Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope

Abstract

Unilamellar liposomes with diameters of 25-100 microns were prepared in various physiological salt solutions, e.g., 100 mM KCl plus 1 mM CaCl2. Successful preparation of the giant liposomes at high ionic strengths required the inclusion of 10-20% of a charged lipid, such as phosphatidylglycerol, phosphatidylserine, phosphatidic acid, or cardiolipin, in phosphatidylcholine or phosphatidylethanolamine. Three criteria were employed to identify unilamellar liposomes, yielding consistent results. Under a phase-contrast microscope those liposomes that showed the thinnest contour and had a vigorously undulating membrane were judged unilamellar. When liposomes were stained with the lipophilic fluorescent dye octadecyl rhodamine B, fluorescence intensities of the membrane of individual liposomes were integer multiples (up to four) of the lowest ones, the least fluorescent liposomes being those also judged unilamellar in the phase-contrast image. Micropipette aspiration test showed that the liposomes judged unilamellar in phase and fluorescence images had an area elastic modulus of approximately 160 dyn/cm, in agreement with literature values. The giant liposomes were stable and retained a concentration gradient of K+ across the membrane, as evidenced in fluorescence images of the K(+)-indicator PBFI encapsulated in the liposomes. Ionophore-induced K+ transport and associated volume change were observed in individual liposomes.

Dynamic microcompartmentation in synthetic cells

“…As is typical for preparations of giant unilamellar vesicles by the ''gentle swelling'' method used here (24,35), we observe a variety of sizes and morphologies of vesicles in our synthetic cell preparations (Fig. 6, which is published as supporting information on the PNAS web site).…”

“…Lipid vesicles containing the ATPS solution were prepared similarly to our previously reported modification (22) of standard lipid hydration procedures (24,25). A key difference is that we did not routinely place synthetic cells in sucrose solutions before observation, as in our previous work (22).…”

“…ATPScontaining GVs were prepared by using modification of the prcedure of Akashi and coworkers (24). Vesicles were prepared by drying a mixture of 90 l of egg PC (10 mg͞ml) with 5 l of DOPG (20 mg͞ml) in CHCl 3 along with 1 l of PE-Rhod (0.2 mg͞ml) under Ar.…”

Phase Boundaries and Biological Membranes

“…Instead, new kinds of experiments have been required for finding phase boundaries in bilayer lipid mixtures. The key experimental technique that led to much of the recent phase diagram results was fluorescence microscopy imaging of giant unilamellar vesicles (GUVs), which in favorable cases reveals both the existence of any coexisting phases and their phase identity (1,3,21). Still, fluorescence microscopy has some deficiencies, including failure to detect domains that are too small or that show too little contrast with a particular dye; difficulty to detect some boundaries accurately (disappearance of a small amount of the minor phase); poor equilibration, especially for samples containing large fractions of solid phase; artifactual light-induced domains (4) (see Sidebar); and difficulty to determine the relative fractions of any coexisting phases.…”

Effects of SiO2 nanoparticles on phospholipid membrane integrity and fluidity

“…GUVs were made using the gentle hydration method [23]. DOPC, DPPG and 16:0 TAP were dissolved in chloroform:methanol (2:1 by volume) to make lipid solutions of 18 mg/mL, 2 mg/mL and 2 mg/mL, respectively.…”