Bioadhesives in Drug Delivery (Adhesion and Adhesives: Fundamental and Applied Aspects) 1119640199, 9781119640196

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Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Adhesion and Adhesives: Fundamental and Applied Aspects The topics to be covered include, but not limited to, basic and theoretical aspects of adhesion; modeling of adhesion phenomena; mechanisms of adhesion; surface and interfacial analysis and characterization; unraveling of events at interfaces; characterization of interphases; adhesion of thin films and coatings; adhesion aspects in reinforced composites; formation, characterization and durability of adhesive joints; surface preparation methods; polymer surface modification; biological adhesion; particle adhesion; adhesion of metallized plastics; adhesion of diamond-like films; adhesion promoters; contact angle, wettability and adhesion; superhydrophobicity and superhydrophilicity. With regards to adhesives, the Series will include, but not limited to, green adhesives; novel and high-performance adhesives; and medical adhesive applications. Series Editor: Dr. K.L. Mittal P.O. Box 1280, Hopewell Junction, NY 12533, USA Email: [email protected] Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Bioadhesives in Drug Delivery

Edited by

K.L. Mittal, I. S. Bakshi and J. K. Narang

This edition first published 2020 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2020 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no rep­ resentations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchant-­ ability or fitness for a particular purpose. No warranty may be created or extended by sales representa­ tives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further informa­ tion does not mean that the publisher and authors endorse the information or services the organiza­ tion, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data Names: Mittal, K. L., 1945– editor. Title: Bioadhesives in drug delivery / edited by K.L. Mittal, I.S. Bakshi and J.K. Narang. Description: First edition. | Hoboken, NJ : Wiley-Scrivener, 2020. | Series: Adhesion and adhesives : fundamental and applied aspects | Includes bibliographical references and index. Identifiers: LCCN 2020018394 (print) | LCCN 2020018395 (ebook) | ISBN 9781119640196 (cloth) | ISBN 9781119640257 (adobe pdf) | ISBN 9781119640264 (epub) Subjects: LCSH: Bioadhesive drug delivery systems. Classification: LCC RS201.B54 B59 2020 (print) | LCC RS201.B54 (ebook) | DDC 615.1–dc23 LC record available at https://lccn.loc.gov/2020018394 LC ebook record available at https://lccn.loc.gov/2020018395 Cover image: Pixabay.Com Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Manila Typesetting Company, Makati, Philippines Printed in the USA 10 9 8 7 6 5 4 3 2 1

Contents Preface xvii Part 1: Fundamental Aspects

1

1 Introduction, Theories and Mechanisms of Bioadhesion Kamla Pathak and Rishabha Malviya 1.1 Introduction 1.1.1 Historical Perspective 1.1.2 Bioadhesion in Biological Systems 1.1.3 Bioadhesive/Mucoadhesive 1.1.4 Factors Affecting Mucoadhesion 1.1.4.1 Molecular Weight of Polymer 1.1.4.2 Concentration of Polymer Used 1.1.4.3 Flexibility of Polymer Chains 1.1.4.4 Swelling 1.1.4.5 pH at Polymer-Mucus Interface 1.1.4.6 Mucin Turnover Rate 1.1.4.7 Stereochemistry 1.2 Bioadhesive Interactions 1.3 The Mechanistic Approach to Bioadhesion 1.4 Factors Controlling Bioadhesion 1.4.1 Chemical Interactions 1.4.1.1 Mussel Adhesion 1.4.1.2 Cell Adhesion to Biomaterials 1.4.2 Surface Morphology Effects 1.4.3 Physiological Factors 1.4.4 Physical and Mechanical Factors 1.4.4.1 Wetting Phenomenon 1.4.4.2 Interpenetration 1.5 Theories of Bioadhesion 1.5.1 Wetting Theory

3 4 4 5 6 6 6 7 7 7 7 7 7 8 9 10 10 10 11 11 12 12 12 12 13 13 v

vi  Contents 1.5.2 Diffusion Theory 1.5.3 Electronic Theory 1.5.4 Adsorption Theory 1.5.5 Fracture Theory 1.6 Stages of Mucoadhesion 1.7 Modulation of Mucoadhesion 1.8 Adhesion Promoters 1.9 Surface Free Energy Analysis of Bioadhesion 1.10 Molecular Biology in Bioadhesion 1.11 Bioadhesives from Marine Sources 1.12 Mucoadhesive Drug Delivery Systems 1.13 Summary References 2 Bioadhesive Polymers for Drug Delivery Applications Kenneth Chinedu Ugoeze 2.1 Introduction 2.1.1 Drug Delivery 2.2 Bioadhesive/Mucoadhesive Drug Delivery Systems 2.2.1 Some Advantages of Bioadhesive/Mucoadhesive Drug Delivery Systems 2.2.2 The General Need for Bioadhesive/Mucoadhesive Drug Delivery Systems 2.3 Mechanism of Bioadhesion 2.4 Requirements for an Ideal Bioadhesive/Mucoadhesive Polymer 2.5 Factors Affecting Bioadhesion/Mucoadhesion 2.5.1 Polymer Related Factors 2.5.1.1 Molecular Weight 2.5.1.2 Chain Length 2.5.1.3 Flexibility 2.5.1.4 Cross-Linking 2.5.1.5 Presence of Functional Groups 2.5.1.6 Concentration of Active Polymer 2.5.2 Environmental Factors 2.5.2.1 pH and Charge on the Polymer 2.5.2.2 Degree of Hydration 2.5.2.3 Initial Contact Time 2.5.2.4 Applied Pressure 2.5.2.5 Swelling 2.5.2.6 Ionic Strength 2.5.2.7 Mucus Gel Viscosity

15 16 16 16 17 18 19 19 20 21 22 23 23 29 30 30 31 32 33 33 34 35 35 36 36 36 36 37 37 37 38 38 38 38 39 39 39

Contents  vii 2.5.3 Physiological Factors 2.5.3.1 Mucin Turnover 2.5.3.2 Disease States 2.6 Bioadhesive Polymers for Drug Delivery Applications 2.6.1 Polymers 2.6.1.1 Natural Polymers 2.6.1.2 Synthetic Polymers 2.6.2 Bioadhesive/Mucoadhesive Polymers 2.6.3 Classification of Mucoadhesive Polymers 2.6.3.1 Classification Based on the Origin of the Polymer 2.6.3.2 Classification Based on Aqueous Solubility of the Polymer 2.6.3.3 Classification Based on the Type of Charge on the Polymer 2.6.4 Natural Polymers 2.6.4.1 Chitosan 2.6.4.2 Starch 2.6.4.3 Gelatin 2.6.4.4 Alginates 2.6.4.5 Hyaluronic Acid 2.6.5 Synthetic Polymers 2.6.5.1 Cellulose Derivatives 2.6.5.2 Polyacrylates 2.6.5.3 Poly (ethylene glycol) (PEG) 2.6.6 Classification Based on Aqueous Solubility of the Polymer 2.6.6.1 Water-Soluble Polymers 2.6.6.2 Water-Insoluble Polymers 2.6.7 Classification Based on the Type of Charge on the Polymer 2.6.7.1 Cationic Polymers 2.6.7.2 Anionic Polymers 2.6.7.3 Non-Ionic Polymers 2.7 Prospects of Bioadhesive/Mucoadhesive Polymers in Bioadhesive Drug Delivery 2.8 Summary Acknowledgements References

39 39 39 40 40 40 40 40 41 41 41 42 42 42 43 44 44 45 45 45 46 46 46 46 46 47 47 47 47 47 48 49 49

viii  Contents 3 In Vitro, Ex Vivo and In Vivo Methods for Characterization of Bioadhesiveness of Drug Delivery Systems Ljiljana Djekic and Martina Martinovic 3.1 Introduction 3.2 Mechanisms of Bioadhesion 3.3 Bioadhesive Drug Delivery Systems (BDDS) 3.3.1 BDDS for Cutaneous Application 3.3.2 BDDS for Buccal Application 3.3.3 BDDS for Peroral Application 3.3.4 BDDS for Vaginal Application 3.3.5 BDDS for Nasal Application 3.3.6 BDDS for Ocular Application 3.4 Methods for Testing Bioadhesive Property of BDDS 3.4.1 In Vitro/Ex Vivo Tests 3.4.1.1 Bioadhesion Strength Tests 3.4.1.2 In Vitro Methods for Characterization of Bioadhesion at the Molecular Level 3.4.2 In Vivo Methods 3.4.2.1 Radiolabelled BDDS Transit Studies 3.4.2.2 Gamma Scintigraphy 3.4.2.3 In Vivo Detachment Tests 3.5 Summary References

57 58 59 62 62 63 64 65 66 67 68 68 68 81 85 86 87 87 89 90

Part 2: Bioadhesive Formulations

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4 Bioadhesive Films for Drug Delivery Systems Kampanart Huanbutta and Tanikan Sangnim 4.1 Introduction 4.2 Theories of Bioadhesion 4.3 Bioadhesive Film-Forming Agents 4.4 Drug Delivery Applications of Bioadhesive Films 4.4.1 Topical and Transdermal Drug Delivery 4.4.1.1 Patches 4.4.1.2 Film-Forming Systems 4.4.2 Mucosal Drug Delivery 4.4.2.1 Buccal Drug Delivery 4.4.2.2 Vaginal Drug Delivery 4.4.2.3 Rectal Drug Delivery 4.4.2.4 Ocular Drug Delivery 4.4.2.5 Nasal Drug Delivery

101 101 102 103 105 105 105 106 106 106 107 107 108 109

Contents  ix 4.4.3 Oral Drug Delivery 4.4.3.1 Orodispersible Films (ODFs) 4.4.3.2 Sublingual Films 4.4.3.3 Oral Colon-Specific Drug Delivery 4.5 Current and Novel Bioadhesive Film Fabrication Techniques 4.5.1 Solvent Casting 4.5.2 Extrusion 4.5.3 Rolling 4.5.4 2D Printing 4.6 Evaluation of Bioadhesive Films 4.6.1 Bioadhesive Strength 4.6.2 Tensile Strength Measurement 4.6.3 Morphology and Thickness 4.6.4 Moisture Content 4.6.5 Permeation 4.6.6 Swelling 4.6.7 Irritation 4.6.8 Stability 4.6.9 Drug Loading and Drug Entrapment Efficiency 4.7 Summary 4.8 Acknowledgements References 5 Redox-Responsive Disulphide Bioadhesive Polymeric Nanoparticles for Colon-Targeted Drug Delivery Erazuliana Abd Kadir and Vuanghao Lim 5.1 Introduction 5.2 Mechanism of Disulphide Bond Formation 5.3 Disulphide Polymers for Colon Drug Delivery 5.4 Colon-Targeted Drug Delivery (CTDD) 5.4.1 Condition of the Colon for Drug Delivery 5.4.2 Approaches for Colon Drug Delivery 5.4.3 Limitations of CTDD 5.5 Nanoformulations of Disulphide Polymers 5.5.1 Thiolated Pectin Polymers 5.5.2 Thiolated Sodium Alginate (TSA) Polymers 5.5.3 Thiolated Chitosan (TCS) Polymers 5.5.4 Thiolated Hyaluronic Acid Polymers 5.5.5 Thiolated Dextran Polymers 5.5.6 Other Thiolated Polymers

109 109 110 110 111 111 111 111 112 113 113 114 114 114 115 116 116 116 117 117 118 118 123 123 124 125 126 127 128 129 130 130 131 134 136 137 138

x  Contents 5.6 Summary Acknowledgements References 6 Bioadhesive Hydrogels and Their Applications Hitesh Chopra, Sandeep Kumar and Inderbir Singh 6.1 Introduction 6.1.1 Bioadhesive Polymer 6.1.2 Hydrogels 6.1.3 Bioadhesive Hydrogels 6.2 Bioadhesive Hydrogel Films 6.3 Bioadhesive Hydrogels for Gastrointestinal Delivery 6.4 Bioadhesive Hydrogels Administered through Injection 6.5 Bioadhesive Hydrogels for Vaginal Delivery 6.6 Bioadhesive Hydrogels for Rectal Delivery 6.7 Mucoadhesive Hydrogels Based Nanoparticles 6.8 Patents and Future Perspectives 6.9 Summary References

Part 3: Drug Delivery Applications

140 140 140 147 147 148 150 155 155 156 156 159 160 161 161 164 164

171

7 Ocular Bioadhesive Drug Delivery Systems and Their Applications 173 Anju Sharma, Mukesh S. Patil, Pravin Pawar, A.A. Shirkhedkar and Inderbir Singh 7.1 Introduction 174 175 7.2 Anatomy and Physiology of the Eye 7.2.1 Anatomy and Function of the Eye 175 7.2.2 Structure of Cornea 176 7.3 Various Bioadhesive/Mucoadhesive Polymers 176 for Ocular Delivery 7.3.1 Chitosan as Ocular Bioadhesive 177 7.3.2 Starch (Drum-Dried Waxy Maize Starch, Pregelatinized Starch) 180 7.3.3 Sodium Hyaluronate (SH) as Ocular Bioadhesive 181 7.3.3.1 Functions of Sodium Hyaluronate  181 7.3.3.2 Viscoelasticity 182 7.3.3.3 Contact Angle 182 7.3.3.4 Adherence to the Mucin Layer (Mucoadhesivity) 183

Contents  xi 7.3.3.5 Water Retention 7.3.3.6 Healing of Superficial Keratitis 7.3.3.7 Free Radical Scavenging 7.3.4 Alginate Based Ocular Bioadhesive 7.3.4.1 General Properties of ALGs 7.3.5 Gellan Gum as Ocular Bioadhesive 7.3.6 Albumin 7.3.7 Collagen Based Ocular Bioadhesive 7.3.8 Xanthan Gum 7.3.9 Guar Gum 7.3.10 Gelatin 7.3.11 Tamarind Seed Polysaccharide (Xyloglucan) 7.3.12 Arabinogalactan 7.3.13 Gum Cordia 7.3.14 Bletilla Striata Polysaccharide (BSP) 7.3.15 Locust Bean Gum (Carob Bean Gum) 7.3.16 Carrageenan 7.4 Summary References 8 Buccal Bioadhesive Drug Delivery Systems and Their Applications Veera Garg and Shammy Jindal 8.1 Introduction 8.1.1 Advantages of a Buccal Bioadhesive System 8.1.2 Disadvantages of a Buccal Bioadhesive System 8.1.3 Ideal Characteristics of a Bioadhesive Dosage Form 8.1.4 Structure of Buccal Mucosa 8.2 Theories of Bioadhesion 8.2.1 Diffusion Theory 8.2.2 Adsorption Theory 8.2.3 Wetting Theory 8.2.4 Electronic Theory 8.2.5 Fracture Theory 8.3 Factors Affecting Bioadhesion 8.3.1 Bioadhesive Polymer Related Factors 8.3.1.1 Molecular Weight of Mucoadhesive Polymer 8.3.1.2 Cross-Linking of Mucoadhesive Polymer 8.3.1.3 Concentration of Mucoadhesive Polymer 8.3.1.4 Mucoadhesive Polymer Chain Length

184 184 184 184 185 188 189 190 192 193 193 195 196 197 197 198 198 199 200 213 213 218 218 219 219 220 221 222 222 222 223 223 224 224 224 224 224

xii  Contents Flexibility of Mucoadhesive Polymer Chain 225 Charge on Mucoadhesive Polymer 225 H-Bonding of Mucoadhesive Polymer 225 Spatial Configuration of Mucoadhesive Polymer 225 8.3.1.9 Swelling of Mucoadhesive Polymer 225 8.3.2 Environment Related Factors 226 226 8.3.2.1 pH 8.3.2.2 Saliva 226 8.3.2.3 Salivary Gland 226 8.3.2.4 Hydration 226 8.3.2.5 Mucin Turnover 227 227 8.3.2.6 Rate of Renewal of Mucoadhesive Cells 8.3.2.7 Disease State 227 8.3.2.8 Buccal Membrane Properties 227 8.4 Mechanism of Buccal Absorption 227 8.5 Buccal Bioadhesive Drug Delivery Systems 229 229 8.5.1 Solid Buccal Bioadhesive Dosage Forms 8.5.1.1 Buccal Tablets 229 8.5.1.2 Microspheres 230 8.5.1.3 Lozenges 230 8.5.1.4 Wafers 230 230 8.5.1.5 Gels 8.5.1.6 Patches 230 8.5.2 Liquid Dosage Forms 231 8.6 Quality Control Tests of Buccal Bioadhesive Dosage Forms 231 231 8.6.1 Moisture Absorption Test 8.6.2 Swelling and Erosion Tests 232 232 8.6.3 Tensile Strength and Elongation at Break 8.6.4 Surface pH 233 8.6.5 In-Vitro Bioadhesive Strength Measurement Test 233 8.6.6 Residence Time 234 8.6.6.1 Ex-Vivo Residence Time 234 8.6.6.2 In-Vivo Residence Time 234 8.6.6.3 Permeation Test 234 236 8.6.6.4 Absorption Test 8.7 Marketed Formulations 236 236 8.8 Summary References 237 8.3.1.5 8.3.1.6 8.3.1.7 8.3.1.8

Contents  xiii 9 Gastrointestinal Bioadhesive Drug Delivery Systems and Their Applications 245 Olufunke D. Akin-Ajani and Oluwatoyin A. Odeku Abbreviations 245 9.1 Introduction 246 9.2 The Mucus Layer 247 9.3 Gastrointestinal Bioadhesive Drug Delivery Systems 247 9.3.1 Solid Bioadhesive Formulations 248 248 9.3.1.1 Tablets 9.3.1.2 Bioadhesive Microparticles/Nanoparticles 249 9.3.1.3 Bioadhesive Patches 251 9.3.2 Semisolid Bioadhesive Formulations 254 9.3.3 Liquid Bioadhesive Formulations 254 254 9.3.3.1 Suspensions 9.3.3.2 Bioadhesive Liquids 255 9.4 Summary 255 References 255 10 Nasal Bioadhesive Drug Delivery Systems and Their Applications 259 Ravindra V. Badhe and Sonali S. Nipate 10.1 Introduction 260 10.1.1 Nasal Route of Administration 260 261 10.1.2 Nasal Cavity 10.1.3 Nasal Route for Brain Drug Delivery 263 10.1.4 Nasal Route for Local and Systemic Drug Delivery 263 267 10.2 Challenges in Nasal Drug Delivery Formulations 10.2.1 Ideal Properties of a Nasal Drug Delivery Formulation 267 10.2.2 Strategies Developed for Improving Nasal Drug Delivery 268 10.3 Mucoadhesion 270 10.3.1 Physiology of Nasal Mucus Layer and Barriers Posed by It 270 271 10.3.2 Factors Affecting Mucoadhesion 10.3.3 Mucoadhesive Polymers Used in Nasal Delivery Formulations 275 10.3.3.1 Chitosan and Its Composites 275 10.3.3.2 Cellulose Derivatives 277 10.3.3.3 Poloxamer or Pluronic 284

xiv  Contents 10.3.3.4 Polyacrylates 10.3.3.5 Lectin - Poly(ethylene glycol)(PEG) Poly(lactic acid)(PLA)/Poly(lacticco-glycolic acid)(PLGA) 10.3.3.6 Miscellaneous Mucoadhesive Agents 10.4 Summary References

285 286 287 289 290

11 Vaginal Bioadhesive Drug Delivery Systems and Their Applications 307 Sanjeevani S. Deshkar, Satish V. Shirolkar and Arun T. Patil 11.1 Introduction 308 308 11.1.1 Advantages of Vaginal Drug Delivery 11.1.2 Limitations 309 11.2 Vaginal Anatomy and Physiology 309 11.2.1 Vaginal Anatomy 309 11.2.2 Physiology of Vagina 310 310 11.2.2.1 Epithelium 11.2.2.2 Vaginal Fluid 311 11.2.2.3 pH 311 11.2.2.4 Microflora 312 11.2.2.5 Cyclic Changes 312 312 11.2.2.6 Enzymes 11.3 Vaginal Absorption of Drug 313 11.3.1 Drugs Administered by Vaginal Route 313 11.4 Conventional Drug Delivery Systems for Vaginal  Application 314 314 11.4.1 Vaginal Rings 315 11.4.2 Vaginal Tablets 11.4.3 Suppositories and Pessaries 315 11.4.4 Semisolid Formulations 316 11.4.5 Limitations of Conventional Vaginal Formulations 316 11.5 Mucoadhesive Drug Delivery Systems 317 11.5.1 Mucoadhesive Polymeric Platforms for Vaginal Drug Delivery 318 11.5.1.1 Poly (acrylic acid) (PAA) Derivatives 318 11.5.1.2 Cellulose Derivatives 319 321 11.5.1.3 Natural Polymers 11.5.1.4 New Generation Mucoadhesive Polymers 324

Contents  xv 11.5.2 Mucaodhesive Polymers as Enzyme Inhibitors and Permeation Enhancers 325 11.5.3 Novel Mucoadhesive Formulations for Drug Delivery to Vagina 326 11.5.3.1 Mucoadhesive Gels 326 11.5.3.2 In Situ Gelling Systems 327 11.5.3.3 Emulgels 337 337 11.5.3.4 Vaginal Films 11.5.3.5 Microparticulate Drug Delivery 338 Systems 11.5.3.6 Nanoparticle Based Drug Delivery Systems 338 11.6 Recent Advancements in Vaginal Drug Delivery  Applications 350 11.6.1 Vaginal Immunization 350 11.6.2 Gene Therapy 350 11.6.3 Mucus Penetrating Nanoparticles 351 11.6.4 Personalized Medicine Using Additive Manufacturing Technology 351 11.7 Summary 352 References 352 12 Pulmonary Bioadhesive Drug Delivery Systems and Their Applications 371 Ridhima Wadhwa, Subhashini Bharathala, Taru Aggarwal, Nikita Sehgal, Nitesh Kumar, Gaurav Gupta, Dinesh Kumar Chellappan, Pawan  Kumar Maurya, Terezinha De Jesus Andreoli Pinto, Trudi Collet, Harish Dureja, Philip M. Hansbro and Kamal Dua 372 12.1 Introduction to Pulmonary Drug Delivery Systems 12.1.1 Deposition of Inhaled Particles 373 12.1.2 Absorption of Inhaled Particles 374 12.1.3 Challenges of Pulmonary Drug Delivery 375 12.2 Bioadhesives in Pulmonary Drug Delivery Systems 376 12.3 Development of Pulmonary Bioadhesive Drug Delivery Systems 378 12.3.1 Nanoparticles 378 381 12.3.2 Microparticles 12.3.3 Liposomes 383

xvi  Contents 12.4 Progress and Clinical Challenges for Bioadhesive Drug Delivery with Future Prospects 12.4.1 Technological Advancements 12.5 Future Prospects and Summary References

384 384 385 386

Index 391

Preface Even a cursory look at the literature will evince that currently there is much interest and brisk research activity in developing efficient target drug delivery routes and systems, and bioadhesives offer many virtues and advantages in this endeavor. Naturally, understanding the phenomenon of bioadhesion, i.e., its theories or mechanism (s) is of critical importance and pertinence in developing optimum boadhesive polymers (used in bioadhesives). Such bioadhesive polymers are the key for exhibiting the process of bioadhesion, controlled/sustained release of drug, and drug targeting. Use of bioadhesives restricts the delivery system to the site of interest and thus offers a useful and efficient technique for targeting a drug to the desired location for a prolonged duration. The information on this important topic of bioadhesives in drug delivery is scattered in many diverse publication media. This book addresses the various relevant aspects of bioadhesives in drug delivery in an easily accessible and unified manner, and thus fills the lacuna in the literature. The book, containing 12 chapters by eminent researchers from many parts of the globe, is divided into three parts: Part 1: Fundamental Aspects; Part 2: Bioadhesive Formulations; and Part 3: Drug Delivery Applications. The topics covered include: Theories and mechanisms of bioadhesion; bioadhesive polymers for drug delivery applications; methods for characterization of bioadhesiveness of drug delivery systems; bioadhesive films and drug delivery applications ; bioadhesive nanoparticles; bioadhesive hydrogels and applications; ocular bioadhesive drug delivery systems; buccal bioadhesive drug delivery systems; gastrointestinal bioadhesive drug delivery systems; nasal bioadhesive drug delivery systems; vaginal drug delivery systems; and pulmonary bioadhesive drug delivery systems. It should be recorded here that all chapters were rigorously reviewed and all were suitably revised (some twice or thrice). So the material presented in this book is of archival value and meets the highest standard of publication. The book is profusely referenced and copiously illustrated. This book should be of immense interest and usefulness to biologists, pharmaceutical xvii

xviii  Preface scientists, polymer chemists; adhesive technologists, materials scientists and those interested in biomaterials, target drug delivery, and controlled drug delivery. Also advanced research students should find this book as a Baedeker to this immensely important area of bioadhesives in drug delivery. As more advanced and more efficient bioadhesives are developed, new application vistas will emerge. Now comes the pleasant task of thanking all those who made this book possible. First and foremost, our sincere and heart-felt thanks go to the authors for their interest, enthusiasm, cooperation and sharing their valuable research experience in the form of written accounts, without which this book would not have seen the light of day. We will be remiss if we fail to extend our thanks to Martin Scrivener (publisher) for his whole-hearted interest in and unwavering support for this book project. Kash Mittal P.O. Box 1280 Hopewell Jct., NY, USA [email protected] Inderbir Singh Bakshi Chitkara College of Pharmacy Chitkara University Rajpura, Patiala, Punjab, India Jasjeet Kaur Narang Khalsa College of Pharmacy Amritsar, Punjab, India April 2020

Part 1 FUNDAMENTAL ASPECTS

1 Introduction, Theories and Mechanisms of Bioadhesion Kamla Pathak1* and Rishabha Malviya2 Pharmacy College Saifai, Uttar Pradesh University of Medical Sciences,  Etawah, Uttar Pradesh, India 2 Department of Pharmacy, School of Medical and Allied Sciences, Galgotias University, Greater Noida, Uttar Pradesh, India

1

Abstract

Bioadhesion refers to adherence of macromolecules (synthetic and natural) to the mucosal layer of the body. The second generation bioadhesives, the biological mucoadhesives, have depicted specific interaction with biological cell surface as well as with the mucin. In this chapter, theories of bioadhesion which include wetting, diffusion, electronic, adsorption, and fracture theories have been described which are important for assessment of bioadhesion. These theories clearly explain the fundamental mechanisms of attachment and elaborate on the formation of bioadhesive bonds. This compilation also summarizes the mechanistic approach to bioadhesion which is a three-step phenomenon, namely: wetting and swelling of mucoadhesive polymer, interpenetration of polymer chains, and finally the formation of weak chemical bonds. Furthermore, various properties of muco­ adhesive polymers, the mechanism(s) controlling bioadhesion, the factors affecting mucoadhesion, mucosal interaction, and biological mucoadhesives have also been elaborated. Keywords:  Bioadhesion, theories, mechanisms, factors affecting mucoadhesion, mucosal interaction, biological mucoadhesives

*Corresponding author: [email protected] K.L. Mittal, I. S. Bakshi and J. K. Narang (eds.) Bioadhesives in Drug Delivery, (3–28) © 2020 Scrivener Publishing LLC

3

4  Bioadhesives in Drug Delivery

1.1 Introduction Bioadhesion can be explained as the attachment of synthetic or natural macromolecules to the mucus and/or epithelial surface for extended period of time. The bond between two materials is governed by interfacial forces. Bioadhesion is quite similar to the conventional adhesion process [1]. The only difference is that bioadhesion involves special characteristics of biological organisms and surfaces. The phenomenon of bioadhesion can be classified into specific and non-specific bioadhesion [2]. The specific bioadhesion invloves mostly polymers or some biological molecules that allow the bioadhesion at cell surface or mucus. For example, lectins, the carbohydrate binding proteins derived from plant sources, have the ability to recognize a particular type of sugar molecule(s) and adhere to it. The adhesion of bacteria to the human gut may be attributed to the interaction of a lectin-like structure (present on the bacterial cell surface) and mucin. The adhering property of the lectins to the cell surface is remarkable and thus these are known to be bioadhesive. Tomato lectin is a good example of a specific bioadhesive. Tomato lectin is a complex glycoprotein that can specifically adhere to the short arrays of N-acetylglucosamine [3]. Bacterial adhesins, fimbrin, wheat germ (Phaseolus vulgaris) agglutinin, etc. are some other specific bioadhesive lectins. The non-specific bio­ adhesive molecules (polycarbophil, chitosan, carbopol, and carbomers) have the ability to bind with both the cell surface and the mucosal layer [4]. The property of bioadhesion has also been observed in the group of marine animals known as ascidians. The development of bioadhesives inspired from marine animals is a promising approach to  generate new tissuecompatible medical components like non-fouling surfaces.

1.1.1 Historical Perspective The use of mucoadhesive polymers for the development of pharmaceutical formulations was reported back in 1947, when attempts were made to develop a mucosal drug delivery of penicillin using gum tragacanth and dental adhesive powders [5, 6]. Improved results were reported when carboxymethyl cellulose and petrolatum were used for the development of the formulation. This research led to the development of a mucoadhesive delivery system comprising of finely ground sodium carboxymethyl cellulose, pectin and gelatin. The formulation was marketed as Orahesive® (Fagron Inc., St. Paul, MN, USA) followed by Orabase® (Colgate Oral Pharmaceuticals, Inc., USA), which is a blend of polymethylene/ mineral oil base. This was followed by the development of a system where a

Mechanisms of Bioadhesion  5 polyethylene sheet was laminated to a blend of sodium carboxymethyl cellulose and polyisobutylene which provided an added advantage of protecting the mucoadhesive layer by the polyethylene backing from the physical interference of the external environment [6-8]. Over the years, various other polymers, e.g., sodium alginate, guar gum, sodium carboxymethyl cellulose, poly(ethylene glycol)s, karaya gum, hydroxyethyl cellulose, methyl cellulose and retene were found to exhibit mucoadhesive property. During the 1980s poly (acrylic acid), hydroxypropyl cellulose and sodium carboxymethyl cellulose were widely explored for the development of mucoadhesive formulations. Since then the use of acrylate polymers for the development of mucoadhesive formulations has increased manifold. Various researchers have investigated the mucoadhesive property of different polymers with varying molecular architecture [9-11]. The voluminous research has concluded that a polymer will exhibit sufficient bioadhesive property if it can form strong intermolecular hydrogen bonds with the mucosal layer, can penetrate into the mucus network or tissue crevices, can readily wet the mucosal layer and has sufficiently long chain. When designed as a matrix (base) the mucoadhesive polymeric matrix should rapidly adhere to the mucosal layer without any change in the physical property of the delivery matrix, offer minimal interference from the release of the active agent, be biodegradable without producing any toxic by-products, inhibit the enzymes present at the delivery site, and enhance the penetration of the active agent if the active agent is meant to be absorbed from the delivery site [12].

1.1.2 Bioadhesion in Biological Systems In biological systems, bioadhesion can be classified into three types: Type 1: Adhesion between two biological components, for example in platelet aggregation and wound healing; Type 2: Adhesion of a biological component to an artificial substrate, for example, cell adhesion to culture dishes and biofilm formation on prosthetic devices and inserts; and Type 3: Adhesion of an artificial material to a biological substrate, for example, adhesion of synthetic hydrogels to soft tissues [13]. Bioadhesion also refers to the utilization of bioadhesive materials to connect two surfaces together which can be beneficial in the surgical and dental applications. Hence, the interest in the bioadhesion research has resulted in the development of new therapies, biomaterials, and other technological products such as biosensors. The literature documents the use of bioadhesive polymeric systems for the development of products for various biomedical applications which include denture adhesives and surgical glue. However, bioadhesion

6  Bioadhesives in Drug Delivery between the materials can be deleterious as well, which is referred to as biofouling [14]. Bioadhesives can also be obtained from living organisms. Example of naturally occurring bioadhesive is mussel adhesive protein (MAP) secreted by mussel, which is comprised of multiple threads that get attached to the glass surface [15].

1.1.3 Bioadhesive/Mucoadhesive The primary goal of development of bioadhesives is to duplicate, mimic, or improve biological adhesives. A bioadhesive material should exhibit durability of adhesion comparable to the natural bioadhesion where required, biodegradability, and non-toxicity. In pharmaceutical sciences, when the adhesive attachment is to mucus or a mucous membrane, the phenomenon is referred to as mucoadhesion. It is the adhesion of polymeric material to a mucosal membrane and is an attractive interaction between mucosal membrane and pharmaceutical dosage form [16]. The polymeric material containing an active pharmaceutical ingredient gets attached to a specifically targeted mucosa for an enhanced period of time as compared to the active pharmaceutical ingredient (API) alone. The extended API residence time on the mucosal surface increases the permeation and also the bioavailability for some APIs. It is very difficult to differentiate between adhesion of molecules to the mucus layer and adhesion of molecules to cell surface, and some bioadhesive molecules bind to both cell surface as well as to mucus layer [17]. Medical researchers deal with the pervasive aspect of the formation of biofilm and bioadhesion on solid surfaces. This phenomenon is equally applicable to catheters and contact lenses. The solid-liquid interface is of great importance because of its involvement in several biological processes. In dental applications, adhesive materials are used to treat tooth damage initiated due to caries and also to secure brackets within the teeth during the orthodontic treatment. In ophthalmology, an anti-adhesive is used in intracorneal implants and intraocular lenses. For the formulation of longer duration wear contact lenses, materials that will bind selectively to specific proteins are used for minimizing the bacterial adhesion [18].

1.1.4 Factors Affecting Mucoadhesion 1.1.4.1 Molecular Weight of Polymer For successful adhesion, a minimum molecular weight of 100,000 daltons is required [13]. In case of a linear polymer, the adhesive property is

Mechanisms of Bioadhesion  7 directly dependent on the molecular weight, but in a non-linear polymer, the adhesive property may or may not depend on the molecular weight.

1.1.4.2 Concentration of Polymer Used The concentration of polymer must be optimum. After an optimum level, the adhesive strength decreases substantially due to the separation of coiled polymeric portion so that the length of the polymer chain available for permeation becomes limited.

1.1.4.3 Flexibility of Polymer Chains Flexibility of the polymer chains basically dictates the diffusion coefficient and viscosity. Therefore, the higher the polymer flexibility, the greater will be the diffusion in the mucus network.

1.1.4.4 Swelling The swelling property depends on the concentration of polymer, presence of water, and the ionic strength. The polymers used in mucoadhesion need hydration to develop and form a macromolecular mesh of required size.

1.1.4.5 pH at Polymer-Mucus Interface The pH influences the charge on the mucosal surface. The surface charges of both mucus and polymers are affected by the pH. The charge density of the mucus membrane would be different depending on the pH, because of difference in the dissociation of functional group on carbohydrate and amino acids of the polypeptide.

1.1.4.6 Mucin Turnover Rate High turnover of mucin occurs most of the time but it is not beneficial because it limits the residence time of the adhesive polymer. The polymer tends to detach from the mucus layer even though the polymer bears good bioadhesive property.

1.1.4.7 Stereochemistry The electron-rich groups such as -OH, -COOH etc. are present in many bioadhesive polymers. These groups cause electronic cloud over functional

8  Bioadhesives in Drug Delivery Molecular weight of polymer Swelling factor, Stereochemistry of polymer

Mucin turnover rate

FACTORS AFFECTING MUCOADHESION pH at polymersurface interface

Flexibility of polymer chains Concentration of polymer used

Figure 1.1  Various factors affecting mucoadhesion.

groups that may be active and responsible for adhesion. The molecular configuration dictates the extent of interaction between the substrate and polymer. Additionally, the orientation of molecules is also responsible for the overall lowering of free energy after binding. Figure 1.1 presents a schematic diagram depicting the factors affecting mucoadhesion.

1.2 Bioadhesive Interactions The bonding and attachment between adhesive polymers and biological surfaces generally occurs via interpenetration followed by secondary non-covalent bonding between them. It has been found that the secondary bonding mainly occurs via the formation of hydrogen bonds [19]. The bioadhesive polymers possess hydrophilic functional groups such as hydroxyl (-OH), sulfate (-SO4H), carboxyl (-COOH), and amino groups (-NH2) that can be considered favorable for target delivery. Therefore, mainly the hydrogen bonds contribute to the formation of a strong network. So the polymers that possess a high amount of hydrogen bonding groups can interact more strongly with the glycoproteins. Polymers that can be used as bioadhesives and can easily get adhered to the mucin epithelial surface should possess the following characteristics. Firstly, the polymer must be adhesive in nature so that it adheres when placed in water. Secondly, it should attach non-specifically, and non-covalently, and thus noncovalent interactions must occur for adhesion. Lastly, the polymer should

Mechanisms of Bioadhesion  9 bind specifically to the receptor sites on the cells or mucosal surfaces. The desirable key attributes of bioadhesive polymers include high molecular weight, appropriate surface tension to spread on mucus layer, and anionic surface charge [20].

