NANOENCAPSULATION OF BILIRUBIN AND ITS EFFECTS ON ISOLATED MURINE PANCREATIC ISLET CELLS Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Bronwyn Anne Fullagar BVSc (Hons) Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2015 Thesis Committee: Christopher A. Adin, Advisor Chen Gilor Alicia Bertone Copyrighted by Bronwyn Anne Fullagar 2015 Abstract Pancreatic islet transplantation would provide a cure for type 1 diabetes mellitus in dogs, but isolation stress and hypoxia cause loss of up to 70% of islets in the first 72h after transplantation. Bilirubin (BR) is a natural antioxidant and can improve survival of murine pancreatic allografts exposed to hypoxic stress. However, its poor bioavailability limits its therapeutic application. Nanoparticle (NP) drug delivery, using biopolymers Pluronic F127 and chitosan, can improve solubility and bioavailability of hydrophobic drugs such as bilirubin. We hypothesized that delivery of bilirubin via Pluronic F127- chitosan nanoparticles (nBR) would improve uptake into murine islets compared to free bilirubin (fBR). We further hypothesized that nBR would improve the viability of islets following exposure to hypoxic stress, compared to fBR or control. Pluronic F127-chitosan NPs were synthesized and bilirubin was nano- encapsulated at a feeding ratio of 1:20. Murine INS-R3 cells, an insulinoma cell line, were incubated in media containing 0-20µM nBR, fBR or empty NP (eNP). After staining with Hoescht and LysoTracker Red, bilirubin uptake was qualitatively studied using Zeiss Apotome (confocal-like) structured illumination microscopy. Further INS-R3 cells were then cultured in media with nBR, fBR or eNP at concentrations of 0-20µM. Cells were exposed to 8h hypoxia (1% O2), followed by 12h recovery (standard conditions). MTT viability assays were performed and cell viability was expressed as ii percentage absorbance of control (untreated) cells. In accordance with the IACUC, pancreatic islets were isolated from female C57BL/6 mice and were incubated with nBR, fBR or eNP at 0-20µM, then exposed to 24h of hypoxia (1% O2). Cells were stained with Propidium Iodide (PI) and Hoescht and imaged using epifluorescent microscopy. Images were analyzed using NIH Image J software to determine the percentage of PI positive cells in each islet. Analysis of cell viability data was via the linear mixed procedure using SPSSv21 software. Significant main effects were investigated using Sidak multiple comparison tests; p≤0.05 was considered statistically significant. Qualitatively, INS-R3 cells showed increased uptake of nBR compared to fBR at all concentrations and active, selective uptake via endocytosis was apparent at 20µM. In INS-R3 cells exposed to hypoxic stress, cells treated with 5µM nBR (113.2% +/- 3.9), had the best viability overall and there was dose-dependent cytotoxicity in all groups. In murine islets exposed to hypoxic stress, islets treated with nBR survived significantly better than other groups (p=0.047). Islets treated with 5µM nBR (18.5% +/- 14.1) survived better than untreated islets (33.5% +/- 17.5%), with reduction of central necrosis. Dose-dependent cytotoxicity at 20µM was seen in all groups. The mechanism of the improved protective effects of nBR is likely due to active, selective uptake of NP via endocytosis. Unencapsulated BR (fBR) had insignificant protective effects on murine islets exposed to 24h hypoxia, which may indicate that its protective mechanisms were overwhelmed by the prolonged hypoxic stress in this study. Central necrosis is a iii characteristic feature of pancreatic islets exposed to hypoxic stress, due to impaired diffusion of nutrients. These effects appear to be ameliorated by treatment with nBR, which may target the metabolically active β-cells residing at the center of murine islets. Empty NP appear to be cytotoxic, which may indicate a pro-inflammatory effect, as chitosan has been shown to induce an IL-1β response. Pluronic F127-chitosan nanoencapsulation of bilirubin is feasible and results in improved cellular uptake and dose-dependent protective effects on murine islets exposed to hypoxic stress. Further investigation into the protective mechanisms of nBR and toxicity of empty NP are warranted. iv This thesis is dedicated to my family, Peter, Nance and Andrea, for their constant love and support, and to my advisor, Dr. Adin, for his endless enthusiasm, encouragement and exceptional mentorship. v Acknowledgments This study was supported by a grant from the Paladin Research Fund. The author would like to acknowledge collaborators Dr. Wei Rao and Dr. Xiaoming (Sean) He at The Ohio State University Department of Biomedical Engineering for their expertise in nanoparticle synthesis