Bilayer Formation Research Articles

Spatiotemporal Kinetics of Supported Lipid Bilayer Formation on Glass via Vesicle Adsorption and Rupture

Supported lipid bilayers (SLBs) represent one of the most popular mimics of the cell membrane. Herein, we have used total internal reflection fluorescence microscopy for in-depth characterization of the vesicle-mediated SLB formation mechanism on a common silica-rich substrate, borosilicate glass. Fluorescently labeling a subset of vesicles allowed us to monitor the adsorption of individual labeled vesicles, resolve the onset of SLB formation from small seeds of SLB patches, and track their growth via SLB-edge-induced autocatalytic rupture of adsorbed vesicles. This made it possible to perform the first quantitative measurement of the SLB front velocity, which is shown to increase up to 1 order of magnitude with time. This effect can be classified as dramatic because in many other physical, chemical, or biological kinetic processes the front velocity is either constant or decreasing with time. The observation was successfully described with a theoretical model and Monte Carlo simulations implying rapid local diffusion of lipids upon vesicle rupture.

Automatic Planar Asymmetric Lipid Bilayer Membrane Formation toward Biological High-Throughput Assay.

This paper describes automation of planar lipid bilayer formation by introducing a stepping motor to a microfluidic device. Planar lipid bilayers or lipid vesicles are useful to understand biological reactions and to investigate the interaction between lipids and proteins. Therefore, to acquire large amount of the information, high-throughput production of planar lipid bilayers or giant vesicles (GVs) is necessary. The droplet split-and-contact method, which enhances the efficiencies of both planar lipid bilayer formation and GV generation, needs to be automated for increasing the throughput. Previous droplet split-and-contact devices were manipulated manually; hence, the influence of manipulation on planar lipid bilayer formation was not evaluated quantitatively. First, to develop an automated system for generating asymmetric planar lipid bilayers, a stepping motor, which allows to control the angular speed of the rotor, is integrated into the droplet split- and-contact device (Fig. $1(\mathrm{b)$). Next, we assessed planar lipid bilayer generation at various angular speeds and found the speed limit for bilayer formation. Finally, we generated asymmetric planar lipid bilayers that have different lipid composition on outer and inner leaflets using this automated device and confirmed the asymmetry of the planar lipid bilayers by generating GVs.

Impact of the protein myristoylation on the structure of a model cell membrane in a protein bound state

The neuronal calcium sensor protein recoverin is expressed in retinal rod and cone cells and is involved in the calcium-dependent control of receptor (rhodopsin) phosphorylation and receptor inactivation. In its Ca2+-saturated form recoverin is attached to membranes by an exposed myristoyl group and responds to oscillating changes of intracellular Ca2+-concentration by performing a so-called Ca2+-myristoyl switch. In this work we analyze changes in a liquid lipid bilayer interacting with myristoylated and non-myristoylated recoverin by employing polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) with electrochemical control. The lipid bilayer is transferred onto a polycrystalline gold electrode using Langmuir-Blodgett Langmuir-Schaefer transfer at the surface pressure π = 30 mN m−1 which ensures, necessary for the lipid-protein interaction, liquid state of the hydrocarbon chains of phospholipids. The model lipid bilayers are adsorbed directly on the Au electrode surface at transmembrane potentials −0.2 < ∆ϕM|S < 0.25 V. The interaction with recoverin leads to a stabilization of the adsorbed state of the lipid bilayer at positive transmembrane potentials. The interaction leads to a decrease in the surface charge density that accumulates on the membrane covered electrode surface, indicating changes in the lateral interactions in the lipid membrane. In situ spectroelectrochemical studies confirm orientation changes in the hydrophobic environment of the model membrane. Insertion of the myristoyl group of recoverin into the membrane is connected with an increase in the tilt of the hydrocarbon chains with respect to the surface normal and decrease in the bilayer thickness. Potential-induced pore formation and desorption of the lipid bilayer from the membrane surface is accompanied by the removal of the acyl chains of recoverin from the membrane.

Reversibly Thermochromic Cyclic Dipeptide Nanotubes.

