7 Pulmonary delivery Pulmonary delivery of drug has advanced substantially in the past decade due to significant inventions in the devises and excipients. Polysaccharides were explored as excipients for pulmonary drug delivery. Inhalable powder formulation of therapeutic protein salmon calcitonin was developed with chitosan and mannitol as excipients. The former is known for its absorption-enhancing property and the latter is incorporated as a protection agent. The stability, physicochemical property of the protein during and after the spray-drying process, the effect of chitosan concentration, in vitro release profiles in acetate and phosphate buffers were evaluated by various spectrometric and chromatographic techniques. A decrease in dissolution rate of calcitonin is observed with the increased proportion of chitosan in spray-drying formulation. However, addition of chitosan in a liquid formulation of the protein affected the recovery slightly. Partially irreversible complex formation between the protein and chitosan during the spray-drying formulation process is indicated [113]. Surface-modified nanospheres of dl-lactide/glycolide copolymer (PLGA) with chitosan were developed for pulmonary delivery of a peptide therapeutic elcatonin, a calcitonin derivative. This nanosphere suspension can be successfully aerosolized for pulmonary delivery of the drug. It was shown that the surface-modified PLGA nanospheres were retained in lungs for a long time and exerted a pharmacological action for 24 h, whereas nonsurface-modified PLGA nanosphere devoid of chitosan has an accelerated elimination from lungs. Surface modification of nanospheres with chitosan is a validated strategy for improving residential time in lugs due to the mucoadhesive property of the chitosan [114]. In a different study, the prospective of chitosan polymers and oligomers as an adjuvant for the peptide drug delivery to the lungs was detailed. Pulmonary administration of aqueous solutions of the oligomers and interferon-α enhanced the absorption of interferon-α in rat. Glucosamine hexamers at a concentration of 0.5% (w/v) showed the highest efficacy in improving the bioavailability when compared to many other oligomers that were tested. It also revealed that chitosan polymers were comparatively less efficient in increasing the systemic level of interferon-α than the oligomers studied; this may be attributed to its poor solubility in lung fluids [115].

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20.4.8 Gene delivery Proficient in vivo therapeutic delivery of nucleic acid remains the major challenge of gene therapy. The difficulties are mostly because of their hydrophilicity, enormous molecular size, and polynegative charges that prevent their transport across the epidermal layers. Free nucleic acids are degraded by nucleases present in the enteric system, which results in low transfection efficiency in oral route of administration. Therefore, parenteral route is preferred for delivering nucleic acids using nonviral vectors composed of cationic lipid complexes, polymers, or peptide carriers for plasmid DNA [116]. Cationic polymers are usually employed as vector-free carriers for nucleic material delivery [117]. Self-assemblies of cationic polymers and nucleic acids (DNA) are also exploited for favorable interactions with negatively charged cellular membrane and this constitutes a promising approach [118]. Zeng et al. employed spontaneous emulsion diffusion method in the preparation of PLGAchitosan nanoparticles for plasmid DNA (pDNA) delivery [119]. The use of chitosan is to improve pDNA-loading efficiency; to increase cellular uptake owing to the cationic charges of chitosan, the nanospheres demonstrate remarkable affinity toward negatively charged cell membranes. They also studied the small interfering RNA (siRNA) mediated gene silencing in slicing Hepatitis B virus in HepG cell lines. Mimi et al. prepared nanohydrogels of gelatin modified by conjugating with branched polyethylene imines to impart the cationic property [120]. They were used to encapsulate siRNA. These cationic nanoparticles demonstrated an increase in transfection efficiency to HeLa cells while effectively protecting the siRNA payload from enzymatic degradation. The nanogel-delivered siRNA demonstrated a 70% silencing of human argininosuccinate synthetase 1 (ASS1) gene expression. Wei et al. prepared a nanoparticulate hydrogel loaded with telomerase reverse transcriptase siRNA for transportation and suppression of tumor growth [121]. The nanogel composed of N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride is intended for oral administration. The cationic polymer coat prevented siRNA from being digested by gastric enzymes while offering to be transported to tumor cells. Oral delivery of siRNA using a pH-responsive nanoparticulate hydrogel fabricated with trimethylchitosan and methacrylic acid copolymer was reported by Dehousse et al. [122]. There was a considerable diminishing in zeta potential for nanoparticulate hydrogel complex containing methacrylic acid with better transfection efficiency in L929 cells in comparison to nanoparticulate hydrogel complex without methacrylic acid. At physiological pH guanidinylated chitosan produced stable complexes with plasmid DNA, and the complex displayed a reduced cytotoxicity, while exerting superior transfection efficiency in comparison to chitosan and 8-fold augment in cellular uptake [123,124]. Guanidinylation of chitosan helped to augment the siRNA gene-silencing activity in comparison to the pure chitosan owing to improved cellular internalization.

