Bruno Sarmento, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal & CICS, Department of Pharmaceutical Sciences, Instituto Superior de Ciências da Saúde–Norte, Gandra, Portugal José das Neves, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal

• List of Contributors xvii Foreword xxiiiMarıa Jose AlonsoPreface xxvAcknowledgments xxviiPart One General Aspects of Chitosan 11 Chemical and Technological Advances in Chitins and Chitosans Useful for the Formulation of Biopharmaceuticals 3Riccardo A. A. Muzzarelli1.1 Introduction 31.2 Safety of Chitins and Chitosans 41.3 Ionic Liquids: New Solvents and Reaction Media 51.4 Chitin and Chitosan Nanofibrils 81.5 Electrospun Nanofibers 101.6 Polyelectrolyte Complexes and Mucoadhesion 121.7 Conclusions and Future Perspectives 162 Physical Properties of Chitosan and Derivatives in Sol and Gel States 23Marguerite Rinaudo2.1 Introduction 232.2 Chitin 242.3 Chitosan 282.4 Conclusions and Future Perspectives 363 Absorption Promotion Properties of Chitosan and Derivatives 45Akira Yamamoto3.1 Introduction 453.2 Effect of Chitosan on the Intestinal Absorption of Poorly Absorbable Drugs 473.3 Effect of Chitosan Derivatives on the Intestinal Absorption of Poorly Absorbable Drugs 473.4 Effect of Chitosan Oligomers on the Intestinal Absorption of Poorly Absorbable Drugs 483.5 Colon-Specific Delivery of Insulin Using Chitosan Capsules 513.6 Conclusions and Future Perspectives 544 Biocompatibility and Biodegradation of Chitosan and Derivatives 57Ahmad Sukari Halim, Lim Chin Keong, Ismail Zainol, and Ahmad Hazri Abdul Rashid4.1 Introduction 574.2 Biocompatibility Evaluation of Chitosan and Derivatives 584.3 Biodegradation of Chitosan and Derivatives 654.4 Conclusions and Future Perspectives 695 Biological and Pharmacological Activity of Chitosan and Derivatives 75Teresa Cunha, Branca Teixeira, Barbara Santos, Marlene Almeida, Gustavo Dias, and Jose das Neves5.1 Introduction 755.2 Biological Activity 765.3 Chitosan's Usefulness in Therapy and Alternative Medicine 825.4 Conclusions and Future Perspectives 846 Biological, Chemical, and Physical Compatibility of Chitosan and Biopharmaceuticals 93Masayuki Ishihara, Masanori Fujita, Satoko Kishimoto, Hidemi Hattori, and Yasuhiro Kanatani6.1 Introduction 936.2 Structural Features of Chitosan and Its Derivatives 946.3 Biocompatibility for Chitosan and Its Derivatives 956.4 Biocompatibility of Photo-Cross-Linkable Chitosan Hydrogel 986.5 Physical and Chemical Compatibility of Chitosan and Its Derivatives 1006.6 Conclusions and Future Perspectives 1027 Approaches for Functional Modification or Cross-Linking of Chitosan 107A. Anitha, N. Sanoj Rejinold, Joel D. Bumgardner, Shanti V. Nair, and Rangasamy Jayakumar7.1 Introduction 1077.2 General Awareness of Chitosan Cross-Linking Methods 1087.3 Modified Chitosan: Synthesis and Characterization 1127.4 Applications of Modified Chitosan and Its Derivatives in Drug Delivery 1187.5 Conclusions and Future Perspectives 118Part Two Biopharmaceuticals Formulation and Delivery Aspects Using Chitosan and Derivatives 1258 Use of Chitosan and Derivatives in Conventional Biopharmaceutical Dosage Forms Formulation 127Teofilo Vasconcelos, Pedro Barrocas, and Rui Cerdeira8.1 Introduction 1278.2 Advantageous Properties of Chitosan and Its Derivatives 1288.3 Oral Administration 1298.4 Buccal Administration 1318.5 Nasal Administration 1328.6 Pulmonary Administration 1328.7 Transdermal Administration 1338.8 Conclusions and Future Perspectives 1339 Manufacture Techniques of Chitosan-Based Microparticles and Nanoparticles for Biopharmaceuticals 137Franca Ferrari, M. Cristina Bonferoni, Silvia Rossi, Giuseppina Sandri, and Carla M. Caramella9.1 Introduction 1379.2 Water-in-Oil Emulsion and Chemical Cross-linking 1389.3 Drying Techniques 1419.4 Ionic Cross-linking Methods 1449.5 Coacervation and Precipitation Method 1519.6 Direct Interaction between Chitosan and Biopharmaceuticals 1529.7 Conclusions and Future Perspectives 15310 Chitosan and Derivatives for Biopharmaceutical Use: Mucoadhesive Properties 159Katharina Leithner and Andreas Bernkop-Schnurch10.1 Introduction 15910.2 Mucoadhesion 16010.3 Chitosan and Its Derivatives 16110.4 Biopharmaceutical Use of Chitosan and Its Derivatives 17110.5 Conclusions and Future Perspectives 17511 Chitosan-Based Systems for Mucosal Delivery of Biopharmaceuticals 181Sonia Al-Qadi, Ana Grenha, and Carmen Remunan-Lopez11.1 Introduction 18111.2 Important Challenges for the Delivery of Biopharmaceuticals by Mucosal Routes 18211.3 Interest in Chitosan for Mucosal Delivery of Biopharmaceuticals 18411.4 Chitosan-Based Delivery