The effect of free cholesterol on the solubilization of cholesteryl oleate in phosphatidylcholine bilayers: A 13C-NMR study

The solubilization of cholesteryl oleate in sonicated phosphatidylcholine vesicles containing between 0 and 50 mol% cholesterol was studied by 13C-NMR using isotopically enriched [carbonyl-13C]cholesteryl oleate. The carbonyl-13C chemical shift from cholesteryl oleate in the phospholipid/cholesterol bilayer was significantly downfield from that for cholesteryl oleate in an oil phase and the peak area, relative to that of the phospholipid carbonyl, was used to determine bilayer solubility of the ester. The solubility (with respect to phospholipid) in the phospholipid bilayer without cholesterol (2.9 mol%) was only moderately reduced (to 2.3 mol%) at cholesterol levels up to 33 mol% but showed a more marked reduction to 1.4 mol% at 40 mol% cholesterol or 1.2 mol% at 50 mol% cholesterol. Since the vesicles containing 50 mol% cholesterol were larger (520 +/- 152 A diameter) than those with no cholesterol (291 +/- 97 A diameter), we measured the solubility of cholesteryl oleate in large vesicles with no cholesterol, prepared by extrusion through polycarbonate membrane filters, and found it similar to that in small, sonicated vesicles with no cholesterol. Therefore, the larger size of vesicles was not the factor responsible for the decreased cholesteryl oleate solubility at high cholesterol contents. A more direct effect of cholesterol is envisioned where the ester becomes displaced to deeper regions of the bilayer.     

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.     

The effects of temperature,salinity, concentration and PEGylated lipid on the spontaneous nanostructures of bicellar mixtures

The self-assembling morphologies of low-concentration (mostly 1 and 10 mg/mL) bicellar mixtures composed of zwitterionic dipalmitoyl (di-C₁₆) phosphatidylcholine (DPPC), dihexanoyl (di-C₆) phosphatidylcholine (DHPC), and negatively charged dipalmitoyl (di-C₁₆) phosphatidylglycerol (DPPG) were investigated using small angle neutron scattering, dynamic light scattering and transmission electron microscopy. A polyethylene glycol conjugated (PEGylated) lipid, distearoyl phosphoethanolamine-[methoxy (polyethyleneglycol)-2000] (PEG2000-DSPE), was incorporated in the system at 5 mol% of the total lipid composition. The effects of several parameters on the spontaneous structures were studied, including temperature, lipid concentration, salinity, and PEG2000-DSPE. In general, nanodiscs (bicelles) were observed at low temperatures (below the melting temperature, T_M of DPPC) depending on the salinity of the solutions. Nanodisc-to-vesicle transition was found upon the elevation of temperature (above T_M) in the cases of low lipid concentration in the absence of PEG2000-DSPE or high salinity. Both addition of PEG2000-DSPE and high lipid concentration stabilize the nanodiscs, preventing the formation of multilamellar vesicles, while high salinity promotes vesiculation and the formation of aggregation. This study suggests that the stability of such nanodiscs is presumably controlled by the electrostatic interactions, the steric effect induced by PEG2000-DSPE, and the amount of DHPC located at the disc rim.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.     

Effect of water-soluble polymers on the state of aggregation, vesicle size, and phase transformations in mixtures of phosphatidylcholine and sodium cholate.

The state of aggregation and the steady-state size of mixed aggregates made of phospholipids and surfactants are both determined by the surfactant/lipid ratio in the mixed aggregates (Re). Water-soluble polymers, such as dextrans and polyethylene glycols (PEGs) of different molecular weights, induce reversible aggregation of phospholipid vesicles, mostly due to dehydration of the vesicle surface and depletion forces, and only at much higher concentrations, PEGs (but not dextran) also induce irreversible size growth of the vesicles. Here we show that the water-soluble polymers dextrans and PEGs do not affect the vesicle-micelle phase boundaries in mixtures of phosphatidylcholine and the anionic surfactant sodium cholate. By contrast, these polymers affect markedly the steady-state size of cholate-containing vesicles. As compared with pure phosphatidylcholine vesicles, the cholate-containing vesicles have a lower tendency to undergo polymer-induced aggregation, probably due to the electrostatic repulsion between the negatively charged vesicles, but a higher tendency to undergo irreversible size growth at relatively low polymer concentrations. Such irreversible size growth was observed not only for PEG but also for dextran, which in the absence of cholate is incapable of inducing vesicle size growth. These findings are consistent with the prevailing concept that the polymer-induced size growth is due to the effect of large structural fluctuations in the bilayers of deformed aggregated vesicles, the surface of which is dehydrated by the polymer. The presence of cholate in the bilayers at sufficiently high concentrations induces such fluctuations, yielding irreversible size growth within the clusters of dehydrated vesicles formed upon mixing with polymers.     