Owing to their capability of forming extensive hydrogen bondings and the facile introduction of chirality, cyclic dipeptides (CDPs) have gained great attention as scaffolds for functional supramolecules. Surprisingly, introduction of a photopolymerizable diacetylene (DA) moiety to the CDP afforded nanotubular structures with enhanced stability and reversible thermochromism. A series of CDP-containing DAs (CDP-DAs) are prepared by coupling 10,12-pentacosadiynoic acid with CDPs, cyclo(-Gly-Ser) and cis/trans cyclo(-Ser-Ser). Fabrication of CDP-DA self-assemblies in a polar chloroform and methanol solvent mixture affords nanotubes comprising single-wall and multiwall structures. The self-assembly behavior and morphology characteristic are examined by scanning electron microscopy and transmission electron microscopy. Next, X-ray diffraction analysis confirms well-ordered lamellar structures with a perfect agreement with the bilayer formation leading to the tubular structure via lamellar scrolling behavior. Upon UV irradiation, monomeric CDP-DA tubular assemblies result in the blue-colored CDP/polydiacetylene (PDA) nanotubes. Interestingly, CDP/PDA nanotubes exhibit a reversible blue-to-red color change for over 10 consecutive thermal cycles. The CDP-DA/PDA supramolecular system demonstrates potential applications in developing stimulus-responsive functional materials.

Unique Interfacial Phenomena on Macroscopic and Colloidal Scales Induced by Two-Dimensional Phase Transitions.

This feature article addresses a variety of unique macroscopic-scale and colloidal-scale interfacial phenomena, such as wetting transitions of oil droplets into molecularly thin films, spontaneous merging and splitting of oil droplets at air-water interfaces, solid monolayer and bilayer formation in mixed cationic surfactant/alkane adsorbed films, switching of foam-film thickness, and oil-in-water emulsion stability. All of these phenomena can be observed using commercial cationic surfactants, liquid alkanes, and water.

Chromium nanostructure formation on the GaAs(1 1 1)–(2 × 2) surface: First principles studies

Structural, electronic and magnetic properties of the chromium (Cr) nanostructure formation on GaAs(1 1 1)–(2 × 2) surfaces are investigated. Studies are performed by first principles total energy calculations within the periodic spin polarized density functional theory plus Hubbard correction (GGA + U) with 1 < U < 5 eV, as developed in the PWscf code of the quantum ESPRESSO package. The chromium adsorption and incorporation into the GaAs(1 1 1)-(2 × 2) atomic structure are investigated on both gallium and arsenic terminated surfaces. Different coverage of Cr is considered from ¼ monolayer (ML) up to a full ML to determine the relaxed configurations and the most favorable structure. High symmetry sites are considered to explore the Cr adsorption and incorporation into the GaAs structure, these are: H3, T4, Top and Bridge. Surface atomic structures contain different number of atoms therefore to determine the most stable atomic geometry the surface formation energies are constructed. On the As terminated surface, under As rich conditions the CrAs bilayer formation is the most favorable structure. On the Ga terminated surface, under Ga rich conditions the Cr trimer formation is the most favorable configuration. Electronic properties of the most stable structures are investigated by calculating the total density of states (DOS) and projected DOS. Results indicate that the most favorable nanostructures exhibit metallic behavior with antiferromagnetic characteristics.

Formation and properties of phospholipid bilayers on fluorine doped tin oxide electrodes

Commercially available fluorine doped tin oxide (FTO) films on glass were functionalized by the octadecyltrichlorosilane (OTS) self-assembled monolayers (SAM), which upon exposure to a multilamellar phospholipid vesicle solutions trigger vesicle fusion and formation of a tethered phospholipid bilayer membrane (tBLM). The formation of tBLMs was monitored in real-time by the Fast Fourier Transform (FFT) electrochemical impedance spectroscopy (EIS), which attested for the completion of the fusion process within 2000 s. We established that highly insulating complete bilayers can be accomplished only in some specific cases of OTS functionalized FTO films. In particular, only OTS functionalized FTO films exhibiting double semicircular features on the Cole – Cole EIS plots produced tBLMs with the specific capacitance below 1 μF cm−2 comparable to capacitances of the free-standing phospholipid bilayers. The double-semicircular features of EIS spectra can be used as a diagnostic tool for predicting the suitability of silanized FTO substrates for the complete vesicles fusion leading to a formation of the tBLMs. The average value of the capacitance of DOPC bilayers containing 40% (mol) cholesterol was of 0.82 ± 0.10 μF cm−2 consistent with the 3.02 nm thick dielectric film with the relative dielectric constant of 2.8. In contrast to the gold substrates, tBLMs on OTS functionalized FTO films can be regenerated at least 3 times by washing off lipid layer and re-assembling tBLM via the vesicle fusion process. EIS provided direct evidence of the susceptibility of the tBLMs to phospholipase A2, which triggered degradation of the films. However, transmembrane reconstitution of the pore-forming toxin vaginolysin (VLY) proteins was not detected. Surface constructs described in this study may be utilized in peripheral protein studies or in applications in which planar phospholipid layers supported on a semiconductor metal oxide surfaces are required.