Nanosystems for Mucosal Delivery of Biopharmaceuticals 18811.5 Conclusions and Future Perspectives 20012 Chitosan-Based Delivery Systems for Mucosal Vaccination 211Gerrit Borchard, Farnaz Esmaeili, and Simon Heuking12.1 Introduction 21112.2 Adjuvant Properties of Chitosan 21212.3 Chitosan in the Delivery of Protein and Subunit Vaccines 21312.4 Chitosan-Based Formulations of DNAVaccines 21512.5 Vaccine Formulations Using Chitosan in Combination with Other Polymers 21612.6 Chitosan Derivatives in Vaccine Carrier Design 21712.7 Conclusions and Future Perspectives 22013 Chitosan-Based Nanoparticulates for Oral Delivery of Biopharmaceuticals 225Filipa Antunes, Fernanda Andrade, and Bruno Sarmento13.1 Introduction 22513.2 Challenges on the Oral Delivery of Therapeutic Proteins 22613.3 Challenges on the Oral Delivery of Genetic Material 22713.4 Role of Chitosan in the Protection of Biopharmaceuticals in the Gastrointestinal Tract 22913.5 Chitosan-Based Nanoparticles for Oral Delivery of Therapeutic Proteins 23213.6 Chitosan-Based Nanoparticles for Oral Delivery of Genetic Material 23413.7 Conclusions and Future Perspectives 23614 Chitosan-Based Systems for Ocular Delivery of Biopharmaceuticals 243Suresh P. Vyas, Rishi Paliwal, and Shivani Rai Paliwal14.1 Introduction 24314.2 Ocular Delivery of Biopharmaceuticals 24414.3 Chitosan: A Suitable Biomaterial for Ocular Therapeutics 24414.4 Chitosan-Based Systems for Ocular Delivery of Biomacromolecules 24514.5 Toxicological and Compatibility Aspects of Chitosan-Based Ocular Systems 24914.6 Conclusions and Future Perspectives 25015 Chemical Modification of Chitosan for Delivery of DNA and siRNA 255You-Kyoung Kim, Hu-Lin Jiang, Ding-Ding Guo, Yun-Jaie Choi, Myung-Haing Cho, Toshihiro Akaike, and Chong-Su Cho15.1 Introduction 25515.2 Hydrophilic Modification 25615.3 Hydrophobic Modification 25715.4 Specific Ligand Modification 25915.5 pH-Sensitive Modification 26415.6 Conclusions and Future Perspectives 269Part Three Advanced Application of Chitosan and Derivatives for Biopharmaceuticals 27516 Target-Specific Chitosan-Based Nanoparticle Systems for Nucleic Acid Delivery 277Shardool Jain and Mansoor Amiji16.1 Introduction 27716.2 Chitosan-Based Nanoparticle Delivery Systems 28316.3 Illustrative Examples of DNAVaccine Delivery 28616.4 Illustrative Examples of Nucleic Acid Delivery Systems for Cancer Therapy 28816.5 Illustrative Examples of Nucleic Acid Delivery Systems for Anti-Inflammatory Therapy 29116.6 Conclusions and Future Perspectives 29417 Functional PEGylated Chitosan Systems for Biopharmaceuticals 301Hee-Jeong Cho, Goen Kim, Hyeok-Seung Kwon, and Yu-Kyoung Oh17.1 Introduction 30117.2 PEGylated Chitosan for the Delivery of Proteins and Peptides 30417.3 PEGylated Chitosan for Delivery of Nucleic Acids 30817.4 PEGylated Chitosan for Delivery of Other Macromolecular Biopharmaceuticals 31117.5 PEGylated Chitosan Used for Cellular Scaffolds 31317.6 Conclusions and Future Perspectives 31318 Stimuli-Sensitive Chitosan-Based Systems for Biopharmaceuticals 319Cuiping Zhai, Jinfang Yuan, and Qingyu Gao18.1 Introduction 31918.2 pH-Sensitive Chitosan-Based Systems 31918.3 Thermosensitive Chitosan-Based Systems 32118.4 pH-Sensitive and Thermosensitive Chitosan-Based Systems 32318.5 pH- and Ionic-Sensitive Chitosan-Based Systems 32518.6 Photo-Sensitive Chitosan-Based Systems 32518.7 Electrical-Sensitive Chitosan-Based Systems 32618.8 Magnetic-Sensitive Chitosan-Based Systems 32618.9 Chemical Substance-Sensitive Chitosan-Based Systems 32718.10 Conclusions and Future Perspectives 32719 Chitosan Copolymers for Biopharmaceuticals 333Ramon Novoa-Carballal, Ricardo Riguera, and Eduardo Fernandez-Megia19.1 Introduction 33319.2 Chitosan-g-Poly(Ethylene Glycol) 33719.3 Chitosan-g-Polyethylenimine 34719.4 Other Copolymers of Chitosan 35719.5 Copolymers of Chitosan with Promising Applications 36319.6 Conclusions and Future Perspectives 368 20 Application of Chitosan for Anticancer Biopharmaceutical Delivery 381Claudia Philippi, Brigitta Loretz, Ulrich F. Schaefer, and Claus-Michael Lehr20.1 Introduction 38120.2 Chitosan and Cancer: Intrinsic Antitumor Activity of the Polymer Itself 38220.3 Chitosan Formulations Developed for Classic Anticancer Drugs 38320.4 Biopharmaceuticals Delivered by Chitosan Preparations 38420.5 Active Targeting Strategies and Multifunctional Chitosan Formulations 38820.6 Conclusions and Future Perspectives 38921 Chitosan-Based Biopharmaceutical Scaffolds in Tissue Engineering and Regenerative Medicine 393Tao Jiang, Meng Deng, Wafa I. Abdel- Fattah, and Cato T. Laurencin21.1 Introduction 39321.2 Fabrication of Chitosan-Based