Experimental conditions for the spontaneous assembly of isolated phospholipid bilayers have been obtained by a systematic thermodynamic study of aqueous lipid dispersions. The analysis utilizes the equilibrium air-water surface film to monitor the physical state of the dispersed lipid phase. At a critical temperature T* the surface film and bulk dispersion simultaneously form the unilamellar state; this state forms only at T*. The critical point is generally higher than Tm, the gel-liquid crystal transition temperature. Thus, the unilamellar state is always liquid crystalline. Examples are presented to show how T* varies in multicomponent and multiphase systems in accordance with the phase rule. The relevance of the critical unilamellar state to membrane bilayer assembly is examined within the context that T* for total lipid extracts of cell membranes is identical with the temperature at which the cells were grown.

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The surface vapor pressure-composition diagrams for a variety of binary lipid mixtures have been determined and shown, from the thermodynamics of the mixing process, to be analogous to those for bulk systems. It was observed that (a) two liquid-condensed or two liquid-expanded lipids gave ideal mixing, and (b) the mixing of a liquid-condensed with a liquid-expanded lipid gave either positive deviations from ideal behavior or phase separation (immiscibility). These results are shown to be consistent with regular solution theory, with the mixing process dominated by the hydrocarbon region of the film. Polar group interactions do not contribute significantly to the mixing process. Regular solution theory predicts that mixtures of cholesterol with either a liquid-expanded or a liquid-condensed lipid will show large positive deviations from "ideal" behavior, or phase separation. This is in agreement with the vapor pressure-composition studies. There is no evidence for specific interactions between cholesterol and neutral lipids in surface films.

Lipid bilayer assembly in cell membranes has been simulated with total lipid extracts from human red blood cells and from mesophilic and thermophilic bacteria grown at several temperatures. Aqueous dispersions of these natural lipid mixtures form surface bilayers, a single bimolecular lipid state, but only at the growth temperature of the source organism. Thus, a single isolated bilayer state forms spontaneously in vitro from lipids that are available in vivo at the growth temperature of the cell. Surface bilayers form at a specific temperature that is a function of hydrocarbon chain length and degree of fatty acid unsaturation of the phospholipids; this property is proposed as an essential element in the control of membrane lipid composition.

The influence of charge density and hydrocarbon chain length on the sponge−vesicle transformation of two bilayer-forming ionic phospholipids, the sodium salts of dilauroyl- and dimyristoylphosphatidylglycerol (NaDLPG and NaDMPG), was examined by measuring the solution properties of the lipids in water. The phase diagrams of these compounds in water indicate they undergo a transformation from a transparent, jelly-like sponge phase to unilamellar vesicles at a critical temperature T*; for NaDLPG T* = 19 °C, and for NaDMPG T* = 31.6 °C. At T > T* multilamellar vesicles form. The Krafft temperature for NaDLPG is ∼5 °C, just above T m, the gel−liquid crystal transition temperature, and for NaDMPG it is ∼32 °C, slightly higher than T*. For both lipids, the charge density of the equilibrium monolayers at the air/water interface decreases dramatically as T increases and approaches T*. This effect is attributed to hydrolysis of the phosphate moiety of the lipid. Changes in solubility consistent with a decrease in bilayer charge density at T* are also observed. The transformation from the sponge phase to vesicles at T* is a structural response to the change in bilayer charge density. The results emphasize the importance of bilayer-localized chemical reactions in the transformation of the sponge state to vesicles.