Characterizing stability properties of supported bilayer membranes on nanoglassified substrates using surface plasmon resonance

Supported bilayer membranes (SBMs) formed on solid substrates, in particular glass, provide an ideal cell mimicking model system that has been found to be highly useful for biosensing applications. Although the stability of the membrane structures is known to determine the applicability, the subject has not been extensively investigated, largely because of the lack of convenient methods to monitor changes of membrane properties on glass in real time. This work reports the evaluation of the stability properties of a series of SBMs against chemical and air damage by use of surface plasmon resonance spectroscopy and nanoglassified gold substrates. Seven SBMs composed of phosphatidylcholine and DOPC+, including single-component, mixed, protein-reinforced SBMs (rSBMs) and protein-tethered bilayer membranes (ptBLMs), are studied. The stability properties under various conditions, especially the effects of surfactants, organic solvents, and dehydration damage on the bilayers, are compared. PC membranes are found to be easily removed from the glassy surfaces using relatively low concentrations of the surfactants, while DOPC+ is markedly more stable toward nonionic surfactant. DOPC+ membranes also demonstrated remarkable air stability while PC films exhibited considerable damage from dehydration. Doping of cholesterol does not improve PC's stability against SDS and Triton but changes the lipid membrane packing enough to protect against dehydration damage. Although rSBMs and ptBLMs improve air stability to a certain degree, they are still quite susceptible to significant damage/removal from ionic and nonionic surfactants at lower concentrations. Overall, DOPC+ has noted higher stability on glass, likely due to the favorable electrostatic interaction between the silicate surface and the lipid headgroup, making it a good candidate for application. Nanoglassy SPR proves to be an attractive platform capable of rapidly screening film stability in real-time, providing critical information for future work using supported membranes for sensing applications.     

Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.     

NSAIDs interactions with membranes: a biophysical approach

This work focuses on the interaction of four representative NSAIDs (nimesulide, indomethacin, meloxicam, and piroxicam) with different membrane models (liposomes, monolayers, and supported lipid bilayers), at different pH values, that mimic the pH conditions of normal (pH 7.4) and inflamed cells (pH 5.0). All models are composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) which is a representative phospholipid of most cellular membranes. Several biophysical techniques were employed: Fluorescence steady-state anisotropy to study the effects of NSAIDs in membrane microviscosity and thus to assess the main phase transition of DPPC, surface pressure-area isotherms to evaluate the adsorption and penetration of NSAIDs into the membrane, IRRAS to acquire structural information of DPPC monolayers upon interaction with the drugs, and AFM to study the changes in surface topography of the lipid bilayers caused by the interaction with NSAIDs. The NSAIDs show pronounced interactions with the lipid membranes at both physiological and inflammatory conditions. Liposomes, monolayers, and supported lipid bilayers experiments allow the conclusion that the pH of the medium is an essential parameter when evaluating drug-membrane interactions, because it conditions the structure of the membrane and the ionization state of NSAIDs, thereby influencing the interactions between these drugs and the lipid membranes. The applied models and techniques provided detailed information about different aspects of the drug-membrane interaction offering valuable information to understand the effect of these drugs on their target membrane-associated enzymes and their side effects at the gastrointestinal level.     

Atomic force microscopy imaging and electrical recording of lipid bilayers supported over microfabricated silicon chip nanopores: lab-on-a-chip system for lipid membranes and ion channels

We describe a silicon chip-based supported bilayer system to detect the presence of ion channels and their electrical conductance in lipid bilayers. Nanopores were produced in microfabricated silicon membranes by electron beam lithography as well as by using a finely focused ion beam. Thermal oxide was used to shrink pore sizes, if necessary, and to create an insulating surface. The chips with well-defined pores were easily mounted on a double-chamber plastic cell recording system, allowing for controlling the buffer conditions both above and below the window. The double-chamber system allowed using an atomic force microscopy (AFM) tip as one electrode and inserting a platinum wire as the second electrode under the membrane window, to measure electrical current across lipid bilayers that are suspended over the pores. Atomic force imaging, stiffness measurement, and electrical capacitance measurement show the feasibility of supporting lipid bilayers over defined nanopores: a key requirement to use any such technique for structure-function study of ion channels. Online addition of gramicidin, an ion-channel-forming peptide, resulted in electrical current flow across the bilayer, and the I-V curve that was measured using the conducting AFM tip indicates the presence of many conducting gramicidin ion channels.     

