Permeability Of Large Unilamellar Vesicles Research Articles

Modulating lipid bilayer permeability and structure: Impact of hydrophobic chain length, C-3 hydroxyl group, and double bond in sphingosine

Sphingosine, an amphiphilic molecule, plays a pivotal role as the core structure of sphingolipids, essential constituents of cell membranes. Its unique capability to enhance the permeability of lipid membranes profoundly influences crucial life processes. The molecular structure of sphingosine dictates its mode of entry into lipid bilayers and governs its interactions with lipids, thereby determining membrane permeability. However, the incomplete elucidation of the relationship between the molecular structure of sphingosine and the permeability of lipid membranes persists due to challenges associated with synthesizing sphingosine molecules. A series of sphingosine-derived molecules, featuring diverse hydrophobic chain lengths and distinct headgroup structure, were meticulously designed and successfully synthesized. These molecules were employed to investigate the permeability of large unilamellar vesicles, functioning as model lipid bilayers. With a decrease in the hydrophobic chain length of sphingosine from C15 to C11, the transient leakage ratio of vesicle contents escalated from ∼ 13 % to ∼ 28 %. Although the presence of double bond did not exert a pronounced influence on transient leakage, it significantly affected the continuous leakage ratio. Conversely, modifying the chirality of the C-3 hydroxyl group gives the opposite result. Notably, methylation at the C-3 hydroxyl significantly elevates transient leakage while suppressing the continuous leakage ratio. Additionally, sphingosines that significantly affect vesicle permeability tend to have a more pronounced impact on cell viability. Throughout this leakage process, the charge state of sphingosine-derived molecule aggregates in the solution emerged as a pivotal factor influencing vesicle permeability. Fluorescence lifetime experiments further revealed discernible variations in the effect of sphingosine molecular structure on the mobility of hydrophobic regions within lipid bilayers. These observed distinctions emphasize the impact of molecular structure on intermolecular interactions, extending to the microscopic architecture of membranes, and underscore the significance of subtle alterations in molecular structure and their associated aggregation behaviors in governing membrane permeability.

Membrane permeabilization induced by Triton X-100: The role of membrane phase state and edge tension

Detergents are widely used to solubilize and separate biomembrane components. It is therefore relevant to study and understand the mechanistic details underlying detergent-lipid interactions using biomimetic systems. Here, we have investigated in detail the process of membrane permeabilization and the nature of pores induced by sub-solubilizing concentrations of the detergent Triton X-100 (TX-100) in bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), sphingomyelin (SM) and binary mixtures of these phospholipids with 30 mol% cholesterol (chol). A fluorescence quenching assay was used to evaluate the permeability of large unilamellar vesicles (LUVs) in the presence of increasing concentrations of TX-100. Confocal microscopy was employed to visualize and quantify the permeability of giant unilamellar vesicles (GUVs) to two fluorescent dyes of different sizes in the presence of TX-100. Both methods showed that POPC, POPC/chol and SM membranes become fully permeable at a specific TX-100 concentration, followed by complete (POPC and SM) and partial (POPC/chol) solubilization at a higher detergent concentration. The confocal microscopy experiments revealed that opening of pores occurs as a well-defined event and that for POPC and POPC/chol the pores were initially selective to the small probe and then grew and allowed passage of the larger dye as well. On the other hand, the insoluble SM/chol membranes exhibited only a mild TX-100-induced permeabilization. The membrane edge tension of the liquid phases was measured from the closure rate of macropores induced by electric pulses in GUVs. Membrane edge tension was shown to be sensitive to membrane composition and to decrease in the presence of TX-100. We propose that extensive permeabilization occurs below a critical membrane edge tension, which is eventually reached in the partially and fully soluble compositions, but not in the insoluble mixture.

Effects of pulsed electric fields on the permeabilization of calcein-filled soybean lecithin vesicles

