Polymer-supported Lipid Bilayers Research Articles

Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers.

Over the last two decades, supported lipid bilayers (SLBs) have been extensively used as model systems to study cell membrane structure and function. While SLBs have been traditionally produced from simple lipid mixtures, there has been a recent surge in compositional complexity to better mimic cellular membranes and thereby bridge the gap between classic biophysical approaches and cell experiments. To this end, native cellular membrane derived SLBs (nSLBs) have emerged as a new category of SLBs. As a new type of biomimetic material, an analytical workflow must be designed to characterize its molecular composition and structure. Herein, we demonstrate how a combination of fluorescence microscopy, neutron reflectometry, and secondary ion mass spectrometry offers new insights on structure, composition, and quality of nSLB systems formed using so-called hybrid vesicles, which are a mixture of native membrane material and synthetic lipids. With this approach, we demonstrate that the nSLB formed a continuous structure with complete mixing of the synthetic and native membrane components and a molecular stoichiometry that essentially mirrors that of the hybrid vesicles. Furthermore, structural investigation of the nSLB revealed that PEGylated lipids do not significantly thicken the hydration layer between the bilayer and substrate when on silicon substrates; however, nSLBs do have more topology than their simpler, purely synthetic counterparts. Beyond new insights regarding the structure and composition of nSLB systems, this work also serves to guide future researchers in producing and characterizing nSLBs from their cellular membrane of choice.

Structural changes in cellular membranes induced by ionic liquids: From model to bacterial membranes

Ionic liquids (ILs) have generated considerable attention recently because of their cytotoxicity and application as antibiotics. However, the mechanism of how they damage cell membranes is not currently well understood. In this paper, the antibacterial activities of two imidazolium-based ILs, namely 1-butyl- 3-methylimidazolium tetrafluroborate ([BMIM][BF4]) and 1-ethyl- 3-methylimidazolium tetrafluroborate ([EMIM][BF4]) have been investigated. The activity of [BMIM][BF4] on gram negative bacteria E. coli is observed to be stronger compared with the short chained [EMIM][BF4]. To explain this observation, the effects of these ILs on the self-assembled structures of model cellular membranes have been investigated. The in-plane elasticity of a monolayer formed at air-water interface by 1,2-dipalmitoyl- sn-glycero- 3-phosphocholine (DPPC) lipids was reduced in the presence of the ILs. The x-ray reflectivity studies on polymer supported lipid bilayer have shown the bilayer to shrink and correspondingly exhibit an increase in electron density. The presence of a certain mol% of negatively charged lipid, 1,2-dipalmitoyl-rac-glycero-3-phospho-L-serine (DPPS), in DPPC mono- and bi-layers enhances the effect considerably.

X-ray Reflectivity Study of the Interaction of an Imidazolium-Based Ionic Liquid with a Soft Supported Lipid Membrane.

Ionic liquids (ILs) are important for their antimicrobial activity and are found to be toxic to some microorganisms. To shed light on the mechanism of their activities, the interaction of an imidazolium-based IL 1-butyl-3-methylimidazolium tetrfluoroborate ([BMIM][BF4]) with E. coli bacteria and cell-membrane-mimicking lipid mono- and bilayers has been studied. The survival of the bacteria and corresponding growth inhibition are observed to be functions of the concentration of the IL. The IL alters the pressure-area isotherm of the monolayer formed at an air-water interface by the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid. The in-plane elasticity of the lipid layer is reduced as a consequence of the insertion of this IL. The X-ray reflectivity study from a polymer-supported lipid bilayer shows strong perturbation in the self-assembled structure of the bilayer due to the interaction. As a consequence, there is a considerable decrease in bilayer thickness and a corresponding increase in electron density. These results, however, depend on the chain configurations of the lipid molecules.

Formation of the layer of influenza A virus M1 matrix protein on lipid membranes at pH 7.0

The kinetics of interaction of influenza A virus M1 matrix protein with hydrophilic polymer-supported lipid bilayers formed by 1,2-diphytanoyl-sn-glycero-3-phosphocholine and 1,2-diphytanoyl-sn-glycerophospho-l-serine at pH 7.0 was studied by surface plasmon resonance spectroscopy. The M1 protein was shown to bind to the lipid bilayer almost irreversibly to form a monolayer; this is in line with the key function of the M1 protein, that is, formation of the protein envelope of the virion. An increase in the percentage of negatively charged lipids from 0 to 30% leads to a considerable increase in the protein monolayer density and a several-fold increase in the binding constant (K a) of the M1 protein with the lipid bilayer up to (3.60±0.11)·108 L mol–1. The negatively charged lipids in the bilayer appear to promote the manifestation of anisotropic properties of the M1 protein, which enable the protein to form reversible non-monolayer structures on the surface. The M1—M1 interaction was found to be reversible and to be characterized by the binding constant K a = (6.3±0.1)·107 L mol–1.

Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer.

Investigations of ligand‐binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity‐related limitations impaired by low signal‐to‐noise ratios. Inspired by affinity capturing of water‐soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane‐protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris‐NTA)–biotin conjugate. A polymer‐supported lipid bilayer containing His6‐tagged β‐secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His6–BACE in the Ni2+–NTA‐modified region of the substrate resulted in a stationary three‐fold increase in surface coverage, and an accompanied increase in ligand‐binding response.

Preserved transmembrane protein mobility in polymer-supported lipid bilayers derived from cell membranes.

Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, β-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms.

A versatile approach to the generation of fluid supported lipid bilayers and its applications.

Establishing supported lipid bilayers with biologically relevant composition, including transmembrane proteins and various classes of lipids, presents a significant challenge. We describe a generic and facile approach to the production of fluid polymer-supported lipid bilayers that allows for the incorporation of a wide variety of lipids and transmembrane proteins. The method is based on the formation of a polymer brush displaying lipid groups, followed by spin-coating of membrane lipids. Subsequentially, transmembrane proteins are incorporated by the fusion of proteoliposomes with the bilayer. Several applications, including the incorporation and single-molecule tracking of transmembrane proteins in a bilayer and the visualization of the fusion of individual, membrane-enveloped viruses with a supported membrane, are demonstrated. Our results suggest that the membrane properties are consistent with those found in physiologically relevant conditions and underscore the wide applicability of our approach to synthetic biology, lab-on-a-chip applications, biophysical and pharmaceutical studies.

Polymer Supported Lipid Bilayers

Lipid bilayers are some of the most fascinating self-assembled structure in living nature. Not only do they serve as the protective boundary of cells and their internal organelles, they also organize and host major parts of the biochemical machinery for cellular communication and transmembrane transport. To study aspects of cellular membranes in a controlled manner, solid supported planar bilayers have served as reliable tools for many decades. They have been used in a large variety of studies ranging from fundamental investigations of membranes and their constituents to the dissection of cellular signaling mechanisms. However, there are limitations to these systems and recently a class of new systems in which the lipid bilayer is supported on a soft, polymer cushion has emerged. Here, we review the different polymer cushioned bilayer systems and discuss their manufacture and advantages.

Native Ligands Change Integrin Sequestering but Not Oligomerization in Raft-Mimicking Lipid Mixtures

Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (αvβ3 and α5β1 integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for αvβ3 and α5β1, respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both αvβ3 and α5β1 sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, αvβ3 sequesters strongly into raft-like domains and α5β1 loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.

PH responsive adhesion of phospholipid vesicle on poly(acrylic acid) cushion grafted to poly(ethylene terephthalate) surface

Polymer-supported lipid bilayer is a key enabling technology for the design and fabrication of novel biomimetic devices. To date, the physical driving force underlying the formation of polymer-supported lipid bilayer remains to be determined. In this study, the interaction between dipalmitoylphosphocholine (DPPC) vesicle and poly(ethylene terephthalate) [PET] surface with or without grafted poly(acrylic acid) [PAA] layer is examined with several biophysical techniques. First, vesicle deformation analysis shows that the geometry of adherent vesicle on either plain PET or PAA-grafted PET surface is best described by a truncated sphere model. At neutral pH, the degree of deformation and adhesion energy are unaltered by the grafted polymerization of acrylic acid on PET surface. Interestingly, the average magnitude of adhesion energy is increased by 185% and −43% on PAA-grated PET and plain PET surface, respectively, towards an increase of pH at room temperature. Our results demonstrate the possibility of tuning the adhesive interaction between vesicle and polymer cushion through the control of polyelectrolyte ionization on the solid support.

Adsorption of Lipid-Functionalized Poly(ethylene glycol) to Gold Surfaces as a Cushion for Polymer-Supported Lipid Bilayers

Inclusion of a polymer cushion between a lipid bilayer membrane and a solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, the properties of a heterofunctional, telechelic PEG lipopolymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N- [3-(2-(pyridyldithio)propionate]) (DSPE-PEG-PDP) adsorbed from ethanol and water solutions onto gold surfaces were studied using a variety of surface-sensitive techniques. X-ray photoelectron spectroscopy showed that the PEG molecules are tethered to the gold surface via thiolate bonds. When adsorbed from water, ethanol, or their mixtures, reflection-absorption infrared spectroscopy showed that amorphous PEG layers with disordered DSPE alkyl chains were formed, independent of adsorption time or solution concentration. On the basis of advancing and receding water and hexadecane contact angles on the lipopolymer films, the DSPE lipid groups appear to segregate from the PEG layer and become exposed at the surface of the polymer films. Swelling observed in surface plasmon resonance experiments and the large contact angle hysteresis observed indicate that highly swellable, mobile films capable of molecular rearrangements are formed. The self-assembling and amorphous properties of these PEG layers make them ideal candidates as polymer cushions for polymer-supported lipid bilayers. The DSPE surface concentration can be controlled, to a limited degree, by varying the adsorption time of DSPE-PEG-PDP from ethanol. A more effective strategy is to coadsorb DSPE-PEG-PDP with a non-lipid-functionalized PEG-PDP from an ethanol/water mixture, which allows the PEG thickness and density to remain constant while decreasing the density of DSPE groups.

