Self assembly peptide amphiphile biomimetic materials for biomedical applications

SELF-ASSEMBLING PEPTIDE-AMPHIPHILE BIOMIMETIC MATERIALS FOR BIOMEDICAL APPLICATIONS LUO JINGNAN NATIONAL UNIVERSITY OF SINGAPORE 2012 SELF-ASSEMBLING PEPTIDE-AMPHIPHILE BIOMIMETIC MATERIALS FOR BIOMEDICAL APPLICATIONS LUO JINGNAN (M. Eng., B. Eng., Tian Jin University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 To my dearest parents and sisters To my beloved, Zhang Ying ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest and most sincere gratitude to my thesis advisor, Professor Tong Yen Wah, for giving me not only the opportunities to learn and grow but also freedom to try and err. I am sincerely grateful to his invaluable patience and advice not only in scientific but also in personal matters, such as career planning and job seeking. Thank you for your patience and guidance. I would like to thank Professor Jiang Jianwen and Professor Yang Kun-Lin for their generous time and guidance during my Ph.D. qualifying examination. I also would like to thank all past and present members of the Tong’s group, but particularly: Khew Shih Tak, Wiradharma Nikken, Koh Shirlaine, Chen Wen Hui, Niranjani Sankarakumar, Liang Youyun, Chen Yiren, Anjaneyulu Kodali, Wang Honglei, Xie Wenyuan, Ajitha Sundaresan, He Fang, Guo Zhi, Wang Bingfang, Sushumitha Sundar, and Lee Jonathan, for unconditional help and invaluable support. I also am grateful to other group members, especially Tan Weiling, Duong Hoang Hanh Phuoc, Deny Hartono, Harleen Kaur, Fong Kah Ee, and Meng Qiao, for providing a pleasant working environment. Additionally, I would like to thank the Department of Chemical and Biomoleclar Engineering, National University of Singapore for providing me the research scholarship and research facilities that make this study possible. Finally, I would like to thank my parents and sisters for their supports on my study. Their unconditional love, support and guidance has made me who I am today. Lastly, I would like to thank my best companion, Zhang Ying, for her care, love and selfless support. i TABLE OF CONTENTS ACKNOWLEDGEMENTS . i TABLE OF CONTENTS ii ABSTRACT . viii LIST OF TABLES . xi LIST OF FIGURES xii CHAPTER . INTRODUCTION 1.1 Background . 1.2 Hypothesis . 1.3 Research objectives . CHAPTER . LITERATURE REVIEW . 2.1 Regenerative medicine and biomaterials . Biomaterials . 10 2.2 The mimicking template: the extracellular matrices (ECM) . 13 Integrins . 14 The extracellular binding to integrins 15 The need of ECM mimics 16 Collagen . 18 Collagen mimics 21 ECM adhesive proteins and their mimics 25 ii 2.3 The mimicking means: molecular self-assembly 26 Self-assembling peptide systems . 26 Peptide amphiphiles . 28 Approaches to program PA self-assembly 31 Functionalization of self-assembled PA nanostructures 33 CHAPTER . 35 MATERIALS AND METHODS 35 3.1 Materials 35 3.2 Experimental section of chapter . 35 Peptide synthesis 35 Critical micelle concentration (CMC) . 36 Self-assembly of CPAs into nanofibers . 37 Transmission electron microscopy 37 Circular Dichroism Spectroscopy 38 Melting studies 38 Cell culture 39 Cell adhesion assay 39 Immunofluorescence staining 40 Statistical analysis . 41 3.3 Experimental section of chapter . 41 Microsphere Preparation 41 Porous polymeric scaffolds . 41 Peptide synthesis 42 Transmission electron microscopy 42 iii Network structure of peptide hydrogel 43 Visualization of cell–nanofiber interaction . 44 Cell culture 44 Cell adhesion assay 44 Cell spreading assay 45 Hybrid gel/scaffold system 46 Hybrid cell/gel/scaffold system . 46 Cell proliferation 47 Albumin secr 48 Statistical analysis . 48 3.4 Experimental section of chapter . 48 Peptide synthesis 48 Fiber formation and gelation of PAs . 49 Transmission electron microscopy 49 Scanning Electron Microscopy 50 Stop-flow analysis . 50 3.4 Experimental section of chapter . 51 Peptide synthesis 51 Preparation of assembled PA nanostructures 51 Transmission Electron Microscopy . 52 Circular Dichroism Spectroscopy 53 Dynamic Light Scattering 54 CHAPTER . 