Preparation of SnPI nanoparticles

SnPI nanoparticles were prepared by the desolvation method using ethanol as a desolvating agent. For this purpose, a solution of 15 mg/mL of freshly prepared SnPI in deionized water was prepared. The pH of the solution was adjusted to 9 by 2 N NaOH, and 0.02% of sodium azide was added as a preservative to all samples. The protein solution was then equilibrated for 2 h at room temperature and stored overnight (8 h) in a refrigerator (4 °C) for hydration of protein molecules. The protein solution was passed through a syringe filter made of polyvinylidene fluoride (0.45 μm) before nanoparticulation. The nanoparticles were obtained upon the dropwise addition of the antisolvent agent (ethanol or methanol) via a syringe pump (JMS SP-500, Japan) at 1 mL/min under mild stirring condition (1200 rpm). The ratio of the desolvating agent to the protein solution was 4:1 in all experiments. After the desolvation process, 20 μL of 0.25% glutaraldehyde solution was added to 1 mL of the protein solution as a crosslinker and crosslinked overnight. Then, the suspension was diluted by distilled water at a ratio of 1:100 in order to eliminate the desolvating agent, and lyophilized (Operon, OPR-FDCF-12012, Korea) and stored at −19 °C.