Phospholipid Bilayers as Molecular Models for Drug-Cell Membrana Interactions. The Case of the Antiinflamatory Drug Diclofenac

Phospholipids are amphipatic molecules with long hydrophobic acyl chains and zwitterionic polar heads which assemble into different types of molecular aggregates. The most relevant is the bilayer because of its relation with cell membranes, which are very complex entities. For this reason, simpler molecular models based on phospholipids bilayers are widely used. We have determined the bilayer structure of phospholipids located in the outer and inner monolayers of most cell membranes, and use them as molecular models to study the way different chemicals of biological interest interact with cell membranes. We present the results of our studies on the nonsteroidal anti-inflammatory drug diclofenac, from which little is known about its effects on human erythrocytes. This report presents the following evidence that diclofenac interacts with the human red cell membrane: a) X-ray diffraction and fluorescence spectroscopy of phospholipids bilayers show that diclofenac interacts with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy studies on human erythrocytes it was observed that the drug induced morphological changes different from their normal biconcave shape.     

Dynamics and instabilities of lipid bilayer membrane shapes

Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. The lipid bilayer represents the fundamental architecture of the cellular membrane with its shapes determined by the Helfrich curvature bending energy. However, the dynamics of bilayer shape transitions, especially their modulation by membrane proteins, and the resulting shape instabilities, are still not well understood. Here, we review in a unifying manner several theories that describe the fluctuations (i.e. undulations) of bilayer shapes as well as their local coupling with lipid or protein density variation. The coupling between local membrane curvature and lipid density gives rise to a ‘slipping mode’ in addition to the conventional ‘bending mode’ for damping the membrane fluctuation. This leads to a number of interesting experimental phenomena regarding bilayer shape dynamics. More importantly, curvature-inducing proteins can couple with membrane shape and eventually render the membrane unstable. A criterion for membrane shape instability is derived from a linear stability analysis. The instability criterion reemphasizes the importance of membrane tension in regulating the stability and dynamics of membrane geometry. Recent progresses in understanding the role of membrane tension in regulating dynamical cellular processes are also reviewed. Protein density is emphasized as a key factor in regulating membrane shape transitions: a threshold density of curvature coupling proteins is required for inducing membrane morphology transitions.     

The cooperative role of membrane skeleton and bilayer in the mechanical behaviour of red blood cells

Red blood cell (RBC) shape, behaviour and deformability can be consistently accounted for by a model for the elastic properties of the RBC membrane that includes the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending. The role of the corresponding energy terms in different RBC shape and deformation situations is analyzed. RBC shape transformations are compared to the shape transformations of phospholipid vesicles that are driven by the difference between the equilibrium areas of the bilayer leaflets (DeltaA0). It is deduced that the skeleton energy contributions play a crucial role in the formation of an echinocyte. The effect of a transformation of the natural biconcave RBC shape into an echinocyte on its resistance to entry into capillary-sized cylindrical tubes is analyzed. It is shown that, during the aspiration of an echinocyte into a pipette, there are two competing skeleton deformation effects, which arise due to skeleton density changes, one due to spicule formation and the other due to deformation induced by micropipette aspiration. Furthermore, the shift of the observed dependence of the projection length on the aspiration pressure of more crenated cells towards higher aspiration pressures can be accounted for by an increase of the equilibrium area difference DeltaA0 and consequent modification of the nonlocal contribution to the cell elastic energy.     

Creation of lipid partitions by deposition of amphipathic viral peptides

Phospholipid vesicles exhibit a natural characteristic to fuse and reform into a continuous single bilayer membrane on hydrophilic solid substrates such as glass, mica, and silica. The resulting solid-supported bilayer mimics physiological tendencies such as lipid flip-flop and lateral mobility. The lateral mobility of fluorescently labeled lipids fused into solid-supported bilayers is found to change upon deposition on the membrane surface of an amphipathic alpha-helical peptide (AH) derived from the hepatitis C virus (HCV) NS5A protein. The binding of the AH peptide to a phospholipid bilayer, with the helical axis parallel to the bilayer, leads to immobilization of the bilayer. We used AFM to better understand the mechanistic details of this specific interaction, and determined that the diminished fluidity of the bilayer is due to membrane thinning. Utilizing this specific interaction between AH peptides and lipid molecules, we demonstrate a novel process for the creation of lipid partition by employing AH peptides as agents to immobilize lipid molecules, thus creating a patterned solid support with partition-defined areas of freely mobile lipid bilayers. This architecture could have a wide range of applications in novel sensing, biotechnology, high-throughput screening, and biomimetic strategies.     

