Cold Dilution Microemulsion for Stealth Nanoparticles

ST-CS NP were prepared using the innovative technique called “cold dilution of microemulsion” [25 ]. This technique involves the preparation of an O/W microemulsion (µE) using a partially water-soluble organic solvent as disperse oil phase. Following the dilution of µE with water, the solubilization of the organic solvent in water occurs, with the consequent NP precipitation. In our experimental conditions, butyl lactate (BL) was chosen as partially water-soluble organic solvent.
Different O/W µE were prepared using cholesterol (CHOL) dissolved in water-saturated BL (s-BL) as internal phase, BL-saturated water (s-water) as external phase, and various surfactants and cosurfactants. ST-CS was pre-solubilized in benzyl alcohol (BA) and then added in the dispersed phase. The resulting µE compositions are reported in Table 1. µE were diluted with water to obtain the precipitation of ST-CS NP.
ST-CS NP suspensions were purified by gel chromatography using agarose cross-linked gel (Sepharose^® CL 4B) as stationary phase. Briefly, 1 mL ST-CS NP was introduced at the head of a 10 mL-column and eluted by gravity with hypertonic PBS (8.0 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na₂HPO₄ 2H₂O, 0.24 g/L KH₂PO₄). Fractions of 1 mL each were collected. The opalescent fractions containing purified ST-CS NP were concentrated under nitrogen up to 1 mL final volume.
To verify the effective presence of ST-CS on NP surface, FITC-ST-CS NP were prepared introducing FITC-ST-CS in µE1.