Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Microcontact printing for creation of patterned lipid bilayers on tetraethylene glycol self-assembled monolayers

Supported lipid bilayers (SLBs) formed on many different substrates have been widely used in the study of lipid bilayers. However, most SLBs suffer from inhomogeneities due to interactions between the lipid bilayer and the substrate. In order to avoid this problem, we have used microcontact printing to create patterned SLBs on top of ethylene-glycol-terminated self-assembled monolayers (SAMs). Glycol-terminated SAMs have previously been shown to resist absorbance of biomolecules including lipid vesicles. In our system, patterned lipid bilayer regions are separated by lipid monolayers, which form over the patterned hexadecanethiol portions of the surface. Furthermore, we demonstrate that α-hemolysin, a large transmembrane protein, inserts preferentially into the lipid bilayer regions of the substrate.     

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle–vesicle and vesicle–substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.     

Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Formation of arenicin-1 microdomains in bilayers and their specific lipid interaction revealed by Z-scan FCS

Z-scan fluorescence correlation spectroscopy (FCS) is employed to characterize the interaction between arenicin-1 and supported lipid bilayers (SLBs) of different compositions. Lipid analogue C8-BODIPY 500/510C5-HPC and ATTO 465 labelled arenicin-1 are used to detect changes in lipid and peptide diffusion upon addition of unlabelled arenicin-1 to SLBs. Arenicin-1 decreases lipid mobility in negatively charged SLBs. According to diffusion law analysis, microdomains of significantly lower lipid mobility are formed. The analysis of peptide FCS data confirms the presence of microdomains for anionic SLBs. No indications of microdomain formation are detected in SLBs composed purely of zwitterionic lipids. Additionally, our FCS results imply that arenicin-1 exists in the form of oligomers and/or aggregates when interacting with membranes of both compositions.     

Poly(dimethylsiloxane)-coated sensor devices for the formation of supported lipid bilayers and the subsequent study of membrane interactions

The development of smooth hydrophilic surfaces that act as substrates for supported lipid bilayers (SLBs) is important for membrane studies in biology and biotechnology. In this article, it is shown that thin films of poly(dimethylsiloxane) (PDMS) formed on a sensor surface can be used as a substrate for the deposition of reproducible and homogeneous zwitterionic SLBs by the direct fusion of vesicles. Poly(dimethylsiloxane) solution (1% w/v) was spin coated on Love acoustic wave and surface plasmon resonance devices to form a thin PDMS layer. Acoustic, fluorescence, and contact angle measurements were used for the optimization of the PDMS film properties as a function of plasma etching time; parameters of interest involve the thickness and hydrophilicity of the film and the ability to induce the formation of homogeneous SLBs without adsorbed vesicles. The application of PDMS-coated sensor devices to the study membrane of interactions was demonstrated during the acoustic and fluorescence detection of the binding of melittin and defensin Crp4 peptides to model supported lipid bilayers.     

Interaction of Polystyrene Nanoparticles with Supported Lipid Bilayers: Impact of Nanoparticle Size and Protein Corona

Polystyrene is one of the most widely used plastics. This article reports on the interaction of 50 and 210 nm polystyrene nanoparticles (PSNPs) with human serum albumin (HSA) and transferrin (Tf), as well as their effect on supported lipid bilayers (SLBs), using experimental and theoretical approaches. Dynamic light scattering (DLS) and atomic force microscopy (AFM) measurements show that the increase in diameter for the PSNP-protein bioconjugates depends on nanoparticle size and type of proteins. The circular dichroism (CD) spectroscopy results demonstrate that the proteins preserve their structures when they interact with PSNPs at physiological temperatures. The quartz crystal microbalance (QCM) technique reveals that PSNPs and their bioconjugates show no strong interactions with SLBs. On the contrary, the molecular dynamics simulations (MDS) show that both proteins bind strongly to the lipid bilayer (SLBs) when compared to their binding to a polystyrene surface model. The interaction is strongly dependent on the protein and lipid bilayer composition. Both the PSNPs and their bioconjugates show no toxicity in human umbilical vein endothelial (HUVEC) cells; however, bare 210 nm PSNPs and 50 nm PSNP-Tf bioconjugates show an increase in reactive oxygen species production. This study may be relevant for assessing the impact of plastics on health.     

