Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Duan, Tianyu
Doctor of Philosophy - PhD
University of British Columbia
Hydrogels are water-containing soft materials made of cross-linked polymer networks. Hydrogels can emulate many features of native tissues and organisms, and have therefore found many applications in biomedical studies. Recently, driven by the needs for stimuli-responsive materials in various biomedical applications, “dynamic” hydrogels that can change their physical, chemical, biological, or mechanical properties have attracted increasing interest. Recombinant proteins are promising hydrogel building blocks due to their biocompatibility, diverse biofunctions, and designable structures. Moreover, stimuli-responsive conformational changes and interactions of proteins provide possibilities for constructing dynamic protein-based hydrogels. This thesis reviews the state-of-the-art studies of dynamic protein-based hydrogels, including the cross-linking methods, the design principles, and the future directions of this field. Driven by the needs for more predictable, sensitive, and reversible hydrogels, we engineer novel protein-based hydrogels in this thesis. An elastomeric protein-based hydrogel is first engineered with thermo-responsive properties, which is controlled by the thermo-responsive in situ phase transition of the polypeptide side chains in the hydrogel network. Then, two dynamic hydrogels are physically cross-linked by a coiled coil interaction and a protein-protein interaction, respectively, which both exhibit time-dependent viscoelastic properties, multiple energy dissipation modes, and injectability. The viscoelastic properties of these two physically cross-linked hydrogels are stimuli-responsive due to the stimuli-responsive nature of their cross-linking methods. Finally, a light-controlled information writing approach is developed by using a light-responsive protein-protein interaction to decorate fluorescent proteins onto protein-based hydrogel blank slates. Consequently, 2D/3D fluorescence images can be produced and stored in protein-based hydrogels. The developed hydrogels are mainly responsive to temperature or light, which are two types of stimuli that have been widely adapted as therapeutic tools in biomedical studies. The stimuli-responsive property changes in the developed hydrogel systems are highly dose-sensitive and reversible, which facilitates customized fine tuning of hydrogel functionality. Therefore, we anticipate that these developed dynamic protein-based hydrogels will find various applications in biomedical studies. Moreover, the results of this thesis are expected to contribute to the quantitative prediction of dynamic hydrogel properties and inspire the bottom-up designs of other dynamic hydrogels.
ENGINEERING DYNAMIC PROTEIN-BASED HYDROGELS by Tianyu Duan B.Sc., Beijing Normal University, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2022 © Tianyu Duan, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Engineering Dynamic Protein-Based Hydrogels submitted by Tianyu Duan in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Hongbin Li, Professor, Chemistry, UBC Supervisor Jayachandran Kizhakkedathu, Professor, Pathology, Chemistry, UBC Supervisory Committee Member Carl Michal, Associate Professor, Physics, Chemistry, UBC University Examiner Mark Thachuk, Associate Professor, Chemistry, UBC University Examiner Additional Supervisory Committee Members: Katherine Ryan, Associate Professor, Chemistry, UBC Supervisory Committee Member Suzana Straus, Professor, Chemistry, UBC Supervisory Committee Member iii Abstract Hydrogels are water-containing soft materials made of cross-linked polymer networks. Hydrogels can emulate many features of native tissues and organisms, and have therefore found many applications in biomedical studies. Recently, driven by the needs for stimuli-responsive materials in various biomedical applications, “dynamic” hydrogels that can change their physical, chemical, biological, or mechanical properties have attracted increasing interest. Recombinant proteins are promising hydrogel building blocks due to their biocompatibility, diverse biofunctions, and designable structures. Moreover, stimuli-responsive conformational changes and interactions of proteins provide possibilities for constructing dynamic protein-based hydrogels. This thesis reviews the state-of-the-art studies of dynamic protein-based hydrogels, including the cross-linking methods, the design principles, and the future directions of this field. Driven by the needs for more predictable, sensitive, and reversible hydrogels, we engineer novel protein-based hydrogels in this thesis. An elastomeric protein-based hydrogel is first engineered with thermo-responsive properties, which is controlled by the thermo-responsive in situ phase transition of the polypeptide side chains in the hydrogel network. Then, two dynamic hydrogels are physically cross-linked by a coiled coil interaction and a protein-protein interaction, respectively, which both exhibit time-dependent viscoelastic properties, multiple energy dissipation modes, and injectability. The viscoelastic properties of these two physically cross-linked hydrogels are stimuli-responsive due to the stimuli-responsive nature of their cross-linking methods. Finally, a light-controlled information writing approach is developed by using a light-responsive protein-protein interaction to decorate fluorescent proteins onto protein-based iv hydrogel blank slates. Consequently, 2D/3D fluorescence images can be produced and stored in protein-based hydrogels. The developed hydrogels are mainly responsive to temperature or light, which are two types of stimuli that have been widely adapted as therapeutic tools in biomedical studies. The stimuli-responsive property changes in the developed hydrogel systems are highly dose-sensitive and reversible, which facilitates customized fine tuning of hydrogel functionality. Therefore, we anticipate that these developed dynamic protein-based hydrogels will find various applications in biomedical studies. Moreover, the results of this thesis are expected to contribute to the quantitative prediction of dynamic hydrogel properties and inspire the bottom-up designs of other dynamic hydrogels. v Lay Summary Hydrogels are water-containing soft materials made of cross-linked polymer networks. This thesis provides a comprehensive understanding of protein-based hydrogels. A series of “dynamic” protein-based hydrogels are engineered, which can change their physical, chemical, or mechanical properties in response to external stimuli including temperature variations and light. Such stimuli-responsive property allows us to readily regulate the mechanical performance of these protein-based hydrogels for desired biomedical applications such as tissue engineering scaffolds. In addition, fluorescence images can be written by light and stored in the light-responsive dynamic protein-based hydrogels for the development of soft smart devices. vi Preface Portions of this thesis benefited from the intellectual and experimental contributions of others. Chapter 2 has been published. [Tianyu Duan] and Hongbin Li. (2020) In Situ Phase Transition of Elastin-Like Polypeptide Chains Regulates Thermoresponsive Properties of Elastomeric Protein-Based Hydrogels. Biomacromolecules. 21:2258-2267. This chapter is incorporated in this thesis with permission of American Chemical Society, Copyright 2020. Dr. Li and I conceived the project. Dr. Li and I designed the experiments together. I am responsible for all the experiments and the data analysis. I wrote the manuscript together with Dr. Li. Chapter 3 has been published. Wenxu Sun, [Tianyu Duan], Yi Cao, and Hongbin Li. (2019) An Injectable Self-Healing Protein Hydrogel with Multiple Dissipation Modes and Tunable Dynamic Response. Biomacromolecules. 20: 4199-4207. This chapter is incorporated in this thesis with permission of American Chemical Society, Copyright 2019. Dr. Li and Dr. Cao conceived the idea and designed the study. Wenxu Sun and I performed the experiments and analyzed the results together. Wenxu Sun and I contributed equally. All the authors discussed the results and wrote and refined the manuscript together. Chapter 4 has been published. [Tianyu Duan], Qingyuan Bian, and Hongbin Li. (2021) Light-Responsive Dynamic Protein Hydrogels Based on LOVTRAP. Langmuir. 37: 10214-10222. This chapter is incorporated in this thesis with permission of American Chemical Society, Copyright 2021. Dr. Li and I conceived the project. Dr. Li and I designed the experiments together. Qingyuan Bian helped with the protein engineering. I performed the remaining vii experiments and analyzed all the data. I wrote the manuscript together with Dr. Li, and Qingyuan Bian helped to revise the manuscript. Chapter 5 is in preparation for publication. [Tianyu Duan], Qingyuan Bian, and Hongbin Li. Protein Hydrogels with Reversibly Patterned Multidimensional Fluorescent Images for Information Storage. Dr. Li conceived the project. Dr. Li and I designed the experiments together. Qingyuan Bian and I performed the protein engineering together. Qingyuan Bian helped with early hydrogel preparation and patterning experiments. I am responsible for the remaining experiments and all the data analysis. I wrote the manuscript together with Dr. Li, and Qingyuan Bian helped to revise the manuscript. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiii List of Figures ............................................................................................................................. xiv List of Symbols .......................................................................................................................... xvii List of Abbreviations ................................................................................................................. xix Acknowledgements .................................................................................................................. xxiii Dedication ...................................................................................................................................xxv Chapter 1: Introduction ................................................................................................................1 1.1 Hydrogels ........................................................................................................................ 1 1.2 Dynamic Protein-Based Hydrogels................................................................................. 2 1.3 General Cross-Linking Methods for Protein-Based Hydrogels ...................................... 3 1.3.1 Chemical Cross-Linking Methods .............................................................................. 4 1.3.1.1 Reactions of Cysteine ......................................................................................... 4 1.3.1.2 Di-Tyrosine Reactions ........................................................................................ 5 1.3.1.3 Reactions of Lysine............................................................................................. 7 1.3.1.4 Covalent Bonds Formed in Protein Assembly .................................................... 8 1.3.2 Physical Cross-Linking Methods ................................................................................ 9 1.3.2.1 Histidine-Metal Coordination ............................................................................. 9 ix 1.3.2.2 Phase Transition of Elastin-Like Polypeptides ................................................. 11 1.3.2.3 Coiled-Coil Interactions .................................................................................... 15 1.3.2.4 β-Sheet Assembly ............................................................................................. 17 1.3.2.5 Non-Covalent Protein Assembly ...................................................................... 19 1.4 Multifaceted Expectations for Dynamic Protein-Based Hydrogels in Biomedical Applications .............................................................................................................................. 21 1.4.1 Sol-Gel Transition ..................................................................................................... 22 1.4.2 Degradation ............................................................................................................... 22 1.4.3 Permeability and Swelling Ratio .............................................................................. 23 1.4.4 Mechanical Properties ............................................................................................... 24 1.4.5 Biocompatibility and Bio-Functionality ................................................................... 25 1.5 General Regulation Strategies to Construct Dynamic Protein-Based Hydrogels ......... 26 1.5.1 Tuning the Number of Cross-Linking Points............................................................ 28 1.5.2 Tuning the Effective Length of Elastically Active Chains ....................................... 31 1.5.3 Molecule Decoration on Protein-Based Hydrogel Blank Slates ............................... 36 1.6 Engineering Dynamic Protein-Based Hydrogels: Goals of Thesis ............................... 40 Chapter 2: In situ Phase Transition of Elastin-Like Polypeptide Chains Regulates Thermo-Responsive Properties of Elastomeric Protein-Based Hydrogels ............................................44 2.1 Synopsis ........................................................................................................................ 44 2.2 Introduction ................................................................................................................... 45 2.3 Materials and Methods .................................................................................................. 48 2.4 Results and Discussion ................................................................................................. 51 2.4.1 Protein Engineering .................................................................................................. 51 x 2.4.2 Temperature-Dependent Phase Transitions of Vn-(GR)4 Proteins ........................... 53 2.4.3 Post-Gelation In Situ Transition of Vn Side Chains Alters Thermo-Responsive Properties of (GR)4+Vn-(GR)4-Hydrogels ........................................................................... 54 2.4.4 Thermo-Responsiveness of (GR)4+Vn-(GR)4-Hydrogels can be Regulated ............ 59 2.4.5 Pre-Gelation Phase Transition of Vn-ELP Chains Compromises Thermo-Responsiveness of (GR)4+Vn-(GR)4-Hydrogels .................................................................. 63 2.5 Conclusions ................................................................................................................... 65 Chapter 3: An Injectable Self-Healing Protein Hydrogel with Multiple Dissipation Modes and Tunable Dynamic Response.................................................................................................66 3.1 Synopsis ........................................................................................................................ 66 3.2 Introduction ................................................................................................................... 67 3.3 Materials and Methods .................................................................................................. 69 3.4 Results and Discussion ................................................................................................. 71 3.4.1 Design of the Hydrogel ............................................................................................. 71 3.4.2 GKE Hydrogels Display Time-Dependent Dynamic Properties .............................. 74 3.4.3 GKE Hydrogels Display Two Dissipation Modes .................................................... 76 3.4.4 Environmental Factors (Temperature, Ionic Strength and Stoichiometry) Affect the Dissipation Modes of the GKE Hydrogels ........................................................................... 78 3.4.5 Injection and Self-Healing of the GKE Hydrogels ................................................... 84 3.5 Conclusions ................................................................................................................... 86 Chapter 4: Light-Responsive Dynamic Protein Hydrogels Based on LOVTRAP .................88 4.1 Synopsis ........................................................................................................................ 88 4.2 Introduction ................................................................................................................... 89 xi 4.3 Materials and Methods .................................................................................................. 91 4.4 Results and Discussion ................................................................................................. 93 4.4.1 Design of LOVTRAP-Based Hydrogels ................................................................... 93 4.4.2 LOVTRAP-Based Hydrogels Display Light-Responsive Viscoelastic Properties ... 98 4.4.3 Dynamic Properties of LOVTRAP-Based Hydrogels ............................................ 101 4.4.4 LOVTRAP-Based Hydrogels Display Shear-Thinning Behaviors and are Injectable Hydrogels ............................................................................................................................ 103 4.4.5 Comparison of LOVTRAP-Based Hydrogels with Other Protein Hydrogels ........ 106 4.5 Conclusions ................................................................................................................. 108 Chapter 5: Protein Hydrogels with Reversibly Patterned Multidimensional Fluorescent Images for Information Storage ...............................................................................................110 5.1 Synopsis ...................................................................................................................... 110 5.2 Introduction ................................................................................................................. 111 5.3 Materials and Methods ................................................................................................ 113 5.4 Results and Discussion ............................................................................................... 116 5.4.1 LOVTRAP can Reversibly Decorate/Release Protein Ligands onto/from Protein Hydrogels ............................................................................................................................ 116 5.4.2 Writing and Erasing Information on Protein Hydrogels by Using Light-Responsive LOVTRAP Interaction ........................................................................................................ 121 5.4.3 Multi-Dimensional Fluorescence Patterns Generated by Using LOVTRAP-Based Method ................................................................................................................................ 125 5.5 Conclusions ................................................................................................................. 130 Chapter 6: Conclusions and Outlook .......................................................................................131 xii 6.1 Conclusions ................................................................................................................. 131 6.2 Outlook ....................................................................................................................... 133 6.2.1 Utilizing Protein Phase Transitions to Construct Nanogels .................................... 133 6.2.2 Adapting Injectable Recombinant Protein-Based Hydrogels as Bioprinting Inks.. 134 6.2.3 Protein-Mediated “4D” Molecule Patterning on Hydrogels ................................... 136 References ...................................................................................................................................138 Appendices ..................................................................................................................................160 Appendix A Amino Acid Sequences of All the Proteins Studied .......................................... 160 Appendix B Python Program for Generating Pseudo Color 3D Heat Maps ........................... 162 xiii List of Tables Table 1.1 Stimuli-responsive cross-linking methods for dynamic protein-based hydrogels ........ 31 Table 2.1. Predicted transition temperature Tt of un-fused Vn-ELP polypeptides ....................... 48 xiv List of Figures Figure 1.1 RuII-mediated di-tyrosine reaction ................................................................................ 6 Figure 1.2 SpyTag-SpyCatcher chemistry ...................................................................................... 9 Figure 1.3 Various modes of histidine-Zn2+ coordination complexes .......................................... 11 Figure 1.4 Phase behaviors of ELPs and hydrogels based on ELP-containing micelles .............. 14 Figure 1.5 Scheme of coiled-coil interactions and a coiled-coil-based hydrogel ......................... 17 Figure 1.6 Schematic of Max1 β-sheet assembly and gelation ..................................................... 19 Figure 1.7 Cph1*-PEG hybrid hydrogel ....................................................................................... 21 Figure 1.8 General regulation strategies to construct dynamic protein-based hydrogels ............. 27 Figure 1.9 Shape memory bilayer hydrogel based on protein folding/unfolding ......................... 34 Figure 1.10 Dynamic hydrogel regulated by the dynamic conformation changes of CaM .......... 36 Figure 1.11 Molecule patterning on protein-based hydrogels through photo-induced oxime reaction .......................................................................................................................................... 40 Figure. 2.1. Protein block copolymers Vn-(GR)4 exhibit temperature-triggered phase behaviours in aqueous media........................................................................................................................... 52 Fig. 2.2. Proposed scheme of hydrogel preparation from (GR)4/Vn-(GR)4 monomer solutions, and in situ phase transition of Vn-ELP side chains in the resultant hydrogels. ............................ 56 Fig. 2.3. The presence of V72-ELP side chains alter the thermo-responsive properties of the (GR)4-based hydrogels. ................................................................................................................. 58 Fig. 2.4. Thermo-responsive properties of (GR)4+Vn-(GR)4s are regulated by Vn side chain length............................................................................................................................................. 60 xv Figure 2.5. Thermo-responsive properties of (GR)4+Vn-(GR)4 hydrogels are regulated by composition ................................................................................................................................... 61 Figure 2.6. Thermo-responsive properties of (GR)4+Vn-(GR)4 hydrogels with different cross-linking density ............................................................................................................................... 62 Fig. 2.7. (A-C) Proposed scheme of hydrogel preparation from Vn-(GR)4 micelle suspensions. 64 Figure 3.1. The design of the GKE hydrogels. ............................................................................. 73 Figure 3.2. Gelation time and stiffness of the GKE gels at different protein concentrations. ...... 74 Figure 3.3. Time-dependent rheological properties of the CCE/CCK gels. ................................. 75 Figure 3.4. Dissipation mechanism of GKE gels. ......................................................................... 79 Figure 3.5. Viscoelastic properties and relaxation spectra of GKE gels at different ionic strength........................................................................................................................................................ 81 Figure 3.6. Dynamic properties of GKE hydrogels at different concentration and CCK: CCE stoichiometry................................................................................................................................. 82 Figure 3.7. Injectability of the GKE Hydrogels. ........................................................................... 85 Figure 3.8. Shear-thinning properties of the GKE hydrogels at different protein concentrations. 86 Figure 4.1.The design principle of LOVTRAP-based hydrogel. .................................................. 95 Figure 4.2. Construction of SpyLOV2 and SpyZdk1 proteins and the formation of LOVTRAP-based hydrogels. ............................................................................................................................ 96 Figure 4.3. Erosion profile of LOVTRAP-based hydrogels. ........................................................ 97 Figure 4.4. Representative rheological characterizations of LOVTRAP-based hydrogels. ......... 99 Figure 4.5. Time-dependent rheological behaviors of 12% LOVTRAP-based hydrogels. ........ 102 Figure 4.6. Injectability of 12% LOVTRAP-based hydrogels. .................................................. 105 xvi Figure 5.1. Schematic of light-controlled LOVTRAP-regulated ligand decoration/release on protein hydrogels. ....................................................................................................................... 117 Figure 5.2. The decoration of YFP-G-LOV2 onto GRG5RG4RGzdk1 hydrogel was specific and reversible. .................................................................................................................................... 118 Figure 5.3. Blank protein hydrogel slate cannot be decorated via non-specific interactions. .... 118 Figure 5.4. The decoration of YFP-G-LOV2 on GRG5RG4RGzdk1 hydrogel only worked in the dark. ............................................................................................................................................ 119 Figure 5.5. Changes of the fluorescence intensity of the GRG5RG4RGzdk1 hydrogel during the decoration-release-redecoration cycle. ....................................................................................... 121 Figure 5.6. light-controlled pattern of LOV2-fused ligand can be produced in GRG5RG4RGzdk1 hydrogels. .................................................................................................................................... 124 Figure 5.7. Ligand patterns can be erased effectively by blue light irradiation. ......................... 125 Figure 5.8. Greyscale image information can be written in a hydrogel in the form of gradient ligand pattern. ............................................................................................................................. 126 Figure 5.9. LOVTRAP can be used to generate multi-dimensional fluorescence images-based 3D codes. .......................................................................................................................................... 128 Figure 5.10. Long-term preservation of fluorescence images on hydrogels. .............................. 129 xvii List of Symbols ∆SR25-50°C the difference between SR at 50 °C and SR at 25 °C C concentration CC an empirical critical concentration in Equation (2.1) E Young’s modulus E50°C/E25°C the ratio between E at 50 °C and E at 25 °C G shear modulus G’ storage modulus G’’ loss modulus G’dark storage modulus in the dark G’lit storage modulus under light irradiation H(τ) relaxation spectrum k an empirical constant in Equation (2.1) k1 scaling factor of G’ versus ω k2 scaling factor of G’’ versus ω L length under tension Linit length without tension m0 initial mass of a hydrogel sample Mc number average molecular weight between cross-linking points ms mass of a fully swollen hydrogel sample xviii n chain length of Vn-ELP (number of VPGVG repeats) n number of samples N cross-linking density p p-value in student t-test S.D. standard deviation SR swelling ratio T Temperature Tt Phase transition temperature Tt,c an empirical critical temperature in Equation (2.1) ρ mass density τ relaxation time τc characteristic relaxation time of a material ω angular frequency in oscillatory rheology ωc critical frequency where G’(ω) = G’’(ω) xix List of Abbreviations [RuII(bpy)3]2+ Ruthenium(II) tris-bipyridyl bication 2D Two-dimensional 3D Three-dimensional 4D Four-dimensional AC10A the triblock protein with a middle random coil domain C10 flanked by two associative leucine zipper domains (A) adoB12 B12 derivative adenosylcobalamin AKe Adenylate kinase APS Ammonium persulfate ASC Apoptosis-associated speck-like protein ATP Adenosine triphosphate BSA Bovine serum albumin CaM Calmodulin CarHC The C-terminal adenosylcobalamin binding domain CARS Coherent anti-Stokes Raman scattering CBD Calmodulin binding domain cELP Cysteine-containing elastin-like polypeptides CFP Cyan fluorescent protein CnaB2 The second immunoglobulin-like collagen adhesion domain Cph1* The Y263F mutant of pytochrome-like protein Cph1 xx CTD The C-terminal domain of spider major ampullate spidroin 1 DLS Dynamic light scattering DNA Deoxyribonucleic acid ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid EGTA ethylene glycol-bis-N,N,N′,N′-tetraacetic acid ELP Elastin-like polypeptides FL Ferredoxin-like protein FPH Fluorescent polymeric hydrogel G GB1 GB1 The B1 binding domain of protein G from Streptococcus GdmCl Guanidinium chloride GE CCE-(GB1)4-CCE GFP Green fluorescent protein GK CCK-(GB1)5-CCK-(GB1)5-CCK GKE hydrogel The hydrogel formed by mixing GE and GK solutions GL5 A mutant of GB1 where five residues (GGGLG) are inserted into the second loop GSH Glutathione GyrB The bacterial gyrase subunit B heptad Periodic seven-amino-acid repeat of coiled coils HisTag Polyhistidine tag (several consecutive histidine residues) xxi HRP Horseradish peroxidase hVEGF Human vascular endothelial growth factor IPTG Isopropyl-1-β-thiogalactoside ITC Isothermal calorimetry LB Lysogeny broth LCST Lower critical solution temperature LED Light-emitting diode LOV2 Light-oxygen-voltage-sensing domain 2 LOVTRAP The interaction between LOV2 and zdk1 MBP Maltose binding protein mCherry A variant of monomeric red fluorescent protein MEP Mutually exclusive protein NPPOC 2-(2-nitrophenyl) propoxycarbonyl OD Optical density oNB Ortho-nitrobenzyl ester PBS Phosphate buffered saline PEG Poly(ethylene glycol) PhoCl Photocleavable domain PMMA Poly(methyl methacrylate) PNIPAM Poly(N-isopropylacrylamide) QR Quick response R Resilin xxii red-ox Reduction-oxidation RNA Ribonucleic acid RTX Repeat-in-toxin RuII Ruthenium(II) Sc SpyCatcher SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SpyLOV2 The protein complex formed between (GSc)3 and G-St-LOV2 SpyZdk1 The protein complex formed between (GSc)3 and zdk1-G-St St SpyTag TEM Transmission electron microscopy THPC Tetrakis(hydroxymethyl) phosphimium chloride THPP β-[tris(hydroxymethyl)phosphino] propionic acid TNfn3 the third fibronectin type 3 domain of human tenascin TPR Tetratricopeptide repeat Tris Tris(hydroxymethyl)aminomethane TSAT Tris-succinimidyl aminotriacetate UCST Upper critical solution temperature UVR8 UV-B resistance 8 protein Vn-(GR)4 (VPGVG)n-(GB1-resilin)4 Vn-ELP (VPGVG)n YFP Yellow fluorescent protein zdk1 The designed binding partner Zdark1 of LOV2 xxiii Acknowledgements I offer my deepest gratitude to my supervisor Dr. Hongbin Li. He gave me the exciting opportunity to pursue my PhD degree in his lab. He always gives valuable advice on my research. He helps me to start my career and encourages me to be an independent researcher. He supported me when I faced difficulties. In himself, Dr. Li has modelled what it is to be a professional, creative, and diligent researcher. I have learned in many ways during my study in his lab. I would like to thank Dr. Jayachandran Kizhakkedathu, Dr. Katherine Ryan, and Dr. Suzana Straus for providing guidance as my supervisory committee. I especially thank Dr. Kizhakkedathu and Dr. Ryan for their critical evaluation of this manuscript. I would like to thank all the colleagues and members of the Li group. I want to particularly thank Dr. Shanshan Lv and Dr. Linglan Fu, who trained me and helped me initiate my first research project. I learned much scientific knowledge from Dr. Yi Cao and Dr. Wenxu Sun in our collaboration. I had many discussions with Dr. Han Wang, Dr. Jiayu Li, Dr. Yongliang Wang, Jiacheng Zuo and Qingyuan Bian about research and technical details. Dr. Jie Fang, Yabin Guo, Dr. Ruidi Wang, Dr. Jiahao Xia, Adam Xiao, Haoquan Lin, Guojun Chen, Sena Arslan, Peiyun Li, Akash Panjabi and Jingqi Lei have provided me suggestions and encouragement. I also would like to thank Dr. Elena Polishchuk and Jessie Chen for their tireless help and technical support. I thank Dr. Margo Lillie and Dr. Roger Shadwick for their generous help with the tensile tests. I thank Dr. Stephen Withers and Dr. Feng Liu for their generous help with the turbidity tests. I thank Dr. Mark MacLachlan for kindly sharing the particle size analyzer of his xxiv lab. I thank Dr. EunKyoung Lee for her assistance with optical microscopy. I thank Brendan Moore for proofreading this thesis. Thanks to all my friends and family members for supporting me over the years. Finally, I own my special thanks to my parents, Zhixun and Limin, for their unconditional love and support. xxv Dedication Dedicated to my parents, Zhixun Duan and Limin Song. 1 Chapter 1: Introduction My research focuses on engineering dynamic protein-based hydrogels. In Chapter 1 I first briefly introduce the concept of hydrogels and the significance of dynamic protein-based hydrogels. Then, I review the general cross-linking methods for protein-based hydrogels in details. Next, the multifaceted expectations for dynamic protein-based hydrogels in their biomedical applications are introduced. Finally, the general strategies for engineering dynamic properties in protein-based hydrogels are discussed. Based on the current limitations of dynamic protein-based hydrogels, I will propose the goals of my thesis. 