Selection of Polyhydroxybutyrate-Co-Valerate (PHBV) as An Improved Polymer Choice in FTY720 Loaded Nanofibers for Islet Transplant

Abstract

Introduction: These studies aim to determine a biodegradeable polymer that can be used within a tissue engineering device that does not reduce the viability of islets to the same degree as poly(lactic-co-glycolic acid) (PLAGA), but can still be successfully electrospun and loaded with FTY720. Drug release profile and in vivo immune response support the new polymer choice. Methods: Nanofibers are electrospun resulting in scaffolds consisting of several types of polymers, including equal blends by weight with polycaprolactone (PCL), with and without FTY720. Images are obtained from dorsal skinfold window chambers where a FTY720 loaded implant and an unloaded implant are placed in the same animal immediately following window surgery. Microvessel metrics are quantified with a MATLAB program. Islets are stained for viability (Propidium Iodide and Fluorescein Diacetate). Release samples are analyzed with Liquid Chromatography-Mass Spectrometry (LC-MS) following lipid extraction. Hematoxylin and Eosin stained slides are obtained from paraffin embedded tissue samples. Results: Human islets tested with PLAGA/PCL nanofibers displayed a decrease in viability compared to the media only controls (68% PLAGA/PCL verses 78% media only). Other polymer types polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) performed better in this assay (75% and 79% respectively). LC-MS assisted in vitro release data indicate that a burst release (>10% loaded drug within 48 hours) is followed by a steady release resulting in 25% of the loaded FTY720 released by 2 weeks. Water droplet absorption time into nanofiber scaffolds indicate differences when FTY720 is added (< 5 sec FTY720 Loaded verses >3 min Unloaded scaffold, until complete wetting) likely related to the amphiphilic character of FTY720. Image analysis of window chambers with dual implants, indicate that after an initial 3 day time period a preferential blood flow in the area surrounding the FTY720 loaded implant exists as measured by both volume of blood in the tissue and length of visible blood vessels. Preliminary histology from subcutaneous implant studies indicate that the PLAGA/PCL fiber scaffolds contain more inflammatory cells and are characterized by tertiary lymphoid tissue development. In contrast, the PHBV/PCL fibers elicit an immune response, but do not result in formation of lymphoid tissue. Conclusion: The monomers of PLAGA are acidic. A decrease in culture media pH may contribute to a loss in islet viability with PLAGA fibers, which is less of an issue with the slower degrading PHBV. FTY720 releases from the fibers at a rate which can drive microvessel remodeling following injury perhaps by the recruitment of microvessel support cells. A depot of FTY720 is maintained however that will release for an extended period of time. Water droplet absorption combined with the burst release suggest that some of the FTY720 is locating near the surface of the fibers. PHBV scaffolds improved viability of islets in culture over PLAGA/PCL scaffolds. Preliminary in vivo studies demonstrate that PHBV/PCL fibers instigate less of an immune response than PLAGA/PCL fibers. In conclusion, PHBV has shown promise as a replacement polymer for PLAGA in FTY720 loaded nanofiber scaffolds for islet transplant.