Molecular dynamics simulations of the Ca2+-pump: a structural analysis

We report large scale molecular dynamics computer simulations, ～100 ns, of the ion pump Ca(2+)-ATPase immersed in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The structure simulated here, E1, one of the several conformations resolved using X-ray diffraction techniques, hosts two Ca(2+)-ions in the hydrophobic domain. Our results indicate that protonated residues lead to stronger ion-residue interactions, supporting previous conclusions regarding the sensitivity of the Ca(2+) behaviour to the protonated state of the amino acid binding sites. We also investigate how the protein perturbs the bilayer structure. We show that the POPC bilayer is ～12% thinner than the pure bilayer, near the protein surface. This perturbation decays exponentially with the distance from the protein with a characteristic decay length of 0.8 nm. We find that the projected area per lipid also decreases near the protein. Using an analytical model we show that this change in the area is only apparent and it can be explained by considering the local curvature of the membrane. Our results indicate that the real area per lipid near the protein is not significantly modified with respect to the pure bilayer result. Further our results indicate that the local deformation of the membrane around the protein might be compatible with the enhanced protein activity observed in experiments over a narrow range of membrane thicknesses.     

Shape fluctuations of large unilamellar lipid vesicles observed by laser light scattering: influence of the small-scale structure

In the present paper, we apply the dynamic laser light scattering technique to investigate the dependence of the characteristic times of thermally induced shape fluctuation of large unilamellar vesicles (LUVs) on bilayer composition. After addressing single-component LUVs made of two common phospholipids, dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC), we investigate the changes in vesicle shape fluctuation times due to the presence of cholesterol and gangliosides (GM1), added in small amounts. The experimental results show that the addition of a second component, even in small amount, to DMPC vesicles induces a change in membrane fluctuation times. Moreover, in the case of ganglioside addition, also the disposition of GM1 within the bilayer is of importance. Quite unexpectedly, the symmetric or asymmetric disposition of GM1 has opposite effects on bilayer dynamics, the first resulting in a "hardening" and the second in a "softening" of the membrane. Those results support that the small-scale structure of the bilayer is important in determining the overall dynamics of the vesicle. They also suggest that the physiological disposition of GM1 in the outer leaflet of real cells has a significative result in mechanical terms, positively affecting the dynamics of the membrane.     

Aqueous dispersions of cubic lipid–water phases

Inverse lipid–water phases such as cubic phases can form kinetically stable dispersions by fragmentation in water. Cubic lipid phases can be dispersed by polar lipids favoring lamellar phases or by block copolymers, which can close the bilayer at the surface so that the hydrocarbon chain core is not exposed to water. Monodisperse particles based on glycerol monooleate, with their bilayer curved as the P-, D- or G-minimal surface, have been prepared in this way. Their inner bilayer conformation and outer shape have been examined, mainly by X-ray diffraction and cryo transmission electron microscopy. There is also a different type of cubic lipid bilayer particles with a periodicity in the micrometer range, which have been identified in phospholipid–water dispersions and in cell membrane assemblies. The mechanism behind formation in vivo of such cubic membranes, which also follow the P-, D- and G-surfaces, is discussed. Other lipid–water dispersions with lower symmetry are finally considered; dispersions formed by the inverse hexagonal phase and the dispersed state of a tetragonal bilayer structure formed by lung surfactants.     

Dewetting-induced membrane formation by adhesion of amphiphile-laden interfaces

We introduce an approach for forming bilayer polymer membranes by adhesion of amphiphile-laden interfaces. This adhesion is induced by a reduction of solvent quality for the amphiphilic diblock copolymers through selective evaporation of good solvent in the solvent mixture. By combining this membrane formation mechanism with a double-emulsion-templated approach for vesicle formation, we fabricate monodisperse polymersomes that exhibit excellent membrane uniformity, and structural stability, using a method that has high encapsulation efficiency. Moreover, we also show that the technique is versatile and can be applied to different block copolymers. The ability to direct the assembly of amphiphiles into a membrane creates new opportunities to engineer the structures of vesicles on the level of the individual bilayer leaflets.     