1.1 Hydrogels Hydrogels are cross-linked hydrophilic polymer networks that absorb but do not dissolve in a large amount of water. The formation of a polymer network requires intermolecular interactions to cross-link polymers. The cross-linking methods can be both covalent chemical bonds and non-covalent physical interactions (hydrophobic interactions, electrostatic interactions, hydrogen bonding, metal-ligand coordination and so on). The resultant cross-linking points confine the hydrophilic polymer chains to maintaining a well-defined three-dimensional structure rather than dissolving, whereas water molecules and other solutes can permeate into the meshes of the network. Owing to their unique features, hydrogels can emulate many features of native tissues or organisms, and have therefore found many biomedical applications including tissue engineering scafolds1-8, drug carriers9-11, biosensors12, 13, contact lenses14, 15, and wearable/implanted electronic devices16-18. 2 1.2 Dynamic Protein-Based Hydrogels The stimuli-responsiveness of hydrogels is critical in many biomedical applications. In tissue engineering, hydrogels should emulate native cellular microenvironments or extracellular matrices (ECMs) that adapt variable cell behaviours and other external stimuli present in vivo. Hydrogels are also expected to convey external mechanical signals to cultured cells to regulate the cell behaviors. In controlled drug delivery, hydrogels should recognize the pathological markers from disease tissues and release drugs accordingly. Hydrogel-based biosensors should respond to bio-signals and convert them into readable electronic signals for diagnosis. Wearable or implanted electronic devices should function in response to electronic signals or other stimuli. Driven by these needs, “dynamic” hydrogels which can change their physical, chemical, biological, or mechanical properties in response to external stimuli have attracted great attention over the last few decades6-8. Dynamic hydrogels are often constructed from stimuli-responsive polymer building blocks. Hydrogels have been constructed from diverse polymers. Early studies have focused on synthetic polymer-based hydrogels. More recently, hydrogels constructed from naturally occurring polymers, including proteins, polysaccharides, and DNAs, have attracted increasing interest due to their biocompatibility. Proteins are large biomolecules that are composed of many amino acids polymerized by peptide bonds. Usually, a protein is composed of at least 50 amino acids, while a shorter amino acid chain is often called a “peptide” or a “polypeptide” instead. Proteins participate in almost all biological processes in living organisms. People have long been harvesting and engineering naturally occurring proteins into artificial functional materials, dated back to the early practice of silkworm silking. Since the 1970s, the development of recombinant DNA technology has allowed for directly translating artificially designed genes and producing 3 “recombinant” proteins in host cells. Proteins are promising hydrogel building blocks, especially for biomedical applications, because 1) they are biocompatible; 2) their sequences and properties can be easily tailored via recombinant DNA technology; and 3) their biological synthesis often results in absolute mono-dispersity, which is difficult for the synthesis of other types of polymers. Moreover, numerous stimuli-responsive conformational changes and interactions of proteins have been discovered, which provide vast possibilities for constructing dynamic protein-based hydrogels. Over the last two decades, many dynamic protein-based hydrogels have been developed19-25. 1.3 General Cross-Linking Methods for Protein-Based Hydrogels Many proteins contain cross-linkable amino acid residues and/or can undergo protein phase transitions or biorecognition-driven protein/peptide self-assembly, so that can be directly cross-linked into protein-based hydrogels22-25. Using recombinant DNA technology, non-cross-linkable protein domains can be conveniently fused with cross-linkable proteins. Therefore, almost all proteins can serve as hydrogel building blocks. Depending on the chemical or physical nature of the cross-linking methods, the resultant cross-linking points can be either permanent or reversible. Permanent cross-linking points typically ensure static properties of hydrogels, while stimuli-responsive, reversible cross-linking points usually potentiate dynamic properties. Moreover, it is not uncommon that two or more types of cross-linking methods are present in one hydrogel system, which may provide both permanent and reversible cross-linking points. Understanding the cross-linking methods is necessary for understanding the design principles of any dynamic protein-based hydrogel. Therefore, herein I provide an overview of the general cross-linking methods for engineering protein-based hydrogels. 4 1.3.1 Chemical Cross-Linking Methods 1.3.1.1 Reactions of Cysteine The disulfide covalent bonds between the thiol groups of cysteine residues have been utilized as cross-linking methods for protein-based hydrogels26-29. Disulfide bond formation is an oxidation reaction of two thiol groups, which occurs slowly in the presence of air oxygen and can be accelerated by additional oxidant such as H2O2. After formation, a disulfide bond can be reversibly cleaved by a reducing agent into two thiol groups again. Therefore, the equilibrium of disulfide formation is controlled by the reduction-oxidation (red-ox) potential. For example, Chilkoti et al. achieved in situ hydrogel formation in mice by co-injection of a H2O2 solution and a solution of cysteine-containing elastin like polypeptide (cELP)27. To gain more controllability over the re-dox potential during the gelation process, Chilkoti and coworkers later excluded H2O2 and instead decorated a photosensitizer Chlorin e6 onto cELP30. In this way, cELP could form intermolecular disulfide bonds and gelate only when Chlorin e6 transformed oxygen to the strongly oxidative singlet form upon 660-nm light. In addition to disulfide bond formation, the thiol group of cysteine can undergo a thio-ene addition reaction with an alkene to form a covalent thioester bond. Thio-ene reactions of cysteine residues have been widely utilized as cross-linking methods for protein-polymer hybrid hydrogels due to their high yield and fast rate31-34. For example, Kiick et al. recombinantly engineered cysteine residues in the sequence of resilin-like protein, and then cross-linked this engineered protein with vinyl sulfone-modified poly(ethylene glycol) (PEG) into a hybrid hydrogel through a thio-ene reaction34. This cross-linking process finished in a few minutes at ambient conditions. Conventionally, thio-ene addition reactions are irreversible. Anseth et al. 5 recently developed a reversible thio-ene addition reaction between a cysteine and an allyl sulfide chain transfer agent in the presence of a photoinitiator activated by 365-nm light35. This modified thio-ene reaction may open opportunity for light-controlled reversible formation of protein-polymer hybrid hydrogels in the future. 1.3.1.2 Di-Tyrosine Reactions Tyrosine plays important roles in various biological processes thanks to its phenol side chain. Many natural biomaterials such as resilin found in insect joints or tendons contain polypeptides that are covalently cross-linked by di- or tri-tyrosine adducts36, 37. Fancy and Kodadek developed a photo-chemical approach that covalently cross-links proteins in proximity through tyrosine residues, as a probe of protein-protein interactions38. This method uses visible light to induce photolysis of [RuII(bpy)3]2+ in the presence of ammonium persulfate (APS), which generates RuIII and sulfate radical that collaboratively oxidize two tyrosine residues in proximity into a permanent di-tyrosine adduct (Figure 1.1 A). Later, Elvin et al. adapted this RuII-mediated di-tyrosine reaction to cross-link a recombinant polyresilin protein, which consisted of 17 repeats of Drosophila resilin elastic motif GGRPSDSYGAPGGGN, into a hydrogel39.The RuII-mediated di-tyrosine reaction took place within several minutes and the resultant hydrogel exhibited the featured blue fluorescence of di-tyrosine adducts (Figure 1.1 B). Since this pioneering work, RuII-mediated di-tyrosine reaction has been widely used for constructing chemical hydrogels from a variety of tyrosine-rich proteins40-48. The dose of light irradiation has proven to influence the equilibrium and kinetics of the RuII-mediated reaction and thus influence the properties of the resultant hydrogel41, 49, 50. Botvinick et al. used confined light irradiation to trigger secondary RuII-mediated di-tyrosine cross-linking and thus locally stiffen selective areas within fibrin 6 hydrogels (natural fibrin gelates spontaneously)50. The stiffness increment was closely determined by the time of the confined light irradiation. In addition to RuII-mediated photo-chemistry, horseradish peroxidase (HRP)-H2O2-mediated di-tyrosine reaction is another well-established approach to cross-link tyrosine-rich proteins into hydrogels51-54. HRP forms a complex with H2O2 which can oxidize a phenol substrate, such as a tyrosine residue, into a phenolic radical55. Two phenolic radicals subsequently couple into a covalent di-tyrosine adduct. Kaplan et al. utilized HRP-H2O2-mediated reaction to cross-link exon-encoded resilin protein into hydrogels51. This enzymatic cross-linking process finished within several minutes at physiological pH and 37 °C. Kawakami et. al. used HRP-H2O2-mediated reaction to cross-link tyrosine-rich proteins with phenol-modified sodium alginate into hybrid hydrogels53. The gelation time of such hybrid hydrogels varied from a few seconds to 6 min through tuning the concentrations of HRP and H2O2. Figure 1.1 RuII-mediated di-tyrosine reaction (A) The phenol side chains of two tyrosine residues were oxidized and coupled into a di-tyrosine adduct at the presence of [RuII(bpy)3]2+ and APS upon light irradiation. (B) Bright field (Left) and fluorescent (Right) photographs of a hydrogel sample cross-linked from polyresilin protein through RuII-mediated di-tyrosine reaction. The blue fluorescence comes from the di-tyrosine adducts39. Copyright 2005, Nature Publishing Group. 7 1.3.1.3 Reactions of Lysine The primary amine side chains of lysine residues participate in various cross-linking methods of proteins. In native ECMs, collagen and elastin are cross-linked through serial mechanisms based on aldehyde form of lysine side chains56. In protein-based hydrogels, reactions of lysine are also widely utilized as cross-linking methods due to the abundance of lysine in many proteins. The Mannich-type reactions between the primary amine side chain of lysine and synthetic cross-linkers, including β-[tris(hydroxymethyl)phosphino] propionic acid (THPP)57-59 and tetrakis(hydroxymethyl) phosphimium chloride (THPC)60, 61, are versatile cross-linking methods for lysine-rich proteins. For example, Kiick et al. used THPP to cross-link resilin-like polypeptides into a stable elastomeric hydrogel58.Tris-succinimidyl aminotriacetate (TSAT) is another synthetic cross-linker for lysine-rich proteins, where their aminotriacetate group can rapidly form amide with the amine group of lysine62. Except for directly participating in cross-linking reactions, the amine group of lysine can be chemically modified with other cross-linkable groups. For example, Fang and Li utilized Bolten-Hunter reagent to transform the amine groups of lysine residues into phenolic groups and cross-linked the derivatized protein into hydrogel through RuII-mediated photochemical reaction63. To avoid using unnatural synthetic cross-linkers, enzymatic reactions of lysine have also been adapted for cross-linking protein-based hydrogels. Two lysine residues can be cross-linked via Schiff base formation catalyzed by lysyl oxidase or plasma amine oxidase64. Moreover, the isopeptide bond formation between lysine and glutamine residues, catalyzed by tissue transglutaminase, is an established cross-linking method for proteins65-68. Barron et al. utilized this transglutainase-mediated reaction to in situ cross-link a lysine-rich protein and a glutamine-rich protein into a hydrogel in vivo65. Li et al. applied 0-20 U/mg transglutaminase to induce the 8 lysine-glutamine reaction in type-A gelatin68. While type-A gelatin gelates spontaneously via non-covalent interactions, the lysine-glutamine reaction further stiffened it by forming secondary covalent cross-linking points. The stiffness increment of the treated type-A gelation could be controlled by the enzyme activity. 1.3.1.4 Covalent Bonds Formed in Protein Assembly Recognition, interaction, and assembly among proteins are ubiquitous in all native organisms. Intermolecular covalent bonds can be formed during some protein assembly processes, which can be used as cross-linking methods for protein-based hydrogels. A representative example is the SpyTag-SpyCatcher chemistry. As the two split fragments of the second immunoglobulin-like collagen adhesion domain CnaB2, SpyTag and SpyCatcher can reconstitute and simultaneously form an isopeptide bond between SpyTag’s aspartic acid and SpyCatcher’s lysine, as shown in Figure 1.269. Thanks to its fast rate without catalysis under ambient conditions, SpyTag-SpyCatcher chemistry has been widely used to chemically cross-link protein into hydrogels70-74. For example, Tirrell et al. mixed two engineered proteins SpyTag-ELP-SpyTag-ELP-SpyTag and SpyCatcher-ELP-SpyCatcher, where ELP was a short elastin like polypeptide linker, to form a covalently cross-linked hydrogel74. At room temperature, such gelation finished in 5 min after the mixing. Unlike the SpyTag-SpyCatcher reconstitution which forms an irreversible isopeptide bond, some binding pairs form disulfide bonds during their assembly, such as the PDZ domains75 and GN/GC reconstitution76, so they allow for re-dox potential-controlled reversible cross-linking. GN and GC are the two split fragments of the GL5 domain, which undergo non-covalent reconstitution and can be further stabilized by an intermolecular disulfide bond. Kong and Li constructed a hydrogel from 9 telechelic proteins (I27-GN-I27)4 and (I273-GC)376. By varying the redox potential, the disulfide bonds between the GN-GC complexes can be reversibly formed and cleaved, thus dynamically tuning the thermal stability of the hydrogel. Figure 1.2 SpyTag-SpyCatcher chemistry The two split fragments of the CnaB2 domain, SpyCatcher and SpyTag, recognize each other and reconstitute into a protein complex that resembles a complete CnaB2 domain. During such reconstitution, the lysine residue of SpyCatcher and the aspartic acid residue of SpyTag form an isopeptide bond69. Copyright 2012, The National Academy of Sciences. 1.3.2 Physical Cross-Linking Methods 1.3.2.1 Histidine-Metal Coordination The metal coordination of the imidazole side chain of histidine is present in many metalloproteins. Several organisms use metal-histidine coordination to cross-link structural proteins into stiff and tough tissues, such as mussel byssus77 and sand worm jaws78. Inspired by these phenomena, metal-histidine coordination has been intensely adapted as cross-linking methods for protein- or peptide-based hydrogels79-83. The affinity of metal-histidine coordination is controlled by the type of metal ions79-81 and other stimuli such as pH83. HisTags (several consecutive histidine residues) are frequently fused in recombinant proteins for their 10 purification84, which are also available to be cross-linked with metal ions for recombinant protein-based hydrogels. An early example from Kopecek et al. used Ni2+-ligand coordination to “glue” the HisTags of the coiled-coil domain CC1 and the iminodiacetate pendants of poly(HPMA-co-DAMA) into a hybrid hydrogel85. Later, taking advantage of the much higher affinity of histidine-Co3+ coordination over histidine-Co2+ coordination, Sun et al. oxidized and cross-linked the mixture of Co2+ and a HisTag-containing protein into a hydrogel79. Blank et al. engineered three histidine residues on each of the two complimentary coiled-coil domains A4H3 and B4H3, so that a soft hydrogel cross-linked by A4H3-B4H3 coiled coil interaction could be further stiffened by secondary histidine-Zn2+ coordination80. Such Zn2+-histidine cross-linking could be reversed upon the addition of competing ligand ethylenediaminetetraacetic acid (EDTA). If Ca2+ was added into the A4H3-B4H3 hydrogel instead of Zn2+, however, very little secondary cross-linking and stiffening were observed, because there was virtually no coordination affinity between Ca2+ and histidine. Moreover, the variable coordination number of metal-histidine complexes can produce variable functionality (the number of elastically active chains connected at one cross-linking point) of the resultant cross-linking points. The Cao group designed three different modes of coordination complexes between Zn2+ and histidine-rich blocks GGH (PH1), GHHPH (PH3) and (GHHPH)2 (PH6)82. As shown in Figure 1-3, PH1, PH3, and PH6 coordinated with Zn2+ in single-ligand mode, multiple-ligand, and tandem multiple-ligand mode, respectively. Isothermal calorimetry (ITC) and single molecule spectroscopy revealed that these three histidine-Zn2+ coordination complexes had distinct binding affinity and mechanical stability. Consequently, the hydrogels cross-linked by the three modes of coordination displayed distinct stiffness and toughness. 11 Figure 1.3 Various modes of histidine-Zn2+ coordination complexes Zn2+ coordinated in single ligand mode with PH1 (Left), in multiple-ligand mode with PH3 (Middle), and in tandem multiple-ligand mode with PH6 (Right). From left to right, the binding affinity and association rate increase82. Copyright 2020, The Authors. Exclusive licensee American Association for the Advancement of Science. 1.3.2.2 Phase Transition of Elastin-Like Polypeptides Elastin is an important structural protein in the body. Derived from the hydrophobic domains of elastin’s precursor tropoelastin, elastin-like polypeptides (ELPs) are composed of multiple tandem repeats of VPGXG pentapeptide motifs, where the X can be any amino acid residue except proline (P)86, 87. ELPs exhibit the lower critical solution temperature (LCST) phase behaviors (Figure 1-4 A): below a specific transition temperature Tt, they adopt relatively hydrophilic structures and dissolve well in aqueous solutions; above Tt, ELPs undergo conformational changes into more hydrophobic structures and aggregate88-92. Such LCST behavior is different from the normal dissolving behaviors of most polymers. Several models have been proposed to explain the LCST behavior of ELPs.93 Urry et al. created the β-spiral model88, 94. They proposed that ELPs undergo conformational changes from random coil to β-spiral upon heating, exposing hydrophobic amino acid side chains to the water, which lead to the aggregation of ELPs due to hydrophobic effect (Figure 1.4 A). However, some more recent studies indicated that ELPs are not completely random coil below Tt, which disagrees with the β-spiral model90, 93. Moreover, some researchers proposed that β-strand and β-sheet play important 12 roles in the conformational changes of ELPs95, 96. Although the exact mechanism of the conformational changes is still in debate, it is widely believed that the aggregation of ELPs following such conformational changes above Tt is an entropy dominated process driven by hydrophobic effect. In this aspect, the LCST behavior of ELPs resembles the LCST behavior of synthetic polymer poly(N-isopropylacrylamide) (PNIPAM), which also aggregates above its Tt in an entropy dominated mechanism97. Because the LCST behavior is collectively controlled by conformational changes and hydrophobic effect, the Tt of ELPs is determined by the various factors including the types of the X residue98, 99, the chain length100, the concentration100, and other factors of the media such as the ionic strength101, 102. The phase transition of ELPs provides a temperature-induced cross-linking method for hydrogels. For example, Kobatake et al. recombinantly engineered a tri-block polyprotein containing a ELP block, a poly(aspartic acid) block, and a coiled-LL domain block, which formed four-arm protein complexes through the coiled-coil tetramerization of the coiled-LL block103. The ELP blocks aggregated above their Tt, and the hydrophilic poly(aspartic acid) blocks regulated the ELP aggregation to form a hydrogel rather than macroscopic precipitates. It is also frequently observed that ELP chains can undergo in situ phase transitions in hydrogel networks62, 104-107. Chilkoti et al. used TSAT to covalently cross-link lysine residues of two types of ELPs, [VPGKG(VPGVG)16]6 and [VPGKG(VPGVG)6]16, into hydrogels62. When heated from 7 to 37 °C, these ELP-based hydrogels de-swelled and were greatly stiffened, indicative of an increased number of cross-linking points. The authors further proposed that such secondary cross-linking came from the in situ phase transition of the ELP chains confined in the network. Later, the aggregates produced by the in situ phase transition of the confined ELP chains in a similar covalently cross-linked network was directly observed by Heilshorn et al. using coherent 13 anti-Stokes Raman scattering (CARS) microscopy105. As a result, the studied hydrogel could be rapidly stabilized after injection in vivo due to the ELP aggregation at 37 °C. The phase behaviors of ELPs have allowed ELP-containing block polymers to form various nanostructures including micelles108-111, nanofibers112, 113, and vesicles109, 114, 115 in aqueous media. Cross-linking such nanostructures produces structurally hierarchical hydrogel networks that are difficult to fabricate from homogenous polymer solutions116-118. Chilkoti et al. engineered a polypeptide that consists of three blocks: a hydrophobic ELP block (VPGVG)60, a hydrophilic ELP block (VPGAGVPGGG)60, and a Zn2+-binding peptide118. As shown in Figure 1-4 B, above the Tt of the (VPGVG)60 block but below that of the (VPGAGVPGGG)30 block, the (VPGVG)60 block aggregated, and the polypeptides formed monodispersed micelles. Such micelles could be reversibly cross-linked by Zn2+-binding into a hydrogel. This micelle-based hydrogel exhibited excellent compression modulus (0.8-1.0 MPa) attributed to its hierarchical structures and the highly entangled micelles cores. Meco and Kempe also reported an ELP coacervate-based hydrogel117. By using THPC to cross-link the coacervates of lysine-containing ELP above its Tt and polymerizing the PEG in the aqueous media at the same time, the ELP coacervates would be captured in the resultant PEG network, thus forming a heterogeneous ELP-PEG hybrid hydrogel. ELPs are the most well-studied type of proteins/peptides that can display LCST phase transition behaviors93, 119, 120. Many other proteins/peptides, especially those containing multiple tandem oligo-peptide repeats, also display LCST behaviours and/or upper critical solution temperature (UCST) behaviours (In a UCST behavior, the polymer aggregates below, rather than above, a Tt)120-122. ELP-analogue peptides, such as (IPGVG)s and (IPAVG)s, have been utilized to construct thermo-responsive hydrogels because of their LCST behaviors104, 123. Xia et al. 14 constructed a protein copolymer (GGRPSDSYGAPGGGN)4(GAGAGS)4 of resilin motifs and silk motifs124. Unlike ELPs that only have one LCST, the polymers of resilin motifs have both an LCST and an upper critical solution temperature (UCST). Therefore, the assembly of the resilin-fused silk blocks was regulated by the LCST and UCST behaviors of the resilin blocks. Consequently, this resilin-silk copolymer selectively gelated near physiological temperature, while formed micelles at lower temperature and precipitated at higher temperature. It is expected that more types of proteins/peptides that can display phase transition behaviors will be utilized for constructing protein-based hydrogels in the future. Figure 1.4 Phase behaviors of ELPs and hydrogels based on ELP-containing micelles (A) Schematic of the β-spiral model for explaining the LCST phase behavior of ELPs, proposed by Urry et al.88, 94 This scheme is a modified version of that drawn by Berthiaume et al92. Copyright 2016, Wiley Periodicals, Inc. (B) Scheme of the tri-block copolymer containing two ELP blocks and a metal-binding sequence. Above the Tt of the hydrophobic ELP block (green), the copolymer forms micelles in aqueous media. The micelles can be further cross-linked by Zn2+-binding into a micelle-based hydrogel118. Copyright 2015, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 15 1.3.2.3 Coiled-Coil Interactions The coiled coil consists of several (most frequently two or three) α-helices that coil together like a strand of ropes. Berger et al. estimated that at least 2-3 % of all protein residues form coiled coils125. The basic coiled coil forming motifs are the periodic seven-amino-acid (heptad) repeats126, often labelled by abcdefg as shown in Figure 1-5 A127. The a and d positions are occupied by highly hydrophobic residues which form a hydrophobic interface on each helix. Such hydrophobic interfaces form the hydrophobic cores and pack several helices into a tight strand. Since leucine is very frequently observed to occupy the a or d positions, such structural motif is also called a leucine zipper. The e and g positions are taken by charged residues, which generate an electrostatic pattern on the outer contacting edges of the coiled coil. The electrostatic attraction and repulsion among the e and g positions prevent the hydrophobic core from massive aggregation and determine the topological structure of the coiled coil: whether the helices form homo- or heterooligomers as well as in the parallel or anti-parallel orientation128, 129. For example, Figure 1-5 A shows the two ways of heptad repeat pairing that generates parallel and antiparallel coiled coil dimers. Because a coiled-coil formation involves both hydrophobic effects and electrostatic interactions, the binding affinity can be regulated by temperature, pH, ion strength, and chemical denaturants130, 131. In 1998, Tirrell and co-workers first reported a protein-based hydrogel constructed from coiled-coil forming domains132. They designed a triblock protein AC10A where the two leucine zipper A domains flank a random coil sequence C10. The coiled coil formation and aggregation among the A domains cross-linked the AC10A concentrated solutions into hydrogels, as shown in Figure 1-5 B. The gelation could be further reversed upon elevated temperature or increased pH. 16 This is because a higher temperature denatured the A helical structures while a higher pH enhanced the electrostatic repulsion among the glutamine residues, both weakening the coiled coil interaction. Kopecek et al. utilized metal-histidine coordination to cross-link the HisTag-containing coiled-coil domain CC1 into a hydrogel85. They observed a dramatic de-swelling by 90% when the hydrogel was heated from 25 to 70 °C and attributed such de-swelling to the thermal unfolding of the CC1 helices which broke the rigid “rod”-like coiled coil bundles in the networks. Similar temperature- and pH-dependent gelation behaviors were also observed in many later reported hydrogels that are cross-linked through other types of coiled-coil interactions133-137. Notably, coiled-coils are highly dynamic cross-linking points which can disconnect and reconnect rapidly, giving coiled-coil-based hydrogels profound viscoelastic properties138-142. Excellent shear-thinning and self-healing properties are often observed in such hydrogels, making them promising injectable ECM-mimetic scaffolds for tissue engineering143. Figure 1-5 C shows the dynamic moduli G’ and G’’ of the AC10A-based hydrogels which were closely influenced by pH138. Blank et al. constructed a series of protein-polymer hybrid hydrogels using the coiled-coil formation between A4 and B4 domains, and conformed that all these hybrid hydrogels exhibited temperature-sensitive viscoelastic properties due to the thermo-responsive affinity of the A4/B4 interaction142. Olsen and co-workers demonstrated that the coiled-coil interaction of domain P4 endowed temperature-responsive diffusion coefficient and creep to the P4-containing hydrogels, which was attributed to the temperature-dependent network dynamics and inter-polymer friction140. 17 Figure 1.5 Scheme of coiled-coil interactions and a coiled-coil-based hydrogel (A) top view of the three-dimensional structure of a coiled-coil dimer (Left). Such coiled-coil dimer can be formed by two coiled-coil forming peptides in the parallel orientation (Middle) or anti-parallel orientation (Right). The letters abcdefg label the residue positions in the heptad repeats127. Copyright 2018, The Royal Society of Chemistry. (B) schematic of the AC10A hydrogel cross-linked by the coiled coil formation of A domains. The AC10A hydrogel can melt at higher temperature or higher pH132. Copyright 1998, The American Association for the Advancement of Science. (C) The dynamic viscoelastic moduli of the AC10A hydrogel at pH 7.0 and pH 8.0138. The solid and open squares represent the G’ and G’’ at pH 7.0, respectively. The solid and open triangle represent the G’ and G’’ at pH 8.0, representatively. Copyright 2007, American Chemical Society. 