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20.5 Conclusion The introduction of distinct crosslinking agents and processed polysaccharide that can undergo gelation facilitated the escalation in diversity of hydrogels that can be fabricated. A variety of hydrogel systems are established with modified porosities and mechanical strengths that can be fine tuned to serve the purpose in a chosen application area. The drug-delivery applications of polysaccharide-based hydrogels are of immense importance due to their demonstrated ability to respond to the surrounding environment in a predictable manner with regard to biocompatibility and biodegradability. We predict that these existing accomplishments will drive the future generation of drug delivery systems as we progress further to gain more insights of the dynamics of mixed polysaccharide chain network systems. The capability of predictable manipulation of the hydrogel-based drug-delivery systems will improve with better understanding of the factors influencing the stability and controlled release. When the design parameters and the resulting properties are well known, efficient ­polysaccharide-based hydrogel drug-delivery systems take a leap toward clinical availability.

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[86] Chang  D, Lei  J, Cui  H, Lu  N, Sun  Y, Zhang  X, Gao  C, Zheng  H, Yin  Y. Disulfide cross-linked nanospheres from sodium alginate derivative for inflammatory bowel disease: preparation, characterization, and in vitro drug release behavior. Carbohydr Polym 2012;88:663–9. [87] Alonso  MJ, Sanchez  A. The potential of chitosan in ocular drug delivery. J Pharm Pharmacol 2003;55:451–1463. [88] Kaur IP, Smitha R. Penetration enhancers and ocular bioadhesives: two new avenues for ophthalmic drug delivery. Drug Dev Ind Pharm 2002;28:353–69. [89] Thanou M, Verhoef JC, Junginger HE. Chitosan and its derivatives as intestinal absorption enhancers. Adv Drug Deliv Rev 2001;50:S91–101. [90] Kotze AF, Luessen HL, de Boer AG, Verhoef JC, Junginger HE. Chitosan for enhanced intestinal permeability: prospects for derivatives soluble in neutral and basic environments. Eur J Pharm Sci 1999;7:145–51. [91] Felt  O, Furrer  P, Mayer  JM, Plazonnet  B, Buri  P, Gurny  R. Topical use of chitosan in ophthalmology: tolerance assessment and evaluation of precorneal retention. Int J Pharm 1999;180:185–93. [92] Cohen S, Lobel E, Trevgoda A, Peled T. A novel in situ-forming ophthalmic drug delivery system from alginates undergoing gelation in the eye. J Control Release 1997;44:201–8. [93] Carlfors J, Edsman K, Petersson R, JoÈrnving K. Rheological evaluation of gelrite in situ gels for ophthalmic use. Eur J Pharm Sci 1998;6:113–9. [94] Thomas  BJ, Finnin  BC. The transdermal revolution. Drug Discov Today 2004;9:697–703. [95] Prausnitz MR, Mitragotri S, Langer R. Current status and future potential of transdermal drug delivery. Nat Rev Drug Discov 2004;3:115–24. [96] Ammar HO, Salama HA, El-Nahhas SA, Elmotasem H. Design and evaluation of chitosan films for transdermal delivery of glimepiride. Curr Drug Deliv 2008;5:290–8. [97] Tsai CJ, Hsu LR, Fang JY, Lin HH. Chitosan hydrogel as a base for transdermal delivery of berberine and its evaluation in rat skin. Biol Pharm Bull 1999;22:397–401. [98] Suedee R, Bodhibukkana C, Tangthong N, Amnuaikit C, Kaewnopparat S, Srichana T. Development of a reservoir-type transdermal enantioselective-controlled delivery system for racemic propranolol using a molecularly imprinted polymer composite membrane. J Control Release 2008;129:170–8. [99] Kulkarni RV, Setty CM, Sa B. Polyacrylamide-g-alginate-based electrically responsive hydrogel for drug delivery application: synthesis, characterization, and formulation development. J Appl Polym Sci 2010;115:1180–8. [100] Fang JY, Huang YB, Lin HH, Tsai YH. Transdermal iontophoresis of sodium nonivamide acetate. IV. Effect of polymer formulations. Int J Pharm 1998;173:127–40. [101] Chen  LLH, Chien  YW. Transdermal iontophoretic permeation of luteinizing hormone releasing hormone: characterization of electric parameters. J Control Release 1996;40:187–98. [102] Fang  JY, Hsu  LR, Huang  YB, Tsai  YH. Evaluation of transdermal iontophoresis of enoxacin from polymer formulations: in vitro skin permeation and in vivo microdialysis using Wistar rat as an animal model. Int J Pharm 1999;180:137–49. [103] Conaghey  OM, Corish  J, Corrigan  OI. Iontophoretically assisted in  vitro membrane transport of nicotine from a hydrogel containing ion exchange resins. Int J Pharm 1998;170:225–37. [104] Miyazaki S, Suisha F, Kawasaki N, Shirakawa M, Yamatoya K, Attwood D. Thermally reversible xyloglucan gels as vehicles for rectal drug delivery. J Control Release 1998;56:75–83.

