Subcutaneous implantation of rapamycin impregnated scaffolds demonstrate the potential for localised islet graft immunosuppression
Francis Kette1,3, Sarah Short1, Sebastian Stead1,3, Juewan Kim1, Chris Drogemuller1,2, Darling Rojas-Canales3, Toby Coates1,2.
1University of Adelaide, Adelaide, Australia; 2RAH, Adelaide, Australia; 3Flinders University, Adelaide, Australia
Introduction: Hepatic islet transplantation continues to serve as the optimal therapeutic modality for treatment refractory Type 1 Diabetes, however is suboptimal for islet function and viability. Subcutaneous sites present attractive alternatives, yet, are currently incapable of supporting islet engraftment without modification. The use of a polyurethane (PU) scaffold has previously been shown to generate a hyper-vascularised dermal layer, and co-opting this may overcome inherent dermal hypoxia and hypo-vascularity. However, as the skin has a highly competent immune system, mitigating this is crucial for successful islet transplantation. Here we evaluated the capability of topical and scaffold loaded immunosuppressant’s to generate a localised immunosuppressive microenvironment supporting islet engraftment.
Methods: PU disks were loaded topically with 2nM of rapamycin. Structural changes to the PU were evaluated using SEM. UV-VIS spectroscopy was utilised to evaluate drug release kinetics. In vitro biocompatibility of the rapamycin-PU was investigated through co-culture with BTC6 murine β-cell line, murine and human islets. Islet viability was evaluated using Fluorescein Diacetate/Propidium Iodide (FDA/PI) staining. T-cell inhibitory properties were evaluated in murine and human cells using CFSE proliferation assay. Murine in vivo biocompatibility of subcutaneously implanted rapamycin-PU was assessed with immunohistochemistry. Porcine in vivo feasibility of rapamycin loaded scaffolds and topically applied immunosuppressant were evaluated with histology, immunofluorescence and RT-QPCR for angiogenesis and immune infiltration.
Results and Discussion: SEM of Rapamycin-PU showed increased porosity with some surface integrity disruption, due to methanol treatment, and crystal deposition. rapamycin-PU release kinetics followed the Higuchi and Ritger-Peppas model over 7 days, characterized by degradation dependent release. No phenotypically, acute, negative effects were observed upon BTC6 cell, murine and human islet viability with exposure PU alone or rapamycin-PU. Murine and human T cell proliferation was potently suppressed by rapamycin released from the scaffold. Murine and porcine in vivo scaffolds were well tolerated, and blood vessel formation was detected in unloaded and rapamycin-PU scaffolds as per CD31 immunofluorescence staining, indicating that the rapamycin loading does not abrogate angiogenesis and is comparable to controls. Porcine CD31 quantification revealed transient hypervascularity, as compared with normal porcine skin, peaking 18 days post insertion.
Conclusion: Islet viability and unperturbed porcine and murine in vivo angiogenesis supports the aim of creating a locally immunosuppressed site for islet transplantation. This model could allow for initial local immunosuppression to prevent early immune infiltration of the graft, reducing the need for systemic immunosuppression.
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