We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.

Fusion between bilayers of mixed egg phosphatidylcholine and soybean phosphatidylethanolamine was induced by freezing and thawing. Contact points between bilayers were observed by freeze fracture electron microscopy, and isotropic molecular motional averaging was detected by phosphorus-31 nuclear magnetic resonance under fusion conditions. A molecular model of point defect structure is proposed as an intermediate stage of fusion.

Salmon, porcine, and human calcitonins interact with phosphatidylglycerol to form water-soluble complexes, but these peptides do not interact with the zwitterionic lipids phosphatidylcholine or sphingomyelin. The calcitonins are more helical in the presence of dimyristoylphosphatidylglycerol than in its absence, but human calcitonin is considerably less helical than the other two, particularly in the presence of the lipid. This may explain the previously reported faster rate of degradation of human compared with salmon calcitonin in vivo. The ability of human calcitonin to solubilize dimyristoylphosphatidylglycerol and to alter the phase transition properties of this phospholipid while maintaining a low content of helix indicates that the presence of an amphipathic helix is not a requirement for these effects. The binding of salmon calcitonin to dimyristoylphosphatidylglycerol has been studied by determining the dependence of the circular dichroism properties of the peptide on the concentration of lipid. At 25 degrees C, salmon calcitonin binds to five molecules of dimyristoylphosphatidylglycerol with an affinity constant of 1 X 10(5) M-1. Little change in these parameters is observed at 38 degrees C, and the complex is stable over a wide range of temperatures both above and below the phase transition temperature. The rate of reaction of salmon calcitonin with dimyristoylphosphatidylglycerol is rapid at or above the phase transition temperature of the lipid but not at low temperatures. Salmon calcitonin also interacts with egg phosphatidylglycerol. These results demonstrate that salmon calcitonin can react with phosphatidylglycerol at or above its phase transition temperature to form complexes which are at least kinetically stable both above and below the phase transition temperature. Salmon calcitonin can solubilize mixtures of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylcholine containing 25% or more of the former phospholipid. The helical content of the peptide in the presence of these lipid mixtures is dependent on the fraction of the lipid which is phosphatidylglycerol, with larger fractions of this lipid leading to the formation of a higher helical content. At 25% phosphatidylglycerol, salmon calcitonin can solubilize the lipid mixture without much increase in the helix content of the peptide, again demonstrating that an amphipathic helical structure is not required for the solubilization of phospholipids. Ionic bonding appears to be an important component in the binding of the cationic calcitonins to phospholipids. Salmon calcitonin binds to the acidic phospholipids phosphatidylinositol and phosphatidic acid, but not to zwitterionic phospholipids. In addition, high concentrations of NaCl cause the dissociation of the complex between salmon calcitonin and dimyristoylphosphatidylglycerol.(ABSTRACT TRUNCATED AT 400 WORDS)

Ribbon-like structures result when amphotericin B interacts with lipid in an aqueous environment. At high ratios of amphotericin to lipid these structures, which are lipid-stabilized amphotericin aggregates, become prevalent resulting in a dramatic attenuation of amphotericin-mediated mammalian cell, but not fungal cell, toxicity. Studies utilizing freeze-etch electron microscopy, differential scanning calorimetry, "P NMR, x-ray diffraction, and optical spectroscopy revealed that this toxicity attenuation is related to the macromolecular structure of the complexes in a definable fashion. It is likely that amphotericin in this specific form will have a much improved therapeutic utility.Amphotericin B, a polyene antibiotic, is the drug ofchoice for a variety of systemic fungal infections that were almost always fatal prior to its introduction (1). The cytotoxic mechanism of this compound has been the subject of intensive investigation over many years and is thought to reside in its ability to form membrane ion channels particularly in the presence of sterols (2). That these channels and associated lethal permeability changes occur at somewhat lower membrane concentrations in the presence of ergosterol (the predominant sterol in fungal cell membranes) rather than cholesterol (the predominant sterol in mammalian cell membranes) most likely forms the basis of the selective toxicity of this drug (3). Still, in its present dosage form, which is a deoxycholate micelle, its clinical utility is profoundly limited by host cell toxicity particularly in those cases (such as in patients with acquired immunodeficiency syndrome) where high-dose therapy is indicated.Much attention has focused on liposome suspensions containing 5-10 mol % amphotericin B because these systems have been shown to produce a significant attenuation of toxicity with little compromise to efficacy (4-7). In fact one such formulation in which amphotericin comprises 5 mol % of a 1,2-dimyristoyl-sn-glycero(3)phosphocholine/1,2-dimyristoyl-sn-glycero(3)phospho (1) PtdGro at a molar ratio of 7:3 was deposited from chloroform as a thin film on the bottom ofa 500-ml round-bottomed flask by rotoevaporation. This film was solubilized in 100 ml of amphotericin B in methanol (0.1 mg/ml) and again rotoevaporated to a thin film. When dry, the film was suspended in isotonic phosphatebuffered saline (PBS) and subjected to bath sonication for 0.5 hr or until particles exhibiting Brownian motion were observed by phase-contrast microscopy.Sucrose Density Centrifugation. Typically, 200 gl of material was layered onto a continuous sucrose gradient in 150 mM NaCI/20 mM Hepes, pH 7.4. The gradient was centrifuged for 22 hr at 22TC in a SW 60 rotor (Beckman) at 230,000 x g. After centrifugation the gradient was fractionated into 150-,ul aliquots and assayed for amphotericin B (from absorbance at 412 nm in dimethylformamide) and phospholipid (from phosphate assay after digestion).In Vivo Toxicity. LD50 values were determined by the method of Reed and Muench (12). Female...

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