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Development of Diverse Hydrogels from the Protean Polymer, Elastin-Like Polypeptide

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Development of Diverse Hydrogels from the Protean Polymer, Elastin-Like Polypeptide Development of Diverse Hydrogels from the Protean Polymer, Elastin-like Polypeptide By Malav S. Desai A dissertation submitted in partial satisfaction of the requirements for the degree of Joint Doctor of Philosophy with University of California, San Francisco in Bioengineering in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Seung-Wuk Lee, Chair Professor Tejal Desai Professor Ting Xu Fall 2017 Development of Diverse Hydrogels from the Protean Polymer, Elastin-like Polypeptide © Copyright 2017 by Malav S. Desai To Elaine & my parents 1 Abstract Development of Diverse Hydrogels from the Protean Polymer, Elastin-like Polypeptide by Malav S. Desai Joint Doctor of Philosophy in Bioengineering with University of California, San Francisco University of California, Berkeley Professor Seung-Wuk Lee, Chair Hydrogels have been used to develop scaffolds to address challenges in tissue engineering and drug delivery for several decades. The major advantage of using hydrogels is that their high water content and low stiffness enables them to mimic tissue- like environments. In my work, I develop four unique hydrogels using a single polymer, elastin-like polypeptide. The first challenge I address is that of hydrogel extensibility. Typical hydrogels cannot undergo large strains, which limits their durability and applicability to active tissues such as muscles. I use ELP to create a rubber-like elastic hydrogel that can stretch to 15 times its original length. Unlike most previous ELP hydrogels, our gels can reach such high strains due to the well-defined structure of our materials with minimal crosslinks. Next, I focus on developing an adhesive hydrogel using ELP. Unlike most adhesives, especially protein-based adhesives, our adhesive hydrogels are flexible and deswell in physiological conditions. This improves their potential for success in practical applications. We also developed a self-healing hydrogel by combining ELP with bioglass. Unlike other strategies that use reversible physical bonds, we use pH driven reversible Schiff base formation to synthesize hydrogels. Bioglass raises the localized pH to yield ideal conditions that favor gelling in our system. We also show that our materials can self-heal after being severed, which makes them durable and ideal for topical applications. Finally, we employ our efforts in the design and synthesis of protein-based beads for biolaser development. These devices are stimuli- responsive, which makes them ideal for optical sensing applications for biomolecule sensing and the flexibility of the hydrogels also makes them suitable for studying cellular biophysics. Through my dissertation, I hope to convey the versatility of ELP and the potential of structural design that can lead to the development of hydrogels with diverse functions. 2 Table of contents Abstract ............................................................................................................................... 2 Table of contents .................................................................................................................. i List of figures ...................................................................................................................... v List of tables ...................................................................................................................... vii Acknowledgments............................................................................................................ viii Chapter 1 Protein biomaterials – hydrogel design and applications ................................... 1 1.1 Hydrogels .................................................................................................................. 1 1.2 Recombinant structural proteins ................................................................................ 2 1.2.1 Elastin & Elastin-like polypeptides (ELP) ......................................................... 3 1.2.2 Collagen & Collagen-like polypeptides (CLP) .................................................. 5 1.2.3 Silk & Silk-like polypeptides (SLP) ................................................................... 7 1.2.4 Resilin & Resilin-like polypeptides (RLP) ....................................................... 10 1.3 Design and applications of protein hydrogels ......................................................... 11 1.3.1 Elastin & Elastin-like polypeptides .................................................................. 11 1.3.2 Collagen & Collagen-like polypeptides ........................................................... 14 1.3.3 Silk & Silk-like polypeptides ........................................................................... 15 1.3.4 Resilin & Resilin-like polypeptides .................................................................. 17 1.4 Tackling materials challenges: A case for ELP ...................................................... 18 Chapter 2 ELP engineering ............................................................................................... 20 2.1 Introduction ............................................................................................................. 20 2.2 Strategy for seamless cloning of ELP ..................................................................... 20 2.2.1 Genetic engineering scheme ............................................................................. 20 2.2.2 Method for cloning ELP genes ......................................................................... 21 2.2.3 Method for construction the expression vector ................................................ 22 2.2.4 ELP Expression and Purification ...................................................................... 23 Chapter 3 Rubber-like hydrogels ...................................................................................... 27 3.1 Introduction ............................................................................................................. 27 3.2 Strategy for improved flexibility ............................................................................. 28 3.3 ELP design and characterization ............................................................................. 30 i 3.3.1 ELP transition temperature ............................................................................... 31 3.4 Thermo-responsive behavior of ELP hydrogels ...................................................... 32 3.5 Uniaxial tensile testing of ELP hydrogels ............................................................... 33 3.6 Cyclic tensile testing of ELP hydrogels for resilience ............................................ 36 3.7 Cyclic tensile tests to determine the extent of elasticity ......................................... 39 3.8 ELP hydrogel resilience is potentially influenced by ELP size .............................. 42 3.9 Conclusion ............................................................................................................... 45 3.10 Methods ................................................................................................................. 46 3.10.1 Swelling Tests ................................................................................................ 46 3.10.2 Tensile Tests ................................................................................................... 46 3.10.3 Resilience Tests .............................................................................................. 47 Chapter 4 Adhesive hydrogels .......................................................................................... 48 4.1 Introduction ............................................................................................................. 48 4.2 Strategy for adhesive synthesis ............................................................................... 49 4.3 ELP synthesis and material design .......................................................................... 50 4.4 Chemical functionalization of V4E125 ................................................................... 51 4.5 Rheological properties of ELP/Cat ......................................................................... 55 4.6 Adhesive properties of ELP/Cat .............................................................................. 59 4.7 Conclusion ............................................................................................................... 61 4.8 Method .................................................................................................................... 61 4.8.1 Functionalizing V4E125 with Dopamine ......................................................... 61 4.8.2 1H NMR ............................................................................................................ 62 4.8.3 Adhesive preparation ........................................................................................ 62 4.8.4 Rheology ........................................................................................................... 62 4.8.5 Skin preparation for adhesion tests ................................................................... 62 4.8.6 Tensile pull-off test ........................................................................................... 63 4.8.7 Lap-shear test .................................................................................................... 63 Chapter 5 Self-healing hydrogels .....................................................................................
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