Chain length and headgroup dependence of phase separation in mixed vesicles of DiA and phospholipids

摘要

Styryl Dyes are used in a variety of biophysical applications since their properties depend on their external environment. In particular, the fluorescence of styryl dyes dramatically increase upon incorporation in to the hydrophobic environment of a lipid membrane compared with the fluorescence in the aqueous phase. These dyes have been used to study endocytosis, pathway of vesicle trafficking networks, synaptic vesicle recycling in neurons and many other phenomena. The method of incorporation of the dyes into cell membranes or vesicles, and whether they are incorporated into only the outer leaflet of the bilayer or in both leaflets is not yet resolved. In addition, whether the dyes segregate into phase separated domains is not yet fully understood. The dye we have investigated is 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA-16), where we have exchanged the I- counterion with the Cl- counterion in DiA-16 to increase water solubility. This enables us to make vesicles composed of only neat DiA-16. In order to investigate the role of headgroup and chain length on the miscibility of DiA-16 with other lipids, we have prepared multilamellar vesicles (MLVs) composed of DMPC, DPPC, DSPC, DPPE, DSPE, and DMPE over the whole composition range of lipid/DiA from molar ratios of 100/1 to 1/100. Nano-diffrential scanning calorimetry (Nano-DSC), fiuorimetry and Langmuir-Blodgett techniques were used to investigate the incorporation of the styryl dyes into multilamellar vesicles (MLVs). Adsorption/exchange experiments were undertaken to investigate the temperature independence of DiA-16 uptake by different lipid vesicles, by comparison with shifts in the gel-to-liquid crystalline phase transition temperature established using calibration curves obtained using vesicles prepared with the dyes already incorporated into the lipid vesicles.