1.3.2.4 β-Sheet Assembly The supramolecular assembly of β-sheets is implicated in the formation of many native proteins aggregates and fibrils such as silk fibres144. A β-sheet consists of multiple β-strands that self-assemble into a pleated sheet. Each β-strand is a 3-to-10-residue extended stretch of peptide that adopts a “zig-zag” backbone configuration where the side chains of adjacent residues point to the opposite directions. Fibrous β-sheet motifs typically contain alternating hydrophilic and 18 hydrophobic residues that point to the opposite directions, making the entire β-sheet strongly amphiphilic145. The amphiphilic β-sheets form bilayers via hydrophobic effects and can further self-assemble into supramolecular fibrils through lateral inter-strand hydrogen bonds. Because a β-sheet assembly process involves hydrophobic effects, electrostatic interactions, and hydrogen bonding in consort, the sizes and morphology of the assembled structures are influenced by temperature, pH, and ionic strength145, 146. β-sheet assembly is the main cross-linking method in regenerated silk fibroin-based hydrogels144. A silk fibroin protein contains hundreds of GAGAGS repeats which form β-strands and pack into the crystalline domains. Kaplan et al. utilized various factors, including concentration, temperature, pH, and metal ions, to regulate the β-sheet assembly in a regenerated silk fibroin and the gelation rate of the resultant silk fibroin-based hydrogels147. β-sheet assembly is also a common cross-linking method for other engineered protein-based hydrogel systems148-152. Pochan et al. designed a β-sheet forming peptide Max1 which contained alternating lysine and valine residues148, 149. At 20 °C, pH 9, and low ionic strength, Max1 remained random coil due to the electrostatic repulsion among the lysine side chains. At 37 °C, pH 7.4, and 150 mM NaCl, the electrostatic repulsion among the lysine residues was screened so that Max1 could spontaneously form β-hairpins (two β-strands connected like a hairpin). As shown in Figure 1-6, the Max1 β-hairpins further underwent a bilayer assembly in the facial direction driven by hydrophobic effects among the valine residues, as well as formed long fibrils through the hydrogen bonds in the lateral direction. Eventually, a hydrogel was formed. Transmission electron microscopy (TEM) directly showed that this Max1-based hydrogels were cross-linked by the highly entangled fibrils at approximately 3-nm diameter. 19 Figure 1.6 Schematic of Max1 β-sheet assembly and gelation The Max1 peptide motifs, which contain alternating valine and lysine residues, fold into β-hairpins from random coil at elevated temperatures (e.g., from 20 to 37 °C). At an appropriate pH and ionic strength, the electrostatic repulsion among the lysine side chains was screened, so the β-hairpins can further assemble. Such assembly involves the facial assembly among the valine side chains driven by hydrophobic effects, as well as the lateral assembly driven by hydrogen bonding. Consequently, large fibres are formed, and a hydrogel is formed by the highly entanglement fibres149. Copyright 2009, American Chemical Society. 1.3.2.5 Non-Covalent Protein Assembly While covalent bonds can be formed during some protein assembly processes, such as in the SpyTag/SpyCatcher reconstitution, most protein-assembly processes are entirely driven by non-covalent interactions. Many protein domains spontaneously assemble into homo-oligomers or hetero-oligomers, driven by hydrophobic effects, electrostatic interactions, and hydrogen bonding. Protein homo-oligomerizations have been widely adapted as physical cross-linking methods in hydrogel construction153-162. For example, protein-based hydrogels could be reversibly formed through the light-responsive tetramerization of fluorescent protein Dronpa153, 154. Weber et al. cross-linked a pytochrome-like protein Cph1* with 8-arm PEG through a Michael-type reaction to form a hybrid hydrogel156. Cph1* was primarily monomerized under far-red light (740 nm) while dimerized and therefore stiffened the hybrid hydrogel under red 20 light (660 nm). Remarkably, the stiffness adjustment of the Cph1*-PEG hybrid hydrogel was highly reversible, wavelength-specific (changing in different amplitudes at different wavelengths) as well as persistent in dark, as shown in Figure 1-7, making this hydrogel promising platform for studying cell responses to environmental mechanical signals. Sun et al. constructed hydrogels from polyproteins containing the C-terminal adenosylcobalamin binding domain CarHC157. CarHC tetramerizes when binding with vitamin B12 derivative adenosylcobalamin (adoB12) in the dark and dissociates into monomers when exposed to green (522 nm) or white light163. Therefore, the gelation of the CarHC polyproteins was induced only by the addition of adoB12 in the dark, and such gelation could be reversed upon green light irradiation. In addition to the homo-oligomerizations, the hetero-oligomerizations of proteins have also been utilized to construct protein-based hydrogels via a two-component approach164-168. Gallivan et al. utilized the Ca2+-sensitive binding between calmodulin (CaM) and calmodulin binding domain (CBDs) to control the formation of protein-based hydrogels164. A CaM-containing protein was only effectively cross-linked with a CBD-containing protein at high Ca2+ concentration, and such cross-linking could be reversed at the presence of EDTA. Similarly, Luo and Sun achieved the reversible gelation of protein-based hydrogels via the reversible association between CaM and M13 peptide in response to Ca2+/EDTA168. Regan et al. constructed a protein-PEG hybrid hydrogel via the assembly of tetratricopeptide repeat (TPR) and its cognate peptides166. As the TPR-peptide interaction was predominantly electrostatic, the hydrogel gelation was closely affected by ionic strength. 21 Figure 1.7 Cph1*-PEG hybrid hydrogel (A) Schematic of the Cph1*-PEG hybrid hydrogels where the photo-responsive dimerization of Cph1* serve as secondary cross-linking methods. (B-D) Modulus measurement demonstrated that the mechanical property changes effectuated by Cph1* dimerization are highly reversible (B), wavelength specific (C), and persistent in the dark (D)156. Copyright 2019, WILEY-VGH Verlag GmbH & Co. KGaA, Weinheim. 1.4 Multifaceted Expectations for Dynamic Protein-Based Hydrogels in Biomedical Applications Dynamic protein-based hydrogels find their applications in various fields of biomedicine. Depending on the tasks, different aspects of stimuli-responsive properties are required for the designs of these dynamic hydrogels. For example, drug delivery studies often focus on the degradation and permeability of hydrogels, while tissue engineering scaffold studies usually pay more attention to the mechanical properties. In this section, I will briefly discuss the multifaceted expectations for dynamic protein-based hydrogels in their biomedical applications. 22 1.4.1 Sol-Gel Transition The sol-gel transition is the transition between liquid solution state and gel state. Gelation can be regarded as the process where cross-linkable moieties continuously connect and disconnect, reaching an equilibrium where there are enough cross-linking points to form a gel network169. External stimuli (which is very often temperature variation170) that alter the equilibrium of the cross-linking methods can greatly reduce the number of cross-linking points and thus cause a gel-to-sol transition. Once the external stimuli are removed, the cross-linking points may recover to its previous equilibrium, allowing for a transition from the sol state back to the gel state. Dynamic, user-controlled sol-gel transitions of hydrogels are important in many biomedical applications, such as in situ tissue engineering scaffold formation, cell/drug delivery, and biosensing171. 1.4.2 Degradation The degradation of a polymer hydrogel is the process where the network loses its mass of polymer to surrounding solutions, usually caused by the hydrolysis of the building blocks. In tissue engineering, implanted hydrogel scaffolds should be degradable in physiological environments and the degradation rate should match the rate of the native tissue regeneration172, 173. In addition, user-controlled degradation rate of hydrogels is a common approach for controlled drug release9-11. Controlled degradation rate can be achieved by using external stimuli to weaken the chemical stability of the cross-linking points or the polymer chains. Other factors including the permeability and the surface area also closely affect the degradation rate. 23 1.4.3 Permeability and Swelling Ratio The equilibrium swelling ratio, often defined by the ratio between the mass of a hydrogel swollen to equilibrium over a reference mass (e.g., the original mass of the newly made hydrogel), is a most fundamental property of any hydrogel. The swelling ratio is the reflection of the hydrogel’s permeability to water and solutes. Permeability can be defined as the product of the partition coefficient and the diffusion coefficient. The partition coefficient describes the thermodynamic equilibrium between the sol phase and gel phase, which is influenced by size effect (the relative size of the solutes and the pores of network) as well as hydrophobic effects between the solutes and the polymers174. A higher polymer hydrophobicity of the network typically reduces the swelling ratio and the permeability of hydrophilic solutes. The diffusion coefficient describes the solute flux in the gel phase, which is affected by size effect and the mesh size of the network175. The cross-linking density largely determines the mesh sizes, and therefore the permeability and the swelling ratio. Other variations of the network topological structures, such as the rigidification of chains via coiled-coil formation85, also greatly influence the swelling ratio. The high swelling ratio makes hydrogels soft and rubbery, and influences their biocompatibility for mimicking soft native tissues or ECMs174 . High-water-content hydrogels were also observed to preserve biomacromolecules which facilitated bio-catalysis176. The volume changes accompanying the swelling ratio changes are often utilized for hydrogel-based actuators177. In drug delivery, the maximal load of drug is primarily controlled by permeability178, while the drug release rate is related to both the drug’s diffusion coefficient and the rate of swelling175. 24 1.4.4 Mechanical Properties The mechanical properties of a material are generally the properties that influence how the material reacts to applied loads, including stiffness, strength, elasticity, toughness, hardness, brittleness, and so on. Tissue engineering scaffolds are required to match stiffness and elasticity to tissues ranging from soft brain tissues to those as stiff as cartilage. Stiff and elastic hydrogels can protect cells or tissues from destruction upon heavy loads. Therefore, fine tailoring of the mechanical properties is important for protein-based hydrogels. In addition, user-applied mechanical property changes are important tools for investigating or controlling cell behaviors, such as using hydrogel stiffening to emulate the breast cancer-related tissue stiffening179-181. In native organisms, the mechanical signals conveyed through the cell-ECM interactions are among the various bio-signals that govern cell behaviors, including spreading, growth, proliferation, migration, and differentiation182-186. These mechanical signals are time-dependent due to the dynamic and adaptable nature of the native ECMs. From a microscopic perspective, the fast and reversible disconnection and reconnection of the ECM networks can respond and/or release the cell-generated traction forces as well as convey any external forces to the cells. At the macroscopic level, such dynamic ECM structures impart viscoelastic properties including stress relaxation, creep, and hysteresis to many soft tissues. As biomaterials, hydrogels are expected to display native ECM-like network dynamics so that they can provide ECM-cell interactions as well as mimic viscoelasticity of native tissues6-8. In this thesis, the stiffness of protein-based hydrogels, that is the extent of their mechanical response to applied force-induced deformation, is of particular interest. The stiffness is quantified by the ratio of stress over deformation strain, termed as the modulus. For example, the Young’s modulus, the ratio of the tensile or compression stress over the strain, is an 25 important gauge of stiffness and can be measured from the stress-strain relationship in a tensile or compression test. Moreover, the mechanical response of a viscoelastic materials under deformation can be classified by the elastic response, in which the stress is proportional to the extent of the strain and the energy is stored in the material, and the viscous response, in which the stress is proportional to the rate of the strain and the energy is dissipated. The elastic and viscous responses of the material are measured by the storage modulus and loss modulus, respectively. The storage modulus and loss modulus are not only measures of stiffness, but also measures of viscoelasticity. If the storage modulus G’ is higher than the loss modulus G’’, the material is regarded as an elastic solid-like material; otherwise, it is a viscous liquid. Oscillatory rheology test where the materials are subject to oscillatory shear force or deformation is usually used to determine the storage modulus G’ and loss modulus G’’ of hydrogels. It should be noted that, a densely cross-linked hydrogel based on rigid chains and stronger cross-linking methods (such as covalent bonds) are usually more elastic and stiffer but less viscoelastic, while a loosely cross-linked hydrogel based on flexible chains and weaker cross-linking methods tends to be more viscoelastic but relatively soft at the same time. Reconciling these two aspects of mechanical properties is still challenging for artificial hydrogels, and trade-offs are always needed depending on intended tasks. 1.4.5 Biocompatibility and Bio-Functionality Biocompatibility is always a primary requirement for any materials that are in direct contact with living organisms. Hydrogels for biomedical applications should not contain any cytotoxic moieties or cause any immune rejections, nor should their precursors or degradation products. In addition to being non-toxic, a hydrogel should be able to provide a friendly 26 environment for various cell behaviors. To this end, bio-functionalization of hydrogels are needed to regulate cell behaviors in designed manners for various tasks. Cell adhesion is a most extensively-studied aspect of hydrogel bio-functionality. In native organisms, cells adhere to ECMs via integrin-ECM interactions. The integrin-binding RGD tripeptide derived from fibronectin187 has been widely incorporated in hydrogels to allow for user-controlled cell adhesion onto these otherwise non-cell-adhesive hydrogels188, 189. Particularly, the RGD peptide or RGD-containing proteins can be conveniently engineered in protein-based hydrogels via recombinant DNA technology. Except for the RGD peptide and other cell-adhesive moieties, protein growth factors have also been widely utilized to functionalize hydrogels via similar methods7. 1.5 General Regulation Strategies to Construct Dynamic Protein-Based Hydrogels In this section, general strategies for using external stimuli to dynamically regulate the properties of dynamic protein-based hydrogels are reviewed. As shown in Figure 1.8, the regulation strategies to construct dynamic protein-based hydrogels can be grouped into three categories: tuning the number of the cross-linking points, tuning the effective length of elastically active chains, and molecule decoration on hydrogel “blank slates”. 27 Figure 1.8 General regulation strategies to construct dynamic protein-based hydrogels (A) Tuning the number of cross-linking points influences the cross-linking density of hydrogels. For hydrogels which are primarily cross-linked by reversible cross-linking methods, the stimuli-responsive disconnection of their cross-linking points can lead to complete degradation or sol-gel phase transition (Left). For hydrogels which contain both permanent and reversible cross-linking methods, the stimuli-responsive disconnection of their reversible cross-linking points changes the cross-linking density but does not cause degradation or sol-gel transition. (Right) (B) Tuning the effective length of elastically active chains between cross-linking points affects the cross-linking density and the physical properties of the hydrogels. (C) The decoration of functional molecules onto inert, static hydrogel blank slates can regulate the chemical functionality of the hydrogels without changing the cross-linking density. 28 1.5.1 Tuning the Number of Cross-Linking Points The cross-linking density closely determines many aspects of hydrogel physical properties, such as the sol-gel transition, the mechanical properties, and the swelling ratio. The cross-linking density describes the number of elastically active chains (the polymer chains between cross-linking points that can bear force and contribute to the elasticity) per unit volume in a cross-linked network, which can be defined by 𝑁 = 𝜌/𝑀𝑐 (1.1) where ρ is the mass density and Mc the number average molecular weight between cross-linking points. The mass density ρ is closely determined by the polymer concentration and the swelling ratio. The Mc is equivalent to the effective length of elastically active chains assuming that all the elastically active chains can be fully extended. For a network of a given total molecular weight, the Mc is inversely related to the number and functionality of the cross-linking points. Therefore, changing the number of cross-linking points can effectively change the cross-linking density and the physical properties of the hydrogels. In the designs of dynamic hydrogels, tuning the number of cross-linking points can be achieved by using stimuli to change the equilibrium of an existed cross-linking methods or by introducing new cross-linking methods. Depending on the network designs, complete hydrogel degradation or gel-to-sol transition can be achieved by dramatically reducing the number of cross-linking points. Alternatively, hydrogels may contain both permanent and reversible cross-linking points, where the permanent cross-linking points ensure an overall non-degradable network while the association/dissociation of the reversible cross-linking points provide a tunable cross-linking density. In section 1.1, the general cross-linking methods for protein-based hydrogels have been reviewed. Many of these cross-linking methods are cleavable or reversible in response to 29 external stimuli, which can be utilized for tuning the cross-linking density of dynamic protein-based hydrogels. Table 1.1 summarizes the available stimuli-responsive cross-linking methods for dynamic protein-based hydrogels, classified by the corresponding types of stimuli. The choice of the cross-linking methods should be made according to the stimuli that present in the applications of the hydrogels. The stimuli may be generated by native organisms. For example, the pathologically high concentration of reducing agent glutathione (GSH) has been observed in cancer tissues, which can be 7-10 folds higher than that in normal tissues190. To specifically deliver anti-cancer drugs to such cancer tissues, re-dox potential-responsive disulfide bonds have been utilized in dynamic hydrogel-based drug carriers191. Yang et al. constructed keratin-based hydrogels through the disulfide bonds between the cysteine residues29. These keratin-based hydrogels degraded faster in response to an elevated GSH concentration due to the cleavage of the disulfide bonds, which also accelerated the release of the encapsulated anti-cancer drug doxorubicin. Alternatively, the stimuli can be manually applied as a means of therapy. As an example, Weber et al. engineered an antibiotic-sensing protein-based hydrogel162. The bacterial gyrase subunit B (GyrB) protein domains can bind an antibiotic coumermycin molecule at 1: 2 stoichiometry and thus be dimerized. The authors grafted GyrB domains onto polyacrylamide and utilized GyrB dimerization to form a hydrogel. Another antibiotic novobiocin could compete with coumermycin and instead bind with GyrB at 1: 1 stoichiometry, therefore dissociating the GyrB dimers. Consequently, the GyrB-based hydrogel degraded faster in response to a higher novobiocin concentration, which can be potentially used for controlled drug release. As a proof of concept, the release profile of a protein drug human vascular endothelial growth factor (hVEGF) from the GyrB-based hydrogel could be controlled by the novobiocin concentration. Moreover, the required novobiocin concentration for effective hVEGF release was comparable 30 with the novobiocin concentration in human plasma 24-72 h after a single oral dose, which demonstrated the feasibility of this method. Many more examples can be raised for stimuli-driven design principles of dynamic protein-based hydrogels. It should be noted that multiple stimuli-responsive cross-linking methods may be present in one hydrogel system for sophisticated regulation. An example was the “dual thermosensitive” protein-based hydrogel developed by Xia et al.192 This hydrogel was constructed from the fusion protein of ELP and the C-terminal domain of spider major ampullate spidroin 1 (CTD). The ELP block displayed an LCST behavior and aggregated at higher temperatures, while the CTD block displayed a UCST behavior and aggregated at lower temperatures. By carefully tuning the ELP/CTD composition, a temperature window was set for the sol-gel transition of this ELP-CTD hydrogel. At 4 °C which was below the Tt of the CTD, the ELP-CTD protein formed a relatively stiff hydrogel (G’ > 1 kPa) which could be used for cell storage; At 25 °C which was between the two Tt values of the CTD and the ELP, the hydrogel completely melted into a liquid and the encapsulated cells could be easily harvested; At 37 °C which was above the Tt of the ELP, the ELP-CTD protein formed a relatively soft hydrogel (G’ ~ 100 Pa) that could be easily injected in vivo. 31 Table 1.1 Stimuli-responsive cross-linking methods for dynamic protein-based hydrogels Type of Stimuli Categories of stimuli-responsive cross-linking methods Examples in dynamic hydrogel designs Temperature Phase transition of polypeptides ELPs62, 105 ELP-derived peptides123 Resilin-derived peptides124 Coiled-coil interactions Leucine zipper A oligomerization132 CC1 oligomerizaion85 A4/B4 hetero-dimerization142, etc. Non-covalent protein assembly CTD assembly192 Light Non-covalent protein assembly Dronpa tetramerization139 UVR8 dimerization155 B12-dependent CarHC tetramerizaiton157 Cph1* dimerization156 Red-ox potential Disulfide bonding Disulfide bonds in keratin29 Disulfide bonds in cELPs27, etc. Metal-histidine coordination Co2+/Co3+-histidine coordination79 Disulfide bond-mediated protein assembly GN/GC reconstitution76 PDZ domain75 pH and ionic strength Coiled-coil interactions Leucine zipper A oligomerization132 CCE-CCK hetero-dimerization133, etc. Non-covalent protein assembly TPR-peptide binding166 Metal ions Metal-histidine coordination Zn2+-histidine coordination80, etc. Non-covalent protein assembly Ca2+-dependent CaM-ligand binding168 Small molecule drugs Non-covalent protein assembly B12-dependent CarHC tetramerization157 Antibiotic-dependent GyrB dimerization162 ASC inflammasome formation158 1.5.2 Tuning the Effective Length of Elastically Active Chains Equation 1.1 assumes that all the elastically active chains between cross-linking points can be fully extended, so the effective chain length of elastically active chains is equivalent to 32 the molecular weight between cross-linking points Mc. However, in many protein-based hydrogels, the elastically active chains include folded globular proteins that are linked among different cross-linking points by two (or more) ends (which are not necessarily the N- and C- termini). Such folded globular proteins are not fully extendable, and their effective length is determined not only by the Mc but also by their three-dimensional folded structures. A folded protein structure generally consists of several local α-helices and/or β-sheets stabilized by hydrophobic effects, electrostatic interactions, and hydrogen bonds in consort, which can be unfolded by extreme temperatures193, chemical denaturants194, or mechanical forces195, 196. Once the stimuli that cause the unfolding are removed, some proteins can refold. In addition to the folding/unfolding, some globular proteins can switch between different folded conformations in response to certain stimuli. The folding/unfolding and other conformational changes can dramatically change the effective length of elastically active chains in globular protein-based hydrogels, and therefore have been widely utilized to regulate the cross-linking density and the physical properties of such hydrogels43-48, 197-204. The force-induced unfolding of globular proteins influences the cross-linking density under applied loads and largely determines the mechanical properties, including elasticity, strength, toughness, and resilience, of globular protein-based hydrogels182. Tuning the mechanical stability of the globular proteins can effectively regulate the mechanical properties of protein-based hydrogels. Dougan et al. demonstrated that using the ligand maltose binding to stabilize the folded structure of the maltose binding protein (MBP) could enhance the mechanical strength of the MBP-based hydrogels45. Lv et al. utilized Ca2+ to tune the mechanical properties of the hydrogels cross-linked by repeat-in-toxin (RTX), because Ca2+ is essential for stabilizing the folded structure of RTX48. In addition to the force-induced folding/unfolding, the chemical 33 denaturant-induced unfolding also plays important roles in globular protein-based hydrogels. The Li group adapted GB1-based hydrogels into an actuator device203. GB1 is the B1 domain of protein G from Streptococcus, which exhibits comparable mechanical stability with the globular domains of giant muscle protein titin and can rapidly refold205. The GB1 domains were first denatured by incubating the GB1-based hydrogel actuator in a denaturant guanidinium chloride (GdmCl) solution. Then the denatured GB1-based hydrogel actuator was transferred into non-denaturing phosphate buffered saline (PBS) and the GB1 domains were allowed to refold. Driven by the refolding of the GB1 domains, the GB1-based hydrogel actuator dramatically de-swelled and contracted, which could do work and lift a weight 50 times heavier than itself. In a following work, the Li group utilized the different denaturant-dependent swelling ratio profiles of GB1- and ferredoxin-like protein (FL)-based hydrogels to develop a bilayer shape memory hydrogel204. As shown in Figure 1.9, the layer of the (GB1)8-based hydrogel and the layer of the (FL)8-based hydrogel displayed distinct swelling ratio and volume changes upon denaturation, so that the bilayer hydrogel varied its shape in response to varying GdmHCl concentration. Such shape changes of the bilayer hydrogel were highly reversible. Khoury and Popa constructed another shape memory hydrogel by cross-linking bovine serum albumin (BSA) domains via RuII-mediated di-tyrosine reaction into a hybrid hydrogel with polyethyleneimine46. The shape of this BSA-based hydrogel can be initially programmed using a mold in a tris(hydroxymethyl)aminomethane (Tris) buffer, then interrupted by using a GdmCl solution to unfold the BSA domains, and finally resumed due to the BSA refolding when the hydrogel was transferred back to a Tris buffer. 