E.A. Disalvo

Effect of glycerol on the interfacial properties of dipalmitoylphosphatidylcholine liposomes as measured with merocyanine 540

Liposomes of dipalmitoylphosphatidylcholine (DPPC) prepared in increasing glycerol/glucose ratios show an increase in the absorbance at 570 nm of merocyanine spectra at temperatures below the phase transition. Since this effect is not observed when liposomes are prepared in solutions containing solely glucose, it is attributed to specific interactions of glycerol with the membrane phase. The increase in the 570 nm absorbance is ascribed to a partial fluidification of the membrane interface and is dependent on the distribution of the dye between the inner and the outer compartments of the liposomes and on their osmotic state. The greatest differences in the absorbance ratio are obtained when merocyanine is added to the external media. In consequence, the changes in the spectra of MC are dependent on the surface state of the liposomes which can be modified by an increase of glycerol or glucose in the external media. The present results are examined in the light of the perturbations that glycerol can induce on the barrier properties of the bilayer.

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.

Properties of gel phase lipid-trehalose bilayers upon rehydration

When dipalmitoylphosphatidylcholine bilayers dried under vacuum in different concentrations of trehalose are rehydrated in buffer without the sugar they show different physicochemical properties in the gel state in comparison to the normal gel state. Dry DPPC/trehalose mixtures are readily dispersed in buffer below the phase transition showing by electron microscopy a morphology similar to liposomes prepared by dispersing the lipids in buffer above the phase transition temperature. In these conditions, an increase in the peak at 570 nm of merocyanine after the dehydration-rehydration process in the presence of the sugar is observed and the water permeation increases to values comparable to those found in the fluid state as indicated by the activation energy values and the osmotic volume. The trehalose-dried liposomes rehydrated in buffer show a similar osmotic response to hypertonic gradient as DPPC liposomes without sugar near the phase transition temperature. In accordance with this behavior the trehalose-dried liposomes are lysed below the phase transition temperature by lysoderivatives. These modifications of the gel state of hydrated phospholipids by trehalose can only be achieved if a drastic dehydration is performed in the presence of the sugar. After rehydration the changes in the gel state can be detected after dyalizing the rehydrated membranes in media without trehalose during at least 24 h. These results suggest that trehalose is still intercalated between the phospholipids after restoring water to the dried liposomes either at temperatures below or above the phase transition.

Leakage from egg phosphatidylcholine vesicles induced by Ca2+ and alcohols.

The results shown in this paper indicate that the permeability properties of egg yolk phosphatidylcholine sonicated vesicles as detected by the leakage of carboxy fluorescein changes according to the Ca2+ content. Vesicles containing Ca2+ show a higher rate of leakage than those containing Na+ solutions in response to the increase of Ca2+ concentration in the outer solution. The results are interpreted in terms of the rigidity promoted by Ca2+ and are compared to those obtained with long and short chain alcohols.

Surface changes induced by osmotic stress and its influence on the glycerol permeability in lipid bilayers

The penetration rate of glycerol across lipid bilayers can be assayed dispersing liposomes filled with a 0.1 M glucose solution in an isotonic or a hypertonic solution of glycerol. The kinetic of glycerol permeation is found to be different in each of those cases. Liposomes dispersed above the phase transition temperature in hypertonic solutions show an increase in the surface polarization as measured by means of merocyanine 540. Under this condition, the permeation of glycerol shows a two-step kinetic which is indicative of a non-fickean diffusion process. In contrast, liposomes dispersed in isotonic solutions of the permeant show a fickean behavior. The changes in polarization of the membrane interface are ascribed to variations in the surface potential due to the osmotic collapse and the glycerol concentration in contact with the outer surface. The permeability of polar molecules can, in consequence, be considered as a function of the surface potential of the liposome which is congruent with previous data in literature reporting that water permeability increases as a function of the zeta potential of liposomes shrunken in hypertonic solutions.

