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Unilamellar liposomes are widely used as model membranes to represent and study the properties of biological membranes and as potential drug delivery systems. It is well established that ethanol and other amphiphiles induce the interdigitated L beta I phase in multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). However, all of the work on this phase has been performed using the hand shaken multilamellar preparations. In the present report, we have studied the induction of interdigitation in a series of unilamellar vesicles prepared by sonication and by extrusion. The methods used to characterize the vesicles were freeze fracture electron microscopy and quasielastic light scattering (QELS). Two fluorescence methods were used to detect interdigitation, the DPH fluorescence quenching method (Nambi, P., Rowe, E.S. and McIntosh, T.J. (1988) Biochemistry 27, 9175-9182) and the pyrene-PC fluorescence method (Komatsu, H. and Rowe, E.S. (1991) Biochemistry 30: 2463-2470). It was found that sonicated vesicles are not stable in the presence of interdigitating concentrations of ethanol; they form higher aggregates at all temperatures examined. The behavior of the extruded vesicles was different from that of the SUV; each size studied was stable in the presence of ethanol, although they exhibited an increase in size. It was shown that extruded vesicles having a 200-nm or greater diameter become interdigitated in the presence of ethanol. The threshold concentration for interdigitation in vesicles is greater than that for MLVs and it decreases with increasing vesicle size, approaching the MLV value for the largest vesicles.

It is well established that ethanol and other amphipathic molecules induce the formation of a fully interdigitated gel phase in saturated like-chain phosphatidylcholines (PC's). We have previously shown that the induction of interdigitation in PC's by ethanol is dependent upon the alcohol concentration, the lipid chain length, and the temperature [Nambi, P., Rowe, E. S. & McIntosh, T. J. (1988) Biochemistry 27, 9175-9182]. In the present study, we have used high-sensitivity differential scanning calorimetry to investigate the transitions of distearoylphosphatidylcholine between the noninterdigitated and the interdigitated phases. The enthalpy of the L beta' to L beta I transition is approximately half that of the L beta' to P beta' transition which occurs in the absence of ethanol. The reversibility of these transitions has also been investigated by employing both heating and cooling scans in order to establish the most stable phases as a function of temperature and ethanol concentration. It has been demonstrated that the transition to the interdigitated phase is reversible as a function of temperature. Kinetic studies on the reverse transition (L beta I to L beta') demonstrate that this transition can be very slow, requiring weeks to reach completion. The rate depends upon temperature and ethanol concentration. The slow phase changes mean that the lipid can exist for long periods of time in a phase structure which is not the most stable state. The biological significance of this type of lipid behavior is the implication that the phase structure of biological membranes may depend not only on the most stable phase structure of the lipids present but also on the synthetic pathway or other kinetic variables.

It is now well established that a number of amphiphilic molecules such as ethanol can induce the formation of the fully interdigitated gel phase in phosphatidylcholines. We have shown earlier that alcohols such as ethanol induce biphasic melting behavior in phosphatidylcholines [Rowe, E. S. (1983) Biochemistry 22, 3299-3305] but not in phosphatidylethanolamines [Rowe, E. S. (1985) Biochim. Biophys. Acta 813, 321-330]. Simon and McIntosh [(1984) Biochim. Biophys. Acta 773, 169-172] showed that the alcohol-induced biphasic melting behavior in phosphatidylcholines is a consequence of acyl chain interdigitation. In the present study we demonstrate the detection of the transition of DPPC and DSPC to the interdigitated phase in the presence of ethanol using the fluorescence properties of the commonly used fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH). By correlating fluorescence and X-ray diffraction results, we have demonstrated the use of fluorescence to study the phase transition from the noninterdigitated to the interdigitated phase. Using this method, we have investigated the temperature and ethanol concentration dependence of the induction of the interdigitated phase in DSPC and DPPC and shown that the induction of interdigitation by ethanol is temperature dependent, with higher temperature favoring interdigitation. The temperature-ethanol phase diagrams have been determined for DPPC and DSPC.

