Lipid-Induced Organization of a Primary Amphipathic Peptide: A Coupled AFM-Monolayer Study

To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a β-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate. Received: 31 July 1998/Revised: 4 November 1998     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

Phase behaviour of stearic acid-stearonitrile mixtures. A thermodynamic study in bulk and at the air-water interface

The solid-liquid phase behaviour of stearic acid (SA) and stearonitrile (SN) in binary mixtures was investigated by differential scanning calorimetry (DSC), and the formation of SA-SN mixed monolayers at the air-water interface was followed by surface pressure-area (pi-A) measurements and by Brewster angle microscope (BAM) observation. The solid-liquid phase diagram is a eutectic type phase diagram, with the eutectic composition 0.90相似文献   

Interaction of differently oriented lipids in monolayer: mixed monolayers of 16-(9-anthroyloxy)palmitic acid with phosphatidylcholine and cholesterol

16-(9-Anthroyloxy)palmitic acid (16-AP) is a bifunctional molecule with carboxyl and 9-anthroyloxy groups attached at both ends of the hydrocarbon chain. At the air-water interface, in a monolayer, the 16-AP molecule has horizontal and vertical orientations, depending on the surface pressure of the monolayer. The miscibilities of 16-AP with dimyristoylphosphatidylcholine (DMPC), cholesterol (CH), and fatty acids in mixed monolayers were evaluated in investigations of monolayer phase transitions. Lipid molecules with flexible hydrocarbon chains, i.e., DMPC and fatty acids, formed homogeneous mixed monolayers with horizontally oriented 16-AP. On the other hand, the rigid molecule, CH, could not accommodate the horizontally oriented 16-AP in a monolayer, and there was a phase separation from 16-AP. In biological and reconstituted membranes, preferential binding of phospholipid to the integral protein and exclusion of cholesterol in close vicinity of the membrane protein have been recognized. On the basis of this work, it can be expected that flexible lipids readily accommodate the rough hydrophobic surface of integral proteins and stabilize the structure of the protein, while rigid lipids such as cholesterol are removed from the immediate environment of the membrane protein, if the protein does not interact specifically with the rigid lipids.     

A long-lived amphiphilic fluorescent probe studied in POPC air-water monolayer and solution bilayer systems.

A novel amphiphilic fluorescent probe (Fluorazophore-L) with a strongly dipolar, nonionic azoalkane as headgroup and a palmitoyl tail has been synthesized and characterized. Pure Fluorazophore-L was found to be sufficiently amphiphilic to form stable air-water monolayers. An analysis of the surface pressure versus area suggests an area per molecule of about 34+/-2 A(2) at 29 mN m(-1). The partitioning into a lipid membrane model was quantified at the air-water interface by spreading 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) monolayers. Measurements with different molar fractions of Fluorazophore-L revealed a small but significant reduction of the mean area in the mixed monolayer. The excess free energy of mixing (-0.5+/-0.1 kT) indicated a weakly attractive interaction slightly above thermal energy, suggesting a good miscibility of the fluorescent probe within the lipid monolayer without major structural modifications. Spectroscopic measurements confirmed the incorporation of Fluorazophore-L into POPC vesicles. The fluorescence lifetime was very long (125+/-5 ns under air) with monoexponential fluorescence decays.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: II. Monolayers of pulmonary surfactant protein SP-C and phospholipids.

The interaction of the hydrophobic pulmonary surfactant protein SP-C with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and DPPC:DPPG (7:3, mol:mol) in spread monolayers at the air-water interface has been studied. At low concentrations of SP-C (about 0.5 mol% or 3 weight%protein) the protein-lipid films collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At initial protein concentrations higher than 0.8 mol%, or 4 weight%, the isotherms displayed kinks at surface pressures of about 50 mN.m-1 in addition to the collapse plateaux at the higher pressures. The presence of less than 6 mol%, or 27 weight%, of SP-C in the protein-lipid monolayers gave a positive deviation from ideal behavior of the mean areas in the films. Analyses of the mean areas in the protein-lipid films as functions of the monolayer composition and surface pressure showed that SP-C, associated with some phospholipid (about 8-10 lipid molecules per molecule of SP-C), was squeezed out from the monolayers at surface pressures of about 55 mN.m-1. The results suggest a potential role for SP-C to modify the composition of the monolayer at the air-water interface in the alveoli.     

