COMPOSITE CONTROLLED RELEASE SYSTEM OF NITRIC OXIDE FOR CARDIOVASCULAR TISSUE ENGINEERING APPLICATION ZHANG QINYUAN NATIONAL UNIVERSITY OF SINGAPORE 2013 COMPOSITE CONTROLLED RELEASE SYSTEM OF NITRIC OXIDE FOR CARDIOVASCULAR TISSUE ENGINEERING APPLICATION ZHANG QINYUAN B.Eng. (Hons), National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Zhang Qinyuan 25 September 2013 -i- Abstract Current synthetic vascular grafts for the reconstructive bypass surgeries face the problem of post-surgery restenosis. Localised sustained release of nitric oxide (NO) is a promising strategy to prevent restenotic events such as vascular smooth muscle cells (vSMCs) over-proliferation, and promote the healing of injured endothelial layer. In this project, a composite controlled release system of NO was developed as a potential material for synthetic vascular grafts via the integration of NO-releasing nanoparticle, hydrogel and polymeric film. A new NO-releasing gelatin-siloxane nanoparticle (GS-NO NP) was developed via S-nitrosothiol modification with good NO-releasing property and excellent cytocompatibility. The potential of freeze-thawed poly(vinyl alcohol) (FT-PVA) hydrogels as carriers for GS-NO NP was evaluated from their property changes with nanoparticle addition, suggesting single FT-PVA hydrogel system might be suitable for applications with moderate addition of GS-NO NPs to maintain their hydrogel properties. GS-NO NP-doped PVA hydrogels were further integrated with biaxial stretched poly(ε-caprolactone) films (µPCL) using electrospinning and freezing-thawing (F/T) technique. The integrated composite release system of NO (PCL/PVA/GS-NO) exhibited prolonged NO release for up to 40 days, and enhanced mechanical properties as compared to single FT-PVA hydrogel system, demonstrating the potential to be used for restenosis prevention and as materials for synthetic vascular graft construction. - ii - Acknowledgements The past few years of PhD study have been an unforgettable and fruitful journey in my life that led me to a world full of exciting challenges and amazing facts. It is even a greater fortune to me that there have been so many people being supportive and accompanying me all along the way. I would like to show my deepest gratitude to my supervisors, Assistant Professor Thian Eng San, Professor Teoh Swee-Hin and Associate Professor Li Jun. I would like to thank them for their teaching and guidance in scientific thinking and writing, and their generous support whenever I encountered difficulties in research work or in daily life. From their constant sharing of knowledge and life experiences, I found not only inspirations in scientific research, but also wisdoms of life. This work would not have been possible without their supervision and support. I would also like to give my appreciations to the past and present colleagues and students from BIOMAT Lab and Supramolecular Biomaterials Lab: Zuyong, Lim Jing, Mark, Erin, Yuchun, Zhiyong, Chang Lei, Poon Nian, Ruey Na, Yi Min, Jason, Freddie, Wang Zhuo, Lan Ying, Chengde, Jingling, Zhao Feng, Yuting, Song Xia, Xiaohong, Ping Yuan, and Yin Hui, for their help and the pleasure of their company. My special thanks go to Dr Wen Feng for his advice and constant support on the experimental work. I am also - iii - Publications grateful to Zhao Jing, Tao Li, and