Biopharmaceuticals Scaffolds 39521.3 Applications of Chitosan-Based Biopharmaceutical Scaffolds in Tissue Engineering and Regenerative Medicine 40321.4 Future Trends: Regenerative Engineering 41621.5 Conclusions and Future Perspectives 41722 Wound-Healing Properties of Chitosan and Its Use in Wound Dressing Biopharmaceuticals 429Tyler G. St. Denis, Tianhong Dai, Ying-Ying Huang, and Michael R. Hamblin22.1 Introduction 42922.2 Brief Review of Wound Repair 43022.3 Wound-Healing Effects of Chitosan 43322.4 Chitosan for Wound Therapeutics Delivery 44022.5 Conclusions and Future Perspectives 444Part Four Regulatory Status, Toxicological Issues, and Clinical Perspectives 45123 Toxicological Properties of Chitosan and Derivatives for Biopharmaceutical Applications 453Thomas J. Kean and Maya Thanou23.1 Introduction 45323.2 In Vitro Toxicity of Chitosan and Derivatives 45423.3 In Vivo Toxicity of Chitosan and Derivatives 45723.4 Conclusions and Future Perspectives 459 24 Regulatory Status of Chitosan and Derivatives 463Michael Dornish, David S. Kaplan, and Sambasiva R. Arepalli24.1 Introduction 46324.2 Source 46424.3 Characterization 46424.4 Purity 46524.5 Applications of Advanced Uses of Chitosan 46624.6 Regulatory Considerations for Chitosan and Chitosan Derivatives in the European Union, and Medical Devices or Combination Products with Medical Device (CDRH) Lead 46824.7 Regulatory Pathways 46924.8 Chitosan Medical Products: US Regulatory Review Processes for Medical Devices or Combination Products with CDRH Lead 46924.9 Chitosan Wound Dressings 47024.10 The European Regulatory System: The European Medicines Agency (EMA) and European Directorate for the Quality of Medicines (EDQM) 47424.11 Further Regulatory Considerations 47524.12 Conclusions and Future Perspectives 47724.13 Disclaimer 47825 Patentability and Intellectual Property Issues Related to Chitosan-Based Biopharmaceutical Products 483Mafalda Videira and Rogerio Gaspar25.1 Introduction 48325.2 Setting the Scene: The Role of Chitosan as a Pharmaceutical Excipient 48425.3 Addressing the Drivers for Scientific Progress on Chitosan: Innovation and Inventability 49525.4 Conclusions and Future Perspectives 49626 Quality Control and Good Manufacturing Practice (GMP) for Chitosan-Based Biopharmaceutical Products 503Torsten Richter, Maika Gulich, and Katja Richter26.1 Introduction 50426.2 Regulatory Requirements for Production 50526.3 Manufacturing GMP: Fundamental Considerations 50826.4 Requirements for Rooms, Personnel, and Equipment 51126.5 Qualification and Validation 51126.6 Quality Control 51326.7 Monitoring and Maintenance of a GMP System 51926.8 Conclusions and Future Perspectives 52227 Preclinical and Clinical Use of Chitosan and Derivatives for Biopharmaceuticals: From Preclinical Research to the Bedside 525David A. Zaharoff, Michael Heffernan, Jonathan Fallon, and John W. Greiner27.1 Introduction 52527.2 Chitosan as a Parenteral (Subcutaneous) Vaccine Platform 52627.3 Chitosan as an Immunotherapeutic Platform 53027.4 Conclusions and Future Perspectives 537References 539Index 543

Bruno Sarmento, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal & CICS, Department of Pharmaceutical Sciences, Instituto Superior de Ciências da Saúde–Norte, Gandra, Portugal José das Neves, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal

• List of Contributors xvii Foreword xxiiiMarıa Jose AlonsoPreface xxvAcknowledgments xxviiPart One General Aspects of Chitosan 11 Chemical and Technological Advances in Chitins and Chitosans Useful for the Formulation of Biopharmaceuticals 3Riccardo A. A. Muzzarelli1.1 Introduction 31.2 Safety of Chitins and Chitosans 41.3 Ionic Liquids: New Solvents and Reaction Media 51.4 Chitin and Chitosan Nanofibrils 81.5 Electrospun Nanofibers 101.6 Polyelectrolyte Complexes and Mucoadhesion 121.7 Conclusions and Future Perspectives 162 Physical Properties of Chitosan and Derivatives in Sol and Gel States 23Marguerite Rinaudo2.1 Introduction 232.2 Chitin 242.3 Chitosan 282.4 Conclusions and Future Perspectives 363 Absorption Promotion Properties of Chitosan and Derivatives 45Akira Yamamoto3.1 Introduction 453.2 Effect of Chitosan on the Intestinal Absorption of Poorly Absorbable Drugs 473.3 Effect of Chitosan Derivatives on the Intestinal Absorption of Poorly Absorbable Drugs 473.4 Effect of Chitosan Oligomers on the Intestinal Absorption of Poorly Absorbable Drugs 483.5 Colon-Specific Delivery of Insulin Using Chitosan Capsules 513.6 Conclusions and Future Perspectives 544 Biocompatibility and Biodegradation of Chitosan and Derivatives 57Ahmad Sukari Halim, Lim Chin Keong, Ismail