Fucosyled neoglycolipids: synthesis and interaction with a phospholipid

The interfacial behavior of the neoglycolipids formed of Guerbet alcohol (G(28)) bound to a triethylene glycol spacer (E(3)) and to a sugar moiety (alpha- and beta-fucose) spread at the air/water interface has been studied under dynamic conditions of compression. Although the alpha (alpha-FucE3G28)- and beta-fucose (beta-FucE3G28) derivatives possessed the same chemical structure, the positioning of the sugar moiety relative to the whole molecule had a significant influence on the organization of neoglycolipid molecules in the spread monolayers. Thus, beta-fucose molecules exhibited higher compressibilities and larger molecular areas than a alpha/beta (84/16%) mixture (alpha(84)-FucE3G28). The comparison of the compressional behavior of the fucose derivatives with that of Guerbet alcohol in the absence and in the presence of the triethylene glycol spacer shows that the presence of the E(3) chain is necessary to stabilize the lipid at the interface and that the incorporation of a sugar moiety into the molecule resulted in an important expansion of a monolayer. Despite their different interfacial behaviors, the two sugar derivatives formed ideal mixtures when cospread at the air/water interface. Conversely, in the presence of a phospholipid, such as DMPC, repulsive interactions were observed and appeared to be stronger for DMPC/alpha(84)-FucE3G28 mixed monolayers. The membrane fluidity of DMPC liposomes bearing the studied amphiphilic molecules was assessed by fluorescence depolarization measurements. The results reveal that whereas G(28) was deeply inserted into the liposome bilayers, the presence of a E(3) chain and of a sugar moiety in these bilayers induced a transfer of the amphiphilic derivatives from the hydrophobic core towards polar headgroups of phospholipid molecules.     

Study of calix[4]resorcinarene-dopamine complexation in mixed phospholipid monolayers formed at the air-water interface

We have studied the physical properties of monolayers formed by calix[4]resorcinarene and in mixtures with dipalmitoyl phosphatidylcholine (DPPC) in various molar ratios formed at the air-water interface and at presence of dopamine in water subphase by means of measurements of surface pressure and dipole potential. We showed that both calix[4]resorcinarene as well as its mixture with DPPC form stable monolayers at the water subphase. The presence of dopamine resulted in an increase of the mean molecular area and in a decrease of the compressibility modulus of the monolayers. For mixed monolayers at higher content of calix[4]resorcinarene (> 0.2 molar fraction) a deviation from ideal miscibility took place especially for monolayers in a solid state. This can be connected with formation of aggregates of calix[4] resorcinarene. Lowest miscibility and weakest interaction of dopamine with a monolayer was observed for calix[4]resorcinarene molar fraction of 0.33 in the monolayer.     

Nanometer scale organization of mixed surfactin/phosphatidylcholine monolayers.

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and of dipalmitoyl phosphatidylcholine (DPPC) were deposited on mica and their nanometer scale organization was investigated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). AFM topographic images revealed phase separation for mixed monolayers prepared at 0.1, 0.25, and 0.5 surfactin molar ratios. This was in agreement with the monolayer properties at the air-water interface indicating a tendency of the two compounds to form bidimensional domains in the mixed systems. The step height measured between the surfactin and the DPPC domains was 1.2 +/- 0.1 nm, pointing to a difference in molecular orientation: while DPPC had a vertical orientation, the large peptide ring of surfactin was lying on the mica surface. The N/C atom concentration ratios obtained by XPS for pure monolayers were compatible with two distinct geometric models: a random layer for surfactin and for DPPC, a layer of vertically-oriented molecules in which the polar headgroups are in contact with mica. XPS data for mixed systems were accounted for by a combination of the two pure monolayers, considering respective surface coverages that were in excellent agreement with those measured by AFM. These results illustrate the complementarity of AFM and XPS to directly probe the molecular organization of multicomponent monolayers.     