1.3 The Mechanistic Approach to Bioadhesion The mechanistic approach to bioadhesion can be related to the polymer based on its physico-chemical properties [21]. The three fundamental steps during the mucoadhesive process are as follows: firstly the wetting and swelling of the polymer should allow an intimate contact with the tissue; secondly, the interpenetration of the polymer chains and entanglement between the polymer and the mucin chains should be attained; and finally, the formation of weak chemical bonds should be possible [22]. The polymeric hydrogels also exhibit mucoadhesive property. Hydrogels possess enhanced mucoadhesive property and based on this property, the characteristics that define hydrogel as a promising system include: the presence of a high amount of hydrogen bonding chemical groups, such as hydroxyls and carboxyls, anionic surface charge, high polymer molecular weight, and high polymer chain flexibility. These characteristics induce spreading of hydrogels onto the mucus layer. There are three main types of interactions between a polymer and the mucous layer. They are physical or mechanical bonds, secondary chemical bonds, and covalent chemical bonds. Physical bonds imply the entanglement of mucin glycoproteins with the polymer chains and the interpenetration of the mucin chains in the polymer matrix. This interpenetration of macromolecules will depend on their respective chain flexibilities and diffusion coefficients. Secondary chemical interactions include, van der Waals interactions and hydrogen bonding. Hydrogen bonding is probably the most important secondary chemical interaction in mucoadhesion because it forms link between the functional groups and the mucoadhesive polymer. Some of the functional groups such as hydroxyls, carboxyls, sulfate and amino groups involved in hydrogen bond formation will establish good mucoadhesive property. The polymers such as poly (vinyl alcohol), poly(acrylic acid), poly(hydroxyalkyl methacrylate) have shown good mucoadhesive property in the past. Formulations possessing mucoadhesive property can increase the retention time of dosage forms in the GI tract [23]. Even these types of forces are weak and only numerous interaction sites lead to strong mucoadhesion. Additionally, covalent bonds may form by chemical reaction between the mucoadhesive polymer and the

10  Bioadhesives in Drug Delivery mucus substrate. Though a stable bond is permanent, the mucus turnover and the epithelial desquamation would result in the detachment and loosening of the polymer from the tissue [24, 25].

1.4 Factors Controlling Bioadhesion The mechanism of biological adhesion is controlled by various key factors that can be classified into four broad categories. These include chemical interactions, surface morphology, physiological factors, and physical or mechanical bonds.

1.4.1 Chemical Interactions Chemical interactions between the protein and the surface are influenced by the charges on both protein and surface. The interaction between chemically active surfaces promotes the adhesion process. If the two chemically active surfaces are able to form bonds like covalent or ionic, then strong adhesion can occur. Similarly, if the mating surfaces form weak bonds such as dipole-dipole, hydrogen bonding or induced dipoles, then weaker adhesion will occur [26-28]. Interestingly, studies on the interaction between protein and solid surfaces have led to the assumption of uniform surfaces and existence of only a single type of interaction between the surface and protein. The strength of protein adsorption relies on polarity and the net charge on the protein. The protein and substrate can be positively charged, negatively charged, hydrophobic, or neutral hydrophilic. The bonding of a neutral hydrophilic portion of protein with the biological surface which possesses almost the same polarity is the basic reason behind weak adsorption mechanism [29]. Moderate adsorption occurs when there is ionic interaction between proteins and substrate surfaces. However, strong adsorption occurs only when the interactions occur between hydrophobic protein moieties and the substrate. Specific examples to illustrate chemical interactions are detailed below.

1.4.1.1 Mussel Adhesion Mussel adhesion is one of the examples that show how the chemical composition of the surface changes the adhesion mechanism. The interaction between mussel and the underwater surfaces that involves the removal of weak layers (mostly water) occurs in two ways. In the first case, the

Mechanisms of Bioadhesion  11 Mussel Weak Dispersion Force

Mussel Adhesive Protein

Water Molecules

Figure 1.2  Interaction between water molecules and mussel adhesive protein.

underwater surface is non-polar. So, the water boundary layers interact via weak dispersion forces. Because the mussel adhesive protein is larger than the water molecule, the protein experiences larger dispersion interaction with a non-polar surface that leads to the removal of boundary water layer and adhesion of the protein (Figure 1.2). In the second case where the underwater surface is polar, the boundary layer of water cannot be easily displaced. So in this case the mussel adhesive protein utilizes its hydrophilic amino acid side chains, which consist of groups like aminoalkyl, hydroxyalkyl, phenolic to form strong hydrogen bonds. Therefore, the mussel adhesive protein is capable of displacing water and gets adhered to polar underwater surfaces as well [30, 31].

1.4.1.2 Cell Adhesion to Biomaterials Adhesion of cells to biomaterial surfaces is a complex phenomenon. It has been found that the microscale surface topography has a direct impact on cell proliferation and adhesion. The composition of chemical present on biomaterial surfaces plays an important role in the cell proliferation process and cell adhesion. Cell adhesion is affected by the integral group of cell surface receptors and thus it depends on the structural conformation of the extracellular matrix protein which seems to be sensitive to the surface [32].

1.4.2 Surface Morphology Effects The adhesion of cells to the surfaces of synthetic biomaterials is significant in the designing and performance of body implants. When a biomaterial is placed into a living host, cells are not directly connected to the biomaterial surface. Instead, the biomaterial is enclosed into a protein layer. Various models have been utilized to study surface morphology effects on cell adhesion and on protein conformation [33]. Micro/nanoscale topography is important to characterize the adhesion of cells to biomaterials which

12  Bioadhesives in Drug Delivery is attained by different approaches. The first approach is the top-down or lithographic method or also known as a dry etching method. The second approach is the bottom-up or self-assembly process [34].

1.4.3 Physiological Factors Physiological factors also play a significant role in bioadhesion and are important in surgical procedures, e.g. when fibrin tissue adhesive is used in surgical applications. This adhesive is applied below the dermis for skin grafts, flaps and also for laparoscopic surgeries. These adhesives are generally packed in two different packages, which are mixed during the surgery. One package contains fibrinogen, plasma glutaminase and calcium chloride. The second package contains thrombin and anti-fibrinolytic agent. This bioadhesive works on the basis of the physiology of the blood coagulation process. Thrombin divides the protein fibrinogen into smaller subunits of fibrin during clotting. The smallest unit is fibrin which causes end-to-end polymerization. The cross-linking of subunits of fibrin results into clot formation in the presence of calcium [35, 36].

1.4.4 Physical and Mechanical Factors The physical and mechanical factors in bioadhesion are generally affected by the interaction between the polymer chain. Wetting and interpenetration are the two factors that affect physical and mechanical interactions.

1.4.4.1 Wetting Phenomenon For successful bioadhesion, interfacial free energy is an important determinant. In systems where bioadhesion takes place, the liquid environment plays an important role. The liquid environment influences the spreading of one material over another; for example, adherence of the adhesive polymer to the mucous layer of a biological membrane which is immersed in a liquid medium. At the time of adhesion, an interface between mucus, liquid and the polymer is formed and the liquid layer disappears thus forming a bond between polymer and mucus layer [37].

1.4.4.2 Interpenetration In the initial stages of bioadhesion, interfacial contact and chemical bonding phenomena are the important considerations to maintain bioadhesion. On the other hand, the adhesive bond will be maintained by interpenetration

Mechanisms of Bioadhesion  13 of molecules between the two contacting surfaces. In the two contacting surfaces, if one is considered as a polymer which is involved in the process of adhesion then the inter-diffusion process involves the property of the single chains and their involvement in the opposing membrane. Similarly, in the case of the swelling polymers the inter-diffusion process is governed by the ratio of wet and dry weights [38].

1.5 Theories of Bioadhesion The important parameters for assessing mucoadhesion are: (i) mucus substrate characteristics, (ii) composition of mucoadhesive material, (iii) functional characteristics of the substrate, and (iv) associated applied force between the mucoadhesive and the mucosal surface or the biological surface [39]. Bonding involved in the process occurs chiefly through both physical and weak chemical bonds. Physical or mechanical bonds result from entanglement of the adhesive material and the extended mucus chains. In this regard, mutual diffusion of the mucoadhesive polymer and mucin chains will result in the maximum attachment. Chemical bonding may be classified as a primary or secondary type. Primary bonds are due to covalent bonding while secondary bonds may be due to electrostatic, hydrophobic, or hydrogen bonds. Electrostatic interactions and hydrogen bonding appear to be important as a result of a large number of charged species e.g., hydroxyl (-OH), carboxyl (-COOH) and amino (-NH2) groups present on the mucosal surface. Hydrophobic bonding occurs when non-polar groups associate with each other in an aqueous solution due to the tendency of water molecules to exclude non-polar molecules. The van der Waals attraction between hydrophobic groups has binding energies between 1-10 kcal/mol, whereas hydrogen bonds between hydrophilic groups have energy of about 6 kcal/mol. Hydrophobic bonding is generally considered to be a key factor in bioadhesion. There are various putative theories in general to explain the fundamental mechanism(s) of attachment [40, 41]. In a particular system, one or more theories (wetting, diffusion, electronic, adsorption and fracture) can equally explain or contribute to the formation of bioadhesive bonds [42].

1.5.1 Wetting Theory It is one of the oldest and well established theories of adhesion. This theory best describes the adhesion of liquids or low viscosity bioadhesives to a biological surface. The adhesion can be expressed in terms of surface and

14  Bioadhesives in Drug Delivery interfacial tensions [41]. When an interface is formed, there is a release of energy per cm2 that can be defined as the work of adhesion. The wetting theory deals with the contact angle and the thermodynamic work of adhesion [43]. The work of adhesion is given by Dupre’s equation (1.1) [13]:

Wa = γa + γb − γab

(1.1)

Where Wa is the specific thermodynamic work of adhesion and γa, γb, and γab represent, the surface tension of the bioadhesive polymer, surface tension of the biological substrate, and the interfacial tension between the polymer and substrate, respectively. The work of cohesion is represented by equation (1.2):

Wc = 2γa or 2γb

(1.2)

When a bioadhesive material (a) spreads on a biological substrate (b), the spreading coefficient can be calculated by the following equation (1.3),

Sa/b = γb – (γab + γa)

(1.3)

The value of Sa/b should be positive for the bioadhesive material to spread on the biological substrate. When a bioadhesive liquid a, spreads on the biological substrate b, the contact angle is given by equation (1.4).

 γ  γ  cosθ =  b– ab   γa 



(1.4)

Figure 1.3 shows the key components involved in spreading of bioadhesive liquid over a soft tissue surface [44]. γa Air

Liquid γb

θ

γab

Tissue

Figure 1.3  Spreading of bioadhesive liquid over a typical soft tissue surface.

Mechanisms of Bioadhesion  15

1.5.2 Diffusion Theory According to this theory, the polymer chains bind to the mucus and comingle to a sufficient depth to create a semipermanent adhesive bond [45]. There is a close interaction of contact between the bioadhesive material and glycoprotein (present in the mucus membrane). The polymer chains penetrate the mucus; the exact depth to which these penetrate to achieve sufficient bioadhesion depends on the diffusion coefficient, time of contact, and other experimental variables. The diffusion coefficient depends on molecular weight and decreases rapidly as cross-link density increases [46]. This suggests that the flexibility and chain segment mobility of the bioadhesive polymer and mucus glycoprotein molecules are important parameters to control inter-diffusion. During chain interpenetration, a concentration gradient is established. The bioadhesive polymer chain penetration depends on the diffusion coefficient of the macromolecule and the chemical potential gradient. In the case of cross-linked polymers, the interpenetration of large chains occurs with great difficulty [47]. The exact penetration depth needed for good bioadhesive bonds is not clearly established, but it is estimated to be in the range of 0.2–0.5 μm. The mean diffusional depth (s) of the bio­ adhesive polymer segments is calculated by equation (1.5),

s = 2tD



(1.5)

Where D is the diffusion coefficient and t is the contact time. Figure 1.4 is a schematic diagram for interdiffusion of the polymer chain and mucin chain. (a) (b) (c)

Mucus

Polymer chain Mucin chain

Mucus membrane

Figure 1.4  Interactions resulting from inter-diffusion of polymer chains of bioadhesive system and mucus membrane. (a) Polymer chains before diffusion, (b) contact between polymer chains and mucin chains and (c) inter-diffusion of mucin chains and polymer chains.

16  Bioadhesives in Drug Delivery

1.5.3 Electronic Theory According to this theory, transfer of electrons takes place when an adhesive polymer comes in contact with a mucus glycoprotein network because of differences in their electronic structures. This leads to the formation of an electrical double layer at the interface. Such a system behaves analogously to a capacitor, which is charged when two surfaces come in contact, and discharged when they are separated [19]. This theory is also applicable since both the biological substrate and the mucoadhesive material possess some electrical charges that are opposite to each other. Therefore, when these two materials come in contact, they transfer electrons, which form the electrical double layer at their interface. The attractive force present within this newly formed electrical double layer determines the muco­ adhesive strength [1].

1.5.4 Adsorption Theory According to this theory, after the initial contact of the two surfaces, the materials will adhere because of the surface forces acting between the atoms in the two surfaces. According to this theory, after the initial contact of the two surfaces, the mucoadhesive material will adsorb on the biological surface due to forces acting between them. In adsorption, the weak forces like van der Waals interaction play an important role at the interface [39]. The chemical bonds include primary and secondary bonds. Primary chemical bonds (covalent in nature) are undesirable in bioadhesion because of their high strength that causes the formation of permanent bonds. Secondary chemical bonds involve forces of attraction, including electrostatic forces, van der Waals forces and hydrogen and hydrophobic bonds [19].

1.5.5 Fracture Theory The fracture theory of adhesion is related to the separation of two surfaces after adhesion [48]. The fracture strength σ is directly proportional to adhesion strength and is given by the following equation (1.6),



σ = (Eε/L)1/2

(1.6)

Where E is Young’s modulus of elasticity, ε is the fracture energy, and L is the critical crack length when two surfaces are separated. The work done

Mechanisms of Bioadhesion  17 Mucoadhesive Fracture in hydrated layer of the system Fracture at the mucoadhesivemucous layer interface Fracture in mucous layer Mucous membrane

Figure 1.5  Regions that represent rupture of the mucoadhesive bond.

by an elastomeric network to cause fracture, Gc, can be expressed as equation (1.7):

Gc = K(Me)1/2

(1.7)

K is a constant which depends on the density of the mucoadhesive polymer, effective mass, length, the flexibility of a single mucin chain bond, and bond dissociation energy. Gc of an elastomeric network increases with molecular weight Me of the network strands [49]. Figure 1.5 is a schematic representation of the rupture of mucoadhesive bonds [50].

1.6 Stages of Mucoadhesion For a better understanding of the broad concept of mucoadhesion, the process of mucoadhesion can be differentiated into three stages: wetting, interpenetration, and interaction of mucoadhesive with biological substrate. The mucoadhesive must wet the substrate to develop an intimate contact between the mating partners. The hydrophilic property of the mucoadhesive polymer is an important consideration in mucoadhesion because mucoadhesive interaction occurs in the presence of water (mucus consists of 95% water) [51]. Low contact angle between water and the polymer will encourage the hydration of the mucoadhesive polymer chains and increase the segmental mobility. Spreading of the mucoadhesive polymer over the mucus also promotes intimate contact. So, it is important that the mucoadhesive polymer chains are able to diffuse into the mucus network so that interdigitation between the interacting materials may occur.

18  Bioadhesives in Drug Delivery The mucoadhesive polymer must have an enough linear chain length to ensure interpenetration during mucoadhesion [51]. The segmental mobility of the mucoadhesive polymer chain is of great importance in inducing entanglement between the interacting agents. The proposed mechanism of adhesion of hydrocolloids suggests that upon hydration the synthetic mucoadhesive polymer molecules become more mobile and are even able to orient themselves at adhesive sites of the substrate. As the level of hydration increases, adhesion strength reduces since the mucoadhesive bonds become more extended. Based on the rate of diffusion of mucoadhesive polymer through mucus networks and the depth of penetration required for mucoadhesion, interdigitation alone cannot account for the mucoadhesive interaction because of the time dependency of the process. Therefore, secondary bond formation is assumed to play a significant role. Mucus layer acts as defensive covering to protect cells and control drug delivery.

1.7 Modulation of Mucoadhesion A large number of variables influence the chemical and physical attributes of the mucin or mucoadhesive polymer that affect the extent of mucoadhesion. Depending on the mucoadhesive polymer, there is the possibility to modulate the mucoadhesive property of the complex system [52]. Surface free energy modulation can influence the interaction of mucin with mucosal liquids as listed below [53]: a) Various substances interact with the mucin and are known as mucous thickening/thinning agents. Accumulation of agent (thickening/thinning) depends upon electrostatic charges and composition of the mucin. b) Calcium precipitates mucin, and when mucin is used to vary the tonicity (osmotic pressure gradient) of the medium, it decreases shear stress. A higher value of shear stress shows better bioadhesion between polymer and mucus. c) The level of hydration of mucoadhesive declines with a consequent reduction in tensile stress, and thus expanding the ionic nature of the medium. d) The mucin can be altered by the action of mucolytic agents. The mucolytic agents decrease the thickness of mucus by adjusting the organization of mucus through a burst of disulfide bonds or by the proteolytic activity of the catalyst.

Mechanisms of Bioadhesion  19 Disulfide bond breaking leads to splitting of the disulfide bridges. e) Structural breakdown of the mucus polypeptide by sodium deoxycholate and lysophosphatidylcholine. f) Duodenogastric reflux causes alteration in the structure of mucin and leads to gastric ulcer. g) Some disease states disturb the integrity of the mucin layer. For example, ulceration and irritation of the digestive system cause rupture of the mucin layer, while cystic fibrosis causes thickening of the mucin layer and may lead to hindrance to bronchi [54].

1.8 Adhesion Promoters Adhesion promoters are used for the improvement of mucoadhesion. Adhesion promoters are used to modulate the macroscopic adhesion property of engineered hydrogels and affect the polymer chain dispersion. Tethered polymer chains are polymer chains with one of their terminals appended to a 2-dimensional surface. Attaching of long polychains on polyhydrogels and their copolymers can be accomplished by grafting reactions or by copolymerization. The hydrogels display mucoadhesive property because of enhanced anchoring of the chains with the mucosa. Engineered hydrogels include many distinguishing features: (i) one of their terminals is covalently bonded with the hydrogels that tends to increase the quality of adhesion bond, and (ii) charge of the tethered chains imparts capacity to cling to the surface [18].

1.9 Surface Free Energy Analysis of Bioadhesion Surface free energy is an important physicochemical property responsible for bioadhesion. The surface offers the opportunity for potential bonds because the molecules and atoms present at the surface can react with the same or different types of atoms and molecules [55]. When water interacts with the surface molecules/atoms, it alters the surface free energy. Interactions occur through hydrogen bonds, van der Waals interaction, hydrophobic interaction, electrostatic and polar interaction. The work of adhesion can thus be explained as the work needed to detach the adhesive

20  Bioadhesives in Drug Delivery from the substratum and is equivalent to the sum of surface tension of liquid and surface free energy of solid minus interfacial free energy between the liquid and solid. It has been studied that the increase in the concentration of dihydroxyphenylalanine (DOPA) increases the bioadhesive interaction [56]. The literature depicts that biomaterials are utilized for cardiovascular devices. The main problem in the establishment of biocompatibility of cardiovascular devices is the interfacial interaction between biomaterial and blood. The reports by researchers also depict that there exists a relation between implant surface free energy, blood flow and thrombosis. High surface free energy promotes bioadhesion [55, 57].

1.10 Molecular Biology in Bioadhesion In the field of molecular biology, bioadhesion refers to the ability of a living organism to adhere to a surface either permanently or temporarily. Molecular biology is beneficial in bioadhesion in terms of isolation of genes, the study of gene expression and gene function [58]. Tremendous research in recent years has led to advances in the sequencing of DNA, RNA as well as in the analysis of proteins. These recent advancements in technologies motivate the researchers to shift their study from a single gene to complete sets of genes followed by examining the genes that are expressed at a time. The advancements in molecular biology provide a large number of research techniques and devices that are productive for the researchers who may work on bioadhesion involving certain particular organisms. The bioadhesion research is based on the techniques used in biochemistry, histology and mechanics but there are certain model approaches for molecular biology [59]. The models are mussels, barnacles, sandcastle worms, flatworms, starfishes, etc. However, there are four different steps that show how to narrow the number of candidate transcripts involved in adhesion. These steps include (i) generation of transcriptome and differentially expressed cDNA that is enriched in adhesion-related transcripts; (ii) setting up a basic local alignment search tool (BLAST); (iii) performing an in-situ hybridization screen, and (iv) functional analysis of selected genes using RNA interference. These steps are detailed below. For the generation of a transcriptome that consists of only adhesionrelated genes of a living organism, it is very necessary to select only the particular type of tissue that contains bioadhesive organs. Selection of proper bioadhesive organs is necessary to minimize the complexity of the

Mechanisms of Bioadhesion  21 transcriptome while expressing genes and minimizing the cost. The steps for the generation of a transcriptome include isolation of total RNA and selection of polyRNA. After selection, the next step is fragmentation of RNA into 200-300 base pairs and reverse transcription into complementary DNA [60]. Then, sequencing adapters bind chemically by a standard protocol. On the other hand, strand-specific sequencing can be executed. Selection of particular size range base pairs (200) followed by polymerase chain reaction-based amplification leads to the generation of DNA [61]. Finally, bioinformatic data analysis is done which includes error correction, de-multiplexing, artifact removal, etc. In the end, all the data are assembled into a hypothetical transcript that finally results in the transcriptome of the organism that was selected [62]. Next is setting up a basic local alignment search tool (BLAST) which is basically a software for the analysis of sequence databases. This software has been widely used to find candidate genes for analysis by utilizing molecular approaches. From this software, adhesion-related transcripts of the organism can be analyzed as well as mass spectrometry results for peptide sequences can be compared with the predetermined transcriptome database [63]. In situ hybridization is a ubiquitous and simple method to detect the spatial and temporal genes expression present within a tissue. The purpose is to investigate different kinds of nucleic acids. The principle of in situ hybridization includes pretreatment (by paraformaldehyde and proteinase K) of the tissue or the organism with an adhesive organ that consists of staining the organisms [64]. After pretreatment of the tissue, digoxigenin-labeled RNA probe is added which gets bound to the complementary messenger RNA. The bound products are analyzed via an antibody (anti-digoxigenin complex) formation. Nitro blue tetrazolium chloride is added that leads to the formation of blue color in cells because of the bound anti-digoxigenin antibody. The blue staining reveals that the cells with the target gene expression are present in the adhesive organ. For evaluating whether a transcript is expressed into an adhesive organ of the organism or not, functional analysis of both protein and the gene is necessary. Of the various techniques to detect the role a gene, RNAi gives fast and direct results.

1.11 Bioadhesives from Marine Sources The process of developing bioadhesives from marine animals is a promising strategy for generating new materials like advanced glues or

22  Bioadhesives in Drug Delivery fouling-resistant surfaces. To develop advanced glues or fouling-resistant surfaces, it is important to know about the structural, mechanical and molecular properties of the adhesive organs of selected species. Ascidians (sea squirts) are the groups of marine organisms that are considered interesting in the context of bioadhesion. The ascidians are important foulers, and the larvae as compared to adult ascidians show adhesive property. The ascidians are also important for the stepwise building of adhesive organ at both cellular and molecular levels [65]. Ascidians that develop adhesives are mostly marine vertebrates. This property results in their attachment to any surface that may undergo metamorphosis and results in permanently sessile adults [66]. They are popular as model organisms for developmental biology [67]. Adhesives developed from marine organisms can be useful in the medical field because these have distinctive ability to cure in wet environments and are also compatible with the tissues. Formation of multicomponent bioadhesives in marine organisms helps in their survival in unfavourable environmental conditions [68]. The secretions from the adhesive animals are based on proteins and vary widely between different adhesive organisms. As mussel byssus and barnacle have maximum adhesive secretions and contain different types of adhesive proteins. In the case of adult mussels, they bind to the surfaces after secreting a bundle of threads from the glands present in the foot of mussel. The first two proteins that are secreted on the surface of the substrate are Mfp/3 and Mfp/5. These proteins contain a high content of 3,4-dihydroxyphenyl-L-alanine (DOPA) [68]. However, in biomimetic engineering, catechol components that duplicate adhesive property are functionalized with synthetic polymers that possess sealant, coating and adhesive properties.

1.12 Mucoadhesive Drug Delivery Systems These days mucoadhesion is garnering considerable attention for development of safe and useful commercial dosage forms. While single unit mucoadhesive dosage forms are yet to place themselves, particulate drug delivery systems like microparticles, microspheres, nanoparticles or liposomes have demonstrated some interesting and useful features like uniform circulation at the target site and reproducible medication adsorption [69, 70]. Different drug delivery carriers based on the mucoadhesive property of the polymers are discussed in the other chapters in this book.

Mechanisms of Bioadhesion  23

1.13 Summary An understanding of the fundamental mechanisms that govern bio­ adhesion is of great interest for researchers in various fields. One area of research focuses on natural adhesive materials produced by or extracted from plants, animals, fungi and bacteria. The second area of research in the field of bioadhesives focuses on man-made materials that aim to mimic the remarkable adherence capability of natural adhesives. To date, a variety of materials have been fabricated that find applications in drug delivery. The term mucoadhesion may be used synonymously with bioadhesion to describe these systems. The concept of bioadhesion opens a new area of research in the biomedical sciences and attracts pharmaceutical industries to develop new formulations for improved therapeutic effects.

References 1. P. Tangri and N.S. Madhav, Oral mucoadhesive drug delivery systems: A review. Intl. J. Biopharmaceutics 1, 36-46 (2011). 2. P. Kingshott and H.J. Griesser, Surfaces that resist bioadhesion. Current Opinion Solid State Mater. Sci. 4, 403-412 (1999). 3. C.M. Lehr, J.A. Bouwstra, W. Kok, A.B. Noach, A.G. de Boer and H.E. Junginger, Bioadhesion by means of specific binding of tomato lectin. Pharmaceutical Res. 4, 547-553 (1992). 4. V. Gavini, B. Ragini and K. Kaumudi, Mucoadhesive microspheres - A novel approach of drug targeting. World J. Pharmacy Pharmaceutical Sci. 3, 310335 (2014). 5. S.E. Harding, S.S. Davis, M.P. Deacon and I. Fiebrig, Biopolymer mucoadhesives. Biotechnol, Genetic Eng. Reviews 1, 41-86 (1999). 6. C.A. Scrivener and C.W. Schantz, Penicillin: New methods for its use in dentistry. J. Amer. Dental Assoc. 35, 644-647 (1947). 7. J.T. Rothner, H.M. Cobe, S L. Rosenthal and J. Bailin, Adhesive penicillin ointment for topical application. J. Dental Res. 6, 544-548 (1949). 8. A.H. Keutscher, E.V. Zegarelli, F.E. Beube and N.W. Chiton, A new vehicle (or a base) for the application of drugs to the oral mucous membranes. Oral Pathology 9, 1080-1089 (1959). 9. J.L. Chen and G.N. Cyr, Compositions producing adhesion through hydration. In: Adhesion in Biological Systems, R.S. Manly (Ed.), pp. 163-181. Academic Press, New York, (1970). 10. J.B. Park, Acrylic bone cement: in vitro and in vivo property-structure relationship - A selective review. Annals Biomed. Eng. 11, 297-312 (1983).

24  Bioadhesives in Drug Delivery 11. J.D. Smart, I.W. Kellaway and H.E.C. Worthington, An in-vitro investigation of mucosa-adhesive materials for use in controlled drug delivery, J. Pharmacy Pharmacology 5, 295-299 (1984). 12. Y. Sudhakar, K. Kuotsu and A. Bandyopadhyay, Buccal bioadhesive drug delivery-A promising option for orally less efficient drugs. J. Controlled Release 1, 15-40 (2006). 13. T.R. Rahamatullah Shaikh, M.J. Garland, A.D. Woolfson and R.F. Donnelly, Mucoadhesive drug delivery systems. J. Pharmacy Bioallied Sci. 3, 89-100 (2011). 14. D.L. Schmidt, R.F. Brady, K. Lam, D.C. Schmidt and M.K. Chaudhury, Contact angle hysteresis, adhesion, and marine biofouling. Langmuir 20, 2830-2836 (2004). 15. M.L. Palacio and B. Bhushan, Bioadhesion: A review of concepts and applications. Phil. Trans. Royal Soc. A 370, 2321-2347 (2012). 16. R. Malviya, P.K. Sharma and S.K. Dubey, Modification of polysaccharides: Pharmaceutical and tissue engineering applications with commercial utility (patents). Mater. Sci. Eng. C 68, 929-938 (2016). 17. A. Alexander, S. Ajazuddin, D.K. Tripathi, T. Verma, J. Maurya and S. Patel, Mechanism responsible for mucoadhesion of mucoadhesive drug delivery system: A review. Intl. J. Appl. Biology Pharmaceutical Technol. 2, 434-445 (2011). 18. C. Hannig and M. Hannig. The oral cavity- a key system to understand substratum-dependent bioadhesion on solid surfaces in man. Clinical Oral Investigations 13, 123-139 (2009). 19. S. Punitha and Y. Girish, Polymers in buccal mucoadhesive drug delivery system - A review. Intl. J. Res. Pharm. Sci. 2, 170-186 (2010). 20. S.U. Zate, P.I. Kothawade, G.H. Mahale, K.P. Kapse and S.P. Anantwar, Gastro retentive bioadhesive drug delivery system: A review. Intl. J. Pharm Technol. Res. 5, 1227-1235 (2010). 21. F.C. Carvalho, M.L. Bruschi, R.C. Evangelista and M.P. Gremiao, Mucoadhesive drug delivery systems. Brazilian J. Pharm. Sci. 1, 1-7 (2010). 22. H. Gerstrom, K. Edsman and M. Stromme, Low‐frequency dielectric spectroscopy as a tool for studying the compatibility between pharmaceutical gels and mucous tissue. J. Pharm. Sci. 92, 1869-1881 (2003). 23. R. Sankar and S.K. Jain. Development and characterization of gastroretentive sustained-release formulation by combination of swelling and mucoadhesive approach: A mechanistic study. Drug Design, Devel. Therapy 7, 1455-1469 (2013). 24. D. Brahmbhatt, Bioadhesive drug delivery systems: Overview and recent advances. Intl. J. Chemical Life Sci. 3, 2016-2024 (2017). 25. V.V. Khutoryanskiy, Advances in mucoadhesion and mucoadhesive polymers. Macromol. Biosci. 6, 748-764 (2011). 26. X. Yang and J.R. Robinson, Bioadhesion in mucosal drug delivery, In: Biorelated Polymers and Gels, T. Okano (Ed.), pp. 135-192, Academic Press, San Diego, CA (1998). 27. B. Bhushan, Introduction to Tribology. Wiley, New York (2002).

Mechanisms of Bioadhesion  25 28. B. Bhushan, Adhesion and stiction: Mechanisms, measurement techniques, and methods for reduction. J. Vac. Sci. Technol. 21, 2262-2296 (2003). 29. J.D. Andrade, V. Hlady and A.P. Wei, Adsorption of complex proteins at interfaces. Pure Appl. Chem. 64, 1777-1781 (1992). 30. J.H. Waite, Nature’s underwater adhesive specialist. Intl. J. Adhesion Adhesives 7, 9-14 (1987). 31. J.L. Dalsin, B.H. Hu, B.P. Lee and P.B. Messersmith, Mussel adhesive protein mimetic polymers for the preparation of nonfouling surfaces. J. Amer. Chem. Soci. 125, 4253-4258 (2003). 32. B.G. Keselowsky, D.M. Collard and A.J. Garcia, Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion. J. Biomed. Mater. Res. 66, 247-259 (2003). 33. L. Vroman, Effect of adsorbed proteins on the wettability of hydrophilic and hydrophobic solids. Nature 196, 476-477 (1962). 34. P. Turbill, T. Beugeling and A.A. Poot, Proteins involved in the Vroman effect during exposure of human blood plasma to glass and polyethylene. Biomaterials 17, 1279-1287 (1996). 35. S.R. Mobley, J. Hilinski and D.M. Toriumi, Surgical tissue adhesives. Facial Plastic Surgery Clinics North America 10, 147-154 (2002). 36. T.K. Vaidyanathan and J. Vaidyanathan, Recent advances in the theory and mechanism of adhesive resin bonding to dentin: A critical review. J. Biomed. Mater. Res. 88, 558-578 (2009.) 37. N.A. Peppas and P.A. Buri, Surface, interfacial and molecular aspects of polymer bioadhesion on soft tissues. J. Controlled Release 2, 257-275 (1985). 38. G. Buckton, Interfacial Phenomena in Drug Delivery and Targeting, pp. 225232, CRC Press, Boca Raton, FL (2000). 39. P Andrews, P.L. Gavin and S.J. David, Mucoadhesive polymeric platforms for controlled drug delivery. European J. Pharm. Biopharm. 3, 505-518 (2009). 40. D.J. Gardner, Theories and mechanisms of adhesion, in: Handbook of Adhesive Technology, third edition, A. Pizzi and K.L. Mittal (Eds.) pp. 3-18, CRC Press, Boca Raton, FL (2018). 41. K.L. Mittal, The role of the interface in adhesion phenomena, Polym. Eng. Sci. 17, 467-473 (1977). 42. R.B. Gandhi and J.R. Robinson, Oral cavity as a site for bioadhesive drug delivery. Advanced Drug Delivery Reviews 2, 43-74 (1994). 43. S. Naskar, S.K. Roy and K. Kuotsu, Drug delivery based on buccal adhesive systems - A review. Intl. J. Pharma Bio Sciences 3, 240-256 (2013). 44. U.M. Trivedi, V.M. Patel, A. Mahajan and P. Mitesh, A review on mucoadhesion, mucoadhesive polymers and mucoadhesive sites. Intl. J. Institutional Pharmacy Life Sciences 1, 1-18 (2011). 45. A. Anil and P. Sudheer, Mucoadhesive polymers: a review. J. Pharm. Res. 17, 47-55 (2018). 46. S.M. Patil and S.S. Kulkarni, Review on buccal mucoadhesive drug delivery systems. Intl. J. Pharmaceutical Life Sci. 6, 101-128 (2013).

26  Bioadhesives in Drug Delivery 47. V.A. Chaudhari, S.M. Sarode, B.S. Sathe and G.P. Vadnere, Mucoadhesive buccal drug delivery system: A review. Intl. J. Pharmaceutical Sci. 2, 142-159 (2014). 48. A. Lohani and G.P. Chaudhary, Mucoadhesive microspheres: A novel approach to increase gastroretention. Chronicles Young Scientists 2, 121-128 (2012). 49. D. Dodou, P. Breedveld and P.A. Wieringa, Mucoadhesives in the gastrointestinal tract: Revisiting the literature for novel applications. European J. Pharm. Biopharm. 1, 1-16 (2005). 50. J. Schultz and M. Nardin, Theories and mechanisms of adhesion, in: Handbook of Adhesive Technology, A. Pizzi and K.L. Mittal (Eds.) pp. 19-33, Marcel Dekker, New York (1994). 51. S. Rossi, M.C. Bonferoni, F. D’Autilia, G. Sandri, F. Ferrari, C. Caramella, E. Mortara, V. Giannini and F. Gasparri, Associations of natural polymers to modulate mucoadhesion of vaginal rinse-off and leave-on formulations. J. Drug Delivery Sci. Technol. 5, 496-502 (2014). 52. J. das Neves, M.F. Bahia, M.M. Amiji and B. Sarmento, Mucoadhesive nanomedicines: Characterization and modulation of mucoadhesion at the nanoscale. Expert Opinion Drug Delivery 8, 1085-1104 (2011). 53. L. Bacakova, E. Filova, M. Parizek, T. Ruml and V. Svorcik, Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants. Biotechnology Advances 6, 739-767 (2011). 54. F. Brochard-Wyart, P.G. de Gennes, L. Leger, Y. Marciano and E. Raphael, Adhesion promoters. J. Phys. Chem. 98, 9405-9410 (1994). 55. J. Wang, M.N. Tahir, M. Kappl, W. Tremel, N. Metz, M. Barz, P. Theato and H.J. Butt. Influence of binding site density in wet bioadhesion. Adv. Mater. 20, 3872-3876 (2008). 56. M. Rodrigues, B. Lengerer, T. Ostermann and P. Ladurner, Molecular biology approaches in bioadhesion research. Beilstein J. Nanotechnology 5, 983-993 (2014). 57. F.M. Etzler, Determination of the surface free energy of solids: A critical review, Rev. Adhesion Adhesives 1, 3-45 (2013). 58. S.Y. Yang, E.D. O’Cearbhaill, G.C. Sisk, K.M. Park, W.K. Cho, M. Villiger, B.E. Bouma, B. Pomahac and J.M. Karp, A bio-inspired swellable microneedle adhesive for mechanical interlocking with tissue. Nature Communications 4, 1-10 (2013). 59. R.J. Stewart, Protein-based underwater adhesives and the prospects for their biotechnological production. Appl. Microbiol. Biotechnol. 1, 27-33 (2011). 60. B.T. Wilhelm and J.R. Landry, RNA-Seq-quantitative measurement of expression through massively parallel RNA-sequencing. Methods 3, 249-257 (2009). 61. Z. Wang, M. Gerstein and M. Snyder, RNA-Seq: A revolutionary tool for transcriptomics. Nature Rev Genetics 1, 57-63 (2009).

Mechanisms of Bioadhesion  27 62. C. Camacho, G. Coulouris, V. Avagyan, N. Ma, J. Papadopoulos, K. Bealer and T.L. Madden, BLAST: Architecture and applications. BMC Bioinformatics 10, 1-9 (2009). 63. A.Y. Chen and A. Chen, Fluorescence in situ hybridization. Investigative Dermatology 5, 1-4 (2013). 64. R. Pennati and U. Rothbächer, Bioadhesion in ascidians: A developmental and functional genomics perspective. Interface Focus 1, 1-10 (2015). 65. H. Lee, B.P. Lee and P.B. Messersmith, A reversible wet/dry adhesive inspired by mussels and geckos. Nature 448, 338-341 (2007). 66. L. Petrone, Molecular surface chemistry in marine bioadhesion. Adv. Colloid Interface Sci. 195, 1-8 (2013). 67. G. Lambert, Invasive sea squirts: A growing global problem. J. Expl. Marine Biol Ecol 342, 3-4 (2007). 68. K. Kamino, Underwater adhesive of marine organisms as the vital link between biological science and materials science. Marine Biotechnol. 10, 111121 (2008). 69. I. Singh, P. Pawar, E.A. Sanusi and O.A. Odeku, Mucoadhesive polymers for drug delivery systems, in: Adhesion in Pharmaceutical, Biomedical and Dental Fields, K.L. Mittal and F.M. Etzler (Eds.) pp. 89-114, Wiley-Scrivener, Beverly, MA (2017). 70. A. Khare, K. Grover, P. Pawar and I. Singh, Mucoadhesive polymers for enhancing retention in ocular drug delivery: A critical review, Rev. Adhesion Adhesives, 2, 467-502 (2014).