Zainol, and Ahmad Hazri Abdul Rashid4.1 Introduction 574.2 Biocompatibility Evaluation of Chitosan and Derivatives 584.3 Biodegradation of Chitosan and Derivatives 654.4 Conclusions and Future Perspectives 695 Biological and Pharmacological Activity of Chitosan and Derivatives 75Teresa Cunha, Branca Teixeira, Barbara Santos, Marlene Almeida, Gustavo Dias, and Jose das Neves5.1 Introduction 755.2 Biological Activity 765.3 Chitosan's Usefulness in Therapy and Alternative Medicine 825.4 Conclusions and Future Perspectives 846 Biological, Chemical, and Physical Compatibility of Chitosan and Biopharmaceuticals 93Masayuki Ishihara, Masanori Fujita, Satoko Kishimoto, Hidemi Hattori, and Yasuhiro Kanatani6.1 Introduction 936.2 Structural Features of Chitosan and Its Derivatives 946.3 Biocompatibility for Chitosan and Its Derivatives 956.4 Biocompatibility of Photo-Cross-Linkable Chitosan Hydrogel 986.5 Physical and Chemical Compatibility of Chitosan and Its Derivatives 1006.6 Conclusions and Future Perspectives 1027 Approaches for Functional Modification or Cross-Linking of Chitosan 107A. Anitha, N. Sanoj Rejinold, Joel D. Bumgardner, Shanti V. Nair, and Rangasamy Jayakumar7.1 Introduction 1077.2 General Awareness of Chitosan Cross-Linking Methods 1087.3 Modified Chitosan: Synthesis and Characterization 1127.4 Applications of Modified Chitosan and Its Derivatives in Drug Delivery 1187.5 Conclusions and Future Perspectives 118Part Two Biopharmaceuticals Formulation and Delivery Aspects Using Chitosan and Derivatives 1258 Use of Chitosan and Derivatives in Conventional Biopharmaceutical Dosage Forms Formulation 127Teofilo Vasconcelos, Pedro Barrocas, and Rui Cerdeira8.1 Introduction 1278.2 Advantageous Properties of Chitosan and Its Derivatives 1288.3 Oral Administration 1298.4 Buccal Administration 1318.5 Nasal Administration 1328.6 Pulmonary Administration 1328.7 Transdermal Administration 1338.8 Conclusions and Future Perspectives 1339 Manufacture Techniques of Chitosan-Based Microparticles and Nanoparticles for Biopharmaceuticals 137Franca Ferrari, M. Cristina Bonferoni, Silvia Rossi, Giuseppina Sandri, and Carla M. Caramella9.1 Introduction 1379.2 Water-in-Oil Emulsion and Chemical Cross-linking 1389.3 Drying Techniques 1419.4 Ionic Cross-linking Methods 1449.5 Coacervation and Precipitation Method 1519.6 Direct Interaction between Chitosan and Biopharmaceuticals 1529.7 Conclusions and Future Perspectives 15310 Chitosan and Derivatives for Biopharmaceutical Use: Mucoadhesive Properties 159Katharina Leithner and Andreas Bernkop-Schnurch10.1 Introduction 15910.2 Mucoadhesion 16010.3 Chitosan and Its Derivatives 16110.4 Biopharmaceutical Use of Chitosan and Its Derivatives 17110.5 Conclusions and Future Perspectives 17511 Chitosan-Based Systems for Mucosal Delivery of Biopharmaceuticals 181Sonia Al-Qadi, Ana Grenha, and Carmen Remunan-Lopez11.1 Introduction 18111.2 Important Challenges for the Delivery of Biopharmaceuticals by Mucosal Routes 18211.3 Interest in Chitosan for Mucosal Delivery of Biopharmaceuticals 18411.4 Chitosan-Based Delivery Nanosystems for Mucosal Delivery of Biopharmaceuticals 18811.5 Conclusions and Future Perspectives 20012 Chitosan-Based Delivery Systems for Mucosal Vaccination 211Gerrit Borchard, Farnaz Esmaeili, and Simon Heuking12.1 Introduction 21112.2 Adjuvant Properties of Chitosan 21212.3 Chitosan in the Delivery of Protein and Subunit Vaccines 21312.4 Chitosan-Based Formulations of DNAVaccines 21512.5 Vaccine Formulations Using Chitosan in Combination with Other Polymers 21612.6 Chitosan Derivatives in Vaccine Carrier Design 21712.7 Conclusions and Future Perspectives 22013 Chitosan-Based Nanoparticulates for Oral Delivery of Biopharmaceuticals 225Filipa Antunes, Fernanda Andrade, and Bruno Sarmento13.1 Introduction 22513.2 Challenges on the Oral Delivery of Therapeutic Proteins 22613.3 Challenges on the Oral Delivery of Genetic Material 22713.4 Role of Chitosan in the Protection of Biopharmaceuticals in the Gastrointestinal Tract 22913.5 Chitosan-Based Nanoparticles for Oral Delivery of Therapeutic Proteins 23213.6 Chitosan-Based Nanoparticles for Oral Delivery of Genetic Material 23413.7 Conclusions and Future Perspectives 23614 Chitosan-Based Systems for Ocular Delivery of Biopharmaceuticals 243Suresh P. Vyas, Rishi Paliwal, and Shivani Rai Paliwal14.1 Introduction 24314.2 Ocular Delivery of Biopharmaceuticals 24414.3 Chitosan: A Suitable Biomaterial for Ocular Therapeutics 24414.4 Chitosan-Based Systems for Ocular Delivery of Biomacromolecules 24514.5 Toxicological and Compatibility Aspects of Chitosan-Based Ocular Systems 24914.6 