Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2006 Engineering polyanhydride microspheres for the stabilization and controlled release of proteins Amy Sywassink Determan Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biomedical Engineering and Bioengineering Commons, and the Chemical Engineering Commons Recommended Citation Determan, Amy Sywassink, "Engineering polyanhydride microspheres for the stabilization and controlled release of proteins " (2006). Retrospective Theses and Dissertations. 1809. https://lib.dr.iastate.edu/rtd/1809 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Engineering polyanhydride microspheres for the stabilization and controlled release of proteins by Amy Sywassink Determan A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Chemical Engineering Program of Study Committee: Balaji Narasimhan, Co-major Professor Marit Nilsen-Hamilton, Co-major Professor Surya K. Mallapragada Monica H. Lamm Michael J. Wannemuehler F. Chris Minion Iowa State University Ames, Iowa 2006 Copyright © Amy Sywassink Determan, 2006. All rights reserved. UMI Number: 3217264 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3217264 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Amy Sywassink Determan has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. For the Major Program iii TABLE OF CONTENTS ACKNOWLEDGEMENTS vii ABSTRACT ix CHAPTER 1 INTRODUCTION 1 1.1. Introduction 1 1.2. References 5 CHAPTER 2 ENGINEERING SURFACE-ERODIBLE POLYANHYDRIDES WITH TAILORED MICROSTRUCTURE FOR CONTROLLED RELEASE 9 2.1 Introduction 9 2.2 Polymer Synthesis 9 2.2.1 Melt polycondensation 11 2.2.2 Solution polymerization 13 2.2.3 Ring opening reactions 15 2.3 Polymer Characterization 17 2.3.1 Chemical composition 17 2.3.2 Molecular mass 20 2.3.3 Crystallinity and thermal properties 21 2.3.4 Stability 23 2.3.5 Biocompatibility 24 2.4 Structures 25 2.4.1 Aliphatic 26 2.4.2 Aromatic 26 2.4.3 Unsaturated 27 2.4.4 Aliphatic-aromatic homopolymers 28 2.4.5 Amino acid-based 28 2.4.6 Fatty acid-based 30 2.4.7 Poly(ester-anhydrides) and poly(ether-anhydride) 31 2.4.8 Crosslinking 33 2.4.9 Branched 34 2.4.10 Blends 35 2.5 Characterization of Polymer Erosion 36 2.5.1 Polymer degradation 37 2.5.2 Polymer erosion 38 2.6 Mechanisms of Protein Denaturation 41 2.6.1 Disulfide formation & aggregation 42 2.6.2 Deamidation 43 2.6.3 Oxidation 43 2.6.4 Degradation by proteases 44 2.7 Microsphere Fabrication 44 2.7.1 Hot-melt microencapsulation 45 iv 2.7.2 Solvent removal microencapsulation 45 2.7.3 Spray drying microencapsulation 48 2.8 Protein-Loaded Microsphere Characterization 51 2.8.1 Confocal laser scanning microscopy 52 2.8.2 Analysis of secondary structure 52 2.8.3 Analysis of primary structure 53 2.9 In Vitro Release 54 2.9.1 In vitro release kinetics of protein-loaded microspheres 54 2.9.2 In vitro effect of polymer degradation products on protein stability 56 2.10 Summary 56 2.11 References 57 CHAPTER 3 RESEARCH OBJECTIVES & ORGANIZATION 67 3.1. Research Objective 67 3.2. Dissertation Organization 69 CHAPTER 4 ENCAPSULATION, STABILIZATION, AND RELEASE OF BSA-FITC FROM POLY ANHYDRIDE MICROSPHERES 70 4.1 Abstract 70 4.2 Introduction 71 4.3 Materials and Methods 74 4.3.1 Materials 74 4.3.2 Polymer synthesis 75 4.3.3 Polymer characterization 75 4.3.4 Microsphere fabrication 76 4.3.5 Microsphere characterization 77 4.3.6 Protein loading 77 4.3.7 In vitro protein release 78 4.3.8 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 78 4.3.9 Fourier transform infrared spectroscopy (FTIR) 79 4.4 Results and Discussion 80 4.4.1 Microsphere characterization 80 4.4.2 Protein loading 84 4.4.3 In vitro release 84 4.4.4 SDS-PAGE analysis 86 4.4.5 FTIR analysis 88 4.5 Conclusions 91 4.6 Acknowledgements 92 4.7 References 93 V CHAPTER 5 PROTEIN STABILITY IN THE PRESENCE OF POLYMER DEGRADATION PRODUCTS: CONSEQUENCES FOR CONTROLLED RELEASE FORMULATIONS 97 5.1. Abstract 97 5.2 Introduction 98 5.3 Materials and Methods 100 5.3.1 Materials 100 5.3.2 In vitro incubation 101 5.3.