2 Bioadhesive Polymers for Drug Delivery Applications Kenneth Chinedu Ugoeze* Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Port Harcourt, Nigeria

Abstract

The bioadhesive polymers applicable in drug delivery systems are considered in this chapter. The limitations encountered with conventional drug delivery systems are tackled through bioadhesive/mucoadhesive drug delivery systems which curtail drug dosing frequencies, offering extended and consistent drug release with better drug bioavailability. In bioadhesive drug delivery, a polymeric drug carrier is trapped to epithelial tissue while mucoadhesion entails the sticking of a polymeric drug carrier to mucous membranes. Epithelial tissues or mucous membranes are components of various parts of the body and constitute potential sites for bonding of a bioadhesive/mucoadhesive drug carriers and have become necessary in the application of bioadhesive/mucoadhesive drug delivery systems. These mode of drug deliveries have become valuable in handling drugs that are not flexible in oral or other conventional mode of administration especially those that are acid degradable or experience the first-pass metabolic pathway. The capability of a dosage form to fit into the bioadhesive/mucoadhesive drug delivery systems is based on several factors, including the nature of the mucosal tissue and the physicochemical behaviour of the polymeric formulation. This chapter reviews several aspects of bioadhesion/mucoadhesion, their mechanisms, bioadhesive/­ mucoadhesive polymers, factors affecting them and their classification among others. Keywords:  Polymers, bioadhesive, mucoadhesive, drug delivery systems

*Email: [email protected]; [email protected] K.L. Mittal, I. S. Bakshi and J. K. Narang (eds.) Bioadhesives in Drug Delivery, (29–56) © 2020 Scrivener Publishing LLC

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30  Bioadhesives in Drug Delivery

2.1 Introduction 2.1.1 Drug Delivery Drug delivery deals with the carriage of medicinal agents through the physiological systems of humans or animals to get to their targeted site of pharmacological action and also to realize the expected therapeutic outcomes [1, 2]. Drug delivery systems (DDS) are pharmaceutical measures or usefully devised means that support the realization of targeted delivery and/or controlled-release (CR) of therapeutic agents in the body. Over the years, drugs have been conveyed through numerous conventional methods such as the oral, parenteral (via injections), transdermal, inhalation, implant, suppository, ophthalmic and the otic drug dosage forms. Drugs given by these orthodox channels may bring about unwanted side effects, occasionally due to their buildup following recurring usage. These undesirable outcomes may arise depending on the body physiology of the user of the drug, nature of the drug, the mode of its delivery into the body and particularly the interaction of the drug with components of the body that are not the target for the drug. These are the key shortcomings of conventional drug delivery approaches even with their several benefits. Due to these challenges, the need for drug delivery systems that will improve patient compliance to drug product usage becomes imperative to reduce multiple drug doses needed to maintain an expected therapeutic outcome. Owing to this need, scientists have contributed to increased utilization of diverse physiological barriers in effective delivery of drugs in the circulatory system and drug transport through cells and tissues. They have come up with innovative methods for transporting medicinal ingredients such as targeted delivery, drug modification by chemical means, drug entrapment in small vesicles that are injected into the bloodstream, drug entrapment within pumps or polymeric materials that are placed in desired bodily compartments, etc. [1, 3]. The delivery of drugs using the innovative procedures enhances drug administration outcome, particularly with reduced undesirable drug activity and providing an effective and safe application of medications to the patient [2-5]. A model DDS assures that the active drug is brought to the site of action in line with the need of the patient for a predictable time frame. The DDS also enhances the realization of required drug concentration at the right site and permits its regularity in the therapeutic window, i.e., between the minimal effective concentration (MEC) and the minimum toxic concentration (MTC).

Bioadhesive Polymers for Drug Delivery  31 The processes of drug delivery involve formulations of the active pharmaceutical ingredient (API), considering the route of drug usage. In several formulations, the API is enmeshed in an environment of drug carriers or excipients which helps in the achievement of drug stability, consistency in drug transport procedures as well as the attainment of the target therapeutic outcomes [2]. These aids may include the inert materials noted as excipients. Among the various excipients and their applications, the polymers are used in drug delivery. The polymers with relevance in bioadhesive drug delivery systems will be discussed in this chapter.

2.2 Bioadhesive/Mucoadhesive Drug Delivery Systems Bioadhesion is defined as the attachment of synthetic or natural polymers to a biological substrate, e.g. epithelial surface for a long time. Likewise, the word bioadhesion is designated as ‘mucoadhesion’ when the polymer is attached or bonded to mucous surface. In discussing bioadhesive drug delivery, mucoadhesion could suggest the attachment of a polymeric substance to a mucous membrane (mucoadhesion) or to an epithelial tissue (bioadhesion) [6-8]. For the purposes of drug delivery, bioadhesion is defined as the ability of the drug carrier system, often a polymeric material, to stick to a biological tissue for a prolonged interval of time. This activity brings about improved drug concentration gradient at the absorption site and, therefore, enhanced bioavailability of a systemically delivered drug [9]. The membranes of the inner areas of the body are filled with a thick gel-like assembly identified as mucin which is produced by goblet cells and special exocrine glands with mucous cell acini. Mucous membrane is the sheath lining the body hollows and cavities, mostly the respiratory, digestive, urogenital paths as well as the mouth, nose, eyelids, trachea, lungs, stomach/intestines, the ureters, urethra and urinary bladder. These internal portions of the body that are covered with the mucous membrane are potential sites for affixing bioadhesive drug carriers in bioadhesive drug delivery systems. Since the word mucoadhesion is the ideal term when adhesion of a bioadhesive material occurs to a biological mucous membrane, and the mucosal layers line several regions of the body that constitute potential sites for the attachment of bioadhesive drug carriers, the terms such as mucoadhesion, mucoadhesive polymers or mucoadhesive drug delivery systems will be used alongside the terms bioadhesion, bioadhesive polymers or bioadhesive drug delivery systems in this chapter [10].

32  Bioadhesives in Drug Delivery

2.2.1 Some Advantages of the Bioadhesive/Mucoadhesive Drug Delivery Systems The benefits of bioadhesive drug delivery systems, among others, are [11-13]: i.

The bioadhesive drug delivery systems have been utilized to target disease states at the mucosal surface. With this technique, multiple systemic application of frequent and elevated doses of drugs is avoided which would have led to unwanted drug actions. ii. The use of bioadhesive drug delivery systems enables the extension of the residence time at the site of drug absorption. This enhances absorption and the therapeutic effectiveness of the drug and also decreases the frequency of dosing which renders the patient compliance to drug usage very easy. iii. More rapid commencement of action of the drug is realized owing to the mucosal surface. iv. The strong adhesion of the drug delivery system (DDS) with the absorptive mucosa or biological site generates a steeper concentration gradient which causes an enhanced drug absorption rate [14]. v. Some mucoadhesive polymers may modulate the absorptivity of epithelial tissues by relaxing the tight intercellular junctions [15, 16]. vi. Some mucoadhesive polymers act as inhibitors of proteolytic enzymes, thus enhancing the stability of the active drug component. vii. The utilization of this form of drug delivery system (DDS) assists with the confinement of drugs to the target site. viii. With the bioadhesive drug delivery system, the firstpass metabolism is averted, causing an increased drug bioavailability. ix. The use of a bioadhesive drug delivery system to a mucosal surface may be beneficial in terms of delivering drug molecules that are not suitable for the oral route, such as those that suffer acid degradation or extensive first-pass metabolism as they are protected from degradation in the acidic environment in the gastrointestinal tract [17].

Bioadhesive Polymers for Drug Delivery  33

2.2.2 The General Need for Bioadhesive/Mucoadhesive Drug Delivery Systems The oral route of drug delivery is a common application because of its exceptional benefits such as sustained and controllable delivery, ease of administration, possibility of formulating solid dosage forms, patient compliance, etc. However, orally administered drugs encounter slow absorption as well as the numerous obstacles they need to deal with such as the first-pass hepatic metabolism, enzyme degradation, patient noncompliance owing to such problems as aphagia, e.g., challenge of letting solids down the throat. In contrast to the orally controlled release systems, the bioadhesive/mucoadhesive drug delivery systems have the potential of being used for controlled release drug delivery for prolonged period of time, allowing active absorption and higher bioavailability of the drugs owing to a high surface to volume ratio and permitting an ample close contact with the mucous layer. It could be designed to suit any mucosal tissue surrounding those in the stomach and permitting the predictions of the systemically controlled release of drugs. The use of bioadhesive/ mucoadhesive drug delivery systems to the mucosal tissues of gastric epithelium is convenient in the control of drugs for regulated or targeted action. These types of drug delivery systems are mostly used since they release the drug for an extended interval. They also reduce the frequency of drug administration and equally increase the patient responsiveness to the required drug doses. With the introduction of numerous innovative medicinal substances owing to drug discovery, bioadhesive/mucoadhesive drug delivery will have even more vital applications in delivering these novel molecules [18-23].

2.3 Mechanism of Bioadhesion The process involved in the development of bioadhesive bonds could be described in two stages: the contact stage and consolidation stage. In the contact stage, close contact is established between the mucoadhesive polymer and the mucous membrane surface, attainable either from good wetting by the mucoadhesive or from the swelling of the mucoadhesive. The moisture that has been absorbed results in the swelling of the dosage form formulation. The consolidation stage defines the beginning and the bonding of bioadhesive/mucoadhesive polymer, realizable through the penetration of the mucoadhesive into the clefts of the tissue surface

34  Bioadhesives in Drug Delivery or interpenetration of the chains of the mucoadhesive with those of the mucous membrane. At this point, weak chemical bonds are formed by weak van der Waals and hydrogen bonds [24, 25].

2.4 Requirements for an Ideal Bioadhesive/ Mucoadhesive Polymer The following characteristics are desired for a model polymer for use in bioadhesive drug delivery: i.

It should be able to bond rapidly to a biological surface such as the mucosa and site-specificity will be an added advantage. ii. The polymer should not create any unwanted influence on the drug molecule, especially on its release. iii. The polymer is anticipated to wet the mucous surface and exhibit an elevated swelling index. iv. The polymer for a bioadhesive drug delivery systems is expected to be biodegradable. v. The presence of food, pH alterations, etc. should not produce any undesirable effect on the polymer. vi. The bioadhesive polymer must be easily incorporated into various dosage forms and should not interfere with release of the drug. vii. The bioadhesive/mucoadhesive polymer should be able to deter the negative impact of any localized enzyme on the incorporated drug molecule. viii. Such polymer is expected to exhibit a consistent viscosity at the point of its contact. ix. It is anticipated that the bioadhesive polymer as well as its degradation products should not generate toxicity, should not be absorbable in the gastrointestinal region and should not constitute a source of irritation to the mucous membrane. x. The polymer should remain stable during its storage and through the shelf-life of the dosage formulation. xi. The bioadhesive polymer should be affordable to avoid escalating the cost of production of the drug product [26-28].

Bioadhesive Polymers for Drug Delivery  35 In bioadhesive drug delivery systems, an ideal polymer is also expected to obey the following facts: i.

Cationic and anionic polymers bind better than neutral polymers. ii. Polyanions are superior to polycations when considering their binding characristics as well as their potential for toxicity. Moreover, water-insoluble polymers are more flexible in dosage form design in contrast with rapidly or slowly dissolving water-soluble polymers. iii. Anionic polymers with sulfate groups bind more effectively than those with carboxylic groups. iv. The degree of binding is consistent with the charge density on the polymer. v. Highly binding polymers include carboxymethyl cellulose, gelatin, hyaluronic acid, carbopol, and polycarbophil [29]. vi. In terms of molecular characteristics, an ideal bioadhesive/mucoadhesive polymer should possess the following traits [30, 31]: a. Strong hydrogen bonding groups (-OH, - COOH). b. Strong anionic charges. c. Sufficient flexibility to penetrate the mucous network or tissue crevices. d.  Proper surface tension for wetting mucus/mucosal tissue surface. e. High molecular weight.

2.5 Factors Affecting Bioadhesion/Mucoadhesion The factors that affect the bioadhesive/mucoadhesive behaviour of polymers are categorized as [32]: polymer related factors, environmental factors, and physiological factors.

2.5.1 Polymer Related Factors These can also be expressed as ‘intrinsic factors’. These refer to the structural characteristics of the polymer that govern its fundamental properties. These comprise the molecular weight, cross-linking and the existence of functional groups as well as the bioadhesive/mucoadhesive spatial conformation [32].

36  Bioadhesives in Drug Delivery

2.5.1.1 Molecular Weight High molecular weight improves the cohesiveness of the polymer and enhances the viscosity it could generate. The interpenetration of polymer molecules into the mucous surface varies. For polymers possessing low molecular weight, penetration is higher than for the polymers with high molecular weight because entanglements are favoured in polymers with high molecular weight. Extremely high molecular weight diminishes the flexibility of the molecule and hence its diffusion. There is an optimal molecular weight for each class of polymer that determines the likely bioadhesive strength. For instance, poly (acrylic acid) has an optimal molecular weight of about 750,000, while poly(ethylene oxide) has an optimum molecular weight close to 4,000,000 [32, 33].

2.5.1.2 Chain Length An increase in the chain length of the polymer is expected to improve the mucoadhesive characteristic of the polymer [34].

2.5.1.3 Flexibility If the polymer chains are flexible, then infiltration and entanglement of the polymer chains with those of the mucosal layer are easy, thus improving the bioadhesive characteristic. Cross-linking and hydration of the polymer affect its elasticity. An extreme cross-linking lowers the plasticity of the polymer chains, hence, the amount of cross-linking should be at an optimum level [34].

2.5.1.4 Cross-Linking Cross-linking of a polymeric material, to a very large extent, dictates how flexible it would become and the extent of its resistance to dissolution as moisture penetrates it. A normally cross-linked hydrophilic polymer would swell but retain its structure when exposed to the water, while a comparable high molecular weight non-cross-linked (linear) hydrophilic polymer would only swell. In mucoadhesive studies, swelling is favourable as it permits more control of drug release and increases

Bioadhesive Polymers for Drug Delivery  37 the surface area for polymer/mucus interpenetration. An elevation in the extent of cross-linking decreases the mobility of the polymer as well as the actual chain length that can penetrate the mucus layer, thus decreasing its mucoadhesive strength. Chain flexibility, for this reason, is a serious factor to consider in discussing the interpenetration and entanglement within the mucus gel. Improved chain mobility implies elevated interpenetration and entanglement of the polymer in the mucous network [32, 33].

2.5.1.5 Presence of Functional Groups For mucoadhesion to take place, the preferred polymers essentially should possess functional groups that can form hydrogen bonds. The hydrophilic functional groups accountable for developing hydrogen bonds are the hydroxyl (-OH) and carboxyl (-COOH) groups. Therefore, the presence of a functional group to form hydrogen bond affects the degree of adhesion obtainable. The attachment of bioadhesive polymers to biological substrates implies interpenetration. This is followed by the development of secondary non-covalent bonding in the form of hydrogen bonds with the substrates [32, 33].

2.5.1.6 Concentration of Active Polymer When the concentration of the polymer is low, the amount of penetrating polymer chains per unit volume of the mucus is lesser and the interaction between polymer and mucus is not very firm. A more concentrated polymer leads to extended penetrating chain length and improved adhesion. Thus, increasing the amount or concentration of a mucoadhesive polymer implies increasing the number of functional groups accessible to form molecular bonds and this enhances mucoadhesion. It is possible up to an optimum concentration since each polymer has an optimal concentration for mucoadhesion. When this optimum concentration level is exceeded, the mucoadhesion ability is lowered [34].

2.5.2 Environmental Factors These could also be described as the external influences that affect the properties of the bioadhesive polymers and the factors such as the

38  Bioadhesives in Drug Delivery pH/charge, degree of hydration, initial contact time, temperature and shear rate or applied pressure are worth mentioning [32-34].

2.5.2.1 pH and Charge on the Polymer At a low pH, ideal mucoadhesion occurs, but at an elevated pH, there is an increase in mucoadhesion. When the pH is increased, polymers possessing a positive charge, like the chitosan, form polyelectrolyte complexes with mucus while showing increased mucoadhesion strength. The external mucous surface of a biological substrate is negatively charged and as such, a positively charged polymer should enable the mucoadhesion process. Perhaps the first stage of mucoadhesion of a positively charged polymer to a biological surface is through electrostatic attraction followed by mechanical intertwining of polymer chains, van der Waals forces, and hydrogen bonds [35].

2.5.2.2 Degree of Hydration Various polymers may exhibit adhesive property in the presence of water. Adhesive characteristics of a polymer differ depending on the extent of hydration. There is an optimal degree of hydration required to realize an actual bioadhesion. Hydration above the optimum level leads to loss of stickiness since a slimy mass is developed, constituting a non-adhesive mucilage which occurs in presence of a massive amount of water at or near the interface. In this situation, it may be necessary to employ a cross-linked polymer which would allow only a certain degree of hydration to achieve an extended mucoadhesive effect.

2.5.2.3 Initial Contact Time The initial interaction time between the mucoadhesive polymer and the mucus layer governs the extent of swelling and the interpenetration of the polymer chains. The strength of mucoadhesion improves as the initial contact time is increased.

2.5.2.4 Applied Pressure The pressure applied to the mucoadhesive tissue at the point of interaction can affect the degree of interpenetration. If increased pressure is applied for an extended interval of time, polymers become mucoadhesive even when they do not have attractive interaction with mucins.

Bioadhesive Polymers for Drug Delivery  39

2.5.2.5 Swelling The degree of swelling obtainable will depend on the quantity of polymer, ionic concentration and the amount of water. Excessive hydration will result in the development of a slimy mucilage which will have decreased adhesion.

2.5.2.6 Ionic Strength The presence of localized ions affects the contact between the bioadhesive polymer and the mucus. Thus, the strength of the ions present determines the degree of the bioadhesion achievable in the system since the existence of the ions decreases mucoadhesion due to several functional sites accessible for the adhesion process [33].

2.5.2.7 Mucus Gel Viscosity The stickiness of mucus differs all through the GIT and in various disease states. Decreased thickness of the mucus results in a weak bioadhesive bond, whereas an enormously thick mucus layer breaks down easily and reduces the bioadhesion owing to a diminished rate of interpenetration. This implies that there should be an optimal level of viscosity of the mucous layer to enable the achievement of strong bioadhesion [33].

2.5.3 Physiological Factors The physiological factors that may influence the properties of a mucoadhesive polymer in the drug delivery system include:

2.5.3.1 Mucin Turnover The rate at which the mucin molecules are produced naturally from the mucus layer is important for consideration since the rate of mucin turnover is estimated to be the rate-determining step for the residence time of mucoadhesive dosage form on the mucous layer. Mucin turnover results in large amounts of soluble mucin molecules which interact with the mucoadhesive before they have a chance to interact with the mucus layer.

2.5.3.2 Disease States When the physiological system is infected, there is bound to be adjustments in the rate at which the mucus is generated [36].

40  Bioadhesives in Drug Delivery

2.6 Bioadhesive Polymers for Drug Delivery Applications 2.6.1 Polymers Generally, polymers are large molecules or macromolecules comprising of large numbers of recurrent single units (monomers) bonded through a chemical procedure described as polymerization [37, 38]. Polymers are classified as either natural or synthetic.

2.6.1.1 Natural Polymers The natural polymers are derivable from natural sources of plants or animals and include polysaccharides, nucleic acids and proteins [39]. Some popularly used polysaccharides in the pharmaceutical and food industries include chitosan, alginates, xanthan gum, tragacanth, acacia, etc. [40].

2.6.1.2 Synthetic Polymers The synthetic polymers are man-made products obtained through chemical manipulations of other polymers, even the natural polymers. They include thermoplastics, thermosets, elastomers and synthetic fibres. The synthetic polymers with common applications in the pharmaceutical and food industries include the vinyl polymers (e.g. Eudragit®), polyvinyl­ pyrrolidone (PVP) (Povidone), carbomers, cellulose ethers, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC), polyethers and silicones [41, 42]. Generally, polymers have been very useful in drug formulations as diluents, binders, disintegrants, films or enteric coatings of solid dosage forms, drug release enhancers/retardants, viscosity enhancers in suspensions, emulsions, production of encapsulation devices, preparation of micro-beads, micro-particles, pharmaceutical packaging, etc. [41]. The innovative techniques employed in drug delivery currently have relied mainly on the applications of polymers in various ways. In this chapter, emphasis will be placed on the polymers that have found special applications in bio-adhesive drug delivery systems.

2.6.2 Bioadhesive/Mucoadhesive Polymers Mucoadhesive drug delivery systems can be adopted in most dosage form designs to enable prolonged retention at the site of application, providing

Bioadhesive Polymers for Drug Delivery  41 a controlled rate of drug release for improved therapeutic outcome. Mucus dehydration and polymer hydration at the same time can increase the cohesive property of the mucous layer which assists in the process of mucoadhesion. The capacity of the polymer to swell is the main influence causing chain flexibility of polymer and interpenetration between the mucin chains and polymer. Therefore, based on the type of dosage form in question, several polymers displaying various characteristics ought to be looked into [43-45]. Bio/mucoadhesive polymers could be water-soluble or water-insoluble. They constitute networks that can swell and could also be joined by crosslinking agents through processes such as wetting, adsorption and interpenetration of polymer and mucus. These polymers retain optimal polarity to ensure that they allow adequate wetting of the mucus and optimal fluidity that encourages the adsorption and interpenetration of the polymer and mucus to occur. Such polymers that adhere to the mucin/epithelial surface can be conveniently divided into three broad classes [13, 46, 47]: Polymers that become sticky when placed in an aqueous medium and can become bioadhesive. ii. Polymers that adhere through nonspecific, non-covalent interactions and are predominantly electrostatic in origin. iii. Polymers that interact with specific receptor sites on the cell surface. i.

2.6.3 Classification of Mucoadhesive Polymers The bioadhesive/mucoadhesive polymers could be grouped as follows [27, 28, 30, 46, 48–50]:

2.6.3.1 Classification Based on the Origin of the Polymer This category is further subdivided into: i. Natural polymers ii. Synthetic polymers

2.6.3.2 Classification Based on Aqueous Solubility of the Polymer This group includes the following: i. Water-soluble polymers ii. Water-insoluble polymers

42  Bioadhesives in Drug Delivery

2.6.3.3 Classification Based on the Type of Charge on the Polymer This class includes: i. Cationic ii. Anionic iii. Non-ionic

2.6.4 Natural Polymers This class includes chitosan, starch, gelatin, hyaluronic acid, gums (guar, xanthan, etc.) and collagen.

2.6.4.1 Chitosan Chitosan is a polysaccharide that is derived from N-acetyl glucosamine and glucosamine through the deacetylation of chitin found in shells of crustacean. Chitosan has –OH and –NH2  groups producing its ability to form hydrogen and covalent bonds which are essential for its muco­ adhesive characteristic. These functional groups also contribute to the solubility of chitosan macromolecules. It is insoluble at neutral or higher pH due to the -NH2 groups resulting from the deacetylation process. It also forms salts with various organic and inorganic acids. At low pH, the amino functional groups undergo protonation, making chitosan macromolecules positively charged. This cationic nature offers a robust electrostatic contact with negatively charged constituents of the mucus and the epithelial surfaces. Chitosan is biocompatible and naturally non-toxic [51]. Chitosan, being cheap, biodegradable and biocompatible, has been highly used as a pharmaceutical excipient in oral, ocular, nasal, parenteral, and transdermal drug delivery systems [52-54]. Chitosan and other substances derived from it have displayed outstanding mucoadhesive behaviour. It was established that coating micro- and nanoparticles with chitosan can enhance drug adsorption to mucosal surfaces, especially in drug delivery to the nasal surfaces [55]. In addition to its hydration in the nasal cavity, the interaction of the positively charged amino groups with the negatively charged sites on the mucosa surface also aids in their mucoadhesion [54, 56, 57]. Chitosan has chemical and biological characteristics of being a bioadhesive/mucoadhesive substance. Its capability to improve nasal absorption is based on the nature of its derivatives, molecular weight and amount of deacetylation. When the pH is neutral, a large number of chitosan molecules lose their charge and are precipitated from their solution because

Bioadhesive Polymers for Drug Delivery  43 chitosan is soluble in an acidic medium where the amino groups located at the C-2 are protonated [58]. Chitosan has been utilized in the transport of DNA due to its positive charge in a weakly acidic medium. It could as well be used to deliver a negatively charged DNA via nasal mucosa [59]. It has been very useful in drug delivery, exhibiting improved absorption of hydrophilic drugs possessing large molecules. Chitosan could serve as a good drug transporter for colon drug delivery because of its insolubility at pH less than 6.5. The mucoadhesive behaviour of chitosan is based on its structure which determines its extent of interaction with the mucin. Electrostatic binding is the major way of intermingling which occurs between the chitosan and mucin. Hydrogen bonding and hydrophobic interactions equally contribute to the mucoadhesion of chitosan. A drawback in the mucoadhesive usefulness of chitosan is its low aqueous solubility at neutral and basic pH. To improve the mucoadhesive behaviour of chitosan for controlled drug delivery, various alterations have been done in various ways to overcome the above drawbacks. The derivatives of chitosan include trimethyl chitosan, carboxymethyl chitosan, thiolated chitosan, chitosan-enzyme inhibitors, chitosan-ethylenediaminetetraacetic acid (chitosan-EDTA), half-acetylated chitosan, acrylated chitosan, glycol chitosan, chitosan-catechol, methyl pyrrolidinone-chitosan, cyclodextrinchitosan and oleoyl-quaternised chitosan [60, 61].

2.6.4.2 Starch Plants synthesize and store starch in their structure as an energy reserve which is entirely deposited in the form of small granules or cells with diameters between 1-100 μm. Starch is a polysaccharide and has been used in drug delivery systems to deliver mucoadhesive drugs [62]. It is useful in enhancing the absorption of both small hydrophobic and large hydrophilic drugs, being one of the most used biocompatible bio­ adhesive/mucoadhesive conveyors of drugs [63]. Since the starch can absorb moisture, it becomes easy for it to form a mucoadhesive gel-like structure which causes dehydration of the mucosal membrane and leads to the delivery of the drug moiety through a paracellular tight intersection [64]. Among the starches, maize starch is the most preferred as a pharmaceutical-grade excipient, especially the drum-dried grade since it has better bioadhesive behaviour [65]. Starch has been found useful in mucoadhesive drug delivery systems especially as a carrier in nasal drug delivery as microspheres, powders or nanoparticles. Such microspheres have been prepared by the process of emulsion polymerization which is achieved by cross-linking starch with epichlorohydrin. Due to the mucoadhesive property of the

44  Bioadhesives in Drug Delivery microspheres, these can improve drug absorption and extend the dwell time of medicinal substance [66, 67].

2.6.4.3 Gelatin Ordinarily, gelatin is a natural polymer that is soluble in water and is usually prepared by denaturation of collagen [68]. Its outstanding characteristics define it as a pharmaceutical and medical agent useful as a biodegradable and biocompatible polymer in drug delivery. It is a useful material for gene delivery, cell culture and lately tissue engineering [69-72]. Gelatin, being a polyelectrolyte, has a net charge which depends both on the pH and the kind of gelatin. There are many types of gelatins. Type A gelatin is obtained by acidic hydrolysis of collagen. This has an isoelectric point between 7.0 and 9.0. Type B gelatin is developed by alkaline hydrolysis having an isoelectric point between 4.7 and 5.3. It has been reported that aminated microspheres of gelatin retain a greater gastric mucoadhesion than gelatin microspheres used alone. A higher number of the amino groups indicates enhanced flexibility of the chain [73].

2.6.4.4 Alginates The poly (alginic acid) is ordinarily known as the alginate. It is a poly­ saccharide found in nature and is an example of a naturally occurring linear polysaccharide extracted from seaweed, algae, and bacteria [71, 74, 75]. The fundamental chemical structure of an alginate is constituted of (1–4)-β-D-mannuronic acid (M) and (1–4)-α-L-guluronic acid (G) units in the form of homopolymeric (MM- or GG-blocks) and heteropolymeric sequences (MG or GM-blocks) [76]. It is useful as a viscosity enhancer in liquid disperse systems, such as suspensions, creams, pastes, gels, etc. It belongs to the class of anionic mucoadhesive polymers which through carboxyl-hydroxyl interactions with mucin glycoproteins forms durable hydrogen bonds. It is a linear polysaccharide that is soluble in water and has been applied in various pharmaceutical and biotechnological fields. The alginate as well as its derivatives, e.g. the thiolated sodium alginate, have been used in mucoadhesive micro-beads for the management of periodontal ailments and extended drug release, all having been experiential in post-application which shows its usefulness in the management of periodontal disorders. The alginates are also useful in the preparation of micro-particles for drug delivery. Its ability to swell has been utilized in applying it for the management of wounds in the human body, especially

Bioadhesive Polymers for Drug Delivery  45 because of its biocompatibility [77, 78]. The alginates and their derivatives are widely used by many pharmaceutical scientists for drug delivery and tissue engineering applications due to their many properties such as biocompatibility, biodegradability, low toxicity, non-immunogenicity, water solubility, relatively low cost, gelling ability, stabilizing properties, and high viscosity in aqueous solutions [79].

2.6.4.5 Hyaluronic Acid The hyaluronic acid is anionic in nature and is found all over the epithelial, connective and neural tissues. Its presence constitutes a distinct aspect of the synovial fluid as it contributes to the high viscosity of the synovial fluid. With the decreasing molecular weight of the hyaluronic acid, muco­ adhesive behaviour is improved [80].

2.6.5 Synthetic Polymers These include cellulose derivatives. Examples of cellulose derivatives are carboxyl methyl cellulose, sodium carboxymethyl cellulose, poly (acrylic acid) based polymers (e.g. carbopol, polyacrylates, poly (ethylene glycol). Various cellulose derivatives and their by-product such as chitosan, hyaluronic acid, guar gum, etc. have been used in several mucoadhesive delivery systems. Polymers in this class have been evaluated for mucoadhesive drug delivery into the eye. Sodium carboxyl methyl cellulose (SCMC) has displayed exceptional ocular mucoadhesive property among all muco­ adhesive cellulose derivatives. Several experiments have shown that the surface dynamic characteristics of the cellulose and its derivatives help in their film-forming ability [81, 82].

2.6.5.1 Cellulose Derivatives Cellulose is a class of most available polysaccharides, containing 8000– 10,000 glucose residues linked by β-1, 4 glycosidic bonds [83]. A good number of cellulose derivatives are of pharmaceutical grade and have been extensively utilized in drug deliveries. These include soluble cellulose derivatives such as hydroxypropyl methyl cellulose (HPMC), methyl cellulose (MC), hydroxypropyl cellulose (HPC), and carboxy methyl cellulose (CMC). Others are the insoluble cellulose derivatives such as ethyl cellulose (EC) and microcrystalline cellulose (MCC). They show mucoadhesive behaviour and, therefore, are capable of offering extended dwell time [84].

46  Bioadhesives in Drug Delivery

2.6.5.2 Polyacrylates Polyacrylates have outstanding mucoadhesive and gel-forming abilities and, therefore, have been considered in various drug delivery routes, including the transdermal [85, 86], ocular [87], oral [88] and nasal drug delivery systems [89]. Some polyacrylates, such as the carbomers and polycarbophil, vary in the degree of cross-linking and viscosity and are extensively applied in nasal mucoadhesive drug delivery systems. Polyacrylates can attach to the mucosal surface, and can enhance the dwell time of the drugs [90].

2.6.5.3 Poly (ethylene glycol) (PEG) The PEG is a polymer with a mucoadhesive property that is non-toxic, non-immunogenic as well as being non-antigenic. It dissolves in an aqueous medium [91]. Its capacity to form hydrogen bonds with sugar on glyco­ sylated proteins forms the basis for its mucoadhesive behaviour [92, 93].

2.6.6 Classification Based on Aqueous Solubility of the Polymer 2.6.6.1 Water-Soluble Polymers Some of the polymers belonging to this class include carbopol, sodium carboxymethyl cellulose, sodium alginate. Carbomers (carbopol) are broadly utilized in controlled drug delivery systems and have been much exploited in oral mucoadhesive drug delivery due to their capability to form networks with the mucous glycoprotein [94]. Carbopol 934P has shown its mucoadhesive capability in the formulation of micro-beads of glipizide for its role in controlling blood sugar in diabetic patients. The mucoadhesion studies confirmed a firm adherence of the beads to the mucosal membrane at a pH of 1.2 for an extended time. Since carbopol 934P could maintain a mucoadhesive function, an extended-release of glipizide and a decrease of fasting blood sugar have been observed in the animal model to reasonable levels, making it easier to control diabetes using this novel product design [95].

2.6.6.2 Water-Insoluble Polymers The main examples in this category are chitosan, ethyl cellulose, polycarbophil.

Bioadhesive Polymers for Drug Delivery  47

2.6.7 Classification Based on the Type of Charge on the Polymer 2.6.7.1 Cationic Polymers Of all the mucoadhesive polymers that are cationic in origin, chitosan is prominent. Its studies have been very widespread and it has been useful in the formulation of various products in its pure or derived products. The distinctive characteristics of chitosan have made it very useful in various areas of application in the pharmaceutical industry as an antipathogenic agent, film-forming agent, as well as in cosmetics [96, 97].

2.6.7.2 Anionic Polymers These include chitosan, carbopol, pectin, sodium alginate, and xanthan gum. They are extremely utilized and are a very popular choice in the pharmaceutical sector. They show potential in bioadhesive/mucoadhesive drug delivery and exhibit a minimal degree of toxicity. Their ability to form very strong hydrogen bonds with the mucosal mucin provides their exceptional mucoadhesive behaviour [98]. Carboxymethyl cellulose is generally deployed for mucoadhesive drug delivery and is an example of an anionic polymer. Another good example is pectin. Pectin is a polysaccharide and anionic in nature. It is precisely a heteropolysaccharide which is mainly found in the major cell walls. Its structure contains carboxyl groups which offer it the mucoadhesive property and which enables it to mingle with the mucous membrane. When pectin is hydrated, it forms a hydrogel with an increased viscosity which enables mucoadhesion [99].

2.6.7.3 Non-Ionic Polymers This category includes hydroxyl ethyl starch, poly (vinyl alcohol), polyvinylpyrrolidone (PVP), hydroxypropyl methyl cellulose (HPMC), and methyl cellulose (MC). These are useful as mucoadhesive polymers [72, 100].

2.7 Prospects of Bioadhesive/Mucoadhesive Polymers in Bioadhesive Drug Delivery Over the past few decades, mucosal drug delivery has attracted considerable attention in applying the bioadhesive/mucoadhesive drug delivery systems with their great potential for controlled release drug delivery over

48  Bioadhesives in Drug Delivery a long period, allowing active absorption and better bioavailability of the drugs owing to a high surface to volume ratio and permitting an ample close contact with the mucous layer. The bioadhesive/mucoadhesive drug delivery systems have since been extended to several routes such as the buccal, oral, vaginal, rectal, nasal and ocular. With the entry of various innovative medicinal substances owing to drug discovery, bioadhesive/ mucoadhesive drug delivery will have an even more vital applications in delivering these novel molecules. Mucoadhesive-centered topical formulations have revealed superior bioavailability of drug products. It is envisaged that the mucosal drug transport system will be of significance in the delivery of various emerging high-molecular-weight molecules. Currently, researchers are thinking beyond conventional polymers, i.e. nextgeneration mucoadhesive polymers such as the lectins, thiols, etc. which are anticipated to provide better contact in retaining dosage forms. Eventually, these novel mucoadhesive formulations will necessitate considerable work to deliver clinically for the management of both topical and systemic diseases [18].

2.8 Summary Bioadhesion has been described as the attachment of a synthetic or biological macromolecule to an epithelial tissue for an extended time. It is important in bioadhesive drug delivery systems to achieve an elongated drug release and improved drug bioavailability. It is understood that in bioadhesive drug delivery, a polymeric drug carrier is attached to epithelial tissue. The term mucoadhesion has been accepted to be applicable when the polymeric drug carrier is rather attached to a mucous membrane. Several inner surfaces of the body lined with epithelial tissues or mucous membranes constitute potential sites for bonding of a bioadhesive drug carrier, and thus, have become important in advancing the approach using bioadhesive/mucoadhesive drug delivery systems. The bioadhesive or mucoadhesive ability of a dosage form relies on several factors, including the nature of the mucosal tissue and the physicochemical behaviour of the polymeric formulation. Such factors as the mechanism of bio­adhesion/mucoadhesion, nature of mucoadhesive polymers, factors affecting bio­adhesive/mucoadhesive polymers, and the classification of the bio­adhesive/mucoadhesive polymers are all important in achieving robust bioadhesive drug delivery.

Bioadhesive Polymers for Drug Delivery  49

Acknowledgements I am highly honoured and I consider it a very special privilege to be requested by Dr. Inderbir Singh, Professor, and Head of the Department of Pharmaceutics, Chitkara College of Pharmacy, Chitkara University, Punjab, India to contribute a chapter in this book. Thank you, Professor Inderbir Singh. I also thank the team of editors and all who envisioned this book. Above all, my mentor, Professor (Mrs) Oluwatoyin A. Odeku  of the Department of Pharmaceutics & Industrial Pharmacy, and the Dean, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria deserves special appreciation for all her motivation, counselling and guide, especially linking me to the Chitkara College of Pharmacy without which I would not have had the opportunity to be asked to contribute a chapter in this book.