Role of lipid charge in organization of water/lipid bilayer interface: insights via computer simulations

Anionic unsaturated lipid bilayers represent suitable model systems that mimic real cell membranes: they are fluid and possess a negative surface charge. Understanding of detailed molecular organization of water-lipid interfaces in such systems may provide an important insight into the mechanisms of proteins' binding to membranes. Molecular dynamics (MD) of full-atom hydrated lipid bilayers is one of the most powerful tools to address this problem in silico. Unfortunately, wide application of computational methods for such systems is limited by serious technical problems. They are mainly related to correct treatment of long-range electrostatic effects. In this study a physically reliable model of an anionic unsaturated bilayer of 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) was elaborated and subjected to long-term MD simulations. Electrostatic interactions were treated with two different algorithms: spherical cutoff function and particle-mesh Ewald summation (PME). To understand the role of lipid charge in the system behavior, similar calculations were also carried out for zwitterionic bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). It was shown that, for the charged DOPS bilayer, the PME protocol performs much better than the cutoff scheme. In the last case a number of artifacts in the structural organization of the bilayer were observed. All of them were attributed to inadequate treatment of electrostatic interactions of lipid headgroups with counterions. Electrostatic properties, along with structural and dynamic parameters, of both lipid bilayers were investigated. Comparative analysis of the MD data reveals that the water-lipid interface of the DOPC bilayer is looser than that for DOPS. This makes possible deeper penetration of water molecules inside the zwitterionic (DOPC) bilayer, where they strongly interact with carbonyls of lipids. This can lead to thickening of the membrane interface in zwitterionic as compared to negatively charged bilayers.     

Formation and diffusivity characterization of supported lipid bilayers with complex lipid compositions

The moving edge of a hydrodynamically manipulated supported lipid bilayer (SLB) can be used to catalyze SLB formation of adsorbed lipid vesicles that do not undergo spontaneous SLB formation upon adsorption on SiO(2). By removing the lipid reservoir of an initially formed SLB, we show how a hydrodynamically moved SLB patch composed of POPC can be used to form isolated SLBs with compositions that to at least 95% represent that of the adsorbed lipid vesicles. The concept is used to investigate the diffusivity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DHPE) in SLBs made from complex lipid compositions, revealing a decrease in diffusivity by a factor of 2 when the cholesterol content was increased from 0% to 50%. We also demonstrate how the concept can be used to induce stationary domains in SLBs containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol (39:21:40 mol %, respectively). Because the method serves as a means to form SLBs with lipid compositions that hamper SLB formation via spontaneous rupture of adsorbed lipid vesicles, it opens up the possibility for new biophysical investigations of SLBs with more nativelike compositions.     

Detection of supported lipid bilayers using their electric charge

We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.     

Supported lipid bilayer microarrays created by non-contact printing

Arrays of supported lipid bilayers (SLBs) provide great potential for future drug development and multiplexed biological research, but are difficult to prepare due to the sensitivity of both the lipid and protein structural arrangement to air exposure. A novel way to produce arrays of SLBs is presented based on non-contact dispensing of vesicles to a substrate through a thin surface confined water film. The approach presents many degrees of freedom since it is not limited to a specific substrate, lipid composition, linker or controlled environment. The method allows adjustment of spot size (180-360 μm) by repeated dispensing as well as control over the composition of the spots and subsequent analytes. SLB formation by vesicle adsorption and rupture allows for incorporation of membrane proteins through pre-formed proteoliposomes. Dispensing through a dip-and-rinse water film avoids contamination, disruptive drying and the need for complex buffer compositions. Furthermore, no humidity control is necessary which simplifies the production step and prolongs the life-time of the spotting system. We characterize the method with respect to control over spot size, bilayer mobility and the formation process as well as demonstrate the possibility to fuse bilayer spots with subsequently added vesicles. Since complex lipid compositions and multiple spotting nozzles can be used, this novel technique is expected to be a promising platform for future applications, e.g. patterning to monitor peptide/protein-lipid interactions, for glycomics using glycolipids or lipopolysaccharides, and to study mixing of spatially confined lipid membranes.     

Anomalous diffusion in supported lipid bilayers induced by oxide surface nanostructures

Hierarchic structure and anomalous diffusion on submicrometer scale were introduced into an artificial cell membrane, and the spatiotemporal dependence of lipid diffusion was visualized on nanostructured oxide surfaces. We observed the lipid diffusion in supported lipid bilayers (SLBs) on step-and-terrace TiO(2)(100) and amorphous SiO(2)/Si surfaces by single molecule tracking (SMT) method. The SMT at the time resolution of 500 μs to 30 ms achieved observation of the lipid diffusion over the spatial and temporal ranges of 100 nm/millisecond to 1 μm/second. The temporal dependence of the diffusion coefficient in the SLB on TiO(2)(100) showed that the crossover from anomalous diffusion to random diffusion occurred around 10 ms. The surface fine architecture on substrates will be applicable to induce hierarchic structures on the order of 100 nm or less, which correspond to the microcompartment size in vivo.     

