Stability Of Lipid Bilayers Research Articles

Amphiphilic Nanoparticles Control the Growth and Stability of Lipid Bilayers with Open Edges.

Molecular amphiphiles self-assemble in polar media to form ordered structures such as micelles and vesicles essential to a broad range of industrial and biological processes. Some of these architectures such as bilayer sheets, helical ribbons, and hollow tubules are potentially useful but inherently unstable owing to the presence of open edges that expose the hydrophobic bilayer core. Here, we describe a strategy to stabilize open bilayer structures using amphiphilic nanoparticle surfactants that present mixtures of hydrophilic and hydrophobic ligands on their surface. We observe that these particles bind selectively to the open edge of bilayer membranes to stabilize otherwise transient amphiphile assemblies. We show how such particles can precisely control the size of lipid tubules, how they can inhibit the formation of undesirable assemblies such as gallstone precursors, and how they can stabilize free-floating lipid microdiscs.

Amphiphilic Nanoparticles Control the Growth and Stability of Lipid Bilayers with Open Edges

AbstractMolecular amphiphiles self‐assemble in polar media to form ordered structures such as micelles and vesicles essential to a broad range of industrial and biological processes. Some of these architectures such as bilayer sheets, helical ribbons, and hollow tubules are potentially useful but inherently unstable owing to the presence of open edges that expose the hydrophobic bilayer core. Here, we describe a strategy to stabilize open bilayer structures using amphiphilic nanoparticle surfactants that present mixtures of hydrophilic and hydrophobic ligands on their surface. We observe that these particles bind selectively to the open edge of bilayer membranes to stabilize otherwise transient amphiphile assemblies. We show how such particles can precisely control the size of lipid tubules, how they can inhibit the formation of undesirable assemblies such as gallstone precursors, and how they can stabilize free‐floating lipid microdiscs.

Single lipid bilayer deposition on polymer surfaces using bicelles.

A lipid bilayer was deposited on a 3 μm diameter polystyrene (PS) bead via hydrophobic anchoring of bicelles containing oxyamine-bearing cholesteric moieties reacting with the aldehyde functionalized bead surface. Discoidal bicelles were formed by mixing dimyristoylphosphatidylcholine (DMPC), dihexanoylphosphatidylcholine (DHPC), dimyristoyltrimethylammonium propane (DMTAP), and the oxyamine-terminated cholesterol derivative, cholest-5-en-3β-oxy-oct-3,6-oxa-an-8-oxyamine (CHOLOA), in the molar ratio DMPC/DHCP/DMTAP/CHOLOA (1/0.5/0.01/0.05) in water. Upon exposure to aldehyde-bearing PS beads, a stable single lipid bilayer coating rapidly formed at the bead surface. Fluorescence recovery after photobleaching demonstrated that the deposited lipids fused into an encapsulating lipid bilayer. Electrospray ionization mass spectrometry showed that the short chain lipid DHPC was entirely absent from the PS adherent lipid coating. Fluorescence quenching measurements proved that the coating was a single lipid bilayer. The bicelle coating method is thus simple and robust, can be modified to include membrane-associated species, and can be adapted to coat any number of different surfaces.

Arrayed Lipid Membranes on Femtoliter Chambers Allow Highly Sensitive Detection of Ion Translocation Catalyzed by Transporter Protein

Femtoliter reaction chamber array enables highly sensitive and quantitative biological assays such as single-molecule enzymatic assay, and digital PCR. Although femtoliter chamber arrays are very powerful for protein science as well as for biomedical applications, most of them have been in general limited to use for water-soluble proteins, due to the technical difficulties in preparing uniform and stable lipid bilayers. Here, we report an arrayed lipid bilayer chamber system (ALBiC) that displays a sub-million of femtoliter chambers, each equipped with micron-size electrodes, and sealed with a stable lipid bilayer membrane with extremely high efficiency of over 90 %. When reconstituted an ion transporter, FoF1-ATP synthase (FoF1), into the bilayers of ALBiC, proton translocation driven by catalysis or membrane voltage were observed, showing that the highly sensitive detection of ion translocation is performed in ALBiC. Next, to explore the feasibility of single-molecule detection of transporter activity, we conducted the same assay in a condition where only a few molecules (0, 1, or 2 molecules) of FoF1 were reconstituted into each bilayer. The results showed that the response to proton translocation was no longer homogeneous between chambers, i.e. stochastic and quantized proton translocation was observed, demonstrating that the single molecule analysis of ion translocation catalyzed by transporter protein is first achieved in this study. Thus, the new platform, ALBiC, largely extended the versatility of femtoliter chamber arrays, and holds promise for understanding the working mechanism of transporter proteins as well as for further analytical and pharmacological applications.

