The total mixture was then placed in an ice bath and emulsified using a Branson Digital Sonifier (model 250, Connecticut, USA) with 55 W of energy output for 3 min to obtain an oil-in-water emulsion. An additional 40 mL of the PVA solution was added to the emulsion. The final solution was stirred for 12 h on a magnetic stir plate to allow the evaporation of methylene chloride and to allow the formation of the nanoparticles. The suspension was then centrifuged at 4880 × g for 20 min. The pellet was resuspended in distilled water and centrifuged three more times at 1220 × g for 20 min each. The resulting nanoparticles were collected and frozen at −80 °C for at least 2 h and subsequently freeze dried for 2 days. The freeze dried nanoparticles were stored at 4 °C.