Stability Of Lipid Bilayers Research Articles

Nanodiscs as a therapeutic delivery agent: inhibition of respiratory syncytial virus infection in the lung

There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG). Preparations of nanodiscs containing POPG (nPOPG) antagonized interleukin-8 production from Beas2B epithelial cells challenged by RSV infection, with an IC50 of 19.3 μg/mL. In quantitative in vitro plaque assays, nPOPG reduced RSV infection by 93%. In vivo, nPOPG suppressed inflammatory cell infiltration into the lung, as well as IFN-γ production in response to RSV challenge. nPOPG also completely suppressed the histopathological changes in lung tissue elicited by RSV and reduced the amount of virus recovered from lung tissue by 96%. The turnover rate of nPOPG was estimated to have a halftime of 60–120 minutes (m), based upon quantification of the recovery of the human Apo A-I constituent. From these data, we conclude that nPOPG is a potent antagonist of RSV infection and its inflammatory sequelae both in vitro and in vivo.

Volumetric Stability of Lipid Bilayers

The gel phase of DPPC has been the best characterized lipid bilayer. It has therefore been alarming that recent publications have reported a gradual decrease in lipid molecular volume of DPPC multilamellar vesicle dispersions in the gel phase upon repeated thermal cycling between 10°C and 50°C using a commercial densimeter. The considerable size of this decrease would have significant implications for the physical chemistry of biomembranes. We have confirmed this phenomenon with the same densimeter model. By contrast, neutral buoyancy measurements performed with similar thermal cycling show no gradual change in lipid volume in the gel phase at 20°C. Remixing the lipid in the densimeter shows that the apparent volume decrease is an artifact. We conclude that volumes obtained by neutral buoyancy measurements remain accurate and that gel phase DPPC bilayers exist in a volumetrically stable state.This research is accepted for publication in Physical Chemistry Chemical Physics. It has been supported by the U. S. National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM044976 to JFN and STN. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Stable Lipid Bilayers Based on Micro- and Nano-Fabrication as a Platform for Recording Ion-Channel Activities

In this review, we will discuss our recent approaches for the formation of mechanically stable bilayer lipid membranes (BLMs) by combining with BLM formation and micro- and nano-fabrication techniques. BLMs were prepared across a microaperture fabricated in silicon (Si) chips or nanoporous alumina films using a minimized amount of organic solvent. Although BLMs spanned over the porous alumina film showed better electrical properties, such as background current noise and current transient, BLMs suspended in a thin Si(3)N(4) septum showed a much superior BLM stability. The BLMs showed tolerance to a high voltage of ±1 V, a membrane lifetime of >40 h, and tolerance to repetitive solution exchanges. Application to a drug screening system has been examined by using the human ether-a-go-go-related gene (hERG) potassium channel as an illustrative example. The potentiality of the present system as a platform of the high-throughput analysis for ion-channel protein is also discussed.

Polymer-based microfluidic device for measuring membrane protein activities

Functional assays of membrane proteins are becoming increasingly important, both in research and drug discovery applications. The majority of current assays use the patch-clamp technology to measure the activity of ion channels which are over-expressed in cells. In future, in vitro assay systems will be available, which use reconstituted membrane proteins in free-standing lipid bilayers suspended in nano- or micrometer-sized pores. Such functional assays require (1) expression, purification and reconstitution of the membrane protein of interest, (2) a reliable method for lipid bilayer formation and membrane protein integration, and (3) a sensitive detection system. For practical applications, especially for automation, the reliable and controllable transport of fluids is essential. In order to achieve a stable free-standing lipid bilayer, a pore diameter in the micro- to nanometer range is essential. Novel microfluidic devices were developed by bonding a thick (300 μm) polyether ether ketone foil, bearing a channel structure, to a thin (12 μm) foil with a micropore of about 10 μm diameter and then utilized for the formation of stable, free-standing lipid bilayers within the pore. A bacterial voltage-gated potassium channel is integrated therein by fusion and the ion channel activity detected by voltage clamp.