Nanoparticle Wettability Influences Nanoparticle-Phospholipid Interactions.

We explored the influence of nanoparticle (NP) surface charge and hydrophobicity on NP-biomolecule interactions by measuring the composition of adsorbed phospholipids on four NPs, namely, positively charged CeO2 and ZnO and negatively charged BaSO4 and silica-coated CeO2, after exposure to bronchoalveolar lavage fluid (BALf) obtained from rats, and to a mixture of neutral dipalmitoyl phosphatidylcholine (DPPC) and negatively charged dipalmitoyl phosphatidic acid (DPPA). The resulting NP-lipid interactions were examined by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). Our data show that the amount of adsorbed lipids on NPs after incubation in BALf and the DPPC/DPPA mixture was higher in CeO2 than in the other NPs, qualitatively consistent with their relative hydrophobicity. The relative concentrations of specific adsorbed phospholipids on NP surfaces were different from their relative concentrations in the BALf. Sphingomyelin was not detected in the extracted lipids from the NPs despite its >20% concentration in the BALf. AFM showed that the more hydrophobic CeO2 NPs tended to be located inside lipid vesicles, whereas less hydrophobic BaSO4 NPs appeared to be outside. In addition, cryo-TEM analysis showed that CeO2 NPs were associated with the formation of multilamellar lipid bilayers, whereas BaSO4 NPs with unilamellar lipid bilayers. These data suggest that the NP surface hydrophobicity predominantly controls the amounts and types of lipids adsorbed, as well as the nature of their interaction with phospholipids.

Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers.

Detergent-solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar "black lipid membranes" formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step-by-step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single-channel currents. By using preformed GUVs it is possible to omit the membrane solvent. Compared to traditional preparations, the lipid bilayers formed from GUVs provide an environment that more closely resembles the native cell membrane. Also described is an alternate protocol that entails the production of planar lipid bilayers from GUVs onto which proteins in detergent are added. © 2018 by John Wiley & Sons, Inc.

Obtaining antimicrobial bilayer starch and polyester-blend films with carvacrol

Bilayer films using polyester blends (P) and starch (S) were obtained and characterized, incorporating carvacrol as active compound. Carvacrol was incorporated by spraying it between melt blended and compression moulded sheets or through its incorporation into the chloroform P solution used to obtain P cast films. Different PLA-PHBV ratios (75:25 and 65:35) were tested, with and without 15 wt% of PEG1000, whereas the 75:25 ratio with PEG was only used for cast sheets, based on its better overall properties. Mono and bilayers were characterized as to their tensile and water vapour barrier properties and thermal behaviour. Release kinetics of carvacrol in different food simulants and in in vitro antibacterial activity against Listeria innocua and Escherichia coli were also analysed. Incorporating carvacrol by spraying it between the polyester and starch sheets was not effective at retaining the compound in the bilayers. However, the incorporation of carvacrol into cast P films, and the subsequent formation of bilayers with the S sheets, was highly effective at providing practically total carvacrol retention. These active bilayers exhibited highly improved tensile and water vapour barrier capacity with respect to the S monolayer (87% reduction in WVP, 840% increase in elastic modulus) and inhibited the growth of L. innocua and E. coli from both P or S contact sides of bilayers, depending on the internal diffusion of carvacrol through the bilayer and its adequate release of the compound into the culture medium.

High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis.

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

Potent Macromolecule-Sized Poration of Lipid Bilayers by the Macrolittins, A Synthetically Evolved Family of Pore-Forming Peptides.

Pore-forming peptides with novel functions have potential utility in many biotechnological applications. However, the sequence-structure-function relationships of pore forming peptides are not understood well enough to empower rational design. Therefore, in this work, we used synthetic molecular evolution to identify a novel family of peptides that are highly potent and cause macromolecular poration in synthetic lipid vesicles at low peptide concentration and at neutral pH. These unique 26-residue peptides, which we call macrolittins, release macromolecules from lipid bilayer vesicles made from zwitterionic PC lipids at peptide to lipid ratios as low as 1:1000, a property that is almost unprecedented among known membrane permeabilizing peptides. The macrolittins exist as membrane-spanning α-helices. They cause dramatic bilayer thinning and form large pores in planar supported bilayers. The high potency of these peptides is likely due to their ability to stabilize bilayer edges by a process that requires specific electrostatic interactions between peptides.