Polyacrylamide Adsorption from Aqueous Solutions on Gold and Silver Surfaces Monitored by the Quartz Crystal Microbalance

Viscoelastic properties of ultrathin films are important in such applications as polymer-supported lipid bilayers, and the quartz crystal microbalance (QCM) offers the ability to measure these properties in situ. In this study, polymer films are created by adsorbing polyacrylamide to gold and silver surfaces from aqueous solutions. The molecular weight of the polymers ranges from 10 000 to 1 000 000 g/mol, and concentrations range from dilute to the overlap concentration. Using a continuum mechanics viscoelastic film model, we extract film property parameters from frequency and dissipation change measurements for multiple harmonics. The precision of the QCM and the modeling technique are limited, however, leading to large property value ranges for any given film. The adsorbed films range from 10 to 150 Å thick, with moduli ranging from 15 to 228 kPa and viscosities ranging from 1.21 × 10-3 to 2.85 × 10-3 Pa s. Meanwhile, the bulk solution properties of the different molecular weight materials lead to drastically different adsorption profiles. While the viscosity differences between water and 10 000 Mw polymer solutions lead to large frequency and dissipation responses, the large relaxation times of 1 000 000 Mw polymers make their bulk solution viscosity differences from water largely undetectable by the QCM. In addition, the viscosity of the 10 000 Mw solutions may be frequency dependent over the range of operating frequencies, contrary to the standard QCM assumption of frequency independence.

Covalent Coupling and Characterization of Supported Lipid Layers

To investigate protein and cell surface interactions, robust and increasingly complex model surfaces need to be developed to mimic specific aspects of the cell membrane structure. Polymer-supported lipid monolayers and bilayers present an opportunity to develop rugged cell mimetic surfaces that may be stable in a wide range of in vivo and in vitro applications and characterization techniques. We have investigated the stability of dimyristoylphosphatidylethanolamine (DMPE) monolayers grafted to poly(hydroxyethyl methacrylate) (pHEMA) via 1,1‘-carbonyldiimidazole (CDI) chemistry using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and fluorescence microscopy. The results illustrate that it is possible to covalently couple the amine-containing lipid headgroup to the carboxyl and hydroxyl groups of the pHEMA and retain a proportion of the lipids at the surface after the samples are sonicated in ethanol. Most importantly, the cross-linking efficiency and retention of the resulting lipid layer is higher if the lipid is transferred to CDI-activated HEMA by Langmuir−Blodgett deposition rather than by adsorption of the lipid directly from solution. Both factors are critical if these monolayers are to form a stable and reproducible model system.

Adsorption of disulfide-modified polyacrylamides to gold and silver surfaces as cushions for polymer-supported lipid bilayers

Inclusion of a polymer cushion between a lipid bilayer membrane and solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, we evaluate the properties of a disulfide- and lipid-modified polyacrylamide polymer cushion. High molecular weight random copolymers with various degrees of disulfide and lipid substitution were synthesized. X-ray photoelectron spectroscopy (XPS) was used to determine quantitatively the percentage of disulfide groups bound to gold and silver surfaces. A quartz crystal microbalance with dissipation (QCM-D) was used to study the adsorption process and resulting film properties in situ. The presence of backbone–surface interactions leads to a competition between physisorption of the acrylamide backbone and chemisorption of the disulfide side-chains. This competition limits the degree of chemisorption to gold and silver surfaces. For a polyacrylamide with 10 mol% disulfide side-chains, 78% of the side-chains covalently bind to silver and only 41% bind to gold. The undesired physisorption of the acrylamide backbone leads to adsorption of the homopolymer itself. In addition, film thicknesses, as indicated by both XPS and QCM-D, are limited to 15–30 Å. The QCM-D results for all films indicate the formation of relatively rigid films, rather than the soft, deformable films desired for a lipid membrane polymer cushion.