Effects of the Size, Shape, and Structural Transition of Thermosensitive Polypeptides on the Stability of Lipid Bilayers and Liposomes

We performed all-atom and coarse-grained (CG) molecular dynamics (MD) simulations of lipid bilayers grafted with elastin-like polypeptides (ELPs; [VPGVG]n). All-atom simulations of a single ELP in water show that ELPs become more collapsed and folded as the temperature increases from 293 up to 353 K, in agreement with experiments. All-atom simulations of lipid bilayers composed of dipalmitoylglycerophosphocholine (DPPC), cholesterol, and fatty acids grafted with ELPs show that ELPs insert into the bilayer and significantly disorder lipids, to an extent that depends on the ELP length over the temperature range 293–323 K. In the bilayer, ELPs are mainly, but not entirely, random coil in character at temperatures between 293 and 315 K and, in contrast to the behavior in water, become increasing random coil and extended in length over the range 315–323 K, over which the bilayer is in the disordered liquid phase. The insertion of ELPs into the lipid-tail region is mediated by the interaction of hydrophobic Pro...

Biomimetic Membrane System Composed of a Composite Interpenetrating Hydrogel Film and a Lipid Bilayer

AbstractA simple way to form an interpenetrating hydrogel (IPH) by combining a layer‐by‐layer polyelectrolyte membrane with agarose is reported. The formed IPH membrane is more robust and easily manipulated compared to a polyelectrolyte membrane with the same number of layers. The IPH has good diffusion characteristics and retains the advantageous surface charge from the polyectrolyte composition that facilitates the adsorption of a stable lipid bilayer. The stable adsorption of a lipid bilayer on the IPH creates a biomimetic membrane system that is optimized for utilization in a diffusion chamber.

Formation of Schiff Bases of O-Phosphorylethanolamine and O-Phospho-d,l-serine with Pyridoxal 5′-Phosphate. Experimental and Theoretical Studies

Pyridoxal 5'-phosphate (PLP) is a B(6) vitamer acting as an enzyme cofactor in various reactions of aminoacid metabolism and inhibiting glycation of biomolecules. Nonenzymatic glycation of aminophospholipids alters the stability of lipid bilayers and cell function as a result. Similarly to protein glycation, aminophospholipid glycation initially involves the formation of a Schiff base. In this work, we studied the formation of Schiff bases between PLP and two compounds mimicking the polar head of natural aminophospholipids, namely: O-phosphorylethanolamine and O-phospho-D,L-serine. Based on the results, the pH-dependence of the microscopic constants of the two PLP-aminophosphate systems studied is identical with that for PLP-aminoacid systems. However, the rate and equilibrium formation constants for the Schiff bases of the aminophosphates are low relative to those for the aminoacids. A theoretical study by density functional theory of the formation mechanism for the Schiff bases of PLP with the two aminophospholipid analogues confirmed that the activation energy of formation of the Schiff bases is greater with aminophosphates; on the other hand, that of hydrolysis is essentially similar with aminoacids and aminophosphates.

Specific DNA Duplex Formation at an Artificial Lipid Bilayer: towards a New DNA Biosensor Technology

A novel technique is described which comprises a base-specific DNA duplex formation at a lipid bilayer-H(2) O-phase boundary layer. Two different probes of oligonucleotides both carrying a double-tailed lipid at the 5'-terminus were incorporated into stable artificial lipid bilayers separating two compartments (cis/trans-channel) of an optically transparent microfluidic sample carrier with perfusion capabilities. Both the cis- and trans-channels are filled with saline buffer. Injection of a cyanine-5-labeled target DNA sequence, which is complementary to only one of the oligonucleotide probes, into the cis-channel, followed by a thorough perfusion, leads to an immobilization of the labeled complementary oligonucleotide on the membrane as detected by single-molecule fluorescence spectroscopy and microscopy. In the case of fluorescent but non-complementary DNA sequences, no immobilized fluorescent oligonucleotide duplex could be detected on the membrane. This clearly verifies a specific duplex formation at the membrane interface.