34 Figure 1.9 Shape memory bilayer hydrogel based on protein folding/unfolding (A) the three-dimensional structures of GB1 and FL folded domains. (B) The swelling ratio profiles of the (FL)8-based and the (GB1)8-based hydrogels show depending on the concentration of denaturant GdmCl. (C) The highly reversible bidirectional bending deformations of the (FL)8/(GB1)8 bilayer hydrogel strips in response to various GdmCl concentration204. Copyright 2022, The Author(s). Licensed under a Creative Commons Attribution 4.0 International License. Some proteins can dynamically switch between different folded structures in response to various stimuli. When such proteins serve as the elastically active chains in a hydrogel network, the dynamic switching of their folded structures will affect their effective length and the cross-linking density of the hydrogel. Unlike complete folding/unfolding processes, such switches between folded structures involve less dramatic conformational changes, so are relatively moderate and can usually avoid further protein aggregation observed in complete protein unfolding43. The stimuli that induce the dynamic conformational changes are usually more 35 specific, too. As an early example, Murphy et al. cross-linked both termini of CaM with four-arm PEGs to form a hybrid hydrogel198, 199. As shown in Figure 1.10, the Ca2+-bound CaM adopts an extended dumbbell-like conformation in the absence of ligand, while switches to a collapsed conformation at the presence of ligand such as trifluoperazine, accompanying a decrease of end-to-end distance from 5.2 nm to 1.4 nm. As a result, the CaM-PEG hybrid hydrogel de-swelled by 25-90% when exposed to 10 mM TFP. Such de-swelling was largely reversible when the CaM-TFP binding was interrupted by EGTA. Kopecek et al. cross-linked adenylate kinase (AKe) protein with synthetic polymers into a hydrogel through the thio-ene reaction of Ake’s two cysteine residues206. AKe adopts a more compacted structure after binding with adenosine triphosphate (ATP), decreasing the distance between the two cysteine residues. As a result, the Ake-based hydrogel de-swelled in response to an elevated ATP concentration. The Li group adapted mutually exclusive proteins (MEP) into dynamic hydrogels200, 201. A MEP is an engineered artificial protein where the sequence of a guest domain, which has a longer end-to-end distance, is inserted into a short loop of a host domain. Thus, there is a folding tug-of-war between the host and the guest domains, and a MEP can only have one of the domains folded at a given time. In the MEP-based hydrogels, the conformational state of the MEP was governed by a di-sulfide bond and its effective contour length can switch between 2.6 nm under oxidizing conditions and 26 nm under reducing conditions207. Consequently, when the MEP were cross-linked into a hydrogel network, the Young’s modulus of the hydrogel could be reversibly tuned between ~10 kPa (reduced) and ~40 kPa (oxidized) for at least 3.5 cycles200. In a following work, the Li group engineered two histidine residues into the MEP, so that the mechanical properties of the MEP-based hydrogels could also be regulated by Ni2+-histidine coordination201. More recently, DeForest et al. engineered light-oxygen-voltage-sensing domain 2 (LOV2) into a 36 hydrogel202. LOV2 reversibly unfolds its C-terminus Jα-helix under blue light, thus extending its effective length and reversibly softening the LOV2-based hydrogels upon alternating blue light irradiation. Figure 1.10 Dynamic hydrogel regulated by the dynamic conformation changes of CaM A CaM protein domain adopts a collapsed conformation in the absence of ligand while switching to an extended conformation when bound with ligand. Such dynamic conformation change reversibly alters the cross-linking density of the CaM-based hydrogels where CaM domains are cross-linked as elastically active chains199. Copyright 2008, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 1.5.3 Molecule Decoration on Protein-Based Hydrogel Blank Slates Tuning the number of cross-linking points or the effective length of elastically active chains aims to greatly change the cross-linking density and the physical properties of the hydrogels. Sometimes, however, the chemical functionality of the hydrogels is required to be changed while the physical properties are expected to remain unchanged at the same time. To this end, functional molecules can be decorated onto a hydrogel “blank slate”. A hydrogel blank slate is an inert static network that has a fixed cross-linking density. The decoration of functional 37 molecules is often achieved through stimuli-induced conjugation methods. After the conjugation, the functional molecules are usually grafted as “pendants” with one end attached to the polymer chains of the hydrogel network. In this way, the decoration of the molecules does not change the cross-linking density. The dynamic decoration of functional molecules onto natural protein-based hydrogel blank slates is often achieved by the chemical modification of the protein building blocks208-210. However, chemical modifications are inconvenient and may introduce non-natural chemical hazards, so purely recombinant protein-based conjugations methods are preferred. To this end, protein-protein interactions that form hetero-dimers are of particular interest as it is convenient to fuse one of the binding partners with the protein building blocks and the other with the protein ligands via recombinant DNA technology. The Li group constructed hydrogels from SpyCatcher-fused polyproteins and successfully decorated SpyTag-fused functional proteins onto such hydrogel blank slates through SpyTag-SpyCatcher chemistry211. Similarly, Wieduwild and Howarth utilized SnoopCatcher-SnoopTag chemistry to decorate SnoopTag-fused proteins onto Snoop-Catcher-containing ELP-hyaluronic acid hybrid hydrogels212. Like SpyTag and SpyCatcher, SnoopTag and SnoopCatcher are two split fragments of RrgA D4 protein, which can also reconstitute and form an intermolecular isopeptide bond213. Both SpyTag-SpyCatcher chemistry and SnoopTag-SnoopCatcher chemistry are irreversible, so the resultant decorated ligands cannot be released anymore. To reversibly decorate and release protein ligands, the Li group later adapted GN/GC reconstitution as the conjugation method214. The reconstituted GN/GC complex was physically assembled under reductive conditions at 37 °C, while could be stabilized by a disulfide bond formed under oxidative conditions at 4 °C. In this way, a GC-fused TNfn3 (the third fibronectin type 3 domain of human tenascin) could be reversibly decorated onto GN-38 containing hydrogels in response to re-dox potential and temperature variations. Thus, the reversible decoration of the RGD-containing protein TNfn3 allowed for the reversible cell adhesion on such hydrogels. Spatially heterogenous functionalization of hydrogel materials has attracted increasing interest for the needs for mimicking sophisticated native organisms in multidimensions. The advent of multi-channel 3D printing technology has enabled sophisticated spatial assembly of multiple protein-based “inks” into one piece of protein-based hydrogel material215, 216. However, 3D-printing is a one-time manufacture and lacks reversibility. Besides, not all protein-based hydrogels or pre-gel solutions meet the multifaceted requirements for being printable inks217, 218. Light-responsive conjugation methods provide alternative approaches for spatially decorating, or “patterning”, functional molecules on hydrogels after the gelation. Functional ligands can be spatially decorated, or “patterned”, onto a hydrogel blank slate via a light-responsive conjugation method at the guidance of a light pattern produced by irradiation through shadow masks or by other techniques such as two-photon lithography219. For example, Deforest et al. recently developed a method for patterning ligands on protein-based hydrogels, as shown in Figure 1-11, where thee alkoxyamine groups modified on the lysine residues of gelatin or fibrin are first photo-uncaged and then conjugated to the aldehyde-modified protein ligands210. Both 2D and 3D patterns of the model ligand fluorescent protein mCherry could be precisely produced on the gelatin hydrogels. However, this photo-caged alkoxyamine-aldehyde reaction method requires three steps of chemical reactions, which is rather inconvenient. A purely recombinant protein-based light-responsive conjugation approach is needed for patterning proteins on protein-based hydrogels. The protein-protein interaction between LOV2 and its designed binding partner ZDark1 (zdk1), nicknamed “LOVTRAP”, has a much higher affinity in the dark than under blue 39 light220. West et al. engineered LOV2 domains on PEG hydrogels and reversibly patterned zdk1-fused protein ligands onto the PEG-hydrogels using alternating dark/lit conditions221. Another light-responsive protein, the photocleavable domain PhoCl, has a C-terminal chromophore-bearing helical peptide which is attached and hidden in the N-terminal β-barrel in the dark while is permanently cleaved and released from the β-barrel upon exposure to 400-nm light222. DeForest et al. fused the C-terminus of PhoCl with a protein ligand and grafted its N-terminus onto a PEG hydrogel network via click reaction, so that the ligand can be spatially released by light to generate a 2D or 3D pattern223. Although the LOVTRAP- and PhoCl-based ligand patterning methods have only been utilized in synthetic polymer-based hydrogels so far, there seems to be little technical difficulty in incorporating them into recombinant protein-based hydrogels because neither of them requires any post-translational chemical modification. 40 Figure 1.11 Molecule patterning on protein-based hydrogels through photo-induced oxime reaction (A) Three-step reactions for molecule patterning on protein-based hydrogels. The lysine residues on proteins are first modified with NPPOC-caged alkoxyamine groups. Then the alkoxyamine groups are spatially uncaged by photo-cleavage of NPPOC group. Finally, aldehyde-modified protein ligand can be selectively ligated with uncaged alkoxyamine group via oxime reaction. (B) A three-dimensional heart pattern of mCherry fluorescence produced at the guidance of a light pattern generated by two-photon lithography (scale bar = 50 µm)210. Copyright 2021. Published under the PNAS license. 1.6 Engineering Dynamic Protein-Based Hydrogels: Goals of Thesis Indeed, the design and construction of dynamic protein-based hydrogels have greatly progressed. Yet challenges remain for engineering next-generation dynamic protein-based hydrogels. Scientists are setting forth towards three major directions. The first direction is to quantitatively predict the macroscopic hydrogel properties from the microscopic properties of the hydrogel building blocks. Currently, most design principles of dynamic protein-based hydrogels 41 are rather qualitative, and the optimization of their properties largely relies on “screening” many individual conditions. The quantitative theories that bridge the microscopic properties of the building blocks and the macroscopic properties of the hydrogels will greatly facilitate bottom-up hydrogel designs as well as save a lot of manpower. The second direction is to finely tailor the properties of dynamic hydrogels, which can be achieved by increasing the stimuli-dose-sensitivity of the hydrogels. Stimuli-sensitive hydrogels can be easily regulated by moderate external stimuli, which is usually more bio-friendly. In addition, stimuli-sensitivity facilitates the spatial control over the hydrogel properties using a spatial pattern of stimuli intensity. The third direction is to develop reversible dynamic hydrogels which can undergo property changes for multiple times in response to alternating stimuli variations. Reversible dynamic hydrogels are required for many long-term biomedical applications and can facilitate the temporal control over hydrogel properties. Driven by these new directions, this thesis aims to better understand, design, and engineer novel dynamic protein-based hydrogels. The in situ phase transition of the polymer chains is an important cross-linking method that alters the cross-linking density and tunes the hydrogel properties. In Chapter 2, we aim to regulate the network structures as well as the temperature-responsive properties of a protein-based hydrogel through the in situ LCST phase transition of the elastin like polypeptide (ELPs) chains. A series of protein block copolymers are engineered by recombinantly fusing ELP sequences of various lengths to an elastomeric GB1-resilin-based polyprotein. The protein copolymers can form micelles in aqueous media at elevated temperatures. Using RuII-mediated di-tyrosine reaction, the protein copolymer at lower temperatures can be cross-linked into hydrogels, where the tyrosine-free ELP blocks serve as side chains. At elevated temperatures, the ELP side chains undergo in situ aggregation, which reversibly increases the Young’s modulus 42 and reduces the swelling ratio. The amplitude of such modulus reinforcement and de-swelling is influenced by the ELP chain length and the composition. Furthermore, it is also explored to directly use RuII-mediated di-tyrosine reaction to cross-link the copolymer-based micelles into micelle-based hydrogels at higher temperatures. The results of this work enhance the understanding of how the in situ phase transition of polypeptide side chains can be utilized to finely tune the stimuli-responsive properties of dynamic protein-based hydrogels. Understanding the stimuli-responsive dynamic mechanical responses of hydrogels is essential for their applications as ECM mimetics in tissue engineering. In Chapter 3, the dynamic mechanical responses of the protein-based hydrogels cross-linked via a hetero-coiled-coil interaction is investigated. The resultant coiled-coil-based hydrogels exhibit highly time-dependent viscoelastic moduli G’ and G’’, and the calculated relaxation spectra reveal unusual multiple energy dissipation modes. Such dynamic mechanical responses and energy dissipation modes can be tuned by the protein concentration, the stoichiometry of the two binding partners, and temperature. The unique features confer on the hydrogel responsiveness to mechanical stimuli in a broad range of frequencies. Therefore, the hydrogels are injectable due to their shear-thinning properties and can fully recover their mechanical properties within a few seconds thanks to the fast dynamics of the coiled-coil cross-linking methods. In Chapter 4, our goal is to use light as a stimulus to regulate dynamic mechanical properties of protein-based hydrogels, because light can be easily applied in a spatiotemporal manner. The non-covalent LOVTRAP interaction between LOV2 and zdk1 domains is selected as the cross-linking method. Due to the light-dependent affinity of LOVTRAP, LOVTRAP-based hydrogels can reversibly display light-dependent viscoelastic moduli. The calculated relaxation spectra reveal two energy dissipation modes corresponding to the LOVTRAP interaction at dark and lit states. The fast dynamics of 43 LOVTRAP also endow LOVTRAP-based hydrogels shear-thinning and self-healing properties, making them potential injectable biomaterials. The results of Chapter 3 and Chapter 4 contribute to understanding the relationships between the microscopic thermodynamics of the cross-linking methods and the macroscopic viscoelasticity of hydrogels. Multi-dimensional molecule patterning is of great interest for encrypting multi-dimensional information, such as bio-signals or fluorescence, into hydrogel blank slates. As LOVTRAP has proven in Chapter 4 to maintain light-responsiveness when cross-linked in a hydrogel network, it is further adapted as a conjugation method for dynamic molecule patterning on protein-based hydrogels. In Chapter 5, zdk1 and LOV2 are recombinantly fused to a cross-linkable protein and a yellow fluorescent protein (YFP), respectively, so that the YFP can be dynamically patterned onto zdk1-containing hydrogels under blue light irradiation through a shadow mask. It is demonstrated that 2D patterns of YFP fluorescence can be produced which precisely inherit the shape of the shadow mask, including 2D quick response (QR) codes that are readable by cell phone. Moreover, the light-dose-dependency of LOVTRAP enables patterning of greyscale fluorescence intensity, which can generate pseudo 3D greyscale codes that can be further converted into pseudo colored 3D codes. In this way, the information storage capacity of the protein-based hydrogels is greatly enlarged. This work demonstrates the utility of protein-based hydrogels as information storage medium and provides a versatile, purely protein-based information approach for 2D/3D fluorescence or potentially other types of signals in multi-dimensions. 44 Chapter 2: In situ Phase Transition of Elastin-Like Polypeptide Chains Regulates Thermo-Responsive Properties of Elastomeric Protein-Based Hydrogels 2.1 Synopsis Engineering protein-based hydrogels that can change their physical and mechanical properties in response to environmental stimuli have attracted considerable interest due to their promising applications in biomedical engineering. Amongst environmental stimuli, temperature is of particular interest. Most thermally responsive protein hydrogels are constructed from thermally responsive elastin-like polypeptides (ELPs), which exhibit a lower critical solution temperature (LCST) transition, or non-structured elastomeric proteins fused with ELPs. Here we report the engineering of thermally responsive elastomeric protein-based hydrogels by fusing ELPs to elastomeric proteins made of tandemly arranged folded globular proteins. By fusing ELP sequence (VPGVG)n to an elastomeric protein (GR)4, which is made of small globular protein GB1 (G) and random coil sequence resilin (R), we engineered a series of protein block copolymers, Vn-(GR)4. The fusion proteins Vn-(GR)4 exhibit temperature-responsive behaviors in aqueous solution that are different from that of Vn-ELPs, as they did not exhibit the macroscopic phase transitions in the turbidity test. Instead, V48-(GR)4 and V72-(GR)4 form micelles at temperatures higher than *A version of Chapter 2 has been published as [Tianyu Duan] and Hongbin Li. (2020) In Situ Phase Transition of Elastin-Like Polypeptide Chains Regulates Thermoresponsive Properties of 45 Elastomeric Protein-Based Hydrogels. Biomacromolecules. 21:2258-2267. This chapter is incorporated in this thesis with permission of American Chemical Society, Copyright 2020. the transition temperature of V48-ELP and V72-ELP at the same concentration. Using the well-developed ruthenium-mediated photochemical cross-linking method, Vn-(GR)4 polymers can be cross-linked into hydrogels, in which Vn-ELP serve as side chains of the hydrogel network. These hydrogels exhibited thermo-responsive properties due to the temperature dependent phase transition behaviors of the incorporated Vn-ELPs blocks. At elevated temperatures, the Vn-ELPs side chains in the hydrogel network underwent aggregation, leading to secondary physical cross-linking. The aggregation of the Vn-ELPs resulted in higher Young’s modulus and reduced swelling ratio. Furthermore, the amplitude of such property changes can be tuned by side chain length and composition. These results demonstrate that in situ phase behaviors of ELP side chains can regulate thermo-responsiveness of protein-based hydrogels. We anticipate that this method can be applied to other elastomeric proteins for potential biomedical applications. 2.2 Introduction Over the last two decades, engineered recombinant proteins have been employed as building blocks to engineer hydrogels, due to their unique features and advantages1, 2, 6, 7, 20, 23. Recombinant engineered proteins can be conveniently synthesized with well-defined structures (from primary to tertiary), mono-dispersity in molecular weight distribution and desired biological functionalities. Such unique features allow for the engineering of protein hydrogels that can mimic various aspects of cellular microenvironments as well as extracellular matrices, making them promising candidates for various applications in biomedical engineering and tissue engineering2, 6, 7. To better mimic the dynamic nature of biological environments, dynamic 46 protein hydrogels whose physical, mechanical and biological properties can be dynamically tuned, have been engineered using various stimuli, such as temperature62, 85, 105, 224-226, redox potential26, 200, 227-229, light153, 154, 156, 157, 202 and ligand201, 230-232. Amongst the protein building blocks, we have explored the use of elastomeric proteins made of tandem repeats of protein domains with folded globular structures as building blocks to engineer protein hydrogels42, 43, 205. Compared with peptide hydrogels, the use of folded globular domains in hydrogel engineering may offer more possibilities and ease for tuning the biochemical and physical properties of the hydrogels. It has become straightforward to incorporate intact protein ligand/receptors into hydrogels for various studies and applications. The conformational changes of protein building blocks can be readily utilized to regulate the physical and mechanical properties of protein hydrogels. In particular, the folding and unfolding of globular protein domains have provided unique opportunities to allow for engineering protein hydrogels with tailored mechanical properties at the molecular level42, 43. For example, using the artificial elastomeric protein made of a small globular domain GB1 and a non-structured sequence resilin as building blocks, we engineered protein-based biomaterials that exhibit mechanical properties mimicking the passive elastic properties of muscles42. These elastomeric proteins are highly soluble, and the mechanical properties of their hydrogels can be tailored at the single molecule level. Thus, these protein hydrogels provide a useful platform for further tuning their physical and mechanical properties for desired applications. In this vein, here we explore the possibility to endow these protein hydrogels with thermo-responsive properties. Temperature is a stimulus that is commonly used in engineering stimuli-responsive hydrogels for various applications, including tissue engineering and drug delivery233, 234. For this purpose, the thermo-responsive elastin like polypeptides (ELPs) have been widely used.235, 236 47 ELPs, derived from native tropoelastin86, 87, are polypeptides with tandem repeats of pentapeptide motif VPGXG, where the “guest residue” X can be any amino acid residue except proline. ELPs exhibit the lower critical solution temperature (LCST) behavior: below a specific transition temperature (Tt), ELPs adopt mostly disordered, relatively hydrophilic structures and dissolve well in aqueous media; above Tt, ELPs undergo conformational changes into more ordered, hydrophobic structures and aggregate88-91. ELPs have been extensively studied as building blocks for the fabrication of thermo-responsive protein-based hydrogels, because their Tt can be finely tailored by their chain lengths88, 100, types of guest residues88, 98, concentration88, 100 and other conditions of the media such as ionic strength101, 102. Meanwhile, ELPs or ELP-fusion proteins can form various nanostructures including micelles108-111, 118, nano-fibres112, 237, or vesicles109, 114 in aqueous media. Furthermore, it has been shown that ELPs can undergo in situ phase transitions when cross-linked into hydrogels, which can serve as secondary physical cross-links effectuating modulus reinforcement or swelling ratio alternation62, 105, 224, 225. Despite this extensive prior work on ELPs, it remains unexplored if ELPs can be incorporated into highly soluble elastomeric proteins made of folded globular proteins to achieve thermo-responsiveness of the resultant hydrogels. Here we incorporated the canonical type of ELPs, (VPGVG)n (abbreviated as Vn-ELPs), into GB1-resilin-based elastomeric proteins to explore the feasibility to construct thermo-responsive elastomeric protein hydrogels. Meyer and Chilkoti proposed an empirical equation for predicting the Tt of (VPGVG)s100: 𝑇𝑡 = 𝑇𝑡,𝑐 +𝑘𝑛𝑙𝑛 (𝐶𝑐𝐶) (2.1) In Equation 2.1, n is the chain length in polypeptides, C the concentration in µM, Tt,c the critical temperature 20.8 ± 0.3 °C, k the constant 129 ± 3 °C, and CC the critical concentration 25000 ± 4600 µM. In this work, the lengths of Vn-ELP polypeptides were chosen as n = 24, 48 and 72, 48 and their theoretical Tt was estimated to be between 27 and 38 °C at 1 mM (Table 2.1) according to Equation 2.1. By fusing Vn-ELPs to the N-terminus of the polyprotein (GB1-resilin)4 (abbreviated as (GR)4), we engineered a series of di-block co-polymers Vn-(GR)4 (Figure 2.1 A), and used the well-developed ruthenium-mediated photochemical cross-linking method39 to engineer protein hydrogels. The resultant protein hydrogels exhibited thermo-responsive properties due to the temperature dependent phase transition behaviors of the incorporated Vn-ELPs blocks. Our study demonstrated the feasibility to incorporate ELPs into tandem modular protein-based elastomeric proteins to achieve thermo-responsive properties of the hydrogels. Table 2.1. Predicted transition temperature Tt of un-fused Vn-ELP polypeptides Chain Length n Concentration (mM) Predicted Tt 24 0.1 50.5 1.0 38.1 48 0.1 35.6 1.0 29.5 72 0.1 30.7 1.0 26.6 2.3 Materials and Methods Protein Engineering. The genes encoding V48-ELP and V72-ELP cloned in a modified pET25b+ expression vector were purchased from Addgene (Addgene plasmid #68395 and #68938), which were originally from Dr. Andrew MacKay and co-workers238, 239. The gene encoding V24-ELP was custom synthesized by GenScript (Piscataway, NJ, USA). The construction of the gene encoding (GR)4 was described previously42. The gene of (GR)4 carries a 49 5’ BamHI and 3’ BglII and KpnI restriction sites, and closed in the pQE80L vector. Then the (GR)4 gene was cloned into a modified pET29b expression vector with a C-terminal His-Tag to facilitate the purification of full-length protein. The construction the genes encoding Vn-(GR)4, were carried out in a modified pET25b+ expression vector with C-terminal His-Tag and NdeI, EcoRI, BamHI and KpnI restriction sites. The genes encoding Vn-ELPs, (n=24, 48 and 72) were cloned into pET25b+ by NdeI and EcoRI first to obtain pET25b+-Vn-ELP, and then the gene of (GR)4 was cloned into pET25b+-Vn-ELP by BamHI and KpnI, resulting in pET25b+/Vn-(GR)4. The overexpression of (GR)4, Vn-ELPs and Vn-(GR)4 were expressed in E. coli strain BL21(DE3). The starter culture was grown at 37 °C in 2.5% lysogeny broth (LB) with 0.1 g/L ampicillin (with pET25b+/Vn-ELPs and pET25b+/Vn-(GR)4s) or 0.05 g/L Kanamycin (with pET29b/(GR)4) until OD600 reached 0.8-1.0. Protein overexpression was induced by 1 mM isopropyl-1-β-D-thiogalactoside (IPTG) and then continued for 4 h at 37 °C. The bacteria cells were harvested by centrifugation and lysed by 2.5 mg/mL lysozyme. DNAs and RNAs were hydrolyzed by 5 µg/mL DNase I and 5 µg/mL RNase A. After cell lysis (GR)4 and Vn-(GR)4 proteins with His6x-Tag were purified from soluble cell lysate using a Co2+-affinity column. V48-ELP and V72-ELP were purified by inverse transition cycling240 after cell lysis. For V24-ELP, a modified inverse transition cycling method was used: after cell lysis, 2 M NaCl was added to the supernatant to reduce the LCST of V24-ELP to below 40 °C. The purified proteins were subsequently dialyzed against deionized water for 9 h and lyophilized. The yield of the target proteins was 30-60 mg/L of cell culture. Turbidity Tests. Turbidity tests were carried out by a Varian Cary-400 Uv-Vis Spectrophotometer. Protein solution at 0.1 mM in PBS was transferred into a PMMA cuvette (d = 1 cm). Optical density at 350 nm was recorded as turbidity at 0.2 °C interval from 15 to 55 °C 50 at a heating rate of 0.3 °C/min. Tt was determined as temperature corresponding to largest turbidity gradient. Dynamic Light Scattering (DLS) Characterization. The DLS characterization was carried out on a Brookhaven NanoBrook Omni Particle Size Analyzer. Protein solutions after being filtered with a 0.22-µm syringe filters were transferred into a PMMA cuvette (d = 1 cm). DLS data was collected at 90° (duration time = 60 s) heated at 5 °C (sample soak time = 120 s) intervals from 25 to 55 °C. The curve of DLS intensity versus recorded diameter for Rayleigh spheres was fitted by Gaussian distribution. The calculated half mean diameter was taken as the hydrodynamic radius (Rh). Hydrogel Preparation. (GR)4 and (GR)4+Vn-(GR)4 hydrogels were prepared by a well-developed [RuII(bpy)3]2+-mediated photochemical cross-linking method. The lyophilized proteins were mixed and dissolved to 1-3 mM for (GR)4 and 1 mM for Vn-(GR)4 in 1x phosphate buffered saline solution (PBS) before the addition of 50 mM ammonium persulfate (APS) and 0.26 mM [RuII(bpy)3]Cl2. The mixture was rapidly transferred into a custom-made ring-shaped plexiglass mold (inner diameter = 8 mm, outer diameter = 10 mm, height = 3 mm) or a custom-made square-shaped aluminum mold (5 mm × 5 mm, height = 2 mm). The sample in the mold was then irradiated by a 200-W white light source at a height of 10 cm. The sample was then carefully removed from the mold and stored in PBS with 0.02% NaN3. Swelling Ratio Measurement. The swelling ratio measurements were performed with square-shaped hydrogel samples. After removal from the mold, the sample was rapidly and carefully dried and then weighed to obtain the fresh-made sample mass m0. After swelling in PBS with 0.02% NaN3 for at least 40 min at a specific temperature, the sample was taken out 51 from the buffer, dried and weighed again to obtain swollen sample mass ms. The swelling ratio was calculated by (ms-m0)/m0 x 100%. Tensile Tests. Tensile experiments were performed using an Instron-5500R tensometer with a customized force gauge. The method has been described previously42. The sample was kept in PBS buffer on the tensometer (achieved by a water-circulation temperature controller) for at least 40 min prior to and during the tensile test, consistent with the condition of swelling ratio measurements to guarantee swelling equilibration. During the experiment, the ring-shaped sample was fixed at one end and stretched by a hook connected with the force gauge at 10 mm/min rate to ~30% strain. The tensile stress was calculated by the recorded force divided by the cross-section area of sample under force. The tensile strain was calculated by (L-Linit)/Linit where L is the recorded extension and Linit is the sample’s length without tension. The slope at 15% strain on the obtained stress-strain curve was calculated as Young’s modulus at 15% strain. Statistical Analysis. Paired samples student t-tests were carried out in Microsoft Excel. When P-value is larger than 0.05, the difference is deemed statistically not significant. 2.4 Results and Discussion 2.4.1 Protein Engineering Using an iterative cloning strategy, we constructed the genes encoding Vn-(GR)4, where n=24, 48 and 72, in a modified pET25 vector, which encodes a C-terminal His-tag. The proteins were overexpressed in E. coli strain BL21(DE3). Since the Vn-(GR)4 proteins carry a C-terminal His6x-Tag, the overexpressed proteins can be purified readily using Co2+-affinity chromatography. Figure 2.1B shows the Coomassie blue stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) photograph of the purified proteins. The yield 52 of the proteins was 30-60 mg per liter of cell culture with a purity greater than 95%. The solubility of Vn-(GR)4 in aqueous solution at 25 °C was at least 1.0 mM (33 to 63 mg/mL) for V48-(GR)4 and V72-(GR)4, and 4 mM for V24-(GR)4, significantly higher than that of the corresponding Vn-ELP polymers (the saturation solubility is < 0.6 mM for both V48-ELP and V72-ELP, and < 2 mM for V24-ELP). Figure. 2.1. Protein block copolymers Vn-(GR)4 exhibit temperature-triggered phase behaviours in aqueous media. A) Schematic of block co-polymers Vn-(GR)4s. B) Coomassie blue stained SDS-PAGE of engineered Vn-(GR)4 proteins. Lane 1: molecular weight marker; lane 2: (GR)4; lane 3: V24-(GR)4; lane 4: V48-(GR)4 and lane 5: V72-(GR)4. The theoretical molecular weight is 35.0 kDa for (GR)4, 43.7 kDa for V24-(GR)4, 53.6 kDa for V48-(GR)4, 63.4 kDa for V72-(GR)4. C) Temperature-dependent turbidity of Vn-ELPs, (GR)4 and Vn-(GR)4s at 0.1 mM. D) Temperature-dependent hydrodynamic radius (Rh) of (GR)4 and Vn-(GR)4s at 1.0 mM measured by DLS. E) Rh of V72-(GR)4 in a heating-cooling cycle between 25 and 50 °C at 1.0 mM measured by DLS. F) Proposed schematic of micellization of V48-(GR)4 and V72-(GR)4 in aqueous media. 53 2.4.2 Temperature-Dependent Phase Transitions of Vn-(GR)4 Proteins The temperature-dependent phase transitions of Vn-ELPs proteins were characterized in detail in previous studies238, 239, 241. To characterize the temperature dependent behaviors of Vn-(GR)4 proteins, we carried out turbidity tests (Figure 2.1 C). Similar to previous studies, the positive control samples (Vn-ELPs) showed the anticipated temperature-dependent transition,100 and the measured transition temperature Tt is similar to the predicted values (Table 2.1). However, Vn-(GR)4 proteins did not show the anticipated clouding transition at higher temperature (up to 55 °C). This result indicated that the fusion of (GR)4 protein to Vn-ELPs may result in a significant change of the temperature responsive behaviors. Since (GR)4 protein is hydrophilic and highly soluble in water, the fusion of (GR)4 protein to Vn-ELPs may render the resultant fusion proteins amphiphilic. To test this possibility, we carried out dynamic light scattering (DLS) experiments. The solubility of (GR)4 and Vn-(GR)4 was proven to be at least 1.0 mM (33 to 63 mg/mL) in aqueous solution at 25 °C, with a hydrodynamic radius (Rh) of 3.7-9.0 nm indicative of well-dissolved monomers108, 111 (Figure 2.1 D). As temperature increased from 25 to 55 °C, (GR)4 and V24-(GR)4 remained monomers and Rh remained largely unchanged. In contrast, Rh of V48-(GR)4 and V72-(GR)4 gradually increased from 6-9 nm to a plateau of 16-26 nm at 50 °C (Figure 2.1 D), suggesting that these two protein polymers formed nanometer-sized aggregates at elevated temperatures. Such a change in Rh was largely reversible with small hysteresis during repeated heating-cooling cycles (Figure 2.1 E), indicative of a phase transition induced by intermolecular interactions among molecules109. Considering that (GR)4 alone did not show any temperature-triggered aggregation behavior below 55 °C, the formation of V48/72-(GR)4 nanoparticles is likely due to the 54 aggregation among V48/72 blocks. In addition, the size of such nanoparticles was similar to the reported sizes of micelles formed by ELP-containing AB-block copolymers with comparable chain lengths108, 110, 118. Therefore, it is likely that the aggregation of V48-(GR)4 and V72-(GR)4 is due to the reversible micellization behaviours in aqueous media at higher temperatures (Figure 2.1 F). Below Tt, both Vn-ELP and (GR)4 blocks were well solvated and soluble as monomers in aqueous solution. Above Tt, micelles formed as the Vn-ELP blocks locally aggregated into the hydrophobic cores while the (GR)4 blocks were still solvated but dispositioned into the shells. These results clearly showed that V48-(GR)4 and V72-(GR)4 exhibited temperature-dependent phase transition behaviors. The result that V24-(GR)4 remained monomers at higher temperatures is probably a result of its higher hydrophilic-to-hydrophobic ratio111, 242. It is of note that the micellization temperature is higher than the predicted Tt of un-fused V48/72 (30 °C and 27 °C, respectively), This observation is largely consistent with the prediction of the transition temperature Tt of ELP-fused with a globular domain using the method proposed by Chikoti et al.243 Similar behaviors were also observed in the micellization of ELP-containing di-block polymers108. 