While the precipitation efficiency was comparable for both solvents, acetonitrile caused less enzyme aggregation and inactivation in the case of lysozyme. This solvent was therefore chosen for all subsequent CP-690550 mouse work. In order to further optimize the precipitation conditions, we varied the volume ratio of acetonitrile-to-water. Similar to Weber et al. [25] who used ethanol as desolvating agent, we found that a 1:4 water-to-ACN volume ratio was sufficient to precipitate both proteins (data not shown). Next, we tested the effect of the protein concentration on precipitation results

(Table 2). The precipitation yield and particle size increased at increasing protein concentration under otherwise constant precipitation conditions. While for both proteins, no significant amounts of buffer-insoluble aggregates were formed regardless of the protein concentration, the residual activity increased at increasing Gefitinib research buy protein concentration. We interpret this as an indication, that protein molecules close to the solvent-interface are more prone to denaturation than molecules buried in the interior of the precipitates. Such observations have been made before in solid-in-oil-in-water encapsulation procedures [28]. It is apparent that protein concentrations of 20–30▒mg/ml give optimum results. For a-chymotrypsin concentrations higher than 40▒mg/ml, unstable suspensions of the precipitated protein resulted and thus

did not allow for the subsequent encapsulation process. We can surmise from the above that similar to findings by Giteau et al. [19], a variety of precipitation conditions was identified by us leading to nano-particulate enzyme precipitates without causing activity loss or formation of buffer-insoluble aggregates. After optimizing the protein precipitation

conditions, we proceeded to encapsulate the model proteins into PLGA nanospheres. Previously, Giteau et al. precipitated proteins to ensure their stability upon subsequent encapsulation within PLGA microspheres using a solid-in-oil-in-water (s/o/w) technique [ 19]. After protein precipitation with glycofurol, proteins were centrifuged and the pellet dipyridamole suspended in acetonitrile (ACN) containing the polymer and encapsulated within PLGA microspheres. Our method used the same desolvating agent (ACN) to precipitate the protein and to dissolve the polymer. Additionally, several steps in the encapsulation procedure were changed systematically to assure obtaining nanosized PLGA spheres with high protein loading while aiming at avoiding enzyme inactivation and aggregation. Initially, we selected PLGA with a co-polymer ratio of 65% lactic acid and 35% glycolic acid, a theoretical loading of 2% (w/w), and ACN as the diffusing phase. We tested two commonly used emulsifying agents, namely, poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG, MW = 8000) using a set of defined conditions ( Table 3) [ 28].