Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles.

Abstract

Two methods are reported for the formation of large, uniform-sized phospholipid vesicles. (5) or ether injection (6). Vesicles may be formed with phospholipid mixtures, purified phospholipids, or synthetic phospholipids. The methods of preparation and the properties of such vesicles have recently been reviewed (1, 7). Although multilamellar liposomes are easy to prepare, they have been criticized as a model system for the study of membrane phenomena (1). Thus, the kinetics of transport processes are more complex due to multiple diffusion barriers and it is not easy to obtain a uniform-sized population of vesicles. These disadvantages are overcome by the use of small, unilamellar vesicles; however, the surface of these vesicles has a high degree of curvature which affects many of the physical properties of the phospholipid in the vesicles (8, 9). One would then expect the large, unilamellar vesicles to resemble most closely biological membranes. It was previously shown (4) that treatment of 1 mol of egg lecithin with 2 mol of bile salt followed by gel filtration to remove the bile salt results in the formation of a uniform population of single-bilayer vesicles having an average diameter of 300 A. We now report that the treatment of 1 mol of egg lecithin with 0.5 mol of bile salt results in the formation of a uniform population of single-bilayer vesicles having an average diameter of 1000 A. These preparations, which have a large internal volume and are free of multilamellar structures, may be useful in cases in which previous techniques (5, 6) cannot be applied.MATERIALS AND METHODS Egg phosphatidylcholine (10), sodium deoxycholate (11), cytochrome b5 heme peptide (12), the catalytic fragment of NADH-cytochrome b5 reductase (13) (pH 5) or 1% ammonium molybdate (pH 5). One drop of liposomes (30-100 ,uM phospholipid) was placed on a Formvar-coated grid and, after 30 sec, one drop of staining solution was added; 30 sec later, the solution was drained off with filter paper and the grid was allowed to air dry. The grids were examined in a Hitachi llE electron microscope operated at 75 kV. Vesicle diameters were measured on several electron micrographs covering different areas on a grid and then averaged from at least 400 measurements.Method I: Formation of Large Vesicles from Small, Preformed Vesicles. Small, uniform-sized egg lecithin vesicles were prepared as described (11). Any solute to be entrapped was added to the vesicle solution at this point and the vesicles were adjusted to 20 mM phospholipid. The solution was then warmed to 25°C and an aliquot of 250 mM sodium deoxycholate was rapidly added and mixed to give a final mixture containing a ratio of deoxycholate to phospholipid of 1:2. The large vesicles began to form almost immediately, as indicated by. the increase in the light scattering of the solution, a change from nearly clear for the small vesicles to a transparent opalescence for the large vesicles. Vesicle formation was complete within 5-10 min at 250C (vesicles could also be formed at 40C...

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