Preparation and characterization of monodisperse unilamellar phospholipid vesicles with selected diameters of from 300 to 600 nm

A method has been developed for making large unilamellar vesicles (LUV) with low polydispersity. The LUV, constituted of dioleoylphosphatidic acid (DOPA), 300 nm in diameter are made by a modification of the pH adjustment technique (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683-1687). This size is 10 times that (30 nm) of vesicles prepared by prolonged sonication. Vesicle size is increased stepwise by adding cholesterol (to a maximum of 40 mol% cholesterol) to form vesicles in 0.15 M KCl with up to 600 nm diameter. The vesicle size is measured by photon correlation spectroscopy, electron microscopy, and by measurement of the internal volume with cyanocobalamin while calculating the number of DOPA molecules per vesicle. Vesicles are stable for at least three weeks. Sepharose 4B column chromatography of the preparation yields a peak of fractions with the same polydispersity as the original sample and shows that 30 to 40% of the original lipid in a sample is recovered as LUV. Less than 2% of the sample forms small unilamellar vesicles (SUV) (diameter = 30 nm), which emerge from the column in a separate peak. Since the remaining lipid is not suspended in the buffer during vesicle formation, for most purposes the vesicles may be used immediately after titration so that they can be prepared in less than 40 min.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

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     

Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer

The aim of this study was to elucidate the effect of various mole percentages (0-25 mol%) of 2000 Da polyethylene glycol-disteroylphosphoethanolamine (PEG-DSPE) in the presence or absence of 40 mol% cholesterol and the effect of degree of saturation of phosphatidylcholine (PC) on the size and the lipid bilayer packing of large unilamellar vesicles (LUV). Egg PC (EPC, unsaturated) LUV and fully hydrogenated soy PC (HSPC, saturated) LUV partial specific volume, specific compressibility, size, and packing parameter (PP) of lipids were characterized by measurements of density, ultrasonic velocity, specific turbidity, and dynamic light scattering. Liposome size and specific turbidity decreased with increase in temperature and PEG-DSPE mol%, except at 7+/-2 mol%. At this PEG-DSPE mol%, an anomalous peak in liposome size of 15+/-5 nm was observed. We attribute this effect mainly to the change in the spatial structure of the PEG-DSPE molecule, depending on whether the grafted PEG is in the mushroom or brush configuration. In the mushroom regime, i.e., when the grafted PEG is up to 4 mol% in LUV, the PEG moiety did not affect the additive PP of the lipids in the bilayer, and the PP value of PEG-DSPE is 1.044; while in the brush regime, i.e., when the grafted PEG is higher than 4 mol%, the PP of PEG-DSPE decreases exponentially, reaching the value of 0.487 at 30 mol% of grafted lipopolymer. The specific compressibility and additive PP values for the mixture of matrix lipid (EPC or HSPC), cholesterol, and PEG-DSPE for all liposome compositions investigated reached their maximum at 7+/-2 mol% PEG-DSPE, the concentration of PEG-DSPE at which the highest biological stability of the LUV is achieved.     

Drug delivery using vesicles targeted to the hepatic asialoglycoprotein receptor

We assessed the utility of liver-targeted vesicles as a drug delivery system for the treatment of liver diseases. Small, unilamellar vesicles (mean diameter, 60–80 nm) composed of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and digalactosyldiacylglycerol (mol ratios, 40:40:5:15) are rapidly cleared from the blood in rats after intravenous injection. In vivo organ distribution shows that the liver is the major site of vesicles accumulation, with roughly 60–80% of the vesicles contents delivered to the liver. Isolated, perfused rat liver experiments show that the uptake is due to the hepatic asialoglycoprotein receptor, and the uptake process occurs with minimal vesicle leakage. At low doses of the vesicles, the single pass extraction by the liver is around 50%, which means that this vesicle formulation operates close to optimal efficiency as a drug delivery system to the liver. Binding of vesicles to the liver was determined to saturate at 6.5 mg total lipid/kg body weight, with a maximum steady-state turnover rate of vesicles at 37° C of 79 μg lipid/min per kg body weight. This gives a receptor recycling time of around 80 min. We have incorporated this information into a pharmacokinetic model of vesicle distribution which quantitatively predicts the kinetics and dose dependence of vesicle uptake by the liver in vivo. This information can be used to optimize vesicle-mediated drug delivery to the liver.     

Effective encapsulation of proteins into size-controlled phospholipid vesicles using freeze-thawing and extrusion

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-l-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 microm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-)(1) and cooling rate of -140 degrees C min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 +/- 20 nm.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

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%.     

Electrostatics of carboxylated anionic vesicles for improving entrapment capacity

Electrostatic interaction is an important secondary force affecting the structure, stability, and function of lipid vesicles (liposomes). For this study, a negatively charged lipid with carboxylic acid was mixed with phospholipid to produce anionic vesicles. The electrostatics of the carboxylated anionic vesicle (ca. 200 nm diameter) was determined and correlated with entrapment capacity of the vesicles. Correlative analysis revealed the zeta potential of the vesicles as a factor quantitatively affecting the entrapment capacity for a water-soluble marker, in which the entrapment capacity reached its maximum level in less than −30 mV of zeta potential. Transmission electron microscopy (TEM) revealed that the vesicles with high entrapment capacity are composed of a unilamellar membrane. This finding is expected to be useful for efficient encapsulation of water-soluble pharmaceuticals within vesicles.     

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.     

