(706e) Effect of Solvent Properties on Antioxidant Enzyme Polymer Nanocarriers Formed with a Modified Double Emulsion

We evaluated a battery of different solvents used during the formation of polymer nanocarriers (PNCs) that may improve loading and protection of the antioxidant enzyme catalase. The polymers used for carrier formulation were poly(ethylene glycol)-poly(lactic acid) diblock copolymers (PEG-PLA), synthesized by ring opening polymerization of lactide. The polymer number averaged molecular weight was determined via proton nuclear magnetic resonance (¹H-NMR), and the weight averaged MW and corresponding polydispersity index were measured with gel permeation chromatography (GPC). PNCs are formed from these polymers by a freeze-thaw modified water/oil/water double emulsion technique. The aqueous/oil interface has been shown to affect enzyme loading and denaturation, therefore we examined the effects of different solvents on those parameters in formed polymer nanocarriers. Candidate solvents were selected by desirable freezing points, water miscibility, and vapor pressures. Tetrahydrofuran (THF), in particular, was compared directly to dichloromethane (DCM), our established solvent used in previous PNC formulations. THF has a higher miscibility and a lower vapor pressure than DCM. We found that nanocarriers formulated with THF showed a higher percentage of entrapped protein by weight loading when measured using radio-isotope tracing of ¹²⁵I labeled-catalase. Concentration changes in hydrogen peroxide, catalase's substrate, were determined by UV absorbance of the substrate and provided a direct measure of catalase activity. The THF-formed PNCs showed a moderate improvement in activity as a percentage of the initial enzyme activity versus those made using DCM. Size distribution of the particles formed as measured by dynamic light scattering was largely unchanged between the two solvents. Protection of catalase by polymer carriers from proteolysis via the nonspecific protease, pronase, appeared relatively independent of solvent selection and was significantly enhanced over that of non-loaded catalase. We have concluded that solvent selection has a significant impact on formulation of PNC loaded with active enzymes. Specifically, THF results in protease-resistant PNC with a significant increase in encapsulation of the potent therapeutic enzyme, catalase.