2.4.3 Post-Gelation In Situ Transition of Vn Side Chains Alters Thermo-Responsive Properties of (GR)4+Vn-(GR)4-Hydrogels Having characterized the temperature dependent phase transition behaviors of Vn-(GR)4 proteins, we attempted to employ Vn-(GR)4 as building blocks to engineer thermo-responsive protein hydrogels whose mechanical properties can respond to temperature changes using the well-developed [Ru(bpy)3]2+-mediated photochemical cross-linking method. Our previous studies showed that the minimal gelation concentration of (GR)4 aqueous solution is ~50 mg/mL 55 (1.7 mM)42, significantly higher than the solubility of Vn-(GR)4 proteins in aqueous solution (~1 mM). To circumvent this technical issue, we engineered (GR)4 and Vn-(GR)4 composite hydrogels. Both (GR)4 and Vn-(GR)4 are well-dissolved in aqueous solution as monomers at room temperature and mixed well into a homogeneous aqueous solution (Figure 2.2 A). The mixture solutions of both proteins were then photo-chemically cross-linked into solid hydrogels in the presence of APS and [Ru(bpy)3]2+ under white light illumination. Tyrosines from (GR)4 can be cross-linked into di-tyrosine adducts when they are in close proximity, giving rise to chemical cross-linking points in the hydrogel network. The resultant composite protein hydrogels were denoted as (GR)4+Vn-(GR)4-hydrogels, where the tyrosine-free Vn-ELP blocks behaves as side chains of the (GR)4-based network (Figure 2.2 B). For comparison, hydrogels based on pure (GR)4 protein were also prepared. Since Vn-ELPs do not contain any tyrosine residue, Vn blocks should not participate in the cross-linking reaction and function as side chains of the network backbone formed by (GR)4 and (GR)4-blocks. Thus, the formation of the primary network in (GR)4+Vn-(GR)4-hydrogels is determined by the total concentration of equivalent (GR)4 species, or C(GR)4eq. To investigate the effect of temperature on the mechanical properties of (GR)4+Vn-(GR)4 protein hydrogels, we carried out tensile and swelling ratio tests of (GR)4+V72-(GR)4 hydrogels as well as (GR)4 hydrogels under 25 or 50 °C (Figure 2.3). In these experiments, we kept C(GR)4eq at 3 mM (3 mM (GR)4 or mixture of 2 mM (GR)4 + 1 mM V72-(GR)4). 56 Fig. 2.2. Proposed scheme of hydrogel preparation from (GR)4/Vn-(GR)4 monomer solutions, and in situ phase transition of Vn-ELP side chains in the resultant hydrogels. (A) (GR)4 and Vn-(GR)4 proteins can be mixed well into a uniform solution at a concentration sufficient for hydrogel preparation under room temperature. (B) At room temperature, un-fused (GR)4 and (GR)4 blocks of Vn-(GR)4 are co-cross-linked into a hydrogel network via di-Tyr reaction, while non-tyrosine containing Vn-ELP blocks of Vn-(GR)4 are dispositioned into the side chains. (C) When heated to higher temperatures, Vn-ELP side chains undergo reversible in situ aggregation, contributing secondary physical cross-links to the hydrogel. When the temperature increased from 25 to 50 °C, the (GR)4 hydrogel softened as evidenced from its stress-strain curves, and its Young’s modulus showed an obvious decrease (Figure 2.3 A), while the swelling ratio remained largely unchanged (Figure 2.3 B&C). These behaviors are common for synthetic polymer hydrogels233, 234, 244-246. Hydration of many polymers effectively lowers their glass transition temperature below room temperature247-249, rendering resultant hydrogels in rubber elastic region at ambient conditions whose Young’s modulus decreases with rising temperature. It was shown that the storage modulus of chemically cross-linked resilin-based hydrogels gradually decreased when the temperature increased from 2 to 70 °C249. However, (GR)4+V72-(GR)4-hydrogel showed drastically different behaviors. When the temperature increased from 25 to 50 °C, the stress-strain curves of (GR)4+V72-(GR)4-hydrogel clearly showed that the hydrogel stiffened, showing a clear increase in Young’s modulus (Figure 2.3 D). In addition, at 50 °C, the hydrogel de-swelled significantly, exhibiting a much smaller swelling ratio (SR) (SR decreased from -12% to -32% from 25 to 50 °C) (Figure 57 2.3 E). These temperature dependent responses for (GR)4+V72-(GR)4-hydrogels are largely reversible (Figure 2.3 F). Comparing the temperature dependent behaviors of (GR)4 and (GR)4+V72-(GR)4 hydrogels, it is clear that at 50 °C, additional cross-linking, which is reversible, occurred for (GR)4+V72-(GR)4 hydrogels, thereby compensating the softening of (GR)4 primary network and resulting in the enhancement of the Young’s modulus and deswelling of the (GR)4+V72-(GR)4 hydrogel. The additional cross-linking is likely due to the self-association of the V72-ELP chains in the primary hydrogel network at higher temperatures. It has been reported that ELP chains can undergo post-gelation phase transition in hydrogels, which led to reversible modulus reinforcement and/or de-swelling62, 105, 224. The similar behaviors observed in (GR)4+V72-(GR)4-hydrogels suggested that in situ phase transition among V72-ELP side chains has occurred in (GR)4+V72-(GR)4-hydrogels at 50 °C (Figure 2.2 C). In solution, V72-(GR)4 dissolves as monomers at 25 °C at a concentration of 1 mM and forms micelles when the temperature is higher than 40 °C (Figure 2.1 D) due to the self-association of ELP chains. In the hydrogels, similar behaviors occurred. From 25 to 50 °C, V72-ELP chains underwent aggregation due to hydrophobic effect to serve as additional physical cross-links, leading to the increased modulus. In addition, the aggregation of V72-ELP side chains involves the loss of hydration88, 91, resulting in a lower swelling ratio. The aggregation of V72-ELP side chains can be reversed upon cooling, leading to a largely reversible modulus reinforcement and de-swelling. 58 Fig. 2.3. The presence of V72-ELP side chains alter the thermo-responsive properties of the (GR)4-based hydrogels. A and D) Representative stress-strain curves of (GR)4 hydrogels (A) and (GR)4+V72-(GR)4 hydrogels (D) in one heating-cooling cycle between 25 and 50 °C. B and E) Photographs of (GR)4 hydrogels (B) and (GR)4+V72-(GR)4 hydrogels (E) at 25 and 50 °C. C and F) Young’s modulus E (n = 4) and swelling ratio SR (n = 6) of (GR)4 hydrogels (C) and (GR)4+V72-(GR)4 hydrogels (F) in 3 heating-cooling cycles between 25 and 50 °C. The volume changes caused by the swelling ratio changes have been considered when calculating the Young’s modulus. All (GR)4 hydrogels are prepared from 3 mM (GR)4 solution in PBS, and all (GR)4+V72-(GR)4 hydrogels are prepared from a mixture of 2 mM (GR)4 and 1 mM V72-(GR)4 in PBS. Data is presented as average  standard deviation (S.D.). The S. D. of SR of (GR)4+V72-(GR)4 hydrogels is smaller than that of (GR)4 hydrogels, implying that (GR)4+V72-(GR)4 hydrogels are more uniform. The changes of SR for (GR)4 hydrogels between 25 and 50 °C are not statistically significant, while the changes of E for (GR)4 hydrogels, E and SR for (GR)4+V72-(GR)4 hydrogels between 25 and 50 °C are statistically significant (p<0.05, student t-tests). 59 2.4.4 Thermo-Responsiveness of (GR)4+Vn-(GR)4-Hydrogels can be Regulated To better understand the thermo-responsive properties of (GR)4+Vn-(GR)4-hydrogels bestowed by post-gelation in situ phase transition of Vn-ELP side chains, we investigated the relationships of modulus reinforcement and de-swelling versus various experimental parameters. We first examined the effect of the length of Vn-ELP block in the GR+Vn-(GR)4 proteins, where n=0, 24, 48 and 72, while keeping the same C(GR)4eq (3 mM) and Vn-(GR)4 composition (1 mM) (Figure 2.4). The Young’s modulus E and swelling ratio SR of hydrogels with different Vn-ELP side chain lengths did not show significant differences at 25 °C (Figure 2.4 A&B). This behavior is expected, as all four protein polymers with different lengths of Vn-ELP blocks are well-solvated in the hydrogel and do not affect the properties of the primary (GR)4-based networks. On the contrary, the E and SR showed a dependence on Vn-ELP chain length at 50 °C (Figure 2.4 A&B), albeit the change in E and SR is relatively small. To better describe the effect of Vn-ELP chain length on the properties of protein hydrogels, we used two parameters, E50°C/E25°C, the ratio between E at 50 and 25 °C, and ∆SR25-50°C, the change of SR between 25 and 50 °C, to evaluate the thermo-responsiveness of (GR)4+Vn-(GR)4-hydrogels. When Vn-ELP block changes from V0 (un-fused (GR)4) to V72-ELP, E50 °C/E25 °C showed a clear increase (from 0.71 to 1.16) while ∆SR25-50 °C decreased from 0 to -21% (Figure 2.4 C). These trends are consistent with previous findings that longer ELP side chains form larger aggregates and repulse more water in hydrogels88, 91. It is of note that although V24-(GR)4 cannot micellize or aggregate in solution at 50 °C, (GR)4+V24-(GR)4-hydrogels exhibited higher E50 °C/E25 °C than (GR)4 hydrogel (P<0.05). The local aggregation of V24-ELP chains in the 60 hydrogel network at 50 °C might be responsible for this difference, as V24-ELP can aggregate in solution on its own. Fig. 2.4. Thermo-responsive properties of (GR)4+Vn-(GR)4s are regulated by Vn side chain length. (A-B) Young’s modulus E (n = 4) (A) and swelling ratio SR (n = 6) (B) of (GR)4- or (GR)4+Vn-(GR)4-hydrogels (n = 0, 24, 48, 72) measured at 25 °C and 50 °C. (C) Relationships of E50°C/E25°C and ∆SR25-50°C versus side chain length n. The (GR)4-hydrogels are prepared from 3 mM (GR)4 solution in PBS, and all (GR)4+Vn-(GR)4-hydrogels are prepared from a mixture of 2 mM (GR)4 and 1 mM Vn-(GR)4 in PBS. The data from the first heating-cooling cycles was used for the comparison. We then investigated the effect of the ratio between (GR)4 and V72-(GR)4 on the properties of (GR)4+V72-(GR)4 composite hydrogels. V72-(GR)4 was fixed at 1 mM near its 61 saturation concentration, and the concentration of (GR)4 was varied. As V72-(GR)4 composition increased from 25% to 50 %, E50 °C/E25 °C was raised from 1.04 to 1.98, and ∆SR25-50 °C decreased from -15% to -28% (Figure 2.5). Such trends are largely anticipated, as a higher V72-ELP composition facilitates a larger scale in situ aggregation within the hydrogels62, 105. Figure 2.5. Thermo-responsive properties of (GR)4+Vn-(GR)4 hydrogels are regulated by composition Young’s modulus (n ≥ 3) and swelling ratio (n ≥ 6) comparison of (GR)4 and (GR)4 +V72-(GR)4-hydrogels with different compositions. All data for comparison are collected in the first heating-cooling cycles. Moreover, we also investigated the influence of cross-linking density on hydrogels’ mechanical and swelling properties. By lowering APS concentration from 50 mM to 25 mM, the cross-linking density of the resultant hydrogel was lower, leading to the decreased Young’s 62 modulus of (GR)4 and (GR)4+V72-(GR)4 hydrogels at both 25 and 50 °C. However, E50 °C/E25 °C and ∆SR25-50 °C was largely unaffected by reduced cross-linking density (Figure 2.6). Figure 2.6. Thermo-responsive properties of (GR)4+Vn-(GR)4 hydrogels with different cross-linking density Young’s modulus (n ≥ 3) and swelling ratio (n ≥ 6) comparison of (GR)4 and (GR)4+V72-(GR)4-hydrogels prepared with different APS concentrations. The (GR)4-hydrogels are prepared from 3 mM (GR)4 solution in PBS, and all (GR)4+V72-(GR)4-hydrogels are prepared from a mixture of 2 mM (GR)4 and 1 mM V72-(GR)4 in PBS. All data for comparison are collected in the first heating-cooling cycles. These results indicated that the thermo-responsive properties of (GR)4+Vn-(GR)4 hydrogels can be tuned by multiple parameters. The mechanical properties of the hydrogels at lower temperatures, with solvated Vn-ELP side chains, are more related to (GR)4-based networks and mainly determined by C(GR)4eq and cross-linking density. In comparison, the modulus and swelling ratio responses at higher temperatures (E50 °C/E25 °C and ∆SR25-50 °C) can be regulated via 63 the aggregation behaviors of Vn-ELP chains, which are dependent on the phase behaviour of Vn-ELP controlled by the chain length and the relative concentration. 2.4.5 Pre-Gelation Phase Transition of Vn-ELP Chains Compromises Thermo-Responsiveness of (GR)4+Vn-(GR)4-Hydrogels Having demonstrated that post-gelation in situ phase transition of Vn-ELP side chains can regulate thermo-responsive properties of (GR)4+Vn-(GR)4-hydrogels, we further investigated if pre-gelation phase transition can lead to similar thermo-responsiveness of (GR)4+Vn-(GR)4 composite hydrogels. Instead of using aforementioned method to prepare hydrogels from Vn-(GR)4 monomers (Figure 2.2), we prepared (GR)4+Vn-(GR)4 composite hydrogels based on Vn-(GR)4 micelles. The mixture solution of (GR)4 and V72-(GR)4 (Figure 2.7 A) was first heated to 50 °C to allow micellization of V72-(GR)4 (Figure 2.7 B), and then photochemical cross-linking reaction was carried out at 50 °C to construct protein hydrogels (Figure 2.7 C). Then the mechanical and swelling properties of the resultant hydrogels were measured at both 25 and 50 °C. Our results showed that the changes for both E and SR for micelle-based (GR)4+V72-(GR)4-hydrogels are much smaller than those in monomer-based hydrogels (Figure 2.7 D). The E50 °C/E25 °C of micelle-based hydrogels was 0.94 compared to 1.16 of monomer-based hydrogels, and the ΔSR25-50 °C was -6% compared to -21%, respectively (Figure 2.7 E). One possible caveat for micelle-based hydrogels is that the cross-linking may lead to the partial cross-linking of the micelle corona, which is made of (GR)4 blocks, and may in turn prevent the complete dissociation of the micelle in the hydrogel and attenuates the effect of thermo-response of Vn-ELP block on the properties of the hydrogel. Nonetheless, the possibility of constructing protein hydrogels using both monomer and micelles may provide further room 64 for tuning the mechanical properties of protein hydrogels. It is also worth noting that the hydrogels prepared here using cross-linking after phase transition of Vn-ELPs are chemically cross-linking hydrogels, and are different from the ELP hydrogels reported in the literature118, 224, 250, 251 that were prepared above the transition temperature via ELP aggregation. These hydrogels are physically cross-linked gels and most of them undergo gel-sol transition when the temperature is below the transition temperature. Fig. 2.7. (A-C) Proposed scheme of hydrogel preparation from Vn-(GR)4 micelle suspensions. (A) (GR)4 and Vn-(GR)4 mixture solution is heated to induce micellization of Vn-(GR)4 (B). At this high temperature, (GR)4 monomers and Vn-(GR)4 micelles are co-cross-linked into a hydrogel network via the photochemical cross-linking reaction (C). (D) Young’s modulus E (n = 3) and swelling ratio SR (n = 6) of micelle-based (GR)4+V72-(GR)4 hydrogels in 3 heating-cooling cycles between 25 and 50 °C. All samples are prepared from a mixture of 2 mM (GR)4 and 1 mM V72-(GR)4 in PBS. The difference of E between 25 and 50 °C is statistically not significant, but the different of SR is (P<0.001). (E) The comparison of E50°C/E25°C and ∆SR25-50°C between monomer-based and micelle-based (GR)4+V72-(GR)4-hydrogels. 65 2.5 Conclusions By genetically fusing the thermo-responsive Vn-ELPs that shows LCST behaviours to highly soluble (GR)4 elastomeric proteins, we successfully engineered Vn-(GR)4 block protein co-polymers. The engineered Vn-(GR)4 protein polymers showed much improved solubility in aqueous media over Vn-ELP polymers, and displayed temperature-dependent micellization. By using photochemical cross-linking strategies, we constructed (GR)4+Vn-(GR)4 hybrid hydrogels. The Young’s modulus and swelling ratio of the resultant hydrogels showed temperature dependent changes, which result from the temperature-dependent aggregation of the Vn-ELP side chains in the hydrogel network. We anticipate that the approach we demonstrated here can be applied to confer thermo-responsiveness to other elastomeric protein-based hydrogels. Further fine-tuning of the thermo-responsiveness of such elastomeric proteins will expand the toolbox to engineer dynamic protein hydrogels, and opens the possibility for these hydrogels to various biomedical applications, such as tissue engineering, drug and cell delivery and mechanobiological applications. 66 Chapter 3: An Injectable Self-Healing Protein Hydrogel with Multiple Dissipation Modes and Tunable Dynamic Response 3.1 Synopsis Hydrogels with dynamic mechanical properties are of special interest in the field of tissue engineering and drug delivery. However, it remains challenging to tailor the dynamic mechanical response of hydrogels to simultaneously meet diverse application needs. Here, we report a hetero-coiled-coil complex cross-linked protein hydrogel exhibiting unusual multiple energy dissipation modes and tunable dynamic response. Such unique features confer the hydrogel responsive to mechanical stimuli in a broad range of frequencies. Therefore, the hydrogels are injectable due to their shearing–thinning properties at low shear rates of 0.8 rad s-1 and can fully recover their mechanical properties within a few seconds due to the intrinsic fast dynamics of the cross-linkers. Moreover, the dynamic response of these hydrogels can be fine-tuned by the temperature and the hydrogel network structures. We anticipate that these hydrogels are promising candidates for delivering therapeutic drugs, biological molecules and cells in a broad spectrum of biomedical applications. *A version of Chapter 3 has been published as Wenxu Sun, [Tianyu Duan], Yi Cao, and Hongbin Li. (2019) An Injectable Self-Healing Protein Hydrogel with Multiple Dissipation Modes and Tunable Dynamic Response. Biomacromolecules. 20: 4199-4207. This chapter is incorporated in this thesis with permission of American Chemical Society, Copyright 2019. 67 3.2 Introduction Injectable hydrogels have received considerable interests in the field of biomedical engineering due to their wide range of potential applications, ranging from tissue engineering252-254, cell encapsulation255, 256, immune modulation257 to drug delivery258, 259. Particularly, in tissue repair process, the use of injectable hydrogel scaffolds can minimize invasive surgical procedures. Therapeutic drugs, bioactive molecules, such as peptides and proteins, and even live cells can be incorporated into the hydrogel prior to its injection9, 10. After being injected to the target site through a syringe, these mixtures can undergo sol–gel transition quickly to form a hydrogel in situ. In this way, the reformed hydrogels can fill the cavity and be integrated with the surrounding tissues. Injectable hydrogels can be either cross-linked in situ by chemical reactions or through self-assembly driven by non-covalent interactions260, 261. In the latter case, the hydrogels exhibit shear-thinning properties and can become viscous liquid under shear stress (during the injection process), and can quickly recover the hydrogel state after the shear stress is removed. Many self-assembling systems, including colloids262, 263, short peptides264-266, metal-ligand interaction267-270, recombinant proteins23, 153, 154, polymers-nanoparticle systems271-273 and polymer containing host-guest interactions274-279, are found to possess the shear-thinning properties and have been explored as injectable hydrogels. Ideally, the gel-sol transition should take place under moderate shear stress to minimize the negative effect of shearing force on the therapeutics, especially proteins and cells. Moreover, the hydrogels should be able to rapidly rebuild the mechanical properties to avoid the precipitation or leakage of the enclosed drugs. The injectability and self–healing properties of hydrogels are largely determined by the dynamic response of hydrogels at different time scales261, 280, 281. Take syringe–based injection as an example, a central wide plug flow region which is able to ensure the gel materials and cell 68 payloads experience little or no shear rate is essential to keep the cells survival after injection.282 However, a shear zone close to the shear wall where gel and cells were subject to shear deformation is inevitable. Therefore, in order to narrow down the shear zone close to the wall and increase the plug flow region, the hydrogels should be able to undergo shear-thinning at low strain rates. Besides, the amplitude of the viscosity decrease must be large enough to ensure high fluidity during the injection. On the other hand, the hydrogels must be able to quickly recover their mechanical and physical properties after injection283-285. This property requires that the interactions involved in the physical cross-linking in the hydrogels exhibit fast dynamics and can recover quickly. To meet these two requirements, the hydrogels need to be responsive to mechanical forces at both low and high frequencies, which remain a challenge for the design of injectable hydrogels. Previously, protein hydrogels cross-linked by homo-coiled-coil complexes were found to show sequence-dependent intrinsic dynamics over a broad range of time scales138-142. Compared with homo-coiled-coils, hetero-coiled-coils have more structural parameters that could potentially affect their stability and conformational dynamics, and influence the intrinsic dynamic response at different time scales of the hydrogels constructed from such hetero-coiled-coils129, 131, 286, 287. Inspired by the pioneering work on the dynamic response of homo-coiled-coils-based hydrogels, here we investigate the dynamic response of a hetero-coiled-coils-based protein hydrogel, and explore the feasibility to establish a novel injectable protein hydrogel system. For this, we utilized hetero-coiled-coils, CCE and CCK, as a model system. CCE/CCK are coiled-coil domains designed by Kopecek and coworkers (Figure 3.1 a)133. In the heptad repeats of the coiled coils, most of the e and g positions are occupied by glutamic acid in CCE and by lysine in CCK (Figure 3.1 b), respectively. Due to electrostatic repulsion, CCE and CCK 69 predominantly form an antiparallel heterodimer. CCE and CCK were used as a means for physical cross-linking to engineer polymer hydrogels.133 We also employed CCE/CCK to develop two-component protein hydrogels.137 Using this hydrogel system and oscillatory rheology, here we showed that this coiled-coil complex CCE/CCK-based hydrogel exhibits two distinct dissipation modes. Moreover, the dynamic response of the resulting hydrogels can be fine-tuned by temperature, chemical environment and network structures of the hydrogels. We also demonstrate the shear-thinning of the hydrogels at low strain rates and their rapid and reversible self-healing properties after shearing. We anticipate that these hydrogels can be used as ideal candidates for injectable drug, gene and cell delivery systems. 3.3 Materials and Methods Protein Engineering. The engineering of CCE-(GB1)4-CCE (denoted as GE protein) and CCK-(GB1)5-CCK-(GB1)5-CCK (denoted as GK protein) were detailed previously.137 Both proteins were expressed in DH5α strain, purified by Co2+ affinity chromatography and eluted by elution buffer (containing 10 mM PBS, 300 mM NaCl, 250 mM imidazole, pH 7.4). The purified protein solution was dialyzed against water for 9 h (water was changed every 3 h) and then lyophilized. The yield of proteins GE and GK was around 100 mg and 50 mg from 1 L of cell culture, respectively. The purity of the proteins was over 95% as estimated from SDS-PAGE Hydrogel Preparation. To prepare the protein hydrogel, lyophilized GE and GK proteins were dissolved in PBS (1X, pH=7.4) separately to obtain pre-gel protein solutions at desired concentrations. The two solutions were then mixed at a desired CCK: CCE stoichiometry to form a hydrogel. The gelation was fast, as the mixture lost fluidity within 2 min after mixing. 70 The hydrogel was then centrifuged at 2500 rpm for 20 min to remove trapped air bubbles and flatten the sample surface. Rheology Measurement. All rheology measurements were performed on a TA Instruments Discovery HR-2 rheometer. The sample was loaded and tested in the testing gap between a 20-mm diameter flat plate and a bottom flat plate that are parallel to each other. The gap was kept as 50 µm during measurements. The hydrogels were carefully transferred to the temperature-controlled rheology plate. During the measurement, silicone oil was used to cover the gap in contact with air to avoid evaporation. The storage modulus G’ and the loss modulus G’’ were measured using oscillatory shear test. The two parallel plates rotated back and forth to apply an oscillatory shear to the sample. For frequency-dependent measurements: The angular frequency of the oscillatory shear ranged from 0.01 to 100 rad·s-1, while the temperature was precisely controlled at a certain value and the maximal oscillation strain was kept constant at 1%. The relaxation spectrum H(τ) was calculated by fitting the G’/G’’-frequency curves using the rheometer controller software provided by TA Instruments. For temperature-dependent measurements, the temperature ranged from 20 to 59 °C, while the maximal oscillation strain was constantly 1% and the angular frequency was constantly 10 rad s-1. For strain-dependent measurements, the strain ranged from 0.1% to 1000%, while the temperature was fixed at 20 C, and the oscillation frequency was fixed at 10 rad s-1. For self-healing measurements, the temperature was precisely controlled at 20 C, and the oscillation frequency was 10 rad s-1. The strain was set to an amplitude of 300% to disrupt the coiled-coil complexes, then switched back to an amplitude of 1% to monitor the recovery of the mechanical properties. To monitor the gelation process using rheology, a flat plate of 8-mm diameter was used and the gap was kept as 400 µm during measurements. The temperature was precisely 71 controlled at 20 C, the oscillation frequency was 10 rad s-1, and the strain was set to an amplitude of 1%. The measurement time was 600 s. Before collecting the modulus data, suitable amount of GK and GE solution were dropped on the measurement platform and the flat plate, respectively. Then the platform and the plate were approached together. The two components were mixed uniformly by rotating the flat plate. The viscosity was measured in the flow test mode (steady measurements) where the two parallel plates continuously rotated in one direction to apply a steady shear rate. During measurements, the temperature was controlled at 20 C. The shear rate range was 0.1 s-1 to 100 s-1. Points rate per decade is 15. The equilibration time and averaging time is 5 s and 20 s respectively. 3.4 Results and Discussion 3.4.1 Design of the Hydrogel CCE and CCK are two complementary leucine zipper coiled-coil domains, and carry opposite charges at neutral pH: CCE is negatively charged, whereas CCK is positively charged (Figure 3.1 a).133 Because of the electrical repulsion, CCE or CCK cannot form homo-coiled-coil complex. Instead, they can only form anti-parallel hetero-coiled-coil complex by dimerizing with each other (Figure 3.1 b). The two-component hydrogel system we designed to construct the hydrogel is based on two multi-functional elastomeric proteins carrying CCE and CCK functional groups, respectively: CCE-(GB1)4-CCE (denoted as GE) and CCK-(GB1)5-CCK-(GB1)5-CCK (denoted as GK). In these constructs, (GB1)n are tandem modular proteins containing four or five repeats of GB1 and serve as the backbone of the hydrogel network (Figure 3.1 c). By simply mixing the bifunctional GE solution with the trifunctional GK protein 72 solution together at desirable concentrations and molar ratios, the protein hydrogels (denoted at GKE hydrogel) can be prepared readily (Figure 3.1 c). The gelation processes of GKE hydrogels at different protein concentrations were investigated using rheological characterization. The pre-gel solution at 16% concentration was too viscous to be evenly mixed on the rheometer. So we simply excluded this condition in the gelation measurement. However, the gelation process of 16% concentration gels should be completed in a few seconds according to the gelation experiments in a centrifuge tube. When the concentration of protein was larger than 7%, the gelation time became less than 10 s, which is the detection limit of our method (Figure 3.2 a). Such short gelation time was attributed to the intrinsically fast dynamics of the CCE/CCK complexes. The stiffness of the gels also increased with the increase of the protein concentration (Figure 3.2 b). Due to the physical nature of the CCE/CCK mediated cross-linking and the intrinsic binding-unbinding dynamics, we anticipate that such GKE hydrogels can exhibit interesting dynamic responses in response to shearing. 73 Figure 3.1. The design of the GKE hydrogels. a) Amino acid sequence of the reference CCK and CCE polypeptides. b) Structural model (cartoon model) of the antiparallel α-helical coiled coils complex formed by the dimerization of CCK and CCE. The left diagram is the side view and the right diagram is the helical wheel (top view) of the coiled-coil. In the heptad repeat of coiled coils, the e and g positions are occupied by glutamic acid in CCE, and by lysine in CCK; and positions a and d are occupied by hydrophobic residues and form the hydrophobic core of the coiled-coil. c) Schematic diagram of the gelation process and the network of the GKE gel. Hydrogels are cross-linked by the CCE/CCK coiled-coil complex which self-associated from CCE and CCK sequence (blue and green helices). 74 Figure 3.2. Gelation time and stiffness of the GKE gels at different protein concentrations. (a) The detection limit of our method is 10 s. When the protein concentrations were larger than 7%, the gelation time cannot be detected accurately by this method. (b) Storage moduli of GKE gels containing different concentrations of proteins measured at 20 C (oscillation strain 1%, angular frequency 10 rad s-1). Data were shown as average ± S.D. (n=3). 3.4.2 GKE Hydrogels Display Time-Dependent Dynamic Properties To investigate the dynamic properties of the GKE hydrogels, we used oscillatory shear rheology to measure the storage (G’) and loss (G’’) viscoelastic modulus of the protein hydrogels at different oscillatory frequencies. As shown in Figure 3.3, the measured G’ and G’’ of the GKE hydrogel were observed to be highly dependent on the oscillatory frequency, due to the noncovalent, dynamic nature of the CCE/CCK complex. At a particular frequency ωc, G’ and G” were observed to crossover with each other, where G’(ω) = G’’(ω). The reciprocal of ωc is approximately equivalent to the material’s characteristic relaxation time, τc. τc reflects the dynamic response of a soft material under active mechanical loading, and is determined by the molecular relaxation mechanisms of its cross-linking interactions at a molecular level269, 288-290. From Figure 3.3 a & b, it can be seen that when ω is smaller than 1/τc, the GKE hydrogel showed the behaviors of a viscous fluid whose G’(ω) < G’’(ω), and the hydrogel dissipated more energy than it stored. Under this condition, the cross-linking points, which are mediated by the 75 CCE/CCK complex, ruptured at the timescale of deformation, leading to the dissociation of the hydrogel network. However, when the frequency is above 1/τc, the GKE hydrogel showed an elastic solid-like material behavior where G’(ω)>G’’(ω), and the material stored more energy than it dissipated. These properties of the GKE hydrogel are consistent with the behavior of a physically cross-linked network, and demonstrated the dynamic response of the GKE hydrogel network. Figure 3.3. Time-dependent rheological properties of the CCE/CCK gels. (a) and (b) Time-dependent rheological properties of the GKE hydrogel measured by rheology (storage modulus G’ and loss modulus G” as a function of frequency ω) at different temperature. (a) and (b) were measured at 20 C and 37 C, respectively. (c) and (d) The log-normal relaxation spectrum calculated by graph a and c describes the measured dynamic properties of the CCE/CCK coiled-coil complex hydrogel. Two dissipation modes were observed under each temperature (dashed lines are double log-normal fitting results). 