Mechanically Stable Lipid Bilayers in Teflon-Coated Silicon Chips for Single-Channel Recordings

We demonstrate the microfabrication of a low-noise silicon (Si) chip as a platform for suspending mechanically stable bilayer lipid membranes (BLMs). Microapertures with smoothly tapered edges were formed by isotropic etching in a silicon nitride layer deposited on a Si substrate. The surface of the Si chip was coated with insulator layers of Teflon and SiO2. The insulator coating worked to reduce the total capacitance, leading to noise reduction (1-2 pA in peak-to-peak after low-pass filtering at 1 kHz) and elimination of current transients ( < 0.5 ms). Since the tapered edges were necessary to maintain mechanically stable BLMs, the entire chip except for the aperture was coated with the insulators. Owing to this process, the BLMs formed in the Si chips still showed high mechanical stability after coating with the insulator layers. The membranes withstood high applied voltage (±1 V) and mechanical shocks during solution exchanges. The mechanically stable BLMs having electric properties suitable for recording activities of biological channels will open up a variety of applications including high-throughput analysis of ion-channel proteins. Keywords: Planar bilayer lipid membranes, single-channel recording, microfabrication, Teflon fabrication., low-noise silicon, BLMs, Fabrication, Insulator Coatings

Ion Channel Reconstitution

Since the discovery of the possible solubilization of membrane proteins and their isolation from other membrane constituents (purification), different methods were developed to reconstitute ion channel proteins into artificial lipid bilayers. These membrane proteins were then fully functional when correctly oriented and inserted in a lipid bilayer. The reconstitution plays a central role in identifying and characterizing the mechanisms of action of membrane proteins. The activity of the membrane proteins is studied using electrophysiology using different methods e.g., the black lipid membrane. Therefore, the structure-function relationship can be investigated to better understand the biophysical properties of membrane proteins in vivo.Using a glass surface containing a micrometer hole, the fusion of vesicles on the surface becomes an attractive method for electrophysiology and then to reconstitute membrane proteins into the lipid bilayer without denaturation. Then stable lipid bilayers are formed by bursting a GUV on the glass surface, forming a free-standing portion above the hole.With this technique, we studied the biophysical and pharmacological properties of different ion channels, for example potassium channels (KcsA, Kv1.2), sodium channels (NachBac, NaVsp1) as well as other ligand-dependent (IP3 receptor, NMDA receptor), mechanosensitive channels (MscL, TRP channels) and non-specific channels (Cx43, VDAC). I will describe here our methods for incorporation of proteins into the bilayer and the recording of single ion channel current measurements by using a planar patch clamp platform.

Volumetric stability of lipid bilayers

In agreement with recent reports, a commercial densimeter has yielded a gradual decrease in lipid molecular volume of DPPC multilamellar vesicle dispersions in the gel phase upon repeated thermal cycling between 10 °C and 50 °C. The considerable size of this decrease would have significant implications for the physical chemistry of biomembranes. In contrast, neutral buoyancy measurements performed with similar thermal cycling indicate no gradual change in lipid volume in the gel phase at 20 °C. Remixing the lipid in the densimeter shows that the apparent volume decrease is an artifact. We conclude that gel phase DPPC bilayers exist in a volumetrically stable phase.

Simple one-pot fabrication of ultra-stable core-shell superparamagnetic nanoparticles for potential application in drug delivery

Ultrastable superparamagnetic core-shell nanoparticles of average diameter 80 nm have been fabricated via a simple one-pot method involving superparamagnetic iron oxide nanoparticles (SPIONs) core (∼50 nm in diameter) and lipid bilayer shell by high energy ultrasonication. The surface charges (zeta potentials) were measured to be between −15 mV and + 16 mV depending on the batch composition. Anticancer drug mitomycin C (MMC) was loaded into four different samples of variable surface charges in aqueous solution (pH = 6.8) and released in PBS buffer (pH = 7.2) at room temperature. The kinetics of drug loading and releasing data indicated that the stable lipid bilayer coated SPIONs (LBCSPIONs) of nearly neutral surface exhibited the highest loading (10.9 μg of MMC/mg of materials), whereas uncoated or partially coated SPIONs of positive zeta potential exhibited the lowest loading (2.8 and 3.5 μg MMC/mg of materials, respectively). The release behavior of MMC was observed to be highest (5.8 μg MMC/mg of materials) from materials of negative zeta potential compared to materials of near neutral surfaces (3.68 μg MMC/mg of materials). The plausible mechanism of MMC loading and releasing behavior has been explained based on the electrostatic interaction and diffusion through the lipid bilayers. To ensure biocompatibility, the interaction of the prepared SPIONs with human cervical cancer cell line (HeLa) was also investigated using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ROS (reactive oxygen species) production assay and the results confirmed the super-compatibility of LBCSPIONs.

