Egg Lecithin Bilayers Research Articles

Link between Membrane Composition and Permeability to Drugs.

Prediction of membrane permeability to small molecules represents an important aspect of drug discovery. First-principles calculations of this quantity require an accurate description of both the thermodynamics and kinetics that underlie translocation of the permeant across the lipid bilayer. In this contribution, the membrane permeability to three drugs, or drug-like molecules, namely, 9-anthroic acid (ANA), 2',3'-dideoxyadenosine (DDA), and hydrocortisone (HYL), are estimated in a pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in a POPC:cholesterol (2:1) mixture. On the basis of independent 2-5-μs free-energy calculations combined with a time-fractional Smoluchowski determination of the diffusivity, the estimated membrane permeabilities to these chemically diverse permeants fall within an order of magnitude from the experimental values obtained in egg-lecithin bilayers, with the exception of HYL in pure POPC. This exception is particularly interesting because the calculated permeability of the sterol-rich bilayer to HYL, in close agreement with the experimental value, is about 600 times lower than that of the pure POPC bilayer to HYL. In contrast, the permeabilities to ANA and DDA differ by less than a factor of 10 between the pure POPC and POPC:cholesterol bilayers. The unusual behavior of HYL, a large, amphiphilic compound, may be linked with the longer range perturbation of the lipid bilayer it induces, compared to ANA and DDA, suggestive of a possibly different translocation mechanism. We find that the tendency of lower permeabilities of the POPC:cholesterol bilayer relative to those of the pure POPC one is a consequence of increased free-energy barriers. Beyond reporting accurate estimates of the membrane permeability, the present contribution also demonstrates that rigorous free-energy calculations and a fractional-diffusion model are key in revealing the molecular phenomena linking the composition of a membrane to its permeability to drugs.

Linear free-energy relationships for water/hexadec-1-ene and water/deca-1,9-diene partitions, and for permeation through lipid bilayers; comparison of permeation systems

Linear free energy relationships based on Abraham descriptors have been obtained for partition from water and the gas phase into hexadec-1-ene and into deca-1,9-diene, and for permeation through egg lecithin bilayers. Comparison with equations for partition into a large number of organic solvents shows that hexadec-1-ene and deca-1,9-diene are excellent models for permeation through egg lecithin bilayers. Wet octanol is a poor model, as are all polar organic solvents. The egg lecithin bilayer is non-polar and has only weak hydrogen bond acidity and weak hydrogen bond basicity. Comparison of equations for permeation through egg lecithin bilayers, through the human intestinal membrane, through the blood-brain membrane and through human skin shows that these four permeation processes are chemically far apart. It is not possible to obtain any general model for permeation: each permeation process requires a different model system.

Interaction of the antimalarial agents halofantrine and lumefantrine with lipid bilayers.

The effects of antimalarial drugs halofantrine and lumefantrine on the fluoresence anisotropy of diphenylhexatriene (DPH)-containing phospholipid vesicles have been examined. Lumefantrine increases DPH anisotropy, indicating a condensing effect on bilayers of dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidylcholine (DOPC), egg lecithin and mouse erythrocyte membranes (including membranes isolated from plasmodial-infected mice). Its condensing effect is more pronounced in bilayers of lower microviscosity. In contrast, increases or decreases in DPH anisotropy are observed with halofantrine, depending on the nature of the lipid. Decreases in anisotropy, which reflect a perturbing effect, are observed in bilayers of high microviscosity (for example, gel state of DPPC bilayers). Increases in anisotropy are observed in bilayers of low microviscosity (such as DOPC and egg lecithin bilayers). The perturbing effect of halofantrine is further confirmed by the increases in permeability of calcein-containing DPPC vesicles in the presence of the drug. However the perturbative effects of halofantrine are observed to the same magnitude in uninfected and plasmodial-infected erythrocyte membranes, and may not be relevant to the antimalarial action of the drug. In contrast, the condensing effect of lumefantrine is significantly greater in infected erythrocyte membranes and may contribute to its antimalarial action.

