Comparison of the enthalpy state of vesicles of different size by their interaction with alpha-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with alpha-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with alpha-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of alpha-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, delta H, of the interaction of the three vesicle types with alpha-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24 degrees C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic delta H values around 24 degrees C for large vesicles approximates the transition enthalpy of the pure phospholipid.     

Cholesterol transfer from small and large unilamellar vesicles

The rates of transfer of [14C]cholesterol from small and large unilamellar cholesterol/egg yolk phosphatidylcholine vesicles to a common vesicle acceptor were compared at 37 degrees C. The rate of exchange of cholesterol between vesicles of identical cholesterol concentrations (20 mol%) did not differ from the rate of transfer from donor vesicles containing 20 mol% cholesterol to egg yolk PC vesicles. Further, the rate of transfer of [14C]cholesterol from vesicles containing 15 mol% dicetyl phosphate (to confer a negative charge) was not different from the rate of transfer from neutral vesicles. However, the half-time for transfer of [14C]cholesterol from large unilamellar donor vesicles was about 5-times greater (10.2 h, 80 nm diameter) than from small unilamellar vesicles (2.3 h, 23 nm diameter). These data suggest that increased curvature in small unilamellar vesicles reduces cholesterol-nearest neighbor interactions to allow a more rapid transfer of cholesterol into the aqueous phase.     

Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

Effect of Phosphatidylcholine on Acrosome Breakdown and Fertilizing Capacity of Amphibian Spermatozoa

The effect of vesicles of purified egg yolk phosphatidylcholine on the fertilizing capacity and acrosome breakdown of amphibian spermatozoa was studied. When Bufo arenarum spermatozoa were incubated with either small unilamellar vesicles (prepared by sonication) or with large unilamellar vesicles (prepared by reverse-phase evaporation) a decrease in the fertilizing capacity of spermatozoa was found. At the same phosphatidylcholine concentration, large unilamellar vesicles were more inhibitory than small unilamellar vesicles. The inhibition was dependent upon the phospholipid concentration and the length of the incubation period. Small unilamellar vesicles did not modify the time course of acrosome breakdown in Leptodactylus chaquensis , while large unilamellar vesicles markedly accelerated the rate of acrosome breakdown. In both biossays, the charge of the vesicles (made either positive or negative by the addition of 5% stearylamine or 5% phosphatidic acid) did not influence their biological effect. Multilamellar vesicles did not alter the fertilizing capacity nor the acrosome breakdown. We conclude that the size and the structure of the vesicles are important parameters in determining the inhibitory capacity of phosphatidyl choline on amphibian fertilization.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.     

Temperature-induced fusion of small unilamellar vesicles formed from saturated long-chain lecithins and diheptanoylphosphatidylcholine

Small unilamellar vesicles which form when gel-state long-chain phosphatidylcholines are mixed with micellar short-chain lecithins undergo an increase in size as the long-chain species melts to its liquid-crystalline form. Analysis of the vesicle population with quasi-elastic light scattering shows that the particle size increases from 90-A radius to greater than 5000-A radius. Resonance energy transfer experiments show total mixing of lipid probes with unlabeled vesicles only when the Tm of the long-chain phosphatidylcholine is exceeded. This implies that the large size change represents a fusion process. Aqueous compartments are also mixed during this transition. 31P NMR analysis of the vesicle mixtures above the phase transition shows a great degree of heterogeneity with large unilamellar particles coexisting with oligo- and multilamellar structures. Upon cooling the vesicles below the Tm, the original size distribution (e.g., small unilamellar vesicles) is obtained, as monitored by both quasi-elastic light scattering and 31P NMR spectroscopy. This temperature-induced fusion of unilamellar vesicles is concentration dependent and can be abolished at lower total phospholipid concentrations. It occurs over a wide range of long-chain to short-chain ratios and occurs with 1-palmitoyl-2-stearoylphosphatidylcholine and dimyristoylphosphatidylcholine as well. Characterization of this fusion event is used to understand the anomalous kinetics of water-soluble phospholipases toward these unusual vesicles.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

The effects of charge and size on the interaction of unilamellar liposomes with macrophages

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type     

Movement of cholesterol between vesicles prepared with different phospholipids or sizes

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.     

