Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

Acid-induced fusion of liposomes: studies with 2,3-seco-5 alpha-cholestan-2,3-dioic acid

The effect of 2,3-seco-5 alpha-cholestan-2,3-dioic acid on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine is markedly dependent on pH. Above pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid raises the temperature of this transition, i.e., it stabilizes the bilayer phase. At pH 6.56 there is little effect of this sterol derivative on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. However, below pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid markedly lowers the temperature of this transition. The promotion of hexagonal phase formation increases both with increasing mol fraction of this sterol derivative and with lower pH, particularly in the range between pH 6.56 and pH 5.0. Below about pH 6, 2,3-seco-5 alpha-cholestan-2,3-dioic acid also induces vesicle fusion as measured both by lipid mixing as well as by mixing of aqueous contents. For these assays vesicles made of phosphatidylethanolamine (made from egg phosphatidylcholine) and extruded through 0.2 micron pore membranes were used. At higher concentrations or at lower pH the 2,3-seco-5 alpha-cholestan-2,3-dioic acid induces some leakage of the contents of these vesicles. Nevertheless, with vesicles containing only 2 weight% sterol derivative, it was possible to demonstrate substantial mixing of aqueous contents of the vesicles over the pH range 3.5 to 5.5. Several of the properties of 2,3-seco-5 alpha-cholestan-2,3-dioic acid indicate that this compound may be useful in sensitizing vesicles to acid-induced fusion for the purpose of endocytic drug delivery.     

Preparation and characterization of long chain amino acid and peptide vesicle membranes

Four amino acid dicarboxylic amphiphiles which contain cysteine or homocysteine were synthesized. Each forms synthetic bilayer membranes upon hydration. Extensive sonication above the lipid phase transition temperature, 61 to 82 degrees C, produced 1000 A diameter vesicles. Treatment of the vesicles with water-soluble carbodiimides during and after sonication induced oligopeptide formation at the vesicle surface with retention of vesicle size and shape. Size exclusion chromatography indicates the products are predominantly di- to decapeptides. The permeability characteristics of the amino acid and peptide vesicles to [3H]glucose and 6-carboxyfluorescein are reported. The amino acid vesicles are among the least permeable nonpolymerized bilayer vesicles described in the literature to date. Formation of the peptide vesicles increases the membrane permeability, whereas in other polymerizable lipid vesicles the permeability decreases upon polymerization. The amino acid vesicles can be immobilized on Sephadex beads by reaction with carbodiimide. The impermeability, biodegradability, and ease of immobilization make this class of vesicles attractive materials for the encapsulation of reagents.     

Effects of physical states of phospholipids on the incorporation and cytochalasin B binding activity of human erythrocyte membrane proteins in reconstituted vesicles

Proteoliposomes were reconstituted from a Triton extract of human erythrocyte membrane proteins and a mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying ratios. With mixtures of egg PC and soybean PE, the protein/lipid ratio of the reconstituted vesicles was maximal at 25% PC and 75% PE, the composition which is known to have a maximum bilayer disruption (highest occurrence of lipidic particles seen by freeze-fracture electron microscopy). With mixtures of 1-palmitoyl-2-oleoyl-PC and dilinoleoyl-PE, which gave vesicles with few isolated lipidic particles at room temperature, the effect was less pronounced. The specific activity of the cytochalasin B (CB) binding protein in the reconstituted vesicles, on the other hand, was increased monotonically up to severalfold as the PC content was increased in the egg PC/soybean PE mixture. A similar increase was observed when soybean PE was partially substituted by dimyristoyl-PC, cholesterol, or transphosphatidylated PE from egg PC. These findings indicate that preexisting defects in the lipid bilayer promote protein incorporation into the bilayer during reconstitution whereas reduction of the bilayer fluidity facilitates the CB binding activity in the reconstituted vesicles.     

Ion permeation across the bilayer of annealed phosphatidylcholine vesicles at elevated temperatures. Concentration dependence and the micelle-bilayer dynamic equilibrium

The relative stability of the lipid bilayer toward ions above the crystalline to liquid-crystalline phase transition temperature has been studied under isotonic conditions for small annealed vesicles of dilauroyl (DLPC), dimyristoyl (DMPC), diplamitoyl (DPPC), and distearoyl (DSPC) phosphatidylcholine by using lanthanide ions as a probe. The bilayer stability increased as the chain length of the lipid fatty acid increased, and a rapid translocation of ions across the bilayer started at about 60, 70, and 80° C for DMPC, DPPC, and DSPC vesicles, respectively. The bilayer of DLPC vesicles is apparently permeable for the tested ions even at room temperature. Two other important phenomena concomitant with the observed translocation of ions were found. Firstly, the ion leakage occurred in an “an-or-none” fashion, i.e. as soon as the vesicles start to become permeable toward ions, the concentration of ions in the intra-and extravesicular media are equalized within a short time. Secondly, the rate of the relative number of inward facing lipid molecules which become exposed to extravesicularly added paramagnetic lanthanide is a function of the inverse phosphatidylcholine concentration. This feature explicitly excludes the possibilities that the observed ion leakage occurs through a diffusion, pore formation, or through the rupture of vesicle walls induced by vesicle-vesicle collisions. We instead propose as the most probable mechanism that a dynamic equilibrium between the various states of the phosphatidylcholine molecules in water, such as monomers, micelles, vesicles, and multilamellar liposomes, is in fact responsible for the observed phenomena.     