Natural and artificial ion channels for biosensing platforms

The single-molecule selectivity and specificity of the binding process together with the expected intrinsic gain factor obtained when utilizing flow through a channel have attracted the attention of analytical chemists for two decades. Sensitive and selective ion channel biosensors for high-throughput screening are having an increasing impact on modern medical care, drug screening, environmental monitoring, food safety, and biowarefare control. Even virus antigens can be detected by ion channel biosensors. The study of ion channels and other transmembrane proteins is expected to lead to the development of new medications and therapies for a wide range of illnesses. From the first attempts to use membrane proteins as the receptive part of a sensor, ion channels have been engineered as chemical sensors. Several other types of peptidic or nonpeptidic channels have been investigated. Various gating mechanisms have been implemented in their pores. Three technical problems had to be solved to achieve practical biosensors based on ion channels: the fabrication of stable lipid bilayer membranes, the incorporation of a receptor into such a structure, and the marriage of the modified membrane to a transducer. The current status of these three areas of research, together with typical applications of ion-channel biosensors, are discussed in this review.

Understanding non-enzymatic aminophospholipid glycation and its inhibition. Polar head features affect the kinetics of Schiff base formation

Non-enzymatic aminophospholipid glycation is an especially important process because it alters the stability of lipid bilayers and interferes with cell function and integrity as a result. However, the kinetic mechanism behind this process has scarcely been studied. As in protein glycation, the process has been suggested to involve the formation of a Schiff base as the initial, rate-determining step. In this work, we conducted a comparative kinetic study of Schiff base formation under physiological conditions in three low-molecular weight analogues of polar heads in the naturally occurring aminophospholipids O-phosphorylethanolamine (PEA), O-phospho- dl-serine (PSer) and 2-aminoethylphenethylphosphate (APP) with various glycating carbonyl compounds (glucose, arabinose and acetol) and the lipid glycation inhibitor pyridoxal 5′-phosphate (PLP). Based on the results, the presence of a phosphate group and a carboxyl group in α position respect to the amino group decrease the formation constant for the Schiff base relative to amino acids. On the other hand, esterifying the phosphate group with a non-polar substituent in APP increases the stability of its Schiff base. The observed kinetic formation constants of aminophosphates with carbonyl groups were smaller than those for PLP. Our results constitute an important contribution to understanding the competitive inhibition effect of PLP on aminophospholipid glycation.

Fluorescence Microscopy of the Pressure-Dependent Structure of Lipid Bilayers Suspended across Conical Nanopores

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.

Analysis of ion Channel Activities in Lipid Bilayers Suspended Over Microwells on SI Substrates

One of the key techniques to fabricate a nanobio device that works with functioning membrane proteins is the creation of stable lipid bilayers, guaranteeing protein activity and the detection of their functions. In order to develop the measurement system of ion channel activities, here, suspended lipid bilayers were formed over the microwells fabricated on the Si substrate. Giant unilamellar vesicles (GUVs) with and without gramicidin A (gA) were ruptured over the microwells and the pH indicator pyranine was confined in the microwells by a lipid bilayer. Activity of gA ion channels contained in the suspended lipid bilayer was analyzed by the change of fluorescent intensity of pyranine. Increasing the pH outside of the microwells by exchanging solution led to an increase in the fluorescent emission of pyranine confined in the microwells. Sealing of the micro-wells by the lipid bilayer was good enough such that no leakage of pyranine was observed during functional analysis. A clear difference in the rate of fluorescent intensity changes is seen between lipid bilayers with and without gA ion channels, indicating that transport of protons through gA ion channels was observed. Proton leak through the lipid membrane was estimated to be 10 x 10−6 ∼ 10 x 10−5 cm/sec. This successful creation of stable lipid bilayers on Si chips with peptides as biological signal transducers is an important step towards the development of large and versatile sensor arrays which allow parallel single channel recordings.