55 iv SEFL-ASSEMBLY OF COLLAGEN-MIMETIC PEPTIDE AMPHIPHILE INTO BIOFUNCTIONAL NANOFIBER 55 4.1 Introduction . 55 4.2 Results and Discussion 57 Design and synthesis of collagen-mimetic peptide amphiphiles . 57 TEM study of morphological structure . 60 CD spectra . 63 Melting point study 64 Cell adhesion assay 69 Immunofluorescence staining 71 4.3 Conclusions . 72 CHAPTER . 74 THREE-DIMENSIONAL POROUS SCAFFOLD FILLED WITH ECM-MIMETIC HYDROGEL TO OPTIMIZE LIVER CELL DISTRIBUTION, PROLIFERATION AND FUNCTION 74 5.1 Introduction . 74 5.2 Results and Discussion 77 Fabrication of ECM-mimetic fibrous hydrogel . 77 Cell adhesion and spreading on ECM-mimetic nanofibers . 83 Preparation of 3D porous PLGA scaffold . 85 Hybrid gel/scaffold system 88 Cell growth and distribution unto scaffolds 89 Cell proliferation and function 92 5.3 Conclusions . 94 CHAPTER . 95 v HIERARCHICAL SELF-ASSEMBLY OF PEPTIDE AMPHIPHILES INTO FIBER BUNDLES MEDIATED BY THE RGDS CELL-BINDING MOTIF . 95 6.1 Introduction . 95 6.2 Results and discussion . 97 Design of peptide amphiphile 97 Characterization of self-assembled PA fibers . 100 Proposed mechanism of hierarchical self-assembly 101 6.3 Conclusions . 108 CHAPTER . 110 POST-ASSEMBLY POLYMERIZATION OF PEPTIDE AMPHIPHILE NANOFIBERS TO ENHANCE FIBER STABILITY AND CONTROL FIBER LENGTH 110 7.1 Introduction . 110 7.2 Results and discussion . 111 The strategy of post-assembly polymerization and scission . 111 The design of PAs with unsaturated alkyl chain . 112 The stability of PA nanofibers 117 The length control of PA nanofibers . 119 7.3 Conclusions . 120 CHAPTER . 122 CONCLUSIONS AND RECOMMENDATIONS . 122 REFERENCES . 127 APPENDIX A . 149 APPENDIX B . 150 vi vii Reference Janet, A. and H. 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OPTN: Organ Procurement and transplantation Network. http://optn.transplant.hrsa.gov/ (1/1/2012) 148 APPENDIX A APPENDIX A LIST OF AMINO ACIDS Table A.1. Letter codes of naturally occurring and non-natural (marked with *) amino acids. Amino acids letter code letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid/ Aspartate Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid/ Glutamate Glu E Glycine Gly G Histidine His H Hyp* O* Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophane Trp W Tyrosine Tyr Y Valine Val V Hydroxyproline* 149 APPENDIX B APPENDIX B LIST OF PUBLICATIONS Journal Papers: 1. Jingnan Luo, Yen Wah Tong, Self-assembly of collagen-mimetic peptide amphiphiles into biofunctional nanofiber, ACS Nano, 2011, (10), 7739-7747 2. Jingnan Luo, Yen Wah Tong, Hierarchical self-assembly of peptide amphiphiles into fiber bundles mediated by the cell binding motif RGDS, ChemComm, submitted. 3. Jingnan Luo, Yen Wah Tong, Enhancing stability and controlling length of selfassembling peptide nanofibers via polymerization and cutting process, JACS submitted. 4. Jingnan Luo, Yen Wah Tong, Three Dimensional Porous Scaffold Filled with Co-assembled Peptide Amphiphile Hydrogel to Optimize Cell Distribution, Proliferation and Function. To be submitted. Conference Papers: 5. Jingnan Luo, Yen Wah Tong, 2011, AlChE Annual Meeting, Minneapolis, USA 6. Jingnan Luo, Yen Wah Tong, 2011, the 5th WACBE Congress on Bioengineering, Taiwan 7. Jingnan Luo, Yen Wah Tong, 2011, Tissue Engineering & Regenerative Medicine International Society (TERMIS): Asia Pacific Meeting, Singapore 150 APPENDIX B 8. Jingnan Luo, Yen Wah Tong, 2010, TERMIS-Asia Pacific Meeting, Sydney, Australia 9. Jingnan Luo, Yen Wah Tong, 2010, 4t East Asian Pacific Student Work shop on Nano-Biomedical Engineering, Singapore 151 [...]