2807400853 INTERACTION OF BLOOD COMPONENTS WITH POLYMERS AND BIOMIMETIC SURFACES By BRENDA HALL B.Sc A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the University of London February 1990 The Royal Free Hospital School of Medicine Rowland Hill Street LONDON NW3 2PF 1 ProQuest Number: U542841 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U542841 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT INTERACTION OF BLOOD COMPONENTS WITH POLYMERS AND BIOMIMETIC SURFACES By BRENDA HALL The work presented in this thesis describes the use of in vitro tests to monitor the blood compatibility of biomimetic surfaces. Phospholipids which predominate on the outer membrane surface of normal blood cells contain the phosphorylcholine polar head group. These surfaces are naturally non-thrombogenic. Phospholipids and polymers which contain the phosphorylcholine polar head group have been coated onto various substrates and analysed in terms of whole blood clot formation (as measured by the technique of Material Thrombelastography), protein adsorption, platelet and cellular interactions. Using Material Thrombelastography phospholipids which contain the phosphorylcholine group were found to limit the activation of clotting factors and reduce the rate and strength of clot formation in comparison to results obtained by negatively charged phospholipids. Furthermore, a polymerisable diacetylenic phospholipid and some polyesters which contain the phosphorylcholine group showed improved blood compatibility as compared to conventional materials. Phosphorylcholine lipids were also found to limit serum protein adsorption onto liposomes prepared from these lipids in contrast to negatively charged phospholipids. In particular dipalmitoylphosphatidylcholine was found to limit the adsorption of proteins (fibrinogen, & -globulin and serum) onto polyethylene sheets coated with this lipid, and limit platelet and cellular surface interactions. Further studies have also been made to analyse polymer substrates which have been modified in order to covalently link phosphorylcholine groups to the surface. These results have been discussed in relation to the development of novel biomaterials which mimic natural biological membranes. 2 ACKNOWLEDGEMENTS I wish to express my sincere thanks to Professor Dennis Chapman F.R.S. for his supervision and guidance during this project. I would like to thank Mr R. le R. Bird and Miss S. Brown for their contribution to the analysis of the thrombelastography and cell spreading data respectively. I am also grateful to Dr D. Perry and Miss J. Bowles (BP Research Centre) for their help and useful discussions relating to ESCA analysis, to Dr A. A. Durrani for supplies of diacetylenic phosphatidylcholine and phosphorylcholine derivatizing reagents and to Mr I. Azpiazu and Dr G. Davies for assistance in the protein adsorption studies. The technical help of Mrs C. Hall is also gratefully acknowledged. Finally, I acknowledge the financial support of the Science and Engineering Research Council. 3 CONTENTS Page No Abstract 2 Acknowledgements 3 Contents 4 List of Figures 10 List of Tables 15 List of Abbreviations 16 Chapter 1: Blood Coagulation and Biomaterials 17 1.1 Introduction 18 1.2 The Blood Coagulation Pathway 23 1.2.1 Intrinsic Pathway 24 1.2.2 Extrinsic Pathway 27 1.2.3 Common Pathway 27 1.2.4 Control of Coagulation 30 1.3 Polymers in Medicine and Surgery 31 1.4 Testing Methods 37 1.4.1 In vitro tests 39 1.4.2 Ex vivo tests 43 1.4.3 In vivo tests 44 1.4.4 Surface analysis techniques 46 Chapter 2: Biological Membranes andBiomembrane 50 Mimicry 2.1 Biomembrane Structure and Function 51 4 Page No 2.2 Membrane Lipids 54 2.3 Membrane Asymmetry 59 2.4 Biomembrane Mimicry 68 Chapter 3: Thrombelastography 74 3.1 Introduction 75 3.1.1 The principle of thrombelastography 75 3.1.2 Interpretation of the 79 thrombe1astogram 3.1.3 Clinical applications of 82 thrombelastography 3.1.4 Material Thrombelastography 87 3.2 Materials and Methods 91 3.2.1 Dip-coating method 91 3.2.2 Preparation of DPPC liposomes 92 3.2.3 Preparation of polymer surfaces 92 3.2.4 Thrombelastographic technique 95 3.3 Results 97 3.3.1 Standardisation 97 3.3.2 MTEG analysis of DPPC coating 99 3.3.3 MTEG analysis of various lipid 104 classes 3.3.4 MTEG analysis of polymeric 110 materials 3.4 Discussion 120 5 Page No 3.5 Summary 