76 To confirm the critical importance of the CCE/CCK complex in the gelation process, we set up two control groups which only contained a single component (GK protein or GE protein) at the same concentration of GKE hydrogel (7%). In both cases, due to the absence of cross-linking, they behaved as viscous liquid. 3.4.3 GKE Hydrogels Display Two Dissipation Modes Oscillatory shear rheology results can provide insights into the stress relaxation mechanism of the hydrogels. When the frequencies are below 1/τc, the viscoelastic moduli G’ and G” of hydrogels are expected to follow the scaling relations for terminal relaxation: G’(ω)~ ω2 and G’’(ω)~ ω1. Indeed, we observed this scaling relationship for the GKE hydrogels. At 20 C, we measured a scaling factor of 1.78 and 0.92 for G’ and G”, respectively, indicating that GKE hydrogels likely follow the terminal relaxation model that G’ and G’’ scale 2 and 1, respectively, with the frequency (Figure 3.3 a). Similar behaviors were also observed to 37 C, where the scaling factor was 1.88 and 0.92 for G’ and G’’, respectively (Figure 3.3 b). Compared with the spectrum at 20 C, it is of note that the critical frequencies (1/τc) of the GKE hydrogel shifted to higher frequencies, suggesting that the CCE/CCK complex became more dynamic at 37 C. The characteristic relaxation time τc provides a rough picture of the GKE hydrogel’s temporal structure. To better characterize the dynamics of the hydrogels, it is possible to quantify the full distribution of the relaxation times, i. e. the relaxation spectrum H(τ)291 by solving the following integral equations: G′(ω) = ∫ 𝐻(𝜏)𝜔2𝜏21+𝜔2𝜏2∞−∞𝑑𝑙𝑛(𝜏) (3.1) 77 G′′(ω) = ∫ 𝐻(𝜏)𝜔𝜏1+𝜔2𝜏2∞−∞ 𝑑𝑙𝑛(𝜏) (3.2) H(τ), with the unit of pascal, can be comprehended as an infinite number of Maxwell elements that are arranged in parallel269. A Maxwell element is an elastic spring and a viscous damper connected in series, which has a characteristic relaxation time. For an infinite number of Maxwell elements arranged in parallel, the relaxation does not occur at a single time but in a set of times. H(τ) is equivalent to the ensemble amount of energy dissipated under dynamic loading/unloading at a given time scale τ, or in other words, a given frequency 1/τ292. Figure 3.3 c shows the H(τ) calculated from the data in Figure 3.3 a. It is interesting to note that H(τ) showed a bimodal distribution. To quantitatively investigate the dissipation mechanism, we use multi-peak log-normal fitting to describe their relaxation behaviors. As shown in Figure 3.3 c, the H(τ) can be fitted by two-mode forms (dashed lines), where each mode is distributed log-normally with a peak-height A and a center frequency ω0. The relaxation mode with lower frequency (larger relaxation time) was denoted as the ‘slow’ mode, on the contrary, that with higher frequency (smaller relaxation time) was denoted as the ‘fast’ mode. The bimodal distribution of H(τ) suggested that the GKE hydrogels have two distinct dissipation modes at 20 C (i. e. two different dissociation modes for the CCE/CCK complex), with a relaxation frequency 1/τ of 0.2 s-1 and 1 s-1 for the slow and fast modes, respectively. In addition, the slow mode has a much higher amplitude that the fast mode. Moreover, when measured at 37 C, the relaxation spectrum H(τ) showed a similar bimodal distribution, revealing two distinct dissipation modes. In the relaxation spectra, the two relaxation modes both shifted to higher frequencies. In addition, the peak-height of the ‘slow’ mode became lower than that at 20 C, whereas the peak-height of the ‘fast’ mode became 78 higher than that at 20 C (Figure 3.3 d). With the increase of temperature, the response of the two relaxation modes was different, indicating that two relaxation modes corresponded to different relaxation mechanisms. 3.4.4 Environmental Factors (Temperature, Ionic Strength and Stoichiometry) Affect the Dissipation Modes of the GKE Hydrogels To further explore the dissipation mechanism of the GKE hydrogels in more detail, we carried frequency sweep experiments at different temperatures. Following the similar method, we obtained the relaxation spectra on a series of GKE gels. As shown in Figure 3.4 a, increasing temperature led to the increase of the critical frequency, i.e. faster energy dissipation. At each temperature, the relaxation spectrum can be fitted by bimodal log-normal equation at the time scales of interest. The peak position shifted towards higher frequencies upon increasing of the temperature, suggesting faster dissipation rates at higher temperature (Figure 3.4 b). However, the amplitude of the two modes varied dramatically at different temperature. when the temperature was below 30 C, the decrease of the ‘slow’ mode amplitude correlated with the increase the ‘fast’ mode amplitude, suggestive of the inter-conversion of the two dissipation modes. However, when the temperature is higher than 30 C, the trend was reversed: With the increase of temperature, the ‘slow’ mode amplitude increased along with the decrease of the ‘fast’ mode amplitude (Figure 3.4 c). The switch of the temperature-dependency is directly related to the melting of the CCE/CCK complexes. It is noteworthy that a third peak appears at 100-1000 rad·s-1 at 10 and 15 °C. Above 20 °C, this third peak appears to shift to higher frequency (beyond 1000 rad·s-1) (Figure 3.4 a). The 79 origin of this dissipation mode remains unclear, which might be attributed to polymer chain vibrations that have much short relaxation time compared to the coiled coil interactions. Figure 3.4. Dissipation mechanism of GKE gels. (a) Two modes log-normal relaxation spectra H(τ) of the GKE hydrogel at different temperatures. Dashed lines are double log-normal fitting results. Asterisk (*) mark represents the slow dissipation mode and the hash (#) mark represents the fast dissipation mode. (b) Peak center of two dissipation modes of GKE gels under different measuring temperature. Dashed lines are log fitting results. Data were shown as average ± S.D. (n=4) (c) Peak height of the two dissipation modes of GKE gels at different temperatures. Data were shown as average ± S.D. (n=4) Next, we studied the influence of ionic strength on the dynamics of the GKE hydrogels. Increasing ionic strength can lead to the screening of charge-charge interactions and affect the dynamics of the CCE/CCK complexes. Additional NaCl was added to the 1× PBS buffer (which contains 137 mM NaCl) to increase the ionic strength. The time-dependent dynamic properties of the GKE gels were measured by standard frequency sweep experiments (Figure 3.5 a-c). The 80 critical frequencies (ωc) decreased with the increasing of ionic strength, suggesting that the CCE/CCK complexes become less dynamic, which is consistent with the expectation that the increased ionic strength enhances the thermal stability of CCE/CCK coiled-coils. The relaxation spectra were further calculated to study the dissipation mechanism in the gels (Figure 3.5 d). The two dissipation modes coexisted at different temperatures except that the characteristic frequency shifted to lower frequencies. The relaxation spectra were fitted by two log-normal distributions. Then the peak location and peak height were calculated to quantitatively measure the change of each relaxation spectra. Though the temperature where the transition occurred was a bit higher at 1000 mM NaCl (Figure 3.5 e) than that at 137 mM NaCl (Figure 3.4 c), the overall trend was the same. These experiments suggested that the ionic interactions played the same roles in both the ‘fast’ and the ‘slow’ modes. As electrostatic interactions are strongly dependent on distance, we can infer that the interactions involved in CCE/CCK complexes in the two dissipation modes are very similar. 81 Figure 3.5. Viscoelastic properties and relaxation spectra of GKE gels at different ionic strength. (a-c) Time-dependent rheological properties of GKE hydrogels measured at 137 mM NaCl (a), 500 mM NaCl (b) and 100 mM NaCl (c). All the measurements were carried out at 20 °C. The total protein concentration is fixed at 7% in all hydrogels and the molar ratio of CCK to CCE is fixed on 1: 1. (d) Relaxation spectra of GKE hydrogels at 1000 mM NaCl and different temperatures. Dashed line are double log-normal fitting results. Asterisk (*) mark represents the slow dissipation mode and the hash (#) mark represents the fast mode. (e) temperature-dependent peak height of the two dissipation modes of the GKE hydrogel at 1000 mM NaCl. Data are shown as average ± S.D. To further confirm that the network structure played an important role in the dynamics of the hydrogels, we measured the critical frequency (ωc) of the hydrogels at different protein concentrations at different temperatures (Figure 3.6 a). There existed a plateau between 30 C to 40 C in the ωc(T)-T curve at each protein concentration, and this trend was not affected by 82 protein concentrations. In contrast, ωc at each temperature was strongly dependent on the protein concentrations and consistently down-shifted upon increasing protein concentrations. Figure 3.6. Dynamic properties of GKE hydrogels at different concentration and CCK: CCE stoichiometry. (a) The critical frequency (ωc, the inverse of the relaxation time) of the CCE/CCK coiled-coil complex cross-linked hydrogel depends on the concentration of GK and GE polyprotein and temperature. Here, the molar ratio of CCE to CCK sequence is fixed on 1:1. Data were shown as average ± S.D. (n=3). (b) The critical frequency (ωc, the inverse of the relaxation time) of the CCE/CCK coiled-coil complex cross-linked hydrogel depends on the molar ratio of CCE to CCK sequence. Here, the concentration of GK and GE polyproteins is fixed on 7%. Data were shown as average ± S.D. (n=3). The network structure can also be modulated by the relative ratios between CCE and CCK. Figure 3.6 b shows the critical frequency (ωc) of the GKE hydrogels at different CCK: CCE molar ratios. When the CCK: CCE molar ratios were higher than one, the hydrogels 83 became weaker and more dynamic. On the contrary, if the molar ratios were lower than one, the dynamic response of the hydrogels became much slower. This result suggested that the internal cross-linking site versus terminal ones may play different roles in the dynamic response of GKE hydrogels, given that fact the CCE and CCK are largely disordered when they are not complexed. We noticed that, with the change of the molar ratio, the trend of ωc versus temperature has undergone great changes (Figure 3.6 b). This may be attributed to the defects in the structure of hydrogel network caused by the free dangling cross-linking groups (CCE or CCK). The appearance of the two relaxation modes in the GKE protein hydrogels is intriguing. Similar multi-mode dissipation was reported recently in a protein-based hydrogel, which was constructed from homo-coiled coils293. The multimode dissipation was attributed to the different types of molecular association, i.e. aggregation, dimerization and tetramerization.291, 294 However, this mechanism is likely not the origin of two dissipation modes in our system. Although the detailed mechanism underlying this behavior remains unclear, we noted that the temperature range for transition regime of both modes of dissipation seems to coincide with the melting transition regime of the CCE/CCK complex (which is around 38 C in isolation). The melting of isolated CCE/CCK coiled coils is a cooperative transition. However, when attached to polymers or fused in the middle of proteins, it has been shown that the cooperativity of the melting transition is reduced and the melting transition occurs in a broader range of temperatures133, 137. The two modes of dissipation may be directly related to this less cooperative transition of CCE/CCK in the protein hydrogels, as well as the position of the CCE/CCK (terminal versus internal) in the protein polymer backbone. Detailed investigations will be needed to fully elucidate the origin of the multi-mode dissipation in the GKE hydrogels. 84 3.4.5 Injection and Self-Healing of the GKE Hydrogels Having measured the dynamic properties of the GKE hydrogels, we then investigated whether the broad and dual relaxation modes could confer the GKE hydrogels with injectability and self-healing properties. We first investigated the shear rate dependence of the viscosity of the GKE hydrogels. As shown in Figure 3.7 a, the viscosity of the GKE hydrogel exhibited a sharp decrease when the shear rate is higher than 0.8 s-1 at 20 C, resulting in the transition from a hydrogel to a viscous liquid. This is a clear indication of the shear thinning. Besides, the shear stress was decreased sharply as the shear-thinning occurred which indicates the strain-dependent gel-sol transition of the GKE gels. The quick gel-sol transition also gave rise to the plug flow during syringe injection. Similar shear thinning behaviors were also observed at different protein concentrations, despite that the storage moduli of the hydrogels were higher at higher protein concentrations (Figure 3.8). Notably, the GKE gels can be shear-thinned by two to three orders of magnitude in a relatively narrow shear rate range, which is comparable to some of the reported shear-thinning gel systems105, 295-297. The shear-thinning observed for GKE hydrogels at low strain rates made the GKE hydrogels injectable. We also carried out measurements to investigate the self-healing properties of GKE hydrogels (Figure 3.7 b). Under a shear amplitude of 300%, the GKE hydrogels were disrupted, leading to a sharp drop in G’. Then the shear strain was reduced quickly to 1% to monitor the recovery process of the hydrogel. In a few seconds, the disrupted hydrogels self-sealed and recovered its original storage modulus. Adjusting the protein concentration only has a small effect on the self-healing ability property of the GKE hydrogels. The rapid self-healing properties of GKE hydrogels are likely due to the inherently fast association kinetics of the 85 CCE/CCK complexes. Such properties give the GKE gels unique ability to prevent undesired leakage of the encapsulated payloads after injection.167, 298-300 The rapid self-healing qualities, together with excellent shear-thinning abilities, endow the GKE hydrogels injectable properties: As shown in Figure 3.7 c, the preformed GKE hydrogel was injected from a 24-gauge needle onto a clean culture dish at room temperature and the injected GKE solution immediately turned into a stable physical hydrogel. Figure 3.7. Injectability of the GKE Hydrogels. (a) Viscosity measurement on a 7% GKE gel (CCK: CCE molar ratio is 1:1) at 20 C. There is an obvious shear thinning region in the graph. (b) Fast recovery of 7% GKE gel observed in the consecutive destroy-recovery measurement. The strain was set to an amplitude of 300% to destroy the CCE/CCK coiled-coil complex and switched back to an amplitude of 1% to monitor recovery of the mechanical properties. The G’ and G’’ were measured at a frequency of 10 rad s-1 at 20 C. (c) Photographs to show the injection of a 7% GKE gel (molar ratio of CCE to CCK is 1:1) to a clean petri dish at room temperature. 86 Figure 3.8. Shear-thinning properties of the GKE hydrogels at different protein concentrations. All the hydrogels at different concentrations, ranging from 4% to 16 %, exhibited typical shear-thinning behaviors. The molar ratio of CCK to CCE was fixed at 1:1. The measurement temperature was 20 C. 3.5 Conclusions In summary, we have demonstrated an entirely protein-based hydrogel with multi-energy dissipation modes and tunable dynamic response using the heterodimeric coiled coil CCE/CCK as a physical cross-linking mechanism. Due to the temperature dependent association/dissociation of the CCE/CCK complexes and the network structures of the protein network, the hydrogels exhibit broad and dual dissipation modes. And, the dynamic properties of the hydrogels can be further tuned by the protein concentrations and the stoichiometric ratios of two components (CCE- and CCK-containing elastomeric proteins). Moreover, the fast association/dissociation kinetics of the CCE/CCK complexes conferred the protein hydrogel excellent shear-thinning and rapid self-healing abilities, making this protein hydrogel a unique injectable hydrogel system. These unique properties may open up avenues towards a range of 87 biomedical applications for this protein hydrogel. Moreover, we anticipate that the interplay between the cross-linker dynamics and the hydrogel network structures may offer a new perspective for the design of injectable dynamic hydrogels in the future. 88 Chapter 4: Light-Responsive Dynamic Protein Hydrogels Based on LOVTRAP 4.1 Synopsis Protein-based hydrogels can mimic many aspects of native extracellular matrices (ECMs) and are promising biomedical materials that find various applications in cell proliferation, drug/cell delivery and tissue engineering. To be adapted for different tasks, it is important that the mechanical or biochemical properties of protein-based hydrogels can be regulated by external stimuli. Light as a regulation stimulus is of advantage because it can be easily applied in demanded spatiotemporal manners. The non-covalent binding between the light-oxygen-voltage-sensing domain 2 (LOV2) and its binding partner ZDark1 (zdk1), named as LOVTRAP, is a light-responsive interaction. The affinity of LOVTRAP is much larger in dark than that under blue light irradiation. Taking advantage of this light-responsive interaction, herein we endeavored to use LOVTRAP as a cross-linking mechanism to engineer light responsive protein hydrogels. By using LOV2-containing and zdk1-containing multifunctional protein building blocks, we successfully engineered a light-responsive protein hydrogel whose viscoelastic properties can change in response to light: in the dark, the hydrogel showed higher storage modulus; under blue light irradiation, the storage modulus decreased. Due to the non-covalent nature of the LOVTRAP, the engineered LOVTRAP protein hydrogels displayed shear-thinning and self-healing properties, and *A version of Chapter 4 has been published as [Tianyu Duan], Qingyuan Bian, and Hongbin Li. (2021) Light-Responsive Dynamic Protein Hydrogels Based on LOVTRAP. Langmuir. 37: 89 10214-10222. This chapter is incorporated in this thesis with permission of American Chemical Society, Copyright 2021. served as an excellent injectable protein hydrogel. We anticipated that this new class of light-responsive protein hydrogels will broaden the scope of dynamic protein hydrogels and help develop other light-responsive protein hydrogels for biomedical applications. 4.2 Introduction Dynamic response is a hallmark of cellular microenvironments and ECMs, allowing tissues to respond to a wide range of signals and stimuli. To better mimic the dynamic nature of biological systems, it is desirable that the mechanical, physical or biochemical properties of protein-based hydrogels can change in response to external stimuli, so that their performances can be regulated on demand for different tasks6-8. Many types of external stimuli, such as temperature62, 85, 105, 224, 225, redox potential79, 200, 227, and small molecules230-232, have been explored as “triggers” to regulate the properties of protein-based hydrogels. Amongst the environmental stimuli, light is of particular importance because it allows for dynamic regulation of protein hydrogels’ properties in a spatial and/or temporal controlled fashion35, 209, 301-304. Over the last two decades, the field of optogenetics has witnessed tremendous progress, leading to the development of a rich arsenal of light-responsive protein systems that allow for the study of neuronal functions in a light-sensitive fashion305, 306. These amazing optogenetic tools have also attracted the attention of researchers in other fields. Inspired by these optogenetic tools, researchers have started to adapt such tools for engineering light-responsive dynamic protein and protein-polymer hybrid hydrogels in the hope of controlling the physical and mechanical properties of hydrogels by light153-157, 202, 307-309. Light responsive proteins that have been 90 explored to engineer light-responsive protein or protein-polymer hybrid hydrogels include Dronpa153, 154, CarHC (the C-terminal adenosylcobalamin binding domain)157, 308, 309, UVR8 (UV-B resistance 8)155, Cph1 photosensory module156, PhoCl (photocleavable protein)307 and LOV2(light-oxygen-voltage-sensing domain 2)-Jα202. All these proteins show light-responsive homo-oligomerization, with the exception of PhoCl that can be considered as a light-triggered cleavable system.222 UVR8 and Cph1 form homo dimer structures which dissociate in response to UV light (for UVR8)310 or far red light (for Cph1)311, while Dronpa and CarHC are two self-associative proteins whose tetrameric and monomeric states can be regulated by light solely (for Dronpa)312 or B-12 dependent light irradiation (for CarHC)163. LOV2-Jα is an intramolecular associative system, in which the binding of Jα to LOV2 is sensitive to light irradiation.313 Among these photo-responsive hydrogels, the mechanics of LOV2-Jα-based polymer-protein hybrid hydrogels were regulated by the cross-linker length changes arising from the association/dissociation of LOV2-Jα, while the mechanical regulation of other hydrogels were largely achieved by changing hydrogel cross-linking degree based on the light-induced switches of protein oligomerization states or the photo-cleavage of PhoCl. While the regulation in hydrogel mechanics of other proteins-based hydrogels are reversible,153-156, 202 the mechanical property changes of CarHC and PhoCl based hydrogels are irreversible157, 307-309 due to the photophysical or photochemical nature of these proteins. Although these efforts have clearly demonstrated the great potential of these optogenetic tools to engineer dynamic hydrogels, such explorations remain limited. Here we explored the use of a hetero-oligomerization-based optogenetic system LOVTRAP to engineer light-responsive dynamic protein hydrogels. LOVTRAP is an optogenetic tool developed by Hahn and co-workers220 for light-induced protein dissociation based on the light-oxygen-voltage-sensing domain 2 (LOV2) of Avena 91 sativa314 and its designed binding partner ZDark1 (zdk1). LOV2 domain has different conformations in dark and under blue light (i.e. the dark state and lit state) and these two conformations co-exist in a photo-regulated equilibrium315. Zdk1 is a designed fragment of staphylococcal protein A and can bind to LOV2 dark state with a high binding affinity (KD of 26 nM)220 (Figure 4.1 A). Upon irradiation of blue light (450-490 nm), LOV2 dark state converts to its lit state, and its binding affinity to zdk1 decreases dramatically (KD > 4 μM)(Figure 4.1 B).220 The binding affinity change of zdk1 and LOV2 is reversible for at least 10 cycles when alternately exposed to darkness and blue light irradiation, and the dissociation is relatively fast (with a t1/2 = 18 s). Moreover, LOVTRAP has been used as an optogenetic tool in cell studies (both in vivo and in vitro)220, 316, 317 as well as a ligand immobilization tool on polymer hydrogels.221 Considering the unique features of LOVTRAP, here we report the use of LOVTRAP to engineer light-responsive dynamic protein hydrogels. 4.3 Materials and Methods Protein Engineering. Genes encoding LOV2, zdk1, SpyTag (St), SpyCatcher (Sc) and YFP were custom-synthesized (GenScript, Piscataway, NJ, USA). All the genes were flanked with N-terminal BamHI and C-terminal BglII and KpnI restriction sites. The engineering of (GSc)3 making use of the identical sticky ends generated by BamHI and BglII restriction enzyme digestions was detailed previously70. The genes encoding G-St-LOV2 and zdk1-G-St, were constructed by the same method. The genes were each cloned in a pQE80L expression vector containing a N-terminal His6-Tag. Protein overexpression was carried out in E. coli. strain DH5α. The starter culture was grown at 37 °C in 2.5% lysogeny broth (LB) with 0.1 g/L ampicillin until OD600 reached 0.8-1.0. The expression of G-St-LOV2 and YFP-G-LOV2 was 92 induced by 0.25 mM isopropyl-1-β-D-thiogalactoside (IPTG) and then continued overnight at 25 °C in dark. The expression of all other proteins was induced by 1 mM IPTG and last for 4 h at 37 °C. After the expression the E. coli. cells were harvested by centrifugation and lysed by 2.5 mg/mL lysozyme. DNAs and RNAs were hydrolyzed by 5 µg/mL DNase I and 5 µg/mL RNase A. Target proteins with His6x-Tag were purified from soluble cell lysate using Co2+-affinity columns. The purified proteins were subsequently dialyzed against deionized water for 9 h and lyophilized. Hydrogel Preparation. (GSc)3 were first mixed with G-St-LOV2 or zdk1-G-St at St: Sc = 1: 1 stoichiometry and dissolved in PBS to the demanded concentration. The solutions were kept at room temperature for at least 3 h to allow St-Sc reaction to form SpyLOV2 or SpyZdk1 complexes. Then SpyLOV2 and SpyZdk1 solutions were mixed at 1:1 stoichiometry to form a LOVTRAP hydrogel in dark. Air bubbles in the hydrogel were removed by centrifugation at 12000 rpm for 10 minutes. Rheology. The rheological test methods are largely the same as those used in Chapter 4. All rheology measurements were performed on a TA Instruments Discovery HR-2 Rheometer equipped with an 8-mm flat plate (top) and a light guide accessory including a 20-mm quartz plate (bottom). A ThorLabs M470L3 LED light source was connected by a light guide so that it can illuminate the quartz plate and the sample. The LOVTRAP hydrogel sample was carefully transferred to the top surface of the quartz base plate, and then the flat plate was lowered to fit the sample in a gap between two plates. The gap was kept as 400 µm during all the types of tests. Silicone oil was used to cover the gap in contact with air to avoid evaporation. Oscillatory time sweeps were conducted at fixed frequency at 10 rad/s and strain at 1%. Oscillatory frequency sweeps were carried out ranging from 0.01 to 100 rad/s with a constant oscillation strain at 1%. 93 Oscillatory strain sweeps ranging from 0.1% to 1000% were performed at 10 rad/s. Self-recovery characterization was a sequential oscillatory time sweep under alternating 1% and 400% strains at 10 rad/s. The viscosity was measured in the flow test mode (steady measurement) at shear rate ranging from 0.01 to 100 s-1. All these experiments were carried out under room temperature (~23 °C). Erosion Rate Measurement. The erosion rate of hydrogel was measured using a method modified from previous report137. The SpyLOV2 and SpyZdk1 solutions were mixed to form 100 mg of 12% LOVRAP hydrogel in a cylindrical glass tube with a flat bottom (inner diameter = 1.05 cm). The glass tube with the hydrogel was allowed to stand overnight. Then the gel with the glass tube was soaked in 6 mL of PBS buffer, pH 7.4, in a Falcon tube. The whole setup was placed on a compact rocker tilting at 90 rpm at room temperature. The erosion profiles were determined by the protein concentration in the supernatant at successive time points, measured by absorbance at 280 using a Nanodrop UV-Vis spectrometer. 4.4 Results and Discussion 4.4.1 Design of LOVTRAP-Based Hydrogels LOVTRAP is a hetero-dimeric association system.220 Hence, it is feasible to use the two-component approach, which we demonstrated for the engineering of hetero-dimeric coiled coil domains-based proteins hydrogel137, to engineer LOVTRAP-based protein hydrogels by engineering multi-functional telechelic LOV2- and zdk1- containing proteins. In this way, an entirely protein-based hydrogel can be engineered without the use of synthetic polymers or multi-arm cross-linkers. In addition, the two-component approach provides more flexibility and controllability of when and where to form a hydrogel in potential applications, as protein 94 hydrogels can form only when two pre-gel solutions are mixed. However, one caveat of LOVTRAP is that previous studies showed that the fusion of protein domains to the C-terminus of LOV2 can impede the association of LOV2 and zdk1220, making it difficult to use the standard telechelic polymer approach to engineer active multi-functional LOV2-containing proteins. To overcome this challenge, we intended to graft LOV2 domains from their N-termini to an elastomeric protein backbone as pendent groups to engineer trifunctional LOV2-containing proteins (Figure 4.1 C) so that LOV2 domain can retain its binding affinity to zdk1. To achieve this, we used the well-developed SpyTag and SpyCatcher chemistry. SpyTag and SpyCatcher are a pair of proteins that can react with each other specifically and form an isopeptide bond, forming a covalent conjugation69. This chemistry has been widely used as an efficient bioconjugation method in many different applications70, 74, 157, 318-320. For this purpose, we used tri-functional elastomeric protein (G-Sc)3 as the polymer backbone, where Sc represents the SpyCatcher domain and G corresponds to the small globular protein GB1, respectively, and used SpyTag-SpyCatcher chemistry to functionalize (G-Sc)3 to introduce LOV2 as pendant functional groups. Similarly, we can engineer multifunctional zdk1-containing elastomeric proteins (Figure 4.1 C). We expected that the resultant proteins were trifunctional LOV2 and zdk1-containing proteins, which should be able to react with each other to form a hydrogel network via LOVTRAP interactions upon mixing (Figure 4.1 D). 95 Figure 4.1.The design principle of LOVTRAP-based hydrogel. A) Crystal structure of LOVTRAP binding complex between LOV2 (yellow) and zdk1 (purple) in dark (PDB: 5EFW220). B) Zdk1 binds to LOV2 with high affinity in dark and with low affinity under blue light irradiation. C) Scheme of the formation of telechelic LOV2- and zdk1-fused protein complexes SpyLOV2 and SpyZdk1 via SpyTag-SpyCatcher Chemistry. D) Schematics of the entirely protein-based hydrogel constructed using LOVTRAP. By mixing SpyLOV2 and SpyZdk1 solutions at 1:1 stoichiometry, a protein hydrogel can form. Under blue light irradiation (Left), the hydrogel displays a lower cross-linking degree; In dark, the hydrogel displays a higher cross-linking degree, due to the association of LOV2 and zdk1(Right). To demonstrate the feasibility of this design, we first engineered G-St-LOV2 and zdk1-G-St, where St represents the SpyTag, by using standard molecular biology techniques (Figure 4.2 A). To engineer multi-functional LOV2 and Zdk1 containing protein building blocks, we reacted (G-Sc)3 with G-St-LOV2 or zdk1-G-St in a 1: 3 ratio (St: Sc = 1: 1) respectively for 3 hr. As shown in Figure 4.2 B, SDS-PAGE analysis showed that after the SpyTag-SpyCatcher reaction, the molecular weights of the resulted grafted proteins corresponded to the sum of the 96 molecular weights of (G-Sc)3 and three G-St-LOV2 or zdk1-G-St, suggesting that three G-St-LOV2 or zdk1-G-St were successfully grafted covalently to the (G-Sc)3 protein. For convenience, the two resulted grafting proteins were referred to as SpyLOV2 and SpyZdk1, respectively, where LOV2 and Zdk1 were pendant groups attached to the (G-Sc)3 protein backbone. In addition, the UV-Vis spectrum of G-St-LOV2 did not show noticeable change before and after the coupling reaction with (G-Sc)3 (Figure 4.2 C), which suggested that the coupling of St and Sc domains did not affect the photophysical properties of the LOV2 domain. Figure 4.2. Construction of SpyLOV2 and SpyZdk1 proteins and the formation of LOVTRAP-based hydrogels. (A) An SDS-PAGE gel image of purified fusion proteins zdk1-G-St (16.0 kDa), G-St-LOV2 (25.9 kDa) and (G-Sc)3 (58.6 kDa). (B) SDS-PAGE gel image of SpyLOV2 and SpyZdk1 protein complexes formed by SpyTag-SpyCatcher chemistry. (C) UV-Vis absorbance spectrum of SpyLOV2 and G-St-LOV2. (D) Photographs of SpyLOV2 solution (Left), SpyZdk1 solution (Middle) and LOVTRAP hydrogel formed by mixing SpyLOV2 and SpyZdk1 solutions at 1:1 ratio (Right). 