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21

Amit Kumar Nayak⁎, Hriday Bera† ⁎ Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj, India, †Faculty of Pharmacy, AIMST University, Bedong, Malaysia

21.1 Introduction The word, “gel” is derived from the Latin term gelu, which means freeze, frost, or congeal and thus, designates the setting of liquid(s) to form semi-solid and solid-like material(s) [1,2]. Therefore, various gels hold a number of characteristic features of liquid(s) by reason of their semi-elastic and elastic characters. During the 18th century, firstly, the term “gel” was applied to classify semi-solid material(s) based on the phenomenological features relatively than the molecular compositions [3]. According to Almdal et al., the “gel” is a solid-like system composed of a minimum of two constituents, and one of the constituents must be a liquid [4]. According to the Encyclopedia of Polymer Science and Engineering, “a gel is a cross-linked polymer network swollen in a liquid medium. Its properties depend strongly on the interaction of these two components” [3]. In most cases, polymeric gels are semi-solid natured systems comprising of a minimum of two components. Within these, polymer(s) configure a 3-D (three-dimensional) networking by the virtue of covalent/noncovalent bond(s) in the medium of other components of liquid natured, wherein the least amount of the liquid is enough to guarantee having the elastic properties of gels [1,3]. On the other hand, the phrase, “in situ” is a Latin term which means “in process” [5]. Recent years, several in situ processing-based polymeric formulations are being developed, where in situ gelation and/or in situ cross-linking of polymers are being employed for drug delivery [6,7]. Basically, in situ gels are polymeric colloidal systems which are of solution type (i.e., sol form) before the administrations in/onto the body; once administered, these undergo the formation of gel in situ through sol-gel transition [6,8]. The formulation of in situ gels is less complex with low manufacturing expenses [9]. The development of these in situ gelling systems depends on some important issues such as occurrence of ions, alteration of pH, temperature modulation, solvent exchanging, ultra-violet (UV) irradiation, etc. [8,10,11]. From the manufacturing viewpoint, the preparation of in situ polymeric gels is not complex enough and therefore it lowers the manufacturing expenses [8,12]. Over the last few decades, the development of a variety of in situ polymeric gels has received extensive interest and these are being explored for their applications in different biomedical uses including drug delivery [11,13,14]. The in situ polymeric gel-based formulations present some important advantages (Fig.  21.1), such as increased residence time of drugs at the Polysaccharide Carriers for Drug Delivery. https://doi.org/10.1016/B978-0-08-102553-6.00021-0 Copyright © 2019 Elsevier Ltd. All rights reserved.

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Fig. 21.1  Advantages of in situ gel formulations.

application site, decreased frequency of drug administration, low dose required for treatment, minimizing side effects (local and systemic), and improved patient compliance [4,9]. These polymeric gel-based systems are capable of providing localized and prolonged drug delivery. From the early 1960s, a variety of natural, semisynthetic, and synthetic polymers are being researched and developed for the formulations of numerous in situ polymeric gel-based systems [2,9,14]. Recently, various smart polymers are used to develop advanced in situ gels as smart polymers that easily undergo sol-gel transitions once administered [10,12]. Nowadays, various biodegradable and hydrophilic polysaccharides such as alginic acid, chitosan, gellan gum, pectin, xyloglucan, carageenan, etc. are employed to formulate various in situ gels for the use in topical drug delivery, from which drugs get released in a sustained as well as controlled way [6,9]. Mainly, in situ polysaccharide-based topical gels are administered by transdermal, ocular, rectal, vaginal, etc. routes [2,10,14]. The current chapter presents a comprehensive discussion on in situ polysaccharide-based gels for drug delivery applications through various topical drug administration routes like ocular, nasal, transdermal, rectal, and vaginal routes.