Conclusions and Future Perspectives 25015 Chemical Modification of Chitosan for Delivery of DNA and siRNA 255You-Kyoung Kim, Hu-Lin Jiang, Ding-Ding Guo, Yun-Jaie Choi, Myung-Haing Cho, Toshihiro Akaike, and Chong-Su Cho15.1 Introduction 25515.2 Hydrophilic Modification 25615.3 Hydrophobic Modification 25715.4 Specific Ligand Modification 25915.5 pH-Sensitive Modification 26415.6 Conclusions and Future Perspectives 269Part Three Advanced Application of Chitosan and Derivatives for Biopharmaceuticals 27516 Target-Specific Chitosan-Based Nanoparticle Systems for Nucleic Acid Delivery 277Shardool Jain and Mansoor Amiji16.1 Introduction 27716.2 Chitosan-Based Nanoparticle Delivery Systems 28316.3 Illustrative Examples of DNAVaccine Delivery 28616.4 Illustrative Examples of Nucleic Acid Delivery Systems for Cancer Therapy 28816.5 Illustrative Examples of Nucleic Acid Delivery Systems for Anti-Inflammatory Therapy 29116.6 Conclusions and Future Perspectives 29417 Functional PEGylated Chitosan Systems for Biopharmaceuticals 301Hee-Jeong Cho, Goen Kim, Hyeok-Seung Kwon, and Yu-Kyoung Oh17.1 Introduction 30117.2 PEGylated Chitosan for the Delivery of Proteins and Peptides 30417.3 PEGylated Chitosan for Delivery of Nucleic Acids 30817.4 PEGylated Chitosan for Delivery of Other Macromolecular Biopharmaceuticals 31117.5 PEGylated Chitosan Used for Cellular Scaffolds 31317.6 Conclusions and Future Perspectives 31318 Stimuli-Sensitive Chitosan-Based Systems for Biopharmaceuticals 319Cuiping Zhai, Jinfang Yuan, and Qingyu Gao18.1 Introduction 31918.2 pH-Sensitive Chitosan-Based Systems 31918.3 Thermosensitive Chitosan-Based Systems 32118.4 pH-Sensitive and Thermosensitive Chitosan-Based Systems 32318.5 pH- and Ionic-Sensitive Chitosan-Based Systems 32518.6 Photo-Sensitive Chitosan-Based Systems 32518.7 Electrical-Sensitive Chitosan-Based Systems 32618.8 Magnetic-Sensitive Chitosan-Based Systems 32618.9 Chemical Substance-Sensitive Chitosan-Based Systems 32718.10 Conclusions and Future Perspectives 32719 Chitosan Copolymers for Biopharmaceuticals 333Ramon Novoa-Carballal, Ricardo Riguera, and Eduardo Fernandez-Megia19.1 Introduction 33319.2 Chitosan-g-Poly(Ethylene Glycol) 33719.3 Chitosan-g-Polyethylenimine 34719.4 Other Copolymers of Chitosan 35719.5 Copolymers of Chitosan with Promising Applications 36319.6 Conclusions and Future Perspectives 368 20 Application of Chitosan for Anticancer Biopharmaceutical Delivery 381Claudia Philippi, Brigitta Loretz, Ulrich F. Schaefer, and Claus-Michael Lehr20.1 Introduction 38120.2 Chitosan and Cancer: Intrinsic Antitumor Activity of the Polymer Itself 38220.3 Chitosan Formulations Developed for Classic Anticancer Drugs 38320.4 Biopharmaceuticals Delivered by Chitosan Preparations 38420.5 Active Targeting Strategies and Multifunctional Chitosan Formulations 38820.6 Conclusions and Future Perspectives 38921 Chitosan-Based Biopharmaceutical Scaffolds in Tissue Engineering and Regenerative Medicine 393Tao Jiang, Meng Deng, Wafa I. Abdel- Fattah, and Cato T. Laurencin21.1 Introduction 39321.2 Fabrication of Chitosan-Based Biopharmaceuticals Scaffolds 39521.3 Applications of Chitosan-Based Biopharmaceutical Scaffolds in Tissue Engineering and Regenerative Medicine 40321.4 Future Trends: Regenerative Engineering 41621.5 Conclusions and Future Perspectives 41722 Wound-Healing Properties of Chitosan and Its Use in Wound Dressing Biopharmaceuticals 429Tyler G. St. Denis, Tianhong Dai, Ying-Ying Huang, and Michael R. Hamblin22.1 Introduction 42922.2 Brief Review of Wound Repair 43022.3 Wound-Healing Effects of Chitosan 43322.4 Chitosan for Wound Therapeutics Delivery 44022.5 Conclusions and Future Perspectives 444Part Four Regulatory Status, Toxicological Issues, and Clinical Perspectives 45123 Toxicological Properties of Chitosan and Derivatives for Biopharmaceutical Applications 453Thomas J. Kean and Maya Thanou23.1 Introduction 45323.2 In Vitro Toxicity of Chitosan and Derivatives 45423.3 In Vivo Toxicity of Chitosan and Derivatives 45723.4 Conclusions and Future Perspectives 459 24 Regulatory Status of Chitosan and Derivatives 463Michael Dornish, David S. Kaplan, and Sambasiva R. Arepalli24.1 Introduction 46324.2 Source 46424.3 Characterization 46424.4 Purity 46524.5 Applications of Advanced Uses of Chitosan 46624.6 Regulatory Considerations for Chitosan and Chitosan Derivatives in the European Union, and Medical Devices or Combination Products with Medical Device (CDRH) Lead 46824.7 Regulatory Pathways 46924.8 Chitosan Medical Products: US Regulatory Review Processes for Medical Devices or Combination Products with CDRH Lead 46924.9 