Permeability of lipid membranes revised in relation to freeze-thaw processes

Water and solute activity gradients created during freeze-thaw processes produce water and solute fluxes across the cell membrane resulting in volume changes. Under these conditions, osmotic and thermal stresses affect the curvature, the phase behavior, and the surface properties of the lipid bilayer. These structural changes are not considered by the classical formalisms describing permeability of lipid membranes to water and nonelectrolytes such as the Nernst-Planck equation, Eyrings absolute rate theory, and Kedem-Katchalskys thermodynamic of irreversible processes approach. In this paper, the influence of such changes on the glycerol permeation kinetics are reported. The results indicate that osmotic and chemical effects of the cryoprotectant on the membrane properties affect the rate of volume swelling depending on whether the membrane is in the gel or in the liquid crystalline state.

Effect of the asymmetric Ca2+ distribution on the bilayer properties of phosphatidylcholine-sonicated vesicles

The incorporation of Ca2+ in the inner volume of egg phosphatidylcholine vesicles increases the fluorescence anisotropy of a diphenylhexatriene probe. This increase is higher than for Na+ at the same normality. An effect of the same magnitude is induced by Ca2+ when using binary lipid mixture (dioleoylphosphatidylcholine and dipalmitoylphosphatidylcholine) as long as the mixture is maintained below the phase-transition temperature of the saturated species. The influence of Ca2+ may be explained by an asymmetric distribution of the saturated and unsaturated lipids between the internal and the external monolayers.

Ca2+-induced phosphatidylcholine vesicle aggregation in the presence of ferricyanide.

The titration of sonicated vesicles of egg phosphatidylcholine with ferricyanide in the presence of Ca2+ results in the formation of aggregates. The turbidity increase caused by these aggregates cannot be reversed by EDTA treatment. In addition, no rearrangement of the bilayer structure has been found in this process, either measuring leakage of vesicle content or exchange of lipids among the bilayers themselves. The aggregation is dependent on the Ca2+ content of the vesicles, the outer Ca2+ and Fe(CN)3-(6) concentration and the order of addition of Ca2+ and ferricyanide. The results can be explained by a specific adsorption of Fe(CN)3-(6) to bilayers of sonicated vesicles, in contrast to other multivalent anions. In contrast to the stability found with sonicated vesicles, the aggregation causes a leakage of the internal solution when multilamellar liposomes are titrated with Fe(CN)3-(6).

Effect of encapsulated Ca2+ on the surface properties of curved phosphatidylcholine bilayers

In this paper, the influence of Ca2+ on the adsorption properties of 1,8-anilinonaphthalenesulfonate (ANS) and analogous probes to sonicated vesicles of phosphatidylcholine was studied by means of spectrofluorometry. The fluorescence of ANS added to the vesicle dispersion increases with the Ca2+ concentration in the inner media but remains constant when Ca2+ concentration is changed in the outside solution. However, the fluorescence decreases when large anions such as ClO4- are present in the external solution. Ca2+ inside large liposomes promotes a similar behaviour to that found in sonicated vesicles when they are osmotically contracted in hypertonic media. The results can be interpreted in terms of Ca2+ adsorption on the inner interface and a cooperative interaction between the monolayers.

Effect of insulin on the lytic action of lysophosphatidylcholine in lipid bilayers

The effect of insulin on the bilayer properties of dimyristoylphosphatidylcholine liposomes at the gel and the liquid crystalline state was measured by differential scanning calorimetry and absorbance at 450 nm. It is found that insulin promotes a decrease in the enthalpy of the gel-liquid crystalline transition without displacing the transition temperature. Under these conditions the lytic action of monomyristoylphospatidylcholine is enhanced, decreasing the critical lytic concentrations to values comparable to the bilayer at the gel state. The effect of the lysoderivate on liposomes in contact with increasing concentrations of insulin promotes a reorganization of the lipids into smaller particles as inferred from fluorescence dequenching, turbidity and exclusion chromatography assay. It is concluded that the action of lysoderivates can be enhanced, at temperatures above the transition temperature, by proteins that without spanning the lipid bilayers can perturb the bilayer interface.