Osmotic dependence of the lysophosphatidylcholine lytic action on liposomes in the gel state

Multilamellar liposomes of dimyristoylphosphatidylcholine are susceptible to lytic action of lysophosphatidylcholine at the gel state, an effect which is not observed when liposomes are in the liquid crystalline state. The lytic action has been found to be enhanced when liposomes are dispersed in hypertonic solutions. On the contrary, hypotonic solutions decreased the effectiveness of the lysolipid. Shrunken liposomes present surface changes as detected by merocyanine 540 and 1-anilinonaphthalene-8-sulfonic acid which can be ascribed to the spontaneous curvature promoted by shrinkage.     

The effect of gramicidin A on the temperature dependence of water permeation through liposomal membranes prepared from phosphatidylcholines with different chains lengths.

The permeation of water through liposomal membranes composed of various saturated phosphatidylcholine plus gramicidin A was studied as a function of temperature. 1. The presence of gramicidin in the liposomal bilayers caused an increase in water permeability. Below the phase transition temperature this effect could be measured quite clearly in all the systems we tested, but the extent of the increase was largely dependent on the length of the hydrocarbon chains. 2. Increasing amounts of gramicidin caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel-liquid crystalline phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. Differential scanning calorimetry analysis of the system containing these relatively small amounts of gramicidin still showed a clear transition from the liquid crystalline to the gel state with only a slight reduction in the enthalpy change. 3. In liposomes composed of dimyristoyl, dipalmitoyl and saturated egg phosphatidylcholine there was a concomitant decrease in the activation energy of water permeation in the presence of gramicidin below and above the phase transition temperature. The activation energy for water permeation through longer chained distearoyl phosphatidylcholine liposomal bilayers was the same with or without gramicidin in the bilayer. 4. It is concluded that the ability of gramicidin to form conducting channels in a gel state bilayer depends on the thickness of the paraffin core.     

Crystallization of phosphatidylserine bilayers induced by lithium

Utilizing differential scanning calorimetry and x-ray diffraction, 1,2-dimyristoyl-L-glycero-3-phospho-L-serine (DMPS) was shown to form hydrated bilayer membrane structures exhibiting a gel leads to liquid crystalline transition at 39 degrees C (delta H = 7.2 kcal/mol). Addition of up to molar concentrations of the alkali halides NaCl, KCl, Rl Cl, and CsCl produced relatively minor changes in DMPS bilayer structure or stability. For example, in the presence of 0.5 M NaCl, the transition temperature (Tc = 42 degrees C) and transition enthalpy (delta H = 7.0 kcal/mol) show only minor changes. In marked contrast, addition of LiCl results in "'crystallization" of the DMPS bilayer membrane structure. At 0.5 M LiCl, the crystalline DMPS exhibits a bilayer gel leads to liquid crystal transition at 89 degrees C accompanied by a high enthalpy change, delta H = 16.0 kcal/mol. Thus, Li+ induces an isothermal crystallization of DMPS bilayers, the hydrocarbon chains adopting a more ordered packing mode than the "hexagonal" arrangement of the gel state. In view of the widespread use of lithium in the treatment of manic-depressive illness, we also raise the possibility that interaction of Li+ with anionic membrane phospholipids could play a role in its pharmacological action.     

Modulation of the bilayer to hexagonal phase transition and solvation of phosphatidylethanolamines in aqueous salt solutions

Several salts affect the temperature of the bilayer to hexagonal phase transition of phosphatidylethanolamines. Their effects are dependent on the anion as well as the cation of the salt. Salt effects on this transition can be explained by preferential hydration and ion binding. Those salts which are excluded from the solvation sphere of the membrane promote hexagonal phase formation. For example, Na2SO4 promotes preferential hydration and is a hexagonal phase promoter while NaSCN does not do this and is a bilayer stabilizer. Unlike amphiphiles and hydrocarbons, salts can shift the bilayer to hexagonal phase transition temperature without altering the cooperativity of the transition. The effect of these salts on the gel to liquid-crystal transition is opposite to their effect on the bilayer to hexagonal phase transition. We also find that MnCl2 markedly raises the gel to liquid-crystal transition temperature. This effect is due to binding of the cation to the membrane surface. The effect is reduced with MnSO4 because of preferential hydration. Our results demonstrate that the nature of the anion as well as the cation can alter the effect of salts on lipid phase transition properties. The observed effects can be explained as resulting from preferential hydration and ion binding.     