97 To engineer protein hydrogels based on LOVTRAP, aqueous solutions of the two reactive proteins SpyLOV2 and SpyZdk1 were then mixed at 1: 1 molar ratio. It is evident that upon mixing of the two protein solutions, a stable protein hydrogel formed (Figure 4.2 D), suggesting that SpyLOV2 and SpyZdk1 reacted with each other via LOVTRAP interactions to form a hydrogel network, termed as LOVTRAP-based hydrogels. We found that the LOVTRAP-based hydrogels can form at a total protein concentration as low as ~6%. LOVTRAP-based hydrogels were physically stable, as evidenced by their slow erosion profiles in open aqueous solution (Figure 4.3). The measured erosion rate of 12% LOVTRAP-based hydrogels under dark was 6.60 × 10-3 mg·cm-2·min-1, which is similar to that of other stable physically cross-linked protein hydrogels.134, 137, 159, 321 Such slow erosion rate is desirable in biomedical application, as the entire erosion takes ~1.5 days, potentially enabling relatively long-term drug release or tissue repair. Figure 4.3. Erosion profile of LOVTRAP-based hydrogels. Erosion profile of 100 mg 12% LOVTRAP hydrogel with a surface area of 0.865 cm2 at room temperature in 1x phosphate buffer saline (PBS), pH 7.4. The results were averaged from 3 parallel experiments. The mass loss of the hydrogel showed a linear relationship versus time, indicating that its erosion occurred at the surface. A linear regression (solid line) measures an erosion rate of 6.60 × 10-3 mg·cm-2·min-1. 98 4.4.2 LOVTRAP-Based Hydrogels Display Light-Responsive Viscoelastic Properties To investigate the viscoelastic properties of the resultant LOVTRAP protein hydrogels and examine if these properties are responsive to light irradiation, oscillatory shear rheology was employed. We first tested the viscoelastic properties of 12% protein hydrogel under alternating dark and lit conditions (Figure 4.4 B). A 470-nm LED light source, which generates 21.9 µW/mm2 irradiance (at 200 mm distance), was utilized to irradiate the samples on spot to induce the dissociation of LOVTRAP. In the dark state, the LOVTRAP protein hydrogel displayed a storage modulus G’ of ~180 Pa and a loss modulus G” of ~40 Pa, indicating the formation of a soft protein hydrogel. Upon blue light irradiation, the G’ showed a rapid decrease (to ~160 Pa) while the G” increased to ~60 Pa, indicating that the hydrogel was softened by the blue light irradiation. Upon removing the blue light irradiation, the G’ and G” of the protein hydrogel returned to near their original values in the dark state. Although the change of the amplitude in G’ of LOVTRAP hydrogels in response to light is moderate, the difference of G’ is statistically significant (with a p value smaller than 0.0001 for both 12% and 18% gels). The relative change of G’ and G” (ΔG’/G’dark=10% and ΔG”/G’’lit =30%) is comparable to that of a chemically cross-linked protein-PEG hybrid hydrogel based on LOV2-Jα202. These results indicated that the LOVTRAP hydrogels were light responsive, and that the changes in viscoelastic properties of were fully reversible. 99 Figure 4.4. Representative rheological characterizations of LOVTRAP-based hydrogels. The total protein concentrations were 6% in (A), 12% in (B), and 18% in (C). The characterization was carried out under alternating dark/lit conditions. The blue-shaded areas indicate the time periods when LOVTRAP hydrogels were irradiated by 470-nm blue light, while the unshaded areas represent time periods in darkness. This light-responsive viscoelastic properties of the LOVTRAP protein hydrogels can be readily explained by the light-responsive dissociation-association of LOVTRAP. Under blue light irradiation, LOV2 and Zdk1 had a lower affinity and LOVTRAP dissociated, leading to a 100 decrease of the cross-linking degree. In addition, the moduli change in response to light finished in 30-40 sec, consistent to the reported 18 s average half-life of LOVTRAP equilibrium in a previous study220. In addition, in response to alternating dark/lit treatments, the G’ and G’’ of the 12% and 18% gels underwent reversible changes for at least 5 full cycles, consistent with the known high reversibility of LOVTRAP interaction220, 221. Interestingly, after a few dark/lit cycles, both the 12% and 18% gels were gradually stiffened (Figure 4.4 B & C). Such gradual stiffening might be the result of network reconfiguration: the LOVTRAP interactions were repeatedly dissociated and re-formed during the test. This process may gradually reduce the defects in the network and help achieve a higher cross-linking density in hydrogel network over an extended time period. It is of note that under blue light, the LOVTRAP-based hydrogels only softened but did not melt, an observation consistent with the lower binding affinity of LOVTRAP under blue light (KD > 4 μM)220, 322. Clearly, the LOVTRAP-based hydrogels demonstrated light-responsive viscoelastic properties. To further characterize the viscoelastic properties of LOVTRAP-based hydrogels, we examined the influence of protein concentration on G’ and G” of the resultant hydrogels. Clearly, the storage modulus G’ of the LOVTRAP-based hydrogels increased with the increase of the total protein concentration. At 6 %, the protein hydrogel was a very soft one with a storage modulus G’ of ~20 Pa (Figure 4.4 A), while the 18-wt% hydrogel was much stiffer with G’ above 400 Pa (Figure 4.4 C). 18% hydrogel showed a more pronounced light-responsiveness in their G’ and G”, similar to 12% hydrogel. However, the 6% gel did not show obvious modulus changes under dark/lit alternations, probably because its moduli were already very low and its 101 further decrease due to the small decrease of the cross-linking degree of the hydrogel network under blue light was too small to be accurately detected in our rheology measurements. 4.4.3 Dynamic Properties of LOVTRAP-Based Hydrogels Having demonstrated that the stiffness of LOVTRAP protein hydrogels was light-responsive, we then carried out rheological studies to investigate their frequency dependent dynamic properties. As shown in Figure 4.5, the LOVTRAP-based hydrogels exhibited clear frequency-dependent behaviors. In both dark and lit conditions, the relationships between shear moduli and oscillation frequency ω were similar. At the critical frequency ωc, G’ equals to G’’. When ω is lower than ωc, G’ is smaller than G’’, indicative of a viscous liquid-like state of the LOVTRAP-based hydrogels where more energy is dissipated rather than stored during the oscillation tests. In contrast, when ω is above ωc, LOVTRAP-based hydrogels showed elastic solid-like behaviors with G’ larger than G’’, and more energy is stored than dissipated. As aforementioned in Chapter 3, the reciprocal of ωc is approximately equivalent to the material’s characteristic relaxation time τc, which reflects the rate of energy dissipation of the material and is primarily determined by the relaxation time of the cross-linking interactions.269, 288-290 Notably, ωc of the same LOVTRAP-based hydrogel samples was always higher under lit condition (Figure 4.5 B) than in the dark (Figure 4.5 A). For example, the average ωc of 12% LOVTRAP hydrogels were 0.055±0.017 rad/s in the dark and 0.71±0.16 rad/s under light. Such an effect was attributed to the photo-responsive nature of LOVTRAP interactions. Under light irradiation, LOVTRAP became less stable322 and more dynamic, and so did the hydrogels where the LOVTRAP interactions were the primary cross-linking mechanism. 102 To further characterize the relaxation of the LOVTRAP hydrogels, we calculated H(τ)291 by solving Equations (3.1) and (3.2). H(τ) is equivalent to the amount of energy dissipated under dynamic loading/unloading at a given frequency 1/τ. Figure 4.5 C and D shows the H(τ) calculated from the data in Figure 4.5 A and B, respectively. Interestingly, in the dark, H(τ) of the hydrogels showed a bimodal distribution. However, under lit condition, H(τ) showed a unimodal distribution. To investigate the dissipation mechanism, we used multi-peak log-normal fitting to describe the relaxation behaviors of LOVTRAP-based hydrogels. Evidently the H(τ) can be fitted by two-mode forms (dashed lines) for LOVTRAP-based hydrogels in the dark, where each mode is distributed log-normally, suggesting that there existed two distinct dissipation modes for LOVTRAP-based hydrogels in the dark: a dominant slower mode at a relaxation frequency 1/τ of around 0.1 s-1 and a minor faster mode at a 1/τ of around 1 s-1. In comparison, the H(τ) of LOVTRAP-based hydrogels under lit condition can be fitted by a single-mode form with a 1/τ of around 1 s-1. Figure 4.5. Time-dependent rheological behaviors of 12% LOVTRAP-based hydrogels. 103 (A-B) Representative oscillation frequency-dependent storage modulus G’ and loss modulus G’’ measured in dark (A) and lit (B) conditions. In each figure the crossover frequency ωC where G’ = G’’ is the reciprocal of the hydrogel’s characteristic relaxation time τC under the respective condition. When frequency is below ωC, G’ and G’’ show terminal relaxation model-like behavior, where G’ ~ ω2 and G’’ ~ ω1 as indicated. G’ and G’’ are fitted by equation (1) and (2), respectively, and the fitted curves are also shown. (C-D) The log-normal relaxation spectra calculated from the fitting results of data shown in (A-B). Dashed lines in (C-D) are the log-normal fitting results of different dissipation modes. It is important to note that the faster relaxation mode of the hydrogel under dark occurred at a similar frequency as the relaxation mode of the hydrogel under lit condition. In addition, upon blue light irradiation, the slow relaxation mode of the LOVTRAP-based hydrogels under dark largely disappeared. These results suggested that the slow mode which predominates in the dark was resulted from the interaction between LOV2 dark state and zdk1, while the faster mode which predominates in lit reflected the interaction between LOV2 lit state and zdk1. The two relaxation modes under dark suggested that there existed a dynamic equilibrium between the two types of interactions between LOV2 and zdk1 in the dark. 4.4.4 LOVTRAP-Based Hydrogels Display Shear-Thinning Behaviors and are Injectable Hydrogels Highly frequency-dependent behaviours of hydrogels often potentiate injectability of the materials143, 323. The flow behavior, which is an important aspect of injectability, of LOVTRAP-based hydrogels was studied by measuring viscosity versus shear rate (Figure 4.6 A&B). Newtonian plateau was observed at lower shear rates where viscosity remained nearly constant regardless of the shear rate changes, followed by a shear thinning region at elevated shear rates where viscosity dropped sharply as shear rate increased. Notably, both in dark and lit, the 104 viscosity of LOVTRAP-based hydrogels decreased by 2 to 3 orders of magnitude in a narrow range of shear rate, which was similar to the behaviours reported in other injectable materials324-326. From dark to lit conditions, the Newtonian plateau viscosity decreased from thousands to hundreds of Pa·s, and the upper limit of Newtonian plateau region shifted from ~0.1 s-1 to ~1 s-1 (Figure 4.6 A&B). Such an alteration of flow behaviours indicated reduced number of cross-linked polymer chains at lit condition324, which was a result of the photo-induced dissociation between SpyLOV2 and SpyZdk1 proteins. 105 Figure 4.6. Injectability of 12% LOVTRAP-based hydrogels. (A-B) Representative flow sweep test results of LOVTRAP-based hydrogels in dark (A) and under blue light (B). (C-D) Representative strain sweep results that measuring the yielding strain and flow strain of LOVTRAP hydrogels in dark (C) and under blue light (D). (E-F) Self-recovery performances of LOVTRAP hydrogels where their G’ dropped and restored in response to alternating 1% and 400% strain (each last for 180 s) in dark (E) and under blue light (F). (G-H) Photographs to show that a 12% hydrogel sample was injected through a 16 gauge needle (G) and regained its gel state after the injection (H). Yielding and recovery performance against large deformations are also important aspect of hydrogel injectability143. The oscillation strain sweep of LOVTRAP-based hydrogels was 106 carried out (Figure 4.6 C&D). The yielding strain, defined by the offset point of G’, of LOVTRAP-based hydrogels was measured to be ~150%, and the flow strain, where G’ and G’’ crossed over, was ~300%. Between the yielding strain and the flow strain, G’ deeply decreased while G’’ sharply increased, indicative of a brittle fracturing behavior accompanying a sharp transition from a hydrogel state to a viscous liquid-like state. The ratio of the flow strain over the yielding strain was ~2.0, similar to that of other reported injectable hydrogels308. Such brittle fracturing behavior can maintain a moderate shear stress and help encapsulated cells to survive high shear strains during injections in potential cell-delivery applications143, 308. Furthermore, tests under alternating 1% and 400% strain demonstrated self-recovery capability of LOVTRAP-based hydrogels after network failure (Figure 4.6 E&F). At 400% strain, the network was interrupted causing G’ to sharply drop below G’’. When strain resumed to 1%, G’ can be quickly restored and the material recovered to gel state with G’ higher than G’’. Such G’ restoration can be repeated for at least 5 cycles of large strain interruptions. This self-recovery behavior reflected fast reversible association of LOVTRAP in LOVTRAP-based hydrogel networks, demonstrating the suitability of LOVTRAP-based hydrogels as injectable hydrogels. Indeed, as shown in Figure 4.6 G&H, a 12% LOVTRAP-based hydrogel can be readily injected through a 16 gauge needle, and regained its hydrogel state rapidly. 4.4.5 Comparison of LOVTRAP-Based Hydrogels with Other Protein Hydrogels LOVTRAP-based hydrogels are physically cross-linked protein hydrogels engineered based on the interactions between LOV2 and Zdk1. As such, their physical and mechanical properties, including shear thinning and self-healing properties, are comparable to those of many physically cross-linked protein hydrogels constructed based on protein-protein/protein-peptide 107 interactions, 23, 132, 134, 155, 159, 166 such as the prototypical protein hydrogels based on coiled coils132. These properties make LOVTRAP-based hydrogels suitable for a range of potential applications, such as controlled drug release and injectable delivery systems. Compared with these classical physically cross-linked protein hydrogels, the unique feature of LOVTRAP-based hydrogels lies in their light-dependent mechanical properties. As a new class of protein hydrogels emerged over the last few years, light-responsive protein hydrogels have achieved considerable progress. Physically cross-linked light-responsive protein hydrogels have been developed based on photo-responsive proteins. Protein hydrogels constructed from CarHC,157, 308, 309 Dronpa153, 154 and UVR8155 are able to undergo gel-sol transitions upon light irradiation, making such hydrogel suitable for applications such as light responsive cargo release.155, 157 However, LOVTRAP-based hydrogels do not undergo light-induced gel-sol transitions, instead, exhibit light-dependent mechanical properties. In addition, this light-dependent switching in LOVTRAP-based hydrogels mechanics display excellent reversibility, in contrast with the limited reversibility observed in other protein hydrogels.153, 154, 157, 308, 309 Such light-dependent physical properties of LOVTRAP-based hydrogels may find applications in mechanobiological studies, for example, LOVTRAP may be used for constructing matrices for studying mechanics-dependent cellular behaviors.202, 229 Moreover, unlike hydrogels based on CarHC or Dronpa whose G’ can exceed 1 kPa153, 154, 308, 309, LOVTRAP-based hydrogels are relatively soft with G’ smaller than 500 Pa, which may originate from the dynamic equilibrium between the LOV2 dark and lit states. Using other mutants of LOV2 or zdk with higher binding affinity could help improve the stiffness of LOVTRAP-based hydrogels, but potentially at the expenses of sacrificing the sensitivity to light220. 108 It is also worth noting that the current LOVTRAP-based hydrogels have a relatively small affinity change (~150-fold of KD) of LOVTRAP220 under dark and lit conditions, that made the property changes of LOVTRAP-based hydrogels in response to light rather moderate. Upon irradiation, LOVTRAP-based hydrogels only exhibit ~10% of G’ decrease. Although increasing the light intensity may likely enhance the changes of the viscoelastic properties220, using higher light intensity may not be desirable in potential biological applications as higher energy inputs may be more destructive to both the materials and the tissues. Further optimizing the photo physical properties of LOVTRAP interacting pairs via protein engineering will be needed for improving the performance of LOVTRAP and LOVTRAP-based hydrogels. 4.5 Conclusions In this study, we explored the use of photo-responsive LOVTRAP interactions to engineer photo-responsive protein hydrogels. In the LOVTRAP-based hydrogel system, LOVTRAP was adapted as the primary cross-linking mechanism to form a physically cross-linked photo responsive protein hydrogel. The dynamic and reversible nature of LOVTRAP bestowed the LOVTRAP-based hydrogels with shear-thinning, self-healable and injectable properties. The viscoelastic properties of LOVTRAP-based hydrogels can be regulated reversibly by alternative darkness and blue light irradiation. These results clearly demonstrate the usefulness of LOVTRAP in engineering photo-responsive dynamic protein hydrogels, thus enriching the toolbox of protein building blocks for constructing light-responsive biomaterials. Given the feasibility of spatial irradiation, it is anticipated that the viscoelastic properties of LOVTRAP-based hydrogels can be patterned by using patterned light irradiation. Moreover, LOVTRAP-based hydrogels respond to visible light that is non-toxic and non-destructive. 109 Hence, the light-responsive viscoelastic properties of LOVTRAP-based hydrogels may offer opportunities for using such protein hydrogels for potential mechanobiological studies. 110 Chapter 5: Protein Hydrogels with Reversibly Patterned Multidimensional Fluorescent Images for Information Storage 5.1 Synopsis Fluorescent polymeric hydrogels are promising building blocks for constructing soft and wet information storage media that are desirable in developing flexible electronics for various applications. Hydrogels based on engineered proteins have attracted considerable interests. However, their potential utility as soft and wet information storage media has remained largely unexplored. Here we report the engineering of a protein-based hydrogel that can serve as a medium for reversible information storage. Using the light-controlled association and dissociation of LOVTRAP, which consists of the light-responsive protein LOV2 and its designed protein binding partner ZDark1 (zdk1), we developed a novel strategy that enabled us to readily decorate/release fluorescent proteins onto/from a blank protein hydrogel slate in a light-controlled and spatially defined fashion, leading to the generation of fluorescent patterns that can be reversibly written and erased. Such patterns can be used readily to store information, resulting in protein hydrogel-based soft and wet information storage media, such as bar codes and quick response (QR) codes. To increase the information storage capacity, we successfully developed greyscale patterning of protein hydrogels based on LOVTRAP to generate pseudo colored multi-dimensional fluorescence images. The information written on these protein hydrogels is stable and can be erased, representing a novel reversible information storage approach in soft and wet materials. Our results open a new avenue towards developing next-generation protein-based smart materials for information storage and anti-counterfeit applications. 111 5.2 Introduction The rapid advent of information technology calls for the development of novel information storage materials with excellent storage capacity and security performance. To meet the needs for developing next-generation lifelike biomaterials and flexible electronics for wearable, soft robotic and biomedical applications17, 327, 328, developing technologies to enable writing and storing information on soft and wet materials has become desirable, yet remains challenging. Owing to their similarities to biological tissues, hydrogels, which are polymer networks that can swell in water and hold a large amount of water, have emerged as a promising candidate for such applications.16, 329 Fluorescent polymeric hydrogels (FPHs), which are engineered by incorporating fluorescent molecules into polymer hydrogels, have attracted considerable interests, as FPHs can seamlessly integrate unique traits of fluorescent molecules with those of hydrogels. FPHs have been developed for various applications in information storage, such as multi-dimensional information storage and encryption, and large capacity information storage330-332. The fluorescent moieties are often introduced into polymer hydrogels by either physical doping or chemical conjugation, and the information writing in FPHs is mostly irreversible. Developing technologies that are easy to implement and allow for reversible information writing/erasing in FPHs remains challenging. Owing to their biocompatibility and possibility for precision engineering, proteins have become attractive building blocks to engineer materials with unique physical, biochemical and mechanical properties for various applications20-25. The development of recombinant DNA technologies has made it possible to use recombinantly engineered proteins as building blocks for constructing protein-based hydrogels in an economically viable fashion for technological applications. Protein-based FPHs can be readily engineered by using fluorescent proteins, either 112 naturally occurring or recombinantly engineered, as building blocks159, 333-335, or by functionalization of protein hydrogels with fluorescent dyes or fluorescent proteins via bioconjugation211, 336, 337. Using photolithography, protein hydrogels with permanently patterned fluorescence can also be engineered210, 338, 339. However, it remains largely unexplored to engineer protein-based FPHs for information writing and storage applications. Taking advantage of the blank hydrogel slate approach211, 214 and the recently developed light-responsive LOVTRAP-based non-covalent protein-protein interactions220, here we report the engineering of protein-based FPHs with reversibly patterned multidimensional fluorescent images for re-writable information storage. The information storage format we will test is 2D and 3D information codes. 2D bar codes, such as quick response (QR) codes, have been widely used as information storage media that can encode a large amount of information in a small black and white geometric 2D pattern and be readily read and decoded by modern camera cell phones. Built upon traditional black and white 2D codes, 3D color codes, which have higher information storage capacity and better security, have been developed by using multi-color FPHs to form different color matrices340-343. To alleviate the need of multi-color fluorescence, pseudo-3D color codes can be constructed using one color by generating the data matrices with different grey-scaled fluorescence344, 345. In Chapter 4, we have used LOVTRAP220, the non-covalent protein-protein interaction between LOV2 and zdk1 domains, as cross-linking method to engineer dynamic protein-based hydrogels. LOVTRAP has also been used to engineer dynamic fluorescent synthetic polymer-based hydrogels221. The LOVTRAP affinity change is reversible and can be controlled by 113 darkness or blue light irradiation, which has been introduced in Chapter 4. Here we attempted to use the protein hydrogel blank slate approach to engineering LOVTRAP-based protein FPHs. 5.3 Materials and Methods Protein Engineering. The construction of GB1 domain (G), resilin sequence (R) and GRG5RG4R has been described previously42. Genes encoding LOV2, zdk1, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and green fluorescent protein (GFP) were custom-synthesized (GenScript, Piscataway, NJ, USA). All the genes were flanked with N-terminal BamHI and C-terminal BglII and KpnI restriction sites. The gene encoding GRG5RG4RGzdk1 is constructed by tandem ligation of GRG5RG4R, G and zdk1 using the identical sticky ends generated by BamHI and BglII restriction enzyme digestions. The genes encoding GRG5RG4RGLOV2, YFP-G-LOV2, CFP-G-LOV2 and GFP-G-LOV2 were constructed in the same way. Constructed genes were each cloned in a pQE80L expression vector containing a N-terminal His6-Tag. Protein overexpression was carried out in E. coli. strain DH5α. The starter culture was grown at 37 °C in 2.5% lysogeny broth (LB) with 0.1 g/L ampicillin until OD600 reached 0.6-0.8. The expression of YFP-G-LOV2, CFP-G-LOV2 and GFP-G-LOV2 was induced by 0.25 mM isopropyl-1-β-D-thiogalactoside (IPTG) and then continued overnight at 25 °C in the dark. The expression of GRG5RG4RGzdk1, GRG5RG4R and GRG5RG4RGLOV2 was induced by 1 mM IPTG and last for 4 h at 37 °C. After the expression the E. coli. cells were harvested by centrifugation and lysed by 2.5 mg/mL lysozyme. DNAs and RNAs were hydrolyzed by 5 µg/mL DNase I and 5 µg/mL RNase A. Target proteins with His6x-Tag were purified from soluble cell lysate using Co2+-affinity resin columns. GRG5RG4RGzdk1, 114 GRG5RG4R and GRG5RG4RGLOV2 were further dialyzed against deionized water for 9 h and lyophilized to facilitate hydrogel preparation. Hydrogel Preparation. GRG5RG4RGzdk1, GRG5RG4R and GRG5RG4RGLOV2 were cross-linked into hydrogels via [RuII(bpy)3]-mediated photochemical di-tyrosine reaction39. Lyophilized proteins were dissolved in 1× phosphate buffered saline (PBS) to 100 mg/mL and mixed with 0.26 mM [RuII(bpy)3]Cl2 and 50 mM ammonium persulfate. 4-20 µL of this solution was immediately injected onto a plexiglass surface, and then a glass cover slip was placed on top of the injected solution, with two layers of tapes in between as spacers. Subsequently, the sample was irradiated by a 200 W white light source at a height of 10 cm. Then the resultant hydrogel, at a thickness of ~ 80 µm, was detached from the plexiglass surface and stuck on the cover slip. Ligand Decoration, Release and Patterning. The in bulk YFP decoration was carried out by soaking a GRG5RG4RGzdk1 hydrogel sample in 150-300 µL 3.6 mg/mL YFP-G-LOV2 solution in dark for 1 hr. The decoration of CFP and GFP was done in the same way with 3.6 mg/mL CFP-G-LOV2 and 2.0 mg/mL GFP-G-LOV2 solutions, respectively. The release of the ligand in bulk was performed by irradiating a decorated hydrogel sample using a ThorLabs M470L3 LED light source (21.9 µW/mm2 irradiance at 200 mm distance) at a height of 1 cm for 10-20 min. The one-step ligand patterning was achieved by soaking a blank hydrogel slate sample in ligand solution on top of the cover slip, while at the same time irradiating the sample using the LED through a photo mask at 1 cm below the cover slip. The two-step ligand patterning was carried out by first soaking a blank hydrogel slate in ligand solution in bulk in dark for 1 hr, and then irradiating the sample after the removal of ligand solution using the LED through a photo mask at 1 cm below the cover slip. During both one-step and two-step patterning, the photo mask was sealed on the bottom side (opposite side of the sample) of cover 115 slip to avoid direct physical contact with the sample. After every decoration, release and patterning treatment, the samples were immediately washed for multiple times in fresh PBS to remove non-specifically bound ligands. The resultant samples were stored in PBS in the presence of Penicillin-Streptomycin, or air dried, under ambient temperature and indoor light. Photo Mask Preparation. The photo masks that were used to form the hexagonal grid patterns were SPI Supplies 100 mesh hexagonal TEM copper grids (Item ID 2470C-XA). The digital images of the black-and-white QR code (0.8 cm × 0.8 cm), the greyscale Einstein portrait (1.0 cm × 1.0 cm), and the 32/128 bit codes (1.0 cm x 1.0 cm) were first converted color, and then printed on a 3M transparency film (CG3480) using a Xerox AltaLink C8055 printer. In the patterning experiments, two layers of printed transparency films were overlapped together as photo mask. Fluorescence Imaging. The fluorescence images were taken either at ×10 magnification using a Nikon Eclipse Ti florescence microscope or at x4 magnification using an Olympus BX53 light/fluorescence microscope. The cm-scale images were taken using the tiling function of the Olympus BX53 microscope. The fluorescence intensity profiles were measured using the NIS-Elements software incorporated with the Nikon Eclipse Ti microscope. The histograms of grey values were measured using the ImageJ software. The greyscale heat map was generated using a modified Python programme344 (Appendix B). 116 5.4 Results and Discussion 5.4.1 LOVTRAP can Reversibly Decorate/Release Protein Ligands onto/from Protein Hydrogels We first tested the feasibility to construct protein-based FPHs by decorating the protein hydrogel blank using fluorescent proteins by using LOVTRAP. The blank protein hydrogel we engineered is based on our well-developed protein hydrogel based on the polyprotein GRG5RG4R42, where G represents the GB1 domain from Streptococcus205 and R is the 15-residue consensus sequence of elastic protein resilin from D. drosophila39. To facilitate the decoration of fluorescent proteins to the blank hydrogel, we fused zdk1 to the C-terminus of GRG5RG4R to create GRG5RG4RGzdk1. Then we used the well-developed RuII-mediated photo-chemical cross-linking strategy39 to cross-link the aqueous solution of GRG5RG4RGzdk1 into a solid protein hydrogel. Since GRG5RG4RGzdk1 did not contain functional protein ligand, its protein hydrogel can thus serve as a blank slate. To functionalize the protein hydrogel blank slate, we could use the high binding affinity between zdk1 and LOV2 in the dark as a bioconjugation method (Figure 5.1). The significantly lower affinity between zdk1 and LOV2 under blue light should allow for the release of the decorated protein ligands. To test this strategy, we engineered a yellow fluorescent protein (YFP) fused with LOV2 as a model protein ligand, YFP-G-LOV2, in which a GB1 domain was included to improve the protein expression346 and served as a spacer. 