and experimental methods, Feng Xu for her technical expertise and patience in teaching me laboratory techniques and Dr. John Bongaura for his assistance with the statistical analyses. vi Vita 2003-2007 ............................................. Bachelor of Veterinary Science, University of Queensland, Brisbane, Australia 2011-2012 ............................................. Small Animal Rotating Internship, Calgary Animal Referral and Emergency Centre, Alberta, Canada 2012 to present ..................................... Graduate Teaching Associate and Small Animal Surgery Resident, Department of Veterinary Clinical Sciences, The Ohio State University Fields of Study Major Field: Comparative and Veterinary Medicine vii Table of Contents Abstract…………………………………………………………………………………… ii Acknowledgments……………………………………………………………………….. vi Vita……………………………………………………………………………………… vii Fields of Study…………………………………………………………………………... vii List of Tables…………………………………………………………………………….xii List of Figures…………………………………………………………………………...xiii Chapter 1: Nanotechnology in veterinary surgery - applications and future directions………………………………………………………………………………….1 Introduction………………………………………………………………..................... 1 Nanomaterials………………………………………………………………………….. 2 Organic (polymeric) nanoparticles………………………………………………….. 2 Inorganic nanoparticles……………………………………………………………… 3 Nanoparticle drug delivery…………………………………………………………….. 4 Drug nanocrystals…………………………………………………………………… 5 Water-soluble polymers……………………………………………………………... 5 Liposomes…………………………………………………………………………… 6 Polymeric nanoparticles……………………………………………………………... 7 Nanoparticle characteristics……………………………………………………………7 viii Preparation of polymeric nanoparticles……………………………………………... 7 Particle size………………………………………………………………………….. 8 Surface properties…………………………………………………………………… 9 Drug loading……………………………………………………………………….. 10 Drug release………………………………………………………………………... 10 Triggered drug release……………………………………………………………... 11 Organ/cellular targeting……………………………………………………………. 12 Intracellular targeting………………………………………………………………. 13 Current and future applications in veterinary surgery……………………………….. 13 Surgical oncology………………………………………………………………….. 13 Thyroidectomy and parathyroidectomy…………………………………………… 18 Wound healing and burns………………………………………………………….. 19 Tendon and ligament injuries……………………………………………………… 21 Bone diseases and bone regeneration……………………………………………… 22 Hemostasis and trauma…………………………………………………………….. 23 Transplant………………………………………………………………………….. 24 Other applications in veterinary medicine………………………………………… 26 Nanoparticle toxicity…………………………………………………………………... 27 Conclusion……………………………………………………………………………. 29 Chapter 2: Nano-encapsulated bilirubin protects murine pancreatic islet cells exposed to hypoxic stress in vitro……………………………………………………… 31 Introduction…………..……………………………………………………………………… 31 ix Materials and methods………………………………………………………………………...34 Materials…………………………………………………………………………… 34 Synthesis of Pluronic F127-chitosan nanoparticles………………………………... 35 Encapsulation of bilirubin to obtain nanoparticle-encapsulated bilirubin (nBR)….. 37 Spectrophotometric analysis of bilirubin content within nanoparticles…………….38 In-vitro release studies……………………………………………………………... 38 Cellular uptake and intracellular distribution of nBR in INS-R3 cells…………….. 39 Effect of nBR on viability of INS-R3 cells exposed to hypoxic stress……………..41 Effects of nBR on viability of murine islets exposed to hypoxic stress…………… 41 Statistical analyses…………………………………………………………………. 42 Results………………………………………………………………………………… 43 Physiochemical characterization of nanoparticles…………………………………. 43 Bilirubin release characteristics……………………………………………………. 44 Cellular uptake of bilirubin in INS-R3 cells……………………………………….. 45 Effects of nBR on INS-R3 cells under hypoxic conditions………………………... 46 Effects of nBR on murine islets under hypoxic conditions………………………... 48 Discussion…………………………………………………………………………….. 51 Release characteristics of nBR and fBR…………………………………………… 51 Improved uptake of nBR by murine islet cells…………………………………….. 53 Effect of nBR on INS-R3 cells exposed to hypoxic stress………………………… 54 Protective effects of nBR on murine islets following hypoxic stress……………… 55 Toxicity of eNP to murine islets and INS-R3 cells…………………………………56 x Negligible protective effects and dose-dependent toxicity of fBR………………… 57 Limitations and future directions…………………………………………………... 58 Conclusion……………………………………………………………………………. 58 References……………………………………………………………………………….. 60 xi List of Tables Table 1: Encapsulation efficiency (EE) and loading content (LC) of bilirubin together with diameter of the resultant NP-encapsulated bilirubin (nBR), determined by dynamic light scattering (DLS): all data are presented as mean