A new preparation method for protein loaded poly(D, L-lactic-co-glycolic acid) microspheres and protein release mechanism study.

A new method for encapsulating a model protein, lysozyme into hydrophilic uncapped poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres was developed using an oil/water (O/W) single emulsion technique. Lysozyme powder, which was prepared from lyophilization after adjusting a lysozyme solution pH at 3, was molecularly dissolved in a co-solvent system composed of dimethylsulfoxide (DMSO) and methylene chloride. The resulting organic solution containing PLGA was directly emulsified into an aqueous phase, and the organic solvent phase was extracted and evaporated. Various lysozyme-loaded PLGA microspheres having different morphologies were obtained depending on the relative mixing ratio of the two co-solvents used. In vitro release experiments indicated that an initial lysozyme release rate from the microspheres was mainly controlled by ionic interaction between basic amino acid residues in lysozyme and free carboxylate groups in PLGA polymer chain ends, which was probed by incubating the microspheres in a series of media having different NaCl concentrations. However, the protein release leveled off after about 15 days' incubation. To determine the reason for the protein 'no-release' from biodegradable microspheres, a systematic analysis was carried out. By separately adding 0.5 M NaCl, 5 M guanidine HCl, or 5 mM sodium dodecyl sulfate into the release media during the non-release period, it was possible to selectively identify a specific protein non-release mechanism: ionic interaction, non-covalent aggregation, and/or surface adsorption, respectively. It was found that non-covalent aggregation and surface adsorption of lysozyme within the microspheres were the main cause of no further release, whereas ionic interaction between degrading polymer and protein played an insignificant role in the later stage of the release period. The greater amount of additional lysozyme release by sodium dodecyl sulfate than by guanidine hydrochloride suggested that protein surface adsorption was a more critical factor in protein release than aggregation.