Formation of spherical protein nanoparticles without impacting protein integrity

Abstract

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-β-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.

Improved Enzyme Activity and Stability in Polymer Microspheres by Encapsulation of Protein Nanospheres

“…HRP nanoparticles were prepared as we described in detail by (9). Briefly, HRP (40 mg) and MβCD (160 mg) were codissolved in 10 ml of nanopure water, and this solution was frozen in liquid N 2 and lyophilized for 48 h using a Labconco FreeZone 6 L freeze drier at a condenser temperature of −45°C and a pressure of <60 μm of Hg.…”

“…We have shown previously that nano-sized dry protein-spheres can be obtained by co-lyophilization of various proteins with methyl-β-cyclodextrin (MβCD) followed by dispersion in ethyl acetate (9). The formulation is scalable because the protein particle size solely depends on the ratio of protein-to-additive during lyophilization (9).…”

“…We have shown previously that nano-sized dry protein-spheres can be obtained by co-lyophilization of various proteins with methyl-β-cyclodextrin (MβCD) followed by dispersion in ethyl acetate (9). The formulation is scalable because the protein particle size solely depends on the ratio of protein-to-additive during lyophilization (9). In addition to potentially improving the encapsulation efficiency in s/o/w encapsulation methods, the drug particle size is highly relevant because it can influence bioavailability, release, and stability of the drug (10).…”

“…Our method of nanoparticle formation typically leads to 100% of recovered enzyme activity (9). Horseradish peroxidase was chosen as the model enzyme because we have accomplished protein nanoparticles using it (9); it is very susceptible to denaturation during in vitro release (11) and has been used as a model to study the crossing of proteins through the hemato-encephalic barrier into the brain (12).…”

“…Ethyl acetate was used during encapsulation instead of dichloromethane (the conventional solvent) because it is less toxic (13). Furthermore, ethyl acetate is the solvent most conveniently used to suspend the protein-MβCD co-lyophilizate to obtain protein nanoparticles (9).…”