Stable protein nanostructured particles, produced by spray freezing into liquid (SFL) nitrogen, were encapsulated uniformly into microspheres to reduce the burst release over the first 24 h. The denaturation and aggregation of these bovine serum albumin (BSA) high-surface area particles were minimal due to ultra-rapid freezing and the absence of a liquid-air interface. Upon sonication, these friable highly porous, solid protein particle aggregates broke up into submicron particles. These particles were encapsulated into DL-lactide/glycolide copolymer (PLGA) and poly(lactic acid) (PLA) microspheres by anhydrous solid-in-oil-in-oil (s/o/o) techniques. For 5% loading of protein, the burst release after 24 h was only 2.5-4.1%, that is, values fivefold to tenfold lower than those observed for larger more conventional BSA particles. At a loading of 10%, the burst was only 6 and 13% for PLGA and PLA, respectively, and at 15% loading it was only 12% for PLGA. As shown with confocal and scanning electron microscopy (SEM), the low burst is consistent with a uniform distribution of protein nanoparticles, which were about 100 times smaller than the microspheres. Changes in aggregation and secondary structure, which were monitored by size exclusion chromatography and FTIR, respectively, indicated only slight monomer loss (3.9%) and high structural integrity (38% alpha-helix) in the encapsulated protein. (C) 2004 Wiley-Liss, Inc.