Cholesterol metabolism by purified cytochrome P-450scc is highly stimulated by octyl glucoside and stearic acid exclusively in large unilamellar phospholipid vesicles

Cholesterol side-chain cleavage (CSCC) catalyzed by purified bovine adrenal mitochondrial cytochrome P-450scc is highly dependent on the vesicles that supply cholesterol. Six-fold higher rates are achieved with large unilamellar dioleoylphosphatidylcholine vesicles (diameter 150 nm) prepared by octyl glucoside (OG) dialysis (DOPC-LUV) than with small sonicated vesicles (diameter 30 nm) (DOPC-SUV) (Vmax = 25 and 4 min-1, respectively. Extensive dialysis that may remove OG decreased Vmax rates for DOPC-LUV almost to rates seen with DOPC-SUV. These dialyzed DOPC-LUV were, however, very sensitive to addition of OG (EC50 = 2.5 microM, 4.3-fold stimulation) while DOPC-SUV were only weakly affected (EC50 = 100 microM, 1.6-fold stimulation). This enhancement of CSCC in LUV by OG only occurred when the cholesterol:DOPC exceeded 0.1 and was associated with a 15-fold increase in the Km for cholesterol. Structural changes in both SUV and LUV at high cholesterol:DOPC ratios (0.1-1) were indicated by decreases in internal volume that were insensitive to OG and did not affect the external diameters. Stearic acid produced a similar stimulation of CSCC in LUV (EC50 = 50 microM) and had no effect on SUV. The Vmax for CSCC, produced by OG activation of DOPC-LUV, is comparable to the highest attained for cytochrome P-450scc (Tween 20/cholesterol). In LUV, a minor proportion of OG (1-5% of cholesterol) is thus sufficient to generate a domain of reactive cholesterol that maintains a near-optimum turnover. This increased CSCC was paralleled by increased binding of cholesterol to P-450scc, suggesting that this cholesterol is more readily donated by the membrane to the cytochrome.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

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.     

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.     

Modeling of matrix vesicle biomineralization using large unilamellar vesicles

Stable, large unilamellar vesicles (LUV) have been constructed that model matrix vesicles (MV) in inducing de novo mineral formation when incubated in synthetic cartilage lymph (SCL). Using a dialysis method for incorporation of predetermined pure lipid, electrolyte and protein constituents, the detergent n-octyl beta-D-glucopyranoside enabled formation of stable, impermeable LUV with a diameter ( approximately 300 nm), lipid composition (phosphatidylcholine-phosphatidylserine-cholesterol, 7:2:2, molar ratio) and enclosed inorganic phosphate level (25-100 mM) similar to that of native MV. Mineral formation by these LUVs was measured by 45Ca(2+) uptake and FTIR analysis following incubation in SCL. Addition of the ionophore A23187 to SCL enabled 45Ca(2+) uptake comparable to that of native MV. FTIR analysis revealed that crystalline mineral formed in the LUV during incubation in SCL, but not in the absence of ionophore. This mineral had an IR absorption spectrum like that of the acid-phosphate-rich, octacalcium phosphate-like mineral formed by native MV. Perturbing the LUV membrane with either detergents or phospholipase A(2) following prior incubation in SCL enabled egress of mineral crystallites from the vesicle lumen, stimulating further mineral formation. Annexin V, a major protein in native MV with known Ca(2+) channel activity, incorporated into the LUV lumen or added to the external medium, induced only limited 45Ca(2+) uptake. This indicates that additional factors are required for annexin V to form Ca(2+) channels. Nevertheless for the first time, stable LUVs have been constructed with MV-like lipid, electrolyte, and protein composition and size that induce formation of mineral like that formed by native MV.     

Cholesterol transfer between lipid vesicles. Effect of phospholipids and gangliosides.

The effect of lipid composition on the rate of cholesterol movement between cellular membranes is investigated using lipid vesicles. The separation of donor and acceptor vesicles required for rate measurement is achieved by differential centrifugation so that the lipid effect can be quantified in the absence of a charged lipid generally used for ion-exchange-based separation. The rate of cholesterol transfer from small unilamellar vesicles (SUVs) containing 50 mol% cholesterol to a common large unilamellar vesicle (LUV) acceptor containing 20 mol% cholesterol decreases with increasing mol% of sphingomyelin in the SUVs, while phosphatidylethanolamine and phosphatidylserine have no appreciable effect at physiologically relevant levels. There is a large decrease in rate when phosphatidylethanolamine constitutes 50 mol% of donor phospholipids. Interestingly, gangliosides which have the same hydrocarbon moiety as sphingomyelin exert an opposite effect. The effect of spingomyelin seems to be mediated by its ability to decrease the fluidity of the lipid matrix, while that of gangliosides may arise from a weakening of phosphatidylcholine-cholesterol interactions or from a more favourable (less polar) microenvironment for the desorption of cholesterol provided by the head-group interactions involving sugar residues. If the effect of asymmetric transbilayer distribution of lipids is taken into consideration, the observed composition-dependent rate changes could partly account for the large difference in the rates of cholesterol desorption from the inner and outer layers of plasma membrane. Such rate differences may be responsible for an unequal steady-state distribution of cholesterol among various cellular membranes and lipoproteins.     

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     

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.     

Erratum

The ionophoretic capabilities of dioleoylphosphatidic acid (DOPA) for transporting calcium across phospholipid bilayers have been investigated. Calcium uptake by large unilamellar vesicles is shown to depend on the presence of DOPA. This uptake is sensitive to the nature and concentration of calcium chelators in the vesicle interior, indicating that accumulation results from DOPA-mediated translocation of calcium across the membrane. Further, it is shown that characteristics of DOPA-mediated Ca²⁺ uptake are similar to those observed for the fungal calcium ionophore, A23187.