Protein encapsulation into biodegradable microspheres by a novel S/O/W emulsion method using poly(ethylene glycol) as a protein micronization adjuvant

A new method for preparing protein-loaded biodegradable microspheres by a process involving solid-in-oil-in-water (S/O/W) emulsion was established using poly(ethylene glycol) (PEG). In the first step, a protein solution was lyophilized with PEG, which resulted in the formation of spherical protein microparticles, less than 5 mum in diameter, dispersed in a continuous PEG phase. This process was well explained by the aqueous phase separation phenomenon induced by freezing-condensation. Since this lyophilizate could be directly dispersed in an organic phase containing biodegradable polymer by dissolving PEG with methylene chloride, a conventional in-water drying method could be adopted in the second step. Through this S/O/W emulsion process, horseradish peroxidase was effectively entrapped into monolithic-type microspheres of poly(DL-lactic-co-glycolic acid) (PLGA), without significant loss of activity. Bovine superoxide dismutase (bSOD), as another model protein, could be encapsulated into reservoir-type microspheres by the 'polymer-alloys method' using both poly(DL-lactic acid) (PLA) and PLGA. The initial release of bSOD from this reservoir-type microsphere was efficiently reduced. Further, the bSOD release kinetics could be suitably modified by adjusting the loading amounts of PEG or polymer composition. In this study, the multi-functional nature of PEG was successfully utilized in the preparation and designing of protein-loaded microspheres. (C) 2000 Elsevier Science B.V. All rights reserved.