Effects of PEF on permeabilization of calcein-filled large unilamellar vesicles (LUV) with average diameter of 1705.64±55.91nm and small unilamellar vesicles (SUV) with average diameter of 213.06±5.50nm, suspended in an isosmotic sucrose solution (150mM) were investigated. The suspensions of vesicles were exposed to PEF at various electric field strengths in the interval of 0–50kV/cm and different treatment times ranging from 0 to 4.8ms. The intensity of permeabilization induced by PEF was analyzed in terms of the changes of fluorescence intensity of the suspension solution and the average diameter of vesicles, due to the leakage of self-quench calcein encapsulated in vesicles. Results indicated that the permeability of LUV was increased with increasing intensity of electric field (>10kV/cm) and treatment time, and the release percentage of calcein (R value) was increased by 57.98% (from 0.46% to 58.44%) with the increase of the electric field strength (from 10 to 50kV/cm) at 4.8ms. However, the R value was only increased by 14.62% (from 43.82% to 58.44%) with the increase of the treatment time (from 1.2 to 4.8ms) at 50kV/cm. Average diameter of LUV was sharply decreased from 1705.64±55.91 to 465.60±37.12nm with the maximum value at 50kV/cm and 4.8ms. Results also showed that shrinkage or rupture of LUV happened, as confirmed by the change of size distribution of LUV after treatment by PEF. However, as for SUV, the changes of the permeability of vesicles and average diameter were not observed. In addition, it was demonstrated that the magnitude of the induced transmembrane potential was proportional to the radius of the vesicle, and larger vesicles could be polarized or ruptured at relatively lower field strength than smaller vesicles.

Differential effects induced by α- and β-endosulfan in lipid bilayer organization are reflected in proton permeability

The effects of two insecticides isomers, α- and β-endosulfan, on the passive proton permeability of large unilamellar vesicles (LUV) reconstituted with dipalmitoylphosphatidylcholine (DPPC) or mitochondrial lipids were reported. In DPPC (LUV) gel phase, at 30 °C, the global kinetic constant ( K) of proton permeability (proportional to the proton permeability) initially increased slightly with the increase of α-endosulfan/lipid molar ratio up to 0.143. In the range from 0.143 to 0.286, a discontinuity in the increment occurred and, above this range, the proton permeability increased substantially. In DPPC fluid phase, at 48 °C, the proton permeability showed a behavior identical to that observed in gel DPPC, with a sharp increase for α-endosulfan/lipid molar ratios ranging from 0.143 to 0.286. At these and higher concentrations, α-endosulfan induced phase separation in the plane of DPPC membranes, as revealed by differential scanning calorimetry (DSC). Conversely to α-endosulfan, β-endosulfan induced only a slight increase in the proton permeability, either in the fluid or the gel phase of DPPC, for all β-endosulfan/lipid molar ratios tested. Additionally, the effects of the endosulfan isomers on the proton permeability of mitochondrial fluid lipid dispersions, at 37 °C, are similar to those described for DPPC. The β-isomer induced a very small effect, and α-endosulfan, at low concentrations, increased slightly the proton permeability, but for insecticide/lipid molar ratios above 0.143 the permeability increased substantially. Consequently, the membrane physical state of synthetic and native lipid dispersions, as affected by the structural features of α- and β-endosulfan, influenced the proton permeability. The effects here observed in vitro suggest that the formation of lateral membrane domains may underlay the biological activity of α-endosulfan in vivo, contributing to its higher degree of toxicity as compared with β-endosulfan.

Permeability of large unilamellar digalactosyldiacylglycerol vesicles for protons and glucose – influence of α-tocopherol, β-carotene, zeaxanthin and cholesterol

The permeability of large unilamellar vesicles formed from digalactosyldiacylglycerol for glucose and protons was measured. The vesicle composition was modified by addition of different terpenoids: α-tocopherol, cholesterol, zeaxanthin and β-carotene. The digalactosyldiacylglycerol species composition was dominated by the species 18:2/18:2 and 18:1/18:2. Using the self-quenching properties of the fluorescent probe 6-carboxyfluorescein trapped in the aqueous space of the vesicles, the permeability for glucose was determined with a glucose gradient of 800 mM. The calculated permeability coefficient for glucose was in the range of 4.85.10 –10 to 1.12.10 –9 cm.s –1. For proton permeability measurements, the pH-sensitive fluorescent dye pyranine was used. The proton permeability was measured with a pH gradient of 0.6 pH units from 7.0 to 7.6 with valinomycin present to dissipate any diffusion potential across the membrane. The permeability coefficients for protons were in the range of 3.5.10 –8 to 1.0.10 –7 cm.s –1. Digalactosyldiacylglycerol vesicles with 5 % α-tocopherol or 10 % cholesterol or 2 % zeaxanthin reduced the permeability for protons, the two latter significantly as compared to digalactosyldiacylglycerol vesicles. α-Tocopherol (5 %) decreased the permeability for glucose remarkably and so did cholesterol (10 %). β-Carotene (< than 1 %) and zeaxanthin (2 %) in galactolipid vesicles, however, increased the permeability. The significance of these results is discussed in relation to the physiological functions of galactolipids and terpenoids in chloroplast membranes.