The alterations of lipid bilayer fluidity induced by newly synthesized phenothiazine derivative

Using fluorescence spectroscopy, calorimetry and ESR the interactions of the phenothiazine derivative 2-trifluoromethyl-10-(4-[methylsulfonylamid]buthyl)-phenothiazine (FPhMS) with lipids were studied. Calorimetry showed biphasic effect of FPhMS on main phase transition of DPPC. At molar ratios up to 0.06 drug induced decrease of transition temperature and enthalpy, while at higher concentrations it caused subsequent increase of these parameters. For all concentrations studied we observed gradual broadening of transition peaks. Fluorescence polarization revealed that in FPhMS/lipid mixtures, order in bilayers is decreased in the gel state and increased in the liquid crystalline state. ESR experiment showed that at molar ratio of 0.06, FPhMS reduces the mobility of spin probes located in both polar and hydrophobic regions. Comparing observed effects with those reported for cholesterol/lipid mixtures, we conclude that at higher concentrations FPhMS presumably induces a new mode of bilayer packing. This structure is less co-operative than an unperturbed bilayer, but locally the mobility of lipid molecules is decreased.     

Differential effect of triiodothyronine and thyroxine on the liposomal membrane in liquid-crystalline and gel state

The effect of thyroid hormones on the degree of order or fluidity of dimyristoyl, dipalmitoyl or egg yolk phosphatidyl choline liposomes was evaluated by fluorescence spectroscopy methods. The freedom of molecular motion above the phase transition temperature was decreased, while below the transition, the mobility was actually increased by the incorporation of triiodothyronine to liposomes. While thyroxine decreases the fluidity in the liquid crystalline state, it cannot increase the fluidity in the gel state.A differential effect of triiodothyronine and thyroxine on the release of the liposomal content was found, depending on the liquid crystalline or gel state of the liposomes. These facts were correlated with the differential incorporation of the hormones to liposomes above and below the phase transition temperature of dimyristoyl and dipalmitoyl phospholipid choline. In gel state, a low incorporation of thyroxine compared with triiodothyronine was found.This work was supported by Grants PID 3-013800/89 from Consejo National de Investigaciones Científicas y Técnicas (CONICET), Fundación Antorchas A-12576/1-000065 and Consejo de Investigaciones de la Universidad National de Tucumán (CIUNT). We thank Dr. G. Rotillo for the space filling models.     

The effect of cholesterol on the structure of phosphatidylcholine bilayers

The effect of cholesterol on the structure of phosphatidylcholine bilayers was investigated by X-ray diffraction methods. Electron density profiles at 5 Å resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayer was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12–16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG2000 liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG2000 liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 degrees C or 4 degrees C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Interactions of pyrethroids with phosphatidylcholine liposomal membranes

Interactions of several pyrethroids with membrane lipids in the form of dipalmitoylphosphatidylcholine (DPPC) liposomes have been studied using fluorescent membrane probes. Fluorescence anisotropy values and lifetimes (determined by phase-shift and demodulation techniques) of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, were decreased in gel phase liposomes by pyrethroids at concentrations on the order of 10 microM. The pyrethroids containing a cyano substituent were also observed to cause collisional quenching of diphenylhexatriene fluorescence. Pyrethroids differed in their effectiveness at lowering the phase transition temperature of DPPC, and in their ability to broaden the temperature range of this transition. The fluorescence intensity of DPPC-incorporated chlorophyll a was used to monitor the pretransition of DPPC and the lateral diffusion of a membrane component located in the polar headgroup region. Permethrin did not affect chlorophyll a fluorescence intensity at any temperature. It may be concluded from these results that pyrethroids are preferentially located in the interior hydrophobic regions of the lipid bilayer, and that these compounds can disorder hydrocarbon packing in the bilayer core. However, polar headgroups were not disordered, and diffusion of membrane components in the polar headgroup region was not altered.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG₂₀₀₀ liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG₂₀₀₀ liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 °C or 4 °C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Preservation of freeze-dried liposomes by trehalose