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3 In Vitro, Ex Vivo and In Vivo Methods for Characterization of Bioadhesiveness of Drug Delivery Systems Ljiljana Djekic* and Martina Martinovic University of Belgrade – Faculty of Pharmacy, Department of Pharmaceutical Technology and Cosmetology, Vojvode Stepe 450, 11221 Belgrade, Serbia

Abstract

Bioadhesion to skin and mucous membranes is a key property of bioadhesive drug delivery systems (BDDS) that temporarily enables resistance to their separation. Development of BDDS (conventional as well as on micro- or nanoscale) for cutaneous, buccal, peroral, vaginal, nasal, and ocular application is a valuabe strategy for improving local or systemic therapeutic efficacy of the drugs (small molecules as well as biomacromolecules), in comparison to conventional pharmaceutical formulations. The mechanisms of bioadhesion/mucoadhesion are highly complex and not yet fully understood, so their characterization is a difficult task and has been performed by using various in vitro, ex vivo, and in vivo methods. The most commonly used in vitro/ex vivo tests include: bioadhesion strength tests (performed as vertical or horizontal detachment strength tests, or tests based on the quantification of adhered amount and/or time required for detachment of adhered sample) and methods for assessment of the mucoadhesive interactions at the molecular level (e.g., colloidal gold colouring method, fluorescence probe methods, rheological measurements, turbidimetry, low-frequency dielectric spectroscopy, zeta potential measurement, Biacore test, and atomic force microscopy (AFM)). The in vitro/ex vivo methods are economical, easy to perform, and thus are useful for screening of BDDS candidates for in vivo assessment. In vivo methods, such as radiolabelled BDDS transit studies, gamma scintigraphy, and in vivo detachment tests, are rarely used; however, they enable full characterization of this property, especially in the case of peroral BDDS, where it is affected by many biological variables. The growing interest in development of BDDS encourages consideration of new methods for testing this property. *Corresponding author: [email protected] K.L. Mittal, I. S. Bakshi and J. K. Narang (eds.) Bioadhesives in Drug Delivery, (57–98) © 2020 Scrivener Publishing LLC

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58  Bioadhesives in Drug Delivery Keywords:  Bioadhesion, mucoadhesion, bioadhesive drug delivery systems, in vitro/ex vivo tests, in vivo tests

3.1 Introduction Research interest in bioadhesive drug delivery systems (BDDS) for different routes of application has continuously increased since 1980s. Bioadhesion, as an umbrella term, represents attachment of a natural or synthetic adhesive polymeric material to the surface of a biological tissue, such as skin and mucous membranes, based on formation of attractive bonds that resist their separation during extended time period. When bioadhesion occurs with mucosal membranes (mucus and/or an epithelial surface) it is termed as mucoadhesion [1-3], and cytoadhesion was introduced as a more specific term for the cell-specific bioadhesion via highly specific receptor-mediated interactions [4]. BDDS for dermal, buccal, peroral, nasal, ocular, rectal or vaginal application provide an attractive strategy for improving local or systemic therapeutic efficacy of small molecules and biomacromolecules including proteins, peptides and oligonucleotides, in comparison to conventional pharmaceutical formulations [5-10]. The bioadhesion phenomenon is based on incorporation of bioadhesive excipients in pharmaceutical formulations and drug carriers. The common bioadhesive ingredients are polymers, either natural (chitosan, hyaluronic acid, alginate, carrageenan, gellan, xanthan gum, pectin, tragacanth, gelatin, starch), semi-synthetic (chitosan derivatives, cellulose derivatives), or synthetic (carbomer, polycarbophil, poly(vinyl alcohol), poly(vinyl acetate), polyamides, polycarbonates, poly(methacrylic acid), poly(methyl methacrylic acid), poly(alkyl cyanoacrylate)s, poly(alkylene glycol)s, poly(vinyl ether)s, macrogols, polylactides, polyglycolides, poly(lactide-co-glycolide)s, polyorthoesters, polyphosphoesters, polyanhydrides) [11, 12]. The biadhesive polymers enable localization and close contact of the drug with the site of application providing significant advantages, including: 1) reduced frequency of administration and improved patient’s compliance; 2) lower therapeutic concentration or dose of the drug, which reduces the risk of side effects; 3) maintenance of the therapeutic concentration of the drug substance in plasma in steady state without major fluctuations, thus achieving more effective control of the disease [7, 13-16]. Furthermore, certain BDDS comprising, for example, hyaluronic acid [17], xanthan gum [18], poloxamer 407 [19], sodium carboxymethyl cellulose [20], or carbopol [21], may undergo a phase change from a liquid to a semisolid upon application. In this case, liquid formulations provide efficient spreading and cover a

Characterization of Bioadhesiveness  59 large area while the viscosity enhancement may contribute to sustained drug release [22-24]. It has been found that some mucoadhesive polymers can delay gastrointestinal transit of BDDS, thereby prolonging the time for absorption, some may affect permeability of epithelial tissues by weakening of tight junctions, while some act as inhibitors of proteolytic enzymes [25, 26]. So far bioadhesiveness of many different BDDS (conventional as well as on micro- or nanoscale) has been characterized using various experimental approaches [27]. This comprehesive chapter discusses performances, advantages and shortcomings of in vitro, ex vivo, and in vivo methods that have been used so far for evaluation of bioadhesiveness of BDDS for various routes of administration.

3.2 Mechanisms of Bioadhesion Morphological and physiological properties of skin and mucous membranes significantly influence the mechanisms of bioadhesion. The main difference is that mucous membranes are not keratinized and produce mucus which prevents their drying. Therefore, they behave as hydrophilic substrates for adhesion [28, 29]. In contrast, stratum corneum (SC) is a relatively thin outer layer of skin, consisting of lipophilic and hydrophilic regions, as well as hair follicles and sweat glands. Normally, water content in SC is about 20%, mostly found in layers of keratin between the corneocytes. The mature skin comprises less water content and is less elastic, while the younger skin is more hydrated and flexible [30]. It was suggested that moisture and dirt increase skin surface free energy, while it is lower on clean and dry skin surface, which is thus more suitable for bioadhesion. Hair follicles and sweat glands increase hydration of the skin surface and affect the adhesion strength [31]. Also, it has been shown that the ability of SC to establish hydrogen bonds has an important role in the bioadhesion process [32]. The polarity of the intercellular region of SC, comprising ceramides, fatty acids and other intercellular lipids, is similar to butanol and is available for hydrogen bonding with BDDS [33, 34]. The skin bio­ adhesion mechanism is poorly investigated so far. Mucosal tissues cover the natural cavities of the body, providing the epithelial barriers to the outer environment. Type of epithelial layer (lamina propria or muscular mucosa), roughness of mucosal surface and creases, and presence or absence of carbohydrate molecules can affect the phenomenon of mucoadhesion [35]. The additional factor to consider is friction on the surface of the mucosa. The interaction of the bioadhesive formulation with mucosal membranes under friction can be increased, however, if the shear stress is too high (e.g., at the surface of the eyeball), the time for

60  Bioadhesives in Drug Delivery establishing adhesive bonds is too short [36]. The mucus acts as a physical barrier for chemical and biological agents, and at the same time, as a lubricant which prevents friction damage. It also plays an important role in homeostasis, particularly in regulation of the balance of water and transport of ions, removing the remains of dead cells, and regulation of mucous immunity [37]. Mucus predominantly consists of water (> 95%) which is embedded in a three-dimensional network of randomly connected mucins, a heterogeneous group of glycoproteins, which are directly involved in the phenomenon of mucoadhesion [38]. Mucus is a non-Newtonian, viscoelastic system with viscosity values of 10-103 Pa·s at low shear rates, but highly dependent on the specific structure, anatomical site and physiological or Table 3.1  Proposed theoretical considerations in mechanisms of mucoadhesion [4, 7, 11, 40, 42, 44-50]. Theory

Consideration

Wettability theory (also wetting theory)

Liquid or low viscosity mucoadhesive spreads, penetrates surface deformations, gets hardened and attaches due to a positive spreading coefficient of the adhesive and near-zero contact angle

Adsorption theory

Mucoadhesion is based on covalent (strong) and ionic, hydrogen, or van der Waals (weak) chemical bonding of the molecules of the two surfaces (the mucoadhesive and mucous membrane)

Electronic theory

Attractive bonding is based on electron transfer and formation of electronic double layer between the adhesive and the mucin network

Fracture theory

Adhesive bond strength is equal to the detachment force (i.e., force required for separation of the mucoadhesive from the mucosal surface)

Diffusion theory (also diffusioninterlocking theory)

Time-dependent diffusion of mucoadhesive polymer chains into the mucin chains network with similar solubility parameters to create a semi-permanent adhesive bond

Mechanical theory

Adhesion due to mechanical attachment of the mucoadhesive formulation over the mucous membrane due to filling of irregularities on a rough surface by a liquid mucoadhesive

Characterization of Bioadhesiveness  61 pathological conditions [9, 39]. The mucus on the surface of various mucous membranes varies in terms of pH, thickness, viscosity and rate of recovery, and these properties must be considered when formulating BDDS [40, 41]. In contrast to scarcely investigated mechanism of skin bioadhesion, several theories have been proposed to explain the mechanisms of mucoadhesion, namely wetting theory, adsorption theory, electronic theory, fracture theory, diffusion theory, and mechanical theory (Table 3.1); however, only a combined theoretically based approach allows adequate understanding of the mucoadhesion phenomenon, and different theories should be considered when describing different steps in the process of mucoadhesion. For example, the mucoadhesion mechanism may include initial wetting of mucin, spreading of the adhesive polymer onto mucus, and adhesion due to electron transfer, or adsorption phenomenon [8, 12, 40, 42, 43]. Table 3.2 lists the numerous factors that affect bioadhesive interactions and performaces of BDDS, including characteristics of the Table 3.2  The main factors affecting bioadhesive interactions between BDDS and mucous membranes [4, 11, 23, 43, 51-53]. Biological factors

pH of the biological membrane Mucin turnover rate Blinking, movement of the buccal tissues while eating, drinking, talking and sleeping, sexual stimulations Disease

Characteristics of the bioadhesive polymer

Molecular weight Flexibility of the polymer chains Spatial conformation Charge Hydrogen bonding capacity Hydration (swelling) Concentration pH of polymer

Characteristics of the drug delivery system

pH Presence of water phase Rheological behaviour Spreadability

Method of application

Initial pressure applied at contact place Contact time

Environment-related factors

Presence of water Contaminants

62  Bioadhesives in Drug Delivery biological membrane, the adhesive polymer, the drug delivery system, and the method of application and environment-related factors. The diversity of BDDS and routes of application as well as a high complexity of the bioadhesion phenomenon make development of methodology for bioadhesion characterization a very challenging task.

3.3 Bioadhesive Drug Delivery Systems (BDDS) Conventional solid, semisolid and liquid dosage forms for different routes of application can become bioadhesive by including appropriate excipients into their formulations. Also, the development of micro- and nanoparticulate BDDS for buccal, nasal, ocular and vaginal application has been the focus of numerous research studies [5, 7, 13-16, 22, 53-57].

3.3.1 BDDS for Cutaneous Application Bioadhesive hydrogels for cutaneous application are usually semisolid three-dimensional networks based on hydrophilic homopolymers or copolymers. They can also be formulated as in situ gelation liquids which form semisolid gels at the site of application, enhancing percutaneous drug delivery. For example, Takahashi and coworkers formulated bioadhesive gels in situ from gelling solutions of xyloglucan (partially decomposed β-galactosidase) and poloxamer 407 for transdermal delivery of nonsteroidal anti-inflammatory drugs ibuprofen and ketoprofen. Release of both drugs from the gels was prolonged for 12 h after the initial delay time, and bioavailability in rats was higher compared to the drug delivery system based only on poloxamer 407 [58]. Bioadhesive transdermal films, obtained from solutions or dispersions of film-forming polymers (e.g., ethyl cellulose, polyvinylpyrrolidone [59], poly(vinyl alcohol) [60], sodium carboxymethyl cellulose, chitosan [57]) and plasticizers (e.g., sorbitol [60], glycerol [57]), are flexible dosage forms intended for use on the skin to achieve systemic effect [61]. Plasticizers soften the rigidity of the film and increase the mobility of polymer chains by decreasing intermolecular forces, thereby improving their mechanical properties. Bioadhesive transdermal films become adhesive when applied to wet skin and they are usually formulated as water permeable (nonocclusive), thus overcoming the disadvantages of transdermal patches. Microemulsions, as thermodynamically stable and isotropic colloidal dispersions (nanodispersions), allow incorporation of bioadhesive molecules such as carbomers, polycarbophils, poloxamers, which can increase

Characterization of Bioadhesiveness  63 the contact time between the designed microemulsion hydrogels and mucous membrane or skin [62-64].

3.3.2 BDDS for Buccal Application Mucoadhesive oromucosal preparations are semisolids or solids prepared with hydrophilic polymers, which on wetting with the saliva form mucoadhesive hydrogel and/or slowly erode, dissolve or melt at body temperature, releasing the drug substance. Mucoadhesive buccal tablets can be coated with one or more films. Coating with water-insoluble excipients, such as ethyl cellulose and hydrogenated castor oil, except the surface that is in contact with the buccal mucosa, is suitable to achieve unidirectional drug release. The mucoadhesive buccal tablets are small, flat, and oval, to allow drinking and speaking without major discomfort. They soften, adhere to the mucosa, and are retained in position until they erode, dissolve, or melt. Buccal films are single-layer or multi-layer dosage forms which may include a backing layer to retard the diffusion of saliva into the drug layer, or a release liner. They adhere better to the mucosa and cause minor discomfort than buccal tablets, since they are ultra-thin and flexible. Other oromucosal mucoadhesive dosage forms, such as buccal hydrogels, ointments, pastes, and patches, also have been developed [23]. Mucoadhesive oromucosal hydrogels usually contain mucoadhesive polymers such as sodium carboxymethyl cellulose, carbomers, hyaluronic acid and xanthan gum, which increase viscosity and allow slow release of the drug substance by diffusion or erosion [8]. Hypromellose and hydroxypropyl cellulose have been used as ingredients of bioadhesive oromucosal ointments providing adhesion to the oral mucosa tissue for up to 8 h [23]. Buccal pastes may be based on liquid paraffin gelled with polyethylene or magnesium-stearate, with the addition of suitable mucoadhesive polymers (e.g., hypromellose, hydroxypropyl celullose, gelatin, pectin, sodium carboxymethyl cellulose, carbomers) [23, 65]. The advantage of semisolid dosage forms is good spreadability over the buccal mucosa, however the risk for drug dosing inaccuracy and removal by saliva is higher in comparison to buccal films, tablets and patches [23, 66]. Mucoadhesive buccal patches are similar to transdermal patches and thus consist of a backing nonpermeable layer (to prevent drug loss and deformation or disintegration of the drug delivery system during application) and the matrix type drug-loaded bioadhesive layer or the reservoir for controlled drug release with the surface that allows adhesion to the buccal mucosa. Recently, more complex mucoadhesive buccal films with embedded liposomes have been formulated to prolong the release and improve permeation of

64  Bioadhesives in Drug Delivery hydrophilic, low permeability vitamin B6. The vitamin is encapsulated into the liposomes containing soy lecithin and propylene glycol, which are coated with the films comprising sodium carboxymethyl cellulose and hypromellose [67].

3.3.3 BDDS for Peroral Application Over the last few decades, there have been many publications describing formulation and characterization of various mucoadhesive gastrointestinal dosage forms to improve local and systemic delivery of therapeutic compounds, particularly for the drugs with a narrow therapeutic window [12, 25, 44, 55]. Current research interest in designing of mucoadhesive peroral dosage forms (tablets, beads, microspheres, films, patches) is focused on combining mucoadhesion and floatation. El-Zahaby and coworkers [68] designed and optimized the gastroretentive levofloxacin floating minitablets-in-capsule system based on the matrix-forming hypromellose for controlled drug release (> 8 h) and with total floating time > 24 h. Floating mucoadhesive beads are small, solid, and free-flowing carriers containing dissolved or dispersed drug, mucoadhesive polymers (such as alginate, hypromellose, hydroxypropyl cellulose) and oil [69-71]. Gattani and colleagues [71] developed floating mucoadhesive beads based on alginate, hypromellose and liquid paraffin, with incorporated clarithromycin for treatment of stomach ulcers. The combination of the two polymers had better adhesive effect in the stomach compared with alginate calcium. Mucoadhesive microspheres (microparticles and microcapsules) enable enhancement of the gastric residence time, thereby significantly increasing bioavailability of amoxicillin [72] and cefpodoxime proxetil [73]. Srivalli and coworkers [74] designed a bilayered gastric mucoadhesive system with the drug and controlled release polymers in the upper layer and the mucoadhesive polymers (carbomer (Carbopol® 974P) and poly(ethylene oxide) (Polyox™)) in the lower layer, for localized and unidirectional release of lamotrigine. The gastrointestinal mucoadhesive patch system (GI-MAPS) has been developed by Eaimtrakarn and coworkers for oral delivery of macromolecules [55]. It consists of four layered films contained in an enteric capsule: the backing layer of a water-insoluble polymer (ethyl cellulose), the surface layer of the pH-sensitive polymer (hydroxypropylmethyl cellulose phthalate (HP-55) or methacrylic acid - methyl methacrylate copolymer (Eudragit® L100 or S100)) coated with the adhesive layer, and the middle drug-containing layer made of cellulose membrane. After dissolving the surface layer in the small intestine, the adhesive layer provides mucoadhesion, thus achieving the high concentration gradient of

Characterization of Bioadhesiveness  65 the model substance (fluorescein) between the patch and the enterocytes, and therefore a faster permeation. The GI-MAPS also has been formulated with granulocyte colony stimulation factor (G-CSF) as a protein model active substance for oral administration to beagle dogs. The increase in the number of leukocytes in dog blood, as the pharmacological effect of the applied protein, was observed [55]. Orally administered microparticles or nanoparticles can be immobilized at the intestinal surface or in mucus layer lining the mucosal surface. This will result in a delay in their gastric residence time. Mucoadhesive nanoparticles have a larger surface-to-volume ratio in comparison with the microparticles, thus signifying increase in the surface available to establish interactions with the biological membranes and longer retention of mucoadhesion [75]. Apart from increasing the available surface for interactions, the small size of the nanoparticles also enhances their ability to fit into aqueous cavities and channels in the mucus, and their further penetration and permeation. Therefore, adjusting an appropriate particle size is the important step in modeling the mucoadhesive behavior of nanoparticles for a specific pathway or pathophysiological conditions [36]. Although nanoparticles with diameters above 500 nm are unable to diffuse through the mucin mesh, smaller nonadhering nanoparticles (around 100 nm) in high concentrations can induce the collapse of mucin network and loss of barrier properties and the ability to retain mucoadhesives in situ [76].

3.3.4 BDDS for Vaginal Application Vaginal tablets are usually designed as fast disintegrating dosage forms which rapidly swell and spread as a dispersion or a gel along the vaginal mucosa or as monolithic matrix-type dosage forms with sustained release. Mucoadhesive vaginal tablets, besides usual excipients (diluents, binders, lubricants), comprise mucoadhesive polymers such as xanthan gum, hypromellose, hydroxypropyl celullose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, carbomer (Carbopol® 934P), and polycarbophyl, with the potential improvement in vaginal lumen retention for up to or even more than 24 h. Fast disintegration and controlled drug release from mucoadhesive vaginal tablets require effervescent disintegrants or superdisintegrants as well as optimisation of polymer content and preparation method [22]. Mucoadhesive vaginal pessaries comprise hydrophilic base (polyethylene glycol)s or lipophilic base (hard fats based on glycerides of saturated C12–C18 fatty acids) and one or more hydrophilic mucoadhesive polymers (usually sodium carboxymethyl cellulose or polyacrylates). The presence

66  Bioadhesives in Drug Delivery of mucoadhesive polymers keeps liquefied and dissolved or melted pessaries in the vaginal lumen, thus prolonging the contact of the drug and the vaginal epithelium. Some marketed mucoadhesive vaginal gels based on polycarbophil (Replens®, Miphil®) or hydroxyethyl cellulose (K-Y® jelly) are indicated in menopause and in cases of bacterial vaginosis as lubricants and for regulation of pH [77]. Mucoadhesive oil-in-water emulgels were investigated for vaginal delivery of poorly soluble contraceptives, hormones and antimicrobial drugs loaded in the oil phase (triglycerides, macrogol cetostearyl ether or other lipids and surfactants) and hydrated mucoadhesive polymers (e.g., hydroxyethyl celullose and sodium carboxymethyl cellulose, polyacrylates/polymethacrylates, chitosan, alginate, hyaluronic acid, pectin, tragacanth, and karaya gum) in the aqueous phase. Mucoadhesive gel based on carbomer (Carbopol® 974P) was used as vehicle for acyclovir loaded liposomes with suitable pH and viscosity for local treatment of genital herpes [78]. Berginc and coworkers [79] developed curcumin-loaded liposomes coated with chitosan and carbomer. Mucoadhesive polymers enabled significantly higher interactions of the liposomes with artificial vaginal mucus and isolated bovine mucus, compared to uncoated liposomes. Coating of liposomes with mucoadhesive polymers significantly improved curcumin permeability through artificial and bovine mucous membranes, compared with controls.

3.3.5 BDDS for Nasal Application Incorporation of mucoadhesive polymers into nasal BDDS allows prolonged retention at the site of administration, as well as enhanced nasal drug delivery. Shelke and coworkers [80] formulated thermoreversible nasal gel, in which carbomer (Carbopol® 934) was used as the mucoadhesive polymer, while poloxamer 407 provided thermoreversibility. In vitro drug release rate testing and ex vivo permeation studies of the nasal gel showed that Carbopol® 934, in addition to mucoadhesive property, also had the property of penetration enhancer, which enhanced bioavailability of naratriptan. Patel and associates [81] formulated microemulsions for nasal delivery of risperidone using 4% oleic acid, 30% surfactant mixture (Labrasol®: Cremophor® RH 40 (1:1)): (Transcutol® P) (3:1) and 66% aqueous phase, and in selected formulations incorporated polycarbophil as the mucoadhesive polymer. It was determined in vitro that the retention time of mucoadhesive microemulsion on the agar disk was significantly longer than the time of retention of the microemulsion itself. Sood and coworkers [82] optimized nanoemulsions and mucoadhesive

Characterization of Bioadhesiveness  67 nanoemulsions for intranasal delivery of curcumin using experimental design. Chitosan was used as the mucoadhesive polymer. It has been shown that the developed formulations are safe for intranasal administration. Ex vivo investigation of the drug diffusion from the formulations on the sheep nasal mucosa, using the Franz diffusion cell, indicated that the best permeation was achieved from the mucoadhesive nanoemulsions. Mucoadhesive hypromellose microparticles for nasal delivery of gentamicin sulphate were prepared by spray-drying technique. In vitro results showed that the mucoadhesive ability of the formulated microparticles was satisfactory and has increased with increasing polymer concentration [83]. Intranasal drug administration is receiving increased attention as a delivery method for bypassing the blood–brain barrier and rapidly targeting therapeutics to the brain, however, rapid mucociliary clearance in the nasal cavity is a major hurdle. Pathak and coworkers [84] optimized mucoadhesive microemulsion with a droplet size of 250 nm and zeta potential of −15  mV. In vitro and ex vivo permeation studies showed an initial burst of drug release at 30 min and sustained release up to 6 h, attributable to the presence of free drug entrapped in the mucoadhesive layer. In vivo pharmacokinetic studies in rats showed that the use of the mucoadhesive microemulsion enhanced brain and plasma concentrations of nimodipine.

3.3.6 BDDS for Ocular Application Prolonged retention of ocular BDDS at the site of application may enhance drug delivery into the deeper segments of eye [53]. Wu and coworkers [85] investigated the possibility of formulating eye drops as mucoadhesive in situ gelling solutions, in order to achieve prolonged drug delivery of flavonoid baykalin. Carbopol® 974P was used as a gelling agent, and hypromellose (HPMC E4M) as a viscosity enhancing agent. Rheological characterization showed that the sol-gel transformation occurred with increase in pH above 6. An extended drug release from these systems for 8 hours has been demonstrated. Mucoadhesive nanoparticles can be applied ocularly in the form of eye drops, ophthalmic inserts, or ophthalmic drug delivery systems of contact lens types [36]. Bhatta and associates [86] formulated mucoadhesive nanoparticles based on lecithin and chitosan for extended ocular delivery of natamycin. In vitro turbidimetric and zeta potential measurements results have shown that the formulated carriers can be used as an alternative to conventional pharmaceutical forms, such as eye drops with the suspended drug, since they allow prolonged release of the drug substance, thus extending the dosing interval.

68  Bioadhesives in Drug Delivery

3.4 Methods for Testing Bioadhesive Property of BDDS Despite a lack of generally established and accepted tests for assessment of bioadhesiveness, the relevant literature describes a great variety of in vitro/ ex vivo and in vivo methods that have been used for assessment of this property during development of different BDDS. Many of the described methods are also used for evaluation of mucoadhesion of polymer excipients. The choice of an appropriate method depends on the drug delivery system characteristics or the polymer considered.

3.4.1 In Vitro/Ex Vivo Tests The most commonly used in vitro/ex vivo tests for characterization of bioadhesiveness include: bioadhesion strength tests performed as vertical or horizontal detachment strength tests, bioadhesion tests based on the quantification of adhered amount and/or time required for detachment of adhered sample, as well as different methods for bioadhesion assessment at the molecular level (spectroscopic techniques, atomic force microscopy, rheological measurements etc.). In vitro/ex vivo tests are in most cases relatively simple, fast and repetable and thus are suitable for screening of a BDDS candidate with potential clinical relevance as well as excipients suitable for design of bioadhesive formulations. Moreover, a recent example of a simplified method for early screening of bioadhesive formulations was reported by Djekic and coworkers [87]. In this study biocompatible hydrogels for percutaneous delivery of ibuprofen were formulated with different bioadhesive polymers and compared by evaluating their miscibility rate with artificial sweat as an indicator of bioadhesiveness.

3.4.1.1 Bioadhesion Strength Tests Mechanical tests for quantification of strength of adhesive interactions between an investigated BDDS and an arteficial substrate or a natural substrate (mucin or mucous membrane) upon their contact are the most commonly employed in vitro/ex vivo methods for testing a wide range of solid and semisolid dosage forms (e.g., tablets, films, hydrogels, pastes) and mucoadhesive polymers. Adhesion strength can be determined either from adhesion between the BDDS and the substrate or indirectly derived from their detachment after previous contact. Adhesion strength tests measure mass, time, or force required to detach the BDDS from the substrate and/or the amount of adhered (or non-adhered) BDDS on the substrate.

Characterization of Bioadhesiveness  69 The mucus is usually of animal origin, for example crude pig gastric mucus in a form of a disc. The membrane is a mucosal epithelium, a piece of the animal mucosa (mainly pig nasal mucosa or rat intestinal mucosa), excised cow vagina or porcine vaginal mucosa. The key factors affecting accuracy and reproducibility of such measurements are: test medium; nature, thickness, keratinization, and intercellular lipid nature of the mucosa; presence or absence of mucus; experimental conditions including contact time, detachment rate of BDDS from the substrate/membrane, state of hydration of the polymer prior to contact with the substrate, volume and time of hydration of solid systems prior to the measurement, pH, and ionic strength of the hydration medium. In order to reduce variability in the results, the usage of commercial mucin is recommended [22, 25, 88-92].

3.4.1.1.1 In Vitro Detachment Strength Tests

Deatchment tests allow measurement of force required to detach the BDDS from the biological substrate as a function of the displacement occurring at their interface. The maximum force of detachment (Fmax) (directly measured) and the work of adhesion (Wad) (calculated as the area under the force vs displacement curve) are the parameters used to estimate mucoadhesive potential. Although frequently employed, the important constraint of such tests in many cases is whether the detachment is a result of adhesion failure at the BDDS/substrate interface or cohesive failure within the BDDS (i.e., a loss of cohesion between mucoadhesive polymer molecules). In the latter case, the correlation of the results obtained and bioadhesive performance of the BDDS is poor [22]. The test can be performed in such a way that the separation force is applied vertically (vertical detachment strength tests) or horizontally (horizontal detachment strength tests).

3.4.1.1.1.1 Vertical Detachment Strength Tests

The most common detachment strength tests are based on vertically applied detachment force by using a modified balance, a tensile apparatus, a dynamic contact angle analyzer, a texture analysis apparatus, or an electromagnetic force transducer device. Adaptations of the modified balance test differ from study to study. Pendekal and Tegginamat [93] described evaluation of mucoadhesive buccal patches by measuring the detahcment force using a modified balance. The sheep buccal mucosa was placed over the open mouth of the glass vial filled with phosphate buffer (pH 6.8), while the patch was fixed to the lower side of a rubber stopper. The pans of the balance were balanced and then the patch was allowed to contact the mucosa by removing the weight on

70  Bioadhesives in Drug Delivery the other side. The weight required to detach the patch from the mucous membrane is determined by gradually adding weights to the right side until the detachment takes place (Figure 3.1). A similar method was described in the study of mucoadhesive buccal films [94]. In this method, the film is fixed to the base of a glass beaker, which is placed on a mobile platform (Figure 3.2). The flat polypropylene stopper (substrate) is placed above the disk and counterbalanced with a plastic container. The beaker is then slowly raised until the film contacts the substrate. A weight of 50 g is placed into the stopper and left for 6 minutes to form adhesive bonds. After that the weight is removed and the plastic container is gradually filled with water using a peristaltic pump at a constant rate of 90 mg per second. The weight of water required to detach the two surfaces is recorded (Figure 3.2). The water collected in the container was measured and expressed as weight (gram force) required for the detachment, using the following equation:



detachment force (dyn/cm2) = [m · g/A],

where m - weight of water required to detach the two surfaces (g), (g) gravitational acceleration (980 cm/s2), and A - the area of tissue exposed (cm2) [95, 96]. Varma and coworkers [97] have investigated the bioadhesive property of the calcipotriol emulgel. The right-hand scale is replaced with a 100 ml

Thread

Rubber stopper

Beaker Patch

Weight

Phosphate buffer

Vial

Sheep buccal mucosa

Figure 3.1  Modified balance used to measure mucoadhesive strength (from reference [94], with permission).

Characterization of Bioadhesiveness  71

Polypropylene stopper Glass beaker Peristaltic pump

Film Preload weight

Plastic container

Moving platform

Figure 3.2  Modified two-arm balance to measure the tensile strength (from reference [94], with permission).

container, while on the left side a glass plate was attached (Figure 3.3). To balance, a weight of 20 g is placed above the glass plate. The second glass plate is placed beneath the tilted plate. The cuts of the rat skin, from which the hair was removed, are attached to both plates. The sample of the examined emulgel is placed between the two cuts of the skin. A small pressure is applied in order to establish bioadhesive bonds between the emulgel and the rat skin, and then gradually water is added into the bowl until the rat skin and emulgel are separated. The volume of water is converted into mass, and the bioadhesiveness of the gel expressed in grams. Different devices based on modified Wilhelmy plate method are used to measure detachment force. In such cases, the force acting on a vertically immersed plate is measured. Mathiowitz and associates [98] used a device containing microtensiometer and microbalances for estimating mucoadhesiveness of solid BDDS. The BDDS is attached to the solid support which is connected to a torsion balance, and lowered into the dispersion of mucus and left for a predetermined period to allow adhesion. After that, the solid support is raised at a constant rate until the maximum weight required for detachment is recorded. In a related study, the mucous membrane is placed in a small mobile chamber in which the pH and temperature are controlled. The BDDS is attached to the stationary microbalance. The chamber with the mucous membrane is raised until it contacts the BDDS and, after a

72  Bioadhesives in Drug Delivery

20 g Weight

Glass slide with rat skin on lower side

Beaker Water

Glass slide with rat skin on upper side

Figure 3.3  Schematic diagram illustrating bioadhesion measurement by the modified balance method (from reference [97], with permission).

certain time of contact, returns to its initial position. The advantages of this method are: simulation of physiological conditions (natural substrate, pH, temperature), mucoadhesion analysis at microscopic level, good reproducibility and sensitivity. Shitrit and Bianco-Peled [99] used Lloyd tensile testing machine (Ametek, USA) for estimation of bioadhesive property of chitosan and its derivatives (chitosan conjugated to poly(ethylene glycol) diacrylate and thiolated chitosan) by the tensile test and rotating system involving detachment of polymer tablets from a fresh intestine sample. A fresh small intestine surface was attached to a stainless steel grid. This grid was then fixed to the lower arm of the tensile apparatus. The investigated polymers were firmly attached to a small stainless steel grid, by the cross-linking reaction resulting in hardening of the polymer solution. The grid was fixed to the upper arm of the tensile apparatus via a long stainless steel rod. The tablet and the mucosa were placed in contact with a given pressure for a given time. In order to measure the maximum adhesion force, the machine was set to an extension mode where the upper arm was pulled at a constant rate of 1 mm/min and the force was recorded when detachment occurred. The detachment tests performed using a tensile apparatus allow the determination of the maximal detachment force and also calculation of parameters such as the work of bioadhesion (the area under the detachment force/displacement curve) and the fracture energy (ratio of work of bioadhesion to initial interface area between the tablet and mucosa). In other words, the work of bioadhesion is the detachment

Characterization of Bioadhesiveness  73 force as a function of elongation of the joint constituted by the interpenetrated bioadhesive polymer and mucin chains. The Thermo Cahn Dynamic Contact Angle Analyzer (CAHN DCA Analyzer) (Cahn Instruments, Cerritos, CA, USA) is an instrument for measurement of the surface properties of solid and liquid BDDS using the Wilhelmy plate technique. It consists of a highly sensitive balance, a moving stage mechanism, and a control station. Vasir and coworkers [4] have used CAHN DCA analyzer for evaluation of mucoadhesive microparticles. For the purposes of this test, the analyzer is modified to be able to measure the adhesion microstrength. The instrument measures the force of mucoadhesion between the mucous membrane and the mucoadhesive microparticle mounted on a small-diameter metal wire. Mucous membrane, usually rat intestinum, is placed in a chamber filled with Dulbecco phosphate buffer containing 100 mg/dl of glucose, and whose temperature corresponds to physiological. The chamber is located on the mobile platform, which is raised until the mucous membrane contacts the microparticle. After 7 minutes, the mobile platform is lowered and the resulting force of adhesion between the microparticle and the mucous membrane is recorded as a plot of the load on microsphere versus mobile platform distance or deformation. The CAHN software can analyze the 3 parameters of bioadhesion: resistance to fracture, deformation before release, and the work of bioadhesion [4]. In the methods for assessing bioadhesiveness, the detachment strength is most frequently analyzed using the texture analyzer type devices [13, 88, 89]. Jaipal and coworkers [100] performed in vitro bioadhesion study of the designed buccal discs comprising buspiron chloride, using texture analyzer (Stable Micro Systems TA-XT Plus, UK). The thawed excised porcine buccal mucosa was fixed at the base of the instrument in temperature controlled bath containing simulated salivary fluid (pH 6.75). The buccal disc was attached to the base of the texture analyzer movable probe. The probe was lowered at a speed of 0.5 mm/s. Upon contact of buccal disc with mucosal membrane a contact force of 0.01 N was applied for 300 s. The probe was then dragged in opposite direction and the force required to detach the buccal disc from the mucosal surface was recorded. Although this method is most often applied to test solid pharmaceutical formulations, such as microparticles [101], there are cases where mucoadhesive semisolid formulations and dermal patches have been examined [13, 88, 89, 102]. Bruschi and associates [89] examined the mucoadhesiveness of gels (comprising propolis extract) based on poloxamer 407 and carbomer (Carbopol® 934P), by measuring the force necessary to detach the formulation from the mucin disc by the texture analyzer. Mucin disc was obtained

74  Bioadhesives in Drug Delivery by compressing 250 mg of pig mucosa. The disc is attached to the bottom end of the cylindrical probe and hydrated by immersion in a 5% dispersion of mucus for 30 seconds prior to testing. The test was carried out at 37 °C. The sample of each of the formulations is placed in a cylindrical container, below the probe, which is then lowered until the mucin disc contacts the gel sample. A force of 0.1 N for 30 seconds was applied to allow close contact between the disc and the formulation. The probe is then lifted at a constant speed of 1 mm/s and the force required for separation of the disc from the surface of the formulation was determined. Woolfson and associates [102] formulated a new type of dermal patch with tetracaine in order to achieve effective percutaneous anesthesia. The gels prepared with different concentrations of bioadhesive polymer, viscosity enhancer and tetracaine were poured onto the membrane (that controlled drug release) and dried, and an outer cover layer was placed over the dried gel. The bioadhesiveness of the formulated patch was tested using the method based on the rupture tensile strength measurement using the texture analyzer. The test patch, with one side pre-wetted with 100 μl water for 60 seconds, is placed at the end of the analyzer probe. Adhesion of the wetted side of the patch on a piece of neonate porcine skin is measured at a probe speed of 0.2 mm/s, with a force of 2.5 N applied for 30 seconds. Adhesion is expressed as the force required to detach the patch from the skin surface. Vasir and coworkers [4] also describe testing of mucoadhesiveness of microparticles with an electromagnetic force transducer device. The transducer uses a calibrated electromagnet to separate the magnetically-attached mucoadhesive carrier from the tissue sample. It allows measurement of the adhesion force in terms of the magnetic force necessary to counteract the force of mucoadhesion. First, the microparticle and the mucous membrane contact was established. The magnetic force was then produced by means of an electromagnet placed vertically above the chamber with the mucous membrane. After the computer calculates the position of the microparticle, the tissue chamber slowly descends and moves away from the magnet and the video analysis continuously calculates the position of microsphere until the latter is completely pulled free of the mucous membrane. The results can be graphically represented as the curve of the dependence of the adhesion force with the distance. The advantage of this instrument is that it requires a physical connection of the transducer and microparticle. This enables precise determination of the mucoadhesive property of fine microparticles, which have been implanted in vivo and then excised (along with the host tissue) for measurement. This method is suitable for assessment of the bioadhesion of the polymers to the specific cells and can, therefore, be used to develop BDDS for target-specific application to various mucous membranes [4].

