Encapsulation of lysozyme in a biodegradable polymer by precipitation with a vapor-over-liquid antisolvent

Lysozyme was encapsulated in biodegradable polymer microspheres which were precipitated from an organic solution by spraying the solution into carbon dioxide. The polymer, either poly(l-lactide) (l-PLA) or poly(DL-lactide-co-glycolide) (PGLA), in dichloromethane solution with suspended lysozyme was sprayed into a CO2 vapor phase through a capillary nozzle to form droplets which solidified after falling into a CO2 liquid phase. By delaying precipitation in the vapor phase, the primary particles became sufficiently large, from 5 to 70 microm, such that they could encapsulate the lysozyme. At an optimal temperature of -20 degrees C, the polymer solution mixed rapidly with CO2, and the precipitated primary particles were sufficiently hard such that agglomeration was markedly reduced compared with higher temperatures. More uniform particles were formed by flowing CO2 at high velocity in a coaxial nozzle to mix the droplets at the CO2 vapor-liquid interface. This process offers a means to produce encapsulated proteins in poly(DL-lactide-co-glycolide) microspheres without earlier limitations of massive polymer agglomeration and limited protein solubility in organic solvents.