One of the practical difficulties with the frequently proposed use of liposomes for delivery of water-soluble substances to cells in whole organisms is that liposomes are relatively unstable during storage. We have studied the ability of trehalose, a carbohydrate commonly found at high concentrations in organisms capable of surviving dehydration, to stabilize dry liposomes. With trehalose both inside and outside the bilayer, almost 100% of trapped solute was retained in rehydrated vesicles previously freeze-dried with 1.8 g trehalose/g dry phospholipid. Trehalose is very effective at inhibiting fusion between liposomes during drying, as assessed by freeze-fracture and resonance energy transfer between fluorescent probes incorporated into the bilayer. However, inhibition of fusion alone does not account for the preservation of the dry liposomes, since the concentration of trehalose required to prevent leakage is more than 10-fold that required to prevent fusion. We provide evidence that stabilization of the dry liposomes requires depression of transition temperature and consequent maintenance of the constituent lipids in the dry liposomes in a liquid crystalline phase.     

Dependence on neutral salt concentration of the latency phase in the time course of hydrolysis of dimyristoylphosphatidylcholine liposomes by phospholipase A2.

The time course of the hydrolytic action of porcine pancreatic phospholipase A2 on sonicated dimyristoylphosphatidylcholine liposomes in the presence of variable NaCl concentrations has been studied at temperatures between 17 and 36 degrees C; at these temperatures liposomes are in the gel phase. At a NaCl concentration of 10 mM, the hydrolysis shows a small and constant lag period of 6-8 min at all temperatures within this range. As the temperature is raised into the liquid crystalline range, the latency phase lengthens monotonically so that at 36 degrees C it reaches 55 min. An increase in the NaCl concentration to 1 M makes the lag period longer at all temperatures studied, with the exception of the phase transition range (near 24 degrees C); within this temperature range, a small reduction in the lag time is observed. The increase in the length of the latency period at high salt concentrations may be due to screening of the negative surface charge generated by the nascent fatty acid which seems to be essential for the efficient interfacial binding of the enzyme. In the phase transition range of the lamellae, the unfavorable effect of high salt concentrations on the electrostatic binding of the enzyme appears to be overcome by another type of interaction. Recent findings raise the possibility that this interaction could be hydrophobic in nature.     

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.     

Effects of anesthetic and nonanesthetic steroids on dipalmitoylphosphatidylcholine liposomes: a calorimetric and Raman spectroscopic investigation

The effects of anesthetic and nonanesthetic steroids on dipalmitoylphosphatidylcholine liposomes were studied by use of high sensitivity scanning calorimetry and Raman spectroscopy. Calorimetric measurements indicated that both anesthetic and nonanesthetic steroids depressed and broadened the gel to liquid-crystalline phase transition. There was no correlation between the perturbations by the steroids on the primary gel to liquid-crystalline phase transition temperature and anesthetic potency. The magnitudes of the steroid-induced transition broadening and lowering of the pretransition temperature, however, correlated well with anesthetic potency. This effect appeared to arise from the projection from the plane of the D ring of substituents at the C(17) position of the steroid nucleus. Raman spectroscopic measurements demonstrated that the steroid molecule is localized within the acyl region of the bilayer and that effects of the steroid do not extend to either the head-group or interface regions of the lamellae. The data are consistent with unitary hypotheses relating general anesthesia to lipid perturbations. For model systems, perturbations to the subtle structural and dynamical properties of the bilayer pretransition may provide a more sensitive marker than the main phase transition in assessing the significance of lipid mediation in inducing anesthetic action.     

Thermotropic behavior of monoglucocerebroside--dipalmitoylphosphatidylcholine multilamellar liposomes

The thermotropic behavior of multilamellar liposomes prepared from mixtures of glucocerebroside and dipalmitoylphosphatidylcholine has been studied by high-sensitivity scanning calorimetry. It is shown that glucocerebroside has a marked effect on the gel--liquid crystalline transition of dipalmitoylphosphatidylcholine. The pretransition seen in pure samples of dipalmitoylphosphatidylcholine is undetectable at small mode fractions of glucocerebrosides (less than 10%). The main transition is shifted to higher temperatures and becomes broader and less cooperative in the presence of glucocerebroside. The enthalpy change of the main transition decreases with increasing the glucocerebroside content. However, this decrease is not linear with the glucocerebroside/phospholipid mole ratio. Glucocerebroside itself does not show a separate transition in the temperature range of these studies (10--75 degree C). The origin of these effects and their dependence on the glucocerebroside content suggest that the in-plane distribution of glucocerebroside molecules is affected by the physical state of the lipid bilayer and by the glucocerebroside/phospholipid mole ratio.     

Effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidylcholine liposomes.

Aspects of osmotic properties of liposomes, prepared from synthetic lecithin, above, at and below the gel to liquid crystalline phase transition temperature are described. The experiments show that liposomal membranes with their lipids in the gel state are still permeable to water. The rate of water permeation changes drastically on passing the transition temperature. The water permeation has activation energies of 9.5 +/- 1.28 and 26.4 +/- 0.9 kcal/mol above and below the transition temperature, respectively, indicating that the diffusion processes take place by different mechanisms. With respect to the barrier properties of the liposomes in the vicinity of the transition temperature, the following conclusions can be made. (1) Studying the osmotic shrinkage of liposomes at a fixed temperature near the transition point, the experiments indicate that dimyristoyl phosphatidylcholine membranes are highly permeable to glucose under these conditions, where liquid and solid domains co-exist. Under the same conditions the osmotic experiments did not indicate a strong increase in glucose permeability of dipalmitoyl phosphatidylcholine membranes as compared to the situation above and below the transition temperature. (2) On the other hand, perturbations of the phase equilibrium by temperature varations resulted in a marked increase of the glucose permeation through dipalmitoyl phosphatidylcholine bilayers. Once a new phase equilibrium of liquid and solid regions is established the permeation rate of glucose is much less.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Free radical label: new approach to the study of super-slow molecular dynamics of lipid systems

A new method of EPR-spectroscopy, the recombination of free radicals appearing as a result of indirect radiolysis of biological molecules after a low temperature irradiation, was applied to the study of molecular dynamics of dimyristoyl phosphatidylcholine in mass and in the structure of liposomes above and below the transition temperature. It was shown that the mobility of lipid molecules in crystalline liposomes was lower than in the structure of liquid-crystalline liposomes. The addition of cholesterol in liposome membranes decreased the lateral molecular motion of lipids in crystalline and liquid-crystalline states; in the latter case, the effect of cholesterol addition was more pronounced. The activation energy for the displacement of the fragments of lipid molecules and the lipid molecule as a whole was estimated, and it was shown that the lipid matrix possesses a high degree of heterogeneity. The solubility of oxygen in the lipid bilayer and the mechanism of lipid diffusion are discussed.     

Characterization of the association of Electrophorus electricus acetylcholinesterase with sphingomyelin liposomes. Relevance to collagen-sphingomyelin interactions

Electrophorus electricus acetylcholinesterase is a large polymorphic enzyme. Its native forms 18 S, 14 S and 8.5 S possess a tail having a collagen-like structure. It was suggested that this tail is involved in the anchorage of the enzyme at the terminal of the synapse. Watkins et al. [1] showed that all forms of the enzyme having a collagen segment also bind to sphingomyelin liposomes with almost no binding to phosphatidylcholine (PC) liposomes. In agreement with the above results, the binding of acetylcholinesterase reported here was independent of the following liposomal parameters (a) curvature, (b) the physical state of the bilayer, (c) the gel to liquid crystalline phase transition of sphingomyelin, (d) stereospecificity of the sphingomyelin, (e) acyl chain of the sphingomyelin. The binding was reduced with increasing PC content in sphingomyelin vesicles. The binding has no effect on the bilayer integrity. The enzymatic activity can be released from the vesicles by incubation with collagenase. The association of the enzyme with the liposomes had minimal effect on its kinetic parameters (Km, Vmax). The only detectable effect was increasing enzyme stability at low enzyme concentration. This suggested that the binding of the enzyme to sphingomyelin liposomes reduced its surface denaturation. Such association was not unique to acetylcholinesterase since collagen showed similar behavior. Collagen binding to sphingomyelin liposomes was 5-10-times larger than to PC liposomes. The exact details of the interaction of collagen and collagen-like peptides with sphingomyelin bilayers are yet unknown although it differs from the well documented hydrophobic or electrostatic interactions [7]. This work proposes hydrogen bonding as a third mechanism which involves the interface region of sphingolipids molecules and the collagen or collagen-like tail of acetylcholinesterase. This binding is also of interest due to its correlation to the accumulation of sphingomyelin and collagen during aging and the development of atherosclerosis in blood vessels of mammals.