The specific effect of ethanol on several aspects of the gel-to-liquid crystal transition of dipalmitoylphosphatidylcholine was investigated using two spectrophotometric techniques, one probe method and one direct method. Ethanol shifts the phase-transition temperature to low temperature, demonstrating that ethanol interacts preferentially with the fluid phase. Thermodynamic analysis of the melting point depression leads to a calculated membrane:buffer partition coefficient of 6.25 (mole fraction units) or 0.15 mole of ethanol per kilogram of lipid:mole of ethanol per liter of solution. Careful evaluation of the transition cooperativity with temperature resolution of +/- 0.1 degrees shows that there is no reduction in transition cooperativity, and thus no reduction in size of the cooperative lipid clusters due to ethanol. The implications of these findings for the mechanism of action of ethanol in terms of current theories of anesthetic mechanisms are discussed.

The interactions of butanol and mixtures of butanol and ethanol with dipalmitoylphosphatidyl choline (DPPC) liposomes have been investigated by both spectrophotometric measurements and Fourier transform 13C nuclear magnetic resonance spectroscopy. The spectrophotometric experiments indicate that butanol exhibits the same effects on the thermotropic properties of DPPC as the other short chain alcohols, methanol, ethanol and propanol, which have been shown to be characteristic of the alcohol induced transition of the lipid to the interdigitated state. An additive effect of butanol and ethanol on the induction of the interdigitated phase in DPPC was also observed. A decrease in line width and increase in T1 of the choline methyl signal were observed in the 13C-NMR experiments conducted at 32 degrees C when butanol was added to DPPC in increasing amounts suggesting an increase of disorder in the head group region of the lipid. Addition of ethanol to the NMR sample containing butanol produced hysteresis in the heating and cooling curves characteristic of the interdigitated state. In the interdigitated state, the choline methyl signal exhibited a T1 value equal to that when the lipid is in the fluid state. The increase of mobility in the head group region in the interdigitated gel state relative to the bilayer gel can be rationalized by the increase in surface area in that site when the lipid interdigitates.

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine (DEPE) has been investigated using spectrophotometry and 31P nuclear magnetic resonance (NMR). It has been demonstrated that the bilayer to inverted hexagonal phase transition can be observed by spectrophotometry. The effects of the methanol, ethanol, and propanol on both the gel to liquid crystal transition and the bilayer to inverted hexagonal transition were investigated by spectrophotometry. It was shown that these alcohols shift the gel to liquid-crystalline phase transition to lower temperature, whereas the bilayer to inverted hexagonal phase transition is shifted to higher temperatures by these alcohols. The structural transition between the bilayer and inverted hexagonal phase of pure DEPE was also investigated by 31P-NMR.

It has previously been shown that alcohol has different effects on the gel to liquid-crystal phase transition of phosphatidylcholines (PC's) and phosphatidylethanolamines (PE's) [Rowe, E. S. (1985) Biochim. Biophys. Acta 813, 321-330]. In this investigation, the thermotropic properties of binary PE-PC mixtures were studied in the presence of ethanol in order to determine whether the differential interactions of alcohol with PC and PE would lead to lateral phase separations. Phase diagrams of the dilaurylphosphatidylethanolamine-dipalmitoylphosphatidylcholine [PE(12:0)-PC(16:0)] system were constructed in the presence and absence of ethanol. It was shown that lateral phase separations occur in the gel phase over a certain composition range in the presence of 100 mg/mL ethanol. In the absence of alcohol these two lipids are miscible in both the gel and liquid-crystal states. The data suggest that in the presence of ethanol these lateral phase separations involve the coexistence of regular bilayer gel and the fully interdigitated gel phase, which has previously been shown to occur in pure PC(16:0) under these conditions [Simon, S. A., & McIntosh, T. J. (1984) Biochim. Biophys. Acta 773, 169-172]. The biological implications of these findings are discussed.

Phase transitions of dimyristoylphosphatidylcholine (DMPC) are followed using optical density measurements, as a function of aqueous ethanol concentration. The shift of the phase transition to lower temperatures is interpreted in terms of the thermodynamics of freezing point depressions. This analysis provides a partition coefficient of ethanol into DMPC of 0.07 liter of solution/kg membrane or 2.64 moles of solvent/mole of DMPC.