Investigation on the release of fluorescent markers from w/o/w emulsions by fluorescence-activated cell sorter

The mechanism of release of two fluorescent markers, fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) and fluorescein, from water-in-oil-in-water (w/o/w) emulsions was investigated using a rapid and sensitive method based on fluorescence-activated cell sorting (FACS). The release of FITC-BSA from a w/o/w emulsion was controlled by diffusion rather than by simple breakdown of the multiple droplets or by formation of reverse micelles in the oil phase. In contrast, the release of fluorescein from a double emulsion was controlled by formation of reverse micelles rather than by diffusion or simple breakdown of multiple droplets. A significant difference in the yield and fraction of FITC-BSA and fluorescein released from double emulsions was observed due to their different molecular structure and properties. The yield of FITC-BSA incorporation in a double emulsion increased with increasing FITC-BSA concentration in the internal water phase, while the yield of fluorescein decreased with increasing concentration. The fraction of FITC-BSA released from a w/o/w emulsion after 24 h decreased with an increasing concentration of FITC-BSA in the internal phase. The w/o/w emulsion with internalized FITC-BSA was more stable than that with fluorescein, indicating its further application for sorting or enriching size-controlled double droplets that contained genes and water-soluble drugs.