Complex pattern formation by adhesion-controlled anisotropic wrinkling

We report complex pattern formation and shape control in the confinement-induced wrinkling that occurs when a poly(dimethylsiloxane) (PDMS) mold is placed on a bilayer of metal and polymer and then heated. Various complex structures that are different from the mold pattern form through the self-organization of wrinkles. These complex structures could be inverted in shape by manipulating the work of adhesion at the interface between the mold and the metal surface. Convex wrinkles result when the work of adhesion is relatively large. However, inverted concave wrinkles emerge when it is relatively small. The ratio of the mold period to the intrinsic wrinkling wavelength is another factor that determines the shape. The ability to tailor the shape of a surface is expected to have a broad range of applications in electro-optics and microfluidics.     

Whole-body rocking motion of a fusion peptide in lipid bilayers from size-dispersed 15N NMR relaxation

Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR (15)N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond protein-membrane interactions.     

Growth of giant membrane lobes mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces

We report on the growth of giant membrane lobes that is mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces and a strategy to control the two-dimensional structure of the membrane lobes on a solid surface. The growth of giant membrane lobes was observed on a single-lipid bilayer which spread from a lump of phospholipid deposited on a silica-glass substrate or an oxidized silicon wafer in aqueous solutions of NaCl, KCl, MgCl2, or CaCl2 at relatively high salt concentrations. Most of the membrane lobes were very flat unilamellar tubes elongating from the lump of phospholipid, and their length reached 1 mm in 5 h. Experimental findings clearly indicate that the membrane lobes are adherent to the surface of the single-lipid bilayer and are mechanically elongated from the lump of phospholipid by the sliding motion of the single-lipid bilayer. We could control the two-dimensional structure of the membrane lobes on the substrate by controlling the spreading direction of the single-lipid bilayer using Pt micropatterns that were deposited on the smooth surface of the oxidized silicon wafer.     

Scanning near-field IR microscopy of proteins in lipid bilayers

We use infrared near-field microscopy to chemically map the morphology of biological matrices. The investigated sample is built up from surface-tethered membrane proteins (cytochrome c oxidase) reconstituted in a lipid bilayer. We have carried out infrared near-field measurements in the frequency range between 1600 and 1800 cm(-1). By simultaneously recording the topography and chemical fingerprint of the protein-tethered lipid bilayer with a lateral resolution of 80 nm × 80 nm, we were able to probe locally the chemical signature of this membrane and to provide a local map of its surface morphology.     

Surface tension in bilayer membranes with fixed projected area

We study the elastic response of bilayer membranes with fixed projected area to both the stretching and shape deformations. A surface tension is associated to each of these deformations. By using model amphiphilic membranes and computer simulations, we are able to observe both the types of deformation, and thus, both the surface tensions, related to each type of deformation, are measured for the same system. These surface tensions are found to assume different values in the same bilayer membrane, in particular, they vanish for different values of the projected area. We introduce a simple theory which relates the two quantities and successfully apply it to the data obtained with computer simulations.     

Vesicle shapes from molecular dynamics simulations

Lipid bilayer membranes are known to form various structures such as large sheets or vesicles. When the two leaflets of the bilayer have an equal composition, the membrane preferentially forms a flat sheet or a spherical vesicle. However, a difference in the composition of the two leaflets may result in a curved bilayer or in a wide variety of vesicle shapes. Vesicles with different shapes have already been shown in experiments and diverse vesicle shapes have been predicted theoretically from energy minimization of continuous curves. Here we present a molecular dynamics study of the effect of small changes in the phospholipid headgroups on the spontaneous curvature of the bilayer and on the resulting vesicle shape transformations. Small asymmetries in the bilayers already result in high spontaneous curvature and large vesicle deformations. Vesicle shapes that are formed include ellipsoids, discoids, pear-shaped vesicles, cup-shaped vesicles, as well as budded vesicles. Comparison of these vesicles with theoretically derived vesicle shapes shows both resemblances and differences.     

Solvent-free model for self-assembling fluid bilayer membranes: stabilization of the fluid phase based on broad attractive tail potentials

We present a simple and highly adaptable method for simulating coarse-grained lipid membranes without explicit solvent. Lipids are represented by one head bead and two tail beads, with the interaction between tails being of key importance in stabilizing the fluid phase. Two such tail-tail potentials were tested, with the important feature in both cases being a variable range of attraction. We examined phase diagrams of this range versus temperature for both functional forms of the tail-tail attraction and found that a certain threshold attractive width was required to stabilize the fluid phase. Within the fluid-phase region we find that material properties such as area per lipid, orientational order, diffusion constant, interleaflet flip-flop rate, and bilayer stiffness all depend strongly and monotonically on the attractive width. For three particular values of the potential width we investigate the transition between gel and fluid phases via heating or cooling and find that this transition is discontinuous with considerable hysteresis. We also investigated the stretching of a bilayer to eventually form a pore and found excellent agreement with recent analytic theory.     