Visualizing the solubilization of supported lipid bilayers by an amphiphilic peptide

The effect of the presequence peptide of cytochrome c oxidase subunit IV (p25) on supported phospholipid bilayers (SPBs) was visualized using atomic force microscopy (AFM). The presequence was found to cause the complete disruption of supported bilayers containing neutral lipids. At relatively low concentrations of presequence, the peptide was found to bind to the membrane, coalescing to form microdomains within the liquid-crystalline bilayer that were located predominantly at bilayer-mica boundaries. Further increases in peptide concentration resulted in the formation of holes within the SPB that were spanned by an interpenetrating network of narrower regions of the bilayer, which, at higher applied peptide concentrations, were observed to disappear through a budding process, ultimately leading to the formation of spherical structures at yet higher peptide concentrations. Within this paper, the impact the presequence has upon the structure and order of the membrane is discussed, as is the potential implication of this apparent solubilization process on the translocation of cytochrome c oxidase into the inner mitochondrial membrane.     

QCM-D on mica for parallel QCM-D-AFM studies

Quartz crystal microbalance with dissipation monitoring (QCM-D) has developed into a recognized method to study adsorption processes in liquid, such as the formation of supported lipid bilayers and protein adsorption. However, the large intrinsic roughness of currently used gold-coated or silica-coated QCM-D sensors limits parallel structural characterization by atomic force microscopy (AFM). We present a method for coating QCM-D sensors with thin mica sheets operating in liquid with high stability and sensitivity. We define criteria to objectively assess the reliability of the QCM-D measurements and demonstrate that the mica-coated sensors can be used to follow the formation of supported lipid membranes and subsequent protein adsorption. This method allows combining QCM-D and AFM investigations on identical supports, providing detailed physicochemical and structural characterization of model membranes.     

Atomic force microscopy of model lipid membranes

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

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

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

GM1 clustering inhibits cholera toxin binding in supported phospholipid membranes

The present studies explore multivalent ligand-receptor interactions between pentameric cholera toxin B subunits (CTB) and the corresponding membrane ligand, ganglioside GM1. CTB binding was monitored on supported phospholipid bilayers coated on the walls and floors of microfluidic channels. Measurements were made by total internal reflection fluorescence microscopy (TIRFM). Apparent dissociation constants were extracted by fitting the binding data to both the Hill-Waud and Langmuir adsorption isotherm equations. Studies of the effect of ligand density on multivalent CTB-GM1 interactions revealed that binding weakened with increasing GM1 density from 0.02 mol % to 10.0 mol %. Such a result could be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibited the binding of CTB. Atomic force microscopy (AFM) experiments directly verified GM1 clustering within the supported POPC bilayers.     

Supported lipid bilayers on biocompatible polysaccharide multilayers

Formation of supported lipid bilayers on soft polymer cushions is a useful approach to decouple the membrane from the substrate for applications involving membrane proteins. We prepared biocompatible polymer cushions by the layer-by-layer assembly of two polysaccharide polyelectrolytes, chitosan (CHI) and hyaluronic acid, on glass and silicon substrates. (CHI/HA)(5) films were characterized by atomic force microscopy, giving an average thickness of 57 nm and roughness of 25 nm in aqueous solution at pH 6.5. Formation of zwitterionic lipid bilayers by the vesicle fusion method was attempted using DOPC vesicles at pH 4 and 6.5 on (CHI/HA)(5) films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; a combination of fluorescence and AFM indicated that this was attributable to formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. By contrast, more uniform bilayers with mobile lipids were produced at pH 4. Fluorescence recovery after photobleaching gave diffusion coefficients that were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. These results demonstrate that polysaccharides provide a useful alternative to other polymer cushions, particularly for applications where biocompatibility is important.     