Transport across 1,9-decadiene precisely mimics the chemical selectivity of the barrier domain in egg lecithin bilayers

The barrier domain solubility-diffusion theory of lipid bilayer permeability relates the permeability coefficient (Pm) to the solute's partition coefficient (PCbarrier/w) and diffusion coefficient (Dbarrier) in the ordered chain region of the bilayer that serves as the barrier region for polar permeants. To select the best solvent to mimic the barrier domain, permeability coefficients across a layer of 1,9-decadiene were compared with permeability coefficients from bilayer transport. Rate constants for transport, k, of α-methyl substituted analogues of p-toluic and p-methylhippuric acid were measured across a layer of 1,9-decadiene embedded in a PTFE filter membrane placed between two aqueous solutions in side-by-side diffusion cells. Permeability coefficients (P1,9-decadiene) were normalized to that obtained for p-toluic acid, which was included in donor solutions. The correlation of log(Pbilayer) versus log(P1,9-decadiene) was linear with a slope of 0.99 ± 0.02 SD, indicating that 1,9-decadiene precisely mimics the egg lecithin bilayer barrier domain in its chemical selectivity. Using the decadiene membrane transport method to indirectly estimate partition coefficients for similarly sized permeants extended the range of measurable values beyond those readily attainable by the traditional shake-flask method, allowing measurement of 1,9-decadiene/water PCs as low as 3 × 10−7. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:640–646, 2002

Independence of substituent contributions to the transport of small molecule permeants in lipid bilayers.

To explore the independence of functional group contributions to permeability of nonelectrolytes across egg lecithin bilayers, the transport rates were measured of a series of alpha-substituted p-methylhippuric acids (-H, -Cl, -OCH3, -CN, -OH, -COOH, and -CONH2) across egg lecithin lipid bilayers, in the form of large unilamellar vesicles (LUVs) at 25 degrees C. Intrinsic permeability coefficients (P(HA)) were calculated from apparent permeability coefficients (P(app)) measured as a function of pH. Group contributions to the free energy of transfer from water into the barrier domain Delta (Delta G degrees)P,X, were calculated for the substituents and compared to the contributions of these groups when attached top-toluic acid measured earlier. The Delta (Delta G degrees)P,X values from permeability data were also correlated with Delta (Delta G degrees)PC,X values of partitioning from water into organic solvents to determine the physicochemical selectivity of the barrier domain. P(app) values in LUVs were found to vary approximately linearly with the fraction of neutral permeant over a pH range of 5.5 to 10.5, suggesting that the transport of the ionized species is negligible over this pH range. The Delta (Delta G degrees)P,X values from the 2 series of compounds appear to be the same, indicating that the functional group contributions are independent. 1,9-decadiene was found to be the most similar to the chemical environment of the barrier domain. Functional group contributions to transport across egg lecithin bilayers appear to be independent of the compound to which they are attached, even though the thickness of the barrier domain in lipid bilayers is approximately the same as the extended length of the permeant.

Differential effects of cholesterol and oxidised-cholesterol in egg lecithin bilayers

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.

The pretransition of dipalmitoyllecithin bilayers as probed by the fluorescent pyrrolopyrimidine, U-104067