Long-Range Interaction Forces between Polymer-Supported Lipid Bilayer Membranes

Much of the short-range forces and structures of softly supported DMPC bilayers has been described previously. However, one interesting feature of the measured force-distance profile that remained unexplained is the presence of a long-range exponentially decaying repulsive force that is not observed between rigidly supported bilayers on solid mica substrate surfaces. This observation is discussed in detail here based on recent static and dynamic surface force experiments. The repulsive forces in the intermediate distance regime (mica-mica separations from 15 to 40 nm) are shown to be due not to an electrostatic force between the bilayers but to compression (deswelling) of the underlying soft polyelectrolyte layer, which may be thought of as a model cytoskeleton. The experimental data can be fit by simple theoretical models of polymer interactions from which the elastic properties of the polymer layer can be deduced.

Polymer-supported lipid bilayers on benzophenone-modified substrates.

Solid-supported lipid membranes are important for their roles in fundamental biophysical research as well as in applications such as biosensors. In our study, lipopolymers containing alkyl side chains were synthesized and a mixture of the lipopolymer and free lipids was preorganized at the air-water interface and then transferred to a solid substrate using the Langmuir-Blodgett technique. A photochemical reaction between a substrate-functionalized benzophenone and C-H bonds on the lipopolymer was used to attach the lipopolymers to the substrate. The final assembly of the membrane was completed by vesicle fusion. Langmuir film experiments at the air-water interface indicate tighter molecular packing for the lipopolymers with 28 mol % alkyl side chains than for the ones with 22 mol %. Atomic force microscopy images point to phase separation of lipopolymers on the substrates due to their dewetting from hydrophobic surfaces. However, a mixture of lipopolymers and free lipids formed a smooth film on the same substrate. After the addition of the second lipid layer on the lipopolymer/free lipid layer, the fluorescence images of the polymer-supported bilayer suggested that the distal lipid layer is homogeneous on the micrometer scale. The relaxation of the fluorescent probe lipids was analyzed after application of an electric field to determine their diffusion coefficient; the distal lipid layer was mobile with an average diffusion coefficient of approximately 0.1 microm(2)/s. Moreover, the immobile fraction of the lipids in the distal layer was estimated to be around 15%.

High Electric Resistance Polymer/Lipid Composite Films on Indium−Tin−Oxide Electrodes

We report the design of polymer/lipid composite films on polarizable indium−tin−oxide (ITO) electrodes and the characterization of their electrical properties by impedance spectroscopy. The polymer films separating the solid surfaces and the lipid membranes were composed of “hairy-rod” macromolecules and provided a fluid and homogeneous ultrathin “cushion” for the deposition of lipid membranes. Two types of polymer cushions were designed: First, electrically insulating multilayers of cellulose derivatives with substituted alkyl chains forming a fluid hydrophobic “brush” at the surface. Second, conducting cellulose multilayers exposing hydrophilic surfaces for the deposition of self-healing lipid bilayers. The electrical properties, the defect densities, and the homogeneities of the stratified films were studied by impedance spectroscopy in the frequency range between 90 mHz and 50 kHz. Impedance spectra were analyzed in terms of equivalent circuits with resistances, capacitances, and so-called “constant phase elements”. Resistances of up to 440 kΩ cm2 were achieved for the polymer-supported lipid bilayers, which corresponds to about 10% of the value for black lipid membranes containing hexane. The gramicidin channels reconstituted into the supported lipid bilayers exhibited the expected cation selectivity.

Two-Dimensional Protein Crystallization at Solid−Liquid Interfaces

The two-dimensional crystallization of proteins on floating lipid layers has been documented extensively. However, the transfer of such organized protein arrays to solid supports for structural analysis or for use as sensing elements in devices can damage the fragile crystalline films. In this work, we investigated several parameters that influence the growth of two-dimensional protein crystals directly on solid substrates. With streptavidin as the model protein, we considered the effects of epitaxial coupling, surface roughness, and substrate order on the two-dimensional crystallization of streptavidin on biotinylated lipids supported on those substrata. The substrates investigated included lipid-coated atomically flat mica sheets, etched mica, and rough glass slides; silanized glass coverslips; and polymer-supported lipid bilayers. Polarized, reflected light fluorescence microscopy and fluorescence recovery after photobleaching results demonstrated that crystallization is coupled strongly to the mobile lipid fraction in the biotinylated lipid monolayer. The absence of observable crystals on polymer-supported lipid bilayers suggests that, despite their uniform appearance, the latter are likely rough and irregular on a submicroscopic length scale.

Influence of Anchor Lipids on the Homogeneity and Mobility of Lipid Bilayers on Thin Polymer Films

Polymer-supported lipid bilayers on solid substrates are of particular interest for the investigation of membrane proteins. A great improvement in the homogeneity of such lipid bilayers can be obtained by the introduction of “anchor lipids” into the monolayer facing the substrate. The resulting attached lipid bilayers (tethered supported bilayers) like that shown schematically on the right are interesting biomembrane models.