Influence of Ionol on the permeability of large unilamellar vesicles in the presence of embryonic calf serum

The influence of various concentrations of an antioxidant—Ionol—(incorporated in the bilayer of large unilamellar liposomes) on the stability of lipoid vesicles in the presence of 10% of embryonic calf serum is considered. The kinetics and temperature dependence of the egress of an intraliposomal marker (6-carboxyfluorescein) from the liposomes has been studied.

Calcium Permeability in Large Unilamellar Vesicles Prepared from Bovine Lens Cortical Lipids

The loss of calcium homeostasis in the lens is thought to play an important role in cataract formation. Although both lens Ca2+-ATPase and membrane lipid permeability are essential to calcium homeostasis, membrane permeability has been studied less extensively. In the present study, the calcium permeability of large unilamellar vesicles (LUVs) prepared from bovine lens cortical lipids has been characterized. When a calcium gradient had been established by the addition of CaCl2to the external medium, calcium influx through the membrane was monitored by calcium concentration sensitive changes in Fura-2 fluorescence as a function of time. The calcium permeability coefficient, P, was 4.46×10−13cm s−1at 37°C. This value was about 4 fold higher than that for LUVs prepared from egg phosphatidylcholine and many times higher than that for LUVs prepared from sphingomyelin. These results provide a basis for future studies of factors that influence the permeability of lens cell membranes to calcium.

Effects of ethanol on permeability of phosphatidylcholine/cholesterol mixed liposomal membranes

Effects of ethanol on permeability of large unilamellar vesicles (about 170 nm in diameter), composed of a dipalmitoyl phosphatidylcholine (DPPC)/cholesterol binary mixture, were studied by monitoring leakage of a fluorescent dye, calcein, entrapped in the inner aqueous phase of the vesicles. In the presence of 25 mol% cholesterol, the leakage was dramatically increased, and the permeability at 1.0 M ethanol was about eight times larger than that without ethanol and cholesterol. This effect of ethanol on the membrane permeability was similar to our previous findings concerning mixed membranes of dilauroyl phosphatidylethanolamine and DPPC (H. Komatsu and S. Okada (1996) Biochim. Biophys. Acta 1283, 73–79). It was suggested that the presence of ethanol can lead to high permeability even if the membranes are stable and have a low permeability in its absence.

Ethanol-enhanced permeation of phosphatidylcholine/phosphatidylethanolamine mixed liposomal membranes due to ethanol-induced lateral phase separation

Effects of ethanol on permeability of large unilamellar vesicles (ca. 160 nm in diameter), composed of dipalmitoyl phosphatidylcholine/dilauroyl phosphatidylethanolamine (DLPE) mixture, were studied by monitoring leakage of the fluorescent dye, calcein, entrapped in the inner aqueous phase of the vesicles. In the presence of 2.1 M ethanol, permeabilities of membranes in various phases were G (bilayer gel) phase > L (bilayer liquid-crystalline) phase with a high mole fraction of DLPE and ( I(ethanol-induced interdigitated gel phase) + G) phase > ( I + L) at 20 mol %DLPE. Arrhenius plots of the leakage rate constants demonstrated that the permeability was greater with 2.1 M ethanol than without ethanol, especially in the temperature above 33°C, suggesting that the presence of ethanol can induce lateral phase separation of liposomal membranes and cause them to have a high permeability even if they are stable and have low permeability in its absence.

アルコールによるリポソーム膜の指組み構造相の形成に関する研究

It was now well established that phosphatidylcholines or phosphatidylglycerols can form an interdigitated structural phase (Iphase), in which acyl chains of the lipids fully interpenetrate the hydrocarbon chains of the opposing monolayer in the presence of short chain alcohols and some other amphiphiles. It has been suggested that the interdigitated structure as well as a hexagonal II structure plays an important role in regulating many functions of biological membranes. We have demonstrated the participation ofIphase formation in ethanol-induced liposomal aggregation and fusion. We have also focused on control of permeability as one of the biomembrane functions, and the effects of ethanol on the permeability of large unilamellar vesicles composed of various phospho-lipids were studied by monitoring the leakage of the fluorescent dye, calcein, entrapped in the inner aqueous phase of the vesicles. In the cases of mixed membranes composed of DPPC and dilauroyl phosphatidylethanolamine (DLPE), as well as DPPC liposomal membranes, large permeabilities were observed in the wide range of ethanol concentrations, where the normal bilayer andIphase coexist and the membrane is in a phase-separated state. These results suggested that ethanol can disturb the normal control of biological membrane permeability.