The amphiphilic pyrrolopyrimidine, U-104067, is a fluorophore ideally suited to report on the relative hydrophobicities of different microenvironments. It forms stable monomolecular layers at the air/water interface with a limiting molecular area of 51.9±0.3 Å 2/molecule and a collapse pressure of about 18 dyn/cm. Differential scanning calorimetry of its mixed liposomes with dipalmitoyllecithin shows full solubility of the compound in the liquid disordered phase and insolubility in the solid ordered phase. In aqueous solutions, the compound binds to phospholipid bilayers with a stoichiometry of 13.2±1.2 moles of lipid per mole of U-104067, with K d=0.33±0.05 μM toward egg lecithin/phosphatidylserine bilayers and K d=1.5±0.3 μM toward pure egg lecithin bilayers. In liquid crystalline phospholipid bilayers the compound behaves as two independently emitting species, one accessible to acrylamide and the other one not. Doxyl fatty acid methyl esters quench both species and show that the average position of the fluorophore is at a depth corresponding to that of the 7th carbon of a fatty acyl chain. Dissolved in the liquid disordered (L α ) phase of dipalmitoyllecithin at 45°C, U-104067 shows a single ionizable group, p K a=3.19±0.03 while in the solid ordered (L β′ ) phase it displays two ionizable groups, p K a1=4.99±0.10 and p K a2=6.96±0.13. The most unusual property of this molecule is that it is miscible with the tilted (L β′ ) and liquid (L α ) phases of dipalmitoyllecithin but totally immiscible with the rippled (P β′ ) phase. Because of this, U-104067 is a sensitive reporter for the tilted/rippled phase transition as monitored by its fluorescence anisotropy and its quantum yield changes.

Mutual adhesion of lecithin membranes at ultralow tensions

We present an extensive light-microscopic study of mutual adhesion of egg (and dimyristoyl) lecithin bilayers in highly swollen samples, including adhesion brought about by osmotic inflation of vesicles. The lateral tensions associated with adhesion, if below ca. 3 x 10-4 dyn cm-1 , could be read from a rounding of the membrane next to the contact area. The contact angle of symmetric adhesion, i.e. of two membranes under equal tension, had a maximum of 45°. While showing some scatter, it did not seem to depend on tension down to 3 x 10-6 dyn cm-1, the lowest tension observed. The maximum contact angle of single membranes adhering to bundles of membranes was near 70°. The data indicate that mutual adhesion of lecithin membranes is always induced by lateral tension. The constancy of the contact angles appears to be compatible with undulation theory, but their magnitude compels us to postulate an unknown, optically unresolvable roughness of lecithin membranes which serves as an area reservoir.

Membrane-directed effects of the plant hormones abscisic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid

This study examines two ways plant hormones might influence membrane processes, effects on overall permeability and modifications of specific ion channels. Abscisic acid (ABA) and indole-3-acetic acid (IAA) greatly enhanced erythritol permeability in mixed egg lecithin bilayers. In single component dioleoylphosphatidylcholine bilayers ABA was less effective than IAA, while 2,4-dichlorophenoxyacetate (2,4-D) did not affect either system or alter their ABA response. In Myxicola axons ABA and IAA had no effect, while 2,4-D (10 uM) caused a depolarizing shift of voltage-dependent Na + and K + activation by 25 ± 4 mV and 15 ± 3 mV, consistent with internal negative surface charge changes of −0.002 e −/A 2 and −0.0007 e −/A 2. We conclude that both generalized and ion channel-directed effects may link plant hormones and intracellular regulation.

Orientation and vertical fluctuations of spin-labeled analogues of cholesterol and androstanol in phospholipid bilayers

We have used ESR and NMR linewidth broadening by spin-labels to determine the overall orientation of spin-labeled analogues of cholesterol and androstanol in egg lecithin bilayers. While the cholesterol analogues were found to have a single orientation in each monolayer, with the acyl chain pointing towards the center of the bilayer, the androstanol analogue appeared, at least in sonicated vesicles, to experience two opposite orientations in the same monolayer, very likely with a rapid reorientation. The possibility of rapid vertical fluctuations of the sterol molecules within the phospholipid bilayer is also discussed.

