Size fractionation and size characterization of nanoemulsions of lipid droplets and large unilamellar lipid vesicles by asymmetric-flow field-flow fractionation/multi-angle light scattering and dynamic light scattering.

Asymmetric-flow field-flow fractionation technique coupled to a multi-angle light-scattering detector (AF4-MALS) was used together with dynamic light-scattering (DLS) in batch mode and transmission electron microscopy (TEM) to study the size characteristics of the trioleoylglycerol lipid droplets covered by a monolayer of sphingomyelin and cholesterol, in water phase. These lipid droplet nanoemulsions (LD) were formed by ultrasonication. In parallel, the size characteristics of large unilamellar lipid vesicles (LUV) prepared by extrusion and composed of sphingomyelin and cholesterol were determined. LD and LUV were prepared at two different molar ratios (1/1, 4/1) of sphingomyelin and cholesterol. In AF4-MALS, various cross-flow conditions and mobile phase compositions were tested to optimize the separation of LD or LUV particles. The particle radii, R, as well as the root-mean-square radii, Rrms, of LD and LUV were determined by AF4-MALS, whereas the hydrodynamic radii, Rh, were obtained by DLS. TEM visualization revealed round shape particles of LD and LUV.