ドロップレット型人工細胞膜によるチャネルタンパク質計測

This review describes recent our studies on artificial cell membranes (lipid bilayers) and their applications using microfabrication technologies. The stable and reproducible lipid bilayer formation can be achieved by “droplet contact method” which is readily created the lipid bilayer by contacting two lipid monolayers at the interface of the droplets. This method will be a powerful tool for the high-throughput ion channel measurements and biomimetic sensors.

Aqueous-filled polymer microcavity arrays: versatile & stable lipid bilayer platforms offering high lateral mobility to incorporated membrane proteins.

A key prerequisite in an ideal supported lipid bilayer based cell membrane model is that the mobility of both the lipid matrix and its components are unhindered by the underlying support. This is not trivial and with the exception of liposomes, many of even the most advanced approaches, although accomplishing lipid mobility, fail to achieve complete mobility of incorporated membrane proteins. This is addressed in a novel platform comprising lipid bilayers assembled over buffer-filled, arrays of spherical cap microcavities formed from microsphere template polydimethoxysilane. Prior to bilayer assembly the PDMS is rendered hydrophilic by plasma treatment and the lipid bilayer prepared using Langmuir Blodgett assembly followed by liposome/proteoliposome fusion. Fluorescence Lifetime Correlation Spectroscopy confirmed the pore suspended lipid bilayer exhibits diffusion coefficients comparable to free-standing vesicles in solution. The bilayer modified arrays are highly reproducible and stable over days. As the bilayers are suspended over deep aqueous reservoirs, reconstituted membrane proteins experience an aqueous interface at both membrane interfaces and attain full lateral mobility. Their utility as membrane protein platforms was exemplified in two case studies with proteins of different dimensions in their extracellular and cytoplasmic domains reconstituted into DOPC lipid bilayers; Glycophorin A, and Integrin αIIbβ3. In both cases, the proteins exhibited 100% mobility with high lateral diffusion coefficients.

How Does Ethanol Affect the Stability of Simple Model Yeast Membranes?

Biofuels such as biomass-based ethanol (EtOH) have become increasingly popular as a source of renewable energy, but the higher cost of production and lower energy content make the fuel difficult to compete with petroleum gasoline. Cellulosic biomass can be used to produce bioethanol, but process efficiency is limited by ethanol's cytotoxic nature. One proposed mechanism for EtOH inhibition of yield growth suggests that ethanol diffuses through cell membranes, decreasing the lipid bilayer stability. Bilayers comprising various molar concentrations of 1-palmitoyl-2-oleoylphosphatidlyserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids were simulated using molecular dynamics. Pure POPS, pure POPE, pure POPC, and mixed 30/70 POPS/POPE lipid bilayers were constructed using the CHARMM36 lipid force field. Each lipid bilayer was exposed to molar concentrations of 0.5%, 1%, 2%, 3%, and 4% ethanol EtOH with additional molar concentrations of 5% and 10% EtOH for pure bilayers only. Average bilayer surface area, NMR deuterium order parameters (SCD), and electron density profiles (EDP) were analyzed to interpret bilayer stability. Each analysis indicated decreased membrane stability with increased EtOH concentration, supporting the proposed mechanism. Trends between different lipid bilayer compositions were all similar. Surface area increases resulted from more frequent EtOH bilayer penetrations into the hydrophobic core, causing bilateral expansion of the membrane's walls. SCD values decreased as more EtOH penetrated the bilayer due to the disruption of lipid packing, increasing membrane fluidity and decreasing stabilizing interactions between hydrophobic lipid tails. As overall EtOH concentration increased, EDP plots indicated increased EtOH concentration within the bilayer, past the hydrophilic head region. EDP plots also indicated decreased bilayer thickness. Increased concentration of EtOH within the bilayer is in accordance with SCD results and decreased bilayer thickness supports increased bilayer surface area results.