Elastic Torques about Membrane Edges: A Study of Pierced Egg Lecithin Vesicles

The shape of mechanically pierced giant vesicles is studied to obtain the elastic modulus of Gaussian curvature of egg lecithin bilayers. It is argued that such experiments are governed by an apparent modulus, kappa(app), not the true modulus of Gaussian curvature, kappa. A theory of kappa(app) is proposed, regarding the pierced bilayer vesicle as a closed monolayer vesicle. The quantity measured, i.e. kappa(app)/kappa, where kappa is the rigidity, agrees satisfactorily with the theory. We find kappa(app) = -(1.9 +/- 0.3) . 10(-12) erg (on the basis of kappa = (2.3 +/- 0.3) . 10(-12) erg). The result may have implications for bilayer fusion.

Molecular packing and area compressibility of lipid bilayers.

Knowledge of the molecular packing of lipids and water in lipid bilayers is important for understanding bilayer mechanics and thermodynamics. Information on packing is most often obtained from x-ray or neutron diffraction measurements. Given the d spacing, composition, and partial specific volumes of the lipid and water, it is a simple matter to calculate the area per lipid molecule, bilayer thickness, and bilayer mass density. The partial specific volumes are commonly assumed to be those of bulk water and of lipid in excess water regardless of the degree of bilayer hydration. We present evidence here that these assumptions should be seriously questioned. At low hydrations, we find the head groups of egg and dioleoyl lecithin to be much less tightly packed than previously thought and the partial specific volume of water to be considerably smaller than 1 ml/g. Because the molecular packing affects the mechanical properties of bilayers, we use the results to reevaluate published experiments concerning the elastic area compressibility modulus of egg lecithin bilayers and the repulsive hydration force between bilayers.

Vinblastine sulphate encapsulation in phospholipid vesicles and liposomes

Vinblastine sulphate (VBLS) encapsulation by small phospholipid (mainly egg lecithin (EL)) vesicles and by large egg lecithin liposomes was studied. It is found that liposomes encapsulate 10 times more drug (∼ 2% mol fraction compared to ∼ 2%o mol fraction) than the small vesicles (60–80 nm diameter). The encapsulation of VBLS may be increased by a factor of 5 when the ab initio (in the dry lipid film) ratio VBLS/EL varies from 0.03 to 1. It is assumed that this increase is due to entrapment of segregated drug in or between the EL bilayers. The release of the entrapped drug into fresh buffer is a slow phenomenon.

Effect of CoQ Homologues on the Fluidity of Phospholipid Bilayers as Studied by Fluorescence Polarization of Perylene

The effect of Coenzyme Q analogues on the fluidity of egg lecithin bilayers was studied by fluorescence polarization of perylene. The parameter was clearly affected by the side-chain length of the quinones, by their concentration, and by their redox state. With increasing concentration, short-chain quinones decreased the bilayer fluidity, and did so to an even greater extent in their reduced state. On the contrary, long-chain analogues fluidized the bilayer, the effect being independent of their redox state. Such observations might explain the lower efficiency of short-chain quinones, as compared to the long-chain analogues, in restoring, in vitro, the respiratory activity of CoQ2-depleted mitochondria.

The fidelity of response by nitroxide spin probes to changes in membrane organization: The condensing effect of cholesterol

The response of doxyl fatty acid spin probes in egg lecithin bilayers to added cholesterol is compared with results from 2H-NMR. Large differences are found between the profiles of order parameter vs. label position and cholesterol concentration. At constant cholesterol content, the ESR spin probe order parameter decreases continuously as the label position is moved toward the terminal methyl region of the bilayer whereas an order parameter ‘plateau’ is observed for the upper region of the bilayer by 2H-NMR. In addition, the spin probe order parameters are smaller than those observed by 2H-NMR. Differences are also observed in the profiles of order parameter vs. cholesterol content for each label position. The spin probes detect a maximal response to added cholesterol for the central portion of the chains with much weaker responses near both ends of the chains. In contrast, the 2H-NMR results indicate a large, approximately constant response for the first ten positions in the chains with a decreasing response toward the terminal methyl group. For all the positions examined, the spin probes show a weaker response than that observed by 2H-NMR. A direct measure of the perturbing effect of a spin label is made by comparing the deuterium quadrupole splittings in egg lecithin-cholesterol bilayers for stearic acid with and without an attached doxyl moiety. The spin-labelled fatty acid has a much reduced quadrupole splitting and an opposite response to cholesterol addition.