Chitosan Wound Dressings 47024.10 The European Regulatory System: The European Medicines Agency (EMA) and European Directorate for the Quality of Medicines (EDQM) 47424.11 Further Regulatory Considerations 47524.12 Conclusions and Future Perspectives 47724.13 Disclaimer 47825 Patentability and Intellectual Property Issues Related to Chitosan-Based Biopharmaceutical Products 483Mafalda Videira and Rogerio Gaspar25.1 Introduction 48325.2 Setting the Scene: The Role of Chitosan as a Pharmaceutical Excipient 48425.3 Addressing the Drivers for Scientific Progress on Chitosan: Innovation and Inventability 49525.4 Conclusions and Future Perspectives 49626 Quality Control and Good Manufacturing Practice (GMP) for Chitosan-Based Biopharmaceutical Products 503Torsten Richter, Maika Gulich, and Katja Richter26.1 Introduction 50426.2 Regulatory Requirements for Production 50526.3 Manufacturing GMP: Fundamental Considerations 50826.4 Requirements for Rooms, Personnel, and Equipment 51126.5 Qualification and Validation 51126.6 Quality Control 51326.7 Monitoring and Maintenance of a GMP System 51926.8 Conclusions and Future Perspectives 52227 Preclinical and Clinical Use of Chitosan and Derivatives for Biopharmaceuticals: From Preclinical Research to the Bedside 525David A. Zaharoff, Michael Heffernan, Jonathan Fallon, and John W. Greiner27.1 Introduction 52527.2 Chitosan as a Parenteral (Subcutaneous) Vaccine Platform 52627.3 Chitosan as an Immunotherapeutic Platform 53027.4 Conclusions and Future Perspectives 537References 539Index 543

Bruno Sarmento, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal & CICS, Department of Pharmaceutical Sciences, Instituto Superior de Ciências da Saúde–Norte, Gandra, Portugal José das Neves, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal

• List of Contributors xvii Foreword xxiiiMarıa Jose AlonsoPreface xxvAcknowledgments xxviiPart One General Aspects of Chitosan 11 Chemical and Technological Advances in Chitins and Chitosans Useful for the Formulation of Biopharmaceuticals 3Riccardo A. A. Muzzarelli1.1 Introduction 31.2 Safety of Chitins and Chitosans 41.3 Ionic Liquids: New Solvents and Reaction Media 51.4 Chitin and Chitosan Nanofibrils 81.5 Electrospun Nanofibers 101.6 Polyelectrolyte Complexes and Mucoadhesion 121.7 Conclusions and Future Perspectives 162 Physical Properties of Chitosan and Derivatives in Sol and Gel States 23Marguerite Rinaudo2.1 Introduction 232.2 Chitin 242.3 Chitosan 282.4 Conclusions and Future Perspectives 363 Absorption Promotion Properties of Chitosan and Derivatives 45Akira Yamamoto3.1 Introduction 453.2 Effect of Chitosan on the Intestinal Absorption of Poorly Absorbable Drugs 473.3 Effect of Chitosan Derivatives on the Intestinal Absorption of Poorly Absorbable Drugs 473.4 Effect of Chitosan Oligomers on the Intestinal Absorption of Poorly Absorbable Drugs 483.5 Colon-Specific Delivery of Insulin Using Chitosan Capsules 513.6 Conclusions and Future Perspectives 544 Biocompatibility and Biodegradation of Chitosan and Derivatives 57Ahmad Sukari Halim, Lim Chin Keong, Ismail Zainol, and Ahmad Hazri Abdul Rashid4.1 Introduction 574.2 Biocompatibility Evaluation of Chitosan and Derivatives 584.3 Biodegradation of Chitosan and Derivatives 654.4 Conclusions and Future Perspectives 695 Biological and Pharmacological Activity of Chitosan and Derivatives 75Teresa Cunha, Branca Teixeira, Barbara Santos, Marlene Almeida, Gustavo Dias, and Jose das Neves5.1 Introduction 755.2 Biological Activity 765.3 Chitosan's Usefulness in Therapy and Alternative Medicine 825.4 Conclusions and Future Perspectives 846 Biological, Chemical, and Physical Compatibility of Chitosan and Biopharmaceuticals 93Masayuki Ishihara, Masanori Fujita, Satoko Kishimoto, Hidemi Hattori, and Yasuhiro Kanatani6.1 Introduction 936.2 Structural Features of Chitosan and Its Derivatives 946.3 Biocompatibility for Chitosan and Its Derivatives 956.4 Biocompatibility of Photo-Cross-Linkable Chitosan Hydrogel 986.5 Physical and Chemical Compatibility of Chitosan and Its Derivatives 1006.6 Conclusions and Future Perspectives 1027 Approaches for Functional Modification or Cross-Linking of Chitosan 107A. Anitha, N. Sanoj Rejinold, Joel D. Bumgardner, Shanti V. Nair, and Rangasamy Jayakumar7.1 Introduction 1077.2 General Awareness of Chitosan Cross-Linking Methods 1087.3 Modified Chitosan: Synthesis and Characterization 1127.4 Applications of Modified Chitosan and Its Derivatives in Drug Delivery 1187.5 Conclusions and Future Perspectives 118Part Two Biopharmaceuticals Formulation and Delivery Aspects Using Chitosan and Derivatives 1258 Use of