Fabrication of Radiopaque Poly(vinyl alcohol) Hydrogel Bilayers for Use as Embolization Coils

On purpose to replace the metallic embolization coils with a polymeric material, a series of poly(vinyl alcohol) (PVA) hydrogels was fabricated into the form of bilayers. A hydrogel layer was prepared by mixing aqueous PVA solution with tantalum particles for radiopacity, crosslinking the mixture with glutaraldehyde crosslinking agent, and casting to a film of desired thickness. The properties of the hydrogel films including expansion ratio and modulus were characterized. Two hydrogel films with different crosslinking density were attached to form a bilayer, which formed coils in the water with the diameter dictated by the difference in swelling ratio. The coils and bundles of coils with diameters between 1 to 6 mm were employed in the occlusion tests using glass models approximating brain aneurysms. The number and sequence of coil insertion to give high packing density were investigated. Packing density much higher than that of platinum coils was achieved with the polymer coils fabricated.

Bilayer Formation vs Molecular Exchange in Organic Heterostructures: Strong Impact of Subtle Changes in Molecular Structure

Organic heterostructures are a central part of a manifold of (opto)electronic devices and serve a variety of functions. Particularly, molecular monolayers on metal electrodes are of paramount importance for device performance as they allow tuning energy levels in a versatile way. However, this can be hampered by molecular exchange, i.e., by interlayer diffusion of molecules toward the metal surface. We show that the organic–metal interaction strength is the decisive factor for the arrangement in bilayers, which is the most fundamental version of organic–organic heterostructures. The subtle differences in molecular structure of 6,13-pentacenequinone (P2O) and 5,7,12,14-pentacenetetrone (P4O) lead to antithetic adsorption behavior on Ag(111): physisorption of P2O but chemisorption of P4O. This allows providing general indicators for organic–metal coupling based on shifts in photoelectron spectroscopy data and to show that the coupling strength of copper-phthalocyanine (CuPc) with Ag(111) is in between that ...

Stacking patterns and carrier mobilities of GeS bilayer

Based on the first-principles calculations, we identify four stacking patterns of the GeS bilayer, in which two most stable ones are almost equally stable. The most stable one corresponds to the experimental pattern in bulk GeS. Its interlayer binding is stronger than those in a-phosphorene and graphene, indicating that the material will rather exist in the form of bilayers or multilayers. Our HSE06 band structure calculations show that both patterns are semiconductors with indirect band gaps in the visible region, which are slightly smaller than that of the monolayer. For the monolayer, our refined calculation based on the deformation potential approximation indicates that the electron mobility along the armchair direction amounts to 4.62×10 4 cm 2 V -1 s -1 , which is ~40 times larger than that of the a-phosphorene. The electron mobility of the bilayer is dependent on the stacking pattern. The most stable pattern is expected to exhibit the mobility of 1.69×10 4 cm 2 V -1 s -1 , which is still ~30 times larger than that of the bilayer a-phosphorene. A detailed comparison of the carrier mobilities suggests that both of the mono- and bi-layer will be useful for n-type electronics.

Evaporation-induced monolayer compression improves droplet interface bilayer formation using unsaturated lipids.

In this article, we report on a new experimental methodology to enable reliable formation of droplet interface bilayer (DIB) model membranes with two types of unsaturated lipids that have proven difficult for creating stable DIBs. Through the implementation of a simple evaporation technique to condition the spontaneously assembled lipid monolayer around each droplet, we increased the success rates of DIB formation for two distinct unsaturated lipids, namely 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), from less than 10% to near 100%. Separately, using a pendant drop tensiometer, we learned that: (a) DOPC and POPC monolayers do not spontaneously assemble into their tightest possible configurations at an oil-water interface, and (b) reducing the surface area of a water droplet coated with a partially packed monolayer leads to a more tightly packed monolayer with an interfacial tension lower than that achieved by spontaneous assembly alone. We also estimated from Langmuir compression isotherms obtained for both lipids that the brief droplet evaporation procedure prior to DIB formation resulted in a 6%-16% reduction in area per lipid for DOPC and POPC, respectively. Finally, the increased success rates of formation for DOPC and POPC DIBs enabled quantitative characterization of unsaturated lipid membrane properties including electrical resistance, rupture potential, and specific capacitance.

