Formation of spherical protein nanoparticles without impacting protein integrity.

Protein formulation at the nanoscale is challenging because of protein susceptibility to chemical and physical degradation during processing. Herein, we present a straightforward method to prepare spherical protein nanoparticles by co-lyophilizing five structurally different enzymes (horseradish peroxidase, carbonic anhydrase, lysozyme, subtilisin Carlsberg and α-chymotrypsin) with methyl-ß-cyclodextrin followed by suspension of the powders in ethyl acetate. The size distribution was narrow and varied from 88 ± 14 to 148 ± 16 nm as determined by dynamic light scattering. Scanning and transmission electron micrographs confirmed the size and spherical morphology of the protein nanoparticles. Residual activities for all enzymes tested were 100% upon dissolving the nanoparticles in buffer and no insoluble aggregates were formed.

Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran.

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.