Optimization of preparative conditions for poly-DL-lactide- polyethylene glycol microspheres with entrapped Vibrio cholera antigens.

Poly-dl-lactide-polyethylene glycol (PELA) with different contents of polyethylene glycol(PEG) were synthesized and the PEG content was estimated according to the integral height of hydrogen shown in 1H-NMR. PELA microspheres containing V. cholera antigen, outer membrane protein (OMP) were prepared by a water-in-oil-in-water (W/O/W) based on solvent evaporation procedure. Antigen microspheres with smooth surface, suitable size for oral administration (0.5-5 microm), high loading efficiency (about 60%) and low level of residual solvent (lower than 20ppm) were obtained. Microspheres prepared from PELA with PEG content of about 10% achieved the highest loading efficiency among PELA copolymers and poly-dl-lactide (PLA) homopolymer, which suggested that microspheres size, morphology and the precipitation rate of polymer showed considerable relations with OMP loading efficiency. The regulation of the solvent components of the oil phase contributes to a stable emulsion W/O, and it is concluded that the stable emulsion W/O plays a significant role in improving the protein loading efficiency of obtained microspheres. The addition of stabilizer, such as gelatin and polyvinyl alcohol, into the internal water phase before emulsification produced no significant difference in OMP entrapment and microspheres size. A higher OMP loading efficiency was achieved by adding NaCl or adjusting the pH at the iso-electric point of OMP in the external water phase. It was indicated in vitro that PELA microspheres with smaller size showed larger extent of initial release and higher release rate, whereas microspheres with the diameter of 2.17 microm showed no apparent burst effect.