Nanoscale carbon particles and the stability of lipid bilayers

The transfer of various nano-scale fullerenes into lipid bilayers has been studied using all-atom (AA) and coarse-grained (CG) molecular dynamics (MD) simulations. The free energy change, when C60, C180, and C540 fullerenes are transferred from water to the interior of a lipid [dioleoylphosphatidylcholine (DOPC)] bilayer, has been calculated. Upon entering the lipid bilayer, the largest (2.4 nm diameter) fullerene causes local distortions in the bilayer surface, which were previously observed in carbon nanotube simulations. These local distortions, however, do not lead to any free energy barriers to bilayer entry. The free energy profiles confirm spontaneous absorption of all three fullerenes. Qualitative agreement was observed when comparing fullerene partitioning in water/bilayer systems to water–hexane systems. In contrast to these nonspecific single fullerene properties, extensive CG-MD simulations of fullerene rich lipid bilayers reveal substantial impact of fullerene-size on the bilayer stability. While previous CG-MD simulations indicated that bilayer bound C60 aggregates have little effect on the bilayer structure, the present MD simulations indicate that C540 aggregation has drastic effects. Specifically, the observed destabilization likely has implications for understanding the cytotoxic mechanisms of nano-carbon particles upon uptake by biological cells.

Development and Application of Rapid Fabrication of Teflon Micropores as a Platform of Stable Lipid Bilayer Formation

Lipid bilayers formed on micropores possess excellent properties for electrical measurements given the low membrane capacitance and high voltage stability. However, conventional methods employed in the fabrication of micropores on semiconducting materials require large-scale equipment with many fabrication steps. To address this problem, we have developed a novel method that generates Teflon micropores in a few seconds by imposing a heated tip onto commercial Polyethylene, Polyethylene terephthalate, Polypropylene and Teflon films. These advantages promise to improve the performance of artificial lipid bilayers when employed in the development of flexible biosensors and a combination system with atomic force microscope (AFM). Thus we succeeded in activating a single channel protein by mechanically stimulating it with an AFM probe, indicating our novel technologies have the potential to be a very powerful tool for the analyses of the activation-deactivation dynamics in channel proteins.

Simple and Stable Lipid Bilayer Formation: A Droplets Contacting Method using Parylene Micro-pores for Multiple Ion Channel Recordings

This paper describes multiple ion-channel recordings through membrane proteins reconstituted in bilayer lipid membranes (BLMs) array. The BLMs array can be prepared by “Droplets Contacting Method” which forms BLMs at the interface of two lipid monolayers. Since this method does not require skilled techniques, it is highly reproducible and can be applied to automated system. We used a double well chip (DWC) for the droplets contacting method. We attempted to confine the BLMs forming areas with parylene micro-pore (parylene double well chip, PDWC) to augment the mechanical stability of BLMs. Subsequently, we arrayed the PDWC with electrodes for multiple recordings of channel proteins. We successfully demonstrated 14 channels simultaneous ion channel recordings through α-hemolysin.

A New Method for Measuring Edge Tensions and Stability of Lipid Bilayers: Effect of Membrane Composition

We report a novel and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (eggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of DOPC with cholesterol and dioleoylphosphatidylethanolamine. Our data confirm previous results for eggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of dioleoylphosphatidylethanolamine is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. It is presumed that interlipid hydrogen bonding is the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on bilayers made of stearoyloleoylphosphatidylcholine, suggesting that cholesterol influences the membrane mechanical stability in a lipid-specific manner.