21.2 In situ polysaccharide-based gels for ocular drug delivery Localized ocular drug delivery is employed for different categories of drugs such as anti-inflammatory drugs, anti-fungal drugs, anti-bacterial drugs, autonomic drugs, etc. [10,15–17]. The uses of conventional drug delivery often bring about poor bioavailability and very much lower therapeutic effectiveness due to the fact of high turnover

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and dynamics of tear fluids, which results in hasty drug eliminations from the eyes [16,18,19]. Therefore, to conquer the ocular bioavailability troubles, ocular in situ gel-based systems were developed [18,20,21]. The ocular in situ gel systems comprise of environmentally responsive polymers, which will be structurally changed with some specific circumstances such as changes of temperature, pH, and ionic strength [10,15,16,20]. Ocular in situ gels are liquids during application/installation into/onto the eye followed by undergoing rapid formation of gels in the cul-de-sac of the eye to form the viscoelastic gels via the sol-gel transition by the influence of one or more environmental response(s) [20,22]. As a result, the residence period of ocular in situ gels formed may be widened and the drug releasing from these ocular in situ gel-based systems may occur by a sustained mode [23]. These will be advantageous to achieve better bioavailability, minimal systemic absorption of drugs, and a reduction of frequent dosing. The attainment of these advantages by the ocular application/installation of in situ gel-based systems surely improves patient compliance [17,23]. In addition, some other prospective advantages of the topical ocular use of in situ gel-based systems are ease of administration, accurate dose deliverance, simple preparation process, and expenses [15,24]. For in situ polysaccharide-based gel-based ocular delivery, natural polysaccharides such as gellan gum, alginic acid, xyloglucan, chitosan, etc., are the most commonly used polymers [10,25]. In recent decades, numerous ocular in situ gels made of gellan gum (an anionic marine natural polysaccharide) have been developed and evaluated for ocular delivery of different drugs [26–28]. The drug releasing patterns of these ocular in situ gellan gum-based gels were found to be sustained over a prolonged period because of the attainment of prolonged precorneal contact time by the viscous nature of in situ gellan gum-based gels as compared to that of the conventional ophthalmic drops [27,29]. In a study by Dewan et al. [30], various in situ ocular gels were prepared using gellan gum and poloxamer 407 for the delivery of pilocarpine HCl. The influence of the gellan gum concentration on the thermo-gelation characteristics and the pilocarpine HCl releasing profile of in situ ocular gels. From the preliminary study results, the in situ ocular gels of gellan gum concentration 0.10%–0.30 wt% were found suitable as an in situ gelled ocular drug delivery system. The in vitro pilocarpine HCl releasing from various in situ ocular gels were studied at 37°C in artificial tear fluid by gravimetric method. In case of in situ ocular gels of pilocarpine HCl made of 18 wt% poloxamer 407 (PM), the largest fraction of the pilocarpine HCl was measured to be released within a period of 5 h. However, for gellan gum-based in situ ocular gels of pilocarpine HCl made of 18 wt% poloxamer 407 with 0.10 wt% gellan gum (PMG-1) and 18 wt% poloxamer 407 with 0.30 wt% gellan gum (PMG-2), a decreased tendency in the in vitro drug releasing rate was measured (Fig. 21.2). The gel dissolution rates from these in situ ocular gels were found to be significantly reduced by reason of gellan gum addition. This fact can be attributed not only to the greater viscosity due to addition of gellan gum, but also because of the occurrence of the physical cross-linking process among the sodium ions contained in the gellan gum and tear fluid. In research by Shastri et al. [31], a thermo-reversible in situ gelling mucoadhesive system for ocular delivery of moxifloxacin HCl (a fourth generation fluroquinolone antibiotic) was developed and evaluated. These moxifloxacin HCl releasing in situ

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Cumulative drug release (%)