Caolemeng for their friendship and kind help in the experiments. I would like to thank Ms Sharen Teo in Mechanical Engineering for her kind assistance in the administrative stuff, and Mr Thomas Tan, Mr Hong Wei Ng and Mr Khalim Abdul in Materials Lab for their help in the experiments all these years. Most importantly, I would like to give my heartfelt thanks to my parents for their trust in my decisions, their cares and love shown in many ways, and their encouragement and unwavering support to me especially during my tough times. With love and gratitude, I dedicate this PhD thesis to them. I also dedicate this PhD thesis to Mr Jinliang Yang, who was my mentor and best friend that gave me the courage to begin this wonderful journey. The work of this thesis was carried out in BIOMAT Lab and Supramolecular Biomaterials Lab with the funding support from the Ministry of Education, Singapore (R 265-000-300-112). I would like to thank National University of Singapore for the research scholarship. - iv - Table of Contents Declaration . i  Abstract . ii  Acknowledgements . iii  Table of Contents . v  List of Figures ix  List of Tables . xiii  List of Abbreviations xiv  Chapter Introduction 1  1.1  Background . 1  1.2  Objectives and Hypotheses . 6  1.3  Scope . 8  Chapter Literature Review 9  2.1  Vascular System 9  2.2  Coronary Arteries 9  2.2.1  Structure and Composition 10  2.2.1.1  Endothelial Cells (ECs) 12  2.2.1.2  Vascular Smooth Muscle Cells (vSMCs) . 13  2.2.2  Response to Injuries . 14  2.2.3  Restenosis of Coronary Artery . 16  2.2.3.1  Pathophysiology of Restenosis . 16  2.2.3.2  Therapeutic Control of Restenosis . 19  2.3  Vascular Tissue Engineering (VTE) . 21  2.3.1  Acellular Approach 21  2.3.2  In vitro Tissue Engineered Blood Vessel (TEBV) 22  2.3.3  Cell Self-Assembled TEBV . 23  2.3.4  Limitations of Current VTE Approaches . 24  2.3.5  Composite Vascular Grafts 25  2.4  Nitric Oxide . 27  2.4.1  Properties of NO 27  2.4.2  NO in Cardiovascular System 29  -v- Table of Contents 2.4.3  NO and Restenosis . 31  2.4.4  NO Donors . 31  2.4.4.1  Organic Nitrate/Nitrites 32  2.4.4.2  Iron-Nitrosyl Complexes 33  2.4.4.3  Sydnonimines . 34  2.4.4.4  C-Nitroso Compounds 34  2.4.4.5  Diazeniumdiolates (NONOates) 35  2.4.4.6  S-Nitrosothiols (RSNOs) 36  2.4.5  Approaches for the Controlled Release of NO 37  2.4.5.1  NO-Releasing Polymeric Membranes 38  2.4.5.2  NO-Releasing Hydrogels/Xerogels 41  2.4.5.3  NO-Releasing Particles 44  2.4.6  2.5  Future Directions of NO-Releasing Materials . 48  Materials and Fabrication Techniques 50  2.5.1  Materials 51  2.5.1.1  Gelatin-Siloxane Nanoparticles 51  2.5.1.2  Poly(vinyl alcohol) (PVA) . 52  2.5.1.3  Poly(ε-caprolactone) (PCL) 54  2.5.2  Fabrication Techniques 56  2.5.2.1  Freezing-Thawing of PVA . 56  2.5.2.2  Biaxial Stretching of Polymer Films 58  2.5.2.3  Electrospinning . 60  Chapter Synthesis of Nitric-Oxide-Releasing S-Nitrosothiol-Modified Gelatin-Siloxane Nanoparticles 62  3.1  Introduction . 62  3.2  Materials and Methods 64  3.2.1  Materials 64  3.2.2  Preparation of Gelatin-Siloxane Nanoparticles (GS NPs) . 65  3.2.3  Preparation of RSNO-Modified GS NPs (GS-NO NPs) 65  3.2.4  Morphological Characterisation . 67  3.2.4.1  Dynamic Light Scattering (DLS) . 67  3.2.4.2  Field Emission Scanning Electron Microscopy (FESEM) . 