Experimental and Monte Carlo simulation studies of the thermodynamics of polyethyleneglycol chains grafted to lipid bilayers.

Experimental measurements of the affinity of binding of fluorescent acylated polyethyleneglycol (PEG) conjugates to bilayers containing varying levels of phosphatidylethanolamine-PEGs (PE-PEGs) have been combined with Monte Carlo simulations to investigate the properties of the polymer chains at a PEG-grafted lipid interface. The affinity of binding of such conjugates to large unilamellar phosphatidylcholine/phosphatidylethanolamine (9:1) vesicles decreases 27-fold as the size of the coupled PEG chain increases from 1 to 114 monomer units. Incorporation of increasing amounts of PE-PEG2000 or PE-PEG5000 into the vesicles progressively reduces the affinity of binding of acylpeptide-PEG2000 or -PEG5000 conjugates. Monte Carlo simulations of surfaces with grafted PEG chains revealed no significant dependence of several characteristic properties of the polymer chains, including the average internal energy per polymer and the radii of gyration, on the grafting density in the range examined experimentally. The average conformation of a surface-grafted PEG2000 or PEG5000 chain was calculated to be fairly extended even at low grafting densities, and the projected cross-sectional areas of the grafted PEG chains are considerably smaller than those predicted on the basis of the estimated Flory radius. The experimental variation of the binding affinity of acylated conjugates for bilayers containing varying mole fractions of PE-PEG2000 or -PEG5000 is well explained by expressions treating the surface-grafted PEG polymers either as a van der Waals gas or as a system of rigid discs described by scaled particle theory. From the combined results of our experimental and simulation studies we conclude that the grafted PEG chains exist in a "mushroom" regime throughout the range of polymer densities examined experimentally and that the diminished affinity of binding of acylated-PEG conjugates to bilayers containing PE-PEGs results from occlusion of the surface area accessible for conjugate binding by the mobile PE-PEG polymer chains.     

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG(2000)) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG(2000). At this proportion of DSPE-PEG(2000), the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG(2000) in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

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.     

Merocyanine 540, a fluorescent probe sensitive to lipid packing

Binding of the lipophilic probe merocyanine 540 to artificial bilayers was assessed by measuring the enhancement of fluorescence which results when dye enters the hydrophobic environment of the membrane. Titration of a constant amount of dye with increasing amounts of vesicles revealed that much more dye binds to multilamellar and 1000-Å, unilamellar vesicles which are in the fluid-phase state than to comparable vesicles which are in the gel-phase state. Incorporation of cholesterol into fluid-phase vesicles at levels of greater than 20 mol% reduced dye binding, whereas cholesterol had no effect at any concentration when incorporated into gel-phase vesicles. Sonicated 200–300-Å unilamellar gel-phase vesicles, which because of their reduced radius of curvature resemble fluid-phase bilayers in their more widely spaced exterior leaflet lipids, bound more dye than 1000-Å unilameilar gel-phase vesicles constructed from the same lipid. These results suggest that merocyanine 540 is able to sense the degree of lipid packing of bilayers and inserts preferentially into bilayers whose lipids are more widely spaced.     