Increased permeability of phase-separated liposomal membranes with mixtures of ethanol-induced interdigitated and non-interdigitated structures

It has been suggested by many workers using model membranes that the interdigitated structure formation, in which the acyl chains fully interpenetrate the hydrocarbon chains of the opposing monolayer, plays an important role in regulating many functions of biomembranes. In the present study the control of permeability was focused on as one of the biomembrane functions, and the effects of ethanol on the permeability of large unilamellar vesicles made by the extrusion technique (LUVET) (average diameter: about 250 nm), composed of dipalmitoyl or egg yolk phosphatidylcholines, were studied by monitoring the leakage of fluorescent dye, calcein, entrapped in the inner aqueous phase of the LUVET. The permeability was estimated from the apparent rate constant of calcein leakage at 25°C. Large permeabilities were observed in the region of 0.6 M to 1.3 M ethanol, with a concentration dependence. In this range of ethanol concentrations the normal bilayer and interdigitated structures coexist and the membrane is in a phase-separated state. The large permeability is due to the instability of the boundary regions, the interdigitated membrane being characterized by a thinner structure and more rigid hydrocarbon regions in the layer than its non-interdigitated counter part. These results suggest the possibility of biomembrane-permeability regulation by interdigitated membrane formation.

Proton permeability of large unilamellar vesicles

Proton permeability of large unilamellar vesicles

Phospholipid-dependent changes in membrane permeability induced by staphylococcal δ-lysin and bee venom melittin

The effect of purified staphylococcal δ-lysin and bee venom melittin on the permeability of large unilamellar vesicles (liposomes) composed of structurally defined lipids was investigated by following release of sequestered ATP using a bioluminescent assay. ATP release was linearly related to the logarithm of the peptide concentration. Sensitivity to the lytic peptides, as measured by the release of ATP and the slope of the dose–response curve, depended on the chain length and the degree of saturation of the hydrocarbon substituents of the phospholipid in the vesicles. Neither the sign of the surface charge nor the presence of sterol had any significant effect on the sensitivity of liposomes to peptide-induced lysis. Release of ATP and haemoglobin from intact sheep and cod erythrocytes was also determined after their exposure to δ-lysin and melittin. Both cell types showed similar degrees of sensitivity to each lytic peptide when ATP release was followed, but the release of haemoglobin from sheep erythrocytes by δ-lysin was much less than from cod erythrocytes. The implications of these results on the mode of action of the two agents are discussed.

Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr 3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90° light scattering, occurs at a slightly lower temperature (40.8°C) than that of MLV (42°C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38°C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1–0.2 μm diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.

Glycophorin incorporation increases the bilayer permeability of large unilamellar vesicles in a lipid-dependent manner

(1) The effect of glycophorin incorporation on the permeability properties of lipid bilayers is investigated by employing a new method, in which the trap of a small permeant and a high molecular weight, non-permeable molecule are simultaneously monitored. (2) Glycophorin induces a high permeability to potassium and glucose in large dioleoylphosphatidylcholine vesicles. (3) The glycophorin-induced enhanced permeability is found to be lipid dependent. By monitoring the % ratio of the glucose (permeant)/dextran (impermeant) trap in large unilamellar glycophorin-containing vesicles, it was determined immediately after removal of non-enclosed marker, that glycophorin-containing vesicles composed of dioleoylphosphatidylcholine were highly permeable to glucose (% glucose/dextran trap=1.4), whereas glycophorin-containing vesicles prepared from total human erythrocyte lipids demonstrated a greatly reduced permeability in glucose (% glucose/dextran trap=50). (4) The glycophorin-induced increased permeability appears to be related to transbilayer movement of lipid molecules. Glycophorin induces a fast lipid-transbilayer movement of lysophosphatidylcholine and a high permeability to potassium and glucose in dioleoylphosphatidylcholine vesicles, whereas glycophorin-containing vesicles comprised of total human erythrocyte lipids show no lipid-transbilayer movement and only a slight permeability enhancement to glucose, as compared to the protein-free vesicles.