Probing organization and communication at layered interfaces

We have investigated the local organization intrinsic to a variety of interfacial structures, by both electrochemical and spectroscopic means. Our focus has been on the design and construction of biomimetic interfaces, where a lipid bilayer or a hybrid bilayer membrane can be bound to an interface. The goal of this work is ultimately to create an interface on a transducer surface that can support an enzyme in its active form. To this point, we have examined the extent of organization that is achievable in monolayers that will be used to bind bilayer structures to a transducer surface. Our electrochemical data point to the important role of the substrate surface in determining adlayer organization. We have also investigated the fluidity and structural heterogeneity of lipid bilayers using time-resolved and steady state fluorescence spectroscopy. Our data point to the highly interactive nature of lipid bilayer constituents, where perturbations introduced to one region have significant consequences on other regions of the bilayer. Such information is directly relevant to the existence and properties of lipid raft structures in both model and biological bilayers.     

Pore nucleation in mechanically stretched bilayer membranes

We report a computer-simulation study of the free-energy barrier for the nucleation of pores in the bilayer membrane under constant stretching lateral pressure. We find that incipient pores are hydrophobic but as the lateral size of the pore nucleus becomes comparable with the molecular length, the pore becomes hydrophilic. In agreement with previous investigations, we find that the dynamical process of growth and closure of hydrophilic pores is controlled by the competition between the surface tension of the membrane and the line tension associated with the rim of the pore. We estimate the line tension of a hydrophilic pore from the shape of the computed free-energy barriers. The line tension thus computed is in a good agreement with available experimental data. We also estimate the line tension of hydrophobic pores at both macroscopic and microscopic levels. The comparison of line tensions at these two different levels indicates that the "microscopic" line tension should be carefully distinguished from the "macroscopic" effective line tension used in the theoretical analysis of pore nucleation. The overall shape of the free-energy barrier for pore nucleation shows no indication for the existence of a metastable intermediate during pore nucleation.     

Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization

The dynamics and state of lipid bilayer-internal hydration water of unilamellar lipid vesicles dispersed in solutions is characterized. This study was enabled by a recently developed technique based on Overhauser dynamic nuclear polarization (DNP)-driven amplification of (1)H nuclear magnetic resonance (NMR) signal of hydration water. This technique can, in the full presence of bulk water, selectively quantify the translational dynamics of hydration water within ～10 ? around spin labels that are specifically introduced to the local volume of interest within the lipid bilayer. With this approach, the local apparent diffusion coefficients of internal water at different depths of the lipid bilayer were determined. The modulation of these values as a response to external stimuli, such as the addition of sodium chloride or ethanol and the lipid phase transitions, that alter the fluctuations of bilayer interfaces together with the activation energy values of water diffusivity shows that water is not individually and homogeneously solvating lipid's hydrocarbon tails in the lipid bilayer. We provide experimental evidence that instead, water and the lipid membrane comprise a heterogeneous system whose constituents include transient hydrophobic water pores or water structures traversing the lipid bilayer. We show how these transient pore structures, as key vehicles for passive water transport can better reconcile our experimental data with existing literature data on lipid bilayer hydration and dynamics.     

Aligning nanodiscs at the air-water interface, a neutron reflectivity study

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 ? with a surface coverage of 66 ± 4%. This layer is located about 15 ? below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 ?) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.     

Nanoscale patterning of solid-supported membranes by integrated diffusion barriers

Ultraflat nanostructured substrates have been used as a template to create patterned solid-supported bilayer membranes with polymerizable tethered lipids acting as diffusion barriers. Patterns in the size range of 100 nm were successfully produced and characterized. The diffusion barriers were embedded directly into the phospholipid bilayer and could be used to control the fluidity of the membrane as well as to construct isolated membrane corrals. By using nanosphere lithography to structure the templates it was possible to systematically adjust the lipid diffusion coefficients in a range comparable to those observed in cellular membranes. Single colloids applied as mask in the patterning process yielded substrates for creation of isolated fluid membrane patches corralled by diffusion barriers. Numerous potential applications for this new model system can be envisioned, ranging from the study of cellular interactions or of molecular diffusion in confined geometries to biosensor arrays.