Polymeric Nano-Shielded Pancreatic Islets for Prevention of Immune Reactions against Transplanted Graft

학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2018. 2. Youngro Byun.The advances in exogenous insulin therapy has been able to improve the lifestyle of Type-1 diabetic (T1DM) patients, but no formulation till date is yet to mimic the nyctohemeral rhythms of this hormone. In spite of all engineering progresses, a mechanical replacement of pancreatic β cell is still out of reach. Pancreatic islet or the whole pancreas transplantation are the only realistic approach towards finding the 'cure' of the disease. In pancreas, only 1-1.5% cells are insulin secretingtherefore, islet transplantation arises as a more realistic alternative. After the introduction of 'Edmonton protocol' in 2000, clinical islet transplantation is now considered as one of the safest and least invasive transplantation procedures. The protocol proposed several modifications to the existing procedures, such as transplantation of freshly isolated islets from more than one donor pancreas, and use of a steroid-free immunosuppressive drug protocol, which increased the initial success rate significantly. A five-year follow-up study confirmed improved glycemic control in a significant number of recipients, however, only 7.5% of them have attained insulin independence. Therefore, continuous researches have been carried out to improve the protocol in the preservation of the efficacy of the transplanted islets. In order to have an improved outcome and availability of this technology we still have to tackle with the problems related with donor shortage and immunogenicity. Porcine islet xenotransplantation has arisen as a potential alternative source of clinical islet transplantation due to its structural and anatomical similarities with human islets. Moreover, to get around immunogenicity, islet nano-shielding with biocompatible polymers has offered a great promise to increase survival time in combination with immunosuppressive drugs. In this thesis, islet nano-shielding was performed with polymers, such as gelatin, polyethylene glycol (PEG), heparin (Hep), and tannic acid (TA)and validated in different animal models. Nano-shielding with an artificial extracellular matrix (A. ECM) based material stabilized inherently fragile porcine islets from dissociation after isolation, when PEG was incorporated into the shielding composition, the immunogenicity was significantly reduced. PEG/PEG-Hep abrogated plasma protein adsorption on the nano-shielded surfaces, reduced instant blood-mediated inflammatory reactions (IBMIR) and immune cell activation while exerting no cytotoxic effects on the islets. Finally, nano-shielded non-human primate (NHP) islets demonstrated excellent success rate in both allo- and xeno-recipients with less immune infiltration. One of the benefits of employing nano-shielding technology for islet immunoisolation is it restricts the increment of cell size more than a few nanometers, which makes clinical transplantation feasible through the conventional portal vein. Moreover, the nano-shielding has synergistic effects on increasing survival time with an appropriate immunosuppressive drug regimen. We believe this is the cutting-edge technology with immense potential in clinical transplantation at the moment. If applied with the current immunosuppressive drug protocol, clinical islet transplantation would enter into a new dimension.Chapter 1 Introduction 1 1.1 Diabetes mellitus 1 1.2 Type-1 diabetes mellitus 1 1.2.1 Symptoms 1 1.2.2 Pathophysiology 2 1.2.3 Diagnostic criteria 2 1.2.4 Treatment options 2 1.2.5 Insulin therapy 3 1.2.5.1 Insulin action 3 1.2.5.2 History 3 1.2.5.3 Types of insulin 3 1.2.5.4 Insulin pump 3 1.2.5.5 Pitfalls of insulin therapy 4 1.2.6 Pancreas transplantation 4 1.3 Pancreatic islet transplantation 4 1.3.1 Obstacles to islet transplantation 5 1.3.1.1 Loss of islet viability 5 1.3.1.2 Loss of intraislet vasculature 5 1.3.1.3 Immune rejection 6 1.3.1.4 Instant-blood mediated inflammatory reactions (IBMIR) 6 1.3.2 Strategies for improving the outcome of islet transplantation 9 1.3.2.1 Immunosuppressive drugs 9 1.3.2.2 Immunoisolation of islets 9 1.3.3 Islet nano-shielding with PEG and heparin 10 1.3.3.1 Advantages and disadvantages of nano-shielding 11 1.3.4 Xenotransplantation 12 1.4 Research rationale 12 1.5 References 17 Chapter 2 Combination strategy of multi-layered nano-shielding using artificial extracellular matrix and hyperbranched polyethylene glycol for simultaneous reduction of islet dissociation and improvement in immunoprotection 26 2.1 Introduction 26 2.2 Materials and methods 29 2.2.1 Animals 29 2.2.2 Diabetic modeling 29 2.2.3 Synthesis of SH-6 arm-PEG-Lipid 29 2.2.4 Synthesis of gelatin-catechol 29 2.2.5 Synthesis of SH-6-arm-PEG-FITC, SH-6-arm-PEG-NHS and 6-arm-PEG-catechol 30 2.2.6 Isolation of porcine islets 30 2.2.7 Nano-shielding of pancreatic islets using biomaterials 31 2.2.8 Monitoring islet