Characterization of Bioadhesiveness  75

3.4.1.1.1.2 Horizontal Detachment Strength Tests

Horizontal detachment strength tests are based on measurement of shear strength which leads to slippage of the mucoadhesive sample with respect to the mucus layer in the direction parallel to their contact surface. In the study of Roy and Prabhakar [103] two glass plates coated with the tested muco­adhesive sample and a thin mucus film were used. The literature also describes the method using a mucoadhesive polymer dispersion, liquid or semi-solid mucoadhesive materials (e.g. gel-forming cutaneous solutions and hydrogels) between two glass plates. The weight of 100 g is set on the upper plate to allow uniform distribution of the polymer dispersion between the two plates. One plate is fixed, and the other one is connected to the weight via the thread. After 15, 30 and 60 minutes, the weight was increased until reaching the value necessary for the separation of the two plates [20, 104]. A similar method was used by Shinde and associates [105] for estimation of mucoadhesiveness of niacin tablets formulated with various mucoadhesive polymers. In the tablets, a very small opening is made, through which the thread is pulled, and wrapped around the tablet. The other end of the thread is connected to a container hooked under the burette. The length of the thread is such that the tablet is placed in the middle of the isolated intestinal mucosa. After the tablet is placed on the mucous membrane, it is gently pressed against it by a tweezer, then left to stand for 30 minutes. Then the water is gradually dropped from the burette into the container. Based on the amount of water needed to separate the tablet from intestinal mucosa, the force needed to break the mucoadhesive bonds is calculated. The force in newtons is calculated using the following formula:



F = 0.00981 W/2,

where W is the amount of water in grams. Zhao and associates [106] formulated hydrogels based on deacetylated and carboxymethylated chitin. Bioadhesion was tested ex vivo on porcine dermis. An aluminum plate was placed over the epidermis using cyano­acrylate adhesive. A sample of the investigated hydrogel was fixed to the poly(ethylene terephthalate) film using cyanoacrylate adhesive. The amount of the adhesive applied was carefully controlled to avoid diffusion in the deep bulk region of the hydrogel. The surfaces of the hydrogel and dermis were contacted using a pressure of 0.1 kPa applied in the direction normal to their interaction for 1 minute. The tensile shear force required to detach the hydrogel from the tissue was measured using a tensile tester (Autograph SD-100, Shimadzu, Japan) at a constant crosshead speed of 5 mm/min (Figure 3.4).

76  Bioadhesives in Drug Delivery Polyester film

15

m

m

Al plate

hydrogel

Pig dermis 15 mm

Figure 3.4  Schematic representation of bioadhesion testing by measuring the tensile shear force (from reference [106], with permission).

3.4.1.1.2 Bioadhesion Tests Based on Quantification of Adhered Amount and/or Time Required for Detachment of Adhered BDDS

Among the in vitro/ex vivo methods, the relevant literature has described quantification of the amount of adhered or non-adhered mucoadhesive sample fraction on excised intestinal mucosa or mucin film placed on a support. The amount of non-adhered formulation can be recorded as a function of time. Smart and coworkers [107] have used the goat intestinal mucosa to test the mucoadhesiveness of polymer granules. The small intestine is cut lengthwise and a separate part is placed in a Plexiglas halfcylinder and washed with saline for 30 minutes at a flow rate of 30 ml/min. Then, 25 mucoadhesive polymer granules (N0) were hydrated with a small amount of water and sprinkled on the surface of the mucosa sample. After 20 minutes, during which mucoadhesive bonds between the granules on the mucous membrane were established, the system was washed with phosphate buffer of pH 7.2 for 20 minutes, at a rate of 22 ml/min. The remaining granules related to the mucous membrane (Ns) were counted. The strength of adhesive bond was determined using the following equation:



Strength of the adhesive bond (%) = Ns/N0 × 100

Nielsen and associates [108] applied flushing test (also washability test) in which the mucus membrane (jejunums of the rabbits, porcine ileum, stomach, and buccal mucosa) is placed in a longitudinally cut stainless steel cylindrical tube with the mucosa layer facing upwards (Figure 3.5). The support with the tissue was tilted at an angle of 21° in a cylindrical cell thermostated at 37°C. An accurately weighed amount of pellets of glyceryl monooleate to be tested for mucoadhesiveness (about 125 mg) was

Characterization of Bioadhesiveness  77 1. 2.

5

3

3. 4.

4 6

5.

2

37°C

6.

Thermostatic water flow (40°C) Reservoir containing the washing solution (37°C) Peristaltic pump Stainles steel support Biological membrane Receiver for collecting the washings

1

Figure 3.5  Schematic illustration of in vitro testing of bioadhesion (from reference [108], with permission).

spread evenly over the jejunal mucosa. About 0.5 ml of the buffer solution was dripped in an even layer onto the pellets to ensure hydration and after 10 min the segments were evenly flushed with the washing solution at a constant flow rate of 10 ml/min for 30 minutes. The effluent was collected into a beaker. The amount of the pellets remaining in the tissue was determined by HPLC. Validation of the method showed that the type of the mucous membrane did not affect the results. A similar principle for assessment of bioadhesion of a more complex vesicular BDDS was applied by Berginc and coworkers [79]. They prepared curcumin-containing liposomes coated with natural and synthetic bioadhesive polymers (chitosan and carbomer (Carbopol® 974P)) for vaginal administration. The strength of bioadhesion of the liposomes was evaluated using the isolated bovine mucosa. The tissue was divided into smaller pieces, which were glued to the cork backing by cyano­ acrylate glue and placed on a permeable membrane as a support. Ringer buffer (pH 7.4) with glucose was used to maintain tissue vitality and integrity during the experiment. Concentrated liposome samples were applied on the tissue and incubated at 37 °C in a plate shaker (300 rpm) protected from the light. After 3 h, tissue inserts were rinsed with 10 ml of simulated vaginal fluid containing human serum albumin and the concentration of curcumin in the rinsed samples was determined. The percent of adhered curcumin was calculated by subtracting the amount of curcumin in the washed samples from the initial curcumin amount in the liposomes and normalized to initial donor curcumin amount. Both curcumin solution and non-coated curcumin liposomes served as controls. Polymer coating of liposomes resulted in an increase in their bioadhesiveness.

78  Bioadhesives in Drug Delivery The number of particles that remain adhered to the mucous membrane after rinsing can be determined using the particle counter [101], the turbidimetric method, after suitable modification of the particulate test sample surface (e.g. fluorescent nanoparticles are available for localisation on the mucosa), or the Fourier transform infrared spectroscopy/attenuated total reflection (FTIR/ATR) method for direct quantification of adsorbed nanoparticles on rat intestinal mucosa [92]. When examining semi-solid preparations, the amount of non-adhering fraction can be quantified using the HPLC method [108]. In the study of Sun and coworkers [109] quantification of the amount of the adhered chitosan based polyelectrolyte complexes (PECs) was performed by dynamic light scattering. PECs formed between chitosan derivatives and enoxaparin were prepared by a self-assembly process and their bioadhesion was characterized ex vivo on rat intestinal mucosa. Fresh small intestine (jejunum) of sacrificed male Wistar rats was excised, rinsed with physiological saline and cut into segments of 4 cm length. Each segment was opened lengthwise along the mesentery and immersed into 0.5 ml of freshly prepared solutions of enoxaparin PEC, and then incubated for 2 hours at 37 °C. The bioadhesiveness of different PECs was characterized by the corresponding reduction in the PEC particles mobility at the intestinal surface by adhesion and decrease in intensity of the light scattering signal measured by dynamic light scattering with Malvern Zetasizer 4 (Malvern Instruments, UK). Santos and associates [110] described the everted gut sac technique for evaluation of the mucoadhesiveness of several microspheres. The microspheres comprise poly(caprolactone) and poly(fumaric-co-sebacic anhydride) at different ratios. The mucoadhesiveness was determined by placing of segments of the rat small intestine in test tubes with the microspheres. After mixing and a certain incubation period, the intestine segments were removed, and mucoadhesion was determined from a decrease in the mass of the examined mucoadhesive. Moreover, this passive adhesion test was compared with the CAHN microbalance method which employs a CAHN dynamic contact angle analyzer with modified software to record the tensile force for a single polymer microsphere pulled from the intestinal tissue. This study demonstrates that CAHN and everted sac experiments yield similar results when used to quantify the bioadhesive nature of polymer microsphere systems, so each method alone is a valuable indicator of bioadhesion. The dynamic perfusion techniques assess the duration of adhesion and simulate the detachment of the BDDS. The early study of Lehr and coworkers [111] described a rat ileal loop model (Figure 3.6) to study the

Characterization of Bioadhesiveness  79 Rat in restraining cage

Small catheter

Injector

Fraction collection

Reservoir

Pump

Waterbath for heat-exchanger

Figure 3.6  Experimental set-up to study the intestinal transit of microspheres in a rat ileal loop model (from reference [111], with permission).

intestinal transit of the multiple-unit bioadhesive microspheres of poly (2-hydroxyethyl methacrylate) coated with mucoadhesive polymers (carbomer (Carbopol® 974P), polycarbophil (Carbopol® EX-55 resin), and blends with Eudragit® RL 100). The diameter of microspheres ranged from 315–400 mm. An intestinal segment of approximately 6-8 cm (about 15 cm proximal to the ileo-caecal junction) was isolated with intact blood supply and the loop remained in the peritoneal cavity. After recovery from the operation (2-4 days) the animal was ready for use in perfusion experiments. The loop was perfused with isotonic saline (37 °C, pH 7.2-7.4) with a constant flow of 1.0 ml/min. About 50 microspheres were injected with a small Teflon catheter without interrupting the perfusion. Fractions of the perfusate were collected over a period of 6 hours at intervals of 5 minutes. The particles in the fractions were counted and at the end of the experiment the last particles were removed by flushing the loop. Bioadhesive property was evaluated by recording the mean residence time of the microspheres into the in situ perfused gut segment. The residence time of carbomer-coated microspheres was comparable to the non-coated controls, whereas polycarbophil-coated microspheres initially showed strong bioadhesion. The number of particulate BDDS such as microspheres in effluents collected after perfusion through the isolated intestinal segment over a period of time can be determined by a Coulter Counter, and thus the fraction

80  Bioadhesives in Drug Delivery of particles remaining in the intestinal loop can be calculated. The perfusion technique for a radiolabelled bioadhesive formulation injected with a syringe into the entire segment of intestine allows assessment of radioactivity of the eluted fractions to quantify the adhered amount of BDDS as a function of time. Perfusion tests are more realistic for evaluation of bioadhesive performance of BDDS as well as mimic various factors that influence retention of BDDS on a mucosal tissue following in vivo application, particularly the flux of a physiological fluid (GIT fluids or nasal, eye and vaginal secretions). The major disadvantages of such tests are: complicated and time consuming surgery for the tissue isolation; the amount of mucus that remains on the mucous membrane after rinsing with saline prior to perfusion is not well known; difficulties to achieve uniform flow of the test material over the tissue; the risk of capture of the tested particles within folds of mucous membranes, as well as aggregation within mucus [22, 44, 53, 54, 99]. For assessment of duration of mucoadhesion in a hydrated environment, as it is more relevant, various in-house apparatuses have been proposed. Grabovac and coworkers [51] investigated mucoadhesiveness of nineteen different polymers (thiolated polymers, polyacrylates, natural polysaccharides, cellulose derivatives, polyvinylpyrrolidone and poly(ethylenglycol)) compressed into discs and adhered to the porcine small intestinal mucosa, by measuring the adhesion time (i.e., time needed for the disc detachment from the mucosa). The discs were brought into contact with the mucous membrane for adhesion, after which they were placed in the basket of the test apparatus and immersed in phosphate buffer (pH 6.8) at 37 °C. The basket rotation speed was set to 125 rpm. Changes in the basket were observed and recorded every 30 minutes to determine the time required for disintegration or separation of disc from the mucous membrane. The described method was suggested to be suitable for testing different solid BDDS. A rotating paddle apparatus was also proposed as suitable for assessment of duration of mucoadhesiveness. As demonstrated by Bhavin and coworkers [112], the modified apparatus was used to measure the adhesion strength of buccal mucoadhesive tablets with nifedipine formulated with carbomer (Carbopol® P934), hypromellose (HPMC K4M), carboxy methyl cellulose (CMC), and tamarind seed polysaccharide. The tablet was pressed for 30 seconds onto fresh goat buccal mucosa fixed to a glass plate. The prepared tablet-mucosa-plate sample was immersed in 750 ml phosphate buffer (pH 6.2) at 37 °C. The paddle was placed 5 cm above the tablet, and the speed of rotation was 25 rpm. The time required for complete erosion or detachment of the tablet from the mucosa was recorded, and the best mucoadhesive performance was observed for the tablet containg carbomer

Characterization of Bioadhesiveness  81 and tamarind seed polysaccharide in a ratio of 1:1. Agaiah and coworkers [113] prepared six different formulations of mucoadhesive buccal tablets of simvastatin containing the polymers in various combinations and characterized their mucoadhesion time. The mucoadhesive side of each tablet was wetted with two drop of phosphate buffer of pH 6.8 and pasted by applying a light force with a fingertip for 30 seconds to the freshly cut sheep buccal mucosa attached to the glass slide. The tablet-mucosa-glass slide sample was then placed in a beaker, which was filled with 200 ml of the phosphate buffer pH 6.8 and kept at 37 °C. After 2 minutes, stirring was applied slowly to stimulate the buccal cavity environment, and tablet adhesion was monitored for 7 hours. The time for the tablet to detach from the sheep buccal mucosa was recorded as the mucoadhesion time. Optimization of mucoadhesive formulations can be performed in relation to a predetermined adhesion time, e.g. 6 hours.

3.4.1.2 In Vitro Methods for Characterization of Bioadhesion at the Molecular Level The methods especially suitable for characterization of mucoadhesive nanoscale BDDS are based on estimation of the extent of their interactions with mucin micro- or nanoparticles or mucin chains in solution, upon mixing and incubation for predetermined time periods. The degree of bioadhesion in such mixtures can be measured by colloidal gold colouring method, fluorescence probe methods, rheological measurements, turbidimetry, low-frequency dielectric spectroscopy, zeta potential measurement, Biacore test, and atomic force microscopy (AFM).

3.4.1.2.1 Colloidal Gold Colouring Method

In vitro method of colouring the mucoadhesive sample by a dispersion of red colloidal gold particles stabilized by adsorption of mucin molecules on their surface (mucin-gold conjugate) was proposed by Park [114] for assessment of mucoadhesiveness of the hydrogels of acrylic acid/acrylamide copolymers formed as strips or discs. After incubation of the strips/discs with the mucin-gold conjugate, they were gently washed and poured into the buffer solution. The strength of mucoadhesive interactions between the conjugate and the hydrogels can be estimated spectrophotometrically from the intensity of the red color. The hydrogel sample in the buffer solution that was not exposed to the dispersion of the mucin-gold conjugate was used as a blank. The test gave precise and reproducible results, although the absorbance value was relatively small. Alternatively, the concentration of

82  Bioadhesives in Drug Delivery the mucine-gold conjugate can be measured after removal of the hydrogel strips/discs. In the latter case, a decrease in the absorbance relative to the initial value measured before adding the hydrogel was recorded.

3.4.1.2.2 Fluorescence Probe Methods

The methods for mucoadhesiveness assessment based on the fluorescence probe are suitable for characterization of the nanoscale BDDS as well as the bioadhesive polymer solutions [92]. The early study of Park and Robinson [115] evaluated the interaction of copolymers of acrylate and acrylamide with the membrane of conjunctival epithelial cells with membrane lipids and proteins labeled with pyrene and fluorescein isothiocyanate, respectively. The study was carried out to specify the characteristics of the polymer structure necessary for mucoadhesion, with the aim to design mucoadhesive formulations for oral administration. The cells were mixed with the potential mucoadhesive, and the changes in the fluorescence spectrum were observed. The results highlight the binding of the copolymer molecules as well as the effect of polymer binding on its adhesiveness. Takeuchi and coworkers [116] used fluorophotometry to determine the amount of fluorescence-labeled nanoparticles coated with mucoadhesive polymers that adhered to the everted rat intestinal sac. Prior to measurement, the everted rat intestinal sac was immersed in a suspension of fluorescencelabeled nanospheres and gently agitated for 15 minutes. Additionally, fluorophotometry was used to determine the amount of nanospheres adhered to mucin particles when the suspension of the fluorescence-labeled nanoparticles (0.01 w/v%) was combined with 6 μm mucin particles suspension (0.05 w/v%). The results obtained indicated that both in vitro methods were useful for screening the mucoadhesiveness of the investigated nanoparticles.

3.4.1.2.3 Rheological Characterization

Rheological investigation of mucoadhesion is based on estimation of the changes in the rheological properties, namely viscosity and viscoelasticity, of the mucoadhesive DDS or polymer when mixed with mucus or mucins [22]. Interpenetration of mucoadhesive polymer chains into mucus, and further entanglement, conformational modifications and chemical interactions between polymer and mucin affect the rheological properties of such mixtures. The viscosity of the mixtures is greater than the sum of the viscosities of the individual components involved in mucoadhesion, and the strength of mucoadhesion can be quantified by correlation with rheological parameters. Hassan and Gallo [117] described a simple viscometric

Characterization of Bioadhesiveness  83 method to determine the strength of the mucoadhesive bonds between mucin and polymer. The viscosity of the pig gastric mucus dispersion (15 w/v%) in 0.1 M HCl (pH 1) or 0.1 M acetate buffer (pH 5.5) was measured with a Brookfield viscometer in the absence or presence of the selected neutral, anionic and cationic polymers (0.1-2.5%). The viscosity resulting from bioadhesion (ηb) is calculated from the equation:

ηt = ηm + ηp + ηb, where ηt, ηm, and ηp are the viscosities of the mixture, mucus and the polymer, respectively, determined at the same concentration, temperature, and shear rate. The dispersions of the polymers were Newtonian liquids, and their viscosity was independent of time and shear rate. The strength of mucoadhesion (F) represents an additional intermolecular force per unit area, and is calculated using the following equation:



F = ηb · σ,

where σ is the applied shear rate. At a constant shear rate, ηb can be used to compare mucoadhesiveness of different polymers. Since ηb is reduced with an increase in shear rate, high shear rates are applied in order to discriminate polymers with poor mucoadhesive properties. Hägerström and coworkers [13] have applied a similar method for testing the mucoadhesive property of two mucoadhesive polymers, Carbopol® 934 and Gelrite® (deacetylated gelan gum), in simulated physiological conditions, using an aqueous medium with ionic strength that corresponds to a tear fluid. The gels of these two polymers were prepared in the simulated tear fluid. Also, mixtures of the polymers and type III partially purified pork gastric mucin and type I-S beef submaxillary mucin were dispersed in the simulated tear fluid. The elastic modulus of the prepared dispersions was measured. The elastic component that originates from the interaction of a given polymer with mucin (ΔGʹ) is determined from the equation:



G′mix = G′p + G′m + ∆G′ ,



where G′mix is the elastic modulus of the mixture of the given polymer and mucin, while G′p and G′m are the elastic moduli of the given polymer and mucin, respectively [13]. Rheological measurements are particularly valuable for characterization of mucoadhesive interactions in polymer/mucin mixtures at the molecular level.

84  Bioadhesives in Drug Delivery Although useful for assessment of mucoadhesiveness of novel polymer derivatives, the commercially available purified and freeze-dried mucuses were found not to be representative for the mucus gel layer in vivo. Nevertheless, this method is usually performed as complementary to other in vitro methods e.g., tensile test [99].

3.4.1.2.4 Turbidimetry

Turbidimetry evaluates the formation of an interaction product between polymer and mucin by measuring the turbidity of their mixture. The absorbance in the 500–650 nm wavelength range is measured according to the European Pharmacopoeia (Ph. Eur.). Turbidity of the suspension is thought to be related to the extent of interaction between polymer and mucin chains, and the plot of turbidity versus mucin concentration serves to assess the adhesive interaction potential [22, 116].

3.4.1.2.5 Low-Frequency Dielectric Spectroscopy

Low-frequency dielectric spectroscopy is a suitable technique to investigate the physico-chemical interactions between the polymer molecules and mucin by determination of response of the material to the application of an electric field. The mucoadhesive polymer is exposed to the sinusoidal current, and the response is measured as a function of frequency. From the obtained response are determined the impedance or permittivity. Hägerström and associates [13] have investigated mucoadhesive property of several polymers (carbomer, sodium carboxymethyl cellulose, chitosan hydrochloride, and poloxamer 407) by using this method. Compatibility factor (CF) was calculated from high-frequency resistance of the mucoadhesive polymer gel, the mucous membrane and the mixtures thereof (Rhfgel, Rhfmucosa and Rhf gel+ mucosa, respectively). Based on CF, the possibility of establishing a close surface contact between the mucoadhesive polymer and mucous membrane, as the initial step in the process of mucoadhesion, was considered. The highest CFs were obtained for carbomer gel (2 w/w%) and chitosan gel (7 w/w%), and they were comparable to the results of the tensile test.

3.4.1.2.6 Zeta Potential Measurement

The mucoadhesion process depends on the molecular structures of the mucoadhesive polymer and mucous membrane as well as on their charge. Mucus glycoproteins are negatively charged at physiological pH. By measuring zeta potential, it is possible to understand the electrostatic

Characterization of Bioadhesiveness  85 interactions between mucus and the mucoadhesive polymer. The test consists of dispersing the mucin particles in an appropriate buffer and adding the dispersion of the mucoadhesive polymer. Changes in zeta potential after addition of the mucoadhesive polymer indicate corresponding affinity of the given polymer to mucus [116].

3.4.1.2.7 Biacore Test

Biacore test is an immunological test, but it can also be adapted for testing mucoadhesiveness of polymers. The device works on the principle of an optical phenomenon called Surface Plasmon Resonance (SPR). The test is based on the passage of the dispersion of mucus through the sensor containing the immobilized mucoadhesive polymer. As mucin binds to the polymer in the sensor, the concentration of the suspension and the refractive index on the sensor surface are changed and thus the interaction is measured as response, in resonance units (RU). The sensor is a chip of glass covered with a fine layer of gold, including various functional groups available for adsorption of the polymer [118].

3.4.1.2.8 Atomic Force Microscopy (AFM)

AFM enables the examination of mucoadhesive interactions under different experimental conditions. This technique enables magnification over 109 times, which provides visualization of isolated atoms and captures three-dimensional surface micrographs. The obtained curve of dependence between the force and the distance consists of an upward curve, which shows the approach of the probe to the mucoadhesive sample surface, and a descending curve which illustrates the separation of the probe from the mucoadhesive sample surface. The downward curve shows certain deviation in relation to the upward curve, which corresponds to the force required to separate the probe from the sample surface (i.e., the force of adhesion). This method is particularly suitable for testing the muco­ adhesiveness of polymers with heterogeneous surfaces, such as block copolymers [40, 43].

3.4.2 In Vivo Methods In vivo methods for characterization of bioadhesiveness are rarely used because they are expensive and time-consuming. Also, the ethical aspect must be taken into consideration. However, these methods enable full characterization of this property, especially in the case of peroral BDDS, where it is affected by many biological variables including GIT motility, mucus

86  Bioadhesives in Drug Delivery turnover, presence of enzymes, electrolytes, bile and exogenous materials (e.g. food, drink, drugs). Several methods of evaluating the mucoadhesive property of particulate systems have been reported in the literature, including: radiolabelled BDDS transit studies, gamma scintigraphy, and in vivo detachment tests.

3.4.2.1 Radiolabelled BDDS Transit Studies Investigation of mucoadhesiveness of BDDS in the GIT in vivo is usually carried out by performing radiolabelled transit studies. These involve the use of radioactive carbon (14C), chromium (51Cr), technetium (99mTc), indium (113mIn) and iodine (123I) or contrast medium such as barium sulphate incorporated or encapsulated in BDDS intended for oral delivery, to investigate bioadhesion over the entire length of the GIT as a function of time. In animal tests, the radiolabelled BDDS is placed into a surgically incised stomach of the rat which is then resealed and the animal allowed to regain consciousness. The animals were sacrificed and the stomach and intestines cut into segments and their radioactivity measured by scintillation counting after set time intervals. The major drawback of this technique is the difficulty to distinguish bioadhesion and stagnation of the BDDS in regions of low motility induced by anaesthesia. Moreover, it is difficult to find an animal model with conditions encountered in humans. For example, rats have little mucus in their stomachs, a portion of which is keratinised. Also, dietary and genetic differences can lead to substantial variations from animal to animal [76]. Fecal collection by using an automated apparatus and X-ray examination is a non-invasive method of monitoring the total retention time of BDDS in the GIT, without affecting its normal motility [98]. Also, in another approach, an aqueous dispersion of microspheres of polystyrene and polyanhydride of bis-2-carboxyphenoxypropane and sebacic acid (P(CPP-SA), in the ratio of 20:80) were loaded with barium sulphate and applied in 10 rats. The suspension of pure barium sulphate was given to rats from the control group. At certain time intervals, the rats were subjected to X-radiation for monitoring the distribution of microspheres in the intestinum. It was shown that the microparticles were retained in the stomach for 11 to 16.5 hours, while barium sulphate was eliminated after 9 hours. The bulk of barium sulphate was eliminated from the small intestine after 14-16 hours. Polystyrene microparticles were eliminated from the gastrointestinal tract after the same time period. However, microparticles of polyanhydride could be observed in the small intestine even after 28 hours. Since the normal time of passage through the GIT is between 4 hours and 12 hours, the results obtained with

Characterization of Bioadhesiveness  87 microencapsulated polyanhydride indicate that mucoadhesion prolonged its retention time in the GIT. It has been observed that fewer microparticles have longer retention times in the small intestine [98, 116].

3.4.2.2 Gamma Scintigraphy Gamma scintigraphy is a safe and non-invasive technique used in the development of peroral pharmaceutical preparations, including a few BDDS. The formulations labelled with a radionuclide (commonly 99mTc) are ingested by human volunteers and a gamma camera records the images and thus follows the course of the formulation throughout the GIT. The technique enables determination of the duration of bioadhesiveness in vivo; however, residence of the BDDS within the GIT may not be a direct result of bioadhesion and it can be significantly affected by food and disease [103]. Gamma scintigraphy was also used for characterization of distribution and retention times of hyaluronic acid ester-based technetiumlabelled mucoadhesive microparticles for vaginal application in sheep. After vaginal administration of microparticles, in the form of powder or vaginal pessaries, the sheep’s vagina was shot by a gamma camera at certain time intervals for 12 hours. The recordings showed that microparticles immediately after application were distributed over the entire surface of the vaginal mucosa (valid for both formulations). It was found that the retention time of mucoadhesive microparticles was longer when administered in the form of a powder, without migration of microparticles from the vagina to the upper parts of the genital tract [119]. In vivo spreadability and retention of marketed vaginal semisolid gels Replens® and K-Y® Jelly in humans was characterized by this technique. It was observed that initially the gel covered about two-thirds of vaginal length and surface area, and in 45 min, spreading increased to about three quarters of the surface [77].

3.4.2.3 In Vivo Detachment Tests Skin and buccal mucosa are available for non-invasive in vivo bioavailability tests in human volunteers. To evaluate the bioadhesion of the BDDS for cutaneous application, the peel test for determining the force required to separate the examined formulation from the skin surface in a vertical direction or at an angle of 90°, was used. Repka and McGinity [31] examined the vertical detachment force of different bioadhesive cutaneous films of hydroxypropyl cellulose containing various polymers as additives: poly(ethylene glycol) (PEG 3350), vitamin E TPGS (5%), and sodium starch glycolate (5%), Eudragit E-100 (5%), carbomer 974P (5%), carbomer 971P

88  Bioadhesives in Drug Delivery (5%) and polycarbophil 5%, alone or in combination with the PEG 3350. In vivo bioadhesion was investigated on the epidermis of 12 healthy volunteers, including two ethnic sub-groups (6 Asian and 6 Caucasians), using a Chatillon digital force gauge DFGS50 attached to a Chatillon TCD-200 motorized test stand to determine force of adhesion, elongation at adhesion failure, and adhesive modulus for the 12 films tested. Each film was attached to the upper movable part of the device. The volunteer’s hand was stationed on the lower, immobile part, and disinfected with isopropanol. Films were sprinkled with 0.5 ml of deionized water before fixing to the steel plate to allow their maximum hydration. The movable part with the fixed film was then lowered to the hand of the volunteer, in order to make contact with the skin, using a load of 0.8 kg for 180 seconds. The plate was then raised at a constant speed of 2.5 mm/min. The adhesion force (the force required to separate the film from the skin of the volunteer) and the elongation at adhesion failure (the height to which the movable part needs to be lifted to separate the film from the skin) were measured. The highest adhesion force was observed in films with carbomer 971P and polycarbophil, and these values ​​being much lower in Asians, attributed to differences in skin physiology of members of the two races. Similar example of determination of vertical detachment force was reported by Silva and coworkers [27]. The bioadhesive cutaneous film based on polyelectrolyte complexes of chitosan and polyacrylates was coated with a layer of pressure-sensitive adhesive (PSA) consisting of polyvinylpyrrolidone and macrogol 400, in order to improve bioadhesive property. The bioadhesiveness of the formulated film, expressed as the maximum force of adhesion, was evaluated in vivo using a texture analyzer. The test film was fixed with a double adhesive tape to the moving part of the apparatus, which was then lowered to contact with the skin of the forearm of the subject when a 3 N force was applied for 60 seconds. After that, the mobile part of the apparatus with the film was raised at a constant speed of 10 mm/s until the film was fully separated from the skin. The force required to detach the film from the skin was used as the parameter for evaluation of bioadhesiveness. It was found that the inclusion of PSA in film formulation significantly improved bioadhesive property without significant impact on its flexibility. Guo and associates [120] examined bioadhesiveness of a novel organic-inorganic hybrid film-forming transdermal BDDS based on a modified poly(vinyl alcohol) gel using γ-(glycidyloxypropyl)trimethoxysilane (GPTMS) as an inorganic-modifying agent, poly(N-vinyl pyrrolidone) as a tackifier and glycerol as a plasticizer. They performed the peel test as detachment of the film from the skin at an angle of 90°. The gels can be applied to the skin by a coating method and in situ form very thin and transparent films.

Characterization of Bioadhesiveness  89 Incorporation of appropriate GPTMS (GPTMS/(poly(vinyl alcohol) + GPTMS) in the range of 20-30%) into the poly(vinyl alcohol) matrix can significantly enhance mechanical strength and skin adhesion property of the resultant film. The peel test was performed in vivo, on the inside of the right forearm of 10 healthy volunteers (5 women and 5 men, ages 20-28 years). Various formulations of film-forming gels have been applied to volunteers. After acclimatization to the test conditions, for 10 min or 1 h, a flexible adhesive tape made of cellophane is placed over the applied film. The removal of the film from the skin of volunteers was performed using a suitable device (Testometric AX M350-10KN Materials Testing Machine (Testometric Company, Germany)), at 90° angle and at a speed of 10 mm/min. The adhesion force represented the force required for the removal or separation of the film from the skin of volunteers. Buccal mucosa is available for testing the retention time of mucoadhesive buccal formulations under the influence of common factors such as fluid and food intake as well as secretion of saliva. In vivo testing of mucoadhesion of the buccal film composed of hypromellose and sodium carboxymethyl cellulose with embedded liposomes loaded with vitamin B6 was performed in three volunteers. They were asked to rinse their mouth with distilled water before the film was placed between the cheek and gingiva in the region of the upper canine and gently pressed onto the mucosa for about 30 seconds. The time for complete erosion or detachment of the film from the buccal mucosa was recorded. Volunteers were allowed to eat and drink during the test period. A retention time of 4.4 hours was achieved [67].

3.5 Summary Novel discoveries in the field of bioadhesive/mucoadhesive pharmaceutical dosage forms and carriers as well as a significant number of biocompatible polymers have significant impact on enhancement in drug delivery. An important step in the characterization of BDDS is the evaluation of bioadhesiveness/mucoadhesiveness. The mechanism of bioadhesion/ mucoadhesion is highly complex and not yet fully understood and there are no official methods for its assessment in current pharmacopoeias. The literature describes a number of in vitro/ex vivo methods, as well as their various modifications, and several in vivo techniques that can be used to characterize BDDS. In vitro/ex vivo methods are most often based on the measurement of the strength of the adhesion force. Such methods are generally economical, easy to perform, and provide useful information

90  Bioadhesives in Drug Delivery on the interactions of bioadhesive/mucoadhesive drug carriers or pharmaceutical formulations with different biological membranes. The main disadvantage of such methods is that they do not include variables in biological environment and relate mainly to the macroscopic aspect of the bioadhesion phenomenon. For a more complete understanding of the mechanisms of bioadhesion/mucoadhesion, additional methods, such as spectroscopic measurements, Biacore test and AFM, are necessary. These methods provide information on mucoadhesive interactions at the molecular level. Nevertheless, in vitro methods represent a useful tool for initial screening of BDDS candidates for in vivo evaluation. In vivo methods enable to take into account biological variables such as GIT motility, mucus turnover, presence of endogenous materials (enzymes, electrolytes, bile) and exogenous materials (food, drink) and thus are more reliable for characterization of BDDS. For the majority of the methods currently available, the results largely depend on the applied method, the experimental conditions and the physiological characteristics of the substrate. Therefore, in vitro – in vivo correlation has been questionable so far. The growing interest in formulating bioadhesive/ mucoadhesive carriers is an incentive for development of new methods for testing this property.

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Part 2 BIOADHESIVE FORMULATIONS

4 Bioadhesive Films for Drug Delivery Systems Kampanart Huanbutta* and Tanikan Sangnim Faculty of Pharmaceutical Sciences, Burapha University, Chonburi, Thailand

Abstract

This chapter describes various applications of bioadhesive films for drug delivery systems. The film fabrication techniques and film-forming agents are reviewed and compared. Due to several applications of bioadhesive films, film-forming agents which are mostly hydrophilic polymers have to be properly selected for their purposes. Numerous active ingredients such as chemical drugs, proteins, monoclonal antibodies, and siRNA have been loaded into bioadhesive films for different administration purposes including transdermal, oral and mucosal routes. Preparation processes for the bioadhesive films have been developed for faster manufacturing and better product quality and also for a specific drug delivery route. Mostly, small scale preparation techniques are successful but large scale manufacturing still poses challenges for the bioadhesive films fabrication. The advanced film characterization techniques, such as drug permeation in cell line, play an important role in monitoring and improving film quality. Lastly, there is still a gap between research approaches and market product development which has to be filled to extend application of bioadhesive films. Keywords:  Bioadhesive film, drug delivery, film fabrication and evaluation, bioadhesive film application

4.1 Introduction Bioadhesive films are a film platform that has the property to attach to a biological tissue surface for an extended period of time. Mostly, *Corresponding author: [email protected] K.L. Mittal, I. S. Bakshi and J. K. Narang (eds.) Bioadhesives in Drug Delivery, (101–122) © 2020 Scrivener Publishing LLC

101

102  Bioadhesives in Drug Delivery bioadhesives are natural polymeric materials and might consist of a variety of substances such as proteins and carbohydrates [1]. The term mucoadhesion is used for the case of polymer attached to the mucin layer of mucosal tissue. Bioadhesives have been introduced to the formulation of the films because they tend to be biocompatible and biodegradable. Bioadhesive films have been widely used in the application of drug delivery because these can be modified for controlled drug release and various drugs can be loaded into the films. For local action, bioadhesive films can remain at the pathological site longer than liquid or semisolid formulations. The process or mechanism of bioadhesive film formation on biological surface is composed of three steps which are (i) Wetting and swelling of polymer, (ii) Interpenetration between the polymer chains and the mucosal membrane, (iii) Formation of chemical bonds between the entangled chains.

4.2 Theories of Bioadhesion There are several theories that explain that the phenomenon of adhesion, including bioadhesion, occurs by complex mechanisms [2-5]. These theories include: (i)  E  lectronic theory: This theory describes that the attractive force comes from electrons transferring between the surfaces of the bioadhesive film and the mucous layer. This is because of the difference in their electronic structures, resulting in the formation of an electrical double layer [6]. (ii)  Wetting theory: This theory explains how a low viscosity mucoadhesive system works. Bioadhesive ability depends on the spreading of liquid polymer making intimate contact with the mucous membrane. This affinity can be estimated by measuring the contact angle [7, 8] as demonstrated in Figure 4.1. (iii)  Adsorption theory: According to this theory, adhesion is the result of various interfacial interactions between the polymer and mucous layer. The main interaction/force is the covalent bond and the secondary chemical interactions are van der Waals, hydrogen bonds, and hydrophobic interaction [6]. (iv)   Diffusion theory: This describes the interpenetration between the polymer and mucin chains. A sufficient depth generates a semi-permanent adhesive bond as depicted in

Bioadhesive Films for Drug Delivery Systems  103

θ

θ Mucous membrane

θ

Contact angle

Adhesion

Figure 4.1  Contact angle between a pharmaceutical dosage form and mucous membrane.

Bioadhesive film

Mucous membrane

Figure 4.2  Mucoadhesion by inter-diffusion of the bioadhesive polymer film and mucous membrane.

Figure 4.2. The adhesion strength depends on the degree of penetration of the polymer chain. The penetration rate depends on the diffusion coefficient, flexibility, and nature of the mucoadhesive polymer chains, mobility and contact time [6]. (v)   Fracture theory: The fracture theory is the most used theory in research concerning the mechanical measurement of mucoadhesion. This theory considers the force required to detach two surfaces after contact is formed. This theory is unlikely for mucoadhesion mechanism but it can be used together with the other theories to describe adhesion phenomena [6].