Specific DNA duplex formation at an artificial lipid bilayer: fluorescence microscopy after Sybr Green I staining.

The article describes the immobilization of different probe oligonucleotides (4, 7, 10) carrying each a racemic mixture of 2,3-bis(hexadecyloxy)propan-1-ol (1a) at the 5’-terminus on a stable artificial lipid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The bilayer separates two compartments (cis/trans channel) of an optical transparent microfluidic sample carrier with perfusion capabilities. Injection of unlabeled target DNA sequences (6, 8, or 9), differing in sequence and length, leads in the case of complementarity to the formation of stable DNA duplexes at the bilayer surface. This could be verified by Sybr Green I double strand staining, followed by incubation periods and thorough perfusions, and was visualized by single molecule fluorescence spectroscopy and microscopy. The different bilayer-immobilized complexes consisting of various DNA duplexes and the fluorescent dye were studied with respect to the kinetics of their formation as well as to their stability against perfusion.

Archaeosomes can efficiently deliver different types of cargo into epithelial cells grown in vitro

Archaeosomes are a type of liposomes prepared from the polar lipids of various Archaeobacteria. These have unique structural features that increase the lipid bilayer's stability even under high temperatures, low or high pH, presence of phospholipases and bile salts. This makes them ideal as basis for the development of new drug, gene and vaccine delivery systems. In this study we prepared large unilamellar archaeosomes (400nm size) from Aeropyrum pernix K1 and demonstrated their potential as base for the development of an efficient and universal system for drug or therapy delivery to epithelial cells. Our archaeosomes may be used to deliver small fluorescent molecules (calcein), smaller proteins (60kDa listeriolysin), large protein aggregates (e.g. keratin 14) and plasmid DNA, into epithelial cells grown in culture. The delivery efficiency for small molecules is already quite high at this initial stage of development, around 40%. Our unilamellar archaeosomes are also not toxic to keratinocytes even at high doses (500μg/ml).

A portable lipid bilayer system for environmental sensing with a transmembrane protein.

This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin) was achieved in the field at a high-altitude (∼3623 m). This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.

Pseudo painting/air bubble technique for planar lipid bilayers.

A functional reconstitution of channel proteins in planar lipid bilayers is still very versatile to study structure/function correlates under well-defined conditions at the single protein level. In this study we present an improved planar lipid bilayer technique in which an air bubble is used for stabilizing unstable/leaky bilayers or for removing excess lipids. The bubble can also be used as a tool for reducing the number of channels in the bilayer with the goal of having only one active channel in the membrane. Stable planar lipid bilayers are formed within seconds to minutes. In the case of multiple channel insertion the air bubble can be used to reduce the number of channels within minutes. The simple improvement of the classical folding technique guarantees a very fast creation of stable bilayers even with difficult phospholipids in a conventional vertical bilayer set-up; it requires no modifications of the existing set-up. This technique is very easy to handle and guarantees successful single channel recordings for any kind of planar lipid bilayer experiment.

Water channel formation and ion transport in linear and branched lipid bilayers

Using molecular dynamics simulations, we studied the influence of methyl chain branching on transmembrane potential induced formation of water channels in lipid bilayers and ion transport. We compared the response of a bilayer lipid that has multiple methyl branches diphytanoylphosphatidylcholine (DPhPC) with its straight-chain counterpart dipalmitoylphosphatidylcholine (DPPC) to a transmembrane potential created by an imbalance in ionic charges across the membrane. We found that, compared to the straight-chain DPPC lipid bilayer membranes, branched DPhPC lipid membranes require a higher critical transmembrane potential to break down, followed by water channel formation, and transport of anions and cations through the pore. We demonstrated that the bulkiness of the added methyl branches leads to "barrel-stave" pores in DPhPC membranes which require a higher transmembrane potential to produce than the toroidal pores produced in the straight chain DPPC lipid bilayers. Our results provided a deeper understanding of the water channel formation and ion transport through lipid bilayer membrane and might help explain the increased resistance to charge-induced poration in organisms with membranes abundant in branched lipids.