Phase behaviour of stearic acid-stearonitrile mixtures: A thermodynamic study in bulk and at the air-water interface

The solid-liquid phase behaviour of stearic acid (SA) and stearonitrile (SN) in binary mixtures was investigated by differential scanning calorimetry (DSC), and the formation of SA-SN mixed monolayers at the air-water interface was followed by surface pressure-area (π-A) measurements and by Brewster angle microscope (BAM) observation. The solid-liquid phase diagram is a eutectic type phase diagram, with the eutectic composition 0.90 < X_SN < 0.95 and T^eut = 40.9 °C. The DSC results also suggest that the two components are immiscible in the solid phase but form a liquid mixture with positive deviations to the ideal behaviour. At the air-water interface, the two components form liquid condensed monolayers in the entire range of compositions, at low surface pressures, while solid mixed monolayers only form at high surface pressures for X_SN < 0.8. Thermodynamic analysis indicates that SA and SN are miscible in the liquid condensed phase, with negative deviations from the ideal behaviour. The variation of the collapse surface pressure of mixed monolayers also indicates miscibility at the air-water interface.     

Study of calix[4]resorcinarene-dopamine complexation in mixed phospholipid monolayers formed at the air-water interface

We have studied the physical properties of monolayers formed by calix[4]resorcinarene and in mixtures with dipalmitoyl phosphatidylcholine (DPPC) in various molar ratios formed at the air-water interface and at presence of dopamine in water subphase by means of measurements of surface pressure and dipole potential. We showed that both calix[4]resorcinarene as well as its mixture with DPPC form stable monolayers at the water subphase. The presence of dopamine resulted in an increase of the mean molecular area and in a decrease of the compressibility modulus of the monolayers. For mixed monolayers at higher content of calix[4]resorcinarene (> 0.2 molar fraction) a deviation from ideal miscibility took place especially for monolayers in a solid state. This can be connected with formation of aggregates of calix[4] resorcinarene. Lowest miscibility and weakest interaction of dopamine with a monolayer was observed for calix[4]resorcinarene molar fraction of 0.33 in the monolayer.     

Packing of ganglioside-phospholipid monolayers: an x-ray diffraction and reflectivity study

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structure of mixed ganglioside-phospholipid monolayers was investigated at the air-water interface. Mixed monolayers of 0, 5, 10, 20, and 100 mol% ganglioside GM(1) and the phospholipid dipalmitoylphosphatidylethanolamine (DPPE) were studied in the solid phase at 23 degrees C and a surface pressure of 45 mN/m. At these concentrations and conditions the two components do not phase-separate and no evidence for domain formation was observed. X-ray scattering measurements reveal that GM(1) is accommodated within the host DPPE monolayer and does not distort the hexagonal in-plane unit cell or out-of-plane two-dimensional (2-D) packing compared with a pure DPPE monolayer. The oligosaccharide headgroups were found to extend normally from the monolayer surface, and the incorporation of these glycolipids into DPPE monolayers did not affect hydrocarbon tail packing (fluidization or condensation of the hydrocarbon region). This is in contrast to previous investigations of lipopolymer-lipid mixtures, where the packing structure of phospholipid monolayers was greatly altered by the inclusion of lipids bearing hydrophilic polymer groups. Indeed, the lack of packing disruptions by the oligosaccharide groups indicates that protein-GM(1) interactions, including binding, insertion, chain fluidization, and domain formation (lipid rafts), can be studied in 2-D monolayers using scattering techniques.     

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.     