67  3.2.5  Composition Analysis 67  3.2.5.1  Fourier Transform Infrared Spectroscopy (FTIR) . 67  3.2.5.2  Ultraviolet-Visible (UV-Vis) Spectrophotometry 68  3.2.5.3  Acid Orange (AO7) Assay 68  3.2.5.4  Ellman’s Reaction 69  - vi - Table of Contents 3.2.6  Release Kinetics of NO from GS-NO NPs 69  3.2.7  Cell Culture 71  3.2.8  AlamarBlue® Assay . 71  3.2.9  Confocal Laser Scanning Microscopy (CLSM) 72  3.2.9.1  Fluorescent DAPI Cell Nucleus Staining . 72  3.2.9.2  In Vitro Cellular Uptake of GS-NO NPs 72  3.2.10  WST-1 Assay . 73  3.2.11  Data Analysis . 73  3.3  Results . 74  3.3.1  Synthesis of GS-NO NPs . 74  3.3.2  Morphologies of GS and GS-NO NPs . 74  3.3.3  Chemical Structure of GS-NO NPs . 77  3.3.4  NO Release from GS-NO NPs . 80  3.3.5  Cytotoxicity of GS-NO NPs 81  3.3.6  AoSMC Cellular Responses to GS-NO NPs . 84  3.3.7  HUVEC Cellular Responses to GS-NO NPs . 86  3.4  Discussion . 87  3.5  Summary . 94  Chapter Properties of Physically Crosslinked Poly(vinyl alcohol) Hydrogels with the Addition of Gelatin-Siloxane Nanoparticles 96  4.1  Introduction . 96  4.2  Materials and Methods 98  4.2.1  Materials 98  4.2.2  Preparation of GS NPs . 98  4.2.3  Preparation of FT-PVA/GS Hydrogels 98  4.2.4  Swelling Test . 99  4.2.5  Mechanical Properties 100  4.2.5.1  Tensile Test 100  4.2.5.2  Compression Test . 101  4.2.6  Differential Scanning Caloriometry (DSC) . 102  4.2.7  Field Emission Scanning Electron Microscopy (FESEM) 102  4.2.8  Statistical Analysis . 102  4.3  Results . 103  4.3.1  Swelling Behaviour of FT-PVA/GS Hydrogels 103  4.3.2  Mechanical Properties of FT-PVA/GS Hydrogels 106  4.3.2.1  Tensile Properties . 106  4.3.2.2  Compressive Properties 111  - vii - Table of Contents 4.3.3  Crystallinity of FT-PVA/GS Hydrogels 116  4.3.4  Structural Properties of FT-PVA/GS Hydrogels . 116  4.4  Discussion . 119  4.5  Summary . 126  Chapter Development of a Composite Controlled Release System of Nitric Oxide 128  5.1  Introduction . 128  5.2  Materials and Methods 130  5.2.1  Materials 130  5.2.2  Preparation of µPCL Films 130  5.2.3  Preparation of µPCL Films with Electrospun PCL Fibres (cPCL) 131  5.2.4  Preparation of GS-NO NPs 131  5.2.5  Preparation of PCL/PVA/GS-NO Composites 131  5.2.6  Differential Scanning Caloriometry (DSC) . 132  5.2.7  Water Contact Angle (WCA) . 132  5.2.8  Morphological Characterisation . 133  5.2.8.1  Field Emission Scanning Electron Microscopy (FESEM) . 133  5.2.8.2  Confocal Laser Scanning Microscopy (CLSM) . 133  5.2.9  Swelling Test . 133  5.2.10  Tensile Test 134  5.2.11  Release Kinetics of NO from PCL/PVA/GS-NO Composites 134  5.2.12  Statistical Analysis . 135  5.3  Results . 135  5.3.1  Assembly of PCL/PVA/GS-NO Composites . 135  5.3.2  Surface Hydrophilicity of PCL/PVA/GS-NO Composites 137  5.3.3  Morphological Properties of PCL/PVA/GS-NO Composites 138  5.3.4  Swelling Behaviour of PCL/PVA/GS-NO Composites . 141  5.3.5  Mechanical Properties of PCL/PVA/GS-NO Composites . 142  5.3.6  NO Release from PCL/PVA/GS-NO Composites . 146  5.4  Discussion . 147  5.5  Summary . 152  Chapter Conclusions . 153  Chapter Future Work . 157  References . 160  Appendix A - 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Journal of Biomedical Materials Research Part A, 2013: p. n/a-n/a. 380. de Oliveira, M.G., et al., Thermal Stability of Primary S-Nitrosothiols:  Roles of Autocatalysis and Structural Effects on the Rate of Nitric Oxide Release. The Journal of Physical Chemistry A, 2002. 