100 PM PMG-1 PMG-2

80

60

40

20

0 0

2

4 Time (h)

6

8

Fig. 21.2  In vitro pilocarpine HCl releasing from various ocular in situ gels were studied at 37°C in artificial tear fluid (PM: 18 wt% poloxamer 407, PMG-1: 18 wt% poloxamer 407 with 0.10 wt% gellan gum, and PMG-2: 18 wt% poloxamer 407 with 0.30 wt% gellan gum). From Dewan M, Sarkar G, Bhowmik M, Das B, Chattoapadhyay AK, Rana D, Chattopadhyaya D. Effect of gellan gum on the thermogelation property and drug release profile of Poloxamer 407 based ophthalmic formulation. Int J Biol Macromol 2017;102:258– 65. Copyright @ 2017, with permission from Elsevier B.V.

ocular gels were prepared using Pluronic F68, Pluronic F127 and deacetylated gellan gum (Gelrite). The statistical optimization of the drug delivery formula was carried out by a 32 full factorial design considering deacetylated gellan gum and Pluronic F68 as independent variable factors used in combination with Pluronic F127. On the other hand, tested dependent variable factors selected were viscosity, gel strength, gelation temperature, bioadhesion force, and in vitro releasing of moxifloxacin HCl (after 1 and 10 h). The analyses of the 32 full factorial design-based statistical optimization of the development of in situ gelling mucoadhesive system for ocular of moxifloxacin delivery clearly indicated that the gelation temperature, gel strength, bioadhesion force, and drug release were powerfully dependent on the independent variable factors. The incorporation of Pluronic F68 within Pluronic F127-based in situ gels demonstrated a significant effect on the gelation temperature of the in situ gels containing moxifloxacin HCl. The incorporation of deacetylated gellan gum within Pluronic F127-based in situ gels of moxifloxacin HCl demonstrated an affirmative effect on the bioadhesion force as well as gel strength. These parameters of the in situ ocular gels of moxifloxacin HCl were found to be supportive in controlling the drug release rate. Zhu et al. [32] developed a novel in situ gel-based ocular ketotifen delivery made of deacetylase gellan gum, which showed sustained release of ketotifen over a longer time in both in vitro and in vivo studies. Sechoy et al. [33] evaluated the in situ gel-based system of alginic acid (an anionic natural polysaccharide) for the ocular delivery of carteolol. The measurements of

In situ polysaccharide-based gels for topical drug delivery applications619

i­ntraocular pressure of the rabbit’s eyes after the treatment with carteolol (1%) containing ocular formulations with alginic acid and without alginic acid demonstrated that the ocular formulation containing alginic acid produced a significantly prolonged duration (8 h) owing to the pressure reduction action. The improved ocular bioavailability of 1% carteolol by the use of ocular formulation containing alginic acid produced an equivalent concentration of carteolol in the targeted ocular tissue even though the ocular administration of 1% carteolol (alone) was only once a day in comparison with that of twice a day. On the whole, the results of this investigation clearly suggested the potential of the use of alginic acid as an effectual vehicle material and excellent carrier excipient for drug delivery. Such kinds of in situ gel-based systems of alginic acid could be used for the development of other long-acting ocular in situ gels of other drugs needed for ocular administration. Aminabhavi et al. [34] developed an in situ polysaccharide-based gel systems for ocular delivery of timolol maleate. These in situ polysaccharide-based ocular gel systems were composed of gaur gum, sodium alginate, and Carbopol 940. In this work, rheological characteristics, gel producing ability, and polymer-blend compatibility were evaluated and analyzed. An enhanced viscosity of in situ polysaccharide-based ocular gel systems containing timolol maleate was noticed upon exposure to specific pH, ions, and temperature of the eyeball. Both in vitro and ex vivo drug releasing study results suggested a prolonged releasing pattern of drug (i.e., timolol maleate). In a research, Miyazaki et al. [35] prepared ocular in situ gels of pilocarpine HCl using xyloglucan (a plant derived polysaccharide). This pilocarpine HCl releasing ocular in situ gel system exhibited a sustained drug releasing pattern, in vitro. In vitro drug releasing from the in situ ocular gels formed via warming the 1%, 1.5%, and 2%, w/w xyloglucan sols at the temperature of 34°C was found to follow the root time kinetics over 6 h. The formulated ocular in situ gel systems of pilocarpine HCl were tested for in vivo potential in the rabbit eye. The in vivo results demonstrated a significant mitotic action over 4 h, when the ocular in situ xyloglucan gels containing pilocarpine HCl were instilled into the lower cul-de-sac area of the rabbit’s eye. Gratieri et  al. [36] investigated the ocular drug delivery potential from an in situ gel made of chitosan (a cationic natural aminopolysaccharide extracted from the partial deacetylation of chitin) and poloxamer. This kind of in situ gel made of ­poloxamer-chitosan exhibited a prolonged retention time at the application site. In another study, the same research group also developed a chitosan/poloxamer in situ forming gels for the topical ocular releasing of an antifungal drug, fluconazole [37]. The researchers formulated the chitosan/poloxamer in situ gels of fluconazole using 0.5%, 1% or 1.5% w/w chitosan and 16% w/w of poloxamer 407. These in situ gels of fluconazole were evaluated for ex  vivo drug permeation across the porcine cornea. The results of this evaluation revealed that these in situ gel-based formulations had a permeation-enhancement action, which was found not to be dependent on the chitosan concentration within the range 0.5%–1.5% w/w. The chitosan/poloxamer in situ gels of fluconazole showed a similar pattern of in vivo activity to the 1% chitosan solution containing fluconazole. Both the formulations (chitosan/poloxamer in situ gels of fluconazole and 1% chitosan solution containing fluconazole) demonstrated a sustained drug releasing pattern, which was estimated to be about 3.5-fold greater