117 Figure 5.1. Schematic of light-controlled LOVTRAP-regulated ligand decoration/release on protein hydrogels. Protein ligands are recombinantly fused with LOV2 domain, and hydrogel networks are recombinantly grafted with zdk1 domains. In dark, LOV2 and zdk1 bind at high affinity so the protein ligands are decorated in the hydrogels. The crystal structure of the binding complex between LOV2 and zdk1 in dark is presented on the Right (PDB: 5EFW)220. When exposed to light, the binding affinity drops dramatically, and ligands are released from the hydrogel. Such decoration/release is reversible under alternate dark/lit conditions. Before decoration, the blank GRG5RG4RGzdk1 protein hydrogel slate did not display any yellow fluorescence (Figure 5.2 A). To decorate YFP ligand onto GRG5RG4RGzdk1 protein hydrogel, we incubated the blank hydrogel slate with the aqueous solution of YFP-G-LOV2 (3.6 mg/ml) for 1 hr in dark. It is evident that after 1 hr incubation, the hydrogel displayed strong yellow fluorescence characteristic of YFP, indicative of the successful labeling of the hydrogel by YFP (Figure 5.2 B). The fluorescence of the hydrogel was stable in dark as well as under ambient light. To confirm that the labeling was due to the specific association of zdk1 and LOV2, control experiments were carried out. Incubating GRG5RG4R and GRG5RG4RG-LOV2 hydrogels with YFP-G-LOV2 respectively for 1 hr did not lead to the decoration of YFP on either hydrogel, due to the lack of specific interactions of YFP-G-LOV2 with the hydrogels 118 (Figure 5.3). These results clearly demonstrated that YFP-G-LOV2 specifically recognized zdk1 domains in the dark on the hydrogel and readily led to the construction of protein-based FPHs. Figure 5.2. The decoration of YFP-G-LOV2 onto GRG5RG4RGzdk1 hydrogel was specific and reversible. The blank GRG5RG4RGzdk1 hydrogel slate did not have any yellow fluorescence on itself (A). After soaked in YFP-G-LOV2 solution in dark, the hydrogel exhibited intense yellow fluorescence (B). The YFP-decorated hydrogel was irradiated by blue light and a large portion of YFP-G-LOV2 ligands were released (C). After the irradiation, the hydrogel can be re-decorated by YFP-G-LOV2 in dark again (D). All the images were taken using the same parameters and presented at the same grey scale. Scale bars represent 100 µm. Figure 5.3. Blank protein hydrogel slate cannot be decorated via non-specific interactions. (A-B) Photographs of a blank GRG5RG4R hydrogel before (A) and after soaking in a 3.6 mg/mL YFP-G-LOV2 solution in dark for 1 hr. (B). (C-D) Photographs of a GRG5RG4RGLOV2 hydrogel before (C) and after soaking in 119 3.6 mg/mL YFP-G-LOV2 solution in the dark for 1 hr. After soaking in YFP-G-LOV2 solution, the decoration of these hydrogels by YFP was minimal. Most decoration occurred at the edge of the hydrogel. These results clearly demonstrated that the decoration of YFP-G-LOV2 on GRG5RG4RGzdk1 hydrogels was highly specific. Since LOVTRAP is light responsive, we then examined if the decoration of the protein hydrogel via LOVTRAP is light responsive. We incubated the blank slate of GRG5RG4RGzdk1 hydrogel (Figure 5.4 A) in YFP-G-LOV2 solution for 1 hr under the irradiation of a 470-nm LED light source instead of in dark. The resultant hydrogel displayed negligible yellow fluorescence increment compared with the blank slate (Figure 5.4 B), indicating that the decoration of YFP-G-LOV2 onto GRG5RG4RGzdk1 hydrogel was ineffective because of the low affinity between LOV2 and zdk1 under blue light. After the irradiation was removed, the same blank hydrogel slate can then be effectively decorated by YFP-G-LOV2 in dark, resulting in intense yellow fluorescence (Figure 5.4 C). Figure 5.4. The decoration of YFP-G-LOV2 on GRG5RG4RGzdk1 hydrogel only worked in the dark. When a non-fluorescent blank GRG5RG4RGzdk1 hydrogel slate (A) was soaked in YFP-G-LOV2 solution and irradiated by blue light at the same time, the resultant yellow fluorescence increment was very slight (B). After the irradiation was removed, the hydrogel can be much more effectively decorated by fresh YFP-G-LOV2 solution in dark (C). All the images were taken using the same parameters and presented at the same grey scale. (Scale bar = 100 µm) 120 To test the feasibility of reversible decoration and release of ligands onto/from the hydrogel blank slate by YFP, YFP-decorated protein hydrogels were then irradiated by the 470-nm LED blue light for 10 min to induce LOVTRAP dissociation. As expected, the YFP fluorescence on hydrogels decreased dramatically after blue light irradiation, indicative of pronounced release of YFP from the hydrogel (Figure 5.2 C), effectively turning a protein FPH into a non-fluorescent one. After the release of YFP, the same hydrogel can be redecorated by YFP-G-LOV2 again via another incubation treatment in dark (Figure 5.2 D). This result indicated that the decorated YFP can be released by the blue light-driven dissociation of LOV2-zdk1 complex, and thus the YFP-decoration/release is reversible and can be controlled by light, providing the possibility of reversible writing and erasing of information on the protein hydrogels. Fluorescence intensity quantification of multiple parallel samples (n = 12) further confirmed our results. Upon irradiation of blue light, the YFP fluorescence intensity showed an intensity decrease by ~53% during the 10-min irradiation of the original fluorescence. Upon redecoration, the fluorescence intensity returned to ~106% of the original intensity (Figure 5.5). These results were comparable to those reported by West and coworkers on ligand decoration/release on PEG-hydrogel using LOVTRAP221. 121 Figure 5.5. Changes of the fluorescence intensity of the GRG5RG4RGzdk1 hydrogel during the decoration-release-redecoration cycle. The fluorescence intensity of the GRG5RG4RGzdk1 hydrogel that was decorated by soaking in 3.6 mg/mL YFP-G-LOV2 solution in dark for 1 hr was taken as 1. Then the decorated hydrogel was irradiated by blue light for 10 min at 1 cm distance to release YFP-G-LOV2. Then the hydrogels were redecorated by YFP-G-LOV2 solution in dark for 1 hr. It is evident that fluorescent protein ligand YFP can be effectively decorated onto, release from and redecorated onto the protein hydrogel blank slate effectively. The data were collected from 12 parallel samples, and on each sample 512 pixels were measured. 5.4.2 Writing and Erasing Information on Protein Hydrogels by Using Light-Responsive LOVTRAP Interaction Having demonstrated the light-controlled decoration/release of fluorescence proteins on protein hydrogels using LOVTRAP, we then explored the feasibility of using LOVTRAP to write and erase information on protein hydrogels by creating spatially patterned fluorescence images. Because darkness was required to effectively decorate protein ligands on GRG5RG4RGzdk1 hydrogels, two methods were developed to create fluorescence patterns: one-step method and two-step method. In the one-step method, the blank hydrogel slate is incubated with YFP ligand and irradiated by blue light through a photo mask at the same time. In this way, 122 only the masked areas of the blank slates will be effectively decorated (Figure 5.6 A). In the two-step method, the blank hydrogel slate is first incubated with YFP ligand in dark to decorate the whole hydrogel, and then the decorated fluorescent hydrogel is irradiated through a photo mask to selectively release the ligands only at the un-masked areas (Figure 5.6 B). Both methods will produce a spatial pattern of fluorescence that is defined by the shape of the photo mask. Thus, information encoded in the shape of the photo mask will be written on hydrogels. As proof of concept, YFP-G-LOV2 patterns were produced on blank GRG5RG4RGzdk1 hydrogel slate samples via both one-step (Figure 5.6 C) and two-step methods (Figure 5.6 D) using the same photo mask that has a hexagonal grid pattern. Both methods produced a hexagonal pattern of YFP fluorescence, which closely matched the shape of the photo mask, on the respective hydrogel samples. The areas covered by the photo mask and not irradiated by blue light showed much stronger fluorescence, while the areas exposed to the blue light were much darker. Cross-section analysis of the fluorescence intensity clearly showed the fluorescence intensity changes in the patterned area (Figure 5.7). No significant difference was observed between the patterns produced via the one-step or two-step methods. Due to its simplicity, we mainly utilized the one-step pathway in the following experiments. Similar hexagonal patterns of fluorescence can be generated by using other LOV2 fused fluorescent proteins, such as cyan fluorescent protein (CFP) and green fluorescent protein (GFP) (Figure 5.6 E&F), thus demonstrating that this method is general. The fluorescence pattern generated on the GRG5RG4RGzdk1 hydrogels can be largely erased by irradiating the patterned hydrogel sample with blue light. Line profile analysis of the fluorescence intensity showed that that after 30-min light irradiation, the original patten was 123 almost completely erased (Figure 5.7). The reversibility of LOVTRAP-controlled patterning and erasing potentiates its utility in customized reversible information writing and erasing. To demonstrate the information writing ability of this method, we then used this method to generate information-coding QR codes. As a proof-of-concept, we generated a YFP fluorescence based QR code (0.8 cm × 0.8 cm) on the protein hydrogel, which encoded the website of the Department of Chemistry at the University of British Columbia (Figure 5.6 G). This QR code can be readily read by a cell phone and directed to the designated website. Evidently, the developed LOVTRAP-controlled information writing method fulfilled the needs for spatially precise visual communication. 124 Figure 5.6. light-controlled pattern of LOV2-fused ligand can be produced in GRG5RG4RGzdk1 hydrogels. (A) One-step method: A blank hydrogel slate is soaked in ligand solution on the top and irradiated though a photo mask from the bottom at the same time to produce a ligand pattern that inherits the shape of the mask. (B) Two-step method: A blank hydrogel slate is first decorated in bulk by ligand solution in dark. After the excess ligand solution is removed, the decorated sample is soaked in fresh PBS and irradiated through a photo mask, which also produces a mask-shape pattern. (C & D) Representative florescence images of YFP-G-LOV2 pattern produced by one-step (C) and by two-step pathway (D) using the same type of hexagonal grid shape photo mask (scale bar = 100 µm). (E & F) Representative fluorescence images of GFP-G-LOV2 pattern (E) and CFP-G-LOV2 pattern (F) produced by one-step method using the hexagonal grid shape mask (scale bar = 100 µm). (G) A 0.8 cm × 0.8 cm QR code pattern of YFP-G-LOV2 produced by one-step method using a printed transparency film-based mask (scale bar = 2 mm). This QR code is readable by cell phone which links to the website of Department of Chemistry at the University of British Columbia. 125 Figure 5.7. Ligand patterns can be erased effectively by blue light irradiation. (A-C) Fluorescence images of the hexagonal pattern of YFP-G-LOV2 on a freshly decorated GRG5RG4RGzdk1 hydrogel (A), the decorated hydrogels after 10 min. (B) and 20 min. (C) blue light irradiation. Scale bar: 100 µm. The contrast was reduced dramatically after 10 min. blue light irradiation, and erased after 20 min. irradiation. (D) Line profile fluorescence intensity analysis of images in (A-C). The arrow in (A) indicated the position of the line profile. 5.4.3 Multi-Dimensional Fluorescence Patterns Generated by Using LOVTRAP-Based Method To achieve higher information capacity and security, multi-dimensional fluorescence images-based 3D codes have been developed330-332, 340-345, which can be real multi-colored generated using fluorophores with different colors or pseudo-3D colored generated from varying fluorescence intensities of one fluorophore. Here we explored the feasibility of developing pseudo-3D color codes using the LOVTRAP-based approach. The principle is to use sufficiently different fluorescence intensity (grey scaled) to generate pseudo colored multi-dimensional fluorescence images. 126 Figure 5.8. Greyscale image information can be written in a hydrogel in the form of gradient ligand pattern. (A & B) A greyscale portrait of Prof. Einstein was selected as model input. The portrait was a 2D array of grey values, which is presented in a pseudo colored heat map (B). (C) the color of the original portrait was inverted. (D) The inverted portrait was printed on a 3M transparency film. Two overlapped layers of printed transparency film were imaged at bright field, which was used later as shadow mask. (E & F) Via one-step method, YFP-G-LOV2 was patterned on a GRG5RG4RGzdk1 hydrogel using the transparency film shadow mask (D). Fluorescence image showed that the YFP fluorescence pattern (E) was greyscale and largely resembled the original input portrait (A), which was further confirmed by comparing the pseudo colored heat map of produced pattern (F) and that of the original portrait (B). (Scale bar = 2 mm) To realize this, the key is to generate sufficiently different fluorescence intensity of the decorated fluorescence using the LOVTRAP-based approach. We reasoned that the amount of decorated/released YFP should depend on the intensity of the blue light irradiation. We tested the feasibility of using a variable transmittance shadow mask to generate grey-scaled fluorescence images on the protein hydrogel using the LOVTRAP approach (Figure 5.8). For this, we printed a greyscale portrait of Albert Einstein on a transparency to serve as a shadow mask. Using the one-step method, we generated the YFP-based fluorescence image of Einstein on the hydrogel using the generated shadow mask. Clearly, the generated fluorescence image of Einstein showed different YFP fluorescence intensities in different regions of the portrait, largely resembling the 127 original greyscale portrait. By converting the fluorescence intensity into greyscale intensity, we converted the YFP image into a pseudo colored heat map. It is evident that the generated YFP heat map largely reproduced the key features of the original shadow mask, suggesting that indeed it is possible to generate pseudo colored multi-dimensional fluorescence images using the LOVTRAP approach. Building on this result, we then tested the feasibility of generating multi-dimensional pseudo colored 3D codes. To increase the data density, we created 4 distinct levels of fluorescence intensity as four accessible states (black, dark grey, light grey and white), and used these four states to generate 3D QR codes (Figure 5.9). Compared with the black-white QR codes (which consist of two accessible states: black (0) and white (1)), the pseudo colored QR codes contain four accessible states, representing bits (00), (01), (10) and (11), and thus contains double amount of information. As a proof-of-principle, we generated 3D codes of 32 bits (Figure 5.9 A-D) and 128 bits (Figure 5.9 E-H) using the LOVTRAP-based approach to pattern the YFP fluorescence. The experimental fluorescence can be readily read using a CCD camera under a cyan light source. Four distinct greyscales of fluorescence intensity were observed on the samples (Figure 5.9 I). Using a simple Python code (Appendix B) originally developed by Wolf et al344, the fluorescence was readily converted to a pseudo colored 3D heat map, which matched the theoretic pattern very well. Clearly, our LOVTRAP-based decoration/release approach can be used to write/erase information-containing pseudo 3D codes. These 3D information codes have the potential of serving as covert security features for reversible information storage, which has higher data density and better security than black-white 2D codes without introducing additional types of fluorophores. 128 Figure 5.9. LOVTRAP can be used to generate multi-dimensional fluorescence images-based 3D codes. (A & E) Greyscale masks encoding 32 bit (A) and 128 bit (E) information. Four accessible greyscales allow for information storage at 2 bits per “pixel”. (B & F) Pseudo color 3D code converted from (A) and (E). (C & G) Fluorescence images generated on the protein hydrogels by using the 32 bit and 128 bit greyscale masks. Four levels of fluorescence intensities can be clearly distinguished from the fluorescence images, and the intensity pattern match that of the masks. (D & H) Pseudo color codes converted from the fluorescence images in (C) and (G). Scale bar = 2.5 mm. (I) Histograms of fluorescence intensities of the four level in (C). The four levels can be clearly distinguished from one another. The mean grey value was 65.4 ± 6.6 for (00), 76.4 ± 10.3 for (01), 120.0 ± 12.4 for (10), and 154.1 ± 16.6 for (11). Data is presented as average ± S.D. The data density of the LOVTRAP-based method is comparable to that of the previously reported sulfoxide-based method utilized on synthetic polymer substrates344, as both methods can achieve 2 bits per “pixel” using four accessible greyscales. It is noteworthy that the distinct levels of fluorescence greyscales are dependent on the distinct levels of the input light intensity. 129 Recently, Gu et al. reported that up to 16 levels of fluorescence greyscales could be photochemically written on a synthetic polymeric gel at the guidance of laser-based photolithography that provided 16 distinct levels of input light intensity345, which indicates a promising direction for further improving the data density of the LOVTRAP-based method. The above-mentioned greyscale pattern was preserved for at least 3 days when the hydrogel sample is stored in sterilized PBS under room temperature (20-23 °C) and ambient indoor light (Figure 5.10 A). Fluorescence intensity pseudo color map showed that its grey value distribution remained largely unchanged before and after the storage. If stored in naturally air-dried sample, a similar greyscale pattern could last for even longer, for more than 70 days, in the same indoor environment (Figure 5.10 B). Such long-term storage rooted in the high affinity between LOV2 and zdk1 in the absence of intense blue light. Figure 5.10. Long-term preservation of fluorescence images on hydrogels. (A) A greyscale YFP-G-LOV2 pattern could be stored in 1x PBS (at the presence of Penicillin/Streptomycin) under ambient indoor light for at least 3 days. (B) A greyscale YFP-G-LOV2 pattern could be stored in a naturally aire-dried hydrogel sample for at least 70 days under ambient indoor light. (Scale bar = 2 mm) 130 5.5 Conclusions In this work, we have demonstrated the utility of purely recombinant protein-based FPHs as information storage media. Light-controlled LOVTRAP-regulated ligand patterning method is a highly specific and spatially precise visual information writing approach. The light dose-sensitive nature of LOVTRAP interaction renders greyscale patterning accessibility, which greatly enhances the storage capacity of the protein-based hydrogel media. In addition, the written information is stable at ambient environments but can be erasable using intense blue light, making the approach suitable for customized secure communication. We anticipate that LOVTRAP-controlled information writing approach will contribute to information storage and anti-counterfeit in next-generation protein-based smart materials. 131 Chapter 6: Conclusions and Outlook 6.1 Conclusions This thesis provides a comprehensive understanding of dynamic protein-based hydrogels. Dynamic hydrogels that can change their physical, mechanical, chemical, or biological properties in response to external stimuli find various biomedical applications. Using stimuli-responsive interactions among proteins or stimuli-responsive protein conformational changes, dynamic protein-based hydrogels can be engineered. In the introductory Chapter 1, state-of-the-art studies of this field are reviewed. The general cross-linking methods for protein-based hydrogels are first introduced. Then the general regulation strategies for constructing dynamic protein-based hydrogels are reviewed. Finally, the future directions of the field are discussed. Chapter 2 demonstrates that the in situ phase transition of ELP side chains can regulate the thermo-responsive properties, including swelling ratio and Young’s modulus, of protein-based hydrogels. The in situ aggregation of Vn-ELP side chains at higher temperatures lead to de-swelling and modulus reinforcement of the (GR)4+Vn-(GR)4-hydrogels, and the amplitude of such property changes is controlled by temperature, chain length, and composition. The thermo-responsive swelling ratio and modulus changes can potentially facilitate the applications of the hydrogels in tissue engineering, drug delivery and mechanobiological studies. In addition, this side chain approach can potentially be utilized to construct other types of dynamic hydrogels. Chapter 3 and Chapter 4 investigate the dynamic viscoelastic properties of protein-based hydrogels that are physically cross-linked by a coiled-coil interaction (for GKE hydrogel) and a non-covalent protein-protein interaction (for LOVTRAP-based hydrogel), respectively. Both GKE hydrogel and LOVTRAP-based hydrogel exhibit highly time-dependent viscoelastic properties. GKE hydrogels display multiple energy dissipation modes. The viscoelastic moduli 132 and dissipation modes of GKE hydrogel can be finely tuned by temperature, ionic strength concentration, composition, and temperature. As for LOVTRAP-based hydrogel, it also exhibits two dissipation modes, which is believed to represent the two states of LOVTRAP interaction. The viscoelastic moduli and dissipation modes of LOVTRAP-based hydrogel are controlled by concentration and light irradiance. Moreover, both GKE hydrogels and LOVTRAP-based hydrogel are excellent injectable hydrogels, which are both shear-thinning and self-healing. The tunable dynamic viscoelasticity and injectability of these two developed hydrogel systems facilitates their potential applications in tissue engineering and drug/cell delivery. Chapter 5 demonstrates the utility of recombinant protein-based hydrogels as information storage media. Yellow fluorescence protein (YFP) ligands can be selectively decorated onto protein-based hydrogels through light-responsive LOVTRAP interaction in a spatiotemporal manner. Such decoration is highly light dose-dependent and reversible due to the light-sensitive and reversible nature of LOVTRAP. With the help of a shadow mask, 2D and 3D fluorescence images can be produced using this method. It is anticipated that this LOVTRAP-controlled method will contribute to information storage and anti-counterfeit in next-generation soft smart materials. Overall, the novel dynamic protein-based hydrogels developed in this thesis can potentially serve as platforms for constructing hydrogel-based biomedical materials. On the one hand, the developed hydrogels are mainly responsive to temperature (for Vn-(GR)4-based hydrogel and GKE hydrogel) or light (for LOVTRAP-based hydrogels or decoration method), which are two types of stimuli that have been widely adapted as therapeutic tools in biomedical studies, such as in thermal therapy347 and targeted phototherapy348 for cancer. On the other hand, the stimuli-responsive property changes in the developed hydrogel systems are highly dose-133 sensitive and highly reversible, which is critical for customized fine tuning of hydrogel functionality. To further optimize the developed hydrogels, their biocompatibility and bio-functionality should be investigated and carefully tailored. We expect that, after further optimization, these developed dynamic protein-based hydrogels will find various applications in biomedical studies. Moreover, the relationships between the microscopic properties of the protein building blocks and the macroscopic properties of the resultant hydrogels are discussed in every chapter of the thesis. We hope that these results will contribute to the quantitative prediction of dynamic hydrogel properties and inspire the bottom-up designs of other dynamic hydrogels in the future. 6.2 Outlook 6.2.1 Utilizing Protein Phase Transitions to Construct Nanogels “Nanogels” are nanosized particles formed by swollen cross-linked polymer networks, which share similar properties including high water content, permeability, and biocompatibility with macroscopic hydrogels while display unique features such as large surface area349. Nanogels are superior drug delivery systems than macroscopic hydrogels and other systems, because their small sizes and surface properties allow them to avoid clearance by Phagocytic cells350, reach the smallest capillary vessels, and penetrate to tissues351. There are two general strategies for the formation of nanogels. They can be formed either by cross-linking pre-gel polymers in solution droplets in a water-in-oil emulsion, or by cross-linking nanosized polymer assemblies such as micelles352. In either strategy, the phase behaviors of the pre-gel polymers play essential roles. Chilkoti et al. fabricated protein-based nanogels based on the micellization of di-block polypeptides comprising hydrophobic and hydrophilic ELPs116. The hydrophobic 134 ELP block was engineered with pAzF groups so that the core of the micelles could be photo-chemically cross-linked into nanogels. In Chapter 2, Vn-(GR)4 copolymers were observed to form nanosized micelles. Further cross-linking these micelles via reactive amino acid residues will potentially result in nanogel formation. Stimuli-controlled drug release is a critical expectation for all drug carriers. Ideally, nanogel drug carriers should only release drug when it senses pathological signals from diseased tissues or receive triggers from certain therapy administration349, 353. The in situ phase transition of proteins has proven capable of rendering protein-based hydrogel particles temperature-dependent permeability and drug release behaviors107. Moreover, recombinant proteins provide versatile platforms for conveniently engineering various stimuli-responsive moieties. For example, cysteine disulfide bonds have been widely utilized for controlled drug delivery into cells in response to the higher GSH concentration in cytoplasm than that in extracellular fluids29, 353. There is truly great potential in recombinant protein-based nanogels for researchers to explore. 6.2.2 Adapting Injectable Recombinant Protein-Based Hydrogels as Bioprinting Inks Three-dimensional (3D) bioprinting is to utilize 3D printing-like techniques to combine cells, biomaterials, and/or growth factors to fabricate biomedical parts that usually imitate native tissues215, 216. The combination of cells and biomaterials are often referred to as “bioprinting inks” or simply “bio-inks”. Injectable hydrogels are ideal building blocks for bio-inks because they provide necessary viscosity for extrusion-based printing processes and generate plug flow that protect incorporated cells from extrusion-induced shear stress217. To adapt injectable hydrogels into printable hydrogels, their capability to resist deformation and maintain 135 programmed shapes after extrusion should be considered217, 218. For example, layer-by-layer printing technology requires each printed filament of bio-inks to maintain a fixed, uniform diameter as well as appropriate contact angles with surrounding substrates354. Stimuli-responsive cross-linking mechanisms of bio-inks are also necessary for their post-printing stabilization via secondary cross-linking to finalize the fabrication of the biomedical parts. Many proteins, including naturally accessible gelatin and collagen as well as recombinantly engineered silk- and elastin-derived proteins, have been intensely adapted into bio-inks because they self-gelate into injectable hydrogels at ambient temperatures and are biocompatible355, 356. To improve the printability of these protein-based hydrogels, alginate is frequently incorporated into such bio-inks to increase viscosity for deformation resistance and effectuate secondary alginate-Ca2+ cross-linking for post-printing stabilization357-360. For example, Wang et al. utilized coaxial nozzle extrusion device to print gelatin/alginate bio-ink into prevascular-like hollow fibers with designed uniform diameter360. Nevertheless, it remains challenging to precisely print more sophisticated bio-mimetic 3D parts, such as porous bone marrow mimics358, 359. In addition, most current studies focus on printing precision, while the post-printing bio-functionality is relatively under-explored. It is anticipated that more and more recombinant protein-based injectable hydrogels (such as the injectable hydrogels developed in Chapter 3 and Chapter 4) that will be adapted into bio-inks in the future, because their well-defined chemical composition and ease of engineering will potentially facilitate sophisticated tailoring of bio-ink properties. 136 6.2.3 Protein-Mediated “4D” Molecule Patterning on Hydrogels 3D and pseudo 3D (greyscale 2D) patterning of functional molecules on hydrogels have been widely achieved, usually through light-responsive conjugation methods (such as LOVTRAP reported in Chapter 5) at the guidance of light patterns. “Four-dimensional (4D)” patterning adds time as one more dimension to 3D patterning. More specifically, 4D allows for sequentially generating different patterns of one or multiple types of molecules on the same hydrogel in a user-controlled order. 4D pattern can be controlled by combination of orthogonal stimuli-responsive conjugation methods. Deforest et al. created “logic gates” by connecting reductant-cleavable disulfide bond, enzyme-cleavable peptide, and light-cleavable oNB group in series or in parallel361. Logic gate-tethered molecules were first linked onto hydrogel networks in bulk, and then released to form patterns only when a unique combination of three orthogonal stimuli (reductant, enzyme, and light) is present. Ideally, such logic gates will allow controlled release of drugs only in response to abnormal combination of disease-associated cues. Alternatively, 4D patterning can be achieved via caging/uncaging of conjugation methods. Deforest et al. engineered SPAAC-caged alkoxyamine groups on hydrogels, so that only when the alkoxyamine groups were photo-uncaged that they could be ligated to aldehyde-modified ligands via oxime reaction362. Through multiple rounds of photo-uncaging under photo mask followed by ligand ligation, different patterns comprising different ligands could be sequentially produced, until all the reactive groups were uncaged. So far, 4D patterning has been limited in synthetic polymer-based hydrogels and synthetic conjugation linkers. Organic synthesis of these linkers is usually inconvenient and may brings in toxic chemicals. As more biofriendly choice, protein-mediated 4D patterning can theoretically be realized by recombinantly associating protein-protein interactions, cleavable 137 proteins, or enzymes that respond to orthogonal stimuli. More likely, protein-mediated 4D patterning can be achieved by caging/uncaging reactive moieties via protein-protein interactions or dynamic protein conformations. Protein-protein interaction has been utilized to sterically inhibit enzyme activity363. 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Journal of the American Chemical Society 2021, 143 (23), 8572-8577. 160 Appendices Appendix A Amino Acid Sequences of All the Proteins Studied A.1 Wild type GB1 MDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE A.2 Resilin GGRPSDSYGAPGGGN A.3 Vn-ELP (VPGVG)n (n = 24, 48, or 72) A.4 CCE LGEVSALEKEVSALEKKNSALEKEVSALEKEVSALEKLG A.5 CCK LGKVSALKEKVSALKEEVSANKEKVSALKEKVSALKELG A.6 SpyTag AHIVMVDAYKPTK A.7 SpyCatcher GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI A.8 LOV2 GSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAAKEL A.9 zdk1 MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANLLAEAKKLNDAQAPK 161 A.10 YFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTATGITLGMDELYK A.11 CFP MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT A.12 GFP MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK 162 Appendix B Python Program for Generating Pseudo Color 3D Heat Maps The Python program that converts grey value color maps is modified from a program reported by Wolf and co-workers344. The codes are presented below.
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Li, Hongbin
Engineering dynamic protein-based hydrogels
Text