Incorporation of PEG-lipids in arsonoliposomes results in formation of highly stable arsenic-containing vesicles

We investigated the effect of pegylation on the physical stability, morphology and membrane integrity of arsonoliposomes. Arsonoliposomes composed of distearoylglycerophosphocholine (DSPC), cholesterol (Chol) and the palmitoyl side chain arsonolipid (with concentrations ranging from 0 mol% [DSPC/Chol vesicles] to 53 mol% of total lipid) containing either 4 or 8 mol% DPPE-PEG2000 or DSPE-PEG2000, were prepared by sonication. Arsonoliposome membrane integrity was evaluated by measuring the retention of encapsulated calcein in vesicles (during incubation in buffer or fetal calf serum [FCS]) while physical stability was evaluated by measuring vesicle dispersion turbidity (during incubation in water or CaCl(2)). Vesicle morphology was studied by cryo-electron microscopy. Experimental results show that: (i) PEG-lipids are incorporated in arsonoliposomes (as confirmed by the vesicle zeta potential modulation), (ii) pegylation of arsonoliposomes prevents their aggregation and fusion in the presence of calcium ions and (iii) when 8 mol% of PEG-DSPE is incorporated in arsonoliposomes based on their arsonolipid content, two groups of pegylated vesicles are formed: low content arsonoliposomes (<20 mol% arsonolipid) which are highly leaky and high content arsonoliposomes (>27 mol% arsonolipid) which are highly stable (70% calcein retention after 24h incubation in fetal calf serum [FCS]). In addition to high membrane integrity, the high content pegylated arsonoliposomes are morphologically perfect round-shaped vesicles without the sharp edges typically observed with non-pegylated DSPC-containing arsonoliposomes.     

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes

In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG(2000)-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG(2000)), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)). Transfer of DSPE-PEG(2000) to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG(2000) was transferred efficiently even at 4 degrees C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG(2000) to become evenly distributed between the two liposome populations (T(EQ)) at 4 degrees C and 37 degrees C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.     

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG₂₀₀₀) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG₂₀₀₀. At this proportion of DSPE-PEG₂₀₀₀, the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG₂₀₀₀ in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

Poly(ethylene glycol)-modification of the phospholipid vesicles by using the spontaneous incorporation of poly(ethylene glycol)-lipid into the vesicles

The critical micelle concentrations of 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 microM, respectively, in buffer solutions ([Tris] = 20 mM, [NaCl] = 140 mM, pH 7.4) at 37 degrees C. When these PEG-lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEG-DSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEG-DPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.     

Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.

Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of membrane-bound macromolecules.     

Effect of unesterified cholesterol on the compartmentation of a fluorescent cholesteryl ester in a lipoprotein-like lipid microemulsion.

The access of enzymes and lipid transfer proteins to neutral lipids located predominantly in the core compartment of lipoproteins may be determined to some degree by the solubility of the neutral lipids in the surface monolayer of phospholipid. This report concerns the hypothesis that unesterfied cholesterol can affect the partition of a cholesteryl ester between the surface monolayer of a lipid emulsion and the internal core compartment, thus controlling the degree to which the cholesteryl ester is presented at the emulsion surface. For microemulsions composed of dimyristoyl phosphatidylcholine and cholesteryl oleate, the addition of unesterified cholesterol results in an increase in the particle size from about 170 nm diameter to 210 nm diameter at 13.5 mol% unesterified cholesterol. Fluorescent quenching methods were devised to determine the apparent partition of a fluorescent cholesteryl ester (cholesteryl anthracene-9-carboxylate) between surface and core compartments. The addition of unesterified cholesterol resulted in the movement of the fluorescent cholesteryl ester from the surface monolayer to the core compartment. The apparent partition coefficient, defined as the ratio of the concentration of probe in the monolayer to that in the core, decreased from 1.03 in the absence of unesterfied cholesterol to 0.54 at 28 mol% unesterified cholesterol in the emulsion. In this process, the fluorescent cholesteryl ester becomes less accessible to a quencher (5-doxyl stearate) located in the surface monolayer. The decrease in the surface curvature resulting from incorporation of unesterified cholesterol into the particle does not influence this quenching process. We conclude that the presence of unesterified cholesterol in the emulsion causes the fluorescent cholesteryl ester to become less soluble in the surface monolayer.     

