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

The sustained delivery of proteins from bio-compatible polymers has attracted remarkable interest (1–4). However, the necessary encapsulation of proteins in hydrophobic polymer microspheres remains challenging when using proteins in aqueous solutions (4). For example, when proteins are encapsulated in biocompatible poly(lactic-co-glycolic) acid (PLGA) microspheres using standard water-in-oil-in-water (w/o/w) methods, substantial protein instability is observed. This is primarily due to protein adsorption and subsequent unfolding at the water–oil interfaces (4). Solid-in-oil-in-water (s/o/w) encapsulation procedures avoid the instability problems associated with the first emulsification step in w/o/w methods by employing dry solids (4). However, a common problem with encapsulation methods that use suspensions of dry protein powders is the potentially low encapsulation efficiency which is due to the size and shape of the protein powder particles (5). Companies have gone through quite some investment in method development to overcome this by producing small particles by, e.g., spray- or spray-freeze drying (6,7). Recently, an interesting article showed that several enzymes were formulated as solid nanoparticles by solvent-precipitation and encapsulated in PLGA microspheres by a s/o/w protocol (8). With the use of lysozyme as the model enzyme, good stability after encapsulation and release was obtained. 
 
In this study, we propose an alternative and simple method to encapsulate proteins as solid nanoparticles in PLGA microspheres. 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). 
 
Herein, we encapsulated spherical horseradish peroxidase (HRP) nanoparticles in PLGA microspheres by a s/o/w method. 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).