106(38): p. 8963-8970. - 188 - Appendix A - Publications International Publications 1. QY Zhang, ZY Wang, F Wen, L Ren, J Li, SH Teoh, ES Thian. GelatinSiloxane Nanoparticles to Deliver Nitric Oxide for Vascular Cell Regulation: Synthesis, Cytocompatibility and Cellular Responses. Journal of Biomedical Materials Research: Part A. (Under review) 2. QY Zhang, ZY Wang, J Lim, J Li, F Wen, SH Teoh. Tailoring of Poly(vinyl alcohol) Hydrogels Properties by Incorporation of Crosslinked Acrylic Acid. Defense Science Research Conference and Expo (DSR), 2011, 1-3. (DOI: 10.1109/DSR.2011.6026842) 3. ZY Wang, E Teo, M Chong, QY Zhang, J Lim, ZY Zhang, MH Hong, ES Thian, J Chan, SH Teoh. Biomimetic Three-Dimensional Anisotropic Geometries by Uniaxial Stretch of Poly(ε-caprolactone) Films for Mesenchymal Stem Cell Proliferation, Alignment, and Myogenic Differentiation. Tissue Engineering Part C: Methods. 19 (7), 538-549. 4. ZY Wang, J Lim, YS Ho, QY Zhang, M Chong, M Tang, MH Hong, J Chan, SH Teoh, ES Thian. Biomimetic Three-Dimensional Anisotropic Geometries by Uniaxial Stretching of Poly(ε-caprolactone) Films: Degradation and Mesenchymal Stem Cell Responses. Journal of Biomedical Materials Research Part A. (DOI:10.1002/jbm.a.34899) 5. ZY Wang, L Jing, QY Zhang, E Teo, F Wen, J Chan, SH Teoh, MH Hong. Responses of Human Fetal Mesenchymal Stem Cells to Various Poly(εcaprolactone) Films: A Comparison Study. Defense Science Research Conference and Expo (DSR), 2011, 1-3. (DOI: 10.1109/DSR.2011.6026841) 6. J Lim, ZY Wang, QY Zhang, E Teo, F Wen, SH Teoh. Blending of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and Polycaprolactone: Characterization and Degradation Studies. Defense Science Research Conference and Expo (DSR), 2011, 1-3. (DOI: 10.1109/DSR.2011.6026840) - 189 - Appendix A - Publications International Conferences Oral Presentations 1. QY Zhang, ZY Wang, SH Teoh, J Li, L Ren, ES Thian. Synthesis of Nitric-Oxide-Releasing S-nitrosothiol-modified Gelatin-Siloxane Nanoparticles. 6th East Asian Pacific Student Workshop on NanoBiomedical Engineering. 23-24 March 2013, Singapore. 2. QY Zhang, ZY Wang, J Lim, E Teo, L Ren, CN Lee, J Li, ES Thian and SH Teoh. Nitric-Oxide-Releasing System Based on Gelatin-Siloxane Nanoparticles for Cardiovascular Applications. 9th World Biomaterials Congress. 1-6 June 2012, China. 3. QY Zhang, ZY Wang, J Lim, J Li, F Wen, SH Teoh. Tailoring of Poly(vinyl alcohol) Hydrogels Properties by Incorporation of Crosslinked Acrylic Acid. Defense Science Research Conference and Expo (DSR), 3-5 August 2011, Singapore. 4. ZY Wang, J Lim, QY Zhang, M Chong, E Teo, ZY Zhang, MH Hong, J Chan, SH Teoh. Direct Cellular Contact and Alignment of Human Fetal Mesenchymal Stem Cells and Umbilical Vein Endothelial Cells Based on Micro-patterned Porous Poly(ε-caprolactone) Film for Tissue Engineering Applications. 9th World Biomaterials Congress. 1-6 June 2012, China. 5. ZY Wang, L Jing, QY Zhang, E Teo, F Wen, J Chan, SH Teoh, MH Hong. Responses of Human Fetal Mesenchymal Stem Cells to Various Poly(εcaprolactone) Films: A Comparison Study. Defense Science Research Conference and Expo (DSR), 3-5 August 2011, Singapore. 