Effect of cholesterol on vesicle bilayer geometry of choline plasmalogen and comparison with dialkyl-, alkylacyl- and diacyl-glycerophosphocholines

Small unilamellar vesicles containing alkenylacyl-, alkylacyl-, dialkyl- or diacyl-glycerophosphocholine were prepared by sonication. Their size was determined from the average internal volume after chromatography on Sepharose 2B and from 31P-NMR linewidths. Alkenylacyl glycerophosphocholine (choline plasmalogen) was found to form the largest vesicles. By addition of 30 mol% cholesterol, the size of plasmalogen vesicles, but not of those containing the alkyl and acyl analogue lipids, was significantly increased. The presence of 50 mol% sterol led to highly increased vesicle sizes of alkylacyl, dialkyl and diacyl-glycerophosphocholine. Mixtures of plasmalogens with 50 mol% cholesterol did not form unilamellar vesicles upon sonication. Bilayer thickness and surface area per phospholipid molecule were determined by small angle X-ray scattering and measurement of partial specific volumes. There is little difference between alkenylacyl glycerophosphocholine and the corresponding diacyl-analog, whereas bilayers consisting of dioleoyl glycerophosphocholine are significantly thinner. Correspondingly their molecular surface area is by about 8% larger than that of the mixed-chain diradyl glycerophosphocholine, since the partial molar volumes are similar for all vesicles tested.     

Modulation of myelin basic protein-induced aggregation and fusion of liposomes by cholesterol, aliphatic aldehydes and alkanes

The effect of cholesterol on myelin basic protein-induced aggregation of zwitterionic phospholipid vesicles was studied by turbidimetry, quasi-elastic light scattering and centrifugation techniques. Without cholesterol, the degree of vesicle aggregation caused by myelin basic protein is relatively low and is only slightly increased using cholesterol concentrations up to approx. 25-30 mol%. When the cholesterol content in the bilayer exceeds approx. 30 mol%, there is a dramatic increase in the susceptibility of the vesicles to aggregation in the presence of myelin basic protein. Palmitoyl aldehyde and eicosane, substances resembling products of lipid degradation, increase myelin basic protein promoted fusion of vesicles. The fusion is accompanied by increased leakage of entrapped carboxyfluorescein. In the presence of cholesterol, myelin basic protein-induced fusion of the liposomes becomes much more sensitive to the presence of aliphatic aldehydes or alkanes. The results suggest that cholesterol has an important role in promoting membrane adhesion in biological systems but these structures become unstable in the presence of small amounts of products of lipid degradation. The findings have important implications to the understanding of the stability of the myelin membrane.     

Fusogenic activity of PEGylated pH-sensitive liposomes

The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: distearoylphosphatidylethanolamine (DSPE)-PEG???? was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG???? was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG???? or sterol-PEG???? into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG???? in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.     

Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose

To determine whether polyethylene glycol (PEG) causes growth of liposomes by affecting them directly or indirectly, vesicles composed of phosphatidylcholine were exposed to increasing concentrations of Mr 15 000-20 000 PEG or Mr 40 000 dextran either by direct mixing or across a dialysis membrane. After incubation at room temperature and dilution below at least 5% (w/w) polymer, the vesicles were monitored for fluorescence energy transfer and for absorbance at 400 nm. PEG induced the same levels of dequenching or lipid mixing and increased turbidity, regardless of whether the vesicles had been mixed directly with or dialyzed against PEG. These changes occurred within 5-15 min of polymer application. It is concluded that the increased lipid mixing and/or increased turbidity, indicating vesicle growth, resulted from an indirect effect of PEG on the vesicles--most likely dehydration. Dextran, in contrast to PEG, induced less dequenching and/or less turbidity increase when vesicles were directly mixed with, as opposed to dialyzed against, dextran. Although dextran not in contact with vesicles and with osmotic activity comparable to PEG was able to cause a degree of membrane fusion similar to that of PEG, therefore, the dehydrating effect of dextran could be mitigated if it were allowed to interact with vesicles. In further support of membrane dehydration as a precursor to membrane fusion, lipid mixing among sonicated and sonicated, frozen-thawed vesicles dialyzed against sucrose increased as a function of sucrose concentration. Vesicle morphology generally determined the maximal degree of membrane fusion inducible by the polymers.(ABSTRACT TRUNCATED AT 250 WORDS)