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total fluconazole amount permeated as compared to that of the aqueous solutions of fluconazole. In research, a pH and temperature responsive in situ ocular gel containing timolol maleate was developed and evaluated by Gupta et al. [38]. The in situ ocular gel system of timolol maleate was prepared by using poloxamer and chitosan. This in situ ocular gel system possessed a bioadhesive property and gel-forming characteristics at the ocular pH. In vitro transcorneal permeability measurement was carried out on the excised goat corneal membrane and the results exhibited an improved drug permeation profile after 4 h for the in situ gelling system made of poloxamer-chitosan in comparison with the conventional eye drops. The results of this research demonstrated that the incorporation of chitosan within the in situ gel formula enhanced the transcorneal permeability of timolol. Varshosaz et al. [39] reported that 15% Pluronic F127 lost the gelling ability after the lacrimal fluid dilution, when it was utilized alone. The phase change temperature turned significantly from 39°C to 43°C. They also developed and evaluated a temperature responsive chitosan/Pluronic in situ ocular gel of ciprofloxacin. These ciprofloxacin releasing in situ ocular gel containing 0.1% chitosan and 15% Pluronic F127 was found to be liquid in nature at the nonphysiological milieu (25°C, pH 4) and transferred to gel appearance under the physiological milieu (37°C, pH 7.4). The overall results of this research demonstrated that this temperature responsive chitosan/Pluronic in situ ocular gel of ciprofloxacin could be employed as an effective option to the conventional eye drops of ciprofloxacin. Khan et al. [40] developed a novel kind of temperature responsive poloxamer 407/chitosan in situ gel, in which tobramycin sulfate-loaded mucoadhesive microparticles were dispersed. The incorporation of chitosan in these poloxamer 407/chitosan in situ gel formulations produced viscosity enhancement and mucoadhesivity improvement of the gel nature. These in situ gel containing tobramycin sulfate-loaded mucoadhesive microparticles supported increased in vitro permeability with an improved aqueous humor concentration of tobramycin sulfate, in comparison with commercial ocular drops of tobramycin sulfate without any ocular irritation. Therefore, temperature responsive poloxamer 407/chitosan in situ gel containing tobramycin sulfate-loaded mucoadhesive microparticles can be employed for the treatment of ocular bacterial infections. A chitosan/Carbopol-based pH-responsive in situ gelling system of timolol maleate was developed by Gupta and Vyas [41]. In these in situ gel systems, Carbopol and chitosan were used as a gelling material and a viscosity enhancement material, respectively. At room temperature and pH 6.0, the in situ gel containing 0.5% w/v chitosan and 4% w/v carbopol was found in liquid state. At the tear fluid pH (7.4), this in situ gel underwent rapid transition into the viscous gel state. The in  vitro releasing of loaded drug (timolol maleate) and the in vivo efficacy of the chitosan/Carbopol-based pH-responsive in situ gelling system were compared with 0.4% w/v Carbopol solution (without chitosan), marketed formulation of timolol maleate (Glucomol, 0.25% timolol maleate ophthalmic solution) and liposomal formulation of timolol maleate. The results of in vitro drug releasing and in vivo study noticeably exhibited that formulated chitosan/Carbopol-based pH-responsive in situ gelling system of timolol maleate was found to be therapeutically effective in comparison with other formulations tested. The in vitro drug releasing from these in situ ocular gels demonstrated a Fickian diffusion mechanism over a period of 24 h. In the in vivo evaluations, in case of Glucomol

