Spontaneous Generation of Giant Liposomes from an Oil/Water Interface

Giant liposomes, phospholipid vesicles with a bilayered membrane and a size greater than 1 mm, have attracted a strong scientific interest mainly motivated by trying to synthesize artificial cells. Indeed, giant liposomes have been shown to be the best primary model of cells in terms of size, membrane composition, or the ability to encapsulate biologically relevant molecules. Hence, various methodologies for the preparation of cell-sized liposomes have been proposed, such as natural swelling, freezing and thawing, electroformation, extraction from a cell membrane, transfer of microdroplets, and microfluidic methods. Among them, the widely used natural swelling method, in which liposomes generate from a dry lipid film, is usually described as a spontaneous phenomenon, and its mechanism has been investigated. Here, we report a new spontaneous phenomenon in a biphasic (oil/water) environment that leads to the formation of giant liposomes with a diameter ranging from 1 to 100 mm. We observed that giant liposomes spontaneously formed when a horizontal liquid interface was made between an upper oil phase containing phospholipid and a lower water phase. Our method mainly differs from natural swelling in that liposomes are obtained from a liquid oil/water interface at which the lipid organization and dynamics differ from that on a usual solid substrate. In this study, we used DOPC phospholipid dissolved in mineral oil at various concentrations, and characterized the peculiar phospholipid organization at the interface as well as the obtained giant liposomes by confocal microscopy. An oil/water interface was prepared in a cylindrical observation chamber made of poly(dimethyloxane) (PDMS) walls bound to a glass microscope cover slide (Figure 1). A thin layer of water was placed at the bottom of the observation chamber, which was topped up with a thin layer of mineral oil containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid and a small fraction of 1-oleoyl-2-[12-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC) fluorescent phospholipid. First, the experiment was performed with a DOPC concentration of 1 mm in oil and a temperature of 25 8C. One minute after interface preparation, we observed the formation of small phospholipid aggregates randomly distributed at the oil/water interface, which presented some irregularities. (Figure 2A top) The presence of liposomes was not detected in either phase. With time, the interface became more indented as phospholipid aggregates slowly reorganized. 18 h after interface preparation, phospholipid molecules were arranged into a dotted pattern at the interface, as shown by the fluorescence of NBD-PC, Figure 1. Experimental set-up.