Masataka Chiba

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.

1P212 Measuring the lamellarity of giant liposomes prepared by inverted emulsion method(13A.Biological & Artifical membrane: Structure & Property,Poster,The 51st Annual Meeting of the Biophysical Society of Japan)

The inverted emulsion method of liposome formation is pushing water-inoil droplets through the water/oil interface. This method is efficient for encapsulating proteins and is thus widely used for modeling cells. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively (e.g., dependency on lipid composition). Here we prepared fluorescently labeled liposomes and analyzed the fluorescence intensity of the membrane of individual liposomes under the microscope. By comparing the intensities of the membrane before and after quenching of the fluorescent dye on the outermost monolayer and monitoring the permeability of the inner and the outer buffers in the presence of hemolysin, we concluded that >90% of liposomes were unilamellar.