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 102 5.3.4 Circular dichroism (CD) 103 5.3.5 Fluorescence spectroscopy 104 5.3.6 TT-specific enzyme-linked-immunosorbent-assay (ELIS A) 104 5.3.7 Ovalbumin-specific enzyme-linked- immunosorbent-assay (ELISA) 105 5.3.8 Lysozyme activity test 106 5.4 Results 107 5.4.1 Stability of tetanus toxoid 107 5.4.2 Stability of ovalbumin 112 5.4.3 Stability of lysozyme 118 5.5 Conclusions 122 5.6 Acknowledgments 124 5.7 References 125 CHAPTER 6 THE ROLE OF MICROSPHERE FABRICATION METHODS ON THE STABILITY AND RELEASE KINETICS OF OVALBUMIN ENCAPSULATED IN POLY ANHYDRIDE MICROSPHERES 128 6.1 Abstract 128 6.2 Introduction 129 6.3 Materials and Methods 132 6.3.1 Materials 132 6.3.2 Polymer synthesis & characterization 132 6.3.3 Ovalbumin lyophilization 133 6.3.4 W/O/W microsphere fabrication method 133 6.3.5 W/O/O microsphere fabrication method 134 6.3.6 S/O/O microsphere fabrication method 134 6.3.7 Cryogenic atomization method for microsphere fabrication 135 6.3.8 Microsphere characterization 135 6.3.9 Encapsulation efficiency 136 6.3.10 In vitro release studies 136 6.3.11 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 137 vi 6.3.12 Fourier transform infrared spectroscopy (FTIR) 138 6.4 Results and Discussion 139 6.4.1 Ovalbumin atomization 139 6.4.2 Microsphere characterization 139 6.4.3 Encapsulation efficiency 144 6.4.4 In vitro release of ovalbumin 145 6.4.5 SDS-PAGE analysis 147 6.4.6 FTIR analysis 148 6.5 Conclusions 150 6.6 Acknowledgements 151 6.7 References 151 CHAPTER 7 UTEROCALIN-LOADED POLYANHYDRIDE MICROSPHERES EXPEDITE CELL MIGRATION FOR WOUND HEALING APPLICATIONS 154 7.1 Abstract 154 7.2 Introduction 155 7.3 Materials and Methods 157 7.3.1 Materials 157 7.3.2 Polymer synthesis & characterization 158 7.3.3 Protein purification 158 7.3.4 Protein lyophilization 160 7.3.5 Microsphere fabrication 160 7.3.6 Microsphere characterization 161 7.3.7 Sodium dodecyl sulfate gel electrophoresis (SDS- PAGE) 162 7.3.8 In vitro release 162 7.3.9 Incubation of microspheres for cell migration assay 163 7.3.10 Cell migration assay 164 7.3.11 Data analysis 166 7.4 Results 166 7.4.1 Uterocalin lyophilization 166 7.4.2 Microsphere characterization 167 7.4.3 SDS-PAGE analysis 168 7.4.4 In vitro release kinetics 170 7.4.5 Cell migration assay 171 7.5 Conclusions 173 7.6 Acknowledgments 175 7.7 References 176 CHAPTER 8 CONCLUSIONS AND FUTURE DIRECTIONS 178 8.1 References 183 vii ACKNOWLEDGMENTS Before reading my dissertation, I would like to acknowledge the faculty, staff, and students who helped make my time as a graduate student a little easier. I would first like to thank my graduate advisors Balaji Narasimhan and Marit Nilsen-Hamilton for the guidance they gave me and for the different perspectives that they provided. I would also like to thank the members of Dr. Narasimhan and Dr. Nilsen-Hamilton's research groups. In particular I owe much thanks to Dr. Matt Kipper and Lee Bendickson for the invaluable assistance that they gave me. I also cannot forget to thank all of the undergraduate students who have helped me (Elizabeth Schmerr, Quentin Leigh, Angie Clark, Jennifer Graham, Katherine Pfeiffer, Martin Gran, Lindsey Boland, Michelle Sukup, and Laura Miller) by synthesizing polymers, doing electrophoresis, or washing dishes your time was much appreciated. As you read the following chapters please note the numerous co-authors whose expertise added more depth to my project. Without their help this work would not have been possible. I also want to acknowledge Warren Straszheim and Jerry Amenson in the MARL laboratories at Iowa State University for their assistance with the scanning electron microscope. And I would like to thank Dr. Robert Doyle in the Carver Laboratory at Iowa State University for the assistance he offered me with my confocal and optical microscopy experiments. I would like to thank the members of my POS committee (Dr.