Chitosan and Derivatives in Conventional Biopharmaceutical Dosage Forms Formulation 127Teofilo Vasconcelos, Pedro Barrocas, and Rui Cerdeira8.1 Introduction 1278.2 Advantageous Properties of Chitosan and Its Derivatives 1288.3 Oral Administration 1298.4 Buccal Administration 1318.5 Nasal Administration 1328.6 Pulmonary Administration 1328.7 Transdermal Administration 1338.8 Conclusions and Future Perspectives 1339 Manufacture Techniques of Chitosan-Based Microparticles and Nanoparticles for Biopharmaceuticals 137Franca Ferrari, M. Cristina Bonferoni, Silvia Rossi, Giuseppina Sandri, and Carla M. Caramella9.1 Introduction 1379.2 Water-in-Oil Emulsion and Chemical Cross-linking 1389.3 Drying Techniques 1419.4 Ionic Cross-linking Methods 1449.5 Coacervation and Precipitation Method 1519.6 Direct Interaction between Chitosan and Biopharmaceuticals 1529.7 Conclusions and Future Perspectives 15310 Chitosan and Derivatives for Biopharmaceutical Use: Mucoadhesive Properties 159Katharina Leithner and Andreas Bernkop-Schnurch10.1 Introduction 15910.2 Mucoadhesion 16010.3 Chitosan and Its Derivatives 16110.4 Biopharmaceutical Use of Chitosan and Its Derivatives 17110.5 Conclusions and Future Perspectives 17511 Chitosan-Based Systems for Mucosal Delivery of Biopharmaceuticals 181Sonia Al-Qadi, Ana Grenha, and Carmen Remunan-Lopez11.1 Introduction 18111.2 Important Challenges for the Delivery of Biopharmaceuticals by Mucosal Routes 18211.3 Interest in Chitosan for Mucosal Delivery of Biopharmaceuticals 18411.4 Chitosan-Based Delivery Nanosystems for Mucosal Delivery of Biopharmaceuticals 18811.5 Conclusions and Future Perspectives 20012 Chitosan-Based Delivery Systems for Mucosal Vaccination 211Gerrit Borchard, Farnaz Esmaeili, and Simon Heuking12.1 Introduction 21112.2 Adjuvant Properties of Chitosan 21212.3 Chitosan in the Delivery of Protein and Subunit Vaccines 21312.4 Chitosan-Based Formulations of DNAVaccines 21512.5 Vaccine Formulations Using Chitosan in Combination with Other Polymers 21612.6 Chitosan Derivatives in Vaccine Carrier Design 21712.7 Conclusions and Future Perspectives 22013 Chitosan-Based Nanoparticulates for Oral Delivery of Biopharmaceuticals 225Filipa Antunes, Fernanda Andrade, and Bruno Sarmento13.1 Introduction 22513.2 Challenges on the Oral Delivery of Therapeutic Proteins 22613.3 Challenges on the Oral Delivery of Genetic Material 22713.4 Role of Chitosan in the Protection of Biopharmaceuticals in the Gastrointestinal Tract 22913.5 Chitosan-Based Nanoparticles for Oral Delivery of Therapeutic Proteins 23213.6 Chitosan-Based Nanoparticles for Oral Delivery of Genetic Material 23413.7 Conclusions and Future Perspectives 23614 Chitosan-Based Systems for Ocular Delivery of Biopharmaceuticals 243Suresh P. Vyas, Rishi Paliwal, and Shivani Rai Paliwal14.1 Introduction 24314.2 Ocular Delivery of Biopharmaceuticals 24414.3 Chitosan: A Suitable Biomaterial for Ocular Therapeutics 24414.4 Chitosan-Based Systems for Ocular Delivery of Biomacromolecules 24514.5 Toxicological and Compatibility Aspects of Chitosan-Based Ocular Systems 24914.6 Conclusions and Future Perspectives 25015 Chemical Modification of Chitosan for Delivery of DNA and siRNA 255You-Kyoung Kim, Hu-Lin Jiang, Ding-Ding Guo, Yun-Jaie Choi, Myung-Haing Cho, Toshihiro Akaike, and Chong-Su Cho15.1 Introduction 25515.2 Hydrophilic Modification 25615.3 Hydrophobic Modification 25715.4 Specific Ligand Modification 25915.5 pH-Sensitive Modification 26415.6 Conclusions and Future Perspectives 269Part Three Advanced Application of Chitosan and Derivatives for Biopharmaceuticals 27516 Target-Specific Chitosan-Based Nanoparticle Systems for Nucleic Acid Delivery 277Shardool Jain and Mansoor Amiji16.1 Introduction 27716.2 Chitosan-Based Nanoparticle Delivery Systems 28316.3 Illustrative Examples of DNAVaccine Delivery 28616.4 Illustrative Examples of Nucleic Acid Delivery Systems for Cancer Therapy 28816.5 Illustrative Examples of Nucleic Acid Delivery Systems for Anti-Inflammatory Therapy 29116.6 Conclusions and Future Perspectives 29417 Functional PEGylated Chitosan Systems for Biopharmaceuticals 301Hee-Jeong Cho, Goen Kim, Hyeok-Seung Kwon, and Yu-Kyoung Oh17.1 Introduction 30117.2 PEGylated Chitosan for the Delivery of Proteins and Peptides 30417.3 PEGylated Chitosan for Delivery of Nucleic Acids 30817.4 PEGylated Chitosan for Delivery of Other Macromolecular Biopharmaceuticals 31117.5 PEGylated Chitosan Used for Cellular Scaffolds 31317.6 Conclusions and Future Perspectives 31318 Stimuli-Sensitive Chitosan-Based Systems for Biopharmaceuticals 319Cuiping Zhai, Jinfang Yuan, and Qingyu Gao18.1 