Nano-encapsulated Bilirubin Protects Murine Pancreatic Islet Cells Exposed to Hypoxic Stress

Pancreatic islet transplantation is the only curative, non- invasive treatment for type 1 diabetes mellitus in both dogs and humans; however, isolation stress and hypoxia cause loss of up to 70% of islet mass prior to any immune insult. Bilirubin, an endogenous antioxidant, can improve survival of murine pancreatic allografts during hypoxic stress, but has poor solubility in aqueous solutions. We hypothesized that nano-encapsulation of bilirubin in Pluronic 127-chitosan nanoparticles (nBR) would improve uptake by murine pancreatic islet cells and improve their viability following hypoxic stress. Nano-bilirubin was synthesized and drug release characteristics were studied in vitro. Cellular uptake of nBR was compared to free bilirubin (fBR) in an insulinoma cell line (INS-R3) model using confocal-like structured illumination microscopy. Then, C57BL/6 mouse islets were treated with concentrations of 0-20μM of nBR, fBR or empty NP (eNP), prior to incubation under standard or hypoxic conditions. Cell viability was assessed via Hoescht and propidium iodide. The release of bilirubin was greatest from nBR suspended in protein-rich solution. Increased, selective uptake of nBR by INS-R3 cells was demonstrated. Cell death after hypoxic stress was significantly decreased in murine islets treated with 5μM nBR (18.5% +/- 14.1) compared to untreated islets (33.5% +/- 17.5%), with reduction of central necrosis. The treatment group had a significant effect on glucose stimulation index (p=0.0137) and islets treated with 5μM nBR had the highest SI overall. Delivery of bilirubin using Pluronic F127-chitosan NP improves uptake by murine islets compared to fBR and offers dose-dependent protective effects following hypoxic stress.