Insulin-loaded poly(lactide-co-glycolide) nanoparticles (INS-PLGA-NPs) were prepared by a double emulsion method (w/o/w), using ethyl acetate as organic solvent and poloxamer188 as emulsifier. Experimental parameters such as the emulsifier and PLGA concentrations, the pH and concentration of the insulin solution, the solvent evaporation method and PVA in the internal phase were investigated for the encapsulation efficiency. The results indicated that higher emulsifier concentration, relatively less amount of PLGA and lower insulin concentration would increase the entrapment of insulin. Furthermore, pH of insulin solution approaching to pI (5.3), adding some PVA to the internal phase and a shorter evaporation time helped to enhance the incorporation efficiency of insulin. Optimized preparation parameters led to nanoparticles with well-defined characteristics such as an average size around 149.6 nm, a polydispersity lower than 0.1 and high encapsulation efficiency up to 42.8%.
Stability of Defibrase in various pH buffer solutions was investigated. Enzyme-linked immuno-sorbent assay (ELISA) and coagulating time method were used to assess antigenic stability and coagulating stability, respectively. The change of antigenic activities and coagulating activities of Defibrase in the same buffer solutions (pH 6, 7 and 8, with the exception of pH 3.6) showed similar tendency to decline with the time. Concentrated Defi-brase was relatively stable at neutral pH 6~7, more than 95% of its initial activities (100BUmL-1) was kept after a 10-day storage at 40 oC, whereas in pH 3.6 and pH 9 buffer solutions, diluted Defibrase was very labile. Addition of Triton X-100 or bovine serum albumin could effectively prevent loss of Defibrase by minimizing adsorption of De-fibrase to plastic surface (P<0.005). Concentration of Defibrase could also affect its stability in aqueous solutions.