Dynamics and stability of lipid bilayers modulated by thermosensitive polypeptides, cholesterols, and PEGylated lipids

Lipid bilayers, which consist of dipalmitoylglycerophosphocholines (DPPCs), PEGylated lipids, cholesterols, and elastin-like polypeptides (ELPs; [VPGVG]3) at different molar ratios, were simulated. Simulations were carried out for 2 μs using the coarse-grained (CG) model that had captured the experimentally observed phase behavior of PEGylated lipids and lateral diffusivity of DPPC bilayers. Starting with the initial position of ELPs on the bilayer surface, ELPs insert into the hydrophobic region of the bilayer because of their interaction with lipid tails, consistent with previous all-atom simulations. Lateral diffusion coefficients of DPPCs significantly increase in the bilayer composed of more ELPs and less cholesterols, showing their opposite effects on the bilayer dynamics. In particular, ELPs modulate the dynamics and phase for the disordered liquid bilayer, but not for the ordered gel bilayer, indicating that ELPs can destabilize only the disordered bilayer. In the ordered bilayer, ELP chains tend to have a spherical shape and slowly diffuse, while they are extended and diffuse faster in the disordered bilayer, indicating the effect of the bilayer phase on the conformation and diffusivity of ELPs. These findings explain the experimental observation that the ELP-conjugated liposomes are stable at 310 K (ordered phase) but become unstable and release the encapsulated drugs at 315 K (disordered phase), which suggests the effects of ELPs and cholesterols. Since the cholesterol-stabilized bilayer can be destabilized by the extended shaped ELPs only in the disordered phase (not in the ordered phase), the inclusion of cholesterols is required to safely shield drugs at 310 K as well as allow ELPs to disrupt lipids and destabilize the liposomes at 315 K.

The use of virtual ground to control transmembrane voltages and measure bilayer currents in serial arrays of droplet interface bilayers

The droplet interface bilayer (DIB) is a simple technique for constructing a stable lipid bilayer at the interface of two lipid-encased water droplets submerged in oil. Networks of DIBs formed by connecting more than two droplets constitute a new form of modular biomolecular smart material, where the transduction properties of a single lipid bilayer can affect the actions performed at other interface bilayers in the network via diffusion through the aqueous environments of shared droplet connections. The passive electrical properties of a lipid bilayer and the arrangement of droplets that determine the paths for transport in the network require specific electrical control to stimulate and interrogate each bilayer. Here, we explore the use of virtual ground for electrodes inserted into specific droplets in the network and employ a multichannel patch clamp amplifier to characterize bilayer formation and ion-channel activity in a serial DIB array. Analysis of serial connections of DIBs is discussed to understand how assigning electrode connections to the measurement device can be used to measure activity across all lipid membranes within a network. Serial arrays of DIBs are assembled using the regulated attachment method within a multi-compartment flexible substrate, and wire-type electrodes inserted into each droplet compartment of the substrate enable the application of voltage and measurement of current in each droplet in the array.

Automated Parallel Recordings of Topologically Identified Single Ion Channels

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

Microarrays of Phospholipid Bilayers Generated by Inkjet Printing

We report an efficient and reproducible method to generate a microarray of model biological membranes on a solid substrate by applying the inkjet printing technology. Although inkjet printing is currently widely used for industrial fabrication processes, including biological materials, printing lipid membranes remains technically challenging due to the hydrophobic nature of droplets and instability of the lipid bilayer structure against dehydration. In the present study, we printed lipids onto a glass substrate covered with a micropatterned membrane of a polymeric phospholipid bilayer. Polymeric bilayers were formed by the lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC). After removal of nonpolymerized DiynePC with a detergent solution, natural lipid membranes were incorporated into the polymer-free regions (corrals) by using an electric-field-based inkjet printing device that can eject subfemtoliter volume droplets. To avoid rapid dehydration and destabilization, we preprinted an aqueous solution containing agarose and trehalose onto the corrals and subsequently printed lipid suspensions ("two-step-printing method"). After rinsing, stable lipid bilayer membranes were formed in the corrals. The bilayers were continuous and fluid as confirmed by fluorescence recovery after photobleaching. We could introduce multiple bilayer patches having different lipid compositions into the neighboring corrals. The present results demonstrate that the combination of a patterned polymeric bilayer and inkjet printing technology enables efficient, reliable, and scalable generation of the model membrane microarrays having varied compositions.