Asymmetric incorporation of trisialoganglioside into dipalmitoylphosphatidylcholine vesicles

Results are presented which demonstrate that purified trisialoganglioside spontaneously incorporates into performed phospholipid vesicles. Determinations of the extent of incorporation were made by separating large unilamellar dipalmitoylphosphatidylcholine vesicles containing incorporated ganglioside from micellar ganglioside on a Sepharose-2B column. Incorporation occurs without appreciably altering the vesicular character of the phospholipid bilayer as judged by the maintenance of an outside/inside ratio, determined by 31P NMR, comparable to that of the original vesicles. All of the incorporated ganglioside ias accessible to neuraminidase, indicating that incorporation occurs only on the outer face of the bilayer. The thermotropic behavior of these asymmetric dipalmitoylphosphatidylcholine-trisialoganglioside vesicles, examined by high sensitivity scanning calorimetry, strongly suggests that the incorporated ganglioside is intercalated into the outer monolayer of the vesicle bilayer. Calorimetric studies indicate that the ganglioside stabilizes these vesicular structures by inhibiting the fusion of small vesicles that occurs below the phase-transition temperature. These structures are a representative model system, which like the mammalian plasma membrane contain an asymmetric distribution of glycosphingolipid in the outer surface.     

Reaction characteristics and mechanisms of lipid bilayer vesicle fusion

Small unilamellar lipid bilayer vesicles were prepared from brain phosphatidylserine, egg phosphatidylcholine, and synthetic dipalmitoylphosphatidylcholine, and were fused into larger structures by freezing and thawing, addition of calcium chloride, and passage through the lipid phase transition temperature. Fusion reactions were studied by electron microscopy, light scattering, and use of fluorescent probes. Fusion was accompanied by leakage of lipid vesicle constituents and of water-soluble solutes in the inner vesicle compartments, and by uptake of these types of components from the external solution. Such leakage was greater during fusion by freezing than by Ca²⁺. Passage through the transition temperature produced a moderate degree of fusion, without loss of membrane components. It is concluded that each fusion method gives rise to a characteristic size or narrow range of sizes of fusion products. The fraction of small vesicles fused into larger structure depends on the method of vesicle preparation, composition of the lipid bilayer, and composition of the external solution. Fusion is induced by creation of a discontinuity in the bilayer or by removal of water associated with the bilayer. The amount of water removed controls the extent of fusion. This is maximized in bilayers when in the liquid-crystal phase, as against the gel phase, in vesicles made by ethanol injection, as against sonication, and in charged bilayers, as against neutral ones.     

Asymmetric fusion between phospholipid vesicles and vesicles formed from synthetic di(n-alkyl)phosphates

We have investigated the fusion behavior of a mixed vesicle system consisting of vesicles prepared from the simple synthetic surfactants di(n-dodecyl)phosphate (DDP) or di(n-tetradecyl)phosphate (DTP) and vesicles prepared from the phospholipids phosphatidylserine (PS) or dioleoylphosphatidylcholine (DOPC). Fusion between the vesicles, induced by Ca2+, was determined by a resonance energy transfer assay for lipid mixing, sucrose density gradient analysis, and electron microscopy. We demonstrate that synthetic surfactant vesicles can specifically engage in asymmetric fusion events, provided that the incubation temperature is kept below the gel-liquid crystalline phase-transition temperature (Tc) of the synthetic amphiphile (29 and 48 degrees C for DDP and DTP, respectively) and that the physical state of the target membrane is fluid. Asymmetric fusion of DDP or DTP vesicles was most efficient with PS vesicles, but it also occurred with zwitterionic PC vesicles. In the latter case, fusion proceeded spontaneously, but the process was markedly accelerated upon addition of Ca2+. Furthermore, in contrast to a massive transformation of bilayer into nonbilayer hexagonal HII tubular structures, as occurs upon symmetric Ca(2+)-induced fusion of DDP vesicles, asymmetric fusion with phospholipid bilayers predominantly leads to the formation of larger vesicles. This indicates that both PS and DOPC stabilize the DDP bilayer structure in the fusion product.     