4.3 Bioadhesive Film-Forming Agents Numerous hydrophilic polymers have been used as film-forming agents to achieve bioadhesive property. The functional groups of the polymer such as hydroxyl, carboxyl, amide, and sulfate attach to the mucus by hydrogen bonding and hydrophobic or electrostatic interactions. The names of

104  Bioadhesives in Drug Delivery polymers and their applications are presented in Table 4.1. Each film-forming agent can offer distinct film properties and characteristics [9]. For example, chitosan has good biodegradable, low-toxicological, and biocompatible characteristics. Moreover, chitosan is a cationic polymer which can neutralize anionic charge existing between mucosal cells in the tight junction area, promoting drug permeation [10]. Polyhydroxy methacrylate copolymer or Eudragit® is available in different solubility properties depending on its modified chemical functional group. Many types of polyhydroxy methacrylate copolymers can be dissolved in aqueous or organic Table 4.1  Chemical names and applications of some bioadhesive film-forming agents used in drug delivery [13]. Chemical name

Application

Reference

Hydroxypropyl methyl cellulose (HPMC)

Bioadhesive gel containing gelatin nanoparticles

[14]

Poly(vinyl alcohol) (PVA)

Novel organic-inorganic hybrid gel for transdermal drug delivery system

[15]

Poly(ethylene glycol) (PEG)

Controlled drug release film

[16]

Poly(acrylic acid) (PAA)

Bioadhesive hydrogel

[17]

Chitosan

Film-former for bioadhesive film for transdermal delivery

[18]

Polyhydroxy methacrylate

Film-forming gel

[19]

Gum

Bioadhesive films for the treatment of candidiasis vaginitis

[20]

Sodium alginate

Excellent gel and film-forming properties and is compatible with most water-soluble thickeners and resins

[21]

Hydroxypropyl cellulose (HPC)

Used to replace synthetic polymers or hydroxypropyl methylcellulose (HPMC) in a polymer matrix with modified starch to enhance solubility

[22]

Bioadhesive Films for Drug Delivery Systems  105 solvents [11]. Therefore, polyhydroxy methacrylate copolymer based films can be prepared in diverse ways. Poly(vinyl alcohol) (PVA), a water-soluble synthetic polymer, provides transparent bioadhesive film with low toxicity, high biodegradability, high bioadhesive nature, and high biocompatibility [12]. To select a film-forming agent from several choices, the purpose of the film application and its fabrication process have to be considered.

4.4 Drug Delivery Applications of Bioadhesive Films 4.4.1 Topical and Transdermal Drug Delivery 4.4.1.1 Patches Topical medicated adhesive patch/film is a formulation placed on the skin to deliver a specific dose of medication through the skin and into the bloodstream or for local therapeutic action which can promote healing to an injured area of the body. An advantage of transdermal drug delivery in the form of film over cream, gel and ointment formulations is that it is nonsticky, non-messy, and has skin occlusive property, thus enhancing drug absorption [23]. Additionally, the film patch can be modified for controlled drug release purposes. Usually, the advanced controlled drug release film is in the form of a porous membrane covering a reservoir of medication or as a drug loaded adhesive membrane layer as presented in Figure 4.3. For example, Padula and coworkers developed and characterized the skin bioadhesive film containing lidocaine. The permeation kinetics across the skin was non-linear, but the polymeric patch acted as a matrix controlling drug delivery [24]. Additionally, the permeation rate increased with drug loading [25]. Another interesting invention is Patch-non-Patch technology which shows potential to be successfully used in the pharmaceutical and cosmetic applications. The Patch-non-Patch is a film that is not self-adhesive in the dry state but becomes adhesive only when applied on wet skin. The film is flexible, invisible and adapts to all skin irregularities. This system has been

Backing film

Drug

Adhesive layer

Membrane

Figure 4.3  Topical medicated adhesive patch/film in different models (adapted from Pastore et al. [25]).

106  Bioadhesives in Drug Delivery shown to be highly efficient, releasing a high percentage of the active in most cases [26].

4.4.1.2 Film-Forming Systems A film-forming system is a novel type of pharmaceutical dosage form which can be applied as an alternative to conventional topical and transdermal formulations. It is classified as a liquid dosage form which suddenly becomes a film after applying or spraying on the skin surface as simulated in Figure 4.4. These systems contain drug and film-forming excipients dissolved in a vehicle which is in solution form. After the solution contacts the skin and is left behind, a film of excipients and the drug forms upon solvent evaporation. The formed film can either be a solid polymeric material that acts as a matrix for sustained release of the drug to the skin or a residual liquid film which is rapidly absorbed in the stratum corneum [23].

4.4.2 Mucosal Drug Delivery 4.4.2.1 Buccal Drug Delivery The buccal administration route has been increasingly evaluated both for systemic drug delivery and local drug targeting. This is because the buccal mucosa has rich supply of blood vessels which is a relatively permeable and excellent site for drug absorption. For these reasons, various buccal adhesive dosage forms such as buccal tablets, bioadhesive wafers, and bioadhesive lozenges have been developed [17]. Among these, films provide higher flexibility and improved patient compliance.

Solution

Becomes film

10s - 60s

Figure 4.4  Working mechanism of a film-forming system.

Bioadhesive Films for Drug Delivery Systems  107 Usually, the bioadhesive films are in the form tiny size which are easy to administer or terminate from the site of application [18]. The application of buccal films to improve the efficacy of several drugs delivery has been demonstrated in the literature. For example, Singh et al. [27] prepared and evaluated buccal bioadhesive films of clotrimazole for oral candidiasis treatment. Sodium carboxymethyl cellulose and Carbopol 974P were used for the preparation of films. Carbopol was used to provide the desired level of bioadhesiveness in the films. The film could inhibit the growth of C. albicans for 6 hours. The drug release mechanism was found to obey non-Fickian diffusion.

4.4.2.2 Vaginal Drug Delivery Pharmaceutical vaginal dosage forms have been developed as a result of their many advantages, such as applicability for local treatment, large surface area for drug absorption, rich blood supply, and avoidance of the first-pass effect. However, there are some limitations of traditional vaginal dosage forms including discomfort and short residence time. Consequently, bioadhesive films for vaginal drug delivery have been invented for reasons of their rapid drug release, enhanced bioadhesive property, negligible vaginal leakage and messiness, the potential for discreet use, low cost, and ease of insertion without an applicator [28, 29]. Comparing with semi-solid formulations such as creams and gels, bioadhesive thin films are effortless for vaginal insertion and the exact drug dose can be administered without dose leakage. This dosage form has been utilized for vaginal administration of the contraceptive/antimicrobial agents, antifungal drugs and the nucleotide reverse transcriptase inhibitor for HIV patients [30-32]. Vaginal films are employed for the delivery of biomolecules such as proteins, monoclonal antibodies, and siRNA [33]. Itraconazole was also loaded into the bioadhesive film for vaginal delivery for treatment against vaginal candidiasis, expecting the drug to remain in the vagina for prolonged intervals [30].

4.4.2.3 Rectal Drug Delivery The rectum is the final straight portion of the large intestine with a length of 10 cm and a surface area of 300 cm2. Most rectal absorption of drugs is achieved by a simple diffusion process through the lipid membrane. Drug delivery via the rectum is a useful alternative route of administration to the oral route for patients who cannot swallow and this administration route

108  Bioadhesives in Drug Delivery can also avoid the first-pass metabolism of drug [34]. The conventional and available pharmaceutical formulations for rectal drug administration are suppositories which are made from body temperature meltable waxes. However, there is inconvenience in using suppositories such as low temperature storage condition, difficult to use and low-stability and these are major limitations of the rectal drug delivery dosage forms. Consequently, bioadhesive film formulation is one of the novel rectal drug delivery systems which has been invented to overcome the above limitations. Thermo-reversible l­ iquidfilm suppository is an example of film preparation using temperature as a trigger to alter the state of the formulation. This formulation is in a liquid form at room temperature and turns into a gel or bioadhesive film instantly at physiological temperature (administered to anus) and it is also mucoadhesive to the rectal tissues without leakage after the dose [35]. Kim and coworkers [36] developed a thermoreversible flurbiprofen liquid suppository base composed of poloxamer and sodium alginate for the improvement of rectal bioavailability of the poorly water-soluble drug. They also utilized cyclodextrin and its derivatives to enhance the aqueous solubility of flurbiprofen.

4.4.2.4 Ocular Drug Delivery An ocular drug delivery system is the most convenient and patient compliant route of drug administration, especially for the treatment of anterior segment diseases. However, poor drug bioavailability is the main weakness of this route of drug administration. There are many factors those decrease the drug bioavailability including ocular anatomy, physiology and inefficient protective mechanism including extensive nasolacrimal drainage, tear dynamics, relative impermeability of the corneal epithelial membrane and the highly efficient protection from the blood-ocular barrier [37]. The common ophthalmic dosage forms are concentrated eye drops associated with low corneal drug absorption and also low ocular and systemic side effects. Consequently, numerous alternative dosage forms have been developed such as solutions, gels, ointments, and aqueous suspensions. The ocular film was also invented to improve bioavailability by increasing contact and retention time of the film. Moreover, an ocular film can be applied for controlled drug delivery film which can ensure drug concentration in the eye and offer accurate drug dosing with less systemic side effects [38]. In addition, the film is in the solid state which offers long shelf-life and provides the advantage of biodegradability or solubility and there is no need to remove it from the eyes. Jafariazar et al. [38] prepared ocular film formulations to achieve controlled drug release. Various polymers such as hydroxypropyl methyl cellulose, polyvinylpyrrolidone and polymethacrylate-based copolymers were used to prepare films by solvent

Bioadhesive Films for Drug Delivery Systems  109 casting method. The optimized formulation and preparation parameters offered controlled and prolonged release, following the zero-order and nonFickian transport.

4.4.2.5 Nasal Drug Delivery Nasal drug delivery via mucosa is an alternate route to achieve faster and higher drug absorption. This administration also avoids liver first-pass and gastrointestinal tract metabolism. This route is non-invasive administration resulting in low risk of infectious disease transmission and ease of convenience for self-medication. Nevertheless, mucociliary clearance in nasal cavity reduces the residence time of drug which affects the bioavailability [39]. This drawback has been overcome by the development of novel formulations such as dry powder inhalers, metered-dose inhalers and sol/gel/thin film formulations [40, 41]. The advantages of bioadhesive films for nasal drug delivery are stability, single dose unit and easy to carry. An interesting example of a film formulation application in nasal drug delivery is in situ gelling nasal inserts. The formulation is in form of a film before administering, then becomes bioadhesive film/gel when attached to the nasal mucosa and there is no need to remove the insert mechanically after it is depleted of the drug loaded film. This delivery system has been utilized for peptide drug or vaccine delivery [40].

4.4.3 Oral Drug Delivery 4.4.3.1 Orodispersible Films (ODFs) Orodispersible films (ODFs) are the formulations which are inherently easy to administer for young and aging patients who have dysphagia problem. Fast dissolving film, fast disintegrating film, and orodispersible film are some synonymous words for this dosage form. Without the need of liquid for administration, this dosage form dissolves in the mouth within 30 seconds [42]. Another advantage of ODFs is the potential to deliver tailored therapies to different patient populations. This is because ODFs can be individually prepared for a person by ink-jet or 2D printing technique [43, 44]. The advantages of film formulation are low risk of choking, convenient and accurate dosing, enhanced stability and rapid onset of action [45]. Sangnim and colleagues [46] prepared ODFs using 2D ink-jet printing technique by applying theophylline solution as ink. The drug was printed on the film, and thus the drug printing size can be altered. Thus, this technique provides accurately adjustable drug dose for an individual

110  Bioadhesives in Drug Delivery patient. The film can be dissolved within 30 seconds and the drug dissolved more than 80% in 3 minutes.

4.4.3.2 Sublingual Films Sublingual drug administration is the placement of the drug under the tongue and the drug is absorbed directly into the bloodstream through the ventral surface of the tongue and floor of the mouth [47]. The pros of this administration are the ease of administration to patients who refuse to swallow a tablet, relatively rapid onset of action, avoidance of first-pass effect and no need for water or chewing for drug administration. Generally, most of the sublingual formulations are in the form of tablets because this platform offers drug stability and easy drug handling. However, film formulations for sublingual administration have been developed to enhance drug solubility. This is because film formulation heightens drug solubility and a film can be divided for dose adjusting purposes. Besides, the use of mucoadhesive polymers in the films will enable them to adhere to the sublingual mucosa for prolonged retention and drug absorption [48]. Recently, Allam and Fetih have prepared and evaluated sublingual fast dissolving films containing metoprolol tartrateloaded niosomes which prolonged release of the drug, whereas the films were used to increase the drug’s bioavailability via the sublingual route [49].

4.4.3.3 Oral Colon-Specific Drug Delivery Delivery of active pharmaceutical ingredients to the colon can offer benefits for various purposes, including peptide or protein drug delivery, reducing drug side effect and local treatments of colonic diseases such as colon cancer and inflammatory bowel disease (IBD). Several mechanisms have been applied to specifically trigger drug release at the colon such as pH, time, enzyme and pressure in GI tract [10]. Film formulation is a novel dosage form which has been introduced to colonic drug delivery because this dosage form can be used in the patients who have dysphagia problem. As for materials used in film formulation for colonic drug delivery, numerous biopolymers such as pectin, chitosan, chondroitin sulfate, galactomannan, and amylose are ideal materials for achieving colon-specific delivery because they can be degraded by the colonic enzymes and are harmless to the organisms. From recent research work, film formulation was modified to deliver the bioactive to the colon. The films were coated with acidenzyme resistant starch to protect the active ingredient from environmental condition in the GI tract. The optimized films showed high potential for accurately targeting bioactive compound delivery to the colon [50].

Bioadhesive Films for Drug Delivery Systems  111

4.5 Current and Novel Bioadhesive Film Fabrication Techniques 4.5.1 Solvent Casting Solvent casting is the most commonly used method for the preparation of bioadhesive films. The main principle of this method is to disperse and/ or dissolve pharmaceutical excipients, polymers (film-forming agents) and drug in a solvent. Then, the prepared solution is poured into a petri dish and the solvent is allowed to evaporate by exposing to high temperature. Drug, excipient and solvent compatibility is a major consideration in the pre-formulation process. The usual problems for film casting are entrapment of air bubbles and uniformity of the films [51]. Consequently, several techniques and methods have been developed to overcome these limitations such as using a vacuum pump to eliminate air bubbles before film drying. In the continuous manufacturing process of the film, a blade is applied to flatten the film solution surface and propel bubbles before the film is moved to the drying station. The viscosity of the film solution before pouring into the casting plate is another vital factor to be concerned. According to the previous work by Sangnim and team [46], concentration of the gum solution varying from 2% to 3.5% provided low viscosity solutions, and as a result, enabled easy casting of films.

4.5.2 Extrusion In hot-melt extrusion, the dry ingredients for the film are heated and homogenized by the propellant force of an extruder screw until all ingredients are melted and blended. The melted material is forced through a flat extrusion die that presses the extrudate into the desired film shape. The thickness and strength of the film are decided by the elongation rollers. A high temperature is utilized in this process; therefore, this film preparation method is not applicable for heat-sensitive active pharmaceutical ingredients (APIs) and excipients. Moreover, the film-forming agent should be meltable and other excipients have to be soluble in the film material. There are many factors to be considered before selection of film manufacturing method, between casting and extrusion as presented in Table 4.2.

4.5.3 Rolling The rolling method is widely used in film manufacturing because it can continuously produce products on a large scale. The preparation starts

112  Bioadhesives in Drug Delivery Table 4.2  Comparison of film preparation using casting and extrusion techniques. Casting

Extrusion

Applicable for heat-sensitive APIs because the required temperatures for solvent removal are relatively low

Not suitable for heat-sensitive APIs because the required temperatures have to be high enough to melt the polymer or wax (film-forming agents)

Might contain trace amounts of residual solvents

Absence of solvent in the preparation process

The film-forming agent should be soluble in the solvent

The film-forming agent should be meltable. All the ingredients that are used in a hot-melt film must also be devoid of water or any other volatile solvents.

Uses several steps in the film preparation process

Shorter preparation time and process

Requires solvent to dissolve drug and excipients

No need to use a solvent or water in the process

Applicable for only solvent soluble drugs

Good dispersion mechanism for poorly soluble drugs

with rolling a solution/suspension containing the active ingredient on a backing film. Mainly, the solvent of the system is water or a mixture of water and alcohol. The film is dried on the rollers and cut into desired sizes and shapes. The key process of this method is the mixing and dissolving/dispersing of API and excipients in the solvent. The API solution/ suspension should be homogeneous; therefore, a metering pump can accurately feed the solution/suspension to the plate. The film thickness can be controlled by a leveling roller. The bottom drying technique is preferable to avoid external air currents or heat on the surface of the film [52].

4.5.4 2D Printing Two-dimensional printing technologies, such as inkjet and flexographic printing, offer possibilities to deposit a variety of functional materials onto different types of carrier surfaces or substrates. The main advantage

Bioadhesive Films for Drug Delivery Systems  113 of inkjet printing is the ability to dispense uniform and accurate volume of ink droplets on the particular area. The ink formulation (dissolved drug and excipient in solvent) properties such as viscosity, surface tension, and conductivity are the most important key factors to print a film. Another advantage of 2D printing in film preparation is the possibility to prepare individual dosage for each patient. This is because this method offers finely adjustable drug loading with an accurate drug amount [46]. Consequently, the 2D printing technology has been adapted for application in the pharmaceutical field [53]. To fabricate a film, various drugs have been printed together with the film-forming agent and other excipients for formulation of orodispersible films and controlled release films [54].

4.6 Evaluation of Bioadhesive Films 4.6.1 Bioadhesive Strength Bioadhesive strength is the force required to detach the bioadhesive films from the mucosal surface. This force is used to define bioadhesive performance. Several types of equipments and models have been applied to evaluate the bioadhesive strength of films. Parodi et al. [55] used a two-arm physical balance in which the right pan had been replaced by a formulation holding glass plate and counterbalanced by a water collecting pan suspended from the left arm as depicted in Figure 4.5. After the film starts to detach from the holding glass, the weight of collected water on the other side is recorded.

Water bottle

Bioadhesive film Beaker

Figure 4.5  Bioadhesive film strength test assembly.

Stomach musoca

114  Bioadhesives in Drug Delivery

4.6.2 Tensile Strength Measurement Mechanical properties evaluation is a fundamental materials science and engineering task in which a sample is subjected to a controlled tension until failure. Generally, mechanical properties of a film are measured in terms of different tensile test parameters which are ultimate tensile strength, breaking strength and maximum elongation. From these measurements the following properties can also be determined: Young’s modulus, Poisson’s ratio, yield strength, and strain-hardening characteristics. These are vital properties of the film which can guide the formulator to modify formulation for different purposes. Each film application requires different film mechanical properties. For example, topical bioadhesive films demand high flexibility and bioadhesive force. On the other hand, orodispersible films need lower flexibility and bioadhesiveness but fast disintegration instead.

4.6.3 Morphology and Thickness Morphology and thickness of the film are crucial properties which control and monitor the quality of film preparation. Many techniques have been utilized to observe film surface and thickness. Scanning electron microscopy (SEM) is commonly used to observe surface of film and thickness in micro/nanometer level and thus this technique is also applied for imaging of film cross section. Another option to evaluate film surface which can magnify better than the SEM is the atomic force microscopy (AFM). Both techniques can observe surface appearance and can also check the solubility of drug in the film. However, the film should be in a dry state during testing [56]. To monitor dynamic change of film in both dry and wet conditions, laser scanning confocal microscopy (LSCM) seems to be a proper choice for this. Sung and colleagues have successfully used LSCM to characterize the changes in film thickness and local surface morphology of polymer film during the UV degradation process [57]. Apart from image analysis, electronic digital calipers are also used to measure film thickness. The applicable range for the caliper is around 0 to 150 mm with an accuracy of ± 0.03 mm which works well with non-thin film measurement [13].

4.6.4 Moisture Content Moisture content of the film has an immense effect on mechanical and optical (clarity and transparency) properties because water is an efficient plasticizer and also affects refractive index. Therefore, the film moisture content must be carefully monitored and controlled to meet the required

Bioadhesive Films for Drug Delivery Systems  115 mechanical properties of the film for its specific applications [58]. Moisture content uptake behavior of the film is useful information to indicate film storage conditions and predict product stability. Hydrophilic polymers trend to have hygroscopic property more than the hydrophobic. In general, moisture content is measured using a moisture balance. The principle of moisture measuring is to monitor weight change from water loss of film during heat supply from the equipment. Normally, halogen is used as a heat generator and different sensitivitises of balance can be used to detect several ranges of weight.

4.6.5 Permeation Assessment of the permeation of drug molecules following application of a mucoadhesive film involves the measurement of drug absorption across dermal epithelium or buccal barrier to the systemic circulation. Physicochemical properties of the drug play a vital role in drug transportation efficiency into and through the membrane. Normally, the permeation test is conducted as in vitro or ex vivo studies which aims to simulate actual physiological conditions (in vivo study). Several models have been invented for the permeation test on a mucoadhesive film but the main components of the test are a receiver, membrane, donor and sampling ports as shown in Figure 4.6 [59]. Alternatively, several types of epithelial cell cultures have also been proposed for a useful in vitro permeation investigation for films and topical dosage forms [60, 61]. Nevertheless, to utilize these culture cells for evaluation of drug transport, the number of differentiated cell layers and

Donor chamber Bioadhesive film Epithelial membrane

Water jacket to control temperature

Sampling port

Receiver chamber

Figure 4.6  Schematic representation of a vertical Franz diffusion cell.

116  Bioadhesives in Drug Delivery the lipid composition of the barrier layers must be well characterized and monitored.

4.6.6 Swelling Most of the bioadhesive films are made of hydrophilic polymers which can swell in an aqueous solution. Consequently, swelling and erosion properties of films are important factors to be concerned. Actually, there are many methods to observe polymer swelling behavior such as magnetic resonance imaging (MRI), X-ray microtomography (XμT), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), fluorescent, texture analyzer, and ultrasound techniques [62] but the most common method for a bioadhesive film is weight change monitoring or water absorption which can be revealed by the swelling index calculated as:



Swelling index = (W2-W1)/W1

where W2 is the weight of the swollen film and W1 is the initial dry weight of the film.

4.6.7 Irritation Using a bioadhesive film in very sensitive areas such as an oral cavity, wound, vagina or even skin might cause irritation or injuries. As a result, film irritation test is very significant. The general method to observe reversible damage to the skin is the Organization for Economic Cooperation and Development (OECD) testing guideline 404. It is generally assessed by the potential of a certain substance to cause erythema/eschar and/or oedema after a single topical application on rabbit skin and is based on the Draize score. Apart from the OECD testing, several prevalidation efforts have been carried out for in vitro alternative methods such as the non-perfused pig ear model and the in vitro mouse skin integrity function test (SIFT) which are under the European Centre for the Validation of Alternative Methods (ECVAM) from the science and knowledge service of the European Commission [63].

4.6.8 Stability As mentioned before a bioadhesive film is a hydrophilic polymer and is made of bio/synthetic polymers or their mixture. Some of them might have to be kept in hydrated state. These internal and environmental factors decrease the stability of films. Hence, stability testing is required before product

Bioadhesive Films for Drug Delivery Systems  117 launching in the market. Mainly, two storage conditions have been applied in the stability test which are accelerated and regular conditions. Temperature and ambient humidity are two controlled environmental factors which are 40°C and 75% RH for the accelerated condition and 25°C (might be 30°C for tropical countries) and 75% for the regular condition [64]. Active ingredient degradation/interaction and physical properties of the film such as appearance, tensile strength, elongation, and moisture content have to be monitored during the test period. This information can predict shelf-life of the product and these are important data for product registration.

4.6.9 Drug Loading and Drug Entrapment Efficiency Drug loading and drug entrapment are noteworthy to assess efficiency of the film system and preparation method. There are different definitions of drug loading and drug entrapment efficiency. Drug loading efficiency or capacity is the portion of loaded drug in the film system which can be expressed as:

Drug loading efficiency = Amount (weight) of loaded drug weight of polymer and excipient in the film system Drug entrapment efficiency indicates the potential for preparation and formulation of the film. It is the amount of entrapped drug in the film compared to the added drug in the film preparation. This can be expressed as:

Drug entrapment efficiency = Actual amount of entrapped drug Added drug in the formulation

4.7 Summary The formulation of bioadhesive films for drug delivery has been of interest in recent years. Many drawbacks of film formulation, for instance, inconvenience of administration, lower bioavailability, and patient non-compliance have been resolved and novel polymeric films have been developed as drug delivery platforms for various applications and routes of administration. Several film preparation methods have been invented and applied for better film properties, and personalized medication purposes. However, the novel bioadhesive film dosage forms encounter manufacturing challenges such as large scale production and

118  Bioadhesives in Drug Delivery product stability. Finally, the future looks very promising for the bioadhesive film technology in drug delivery to serve the different requirements of patients.

4.8 Acknowledgements The authors acknowledge Prof. Dr. Pornsak Sriamornsak, Assoc. Prof. Dr. Jurairat Nunthanid and Assoc. Prof. Dr. Sontaya Limmattavapirat for their support and encouragement, and the Faculty of Pharmaceutical Sciences, Burapha University for database support for this chapter.

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Bioadhesive Films for Drug Delivery Systems  119 11. K. Huanbutta, T. Nernplod, P. Akkaramongkolporn, and P. Sriamornsak, Design of porous Eudragit® L beads for floating drug delivery by wax removal technique. Asian J. Pharm. Sci. 12, 227-234 (2017). 12. T. Sangnim, S. Limmatvapirat, J. Nunthanid, P. Sriamornsak, W. Sittikijyothin, S. Wannachaiyasit, and K. Huanbutta, Design and characterization of clindamycin-loaded nanofiber patches composed of polyvinyl alcohol and tamarind seed gum and fabricated by electrohydrodynamic atomization. Asian J. Pharm. Sci. 13, 450-458 (2018). 13. S. Karki, H. Kim, S.-J. Na, D. Shin, K. Jo, and J. Lee, Thin films as an emerging platform for drug delivery. Asian J. Pharm. Sci. 11, 559-574 (2016). 14. S. Manna, U.S. Lakshmi, M. Racharla, P. Sinha, L.k. Kanthal, and S.P.N. Kumar, Bioadhesive HPMC gel containing gelatin nanoparticles for intravaginal delivery of tenofovir. J. Appl. Pharm. Sci. 6, 22-29 (2016). 15. R. Guo, X. Du, R. Zhang, L. Deng, A. Dong, and J. Zhang, Bioadhesive film formed from a novel organic–inorganic hybrid gel for transdermal drug delivery system. Eur. J. Pharm. Biopharm. 79, 574-583 (2011). 16. Q. Wang, Z. Dong, Y. Du, and J.F. Kennedy, Controlled release of ciprofloxacin hydrochloride from chitosan/polyethylene glycol blend films. Carbohydr. Polym. 69, 336-343 (2007). 17. T. Ito, M. Eriguchi, and Y. Koyama, Bioabsorbable bioadhesive hydrogel comprising poly(acrylic acid) and poly(vinylpyrrolidone) for adhesion barrier and hemostatic device. MRS Commun. 5, 291-295 (2015). 18. B. Rasool, U. Aziz, O. Sarheed, and A. Rasool, Design and evaluation of a bioadhesive film for transdermal delivery of propranolol hydrochloride. Acta Pharm. 61, 271-282 (2011). 19. N.N. Vij and R.B. Saudagar, Formulation, development and evaluation of film-forming gel for prolonged dermal delivery of terbinafine hydrochloride. Int. J. Pharm. Sci. Res. 5, 537-554 (2014). 20. P. Bassi and G. Kaur, Polymeric films as a promising carrier for bioadhesive drug delivery: Development, characterization and optimization. Saudi Pharm. J. 25, 32-43 (2017). 21. J.O. Morales and J.T. McConville, Manufacture and characterization of mucoadhesive buccal films. Eur. J. Pharm. Biopharm. 77, 187-199 (2011). 22. A.F. Borges, C. Silva, J.F.J. Coelho, and S. Simões, Oral films: Current status and future perspectives: I — Galenical development and quality attributes. J. Control. Release 206, 1-19 (2015). 23. K. Kathe and H. Kathpalia, Film forming systems for topical and transdermal drug delivery. Asian J. Pharm. Sci. 12, 487-497 (2017). 24. C. Padula, G. Colombo, S. Nicoli, P.L. Catellani, G. Massimo, and P. Santi, Bioadhesive film for the transdermal delivery of lidocaine: in vitro and in vivo behavior. J. Control. Release 88, 277-285 (2003). 25. M.N. Pastore, Y.N. Kalia, M. Horstmann, and M.S. Roberts, Transdermal patches: History, development and pharmacology. Br. J. Pharmacol. 172, 2179-2209 (2015).

120  Bioadhesives in Drug Delivery 26. C. Padula, S. Nicoli, V. Aversa, P. Colombo, F. Falson, F. Pirot, and P. Santi, Bioadhesive film for dermal and transdermal drug delivery. Eur. J. Dermatol. 17, 309-312 (2007). 27. S. Singh, S. Jain, M.S. Muthu, S. Tiwari, and R. Tilak, Preparation and evaluation of buccal bioadhesive films containing clotrimazole. AAPS PharmSciTech 9, 660-667 (2008). 28. R.K. Malcolm, A.D. Woolfson, C.F. Toner, R.J. Morrow, and S.D. McCullagh, Long-term, controlled release of the HIV microbicide TMC120 from silicone elastomer vaginal rings. J. Antimicrob. Chemother. 56, 954-956 (2005). 29. R.M. Machado, A. Palmeira-De-Oliveira, J. Martinez-De-Oliveira, and R. Palmeira-De-Oliveira, Vaginal films for drug delivery. J. Pharm. Sci. 102, 2069-2081 (2013). 30. N.B. Dobaria, A.C. Badhan, and R.C. Mashru, A novel itraconazole bioadhesive film for vaginal delivery: Design, optimization, and physicodynamic characterization. AAPS PharmSciTech 10, 951-959 (2009). 31. S. Garg, K. Vermani, A. Garg, R.A. Anderson, W.B. Rencher, and L.J.D. Zaneveld, Development and characterization of bioadhesive vaginal films of sodium polystyrene sulfonate (PSS), a novel contraceptive antimicrobial agent. Pharm. Res. 22, 584-595 (2005). 32. W. Zhang, M. Hu, Y. Shi, T. Gong, C.S. Dezzutti, B. Moncla, S.G. Sarafianos, M.A. Parniak, and L.C. Rohan, Vaginal microbicide film combinations of two reverse transcriptase inhibitors, EFdA and CSIC, for the prevention of HIV-1 sexual transmission. Pharm. Res. 32, 2960-2972 (2015). 33. A.S. Ham, M.R. Cost, A.B. Sassi, C.S. Dezzutti, and L.C. Rohan, Targeted delivery of PSC-RANTES for HIV-1 prevention using biodegradable nanoparticles. Pharm. Res. 26, 502-511 (2009). 34. T.J. Purohit, S.M. Hanning, and Z. Wu, Advances in rectal drug delivery systems. Pharm. Dev. Technol. 23, 942-952 (2018). 35. H.-G. Choi, J.-H. Jung, J.-M. Ryu, S.-J. Yoon, Y.-K. Oh, and C.-K. Kim, Development of in situ-gelling and mucoadhesive acetaminophen liquid suppository. Int. J. Pharm. 165, 33-44 (1998). 36. J.-K. Kim, M.-S. Kim, J.-S. Park, and C.-K. Kim, Thermo-reversible flurbiprofen liquid suppository with HP-β-CD as a solubility enhancer: Improvement of rectal bioavailability. J. Incl. Phenom. Macrocycl. Chem. 64, 265-272 (2009). 37. A. Patel, K. Cholkar, V. Agrahari, and A.K. Mitra, Ocular drug delivery systems: An overview. World J. Pharmacol. 2, 47-64 (2013). 38. Z. Jafariazar, N. Jamalinia, F. Ghorbani-Bidkorbeh, and S.A. Mortazavi, Design and Evaluation of ocular controlled delivery system for diclofenac sodium. Iran J. Pharm. Res. 14, 23-31 (2015). 39. S. Dey, B. Mahanti, B. Mazumder, A. Malgope, and S. Dasgupta, Nasal drug delivery: An approach of drug delivery through nasal route. Pharmacia Sinica 2, 94-106 (2011).

Bioadhesive Films for Drug Delivery Systems  121 40. R.M. Farid, M.A. Etman, A.H. Nada, and A.E.A.R. Ebian, Formulation and in vitro evaluation of salbutamol sulphate in situ gelling nasal inserts. AAPS PharmSciTech 14, 712-718 (2013). 41. S. Upadhyay, A. Parikh, P. Joshi, U.M. Upadhyay, and N.P. Chotai, Intranasal drug delivery system- A glimpse to become maestro. J. Appl. Pharm. Sci. 1, 34-44 (2011). 42. A. Deshpande and C. Gijare, Orodispersible Films: A systematic patent review. Recent Patents Drug Deliv. Formul. 12, 110-120 (2018). 43. M. Scarpa, S. Stegemann, W.-K. Hsiao, H. Pichler, S. Gaisford, M. Bresciani, A. Paudel, and M. Orlu, Orodispersible films: Towards drug delivery in special populations. Int. J. Pharm. 523, 327-335 (2017). 44. M. Irfan, S. Rabel, Q. Bukhtar, M.I. Qadir, F. Jabeen, and A. Khan, Orally disintegrating films: A modern expansion in drug delivery system. Saudi Pharm. J. 24, 537-546 (2016). 45. D. Sharma and R. Kohad, Update review on oral disintegrating film. Int. J. Creative Res. Thoughts 6, 92-102 (2018). 46. T. Sangnim, P. Sthiraphan, P. Teerasaksopon, S. Treewechvinit, and K. Huanbutta, Development of theophylline orodispersible film fabricated by 2D printing technique. Isan J. Pharm. Sci. 13, 189-196 (2017). 47. A.K. Bind, G. Gnanarajan, and P. Kothiyal, A review: Sublingual route for systemic drug delivery. IJIRSET 3, 31-36 (2013). 48. R.C. Mashru, V.B. Sutariya, M.G. Sankalia, and P.P. Parikh, Development and evaluation of fast-dissolving film of salbutamol sulphate. Drug Dev. Ind. Pharm. 31, 25-34 (2005). 49. A. Allam and G. Fetih, Sublingual fast dissolving niosomal films for enhanced bioavailability and prolonged effect of metoprolol tartrate. Drug Des. Dev. Ther. 10, 2421-2433 (2016). 50. J. Chen, X. Li, L. Chen, and F. Xie, Starch film-coated microparticles for oral colon-specific drug delivery. Carbohydr. Polym. 191, 242-254 (2018). 51. T. Parikh, S.S. Gupta, A.K. Meena, I. Vitez, N. Mahajan, and A.T.M. Serajuddin, Application of film-casting technique to investigate drug– polymer miscibility in solid dispersion and hot-melt extrudate. J. Pharm. Sci. 104, 2142-2152 (2015). 52. R. Mishra and A. Amin, Manufacturing techniques of orally dissolving films. Pharm. Technol. 35, 70-73 (2011). 53. N. Genina, D. Fors, H. Vakili, P. Ihalainen, L. Pohjala, H. Ehlers, I. Kassamakov, E. Haeggström, P. Vuorela, J. Peltonen, and N. Sandler, Tailoring controlled-release oral dosage forms by combining inkjet and flexographic printing techniques. Eur. J. Pharm. Sci. 47, 615-623 (2012). 54. W. Jamróz, M. Kurek, E. Lyszczarz, W. Brniak, and R. Jachowicz, Printing techniques: Recent developments in pharmaceutical technology. Acta Polish Pharm. 74, 753-763 (2017).

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5 Redox-Responsive Disulphide Bioadhesive Polymeric Nanoparticles for Colon-Targeted Drug Delivery Erazuliana Abd Kadir and Vuanghao Lim* Integrative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Penang, Malaysia

Abstract

Choosing the right carrier in nanoparticle (NP) formulation (nanoformulation) is crucial in determining the efficiency of colon-targeted drug delivery (CTDD). The selection of the carrier should be based on the drug’s nature, which involves its chemical nature, stability and partition coefficient. Polysaccharide-based polymers, such as chitosan, alginate, pectin, dextran, guar gum and hyaluronic acid, are commonly used for NP formulation in CTDD. Apart from their biodegradability; biocompatibility; and low-immunogenic, unique bioadhesive and non-toxic properties, polysaccharide-based carriers also have a degradation pattern, which allows the prediction of drug release from the encapsulation matrix. This chapter focuses on thiolated polymeric NPs for CTDD. Different thiolations of polymers are discussed to understand the nanoformulation process, redox behaviour and drug release. Here, the drug cellular uptake in colonic cells for some NPs is also described. Keywords:  Disulphide polymers, redox-responsive, polymeric nanoparticles, colon, drug delivery

5.1 Introduction Various kinds of drug delivery systems are currently being researched and established for drug administration due to the vast number of drug *Corresponding author: [email protected] K.L. Mittal, I. S. Bakshi and J. K. Narang (eds.) Bioadhesives in Drug Delivery, (123–146) © 2020 Scrivener Publishing LLC

123

124  Bioadhesives in Drug Delivery candidates in the market. The discoveries of newer potent drugs have increased interest in research for ideal drug release. As most drug-releasing systems use polymers, the therapeutic efficacy is always an issue because of nonspecific targeting potentials. Controlled drug release approach has been ventured to improve conventional drug delivery systems. This approach has steered towards the development of smarter drug release systems to attain drug targeting. In this approach, various kinds of drugs in matrix structures are formulated by polymers that release the drug via a stimulus, known as ‘stimuli-responsive polymers’ [1-2]. The concept of drug targeting a desired site is not new. The interest in treating disorders of the colon and peptide drug delivery to the colon have attracted considerable attention of researchers. Before delivering to the colon, the formulated dosage form has to travel along the upper gastrointestinal tract (GIT) in intact form [3-4]. In biological systems, redox environment is the most useful stimulus because of the distinct redox potentials in extra- and intracellular partitions [5]. In recent years, novel biocompatible nanocarriers based on redox-responsive polymers have become more popular by utilising a special reducing agent called glutathione (GSH) for nanoparticles (NPs) in drug delivery [6]. GSH provides a range of concentration from 2 mM to 10 mM (intracellular partition) compared to the concentration measured in extracellular fluids [2, 7]. The redox potential of the environment determines the internal stability because the formation or cleavage of the disulphide bond requires a suitable electron acceptor or donor [8]. Therefore, developing colon-specific systems using disulphide-containing polymer NPs has become a new research interest. The disulphide bonds are reduced to thiols in the colon with low redox potential, thereby depolymerising and degrading redox-sensitive NPs. Generally, the degradation products and the polymer itself are biologically acceptable and non-toxic to humans (or animals) [9-10].