Cholesterol levels inversely reflect the thermal sensitivity of mammalian cells in culture

Cholesterol is a primary component of the mammalian cell plasma membrane. Although its function is unknown, it may be of major importance in maintaining membrane fluidity and rigidity. In artificial membrane systems, the addition of cholesterol results in a condensing effect--thickening the bilayer and inducing higher order in the acyl chains of the phospholipids. Permeability profiles indicate that the addition of cholesterol into egg-lecithin bilayers increases the half-time of solute transport. In addition, decreased amounts of sterol in the membrane increase glucose permeability, and, in L cells, increase the transport of rubidium. These studies suggest a role for cholesterol in changing the physical characteristics of the membrane resulting in the alteration of membrane permeability. We now provide evidence that cholesterol may act, presumably via changes in physical membrane properties, with yet another biological consequence; regulating the survival sensitivity of mammalian cells to hyperthermic temperatures.

A spin label study of the effects of sterols on egg lecithin bilayers

Paramagnetic resonance of cholestane and three fatty acid probes is used to measure the effects of the addition of cholesterol, 7-dehydrocholesterol and ergosterol to egg phosphatidylcholine bilayers. At low concentrations we find that all three sterols effectively align the bilayers. However, concentrations of ergosterol above 15 mol% disorder and disrupt the bilayers. The observed behavior is explained in terms of a steric model in which the steroid nucleus organizes the bilayer and the bulky egosterol tail disorganizes the bilayer. The three fatty acid spin labels are used to probe the layers at different depths, and the data observed are in agreement with the normal presented.

Diffusion within egg lecithin bilayers resembles that within soft polymers.

An analysis is presented of how the permeability coefficient/octanol:water partition coefficient ratio for 33 different chemical substances crossing egg lecithin bilayers depends on the molecular volume of the substances. From this analysis we conclude that bilayers made from egg lecithin behave as soft polymers in their discrimination between permeants of different sizes and shapes.

Measurement of forces between lecithin bilayers

CELL fusion1 and vesicle-membrane2 or vesicle-synapse3 fusion are currently thought to involve distance-dependent interactions between membranes. We have devised a general method for measuring such forces between planar phospho-lipid membranes, and report here results for the specific case of egg lecithin (phosphatidyl choline) bilayers in water.

Monitoring the permeability profile of lipid membranes with spin probes

The rate of reaction of the ascorbate ion with the nitroxide group of spin probes intercalated in lipid bilayers has been studied to examine the mechanism of transport of solutes across membranes. The loss of electron spin resonance (ESR) signal follows first-order kinetics. For a given bilayer system, the half-time of the process increases with the distance of the reacting group from the aqueous interface, according to an approximately linear permeation profile. The dependence on phospholipid headgroup is that which would be predicted from the net charge; addition of negatively charged headgroups increases the half-time of reaction, and positively charged headgroups decrease it, compared with bilayers having no net charge. Addition of cholesterol, which is known to decrease the fluidity of the hydrocarbon core of the bilayer, is found to increase the half-time of reaction. The results have been analyzed in terms of a partition-diffusion mechanism. It is suggested that the rate-limiting step for partitioning the solute into the bilayer might be removal of water of hydration. Cholesterol increases the activation energy, most probably by increasing the height of the barriers to diffusion. Quantitation of the changes in reaction rates gives an estimate of the change in bilayer surface potential on changing the headgroup composition. Examination of the permeation profile supports a diffusive mechanism, from which it can be estimated that the diffusion coefficient is approximately halved on adding 35 mol% cholesterol to egg lecithin bilayers.