Chemical activity of cholesterol in membranes

Measurements are reported for the rate constants for the release of cholesterol (and dihydrocholesterol) to beta-cyclodextrin from mixtures with phospholipids in homogeneous monolayers at constant pressure at the air-water interface. In each mixture, it is found that the release rate shows a sharp decrease as the cholesterol concentration in the monolayer decreases through a composition corresponding to the stoichiometry of a cholesterol-phospholipid complex. The stoichiometry of the complex was established previously by the position of a sharp cusp in the thermodynamic phase diagram of each mixture and also by a minimum in average molecular area versus composition measurements. A theoretical model used earlier to account for the phase diagrams predicts the chemical potential and chemical activity of cholesterol in these mixtures. The calculated chemical activity also shows a sharp change at the complex stoichiometry in homogeneous monolayers. The similarities in change of observed release rate and calculated chemical activity are expected from reaction rate theory where the release rate is proportional to the cholesterol chemical activity. The chemical activity of cholesterol as determined by complex formation between some phospholipids and cholesterol in the plasma membrane of cells may serve a regulatory function with respect to intracellular cholesterol transport and biosynthesis.     

Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram

Small-angle neutron scattering has been used to study structural features of lamellar bilayer membranes of dimyristoylphosphatidylcholine (DMPC) and DMPC mixed with various amount of cholesterol. The studies were recorded at a fixed hydration level of 17% 2H2O, i.e. just below saturation. Bragg reflections gives information on the ripple structure and on the bilayer periodicity. The crystalline Lc phase, which was stabilized after long time storage at low temperature, exhibits major small angle scattering when cholesterol is mixed into the membrane. The intermediate P beta' gel-phase, which is characteristic by the rippled structure, is dramatically stabilized by the introduction of cholesterol. The ripple structure depends significantly both on the cholesterol content and on the temperature. At high temperatures, T greater than 15 degrees C, the inverse ripple periodicity varies basically linearly with cholesterol content, and approach zero (i.e. periodicity goes to infinite) at 20 mol% cholesterol, approximately. At lower temperatures the correlation is more complex. The data indicate additional phase boundaries below 2 mol% and at approx. 8 mol%. Secondary rippled structures are observed in the low temperature L beta'-phase for cholesterol content below approx. 8 mol%. The data gives detailed insight into the phosphatidylcholine cholesterol phase diagram, which is discussed on the basis of a simple model in which the cholesterol complexes are fixed to the defect stripes of the rippled structure.     

Phase diagram of mixed monolayers of stearic acid and dimyristoylphosphatidylcholine. Effect of the acid ionization

The aim of this work is to study the phase diagram of mixed monolayers composed of dimyristoylphosphatidylcholine (DMPC) and stearic acid (SA) at different ionic strength and bulk pH of the aqueous subphase. In this way, the effect of ionization of SA on the interaction and thus on phase separation with the DMPC matrix can be analyzed. To this purpose, we first determined the ionization state of pure SA monolayers as a function of the bulk subphase pH. The SA monolayers are nearly fully ionized at pH 10 and essentially neutral at pH 4 and the mixture of DMPC and SA was studied at those two pHs. We found that the DMPC-enriched phase admits more SA if the SA monolayer is in a liquid-expanded state, which is highly related to the acid ionization state, and thus to the bulk pH and ionic strength. At pH 4 the molecules hardly mix while at pH 10 the mixed monolayer with DMPC can admit between 30 and 100% of SA (depending on the lateral pressure) before phase separation is established. The addition of calcium ions to the subphase has a condensing effect on SA monolayers at all pHs and the solubility of SA in the DMPC matrix does not depend on the bulk pH in these conditions. The observed phase diagrams are independent on the manner in which the state of the mixed film is reached and may thus be considered states of apparent equilibrium.     

Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung.