5.2 Mechanism of Disulphide Bond Formation The disulphide bond is formed through an oxidative method using spontaneous oxidants. To form the disulphide bond completely, the oxidation process requires high-dilution and slightly alkaline conditions (pH 7.5-8.5), by gentle stirring (or bubbling) through dilute solutions. Air oxidation is the easiest and most commonly used method of exposing the aqueous solution of the thiol compound to the atmosphere. Nevertheless, this procedure takes a significant amount of time to achieve valuable yields of monomeric

Bioadhesive Disulphide Polymeric Nanoparticles  125 products. However, its effectiveness can be increased by using a highly diluted solution of the compound (10–100 µM). Using dimethyl sulfoxide (DMSO)-induced oxidation of thiols allows the formation of disulphide to proceed under conditions with similar mildness as air oxidation [1, 11-12]. The mechanism of oxidation by DMSO involves the formation of a free thiol with the sulfoxide molecule into an unstable thiol sulfoxide compound. Interaction with another molecule of thiol destroys the adduct compound, yielding disulphide, sulphide and water molecules (Scheme 5.1).



RSH  +  R2SO ──────▶  [R2S(OH)SR]

[R2S(OH)SR]  +   RSH ──────▶  RSSR + R2S + H2O Scheme 5.1 Oxidative mechanism by DMSO. The rate of reaction is dependent on the acidity of the thiol.

5.3 Disulphide Polymers for Colon Drug Delivery Although disulphide polymers are secure in the upper part of the GIT, the bonds are vulnerable to degradation in the colon due to the low redox condition and exposure to reducing agent, such as GSH in cancer cells. The high stability of disulphide polymers can be contributed to the oxidation through air by thiol groups (aqueous solution), which causes self-assembly of the disulphide cross-links during the swelling process. This could prevent the premature release of drugs encapsulated by polymers during the transit through the GIT, thus favouring the use of disulphide polymers for orally administered drugs [11]. Disulphide polymers are mucoadhesive polymers as the thiolated polymers (thiomers) form covalent bonds. These polymers can also be cleaved (reversibly) by the colon (low reduction potential) [3]. Also, the standard reduction potential for disulphide bond is approximately 250 mV [11]. Polymers with disulphide bonds are also biodegradable as disulphide reducing agents (e.g. GSH) can degrade polydisulphides into oligomers or small molecules to be secreted later from the body [12]. Polysaccharides are also capable of swelling upon hydration during the passage along the GIT, which creates a barrier around the carrier that prevents drug diffusion. For colon-targeted drug delivery (CTDD), polysaccharide polymers are mostly selected because of their unique bioadhesive and mucoadhesive properties in the colon.

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5.4 Colon-Targeted Drug Delivery (CTDD) CTDD aims for the drug to be absorbed in the colon instead of in the upper GIT, i.e., the stomach and small intestines. This CTDD system is suitable for delivery of substances highly susceptible to chemical and enzymatic degradation in the upper GIT, such as therapeutic proteins and peptides [13]. The drug can be delivered to the colon through oral or rectal route. Rectal administration allows shorter period of drug delivery to the colon via the rectum, but it suffers from poor patient compliance. Oral administration of the drug is preferred over the rectal route as it is more convenient to the patient. The drugs formulated for oral intake are also cheaper as they do not need to be sterilised, lowering the manufacturing cost. The major challenge in formulating oral drugs for CTDD is the need for the drugs to be released in the colon and not earlier in the stomach or small intestines. An efficient CTDD depends on the drug’s physicochemical properties. The suitable drug candidates for CTDD include drugs that are highly eliminated in the first-pass metabolism (e.g. corticosteroids) [14], drugs that degrade easily in acidic and enzymatic conditions in the stomach (peptidebased drugs such as insulin and anticancer agents). The drug must exhibit local effects in the colon for the treatment of intestinal diseases, which include ulcerative colitis (UC), colorectal cancer, Crohn’s disease, irritable

Oesophagus

Stomach Duodenum

Splenic flexure

Hepatic flexure Tansverse colon Jejunum Ascending (proximal) colon Caecum Appendix

Descending (distal) colon Ileum Sigmoid colon Rectum Anus

Figure 5.1  Anatomy of the human GIT.

Bioadhesive Disulphide Polymeric Nanoparticles  127 bowel syndrome and even less severe condition such as constipation. Apart from having local effects, the drugs should have systemic effect for the treatment of some other diseases, including asthma, hypertension and diabetes [15]. The GIT can be divided into two main components. The first part is the upper GIT, which covers the oesophagus, stomach and duodenum. The second part is the lower GIT, covering the small and large intestines (Figure 5.1). The small intestine, which measures approximately 7 m, covers the duodenum, jejunum and ileum. In addition, large intestine stretches from the ileum (distal end) at the ileocecal junction until the anus, dividing into three important sections: the colon, rectum and anal canal. The length of the colon is about 1.5 m and separated into caecum, ascending (proximal) colon, hepatic flexure, transverse colon, splenic flexure, descending (distal) colon and sigmoid colon. The main functions of the colon are to store the faecal material and absorb minerals and water from the digested food [16].

5.4.1 Condition of the Colon for Drug Delivery The physiological conditions in the colon provide many opportunities to improve drug delivery. The colon has less enzymes that could degrade the drugs compared with other sites in the GIT, such as the duodenum and stomach. The widely used approaches for CTDD are based on the difference in pH along the GIT, transit times and presence of microbial activity in the colon. pH in the GIT is influenced by diet, state of diseases and intestinal motility of the individual. In the stomach, the highly acidic condition provides a pH between 1 and 2, which increases to 6 in the duodenum. In the small intestines (distal), the pH increases gradually from 6 to 7.4 in the terminal ileum [17]. Then, the pH increases again from 6.8 (proximal colon) to 7.2 (distal colon) [18]. Microbial colonisation occurs in the colon because of low redox potentials, slow intestinal motility, and comparatively neutral environment [19]. These ideal conditions for bacterial growth have resulted in the high amount of bacteria found in the colon, approximately 1011–1012 CFU/mL [20]. Approximately 99.9% of more than 400 types of bacterial species in the colon are constraint anaerobes. The prevailing anaerobic bacteria include Bacteroides, Peptostreptococcus, Eubacterium, Bifidobacterium, Ruminococcus, Bacillus, Fusobacterium, Clostridium, Lactobacillus, Enterobacter and Enterococcus [21]. Bacteria contribute largely to a number of intestinal enzymes responsible for the hydrolytic and enzymatic activities in the colon. These activities are involved in the metabolism of xenobiotics and biomolecules, deactivation of harmful metabolites

128  Bioadhesives in Drug Delivery and fermentation of carbohydrates and proteins [21]. Enzymatic activity by anaerobic bacteria in the GIT also causes reduction in redox potentials along the GIT to the level of -415 ± 72 mV in the right (ascending) colon [16]. The transit period of substances plays a crucial role in drug bioavailability. The transit period, which is also known as the transit time of intestinal–colonic, is affected by several factors including diet, mobility, stress level and state of disease. As for small intestine, the average transit time is approximately 3–4 h [22]. Colon takes a longer transit time compared to small intestine, which can range between 20 h and 70 h [18, 23]. The particle size of the substances residing in the colon also affects the transit period, wherein small particles take longer to transit compared with large particles.

5.4.2 Approaches for Colon Drug Delivery Oral administration of the drug in the form of prodrug could improve its bioavailability and avoid early drug degradation in the upper GIT. Colontargeted prodrug is the inactive derivative of a drug molecule, which becomes an active ingredient upon enzymatic hydrolysis in the colon. One approach is the formation of disulphide bond conjugates between the prodrug and the carrier that protects the drug. The disulphide bond is stable in the upper GIT and can be broken down by intracellular enzymes and extracellular reduction process by colonic bacteria. This leads to the breakage of the carrier and release of the prodrug into the colon. The use of nanotechnology has drastically improved current drug formulations for enhanced absorption and site-targeted drug delivery. The use of carrier in nanodelivery systems helps in delivering the active compound, which is attached to or encapsulated in the carrier, to the diseased tissues in the colon. The use of NPs in drug formulations requires lower dose of drug for therapeutic effect than the amount necessary for conventional dosing. NPs designed with different charges are used for different purposes in treating colonic diseases. For instance, polycation NPs would adhere to negatively charged inflamed tissues of the intestinal mucosal surface in bowel diseases, whereas polyanion NPs interact with positively charged components (e.g. cytokines) in colorectal cancer. At the inflamed areas of the colon, NPs are also prone to be taken up by macrophages, thus allowing the drug to remain in the colon for a longer period. Different kinds of polymers have been used in designing NPs, which are capable of entrapping drugs in the polymer matrices for targeted release in the colon. Polymeric NPs release drugs selectively via the enzymatic

Bioadhesive Disulphide Polymeric Nanoparticles  129 activity of the colonic bacteria or by manipulation of the transit time in the colon for time-based release NPs. Here, the NPs function as a bioadhesive system by keeping the formulation in contact with the organ for a prolonged period, which may later increase drug absorption in the colon. The bioadhesion involves the attachment of anionic NPs to the inflamed tissues by electrostatic interactions with the proteins (positively charged), such as the eosinophil cationic protein and transferrin [24]. The NPs developed as a mucoadhesive system use positively charged nanocarriers, which can adhere to the negatively charged intestinal mucosa [25]. This cationic delivery system can be utilised in GIT targeting for cellular uptake of the drug released into the mucosal surface. This system can also decrease drug elimination during intestinal motility in inflammatory bowel disease (IBD). Polymer modification for CTDD is also performed by designing the polymer into pH-dependent nanocarriers. The polymer does not break in the stomach (acidic pH) and proximal small intestine, therefore protecting the active ingredients residing in the carrier. The pH-sensitive nanocarrier only breaks or degrades in the basic pH environment in the terminal ileum, thus releasing the drug only in the colon. This process creates a delayedrelease profile of the drug formulation. One common polymer that behaves in such way is the methacrylic-based copolymer known as Eudragit® [26]. The efficiency of NPs as a drug carrier is due to their nanometre scale sizes. Nanosized particles allow epithelial permeability and retention effect, which increase selective delivery into the colitis tissues [27], avoids elimination by diarrhoea [28] and increases uptake by the immune cells accumulated at the inflamed region of the colon. The pathological changes in certain colonic diseases affect the efficiency of CTDD. In colon cancer, the pathophysiological changes, such as crypt distortions, mucosal surface alterations, increased mucus production, and ulcers, help in the accumulation of drugs or NPs in these inflamed colonic regions.

5.4.3 Limitations of CTDD Oral administration of drugs is preferable as it enables patients to have full control on self-medication and avoid pain and chance of contamination from parenteral route injections. However, drugs taken orally must travel through the entire GIT to reach the colon at the distal end of the alimentary canal. In this case, the drug will be exposed to acidic condition and various enzymatic activities in the stomach. These conditions pose a risk for drug degradation and destabilisation of the drug formulations. The drug may also undergo peristalsis caused by the muscular contractions in

130  Bioadhesives in Drug Delivery the stomach wall. Subsequently, the drug would face another risk of degradation and inactivation by the activity of pancreatic enzymes, bicarbonate and bile salts from the bile duct. Drugs in the GIT are likely to mix with these enzymes with the help of mechanical pressure in the colon, thus contributing to drug breakdown. Apart from this, the semisolid luminal contents that act as a physical barrier may prevent the drug from reaching the mucosal wall and limit the access to the inflamed areas. CTDD is also influenced by the inconsistency of transit time, pH value and fluid volume in the colon. All these factors are highly dependent on food and metabolic enzymes present in the colon. The variability in transit time can be affected by the type of colonic disease. For example, patients with ulcerative colitis (UC) have approximately 24 h of transit time compared with the average 52 h in healthy individuals [29]. The short transit time may reduce the chance of drug absorption through the mucosal wall in the colon. Variation of colonic pH can be contributed by carbohydraterich diet and polysaccharide-based drugs, which is due to the fermentation of polysaccharides by the microflora in the colon. In the case of IBD, the pH can range from 5.5 to 2.3 from the proximal to distal colon of patients with UC [17] compared with pH 6.8–7.2 in healthy individuals. The difference in the pH values at the colonic sites in individuals with different states of health may alter the efficiency of the CTDD designed to react at the specific pH based on the normal colon condition. This requires the CTDD system to be specifically formulated for each type of colonic disease, which is impractical for general production. The low volume of fluid in the colon causes the formation of high-viscosity luminal content [17]. This condition may affect the dissolution of drugs, thereby limiting the drug bioavailability in local areas. This may also affect the penetration of drugs into the bacteria that are harmful to the colon.

5.5 Nanoformulations of Disulphide Polymers 5.5.1 Thiolated Pectin Polymers Pectin is a type of polysaccharide with negative charges; extracted from beans, leaves or fruits, and contains polygalacturonic acid (partial methyl esters), with approximately 200 kDa in molecular weight [30-33]. Pectin consists of linear chains of α-(1-4)-linked -galacturonic acid residues with occasional α-(1-2)-linked -rhamnose. Some rhamnogalacturonic acid residues and α- -rhamnopyranose by α-1-2 linkage break the continuity of linear chains of (1–4) linked α- -galacturonic acid residues. Given its

Bioadhesive Disulphide Polymeric Nanoparticles  131 biodegradability, biocompatibility and non-toxicity, pectin has great potential use as a CTDD nanocarrier [32-34]. The bioadhesive property towards GI tissues prolongs the retention period in the upper GIT and optimal drug delivery in the colon due to the presence of colonic enzymes [35-36]. Pectin is used for CTDD due to its degradability by colonic pectinolytic enzymes [35], not by gastric or intestinal enzymes. Given the presence of hydroxyl groups, pectin is also naturally mucoadhesive by the electrostatic interactions with the mucosal layer. Thiolated pectin improves the mucoadhesive property and increases swelling ability and adhesion duration. A simple preparation of redox-responsive thiolated pectin polymers is possible using thioglycolic acid conjugate. Ester bonds between galacturonic acid (hydroxyl group) of pectin and thioglycolic acid (carboxyl group) are formed as a result of covalent linkage [36]. This method yields 48% of thiolated pectin through precipitation in an aqueous solution using methanol. Using the Ellman’s method, 0.60 ± 0.04 mmol of thiol groups/g was detected in the thiolated pectin [37]. Pectin thiolation was also conducted on cysteine to improve the mucoadhesive property. This conjugation is biocompatible in Caco-cells with 892.27 ± 68.68 µmol thiol groups immobilised per gram of polymer (Figure 5.2) [38]. The stability of thiolated pectin was further tested on pectin–4-aminothiophenol (4-ATP) conjugates, where it was significantly improved compared with pectin alone. The conjugate recorded 802.63 ± 45.9 µmol thiol groups per gram of polymer. The conjugated polymer yield was 65.2% with white, odourless powder appearance and soluble in water [39]. Cheewatanakornkool et al. documented the synthesis of doxorubicin (DOX)–3,3 -dithiopropionic acid, DOX–pectin and cystamine–pectin conjugates [36] using two methods: disulphide bond formation and disulphide bond exchange. The latter was found to be simple, rapid and adequate for DOX–pectin conjugate synthesis. Thiolated pectin–DOX conjugates were observed to be spherical or ellipsoidal aggregates with sizes of 300–500 nm in the hydrated condition. Significant result was observed for in vitro anticancer activity against mouse colon carcinoma cell lines (CT26) compared with the cells treated with DOX only. Given the marked anticancer activity, the thiolated pectin– DOX conjugates might be suitable for delivering DOX to the targeted colon cancer sites.

5.5.2 Thiolated Sodium Alginate (TSA) Polymers Alginate is isolated from brown seaweed and belongs to polyanionic polysaccharides consisting of α-L-guluronic acid and β-d-mannuronic acid [11, 40]. Given the many benefits of alginates, such as biocompatibility,

132  Bioadhesives in Drug Delivery (a)

COOH

COOCH3

HO

OH

OH

O

O OH

O

O

O

O

O COOH

O

COOCH3

OH HO

O

O

HN

OH

O

O

O

OH

O

O

OH OH

O

O

O

O

O

HO

OH

O OH

O

O

O

NH

O

O

(d)

O

SH

O O

OH

(e)

S

S

CH3

O

C

S

NHO

O

O C OH

HO

OH

OCH2 O

O OHO

HO

(f)

OH

NH2

NH2

O

OH

OH

O

SH

CH3

CH3 O

n

O

O HO

HO H

O

HS

(c)

OH O

CH3

OH

OH

O

H

O

H3CO

O

H3C

O

(b)

HO

C

S

NH O

O OH

HO HO

O

OH O

HO

O H3C

O

OH

H3C OH

O HN

O

O

O

S

O

NH2

S

S

S

O

OH O

O

CH3

CH3

NHOH

S

S O O

O

OH

C

NHO

HO

O OCH O OCH O2 O NHO O2 C O C OC OHO OHO OH OH HO HO HO

Figure 5.2  Structures of (a) thiolated pectin, (b) pectin–cysteine conjugate, (c) pectin–4ATP, (d) cystamine–pectin, (e) DOX–3,3 -dithiopropionic acid, (f) DOX–pectin conjugates (adapted from [36-39]).

non-toxicity, biodegradability, chemical versatility and mucoadhesiveness, they are used in oral drug delivery systems. Thiolated alginate was first used by Bernkop-Schnurch et al. [41] to improve the mucoadhesive property of alginate by covalent attachment of cysteine to the polymer. Since then, many attempts have been made to utilise thiolated alginate in improving drug delivery systems. The presence of carboxylic acid groups on the sodium alginate (SA) structure gives pH sensitivity and ion exchange property to the polymer [40], making SA a useful excipient or carrier for drug delivery. Studies have reported that mucoadhesive thiolated polymer can be used as an effective excipient for numerous drug delivery systems as it can prolong residence time and increase stability [41]. Various TSA polymers have

Bioadhesive Disulphide Polymeric Nanoparticles  133 been synthesised for CTDD. Chang et al. [11] synthesised SA with 4-ATP through an oxidative state using sodium periodate, forming a Schiff base after reacting with 4-ATP. The synthesised TSA conjugate provided free thiol groups in the range of 191–441 µmol SH/g polymer [11]. The authors then developed reduction-responsive and pH-sensitive nanospheres made of TSA modified with hydrophobic 4-ATP. The amphiphilic TSA was able to cross-link in deionised water (self-assembly) to form nanospheres with model drug 5-aminosalicylic acid (5-ASA) used for inflammatory bowel disease treatment. The 170 nm core–shell nanospheres showed no apparent toxicity towards Caco-2 cells. In vitro drug release study showed that 25% of 5-ASA was released from the nanospheres in 15 h in the medium without GSH but displayed a marked increase in drug release in the pH 6 medium in the presence of GSH. The authors suggested the disulphide cross-linked nanospheres to be capable of improving the 5-ASA delivery to the inflammatory bowel disease sites. A similar synthesis method for thiolated SA was also carried out by Ayub et al. [42] using cysteamine as the conjugator. This conjugation involves a short-chain aminothiol (2 carbons) compared with the benzene ring in 4-ATP conjugates. The cysteamine moieties attach covalently to alginates, making them better redox-responsive disulphide polymers for selfassembly nanoencapsulation [42]. The authors formulated TSA-derived nanospheres to enhance the chemotherapeutic drug paclitaxel (PCX) delivery to the colonic cancer cells. The nanospheres were ­cysteamine-based disulphide cross-linked SA and were prepared using a layer-by-layer approach. The measured average size of the nanospheres was 173.6 ± 2.5 nm with low size variability (polydispersity index of 0.394 ± 0.105). The PCX-loaded nanospheres had 77.1% encapsulation efficiency and 45.1% of cumulative drug release in the colonic medium with GSH as the reducing agent. These fabricated nanospheres were also internalised by the HT-29 cell lines with significantly reduced viability of the cancer cells. The controlled and sustained drug release behaviour of the nanospheres enables them to deliver PCX to the colon-targeted sites. Conjugation with cysteine and poly(acrylic acid)450 was performed by covalent conjugation of L-cysteine. Amide bonds were formed between primary amino acids and carboxylic acid from SA using 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride. The free thiol groups had approximately 300 µmol (low viscosity alginate) and 50 µmol (medium viscosity alginate) disulphide bonds per gram of polymer for alginate–cysteine conjugates, which is much lower than poly(acrylic acid)450–cysteine conjugate with approximately 1,000 µmol free thiol groups and 100 µmol disulphide bonds per gram of polymer [43].

134  Bioadhesives in Drug Delivery (a)

COO– O

O

OH OH

(b)

COO– O

O HS

OH

O

NH O

R1

NH O OH

SH (c)

OH

(d)

HN

O

O

COOH COOH

O HO

SH

N S

O

NH

O NH

O

OH (e)

O O

O R2 OH OH

Na+

Na+

O

–

O OH NH O– HO OH O O O OH O OH O– O n Na+

O

–O

O O

O O

HO

OH

NH

O

n

HS

Figure 5.3  Chemical structures of SA thiomers (a) alginate-4-ATP, (b) alginate-cysteine (R1 = H, R2 = COOH or R1 = COOH, R2 = H), (c) poly(acrylic acid)450–cysteine, (d) S-nitrosoglutathione-alginate, and (e) alginate–cysteamine conjugates (adapted from [11, 42-44]).

Shah et al. [44] developed S-nitrosoglutathione-alginate by cross-linking S-nitrosothiol with alginate to prolong the delivery time of nitric oxide in the intestines. The thiolation occurred through the covalent attachment of alginate to GSH in suitable pH. This conjugate had 711 ± 24 µmol thiol groups per gram of polymer with 481 ± 22 µmol thiol groups attached to the alginate polymer [44]. Figure 5.3 shows the chemical structures of SA thiomers.

5.5.3 Thiolated Chitosan (TCS) Polymers Chitosan is a type of natural aminopolysaccharide copolymer composed of N-acetyl- -glucosamine and -glucosamine, which is linked by 1-4-βglycosidic bonds. The presence of functional groups, such as the primary

Bioadhesive Disulphide Polymeric Nanoparticles  135 amino group at the C-2 or the hydroxyl group at the C-6 position on the glucosamine unit, allows derivatisation of chitosan for modification of the polymer into carriers suited for targeted delivery [45-46]. As a polysaccharide, the amino groups with positive charge interact with the substructures of the mucus (negative) and mediate the mucoadhesive property together with van der Waals and hydrophobic interactions [47, 48]. This property is enhanced by the conjugation of thiol with chitosan to form a disulphide bond [49]. TCS has a superior mucoadhesion property and enhanced permeation capability [50] from the conjugation of disulphide bonds with the cysteine domains of glycoproteins in the mucus. Thiolation of chitosan can be conducted with various thiolating agents, such as thioglycolic acid, cysteine, N-acetyl cysteine, GSH, 4-thiobutylamidine and isopropyl-S-acetylthiocetimidate. A redox-responsive TCS using thioglycolic acid was synthesised for CTDD, which achieved 60% degree of thiol substitution via Ellman’s method of detection [6]. The mucoadhesive property of chitosan was improved with thiolation of homocysteine thiolactone (HT) to p-coumarin acid (pCA)–chitosan conjugate (Figure 5.4). This conjugation (pCA–HT– chitosan) yielded grafted thiol groups of ~17.6 µmol/g [51]. Conjugation of GSH to chitosan was reported to be via covalent attachment between carboxylic acid (GSH) and amide (chitosan). The thiolated conjugate exhibited 687.32 mol/g of sulfhydryl groups [52]. Anitha et al. [53] added TCS-based mucoadhesive polymers into positively charged NPs by ionic cross-linking reaction. The TCS NPs were loaded with either curcumin (CRC) or 5-fluorouracil (5-FU) into spherical SH H2N H 3C

OH O

O

O

NH C=O

n-x

O

C NH O

OH O OH n-y

O NH2

C=O NH

O

O n

O OH n-z

OH

Figure 5.4  Structure of pCA–HT–chitosan conjugate (adapted from [51]).

136  Bioadhesives in Drug Delivery shape NPs at the size of approximately 150 nm for both formulations. The drug entrapment was resulted from the ionic interaction/hydrogen bonding amongst the loaded drug, TCS and pentasodium tripolyphosphate. Both formulations also had high colloidal stability and blood compatibility. The drug release study (in vitro) showed the profile of sustained release for four days in pH 4.5 and 7.4 for both NP formulations with higher release in acidic condition. The combination of both formulations showed enhanced anticancer effects (2.5- to 3-fold) against human colon cancer adenocarcinoma cells (HT-29) compared with individual and without drug treatments and improved in vivo plasma concentrations for both CRC and 5-FU up to 72 h. Akhlaghi et al. [52] prepared core–shell NPs from poly(methyl methacrylate) coated with chitosan–GSH conjugate to encapsulate insoluble paclitaxel using radical polymerisation method. The surface of the NPs was modified with TCS with different molecular weights in the particle shell to enhance the mucosal permeation property by GSH regeneration mechanism [54]. High PCX encapsulation efficiency (maximum value of 98.27%) was achieved. The PTX-loaded NPs also showed sustained release of PCX for up to 10 days. The presence of TCS–GSH on the surface of NPs improved the mucoadhesion behaviour. However, the NPs showed significant cytotoxicity only against NIH 3T3 and T47D breast carcinoma cells, but not towards colon cell lines HT-29 and Caco-2.

5.5.4 Thiolated Hyaluronic Acid Polymers Hyaluronic acid (HA) consists of alternating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine with beta 1,4-interglycosidic linkage [55], and is classified as a macromolecular polysaccharide. HA is found in most organs, synovial fluid and connective tissues of animals and functions as a lubricant due to its peculiar viscoelastic properties. Given the biodegradable and biocompatible characteristics of HA, it is used in pharmaceutical and medical industries, especially in drug delivery [56]. The development of novel drug delivery systems using HA has been ventured in nasal, oral, topical, ophthalmic and gene delivery. Nevertheless, HA is prone to enzyme degradation by hyaluronidase, which is absorbed by various metabolic pathways [10]. The carboxylic acid and hydroxyl moieties have been studied for the thiolation of HA to prolong the transit time. This is conducted through coupling reaction of L-cysteine ethyl ester and HA to form thiolated HA (Figure 5.5). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) plays a role in mediating the

Bioadhesive Disulphide Polymeric Nanoparticles  137 O

NH2 O

HO

HS

OH

OH

O

OH

O

CH2OH O O OH

O

O O

O

SH NH

Cysteine ethyl ester HO

NH−CO−CH3

EDAC

Hyaluronic acid NHS

OH

O

OH

O

CH2OH O OH

NH−CO−CH3

Hyaluronic acid−cysteine ethyl ester

Figure 5.5  Reaction mechanism of thiolated HA by amide bond formation and covalent attachment of HA with L-cysteine ethyl ester. EDAC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, NHS: N-hydroxysuccinimide (adapted from [57]).

coupling reaction by adding N-hydroxysuccinimide [57]. This conjugate had 201.3 ± 18.7 mol immobilised free thiol groups and 85.7 ± 22.3 mol disulphide bonds per gram of polymer as odourless, fibrous and white powder [10]. Laffleur et al. reported similar thiolation with 250.66 ± 0.39 µmol per gram of HA [58].

5.5.5 Thiolated Dextran Polymers Dextran is a polysaccharide that is soluble in water and consists of a linear α-1,6-linked d-glucopyranose residue with traces of α-1,2-, α-1,3- and α-1,4 branching. Dextran is mainly derived from Lactobacillus, Leuconostoc and Streptococcus species of bacteria. Unlike other types of polysaccharides that have various functional groups, dextran has only hydroxyl (-OH) groups. The abundance of -OH groups contributes to the high hydrophilicity of dextran and allows chemical conjugation with other materials for property modification of the polymer, such as thiol substitution to form thiolated dextran [59]. Kiani et al. [4] developed polyelectrolyte complexes (PECs), nanocomplex systems using chitosan, carboxymethyl dextran and thiolated carboxymethyl dextran, as carriers for sensitive biomaterials. Thiolated carboxymethyl dextran with high thiol moieties showed 74.89 to 273 µmol thiol groups per gram of polymer [4, 60]. PECs were used in nanoformulation of hSET1 antisense using complex coacervation method at acidic pH for injectable or oral delivery. PECs had a size of approximately 115 nm and were positively charged. The nanoformulation was reported as stable in serum, fasting state of simulated intestinal fluid and simulated gastric fluid as the antisense was protected from degradation.

138  Bioadhesives in Drug Delivery Thiolated PECs also demonstrated cellular penetration and inhibited the proliferation of SW480 colon cancer cells, suggesting the potential of the nanocomplex for efficient delivery of nucleic acids and other gene materials.

5.5.6 Other Thiolated Polymers Poly(vinyl alcohol) (PVA) polymers were used as the backbone for thiol conjugation as part of obtaining mucoadhesive polymers for CTDD. Thiolations of PVA were reported using thiourea and 3-mercaptopropionic acid, forming thiolated PVA conjugates (Figure 5.6). Thiol groups from thiourea replaced the hydroxyl moieties of PVA in acidic condition and yielded insoluble white fibrous material. Thiourea–PVA polymer had 130.44 ± 14.99 µmol immobilised thiol groups per gram of polymer. The second thiolation involves esterification of PVA with 3-mercaptopropionic acid as a coupling ligand, producing water-soluble white fibrous material. This thiolated polymer exhibited 958.35 ± 155.27 µmol immobilised thiol groups per gram of polymer, which was higher compared with unmodified PVA (8.71 ± 1.61 µmol/g) [61]. Mucoadhesive natural polymers, such as starch, have been used in CTDD. The mucoadhesive property of starch could be improved by conjugating cysteamine to starch via oxidative cleavage of vicinal diols [62]. Cysteamine was also conjugated with polysaccharide polymers, such as pullulan, to improve the mucoadhesive property. The pullulan–cysteamine conjugate was synthesised via bromination–nucleophilic substitution and reductive amination, with the latter showing higher coupling rates with 1,522 ± 158 µmol immobilised thiol groups and 280 ± 70 µmol free thiol groups per gram of polymer. To activate the disulphide bonds, pullulancysteamine conjugate was further conjugated with 6-mercaptonico­ tinamide, yielding a yellow, fibrous and hydrophobic conjugate [63]. Cysteamine was also conjugated with hydroxyethyl cellulose to enhance the mucoadhesive and permeation properties [64]. This conjugated polymer had 1548 ± 73 µmol immobilised free thiol groups and 248 ± 30 µmol disulphide bonds per gram of polymer. Apart from carbohydrate polymers, amino acids such as histidine, was also used in the synthesis of stimuli-responsive disulphide polymers. John et al. [65] synthesised a series of poly(L-histidine)n-S-S-polyurethane-SS-poly(L-histidine)n [p(His)n-SS-PU-SS-p(His)n; n = 25, 35, 50, 75] triblock copolymers, which consist of redox-responsive disulphide linker at the PU middle block. This two-step polymerisation provides cleavage property to the disulphide bridge in the intracellular cytosol after being

Bioadhesive Disulphide Polymeric Nanoparticles  139 S

(a) H2N n

1. NaOH

NH2 HCI

2. H2SO4

n

OH

n

NH2CI

S

SH

NH2

(b)

O H+ HS

OH

H2O

n

n O

OH

O

HS

(c)

O CH2 OH OH

(d) CH2OH O

O OH

O

NH OH

O

CH2OH O OH O OH

OCH2CH2OH OCH2CH2OH OH

CH2

O

O O

O OH

S

S

NH HO

N HS

O

NH2

(e)

(f)

n O

O NH

OCH3

HS

OH

S S

O N N H H O H N nH N H N NH

H N O O

O

9

H O N O 7

O N H O H H H N nN N H

S S

N NH

O

Figure 5.6  Synthesis of (a) thiourea–PVA conjugate, (b) 3-mercaptopropionic–PVA conjugate, (c) pullulan–cysteamine with aromatic residue 6-mercaptonicotinamide conjugate, (d) thiolated hydroxyethyl cellulose with cysteamine, (e) Eudragit® L100– cysteine conjugate and (f) p(L-histidine)n-SS-polyurethane-SS-p(L-histidine)n (n = 25, 35, 50, 75) triblock copolymers (adapted from [61, 63-66]).

exposed to high concentration of GSH (Figure 5.6). In another study, the carboxylic group of Eudragit® L100 was thiolated with cysteine. This conjugation yielded 390.3 ± 13.4 µmol thiol groups per gram of polymer [66]. Isolation of the polymer conjugates was conducted in the dark at 10 °C via dialysis process.

140  Bioadhesives in Drug Delivery

5.6 Summary Currently, disulphide polymeric NPs have received considerable attention from researchers because of their various application potentials. Disulphide polymeric NPs play an essential role as an efficient biomaterial for biomedical applications, such as mucoadhesive drug delivery. In recent years, the development of a CTDD system that is based on redox-responsive disulphide polymeric NPs for drug delivery has drawn considerable attention. Modifying thiolated conjugates with different derivatives can affect their sensitivity, redox potential, cellular uptake and drug release. Various natural and synthetic polymeric NPs have been designed with redox responsive characteristic for CTDD application. In the near future, they can be utilised as safe vehicles for the treatment of many diseases.

Acknowledgements The authors would like to thank the Ministry of Higher Education, Malaysia for the financial support through Fundamental Research Grant Scheme [FRGS, 203/CIPPT/6711684] Novel thiolated alginate-PLGA aptamer-­ functionalised targeting nanoparticle as multi-drug carrier: Mechanism of cytotoxicity, cellular uptake and multi drug resistance.

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Bioadhesive Disulphide Polymeric Nanoparticles  143 3,4-dihydroxyphenylglycol with pectin and their potential use for colon targeting. Carbohydrate Polymers.163, 292–300 (2017). 34. H. Andishmand, M. Tabibiazar, M.A. Mohammadifar and H. Hamishehkar. Pectin-zinc-chitosan-polyethylene glycol colloidal nano-suspension as a food grade carrier for colon targeted delivery of resveratrol. Int. J. Biol. Macromol. 97, 16–22 (2017). 35. J.H. Cummings and H.N. Englyst. Fermentation in the human large intestine and the available substrates. Am. J. Clin. Nutr. 45, 1243–1255 (1987). 36. K. Cheewatanakornkool, S. Niratisai and S. Manchun. Thiolated pectin – doxorubicin conjugates: Synthesis, characterization and anticancer activity studies. Carbohydrate Polymers. 174, 493–506 (2017). 37. R. Sharma and M. Ahuja. Thiolated pectin: Synthesis, characterization and evaluation as a mucoadhesive polymer. Carbohydr. Polym. 85, 658–663 (2011). 38. S. Majzoob, F. Atyabi, F. Dorkoosh, K. Kafedjiiski, B. Loretz and A. BernkopSchnürch. Pectin-cysteine conjugate: synthesis and in-vitro evaluation of its potential for drug delivery. J. Pharm. Pharmacol. 58, 1601–1610 (2006). 39. G. Perera, J. Barthelmes and A. Bernkop-Schnürch. Novel pectin – 4-amino­ thiophenole conjugate microparticles for colon-specific drug delivery. J Controlled Release. 145, 240–246 (2010). 40. K. Y. Lee and D. Mooney. Alginate: Properties and biomedical applications. Prog. Polym. Sci. 37, 106–126 (2012). 41. A. Bernkop-Schnurch, C.E. Kast and M.F. Richter. Improvement in the mucoadhesive properties of alginate by the covalent attachment of cysteine. J. Controlled Release 71, 277–285 (2001). 42. A.D. Ayub, H.I. Chiu, S.N.A.M. Yusof, E. Abd Kadir, S.H. Ngalim and V. Lim. Biocompatible disulphide cross-linked sodium alginate derivative nanoparticles for oral colon-targeted drug delivery. Artif. Cells Nanomed. Biotechnol.47, 353–369 (2019). 43. A. Greimel, M. Werle and A. Bernkop-Schnürch. Oral peptide delivery: In-vitro evaluation of thiolated alginate / poly ( acrylic acid ) microparticles. J Pharm Pharmacol.59, 1191–1198 (2007). 44. S.U. Shah, M. Socha, I. Fries and S. Gibaud. Synthesis of S-nitrosoglutathionealginate for prolonged delivery of nitric oxide in intestines. Drug Deliv. 23, 2927-2935 (2016). 45. L. Kaur and I. Singh. Chitosan-catechol conjugates - a novel class of bio­ adhesive polymers: A critical review, Rev. Adhesion Adhesives. 7, 51-67 (2019). 46. S. Chen, Y. Cao, L.R. Ferguson, Q. Shu and S. Garg. Evaluation of mucoadhesive coatings of chitosan and thiolated chitosan for the colonic delivery of microencapsulated probiotic bacteria. J. Microencapsulation. 30, 103–115 (2013). 47. I. Bravo-osuna, C. Vauthier, A. Farabollini, G.F. Palmieri and G. Ponchel. Mucoadhesion mechanism of chitosan and thiolated chitosan-poly (isobutyl cyanoacrylate ) core-shell nanoparticles. Biomaterials. 28, 2233–2243 (2007).