二氧化硅纳米粒子薄膜的制备及光学性能

以二氧化硅胶体和聚二烯丙基二甲基氯化铵(PDDA)为原料,利用静电自组装技术制备了PDDA/SiO2复合薄膜. TEM图象显示,薄膜中的SiO2纳米粒子为密堆积,薄膜均匀、致密;电子衍射实验结果显示,所组装的薄膜为非晶态膜.载玻片表面组装SiO2纳米粒子薄膜后,透射率随薄膜双层数增加呈现周期变化.薄膜具有增透作用,载玻片双面组装薄膜后在一定波长范围内的透射率可提高5％以上. PDDA/SiO2复合薄膜的光学性质主要由SiO2纳米粒子决定,每一双层的平均物理厚度小于SiO2纳米粒子的粒径,薄膜中存在层间穿插现象,逐层组装的复合薄膜具有单层光学薄膜的特性.     

Supported lipid bilayer composition microarray fabricated by pattern-guided self-spreading

We report on the fabrication of a microarray of supported lipid bilayers (SLBs) with different chemical compositions and demonstrate its biosensing application. The technique utilizes the phenomenon of lipid self-spreading on a patterned surface, which offers complete positional selectivity for a supported lipid bilayer. We describe the fabrication of parallel 10-μm-wide lines, each filled with an SLB with a unique composition, at 5 μm intervals. Structures obtained with our new technique are finer and more highly integrated than previously reported structures that employ the vesicle fusion technique on patterned surfaces. We also detected specific binding between biotin and streptavidin with high contrast, indicating that the microarray is valuable for biosensing applications.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: detection by antibiotic membrane probes.

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylcholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmetrically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Double cushions preserve transmembrane protein mobility in supported bilayer systems

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)-conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 x 10(-8) cm2/s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 x 10(-8) cm2/s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.     

Supported lipid bilayers lifted from the substrate by layer-by-layer polyion cushions on self-assembled monolayers

The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11-mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1-palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2–2.1)×10⁻⁹ cm²/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes.     

Substrate effects on interactions of lipid bilayer assemblies with bound nanoparticles

Understanding the interactions of nanoparticles with lipid membranes is crucial in establishing the mechanisms that govern assembly of membrane-based nanocomposites, nanotoxicology, and biomimetic inspired self-assembly. In this study, we explore binding of charged nanoparticles to lipid bilayers, both as liposomes and substrate supported assemblies. We find that the presence of a solid-support, regardless of curvature, eliminates the ability of zwitterionic fluid phase lipids to bind charged nanoparticles.     

A simulation study on nanoscale holes generated by gold nanoparticles on negative lipid bilayers

Understanding the interactions of gold nanoparticles (AuNPs) with cellular compartments, especially cell membranes, is of fundamental importance in obtaining their control in biomedical applications. An effort is made in this paper to investigate the interactions of 2.2 nm core AuNPs with negative model bilayer membranes by coarse-grained (CG) molecular dynamics (MD) simulation. The CG model of lipid bilayer was taken from Marrink et al. ( J. Phys. Chem. B 2004, 108, 750-760 ), whereas the CG AuNPs model was developed on the basis of both atomistic MD simulations and experimental data. It was found that AuNPs functionalized with cationic ligands penetrated into the negative bilayer membranes and generated significant disruptions on bilayers. The lipids surrounding the nanoparticle were highly disordered and the bulk surface of the bilayer exhibits some defective areas. Most importantly, it is observed that a nanoscale hole can be formed and expanded spontaneously on the peripheral regions of the 20 × 20 nm bilayer. The expansion of the hole is on the time scale of hundreds of nanosceonds. The fully expanded hole had a radius of ～5.5 nm and could transport water molecules at a rate of up to ～1100 molecule/ns. However holes could not be formed on a larger bilayer (28 × 28 nm). The factors that can eliminate hole formation on the bilayer also include the decrease of cationic lignads on the AuNP, the reduction of negative lipids in the bilayer, the release of bilayer surface tension, the lowering of temperature, and the addition of a high concentration of salt. The results suggest that a hole can only be formed on living cell membranes under extreme conditions.     

High pressure effect on phase transition behavior of lipid bilayers

Phase behavior of lipid bilayers at high pressure is critical to biological processes. Using coarse grained molecular dynamic simulations, we report critical characteristics of dipalmitoylphosphatidylcholine bilayers with applied high pressure, and also show their phase transition by cooling bilayer patches. Our results indicate that the phase transition temperature of dipalmitoylphosphatidylcholine bilayers obviously shifts with pressure increasing in the rate of 37 °C kbar(-1), which are in agreement with experimental data. Moreover, the main phase transition is revealed to be strongly dependent on lipid area. A critical lipid area of ~0.57 nm(2) is found on the main phase transition boundary. Similar structures of acyl chains lead to the same sensitivity of phase transition temperature of different lipids to the pressure. Based on the lateral density and pressure profiles, we also discuss the different effects on bilayer structure induced by high temperature and high pressure, e.g., increasing temperature induces higher degree of interdigitation of lipid tails and thinner bilayers, and increasing pressure maintains the degree of interdigitation and bilayer thickness.