Hydrophobic pulmonary surfactant protein enriched in SP-C has been mixed in amounts up to 10% by weight with various phospholipids. The lipids used were dipalmitoyl phosphatidylcholine (DPPC), or DPPC plus unsaturated phosphatidylglycerol (PG), or phosphatidylinositol (PI) in molar ratios of 9:1 and 7:3. The protein enhanced the rate and extent of adsorption of each lipid preparation into the air-water interface, and its respreading after compression on a surface balance. Maximum surface pressures attained on compression of monolayers of mixtures of lipids were slightly higher in the presence of protein. The effects on rate and extent of adsorption were proportional to the amount of protein present. Mixtures containing 30 mol% PG or PI adsorbed more readily into the interface than those containing 10% acidic lipid or DPPC alone. Mixtures containing 30% PI were slightly more rapidly adsorbed than those containing 30% PG. The results suggest that mixtures of DPPC with either acidic lipid in the presence of surfactant protein could be effective in artificial surfactants.     

Aggregation of gramicidin A in phospholipid Langmuir-Blodgett monolayers

The aggregation of Gramicidin A (gA) in dipalmitoylphosphatidylcoline (DPPC) monolayers is investigated by both thermodynamic and structural methods. Compression isotherm analysis and atomic force microscopy (AFM) observations are performed. Our experimental results indicate that gA aggregation does occur in DPPC monolayers even at very low gA concentration (about 8 x 10(-4) mol%). At the low gA concentration limit, the aggregation process seems to be mainly horizontal (i.e., side-by-side, into the monolayer plane), following a fractal pattern growth producing the formation of typical, flat (0.5 nm height) "doughnut" structures, with a diameter of approximately 150 nm. These structures appear to be composed of smaller subunits (about 70 nm diameter) showing the same doughnut structure. At a molar fraction of approximately 3.8 mol%, the big doughnuts start to disaggregate and only small doughnuts appear. Above a gA concentration of approximately 4.4 mol%, all doughnuts (large and small) disappear, and the morphology assumes the appearance of a patchwork of two distinct phases: one that, being very flat, can be associated with a gA-free or gA-poor DPPC phase, and a second one, characterized by a more corrugated surface, associated with a gA-rich DPPC phase. At gA concentration of approximately 5 mol%, a percolation transition in the gA-rich DPPC phase occurs. Thermodynamic data indicate that the maximum of miscibility between gA and DPPC molecules occurs at approximately 28 mol%, suggesting that gA could aggregate in hexamers that are, on average, bound to 16 DPPC molecules. At the same concentration, AFM images show a network of small gA aggregation units of a size compatible with gA hexamers.     

Phase behaviour and morphology of binary mixtures of DPPC with stearonitrile, stearic acid, and octadecanol at the air-water interface

The behaviour of dipalmitoylphosphatidylcholine (DPPC), mixed with stearonitrile (SN), was investigated at the air-water interface by surface pressure-area (pi-A) measurements and by direct visualisation of monolayers by Brewster angle microscopy (BAM). The pi-A-X diagram of system DPPC/SN was compared with the corresponding diagrams of systems DPPC/stearic acid (SA) and DPPC/octadecanol (OD) at 20 degrees C. Monolayers of the three systems reach the closest packing of alkyl chains in the 0.4-0.6 range of XDPPC. Thermodynamic analysis indicates miscibility in the three binary systems with negative deviations from the ideal behaviour. Morphological features of system DPPC/SN change significantly with XDPPC and temperature in the range 10-30 degrees C. At 10 and 20 degrees C mixed monolayers form condensed states from low pi all over the composition range. At 30 degrees C, the liquid-expanded (LE)--liquid-condensed (LC) phase transition occurs at increasing pi with XDPPC. The shape and size of condensed domains change with XDPPC and pi. Contrarily to the behaviour of pure components, mixed monolayers of DPPC/SN exhibit orientational order in the 0.2-0.6 mol fraction range of DPPC. BAM observation confirmed the partial miscibility indicated by GE data in a limited range of compositions at 30 degrees C.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.