6. J Lim, ZY Wang, QY Zhang, E Teo, F Wen, SH Teoh. Blending of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and Polycaprolactone: Characterization and Degradation Studies. Defense Science Research Conference and Expo (DSR), 3-5 August 2011, Singapore. Poster Presentations 1. QY Zhang, ZY Wang, F Wen, L Ren, J Li, SH Teoh, ES Thian. Development of Nitric-Oxide-Releasing Gelatin-Siloxane Nanoparticles for Angiogenesis Regulation in Regenerated Bone Tissue. International Bone-Tissue-Engineering Congress, 16-19 December 2013, Singapore. 2. QY Zhang, F Goh, ZY Wang, J Lim, E Teo, J Li, ES Thian, SH Teoh. Properties of Physical Crosslinked Polyvinyl Alcohol Hydrogel for Controlled Release of Nitric-Oxide-Releasing Nanoparticles. 1st International Conference for Young Researchers in Advanced Materials. 1-5 July 2012, Singapore. - 190 - Appendix A - Publications 3. QY Zhang, F Goh, J Lim, ZY Wang, ES Thian, J Li, SH Teoh. Physical Properties of Freeze-Thawed Polyvinyl Alcohol Hydrogel for Cartilage Tissue Engineering Applications. 9th World Biomaterials Congress. 1-6 June 2012, China. 4. ZY Wang, QY Zhang, J Lim, E Teo, MH Hong, J Chan, ES Thian, SH Teoh. Bioresorbable 3D Micro-Topographical Structures for Vascular Cellular Pattern and Differentiation Control. 1st International Conference of Young Researchers on Advanced Materials. 1-5 July 2012, Singapore. 5. ZY Wang, J Lim, QY Zhang, M Chong, E Teo, ZY Zhang, MH Hong, J Chan, SH Teoh. Direct Cellular Contact and Alignment of Human Fetal Mesenchymal Stem Cells and Umbilical Vein Endothelial Cells Based on Micro-patterned Porous Poly(ε-caprolactone) Film for Tissue Engineering Applications. 9th World Biomaterials Congress. 1-6 June 2012, China. 6. ZY Wang, YS Ho, J Lim, QY Zhang, MH Hong, J Chan, SH Teoh. Highly Orientated 3D Micro-Topographical Structures for Bone Tissue Engineering Applications: A Degradation Study. 9th World Biomaterials Congress. 1-6 June 2012, China. - 191 - Appendix B - Awards 1. Oral Presentation Award. 6th East Asian Pacific Student Workshop on Nano-Biomedical Engineering. 23-24 March 2013, Singapore. 2. Young Scientist Award. 9th World Biomaterial Congress. 1-6 June 2012, China. - 192 - [...]... Sodium Hydroxide NO Nitric Oxide NONOate Diazeniumdiolate NOS Nitric Oxide Synthases NP Nanoparticle PBS Phosphate Buffered Saline PCI Percutaneous Coronary Intervention PCL Poly(ε-caprolactone) PCL/PVA/GS-NO Integrated Composite Controlled Release System of Nitric Oxide PDGF Platelet-Derived Growth Factor PDI Polydispersity Index PEG Poly(ethylene glycol) PEI Poly(ethylenimine) PEO Poly(ethylene oxide) ... film The integrated composite system is expected to show not only the ability of controlled release of NO, but also improved mechanical properties as compared to single hydrogel release system, and the potential to be used as a material for synthetic vascular graft construction As NO plays various physiologic roles in the biological system, the successful construction of the composite system also implies...List of Figures Figure 1-1 Schematic illustration of the proposed work 6 Figure 2-1 Anatomy of the coronary arteries of the heart [41]……… 10 Figure 2-2 Layered structure of a coronary artery [45]……………… 11 Figure 2-3 Schematic of the basic physiological functions of nitric oxide in the cardiovascular system (Blue arrows: inhibitive actions; red arrow:... (a, b) FESEM images of the cross-sections of