Population analysis of structural properties of giant liposomes by flow cytometry

We used fluorescence flow cytometry to analyze the structural properties of populations of giant liposomes formed by different preparation methods. The inner aqueous volumes and nominal membrane surface areas of a large number of individual liposomes were measured simultaneously by using fluorescent markers. We compared these properties of liposomes prepared by the natural swelling method, the freeze-dried empty liposomes method, and the water-in-oil (W/O) emulsion method. A two-dimensional contour distribution map of the inner volume and the nominal surface area was used to elucidate the structural properties of liposomes over a wide range of liposome sizes. Lamellarity of liposomes was evaluated as the ratio of the nominal surface area to the theoretical surface area calculated from the liposome inner volume. This population analysis revealed the dependency of lamellarity on liposome volume: while the nominal surface areas of populations of liposomes prepared by the natural swelling and the freeze-dried empty liposome methods were widely distributed, those prepared by the W/O emulsion method had a narrower distribution within small values. Furthermore, with the latter method, the nominal surface area varied in proportion to the two-thirds power of the inner volume ranging for several orders of magnitude, indicating the liposomes had a thin membrane, which was constant for the wide volume range. The results as well as the methodology presented here would be useful in designing giant liposomes with desired properties.