Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques

Lysozyme distribution and conformation in poly(lactic-co-glycolic acid)(PLG A) microspheres was determined using various infrared spectroscopic techniq ues. Infrared microscopy and confocal laser scanning microscopy indicated t hat the protein was homogeneously distributed inside the microspheres in sm all cavities resulting from the water-in-oil emulsification step. Part of t he protein was observed at or near the cavity walls, while the rest was loc ated within these cavities. Attenuated total reflectance (ATR) and photoaco ustic spectroscopy (PAS) also showed that there is hardly any protein at th e surface of the microspheres. Since this microsphere formulation gave a la rge burst release (ca. 50%), this burst release can not be caused by protei n at the surface of the particles. Probably, the protein is rapidly release d through pores in the PLGA matrix. Conformational analysis of lysozyme in the PLGA microspheres by KBr pellet transmission suffered from band shape d istortion and baseline slope. Despite incomplete subtraction of the PLGA ba ckground, a characteristic band of non-covalent aggregates at 1625 cm(-1) w as observed in the second derivative spectrum of the protein Amide I region . The other Fourier-transform infrared (FTIR) methods yielded similar resul ts, indicating that the sample preparation procedure did not introduce arti facts. The observed aggregation signal may correspond to the protein adsorb ed to the cavity walls inside the microspheres. (C) 2000 Elsevier Science B .V. All rights reserved.