128 Chapter 4: Protein Adsorption Studies 130 4.1 Introduction 131 4.1.1 Protein interaction with liposomes 131 4.1.2 Protein interaction with polymers 133 4.2 Materials and Methods 138 4.2.1 Protein adsorption onto phospholipid 138 vesicles 4.2.1.1 Liposome preparation 138 4.2.1.2 Measurement of protein 139 adsorption 4.2.1.3 Total protein assay 139 4.2.1.4 Phosphate assay 140 4.2.1.5 SDS-PAGE 141 4.2.2 Protein adsorption onto polymers 141 4.2.2.1 125I studies 142 4.2.2.2 FTIR studies 143 4.3 Results 146 4.3.1 Liposome studies 146 4.3.2 125I studies 152 4.3.3 FTIR studies 153 4.4 Discussion 164 4.5 Summary 170 6 Page No Chapter 5: Platelet Studies and Measurement 171 of Plasma Clotting Times 5.1 Introduction 172 5.1.1 Platelet structure and function 172 5.1.1.1 Platelet adhesion 175 5.1.1.2 Platelet activation 177 5.1.1.3 Platelet secretion 179 5.1.1.4 Platelet interaction with 180 polymeric surfaces 5.1.2 Plasma clotting time tests 182 5.2 Materials and Methods 184 5.2.1 Platelet studies 184 5.2.1.1 Surface treatment 184 5.2.1.2 Platelet interaction with 184 surfaces 5.2.1.3 Assessment of platelet 185 number 5.2.1.4 Scanning electron 185 microscopic studies 5.2.2 Plasma clotting time measurements 186 5.2.2.1 Surface treatment 186 5.2.2.2 Partial thromboplastin 186 time 5.2.2.3 Activated partial 187 thromboplastin time 5.3 Results 188 5.3.1 Assessment of platelet number 188 7 Page No 5.3.2 Scanning electron microscopy 189 5.3.3 Plasma clotting time measurements 193 5.4 Discussion 198 5.5 Summary 203 Chapter 6: Studies of Derivatized Polymers 205 Using Surface Analysis Techniques 6.1 Introduction 206 6.1.1 Phosphorylcholine derivatization 206 6.1.2 ESCA theory and applications to 210 the study of biomaterials 6.1.3 Fourier Transform Infrared 217 Spectroscopy 6.1.3.1 Attenuated total reflection 217 (ATR) 6.2 Materials and Methods 222 6.2.1 Glycidyl acrylate plasma glow 222 discharge treatment of polymers 6.2.2 Preparation of phosphorylcholine 223 reagents 6.2.3 Phosphorylcholine derivatizationof 224 hydroxylated polymers 6.2.4 ESCA analysis 224 6.2.4.1 Derivatization of 226 hydroxylated polymers 6.2.5 FTIR analysis 226 6.3 Results 228 6.3.1 Hydroxylation of polymers 228 8 Page No 6.3.2 Derivatization of surface 234 hydroxyl groups 6.4 Discussion 247 6.5 Summary 255 Chapter 7: Cell Interactions with Modified 256 Glass I Surfaces 7.1 Introduction 257 7.2 Materials and Methods 260 7.2.1 Surface treatment 260 7.2.2 Cell Culture 260 7.2.3 Cell Spreading 262 7.3 Results 264 7.3.1 Cell spreading on derivatized 264 glass!surfaces 7.3.2 Cell spreading on untreated 276 and lipid-coated glass surfaces 7.4 Discussion 278 7.5 Summary 283 Chapter 8: Conclusions and Future 285 Developments References 291 Appendix: Publications 311 9 LIST OF FIGURES Figure number Title Page No 1.1 Blood - material interactions. 21 1.2 The blood coagulation pathway. 25 2.1 The Fluid Mosaic Model. 53 2.2 The structure of some common 57 phospholipid polar head groups. 2.3 Schematic representation of the asymmetric distribution of 63 phospholipid polar head groups in the erythrocyte and platelet plasma membrane. 2.4 The effect of liposome concen­ tration on clotting time as 72 measured by the Stypven assay. 3.1 Thrombelastography: The principle. 77 3.2 A typical thrombelastogram. 78 3.3 Thrombelastographic patterns for 84 various haemostatic defects. 3.4 The structure of PC-containing 94 polyesters. 3.5 MTEG analysis of standardisation 98 of plastic and metal cuvettes. 3.6 MTEG analysis of DPPC coatings. 101 3.7 Mr and Mma values obtained for DPPC coatings at various surface 102 concentrations. 3.8 MTEG analysis of octacosane and DPPC liposomes added directly to 103 blood. 10 Page No 3.9 MTEG analysis of DMPC coatings. 106 3.10 MTEG analysis of SM and a mixed 107 lipid coating. 3.11 Typical thrombelastographic traces obtained for negatively charged 108 phospholipids. 3.12 MTEG analysis of DPPE and DPPA 111 coatings. 3.13 Typical thrombelastographic traces obtained for PET coating, PTFE 113 and silicone smear. 3.14 MTEG analysis of C25 DAPC. 115 3.15 MTEG analysis of isophthalate 117 and sebacate based polyesters. 3.16 Typical thrombelastographic traces obtained for phathalate based 118 polyesters. 4.1 Adsorption of serum proteins onto multilamellar vesicles composed 148 of saturated lipids of varying compositions: SDS-PAGE 1.