Cord factor (trehalose 6,6′-dimycolate) forms fully stable and non-permeable lipid bilayers required for a functional outer membrane

Cord factor (trehalose 6,6′-dimycolate, TDM) is the major lipid in the outer membrane of Corynebacteria and Mycobacteria. Although its role is well recognized in the immune response phenomena, its membrane biophysical properties remained largely unexplored and TDM has often been described as a detergent. We purified the main components of the outer membrane from Corynebacterium glutamicum and analyzed their membrane forming properties. In mixture with endogenous cardiolipin, but not alone, the spontaneous hydration of TDM produces liposomes. As a pure component, TDM formed vesicles only by the detergent dialysis method. Perdeuterated cardiolipin–TDM mixtures were shown by deuterium nuclear magnetic resonance (NMR) to exhibit a gel to liquid crystalline phase transition over a 273–295K temperature range, for cells grown at 303K, and thus to be in a liquid crystalline state at physiological temperature. Molecular dynamics simulations of hydrated TDM bilayers provided the trehalose average orientation and conformation, the chain order parameters, the area per lipid and the bilayer thickness which was confirmed by electron microscopy. Finally the Porin A–Porin H ion channel from the Corynebacterial outer membrane was reconstituted in TDM liposomes. With properly mycoloylated proteins, it manifested the typical voltage dependent ion channel properties of an outer membrane porin.

A Photodependent Switch of Liposome Stability and Permeability

Liposomes offer a method to encapsulate high concentrations of a drug, protecting the therapeutic upon in vivo administration. With an appropriate mechanism to manipulate lipid bilayer permeability, liposomes have the potential to deliver encapsulated drugs in a spatially and temporally controlled manner. In this investigation, the photosensitizer aluminum phthalocyanine disulfonic acid (AlPcS(2)) is identified as a modulator of the colloidal properties of liposomes. AlPcS(2) adsorption to liposomes stabilizes lipid bilayers and reduces permeability. Spectroscopic data suggests that AlPcS(2) interacts with the phospholipid to increase lipid bilayer stability. In the presence of AlPcS(2), the liposome permeability was five times lower than that without the photosensitizer. This results in more stable liposome systems that contain higher doses of the encapsulated material for longer. Then, upon irradiation of the AlPcS(2)-liposome system with tissue penetrating red light, lipid bilayer permeability increases 10-fold over the baseline. The release is shown to be a singlet oxygen mediated process, due to the type II photodynamic action of AlPcS(2). It is concluded that this activity provides a novel photorelease mechanism for liposome mediated drug delivery.

Effect of saccharides on the stability of lipid bilayer in dry state

Effect of saccharides on the stability of lipid bilayer in dry state

Is the fluid mosaic (and the accompanying raft hypothesis) a suitable model to describe fundamental features of biological membranes? What may be missing?

The structure, dynamics, and stability of lipid bilayers are controlled by thermodynamic forces, leading to overall tensionless membranes with a distinct lateral organization and a conspicuous lateral pressure profile. Bilayers are also subject to built-in curvature-stress instabilities that may be released locally or globally in terms of morphological changes leading to the formation of non-lamellar and curved structures. A key controller of the bilayer’s propensity to form curved structures is the average molecular shape of the different lipid molecules. Via the curvature stress, molecular shape mediates a coupling to membrane-protein function and provides a set of physical mechanisms for formation of lipid domains and laterally differentiated regions in the plane of the membrane. Unfortunately, these relevant physical features of membranes are often ignored in the most popular models for biological membranes. Results from a number of experimental and theoretical studies emphasize the significance of these fundamental physical properties and call for a refinement of the fluid mosaic model (and the accompanying raft hypothesis).