Introduction 31918.2 pH-Sensitive Chitosan-Based Systems 31918.3 Thermosensitive Chitosan-Based Systems 32118.4 pH-Sensitive and Thermosensitive Chitosan-Based Systems 32318.5 pH- and Ionic-Sensitive Chitosan-Based Systems 32518.6 Photo-Sensitive Chitosan-Based Systems 32518.7 Electrical-Sensitive Chitosan-Based Systems 32618.8 Magnetic-Sensitive Chitosan-Based Systems 32618.9 Chemical Substance-Sensitive Chitosan-Based Systems 32718.10 Conclusions and Future Perspectives 32719 Chitosan Copolymers for Biopharmaceuticals 333Ramon Novoa-Carballal, Ricardo Riguera, and Eduardo Fernandez-Megia19.1 Introduction 33319.2 Chitosan-g-Poly(Ethylene Glycol) 33719.3 Chitosan-g-Polyethylenimine 34719.4 Other Copolymers of Chitosan 35719.5 Copolymers of Chitosan with Promising Applications 36319.6 Conclusions and Future Perspectives 368 20 Application of Chitosan for Anticancer Biopharmaceutical Delivery 381Claudia Philippi, Brigitta Loretz, Ulrich F. Schaefer, and Claus-Michael Lehr20.1 Introduction 38120.2 Chitosan and Cancer: Intrinsic Antitumor Activity of the Polymer Itself 38220.3 Chitosan Formulations Developed for Classic Anticancer Drugs 38320.4 Biopharmaceuticals Delivered by Chitosan Preparations 38420.5 Active Targeting Strategies and Multifunctional Chitosan Formulations 38820.6 Conclusions and Future Perspectives 38921 Chitosan-Based Biopharmaceutical Scaffolds in Tissue Engineering and Regenerative Medicine 393Tao Jiang, Meng Deng, Wafa I. Abdel- Fattah, and Cato T. Laurencin21.1 Introduction 39321.2 Fabrication of Chitosan-Based Biopharmaceuticals Scaffolds 39521.3 Applications of Chitosan-Based Biopharmaceutical Scaffolds in Tissue Engineering and Regenerative Medicine 40321.4 Future Trends: Regenerative Engineering 41621.5 Conclusions and Future Perspectives 41722 Wound-Healing Properties of Chitosan and Its Use in Wound Dressing Biopharmaceuticals 429Tyler G. St. Denis, Tianhong Dai, Ying-Ying Huang, and Michael R. Hamblin22.1 Introduction 42922.2 Brief Review of Wound Repair 43022.3 Wound-Healing Effects of Chitosan 43322.4 Chitosan for Wound Therapeutics Delivery 44022.5 Conclusions and Future Perspectives 444Part Four Regulatory Status, Toxicological Issues, and Clinical Perspectives 45123 Toxicological Properties of Chitosan and Derivatives for Biopharmaceutical Applications 453Thomas J. Kean and Maya Thanou23.1 Introduction 45323.2 In Vitro Toxicity of Chitosan and Derivatives 45423.3 In Vivo Toxicity of Chitosan and Derivatives 45723.4 Conclusions and Future Perspectives 459 24 Regulatory Status of Chitosan and Derivatives 463Michael Dornish, David S. Kaplan, and Sambasiva R. Arepalli24.1 Introduction 46324.2 Source 46424.3 Characterization 46424.4 Purity 46524.5 Applications of Advanced Uses of Chitosan 46624.6 Regulatory Considerations for Chitosan and Chitosan Derivatives in the European Union, and Medical Devices or Combination Products with Medical Device (CDRH) Lead 46824.7 Regulatory Pathways 46924.8 Chitosan Medical Products: US Regulatory Review Processes for Medical Devices or Combination Products with CDRH Lead 46924.9 Chitosan Wound Dressings 47024.10 The European Regulatory System: The European Medicines Agency (EMA) and European Directorate for the Quality of Medicines (EDQM) 47424.11 Further Regulatory Considerations 47524.12 Conclusions and Future Perspectives 47724.13 Disclaimer 47825 Patentability and Intellectual Property Issues Related to Chitosan-Based Biopharmaceutical Products 483Mafalda Videira and Rogerio Gaspar25.1 Introduction 48325.2 Setting the Scene: The Role of Chitosan as a Pharmaceutical Excipient 48425.3 Addressing the Drivers for Scientific Progress on Chitosan: Innovation and Inventability 49525.4 Conclusions and Future Perspectives 49626 Quality Control and Good Manufacturing Practice (GMP) for Chitosan-Based Biopharmaceutical Products 503Torsten Richter, Maika Gulich, and Katja Richter26.1 Introduction 50426.2 Regulatory Requirements for Production 50526.3 Manufacturing GMP: Fundamental Considerations 50826.4 Requirements for Rooms, Personnel, and Equipment 51126.5 Qualification and Validation 51126.6 Quality Control 51326.7 Monitoring and Maintenance of a GMP System 51926.8 Conclusions and Future Perspectives 52227 Preclinical and Clinical Use of Chitosan and Derivatives for Biopharmaceuticals: From Preclinical Research to the Bedside 525David A. Zaharoff, Michael Heffernan, Jonathan Fallon, and John W. Greiner27.1 Introduction 52527.2 Chitosan as a Parenteral (Subcutaneous) Vaccine Platform 52627.3 Chitosan as an Immunotherapeutic Platform 53027.4 Conclusions and Future Perspectives 537References 539Index 543