stability 32 2.2.9 Cell viability assay 32 2.2.10 Glucose-stimulated insulin secretion (GSIS) assay 33 2.2.11 In vitro protein adsorption study 33 2.2.12 Nano-shielded islet xenotransplantation 33 2.2.13 Immunosuppressive drug treatment to the recipients 34 2.2.14 Statistical analysis 34 2.3 Results 34 2.3.1 Characterization of artificial ECM grafted islets 34 2.3.2 Maintaining islets stability using artificial ECM 37 2.3.3 Improved immunoprotection by PEG nano-shielding 40 2.3.4 Protein adsorption on artificial ECM and PEG nano-shielded surfaces 40 2.3.5 Effect of artificial ECM + PEG with immunosuppressive drugs on survival prolongation of xenografted porcine islets 40 2.4 Discussion 43 2.5 Conclusion 47 2.6 References 48 Chapter 3 Xenotransplantation of layer-by-layer polyethylene glycol nano-shielded non-human primate islets with a specified immunosuppressive drug protocol 53 3.1 Introduction 53 3.2 Materials and methods 55 3.2.1 Animals 55 3.2.2 Diabetic modeling 55 3.2.3 Isolation of pancreatic islets 55 3.2.4 Synthesis of SH-6-arm-PEG-NHS 56 3.2.5 Synthesis of 6-arm-PEG-catechol 56 3.2.6 Synthesis of FITC labeled linear PEG-SH 57 3.2.7 AFM imaging of nano-shielded surface 57 3.2.8 Protein adsorption on nano-shielded surface 57 3.2.9 Nano-shielding of islets 57 3.2.10 Cell viability assay 58 3.2.11 Glucose-stimulated insulin secretion (GSIS) assay 59 3.2.12 Nano-shielded islet xenotransplantation 59 3.2.13 Immunosuppressive drug protocol 59 3.2.14 Intraperitoneal glucose tolerance test 60 3.2.15 Immunohistochemistry 60 3.2.16 Quantification of insulin, C-peptide and IL-1β concentrations in serum 60 3.2.17 Statistical analysis 61 3.3 Results 61 3.3.1 Synthesis and Characterization of PEGs 61 3.3.2 Nano-shielding reduced protein adsorption 61 3.3.3 Nano-shielding of islets 61 3.3.4 Xenotransplantation of nano-shielded NHP islets in mice 65 3.3.5 The combined effort of nano-shielding and immunosuppressive drugs on immunoprotection 70 3.4 Discussion 70 3.5 Conclusion 76 3.6 References 77 Chapter 4 Effects of transplanted islets nano-shielded with hyperbranched polyethylene glycol and heparin on microenvironment reconstruction and glucose control 83 4.1 Introduction 83 4.2 Materials and methods 84 4.2.1 Animals 84 4.2.2 Isolation of pancreatic islets 84 4.2.3 Synthesis of PEGs and heparin 85 4.2.4 Islet nano-shielding and xenotransplantation 86 4.2.5 Whole-mount staining 86 4.2.6 Immunohistochemistry 87 4.2.7 Cell viability assay 87 4.2.8 Glucose stimulated insulin secretion assay 87 4.2.9 Complement activation 88 4.2.10 Immune cell activation 88 4.2.11 Statistical analysis 88 4.3 Results 88 4.3.1 Loss of islet microenvironment during isolation delays engraftment after transplantation 88 4.3.2 LbL PEG-Hep nano-shielding of islets 89 4.3.3 PEG-Hep nano-shielding inactivates the innate and adaptive immune systems 97 4.3.4 PEG-Hep islets are functional in vivo 97 4.4 Discussion 101 4.5 Conclusion 102 4.6 References 104 Chapter 5 Allotransplantation of polymeric nano-shielded islets with polyethylene glycol and heparin in a non-human primate model 107 5.1 Introduction 107 5.2 Materials and methods 108 5.2.1 Non-human primate model 108 5.2.2 Induction and management of diabetes mellitus 109 5.2.3 Islet isolation and functional assay 110 5.2.4 Synthesis of PEGs and heparin 110 5.2.5 Nano-shielding of islets with PEGs and heparin 111 5.2.6 Viability and functionality of PEG-Hep islets 111 5.2.7 Surface characterization and platelet adhesion on PEG-Hep nano-shielded surfaces in vitro 112 5.2.8 Complement activity on PEG-Hep islet 112 5.2.9 Immune cell activity on PEG-Hep islet 112 5.2.10 Activated Partial Thrombosis Time (APTT) assay 112 5.2.11 Islet transplantation and perioperative management 113 5.2.12 Flow cytometric analysis 117 5.2.13 Histology 117 5.2.14 Statistical analysis 118 5.3 Results 118 5.3.1 Islet nano-shielding with PEG and heparin 118 5.3.2 Islet allograft survival and outcome 119 5.3.3 Lymphocyte subset analysis in the peripheral blood and in the liver 131 5.4 Discussion 131 5.5 Conclusion 136 5.6 References 138 Chapter 6 Islet nano-shielding with a combination of tannic acid, polyethylene glycol and heparin for improved immune protection 144 6.1 Introduction 144 6.2 Materials and methods 145 6.2.1 Animals 145 6.2.2 Isolation of pancreatic islets 145 6.2.3 Synthesis of PEG and heparin 145 6.2.4 LbL islet nano-shielding with TA, PEG and heparin 146 6.2.5 Cell viability assay 146 6.2.6 Glucose-stimulated insulin secretion assay 148 6.2.7 Migration assay 148 6.2.8 Xenotransplantation 148 6.2.9 Immunohistochemistry 148 6.2.10 Quantification of insulin and C-peptide concentrations in serum 148 6.2.11 Statistical analysis 149 6.3 Results 149 6.3.1 Islet nano-shielding with TA, PEG and heparin 149 6.3.2 Xenotransplantation of the nano-shielded islets 149 6.3.3 TA-PEG-Hep nano-shielding decreases immune cell migration in vitro 153 6.4 Discussion 153 6.5 Conclusion 156 6.6 References 157 Chapter 7 Summary 159Docto