Rapid kinetics of Ca2+-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles. The effect of bilayer curvature on leakage

We have employed both small unilamellar vesicles (SUV) and large unilamellar vesicles formed by the reverse phase evaporation technique (REV) to study the initial kinetics of membrane aggregation and fusion. Stopped flow measurements of the calcium-induced changes in the turbidity of SUV and REV, formed from 1:1 (mol/mol) mixtures of bovine phosphatidylserine (PS) and Escherichia coli phosphatidylethanolamine (PE), were used to follow particle aggregation. Simultaneous measurements of the fluorescence resonance energy transfer from N-(7-nitro2,1,3-benzoxadiazol-4-yl) (NBD)-PE to rhodamine (Rho)-PE incorporated into the vesicle bilayers established that 1) both initial aggregation and fusion can be described as a bimolecular process and 2) the rate-limiting step of membrane fusion is aggregation. Thus fusion takes place in the microsecond time domain. Parallel experiments, which simultaneously measured aggregation and the dequenching of encapsulated carboxyfluorescein (CF) in the presence and absence of antifluorescein antibodies in the suspension medium, established that the small unilamellar vesicles rapidly lose their contents of CF as they fuse. On the other hand, the first few cycles of fusion of the large unilamellar vesicles are nonleaky, but leakage develops within 1-2 s as the particles grow in size. Thus the results demonstrate that the SUV are poor models for the study of nonleaky fusion, while the REV must be carefully tested before unambiguous interpretation of fusion assays involving the formation of tight complexes (such as the terbium-dipicolinic assay) can be made. NBD-PE undergoes very rapid, Ca2+-promoted changes in quantum yield which can obscure the resonance energy transfer signals. Thus data from the NBD-PE/Rho-PE energy transfer pair must be carefully scrutinized for artifacts.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles.

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37 degrees C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20 degrees C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20 degrees C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freeze-thaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Spontaneous lipid vesicle fusion with electropermeabilized cells

Fusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process.     

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.     

Binding of insulin to the external surface of liposomes: Effect of surface curvature,temperature, and lipid composition

The binding of insulin to the external surface of phosphatidylcholine liposomes as a function of the temperature, the surface curvature, and the composition of lipids was studied. The amount of the saturated binding of insulin to liposomes was assessed by gel-filtration chromatography. The binding of insulin to small unilamellar vesicles was highly dependent upon the temperature, favoring low temperatures. As the temperature increased, there was a distinct temperature range where the binding of insulin to small unilamellar vesicles decreased. The temperature ranges for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles were found to be 10–20°C and 21–37°C, respectively. These temperature ranges were quite different from the reported ranges of the gel → liquid crystalline phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) for DMPC or DPPC small unilamellar vesicles. In contrast to other proteins, the amount of insulin bound to DMPC and DPPC small unilamellar vesicles was negligible at or above the upper limit of the above temperature ranges, and increased steadily to 6–7 μmol of insulin per mmol of phospholipid as the temperature decreased to or below the lower limit of these temperature ranges. On the other hand, the binding of insulin to the large multilamellar liposomes cannot be detected at all temperatures tested. The affinity of insulin to neutral phosphatidylcholine small unilamellar vesicles appeared to be related to the surface curvature of the liposomes, favoring the liposomes with a high surface curvature. Furthermore, the amount of insulin bound to small unilamellar vesicles decreased as the content of the cholesterol increased. The presence of 10% molar fraction of phosphatidic acid did not appear to affect the binding of insulin to small unilamellar vesicles. However, the presence of 5% molar fraction of stearylamine in DPPC small unilamellar vesicles increased the amount of bound insulin as well as the extent of aggregation of liposomes. The results of the present study suggest that the interstitial regions of the acyl chains of phospholipids between the faceted planes of small unilamellar vesicles below $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ may be responsible for the hydrophobic interaction of insulin and small unilamellar vesicles. The tight binding of insulin to certain small unilamellar liposomes could lead to an overestimation of the true amount of insulin encapsulated in liposomes, if care is not taken to eliminate the bound insulin during the procedure of encapsulating insulin in liposomes.     

Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers

We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Fusogenic activity of δ-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin δ-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of δ-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance

 It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band ³¹P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters ≤30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From ³¹P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles. Received: 2 September 1999 / Revised version: 27 February 2000 / Accepted: 27 February 2000