Preparation and in vitro/in vivo evaluation of insulin-loaded poly(acryloyl-hydroxyethyl starch)-PLGA composite microspheres.

PURPOSE: The purpose of this study was to develop and evaluate a novel composite microsphere delivery system composed of poly(D,L-lactide-co-glycolide) (PLGA) and poly(acryloyl hydroxyethyl starch) (acryloyl derivatized HES; AcHES) hydrogel using bovine insulin as a model therapeutic protein.
METHODS: Insulin was incorporated into the AcHES hydrogel microparticles by a swelling technique, and then the insulin-containing AcHES microparticles were encapsulated in a PLGA matrix using a solvent extraction/evaporation method. The composite microspheres were characterized for loading efficiency, particle size, and in vitro protein release. Protein stability was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, high-performance liquid chromatography, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The hydrogel dispersion process was optimized to reduce the burst effect of microspheres and avoid hypoglycemic shock in the animal studies in which the serum glucose and insulin levels as well as animal body weight were monitored using a diabetic animal model.
RESULTS: Both the drug incorporation efficiency and the in vitro release profiles were found to depend upon the preparation conditions. Sonication effectively dispersed the hydrogel particles in the PLGA polymer solution, and the higher energy resulted in microspheres with a lower burst and sustained in vitro release. Average size of the microspheres was around 22 microm and the size distribution was not influenced by sonication level. High-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, along with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed the retention of insulin stability in the microspheres. Subcutaneous administration of microspheres provided glucose suppression <200 mg/dL for 8-10 days with hyperglycemia recurring by day 16. During the treatment, the time points with higher serum insulin level were consistent with a more significant glucose suppression. The microsphere-treated rats also grew virtually at the same rate as normal control until the insulin level declined and hyperglycemia returned. Multiple dosing given every 10 days demonstrated that the pharmacological effect and serum insulin levels from second or third doses were similar and comparable to that of the first dose.
CONCLUSION: The AcHES-PLGA composite microsphere system provides satisfactory in vitro and in vivo sustained release performance for a model protein, insulin, to achieve 10-day glucose suppression.