... requirement for biomaterials and increasing appreciation of the functionality of biological matrix caused scientists to consider nature for design and fabrication inspiration for new generation of biomimetic materials This thesis was designed to develop new class of biomimetic materials that closely resembled the roles of natural materials and held great potential for a number of biomedical applications, ... great potential to engineer biomimetic materials for biomedical applications 1.2 Hypothesis It is hypothesized that the design and fabrication inspiration that nature offers, such as the ECM and molecular self- assembly, may result in new generation of biomimetic materials that structurally and functionally resemble biological matrices and are of capacities to biomedical applications 3 Chapter 1 1.3... to develop new class of biomimetic materials that closely resemble the features of natural materials for biomedical applications, through using peptides as the building blocks, native ECM as the mimicking template, and selfassembling PA system as the mimicking means The specific aims of the thesis include: 1) Fabricate collagen-mimetic peptide amphiphiles (CPAs) capable of selfassembling into nanofibers... of materials for biomedical applications However, the intrinsic problems of using animalderived proteins, such as poor reproducibility, possible immunogenicity, and potential risk of disease transmission, severely limit their applications in body, thus necessitating the fabrication of biomimetic materials closely resembling native ECM In recent years, the use of peptides to construct biomimetic materials. .. facilities construction of information-rich, intricate architectures in a highly reproducible manner with minimal energy input In recent years, several molecular self- assembling systems, such as selfcomplementary ionic peptides, α-helical coiled-coil peptides, β-hairpin peptides, and single-tail peptide amphiphiles (PAs), have been developed and used to fabricate biomaterials for regenerative medicine... host-biomaterials relationship and identify bioactive components for improving restoration, leading to new design principles and synthetic strategies for biomaterials The “second generation” biomaterials capable of eliciting a desired response from the host tissue have been developed through incorporating the bioactive components into synthetic materials These bioactive materials not only perform mechanical... materials and held great potential for a number of biomedical applications, through using peptides as the building blocks, the extracellular matrix (ECM) as the mimicking template, and peptideamphiphile self- assembly as the mimicking means The first part of the thesis was to fabricate collagen-mimetic peptide amphiphiles (CPAs) to structurally and biologically resemble fibrous collagen that is the... prepared through a co -assembly strategy The hybrid nanofibers will be assessed for the ability to form 3D fibrous network and promote cell adhesion and spreading The fabricated ECM-mimetic materials will further be infused into a 3D porous architecture to form biomimetic scaffold to optimize liver cell distribution, proliferation and function (Chapter 5) 3) Develop a hierarchical self- assemble pathway... collagen fiber bundle formation A hierarchical self- assembly pathway to form PA fiber bundles by inducing and controlling inter-nanofibers interactions using the bioactive motif RGDS will be developed and investigated Mechanism beyond the hierarchical self- assembly of PAs into fiber bundles will be proposed and proved 3D architecture built up from fiber bundles will be assessed for pore size and permeability... Chapter 2 effect of implanted materials in body, engineers, chemists, and biologists, in collaboration with physicians, were formalizing design principles and synthetic strategies for biomaterials The important principle that the release of toxic from implanted materials would adversely affect healing was realized and applied to design of implanted materials Based on the formalized design principles, . SELF- ASSEMBLING PEPTIDE- AMPHIPHILE BIOMIMETIC MATERIALS FOR BIOMEDICAL APPLICATIONS LUO JINGNAN NATIONAL UNIVERSITY OF SINGAPORE 2012 SELF- ASSEMBLING PEPTIDE- AMPHIPHILE. mimicking means: molecular self- assembly 26 Self- assembling peptide systems 26 Peptide amphiphiles 28 Approaches to program PA self- assembly 31 Functionalization of self- assembled PA nanostructures. biomimetic materials. This thesis was designed to develop new class of biomimetic materials that closely resembled the roles of natural materials and held great potential for a number of biomedical applications,