In situ polysaccharide-based gels for topical drug delivery applications621

(the marketed formulation having 0.25% timolol maleate), the timolol maleate amount leaked was estimated as 2.08% after 10 min. The 0.4% w/v carbopol solution and liposomes containing timolol maleate presented some control over the timolol maleate drainage of 1.48% and 0.89%, respectively, which was estimated to be drained after a period of 2 h. However, only 0.86% of timolol maleate was estimated to be leaked from the formulated chitosan/Carbopol-based pH-responsive in situ gelling system of timolol maleate into the circulation after a period of 2 h and that was significantly (P  0.05) were evidenced in the degradation rates of the nanogel without or with chitosan. This result indicated that the chitosan nanoconjugate containing ibuprofen did not interfere with the gelation of gellan gum. The gravimetric alterations in the swelling and degradation profiles of the nanoconjugate indicated that this might support the ibuprofen releasing from the chitosan-ibuprofen-gellan gum ternary nanogels. Chitosan was found to improve the ex  vivo skin permeation and transdermal permeation rate (fourfold increments). This occurrence depended on the rate of ionic interactions of ibuprofen-chitosan and also their concentration. The mechanism of ex vivo permeation of ibuprofen across the pig skin was revealed as the drug diffusion. However, the drug partition as well as drug erosion occurred. From the results of this investigation, it was clear that these novel in situ nanogels are capable of providing controlled transdermal delivery of ibuprofen. In an investigation, Sun et al. [70] studied the transdermal delivery of curcumin and its complexes with hydroxypropyl-β-cyclodextrin from the in situ hydrogels for the use in the treatment of melanoma. In this work, the inclusion complexes of curcumin were prepared by grinding. The in situ hydrogels of curcumin and its inclusion complexes were formulated by employing two different grades of poloxamer: poloxamer 407 and poloxamer 188. The in vitro transdermal permeation was carried out in phosphate buffer saline (pH 7.4) by the Franz-type diffusion cell. The in vitro drug releasing rate of these in situ hydrogels of curcumin was found to be dependent on the drug dissolution. Both the in situ hydrogels containing curcumin inclusion complexes exhibited higher transdermal permeation as compared to that of in situ hydrogels containing curcumin. The cytotoxicity of the in situ hydrogels containing inclusion complexes (tested using mouse melanoma cell line- B16-F10) was found to be higher than the hydrogel containing curcumin, and the cytotoxicity of the melanoma cells was associated with the fact of the blocking of cellular proliferation in the G2/M stage followed by the cellular apoptosis.

21.5 In situ polysaccharide-based gels for rectal and vaginal drug delivery Drug administration through the rectal and vaginal routes is recognized as the effective alternative to the oral route [71]. In situ gels also have the potential uses in the delivery of various drugs through rectal as well as vaginal routes. Rectal drug delivery has been employed efficiently in the treatment of various local diseases in the anorectal zone and to deliver drugs systematically, in particular, to certain populations that are often problematic to treat clinically with oral dosage forms [72,73]. Rectal gels are also