Effect of membrane phase transition on long-time calcium-induced fusion of phosphatidylserine vesicles

Dynamic light scattering has been used to study the temperature dependence of the extent of long-time calcium-induced fusion of sonicated vesicles composed of various natural and synthetic phosphatidylserine with different acyl chains. The vesicles of each composition are found to exhibit a peak temperature in the vicinity of which the extent of fusion shows a distinct maximum. The fusion peak temperature increases as the bilayer gel-to-liquid-crystal phase transition temperature increases. The results suggest a role played by membrane fluidity in determining fusion efficiency.     

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).     

Interaction of dolichol and dolichyl phosphate with phospholipid bilayers

The thermotropic phase transition of dipalmitoylphosphatidylcholine vesicles reconstituted with dolichol or dolichyl phosphate was investigated as a function of the lipid-to-polyisoprenoid ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. At the concentrations studied, dolichol and dolichyl phosphate lowered and broadened the transition temperature of dipalmitoylphosphatidylcholine bilayers. Dolichol was found to increase the motional freedom of the bilayer both below and above the transition temperature as determined by fluorescence depolarization. In contrast, low concentrations of dolichyl phosphate decreased the bilayer motional freedom below the transition temperature while high concentrations increased the motional freedom. Above the transition temperature, dolichyl phosphate decreased bilayer 'fluidity' at all concentrations. The data suggest that these polyisoprenoids perturb the bilayer lattice, with the neutral species dolichol increasing membrane 'fluidity', while dolichyl phosphate acts to 'stiffen' the membrane.     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Thermoelasticity of large lecithin bilayer vesicles.

Micromechanical experiments on large lecithin bilayer vesicles as a function of temperature have demonstrated an essential feature of bilayer vesicles as closed systems: the bilayer can exist in a tension-free state (within the limits of experimental resolution, i.e., less than 10(-2) dyn/cm). Furthermore, because of the fixed internal volume, there is a critical temperature at which the vesicle becomes a tension-free sphere. Below this temperature, thermoelastic tension builds up in the membrane and the vesicle's internal pressure increases while the surface area remains constant. Above this temperature, the vesicle's surface area increases while the tension and internal pressure are negligible. Without mechanical support, the vesicles fragment into small vesicles because they have insufficient surface rigidity. In the upper temperature range we have measured the increase of surface area with temperature. These data established the thermal area expansivity to be 2.4 X 10(-3)/degrees C. At constant temperature, we used either pipet aspiration with suction pressures up to 10(4) dyn/cm2 or compression against a flat surface with forces up to 10(-2) dyn to produce area dilation of the vesicle surface on the order of 1%. The rate of increase of membrane tension with area dilation was calculated, which established the elastic area compressibility modulus to be 140 dyn/cm. The tension limit that produced lysis was observed to be 3-4 dyn/cm (equivalent to 2-3% area increase). The product of the elastic area compressibility modulus, the thermal area expansivity, and the temperature gives the reversible heat of expansion at constant temperature for the bilayer. This value is 100 ergs/cm2 at 25 degrees C, or approximately 5 kcal/mol of lecithin. Similarly, the product of the thermal area expansivity multiplied by the area compressibility modulus determines the rate of increase of thermoelastic tension with decrease in temperature when the area is held constant, i.e., -0.34 dyn/cm/degrees C.     

Effect of tryptophan derivatives on the phase properties of bilayers

Binding of several tryptophan derivatives and tryptophan-containing peptides to bilayers is examined by monitoring fluorescence enhancement as a function of lipid concentration. The thermodynamic and spectral parameters of the solutes in the bilayers of vesicles and liposomes do not exhibit any anomalous dependence upon the gel or the liquid-crystalline phase state of the bilayer. Effects of these solutes on the phase-transition profiles of the bilayers of liposomes and vesicles are examined, and the lowering of the phase-transition temperature is correlated with the mole fraction of the solute in the bilayer. The partition coefficients do not change at the main phase-transition temperature. These observations contradict the thermodynamic explanation of the solute-induced lowering of the phase-transition temperature which is based on the Van't Hoff relationship for distribution of the solute in the two coexisting phases at the phase-transition temperature. It is postulated that solute molecules bound to defect sites in bilayers modulate the phase properties of bilayers. These defect sites are induced in the gel phase of bilayers of liposomes above the subtransition temperature.     

Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol

The spontaneous incorporation of the polyene antibiotic amphotericin B from a micellar solution into phospholipid vesicles was examined as a function of the lipid composition of the vesicles and their physical state. Virtually no insertion of the antibiotic into egg phosphatidylcholine vesicles was observed even when cholesterol was also present in the bilayer. In contrast, rapid incorporation occurred into systems containing an anionic phospholipid such as phosphatidylglycerol or phosphatidylserine with the fastest rates observed for lipids containing the saturated dimyristoyl fatty acyl species. Insertion of amphotericin B into vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3 mole ratio) was rapid either above, below or within the gel-to-liquid-crystalline phase transition temperature (23 degrees C). The ability of amphotericin B to intercalate into lipid vesicles is discussed in relation to their relative bilayer stabilities.     

