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First Line of Title RESILIN-LIKE POLYPETIDE-BASED MICROSTRUCTURED HYDROGELS VIA AQUEOUS-BASED LIQUID-LIQUID PHASE SEPARATION FOR TISSUE ENGINEERING APPLICATIONS by Hang Kuen Lau A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Materials Science and Engineering Fall 2018 © 2018 Hang Kuen Lau All Rights Reserved RESILIN-LIKE POLYPETIDE-BASED MICROSTRUCTURED HYDROGELS VIA AQUEOUS-BASED LIQUID-LIQUID PHASE SEPARATION FOR TISSUE ENGINEERING APPLICATIONS by Hang Kuen Lau Approved: __________________________________________________________ Darrin J. Pochan, Ph.D. Chair of the Department of Materials Science and Engineering Approved: __________________________________________________________ Babatunde A. Ogunnaike, Ph.D. Dean of the College of Engineering Approved: __________________________________________________________ Douglas J. Doren, Ph.D. Interim Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Kristi L. Kiick, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Xinqiao Jia, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Christopher J. Kloxin, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Thomas H. Epps, III, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Kristi Kiick her guidance and support as a mentor, scientist and scholar. I am grateful for her encouraging and guiding me via my time in University of Delaware to improve my research, critical thinking, and writing and presentation skills. This dissertation would not have been possible without her. I also thank my committee members Dr. Thomas Epps from Chemical Engineering, Dr Xinqiao Jia and Dr. Chris Kloxin from Materials Sciences and Engineering for their support and assistance on my dissertation. I am also thankful for our collaborators Dr. Sapun Parekh from Max Planck Institute for Polymer Research, Dr. Alexandra Paul and Dr. Annika Enejder from Chalmers University of Technology for their contribution on CARS characterization on microstructued hydrogels micrcomposition. I would also like to thank Dr. Al Crosby, Shruti Rattan, Hongbo Fu and Dylan Barber from the University of Massachusetts Amherst for help and contribution in micromechanical characterization on microstructued hydrogels. I also thank Dr. Susan Thibeault and Renee King from University of Wisconsin, Madison for their help and contribution on in vivo animal vocal fold injections. I would like to thank Dr. Linqing Li and Dr. Chris McGann for training and guiding me to start on the resilin-like polypeptides projects. Thanks also to Dr. Becca Scott and Ishnoor Sidhu for help in cell culture experiments. I would also like to thank, from the Delaware Biotechnology Institute Bioimaging Center, Dr. Jeff Kaplan, Dr. Michael Moore and Sylvain Le Marchand for training and help with multi-photon and confocal microscopes and also extend thanks to Dr. Chandran Sabanayagam for help with nanoindentation on iv AFM. Thanks to Dr. Shi Bai for help with NMR spectroscopy and Dr. PapaNii Asare- Okai for help and training with mass spectrometry. I would also like to thank all the current and previous Kiick group members Dr. Linqing Li, Dr. Chris McGann, Dr. Nandita Bhadwat, Dr. Eric Levenson, Dr. Tianzhi Luo, Dr. Yingkai Liang, Dr. Prathamesh Kharkar, Dr. Morgan Urello, Dr. Chris Koehler, Dr Becca Scott, Bradford Paik, Ishnoor Sidhu, Haocheng Wu, Yu Tian, Michael Haider, Cristobal Garcia, Jingya Qin, Haofu Huang, Luisa Palmese and Ming Fan. Thanks also to all my friends and colleagues Shuang Liu, Shuyu Xu, Ying Hao, Tugba Ozdemir, Anitha Ravikrishnan, Kevin Dicker, Aidan Zerdoum, Eric Fowler. I would also like to thank the current and previous staff in Department of Materials Science and Engineering Charlies Garbini, Christine Williamson, Naima Hall, Robin Buccos, Kathleen Forwood and Judy Allarey for ensuring lab safety and providing administrative supports. v TABLE OF CONTENTS LIST OF TABLES ........................................................................................................ ix LIST OF FIGURES ........................................................................................................ x ABSTRACT ............................................................................................................... xvii Chapter 1 MULTICOMPONENT HYBRID HYDROGELS IN BIOMEDICAL APPLICATIONS ................................................................................................ 1 1.1 Introduction ............................................................................................... 1 1.2 Hydrogel Network Formation ................................................................... 3 1.3 Mimics of Natural Proteins ....................................................................... 8 1.3.1 Elastin ............................................................................................ 9 1.3.2 Resilin .......................................................................................... 10 1.4 Composite Hydrogels .............................................................................. 13 1.4.1 Nanocrystal-reinforced Matrices ................................................. 14 1.4.2 Particle-reinforced Matrices ........................................................ 15 1.4.3 Fiber-reinforced Matrices ........................................................... 18 1.5 Hybrid Materials with Engineered Biological Functions ........................ 19 1.6 Conclusion and Perspectives ................................................................... 24 1.7 Dissertation Summary ............................................................................. 26 2 AQUEOUS LIQUID-LIQUID PHASE SEPRARATION OF RESILIN- LIKE POLYPEPTIDE/POLYETHYLENE GLYCOL FOR FORMATION OF MICROSTUCTURED HYDROGELS ...................................................... 28 2.1 Introduction ............................................................................................. 28 2.2 Materials and Methods ............................................................................ 31 2.2.1 Materials ...................................................................................... 31 2.2.2 RLP Expression and Purification ................................................ 31 2.2.3 Characterization of RLP/PEG Phase Separation ......................... 32 2.2.4 Characterization of Equilibrium Concentrations ......................... 33 vi 2.2.5 Fluorescent Labeling of RLP and PEG ....................................... 33 2.2.6 Hydrogel Formation .................................................................... 34 2.2.7 Oscillatory Rheology ................................................................... 34 2.2.8 AFM Indentation ......................................................................... 35 2.3 Results and Discussion ............................................................................ 36 2.3.1 RLP/PEG Phase Separation ......................................................... 36 2.3.2 Co-existence Concentration of RLP/PEG ................................... 41 2.3.3 Microstructured Hydrogels .......................................................... 45 2.3.4 Hydrogel Bulk and Micromechanical Properties ........................ 48 2.4 Conclusions ............................................................................................. 53 3 MICROSTRUCTURED ELASTOMER-POLYETHYLENE GLYCOL HYDROGELS VIA KINETIC CAPTURE OF AQUEOUS LIQUID- LIQUID PHASE SEPRARATION .................................................................. 55 3.1 Introduction ............................................................................................. 55 3.2 Materials and Methods ............................................................................ 58 3.2.1 Materials ...................................................................................... 58 3.2.2 RLP Expression and Purification ................................................ 59 3.2.3 RLP Functionalization and Characterization ............................... 59 3.2.4 Characterization of RLP-Ac/PEG-4Ac Phase Separation ........... 60 3.2.5 Characterization of Equilibrium Concentrations ......................... 61 3.2.6 Hydrogel Formation .................................................................... 61 3.2.7 Oscillatory Rheology ................................................................... 61 3.2.8 Polymerization Yield ..................................................................
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