| Protein-loaded poly(lactic-co-glycolic acid) microspheres were prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation technique. Protein inactivation associated with microsphere preparation was thoroughly investigated using three model proteins (i.e., lysozyme, chymotrypsin, and bovine serum albumin). The effects of microsphere preparation conditions (i.e., sonication, water/organic interface, and surfactant) on protein stability and biological activity were studied. A rational stabilization strategy was developed by adding protein stabilizers into the aqueous protein phase. Hydroxypropyl-β-cyclodextrin was found to be a promising stabilizer that could protect proteins during the primary emulsification process. The biological and conformational stabilities of proteins were monitored using enzymatic activity assay and differential scanning calorimetry, respectively. A novel protein extraction technique, involving polyethylene glycol 400, was developed, which allowed us to study protein stability in the water-in-oil emulsions in the presence of PLGA.;Various process variables in microsphere preparation were investigated. We obtained a better understanding of the relationship between the process variables and the characteristics of microspheres such as particle size, surface morphology, encapsulation efficiency, and in vitro release.;Finally, a therapeutic protein, insulin, was selected for delivering at its basal level, using PLGA microspheres. In addition to microspheres, an in situ forming gel system was also studied. The system was prepared by dissolving poly(lactic acid) in a solvent mixture of benzyl benzoate and benzyl alcohol. In vitro release of insulin increased by adding more benzyl alcohol, the hydrophilic component, in the solvent mixture. In vivo absorption insulin from microspheres and the in situ forming gel system was studied in diabetic rabbits. Microspheres provided a serum insulin level in the range of 20∼40 μU/ml up to 40 days. In vivo biocompatibility of microspheres and the in situ forming gel system in rabbits, based on the histological evaluation of the tissue sample retrieved from injection sites at different time points, showed that both of the delivery systems were biocompatible. |