Transdermal delivery of raloxifene HCl via ethosomal system: Formulation, advanced characterizations and pharmacokinetic evaluation

Raloxifene HCl belongs to a class of selective estrogen receptor modulators (SERMs) which is used for the management of breast cancer. The major problem reported with raloxifene is its poor bioavailability which is only up to 2%. The main objective of the present work was to formulate raloxifene loaded ethosomal preparation for transdermal application and compare it with an oral formulation of the drug. Five ethosomal formulations with different concentrations of ethanol and a conventional liposomes formulation were prepared by rotary evaporation method. The prepared systems were characterised by high resolution transmission electron microscopy (HRTEM), force emission electron microscopy (FESEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and 31P NMR study. All these advanced characterization study established that the ethosome formulation was well defined by its size, shape and its bilayer formation. Transdermal flux of the optimized ethosome formulation was 22.14 ± 0.83 µg/ml/cm2 which was 21 times higher when compared to the conventional liposomes. Confocal microscopy study revealed an enhanced permeation of coumarin-6 dye loaded ethosomes to much deeper layers of skin when compared with conventional liposomes. The gel was found to be pseudoplastic with elastic behaviour. In-vivo studies on rats showed a higher bioavailability of RXL (157% times) for ethosomal formulation when compared with the oral formulation. In conclusion, RXL loaded ethosomal formulation via transdermal route showed superior drug delivery properties as compared to oral formulation.

Hybrid Biomimetic Interfaces Integrating Supported Lipid Bilayers with Decellularized Extracellular Matrix Components.

This paper describes the functionalization of solid supported phospholipid bilayer with decellularized extracellular matrix (dECM) components, toward the development of biomimetic platforms that more closely mimic the cell surface environment. The dECM was obtained through a combination of chemical and enzymatic treatments of mouse adipose tissue that contains collagen, fibronectin, and glycosaminoglycans (GAGs). Using amine coupling chemistry, the dECM components were attached covalently to the surface of a supported lipid bilayer containing phospholipids with reactive carboxylic acid headgroups. The bilayer formation and the kinetics of subsequent dECM conjugation were monitored by quartz crystal microbalance with dissipation (QCM-D). Fluorescence recovery after photobleaching (FRAP) confirmed the fluidity of the membrane after functionalization with dECM. The resulting hybrid biomimetic interface supports the attachment and survival of the human hepatocyte Huh 7.5 and maintains the representative hepatocellular function. Importantly, the platform is suitable for monitoring the lateral organization and clustering of cell-binding ligands and growth factor receptors in the presence of the rich biochemical profile of tissue-derived ECM components.

Cholesterol concentration effect on the bilayer properties and phase formation of niosome bilayers: A molecular dynamics simulation study

Niosome bilayers formed by the mixture of sorbitan monostearate (Span60) and cholesterol molecules with different cholesterol concentrations (0 to 70mol% Chol) were studied by molecular dynamics simulations at the temperature of 298K and the pressure of 1bar. Structural properties and dynamics of niosome bilayers were characterized as a function of cholesterol concentration. We found that addition of cholesterol in the niosome bilayer significantly changes the molecular structure and stability. Increasing of cholesterol concentrations causes the area per molecule to increase and bilayer thickness to decrease, suggesting that the niosome bilayer is expanded from the gel to the liquid ordered phase. This contrasts with DOPC or DMPC lipid bilayers which show a condensation effect with increasing cholesterol content. The calculated isothermal area compressibility of the niosome bilayer shows a remarkable increase at moderate concentration (40–50mol% Chol) and slight change at the higher concentrations. This suggests that the preparation of niosome with the higher cholesterol concentrations does not significantly improve the bilayer compressibility. With increasing cholesterol concentration the Span60 tails gradually adopt a more conformation while the orientation and dynamics of the Span60 groups are rather unaffected. The hydrogen bond interactions of the Span60/chol/water and Span60/water system increased with increasing cholesterol concentration, leading to improved bilayer stability.

Effect of Concentration on the Interfacial and Bulk Structure of Ionic Liquids in Aqueous Solution.

Bio and aqueous applications of ionic liquids (IL) such as catalysis in micelles formed in aqueous IL solutions or extraction of chemicals from biologic materials rely on surface-active and self-assembly properties of ILs. Here, we discuss qualitative relations of the interfacial and bulk structuring of a water-soluble surface-active IL ([C8MIm][Cl]) on chemically controlled surfaces over a wide range of water concentrations using both force probe and X-ray scattering experiments. Our data indicate that IL structuring evolves from surfactant-like surface adsorption at low IL concentrations, to micellar bulk structure adsorption above the critical micelle concentration, to planar bilayer formation in ILs with <1 wt % of water and at high charging of the surface. Interfacial structuring is controlled by mesoscopic bulk structuring at high water concentrations. Surface chemistry and surface charges decisively steer interfacial ordering of ions if the water concentration is low and/or the surface charge is high. We also demonstrate that controlling the interfacial forces by using self-assembled monolayer chemistry allows tuning of interfacial structures. Both the ratio of the head group size to the hydrophobic tail volume as well as the surface charging trigger the bulk structure and offer a tool for predicting interfacial structures. Based on the applied techniques and analyses, a qualitative prediction of molecular layering of ILs in aqueous systems is possible.