Pseudomonas exotoxin A. Membrane binding, insertion, and traversal

Using vesicle targets composed of phosphatidylcholine and cholesterol (1:1 molar ratio), we found that Pseudomonas aeruginosa exotoxin A (PTx) binding and insertion are not only dependent on pH (Zalman, L.S., and Wisnieski, B.J. (1985) Infect. Immun. 50, 630-635) but also on ionic strength, reaching a maximum in pH 4 buffer that contains 150-200 mM NaCl. Insertion was monitored by photolabeling with an intramembranous probe. Higher levels of binding and insertion were attained with vesicles that contained 2.5 mol% dicetylphosphate than with neutral vesicles. Positively charged vesicles (2.7 mol% stearylamine) were the least effective targets. At pH 7.4, all binding levels were depressed. While PTx binding increased with increasing temperature, the relative proportion of the vesicle-associated toxin that was photolabeled decreased. The most likely explanation for the decrease is that the bilayer translocation rates increased with increasing temperature, and hence fewer PTx molecules were accessible at the time of photolabeling. At 37 degrees C, binding and insertion both plateaued within 10 min of lowering the pH to 4. After 10 min, the amount of bound toxin decreased slightly with time but there was a dramatic decrease in photolabeling, indicating that inserted PTx had begun to cross the bilayer. This was verified by the finding that when PTx was incubated with vesicles that contained trypsin, cleavage occurred only in those samples in which the pH was shifted down to pH 4. Entry is triggered by an acid-induced conformational change that promotes productive binding and insertion. After insertion, the kinetics of membrane traversal appear to be regulated by the physical properties of the bilayer.     

Interaction of gramicidin S analogs with lipid bilayer membrane.

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.     

Phase separation of miscible phospholipids by sonication of bilayer vesicles.

Sonication of phospholipid vesicles may result, according to their liquid or solid crystal state, in the generation of unilamellar vesicles or structural defects within their bilayers, respectively. The transition temperature Tm of the phospholipid bilayer is usually the threshold temperature delineating the physical effects of ultrasound. However, for vesicles made from a mixture of two miscible phospholipids, this threshold temperature was not found to be the intermediate Tm of the phospholipid mixture bilayers, but the Tm of the lowest melting component. This was due to a simultaneous lateral phase separation of the two phospholipids induced by the sonication as demonstrated by differential scanning calorimetry analysis.     

Characterization of DODAB/DPPC vesicles

Dioctadecyldimethylammonium bromide (DODAB)/dipalmitoylphosphatidylcholine (DPPC) large and cationic vesicles obtained by vortexing a lipid film in aqueous solution and above the mean phase transition temperature (T(m)) are characterized by means of determination of phase behaviour, size distribution, zeta-potential analysis and colloid stability. The effect of increasing % DODAB over the 0-100% range was a nonmonotonic phase behaviour. At 50% DODAB, the mean phase transition temperature and the colloid stability were at maximum. There is an intimate relationship between stability of the bilayer structure and colloid stability. In 1, 50 and 150mM NaCl, the colloid stability for pure DPPC or pure DODAB vesicles was very low as observed by sedimentation or flocculation, respectively. In contrast, at 50% DODAB, remarkable colloid stability was achieved in 1, 50 or 150mM NaCl for the DODAB/DPPC composite vesicles. Vesicle size decreased but the zeta-potential remained constant with % DODAB, due to a decrease of counterion binding with vesicle size. This might be important for several biotechnological applications currently being attempted with cationic bilayer systems.     

Tamoxifen perturbs lipid bilayer order and permeability: comparison of DSC, fluorescence anisotropy, laurdan generalized polarization and carboxyfluorescein leakage studies

The perturbation of the lipid bilayer structure by tamoxifen may contribute to its multiple mechanisms of anticancer action not related to estrogen receptors. This study evaluates the effect of tamoxifen on structural characteristics of model membranes using differential scanning calorimetry (DSC), fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-[trimethylammonium)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), as well as 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) generalized polarization. The comparative measurements in multilammelar vesicles (MLV) prepared from dipalmitoylphosphatidylcholine (DPPC) revealed that tamoxifen decreases the phase transition temperature (Tm) paralleled by a broadening of the phase transition profile. In large unilamellar vesicles (LUV) prepared from egg yolk phosphatidylcholine (EPC), tamoxifen increased the lipid bilayer order predominantly in the outer bilayer region. From membrane permeability measurements, we conclude that the tamoxifen-induced release of entrapped carboxyfluorescein (CF) results from a permanent bilayer disruption and the formation of transient holes in the lipid bilayer.