PCL/PVA/5GS-NO, (c, d) FESEM images of the cross-sections of PCL/PVA/10GS-NO and (e) CLSM image of the cross-section of hydrated PCL/PVA/5GS-NO (Red arrows: GS-NO NPs; white arrows: electrospun PCL fibres; scale bar = 100 μm for a and c, 10 μm for b and d, and 500 μm for e) 140 - xi - List of Figures Figure 5-5 Dynamic swelling of (a) PCL/PVA, (b)... demonstrates the feasibility of assembling components with superior properties in different fields to improve the systematic performance, which may provide some inspirations in engineering design of biomedical devices such as synthetic vascular grafts -5- Chapter 1 Introduction 1.2 Objectives and Hypotheses The schematic illustration of the proposed composite controlled release system of NO is depicted in... be adsorbed by PCL fibrous matrix with the aid of a vacuum The composite will then be subjected to freezing-thawing (F/T) cycles to crosslink the hydrogel as the host of GS-NO NPs Figure 1-1 Schematic illustration of the proposed work -6- Chapter 1 Introduction The primary goal of this study is therefore to construct a composite controlled release system of NO using nanoparticles, tailored PVA hydrogel... literature reviews on the pathology of restenosis, the biology and role of NO in cardiovascular system, current therapeutic applications of NO, and approaches for controlled release of NO Literature reviews on gelatin-siloxane nanoparticles (GS NPs), PVA and PCL materials and the relevant fabrication techniques are also included Chapter 3 presents the synthesis and properties of the novel NOreleasing gelatin-siloxane... use of nanoparticles in controlled release of NO Fumed silica nanoparticles and gold nanoparticles have been modified to release NO through derivatisation of the particle’s surface with functional groups [33-35] A unique advantage of this approach is that these particles can be blended into a wide variety of biomedical polymers without altering the fundamental chemistry of the polymer backbone Therefore,... details the development of a composite controlled release system of NO as an improved material for synthetic vascular graft construction, which is capable of releasing NO in a slow and sustained manner to prevent restenotic events such as vSMC over-proliferation A novel NO-releasing gelatin-siloxane nanoparticle (GS-NO NP) is synthesised and incorporated into a composite architecture formed by poly(vinyl... intermediate; and 4 The integrated composite controlled release system of NO consisting of NO-releasing nanoparticles, PVA hydrogel and supporting PCL film has improved systematic properties and performance than each single component -7- Chapter 1 Introduction 1.3 Scope This dissertation includes seven chapters Chapter 1 gives the background information to the field of research and the research proposal, . COMPOSITE CONTROLLED RELEASE SYSTEM OF NITRIC OXIDE FOR CARDIOVASCULAR TISSUE ENGINEERING APPLICATION ZHANG QINYUAN NATIONAL UNIVERSITY OF SINGAPORE 2013 COMPOSITE. COMPOSITE CONTROLLED RELEASE SYSTEM OF NITRIC OXIDE FOR CARDIOVASCULAR TISSUE ENGINEERING APPLICATION ZHANG QINYUAN B.Eng. (Hons), National University of Singapore . healing of injured endothelial layer. In this project, a composite controlled release system of NO was developed as a potential material for synthetic vascular grafts via the integration of NO-releasing