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6 Bioadhesive Hydrogels and Their Applications Hitesh Chopra1, Sandeep Kumar2 and Inderbir Singh1* Chitkara College of Pharmacy, Chitkara University, Punjab, India 2 ASBASJSM College of Pharmacy, Bela, Ropar, Punjab, India

1

Abstract

Bioadhesive drug delivery systems are being used for the delivery of active pharmaceutical ingredients through various routes of administration. Bioadhesive hydrogels combine the properties of bioadhesion and extensive swelling ability of hydrogels. Bioadhesive hydrogel films, tablets and nanoparticles for delivering drugs via buccal, transdermal, gastrointestinal, parenteral, vaginal and rectal routes have been discussed systematically in this chapter. The aim of this chapter is to present a comprehensive review on the applications of bioadhesive hydrogels for different routes of drug administration. Patents filed on bioadhesive hydrogels have been compiled and tabulated. Biomimetic hydrogels, physiologically responsive hydrogels, composite hydrogels, and 3D construct hydrogels can be seen as the future of bioadhesive hydrogels for developing targeted drug delivery systems. Keywords:  Hydrogels, bioadhesive hydrogels, films, nanoparticles

6.1 Introduction Bioadhesion is the phenomenon that describes attachment of two different materials out of which one should be biological in nature and the adhesion should prevail for long periods of time [1]. Bioadhesion term can also be used in drug delivery and dental and surgical applications. Therefore, due to its wide applications it has attracted researchers across the globe.

*Corresponding author: [email protected] K.L. Mittal, I. S. Bakshi and J. K. Narang (eds.) Bioadhesives in Drug Delivery, (147–170) © 2020 Scrivener Publishing LLC

147

148  Bioadhesives in Drug Delivery In biological systems, bioadhesion can be classified into 3 types [2]: • Type 1, adhesion between two biological phases, for example, platelet aggregation and wound healing. • Type 2, adhesion of a biological phase to an artificial substrate, for example, cell adhesion to culture dishes and biofilm formation on prosthetic devices and inserts. • Type 3, adhesion of an artificial material to a biological substrate, for example, adhesion of synthetic hydrogels to soft tissues and adhesion of sealants to dental enamel [3]. In terms of drug delivery, bioadhesion may be defined as the attachment of drug carrier system to a certain biological location. The biological location referred here can be an epithelial tissue or a mucus layer on a tissue surface. Bioadhesion can be defined as the binding of a natural or synthetic polymer to a biological substrate. When this substrate is a mucous layer, the term used is mucoadhesion. Leung and Robinson had defined mucoadhesion as the interaction between a mucosal surface and the polymer [4]. The mucosal layer lines a number of regions of the body including the gastrointestinal tract, the urogential tract, the airways, the ear, nose and eye. Hence, mucoadhesive drug delivery systems could be designed for potentially targeting buccal, oral, vaginal, rectal, nasal and ocular routes of administration.

6.1.1 Bioadhesive Polymer A polymer is said to be an ideal bioadhesive if it satisfies the following properties [5, 6]: 1. The polymer itself and its degradation moieties should be non-toxic, biodegradable and non-absorbable. 2. It should be capable of forming strong bonding with the mucus or any other biological surface. 3. It should be capable of adhering quickly and strongly to the surface. 4. It should be easy to formulate with the drug and should not interfere with the drug release pattern. The different mucoadhesive polymeric systems and their routes of administration have been described in Table 6.1.

Bioadhesive Hydrogels and Their Applications  149 Table 6.1  Different mucoadhesive polymeric systems and their application. Route of administration Buccal

Carrier/ polymeric system

Drug used

Purpose/ application

Mucoadhesive tablets

Miconazole nitrate, Ketoconazole

For HIV-positive patients suffering from oropharyngeal candidiasis

Tablets (Nitrogard®)

Nitroglycerin

To take care of angina attack

Patches

Verapamil hydrochloride

Preventive medication for migraine

Carvedilol

To prevent left ventricular dysfunction following myocardial infarction

Glipizide

Most potent of the sulfonylurea antidiabetic agents

Clotrimazole

For oral Candida infections

Glibenclamide

Used in the treatment of maturityonset diabetes

Films

(Continued)

150  Bioadhesives in Drug Delivery Table 6.1  Different mucoadhesive polymeric systems and their application. (Continued) Route of administration

Carrier/ polymeric system

Drug used

Ophthalmic

Liposomes

Pilocarpine HCl

For increased miotic response and ocular bioavailability of the drug

Nanoparticles

Amikacin, Metipranolol, Indomethacin

For the treatment of respiratory diseases and to treat glaucoma

Suppositories

Acetaminophen

Antiinflammatory analgesic

Solid dispersions and tablets

Benzydamine

–

Clotrimazole

Antifungal activity, antifungal chemotherapy

Liposomes

Acyclovir

Anti-HIV

Aqueous solutions

Apomorphine

To treat Parkinson’s disease

Microspheres

Gentamicin

Antibiotic

Vaginal

Nasal

Purpose/ application

Reproduced with permission from Elsevier [7].

6.1.2 Hydrogels Hydrogels are three-dimensional structures that have the ability to absorb large amounts of water, causing swelling of the network [8]. The term Hydrogel when searched on Google produced about 11,90,00,000 results. The discovery of hydrogels was serendipitous. A researcher named Lim

Diameter of 3 mm and length of 3.5 cm

Cylinders

Reproduced with permission from Elsevier [30].

Diameter of 14 mm and thickness of 0.8 mm

Discs

Implants

Diameter of 2 mm and total weight of 1 mg (round-shaped)

Circular inserts Variable

N/A

Suspensions, Ointments

Dressings

Hydrogel particles present in the eye drops must be smaller than 10 μm

Drops

Transdermal

Conventional dimensions (typical diameter ≈12 mm)

Contact lenses

Length of 30 mm and thickness of 10 mm

Torpedo-shaped pessaries

Ocular

Height of 2.3 cm, width of 1.3 cm and thickness of 0.9 cm

Vaginal tablets

Vaginal

Conventional adult suppositories with length ≈32 mm, a central cavity of 7 mm and wall thickness of 1.5 mm

10–1000 nm

Nanoparticles

Suppositories

Diameter of 0.8 cm and thickness of 1 mm

Discs

Rectal

1 μm to 1 mm

Spherical beads

Peroral

Typical dimensions

Shape

Route of administration

Table 6.2  Main types of hydrogel-based products applied via different routes of drug administration.

[28, 29]

[27]

[26]

[25]

[24]

[23]

[22]

[21]

[20]

[19]

[18]

[17]

[15, 16]

References

Bioadhesive Hydrogels and Their Applications  151

152  Bioadhesives in Drug Delivery was working on the formulation of soft contact lenses using methacryloylated poly (vinyl alcohol), and was trying to synthesize a polymer that could hold water and was physically or optically transparent. He found that he had formulated such polymer which could hold 80-90% of water but the formulation failed to handle stress [9]. The water absorbing capacity of a hydrogel is directly related to the presence of functional groups attached to the backbone of the molecule. Due to the presence of different functional groups, the hydrogels possess a variety of different characteristics such as porosity, structural softness, swelling character and elasticity. The hydrogel when swollen provides flexibility comparable to natural tissues. Adding more water-loving neutral moieties also tends to increase the degree of volume transition of corresponding hydrogels. Hydrogels also exhibit drastic volume changes in response to external stimuli such as temperature, solvent, pH and electric field [10, 11]. Degree of swelling is an important parameter for characterising hydrogels. The degree of swelling is dependent upon network density, nature of solvent and polymer-solvent interaction [12, 13]. It has been found that the hydrogels containing more hydrophilic functional groups have stronger interaction with the swelling medium which is the leading cause of expansion of hydrogels. However, the cross-linking is up to a certain limit, after that it prevents its expansion and contraction [14]. Hydrogels have been explored for the delivery of therapeutically active agents through different routes, viz. peroral, ocular, transdermal, vaginal, and rectal (as shown in Table 6.2). The gel strength is provided via connections based on physical crosslinking or chemical cross-linking. Once the gel comes in contact with the aqueous phase the gel turns into a solid and becomes insoluble and chains

Hydrogels

Preparation

Ionic charge

Source

Cross-linking

Homo polymeric

Cationic

Natural

Physical

Co-polymeric

Anionic

Synthetic

Chemical

Interpenetrating

Non ionic

Hybrid

Response

Chemical

Biochemical

Physical

pH

Ligand

Temperature

Glucose

Antigen

Light

Oxidant

Enzyme

Pressure

Figure 6.1  Different classification criteria for categorization of hydrogels.

Catechol conjugated chitosan cross-linked with thiolated Pluronic F-127

HA–catechol conjugation via 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)

Dopamine conjugated HA and thiol-terminated Pluronic F-127 cross-linked by catechol–thiol reaction

Self-healing in catechol-functionalized acrylic polymers initiated by catechol-mediated hydrogen bonding

DOPA/iron coordination in response to pH in DOPA-functionalized polyallylamine

Hyaluronic acid (HA)

HA & Pluronic F-127

Polyacrylate & polymethacrylate

Polyallylamine

Mechanisms of self-healing and/or environmental sensitivity

Chitosan & Pluronic F-127

Polymers

[37]

[38]

Temperature sensitive, rapid and reversible sol–gel transition, and excellent tissue adhesion Metal-free underwater self-healing

(Continued)

[39]

[36]

Biocompatible with neural stem cell and pH-sensitive adhesion

High mechanical strength and fast self-repair behavior in alkaline condition

[35]

Reference(s)

Temperature sensitive, wet adhesion, and hemostasis

Properties

Table 6.3  Self-healing, pH-sensitive and temperature-sensitive mussel inspired hydrogel tissue adhesives.

Bioadhesive Hydrogels and Their Applications  153

[46]

Rapid pH-sensitive gelation through Fe– catechol complexes

Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.

PU hydrogels containing hexamethylene diisocyanate as hard segment, PEG as soft segment, and lysine–dopamine as side chain

Polyurethane (PU)

[45]

[44]

Provide on-demand photocuring and strong adhesion

Highly branched PEG–catechol based thermoresponsive copolymers synthesized via a onepot Reversible addition-fragmentation chain transfer (RAFT) polymerization

Rapid self-repair adhesive polymer and multiple stimuli-responsive behavior

[42, 43]

pH dependent adhesive performance

Four-armed PEG end-capped with dopamine

Boronate ester linkages provide self-healing property in alkaline condition

[41]

Self-healing hydrogels with pH, glucose, and dopamine responsiveness and biocompatibility

Phenyl borate–catechol complexation in hydrogels containing dopamine functionalized 4-armed PEG and phenylboronic acid modified 4-armed PEG

Reference(s) [40]

Properties Cross-linked network with elastic modulus approaching covalently cross-linked gels and with self-healing

3+

pH-induced metal–ligand (catechol–Fe ) cross-linking in DOPA-modified PEG

Mechanisms of self-healing and/or environmental sensitivity

Poly(dopamine methacrylateco-N-isopropylacrylamide)

Poly(ethylene glycol) (PEG)

Polymers

Table 6.3  Self-healing, pH-sensitive and temperature-sensitive mussel inspired hydrogel tissue adhesives. (Continued)

154  Bioadhesives in Drug Delivery

Bioadhesive Hydrogels and Their Applications  155 get interlinked to form a network. The hydrogels can be further divided into different categories as shown below. Hydrogels can be classified based on different criteria such as preparation method, ionic charges, sources, cross-linking properties, responses, physical properties (as described in Figure 6.1).

6.1.3 Bioadhesive Hydrogels The hydrogels having capability of getting attached to the biological membranes are called bioadhesive hydrogels [31-34]. They are also known as mucoadhesive hydrogels when the biological surface is the mucous membrane. The bioadhesive hydrogels have advantages over the conventional hydrogels as they provide a longer residence time compared to the other delivery systems, maintaining a high drug concentration at the target site and hence providing longer action period and reduced dose frequency. The bioadhesive hydrogels investigated by various researchers have been compiled in Table 6.3.

6.2 Bioadhesive Hydrogel Films Bioadhesive hydrogel films have been investigated by a number of researchers for various drug delivery applications. Pagano et al. [47] had formulated usnic acid (UA) based hydrogel films for wound burn treatment. The UA is a lichenic metabolite that has low solubility and high permeability (BCS Class II). UA has been found to have a good antimicrobial property against infection causing strains such as both Gram-positive bacteria (such as E. faecalis, E. faecium, S. aureus, S. epidermidis, S. mutans, S. pyogenes) and anaerobic bacteria (such as Bacteroides fragilis, Bacteroides ruminicola ssp. Brevis, Bacteroides thetaiotaomicron, Bacterioides vulgatus, Clostridium perfringens, Propionibacterium acnes). The films thus formed were found to increase the bioavailability of UA. Hydrogel films were formulated employing sodium carboxymethyl cellulose 2% alone (F1), sodium carboxymethyl cellulose mixed with PVP K90 0.1% (F2) or with Carbopol 971P 0.1% (F3). The ex vivo studies were conducted on pig skin and the results showed that films with formulation F2 showed greater antimicrobial activity [47]. Ito et al. formulated water swellable film of poly(acrylic acid) (PAA) and poly vinylpyrrolidone (PVP) cross-linked via hydrogen bonding. Simply mixing the PAA and PVP resulted in the formation of hydrogel film [48]. The PAA solution was dried at room temperature to form the film on a plate and later the PVP solution was added to the initially formed PAA

156  Bioadhesives in Drug Delivery film. Drying resulted in the formation of a transparent film that swelled in aqueous media. The hydrogel thus formed slowly dissolved in body tissues. However, the dissolution of films was found to be related to the molecular weight and cross-link density of the polymers. The bioadhesive hydrogels could significantly absorb blood or body fluids and are useful for medical devices such as hemostats, wound dressings, and drug release devices.

6.3 Bioadhesive Hydrogels for Gastrointestinal Delivery Gastroretentive drug dosage forms (GRDDFs) are a boon due to their long residence time for the class of drugs that are absorbed in the proximal part of GI tract. After ingesting the drug loaded GRDDFs, these are retained in the gastrointestinal tract and release the drug in a sustained manner. This leads to increase in residence time and maintains the drug for long periods at therapeutic levels [49]. There are three practically applicable designs of GRDDFs: (1) mucoadhesion to the gastric mucosa to extend the residence time of GRDDFs in the stomach; (2) density modification (floating or sinking) to make the dosage form float or sink and prevent it from leaving the stomach due to limited access to the pylorus; and (3) expansion (swelling) of a GRDDF to a size that is too large to pass through the pylorus, thus prolonging gastric retention [50]. Su et al. formulated complex hydrogels of chitosan and ring opened polyvinylpyrrolidone (PVP) to enhance the bioavailability of biphosphonates [51]. The biphosphonates have the drawback that they cause irritation of mucous membrane of upper GIT, and have lower bioavailability when administered orally. The researchers formulated polyionic complexed hydrogels of chitosan (CS) and PVP that possessed mucoadhesive and swelling ability for prolonged release of alendronate in the upper GIT. The positive and negative charge based interaction between alendronate sodium and chitosan decreases irritation of upper digestive mucosa.

6.4 Bioadhesive Hydrogels Administered through Injection Surgical site infection results in prolonged wound healing, abscess formation, dehiscence, and sepsis also in many cases. The patients with such issues are re-admitted many times in the intensive care units [52]. To counter such concerns many researchers have formulated injectable bioadhesive

Bioadhesive Hydrogels and Their Applications  157 Bioadhesive component O

O

HO HO

O

O OH

Antimicrobial component H2N

NH3 O H2N

O

N

N NH H

N H2

O

NH

H2N

Polydextran aldehyde (PDA)

H2 N

N

NH3

NH2

n

Ployethylenimine (PEI)

O O N

N

O

O

O

O

N N H2 NH

H N

NH2

N

Figure 6.2  The precursor components of the adhesive, PDA and PEI, define the material’s bioadhesive and antibacterial properties, respectively.

hydrogels. Giano et al. described the design, synthesis and evaluation of a novel syringeable bioadhesive hydrogel system prepared from polydextran aldehyde and branched polyethylenimine as shown in Figure 6.2 [53]. The hydrogel thus formed was shown to be perfect dead space filler for wound closure and to prevent bacterial growth and infection. The filler was able to kill Gram-positive as well as Gram-negative bacteria but did not kill human erythrocytes. Animal study was conducted and murine infection model showed that formulation was able to kill streptococcus pyrogenes. Mohammadreza et al. formulated citric acid based mussel inspired bioadhesive hydrogels that can be used for the sutureless wound closure [54]. The citric acid was used here to accelerate the degradable ester bond formation and it also increases the hemocompatibilty and hydrophilicity of the polymer (as described in Figure 6.3). The iCMBAs are strong wet tissue adhesives that can seal the open wound and stop bleeding immediately. These hydrogels can be of interest in sutureless wound closure where there is lack of collagen structure and fascia. Jin et al. formulated injectable hydrogel based on hyaluronic acid and poly(ethylene glycol) that can act as a biodegradable matrix for cartilage

158  Bioadhesives in Drug Delivery (a)

(c) Component 2 (Initiator solution)

H N

O OH

N

iCMBA Network

O

Bonding to Tissue Component 1 (iCMBA solution)

(b)

N

H O

C

O H

O

Figure 6.3  Schematic of injectable citrate-based mussel-inspired bioadhesives (iCMBAs) application for wound closure. (a) Preparation and application of 2-component adhesive: iCMBA and oxidizing (sodium periodate) solutions. (b) Schematic representation of iCMBA utilized for sutureless wound closure. (c) Proposed mechanism of iCMBA adhesion to tissue. Reproduced with permission from Elsevier [54].

tissue engineering [55]. Initially the hyaluronic acid and poly(ethylene glycol) were thiolated and then cross-linked via Michael addition reaction to form three-dimensional hydrogel. The gelation time was found to vary from 14 min to 1 min depending on grades of hyaluronic acid and poly(ethylene glycol) used. In vitro study showed that chondrocytes sustained for a period of 3 weeks and cell division also took place. Hoque et al. reported the preparation of injectable hydrogels capable of inherent bioadhesive, anti-bacterial and hemostatic capabilities for wound closure applications [56]. The hydrogels were prepared in situ using N-(2hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) and a bioadhesive polymer polydextran aldehyde. The gels thus formed were found to be active against both Gram-positive and Gram-negative bacteria and also against drug resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE) and beta-lactam-resistant Klebsiela pneumoniae. The mechanism of action against bacteria was disclosed and it was found that they acted by disrupting the membrane. The gels were found to be effective for preventing sepsis in a cecum ligation and puncture model in mice.

Bioadhesive Hydrogels and Their Applications  159 Lowman and coworkers formulated an injectable bioadhesive hydrogel as a synthetic replacement for the nucleus pulposus of the intervertebral disc or as an annulus closure material [57]. In the study, the copolymers of poly(N-isopropylacrylamide) PNIPAAm and poly(ethylene glycol) were blended with the polyethylenimine (PEI), to forms gels at physiological temperature and injected. This will be followed by the injection of aqueous dialdehyde solution into the gel core, which will cross-link PEI and then continue to diffuse through the gel and cross-link it to the tissues in contact with the implant. Houque et al. prepared vancomycin loaded dual function injectable bioadhesive hydrogels for treatment of infection in avascular or necrotic tissues [58]. The hydrogels were prepared from N-(2-hydroxypropyl)3-trimethylammonium chitosan chloride with vancomycin. The encapsulation occurs due to the presence of irreversible pH-sensitive imine bond present between vancomycin and polydextran aldehyde. The gels have been able to kill the bacteria on coming in contact with them and release the antibiotics.

6.5 Bioadhesive Hydrogels for Vaginal Delivery The vaginal cavity is one of the important sites for the administration of drugs; this route offers many advantages such as avoiding first-pass effect. Many of the studies have shown a higher potential of vaginal delivery in comparison to oral drug delivery. A vaginal bioadhesive system has many advantages compared to the conventional one such as easy localization in the region of application and improved bioavailability of drugs. Timur et al. formulated chitosan based nanoparticles for treatment of HIV contacting anti- viral agent Tenofovir [59]. The mucoadhesion is caused via blends of Poloxamer 407 and chitosan mixed in different ratios. A certain amount of Tenofovir was entrapped in the gel, with the aim of controlling the leakage of drugs from the nanoparticles. The in vitro analysis evidenced an initial burst release of Tenofovir from the formulation made up of nanoparticles and Poloxamer gel, compared to the gel containing the active compound in the free form or to a solution of the drug. The total amount of Tenofovir released from the nanoparticle/gel system was 85% in 24 h, while that of the drug contained in the Poloxamer gel was ~95%. Both of these values were dramatically better than that obtained by the drug solution, which evidenced full leakage of the active compound over a 3 h period. The entrapment of Tenofovir within the nanoparticles retained by the Poloxamer-gel had two special features: (1) the burstrelease effect, induced by the presence of the free drug within the gel

160  Bioadhesives in Drug Delivery network, and (2) sustained drug leakage for up to 24 h due to the presence of the colloidal system in the polymeric matrix. Demiröz et al. developed mucoadhesive and thermosensitive gels for vaginal delivery that were able to provide a controlled release of the model drug, cidofovir [60]. The drug loaded vaginal gel was prepared using different types of polymers such as Carbopol 974P, HPMC and Poloxamer 407. It was found that the formulation containing HPMC had the highest visocity, adhesiveness, cohesiveness and mucoadhesion property. For in vitro model study the herpes virus type 1 was used to observe the antiherpetic action of gel. Gafiţanu et al. developed superporous hydrogels based on freeze-drying method [61]. The resulting porous microarchitectures were influenced by the composition of hydrogel formulation and temperature. The anise based hydrogels were prepared in liquid nitrogen by freezing them and generating a regular assembly of polyhedral pores. For the hydrogels of Carbopol 934, the hydrogels showed solid-like behaviour due to dense network structure and entanglement, while formulation of sodium alginate showed the viscoelastic behaviour due to formation of pseudo-gel structure.

6.6 Bioadhesive Hydrogels for Rectal Delivery Jinke et al. formulated a mucoadhesive hydrogel to improve the efficacy of rectal sulfasalazine (SSZ) administration [62]. The mucoadhesive gel was prepared from catechol modified chitosan cross-linked with genipin. Animal model mouse was used for evaluation of its effect on ulcerative colitis. It was found that the animal group which was treated with rectal formulation showed better results compared to oral delivery. A pharmacokinetic study of acetaminophen loaded suppository made up of Carbopol P407 and P188, and sodium alginate (as mucoadhesive agent) exhibited faster absorption of drug when compared to conventional suppositories which was attributed to significant bioadhesive features [63]. Recently, Liu et al. developed a thermo-sensitive in situ gel made up of Poloxamer 407, HPMC, and sodium alginate for the rectal delivery of ibuprofen [64]. The ibuprofen belongs to the poorly soluble category. The HPMC and sodium alginate used decreased the sol-gel transition temperature of Poloxamer while increased the strength of gel. In situ gel supposi­ tories exhibited improved pharmacokinetic performance as compared to solid suppositories for rectal delivery of ibuprofen.

Bioadhesive Hydrogels and Their Applications  161

6.7 Mucoadhesive Hydrogels Based Nanoparticles A tough polydopamine-clay-polyacrylamide adhesive hydrogel was prepared by Han et al. [65]. The hydrogel was developed by a two-step procedure. Firstly, dopamine (DA) was intercalated into layers of clay nanosheets where its oxidation was limited in the confined nanospace, resulting in polydopamine intercalated clay nanosheets with free catechol groups. Secondly, acrylamide (AM) monomers were added and polymerized in situ by free radical polymerization in the presence of reaction initiator and cross-linker leading to the formation of adhesive PDA-clay-PAM hydrogels. The hydrogels exhibited high mechanical strength and extensibility and could be adhered to human skin without any damage. Similarly conductive adhesive hydrogels were prepared using carbon nanotubes [66] and graphene oxide [67]. They have been used as wearable or implantable bioelectronic devices [68]. Szymańska and co-workers developed mucoadhesive gelling system of silver nanoparticles modified with tannic acid for the treatment of herpes virus infection [69]. The silver nanoparticles thus formed were found to have a size of 33 ± 13 nm and zeta potential of −52 ± 8 mV (examined using dynamic light scattering Nano ZS zetasizer system (Malvern Instruments, Worcestershire, UK). In this the different concentrations of Carbopol 974P (0.2, 0.25, 0.3, 0.375, 0.5, 0.525 and 0.75% (w/w)) were used to evaluate the rheological and mechanical properties. The results showed that the hydrogels were able to effect the viral attachment, impede penetration and cell to cell transmission and were effective against HSV-2 virus. Marques et al. formed nanostructured lipid carriers (NLCs) to develop mucoadhesive hydrogels of ibuprofen for buccal delivery. The researchers used Carbopol 980 and polycarbophil to prepare hydrogels. The results showed that the NLC dispersion thus obtained had a nanometer size and a low polydispersity index [70].

6.8 Patents and Future Perspectives In the past decade many researchers were able to file patents for the leading edge technologies in the field of bioadhesive hydrogels. Table 6.4 highlights some of the patents filed and granted by official agencies. 3D bioprinting is a rapidly developing discipline merging diverse applications of biomedicine, tissue engineering and materials science for developing customized drug delivery systems for reconstructive and

K.D. Park, Y.K. Joung, K.M. Park, and E.G. Lih

In situ-forming hydrogel for tissue adhesives and biomedical use thereof

Bioadhesive compositions

WO2011028031A2

USOO6683120B2

H.S. Munro

K.B. Kita, N.G. Smith, A.M. Lowman and G.W. Fussell

Bioadhesive hydrogels

EP2227262B1

First Water Ltd., United Kingdom

2004

2011

[75]

[74]

[73]

2017

Synthes GmbH, Germany and Drexel University, USA  Ajou University, Korea

[72]

2016

Case Western Reserve University, USA

E. Alsberg

Bioadhesive hydrogels

US20160022862A1

Reference [71]

Year of publication 2007

Mantra International Ltd, Hong Kong

M. Yasin

Bioadhesive compositions and their use in medical electrodes

US20070196320A1

Assignee

Inventor

Title of patent

Patent no.

Table 6.4  Patents filed by different inventors on bioadhesive hydrogels.

(Continued)

162  Bioadhesives in Drug Delivery

Title of patent

Hydrogel compositions including fibers and methods of use thereof

Bioadhesive hydrogel with surface-modified nanofiber

Bioadhesive compositions and biomedical electrodes containing them

Bioadhesives for corneal repair

Patent no.

WO2018197946A1

US20170072091 A1

US7076282B2

US20190022280A1

[79]

2007

2019

First Water Ltd., United Kingdom

Brigham and Women’s Hospital, Boston, USA

H.S. Munro and M. Yasin  

A. Khademhosseini, N. Annabi, R. Dana, and A. Kheirkhah

[78]

2013

POSTECH Academy Industry Foundation, Pohang University of Science and Technology, Korea

D.S. Hwang, D.Y. Oh, D.H. Lee and J. Jung 

[77]

[76]

2018

Meital Zilberman, Israel

Reference

M. Zilberman

Year of publication

Assignee

Inventor

Table 6.4  Patents filed by different inventors on bioadhesive hydrogels. (Continued)

Bioadhesive Hydrogels and Their Applications  163

164  Bioadhesives in Drug Delivery transplantation surgery, and implantable medical devices. A primary challenge of 3D bioprinting is the pursuit of appropriate biocompatible materials. The materials must have appropriate blend of biological and mechanical properties for use as liquid bioink. 3D printing of bioadhesive hydrogels for cellular tissue engineering applications is the focus area for future research on bioadhesive hydrogels. Clinical use of such devices needs to be explored from regulatory and toxicological viewpoints.

6.9 Summary Hydrogels are three-dimensional polymeric networks capable of absorbing large amounts of water and/or biological fluids. Hydrogels have potential applications in pharmaceutical sector because of their biocompatibility, good mechanical properties, high water content, soft consistency and tuneable drug release behaviour. Bioadhesive hydrogels are comprised of a polymer having bioadhesive property providing prolonged retention, enhanced stability and customized delivery of drug from the system. Bioadhesive hydrogels are gaining importance for the delivery of therapeutic agents through transdermal, gastrointestinal, parenteral, vaginal and rectal routes. The future of bioadhesive hydrogels for developing targeted drug delivery systems lies in biomimetic hydrogels, physiologically responsive hydrogels, composite hydrogels and 3D construct hydrogels. Toxicity, compatibility and therapeutic applications of bioadhesive hydrogels need to be studied and optimized from regulatory point of view. With the advent and growth of 3D printing technology, bioadhesive hydrogels will undergo revolutionary changes in the future.

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Part 3 DRUG DELIVERY APPLICATIONS

7 Ocular Bioadhesive Drug Delivery Systems and Their Applications Anju Sharma1, Mukesh S. Patil2, Pravin Pawar3, A.A. Shirkhedkar4 and Inderbir Singh1* Chitkara College of Pharmacy, Chitkara University, Patiala, Punjab, India Shri D.D. Vispute College of Pharmacy and Research Centre, Devad-Vichumbe, Panvel, Navi Mumbai, Maharashtra, India 3 Annasaheb Dange College of B. Pharmacy, Ashta, Sangli, Maharashtra, India 4 R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Maharashtra, India 1

2

Abstract

Amongst various routes of drug delivery, ocular drug delivery has been one of the most interesting and challenging endeavors encountered by the pharmaceutical scientists for many years. As an isolated organ, the eye is very difficult to study from a drug delivery point of view. Despite these limitations, improvements have been made with the objective of maintaining the drug in the biophase for an extended period. In this chapter, we have summarized the different types of polymers used for ophthalmic formulations. The eye is the most sensitive body organ responsible for vision. So, it is important to carefully deliver the drugs through this route. Natural polymers are promising carriers of drugs due to their favorable properties and can be used to prolong the contact time. The major problem with the ocular disease treatments is to provide and maintain an adequate concentration at the site of action for a long time. The solutions show a very short residence time in the ocular region due to rapid clearance and nasolachrymal drainage. Different formulations have been prepared with polymers to overcome the problems associated with the ocular delivery. Keywords:  Eye, ocular drug delivery, natural polymers, bioadhesion, ocular bioavailability

*Corresponding author: [email protected] K.L. Mittal, I. S. Bakshi and J. K. Narang (eds.) Bioadhesives in Drug Delivery, (173–212) © 2020 Scrivener Publishing LLC

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7.1 Introduction Since the 1980s, the concept of bioadhesives has gained much interest in ocular drug delivery systems. The topical application of dosage forms such as aqueous ophthalmic solutions and eye drops is most suitable for the treatment of various eye disorders. The major drawback of this type of drug delivery is the precorneal elimination of drugs caused by naso-lachrymal drainage and high tear fluid turnover. Certainly, very limited quantity of active pharmaceutical ingredients (about 1-5%) applied on the surface of eye penetrates corneal tissue and sclera and reaches intraocular space of the eye cavity. However, to attain the maximum therapeutic level of the drugs in aqueous humor of the eye cavity is most challenging task for the researchers due to unique physiology of eye and different barriers present in ocular system [1]. Some of the natural protective mechanisms of the eye offer significant barrier to the diffusion of the drug molecules [2]. Multilayed compostion of the cornea offers anatomical barrier, whereas physiological ocular barrier could be due to precorneal factors such as solution drainage, tear dilution, tear turnover, and increased lacrimation. Moreover, the lipophilic nature of corneal epithelium drastically limits the entry of hydrophilic molecules [3]. Further, the physicochemical properties of drug molecules like solubility, liophilicity, molecular weight and size also affect the penetration through corneal surface [4]. Repeated administration of ophthalmic formulations into the ocular tissue for maintaining the desired therapeutic concentration of drug molecule promotes the toxic effect leading to tissue injury [5, 6]. To overcome this problem, increasing the corneal residence time is one of the suitable approaches. To achieve this, suitable penetration enhancers and viscosity promoters are used in the ophthalmic formulations. Conventional approaches for ocular drug administration include eye drops, invasive injections or surgery-implanted cannulas [7]. However, such attempts also necessitate repeated injections to retain the therapeutic concentration that may result in associated clinical complications. Hydrogels, contact lenses and bioadhesive formulations are the latest developments for overcoming disadvantages of conventional ocular drug delivery systems [1, 3]. Sustained, controlled and extended dosage forms can overcome the limitation of repeated drug administration [8]. Many advantages are possible by employing this kind of approach such as a decreased dosing frequency which is the most common problem associated with ocular formulations [9]. In the past two decades, the formulation

Ocular Bioadhesive Drug Delivery Systems  175 of bioadhesive and mucoadhesive based dosage forms for the treatment of ocular disorders has received much attention. The drug release pattern from polymer based dosage forms is significantly affected by the nature of polymer, surface charge on the polymer and solubility of polymer material. On the basis of above information, the objective this book chapter is to summarize various natural and synthetic bioadhesive and mucoadhesive polymers and their application in ophthalmic formulations.

7.2 Anatomy and Physiology of the Eye 7.2.1 Anatomy and Function of the Eye The eye is the most marvelous sense organ as it makes us aware of various objects all around us, near and far away. The eye is nearly spherical in shape except that its front portion i.e., transparent cornea bulges slightly forward. The eye is protected by the eyelashes, eyelids, tears and blinking. The eye lashes catch foreign materials and the blink reflex prevents injury by closing the lids, blinking occurs frequently during waking hours to keep the corneal surface free of mucus and keep it moistened by the tears secreted by the lacrimal glands. Tears wash away irritating agents and are bactericidal, preventing infections. The protective operations of the eye lids and lacrimal system are such that there is a rapid removal of material instilled into the eye unless the material is suitably small in volume and is chemically and physiologically compatible with surface tissues. The eye is one of the most delicate and yet most valuable of the sense organs and is a challenging subject for topical administration of drugs to the eye [10]. In the anterior portion, cornea is the outermost part of the eye offering resistance to the passage of most drugs due to the presence of layers: hydrophobic epithelium, hydrophilic stroma and hydrophobic endothelium [11]. In ophthalmic formulations, the cornea act as reservoir of drugs and rate limiting membrane for the hydrophilic and hydrophobic molecules due to typical characteristics (hydrophilic and hydrophobic) of both epithelium and stroma [12]. Due to the presence of large surface area, the conjunctiva acts as a more favorable site for the penetration of the topically administered drugs. The tear film consists of three layers: the mucoid, aqueous and oily layers. The mucoid layer is produced by the goblet cells and lies adjacent to the corneal epithelium. It improves the wetting properties of the tears.

176  Bioadhesives in Drug Delivery The watery (aqueous) layer is produced by the lacrimal gland and is composed of electrolytes, proteins, lysozyme, immunoglobulins, glucose and dissolved oxygen. The oily surface (surface layer of the tear film) is produced by the meibomian glands (modified sebaceous glands) of the eyelid margins. This oily layer helps maintain the vertical column of tears between the upper and lower lids and prevents excessive evaporation. The tears normally flow away through a drainage system formed by the puncta (inferior and superior), canaliculi (inferior and superior), the common canaliculus (opening into the lacrimal sac) and the nasolacrimal duct (which drains into the nose) [12, 13].

7.2.2 Structure of Cornea The clear and transparent cornea situated at the front of the eye conveys images to the back of the nervous system. The cornea consists of five layers i.e. epithelium, Bowman’s layer, stroma, descemet’s membrane and endothelium. The corneal thickness is 0.5-0.7 mm in the central region and the epithelium acts as the main barrier to drug absorption due to its 5 to 6 cell-type layers. The cornea is made of basal cells, which are packed with a tight junction and form not only an effective barrier to dust particles and most microorganisms, but also for drug absorption. The drug transport through the cornea occurs mainly by transcellular or paracellular pathway. The transcellular route is mainly responsible for the lipophilic drugs and paracellular route is for the hydrophilic drugs (passive or altered diffusion through intercellular spaces of the cells).

7.3 Various Bioadhesive/Mucoadhesive Polymers for Ocular Delivery The important points to be considered during the fabrication of ophthalmic formulations, e.g., properties of drug molecule and polymer which affect the release rate are discussed. Conventional polymers such as Chitosan, Starch, Sodium Hyaluronate, Sodium Alginate, Gum, Albumin, Collagen, Xanthan Gum, Guar Gum and Gelatin besides these novel polymers, like Xyloglucan, Arabinogalactan, Gum Cordia, Bletilla Striata Polysaccharide, Locust Bean Gum and Carrageenan have demonstrated the potential to safely deliver drugs at a controlled rate in different ophthalmic formulations.

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7.3.1 Chitosan as Ocular Bioadhesive Chitosan is deacetylated chitin which is the structural element in the exoskeleton of insects, crustaceans (mainly shrimps and crabs) and cell walls of fungi. It is the second most abundant natural polysaccharide after cellulose. Chitosan is composed of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine randomly distributed within the polymer. The interesting characteristics of chitosan such as biocompatibility, nontoxicity, low allergenicity and biodegradability allow it to be used in various applications [14]. It is obtained by the alkaline deacetylation of chitin present in the crustacean shells of crimps, lobster and crab. Practically, an 85% degree of deacetylation or higher is preferred due to its strong mucoadhesive property and high biocompatibility [15]. In earlier study, the effect of deacetylation was studied and it was suggested that increasing the molecular weight and decreasing the deacetylation degree led to increased irritation scores in rabbits [16]. The strength of chitosan bioadhesion is due to strong electrostatic interactions between the positively charged amino groups present in chitosan and negatively charged sialic acid residues present in mucus [17]. It is a nontoxic and biodegradable polymer. Charges are induced in chitosan molecules in acidic and basic media which leads to its swelling but it does not swell in the neutral media [18]. Chitosan is soluble at acidic pH (pH