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helpful to use for such patients (such as, infants, elderly, and unconscious patients), who are unable to swallow the oral dosage [74]. During the past few decades, numerous topical rectal in situ gels have been developed and evaluated for their clinical efficacy. These rectal in situ gels provide some potential advantages over the conventional rectal suppositories [72,73]. These advantages are ease of dosage administration and the control of exact dose volume as the liquid at the room temperature turns instantly into gel form at the physiological temperature. Recently, various polysaccharides are being utilized to formulate these in situ gels for the rectal delivery of different drugs. Sometimes, these polysaccharide-based in situ gels add the mucoadhesivity with the tissues of rectal areas without any leakage after the dosing [73]. Miyazaki et al. [75] studied the efficacy of xyloglucan-based temperature responsive in situ gels for the rectal delivery of indomethacin (a nonsteroidal anti-­inflammatory drug). The rectal administration of xyloglucan-based in situ gels containing indomethacin to the rabbits showed a broad peak suggesting the drug absorption with a prolonged residence period of the dosage in comparison with that resulting after the rectal administration of marketed suppository of indomethacin. Moreover, a ­significant decrease of drug Cmax was noticed after the rectal administration of the in situ polysaccharidic system made of xyloglucan. Yuan et al. [73] formulated temperature responsive mucoadhesive rectal in situ gels of nimesulide (a nonsteroidal anti-­ inflammatory drug) using sodium alginate (a natural polysaccharide) and HPMC (a semisynthetic polysaccharide) as mucoadhesive agents. These temperature responsive rectal in situ gels of nimesulide contained 2% nimesulide, 0.5% mucoadhesive polysaccharide(s), and 18% poloxamer 407. In vitro nimesulide releasing from the in situ polysaccharide-based gels was carried out by the USP paddle type dissolution method using a semipermeable dialysis bag. In addition, polyethylene glycol was employed to modify the gelation temperature and nimesulide releasing characteristics. The gelation temperature was found to be significantly increased with the incorporation of drugs (i.e., nimesulide) within the solution of poloxamer 407, while the incorporation of polyethylene glycol improved the gelation temperature and nimesulide releasing rate. Among these temperature responsive rectal in situ gels, rectal gels containing 2% nimesulide, 18% poloxamer 407, 0.5% sodium alginate, and 1.2% polyethylene glycol showed the desirable gelation temperature and nimesulide releasing with a sufficient period of rectal retention at the site of application. In addition, microscopic morphology of the rectal mucosa in male albino rabbits after the rectal administration of nimesulide contained rectal in situ gels given at the 20 mg/kg, was found safe without any irritation at the rectal mucosa (Fig.  21.6). Moreover, these in situ ­polysaccharide-based gels resulted in significantly higher initial serum concentrations (Fig. 21.7) and the bioavailability, in vivo. Topical vaginal administration comprises the application of dosage forms onto the vagina, often, using an applicator [76]. Vaginal drug delivery systems are being employed to attain localized action and to deliver various drugs systemically devoid of hepatic first-pass metabolism [77]. The prime benefits of the vaginal route of drug administration is that various diseases of the vagina can be effectually treated locally through facilitating higher drug concentrations at the diseased site allowing a potential minimization of the adverse effect chances [76,77]. During the past few decades,

In situ polysaccharide-based gels for topical drug delivery applications631

Fig. 21.6  Microscopic morphology of rectal mucosa of rabbits after the rectal administration of nimesulide in situ gel (A) before the dose and (B) after the dose. From Yuan Y, Ying C, Li Z, Hui-ping Z, Yi-Sha G, Bo Z, Xia H, Ling Z, Xiao-hui W, Li C. Thermosensitive and mucoadhesive in situ gel based on poloxamer as new carrier for rectal administration of nimesulide. Int J Pharm 2012;430:114–9. Copyright @ 2012, with permission from Elsevier B.V.

Serum concentration (mg/mL)

30 Conventional suppository Retal in situ gel 20

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Fig. 21.7  Mean serum concentration-time profiles following rectal administration of nimesulide in situ gel and conventional solid suppository. In situ gel was composed of 2% nimesulide, 18% poloxamer 407, 0.5% sodium alginate and 1.2% polyethylene glycol. Conventional solid suppository of nimesulide was composed of. From Yuan Y, Ying C, Li Z, Hui-ping Z, Yi-Sha G, Bo Z, Xia H, Ling Z, Xiao-hui W, Li C. Thermosensitive and mucoadhesive in situ gel based on poloxamer as new carrier for rectal administration of nimesulide. Int J Pharm 2012;430:114–9. Copyright @ 2012, with permission from Elsevier B.V.

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n­ umerous topical vaginal gels have been researched and developed because of their ease of application, good spreading, and retention capabilities so that these cause minimal or no irritation to the patient [78]. In recent years, various vaginal in situ gels made of polysaccharides have been investigated by various research groups and most of these in situ polysaccharidic topical vaginal gels were reported to have improved retention time and desirable mucoadhesive properties [79–81]. Patel and Patel [82] formulated and evaluated in situ gel of clindamycin HCl for vaginal application. These in situ gels of clindamycin were made of gellan gum and HPMC using 0.9% NaCl as an isotonic agent by means of the cold method. Gel persistent spreadability of polysaccharidic in situ gels of clindamycin HCl was measured to have minimum 8 h and maximum