Nanoscale patterning in mixed fluorocarbon-hydrocarbon phospholipid bilayers

A growing body of literature suggests that fluorocarbons can direct self-assembly within hydrocarbon environments. We report here the fabrication and characterization of supported lipid bilayers (SLBs) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a synthetic, fluorocarbon-functionalized analogue, 1. AFM investigation of these model membranes reveals an intricate, composition-dependent domain structure consisting of approximately 50 nm stripes interspersed between approximately 1 microm sized domains. Although DSC of 1 showed a phase transition near room temperature, DSC of DPPC:1 mixtures exhibited complex phase behavior suggesting domain segregation. Finally, temperature-dependent AFM of DPPC:1 bilayers shows that, while the stripe structures can be melted above the Tm of 1, the stripes and domains result from immiscibility of the hydrocarbon and fluorocarbon lipid gel phases. Fluorination appears to be a promising strategy for chemical self-assembly in two dimensions. In particular, because no modification is made to the lipid headgroups, it may be useful for nanopatterning biologically relevant ligands on bilayers in vitro or in living cells.     

Characterization of the physical properties of model biomembranes at the nanometer scale with the atomic force microscope

Interaction forces and topography of mixed phospholipid-glycolipid bilayers were investigated by atomic force microscopy (AFM) in aqueous conditions with probes functionalized with self-assembled monolayers terminating in hydroxy groups. Short-range repulsive forces were measured between the hydroxy-terminated probe and the surface of the two-dimensional (2-D) solid-like domains of distearoyl-phosphatidylethanolamine (DSPE) and digalactosyldiglyceride (DGDG). The form and range of the short-range repulsive force indicated that repulsive hydration/steric forces dominate the interaction at separation distances of 0.3-1.0 nm after which the probe makes mechanical contact with the bilayers. At loads < 5 nN the bilayer was elastically deformed by the probe, while at higher loads plastic deformation of the bilayer was observed. Surprisingly, a short-range repulsive force was not observed at the surface of the 2-D liquid-like dioleoylphosphatidylethanolamine (DOPE) film, despite the identical head groups of DOPE and DSPE. This provides direct evidence for the influence of the structure and mechanical properties of lipid bilayers on their interaction forces, an effect which may be a major importance in the control of biological processes such as cell adhesion and membrane fusion. The step height measured between lipid domains in the AFM topographic images was larger than could be accounted for by the thickness and mechanical properties of the molecules. A direct correlation was observed between the repulsive force range over the lipid domains and the topographic contrast, which provides direct insight into the fundamental mechanisms of AFM imaging in aqueous solutions. This study demonstrates that chemically modified AFM probes can be used in combination with patterned lipid bilayers as a novel and powerful approach to characterize the nanometer scale chemical and physical properties of heterogeneous biosurfaces such as cell membranes.     

AFM-based force-clamp monitors lipid bilayer failure kinetics

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. The elucidated information not only contributes to a better understanding of such relevant process, but also proves the suitability of AFM-based force clamp to study model structures as lipid bilayers. These findings on the kinetics of lipid bilayers rupture could be extended and applied to the study of other molecular thin films. Furthermore, systems of higher complexity such as models mimicking cell membranes could be studied by means of AFM-based force-clamp technique.     

Spectral Förster resonance energy transfer detection of protein interactions in surface-supported bilayers

F?rster resonance energy transfer (FRET), a fluorescence detection technique, is often used for sensing molecular interactions in solution and in membranes. Here we show that (1) FRET spectra can be recorded in single bilayers, supported on a surface, and (2) the fluorescein/rhodamine dye pair is an adequate reporter of FRET when spectral detection is used. Thus, measurements pertaining to molecular interactions in membranes can be carried out in supported bilayers. Spectral FRET has advantages over imaging FRET, which monitors only signal amplitudes at certain wavelength. There are also advantages to performing spectral FRET measurements in supported bilayers as compared to free liposomes in suspension. However, the spectral properties of dyes can be altered in an unexpected manner in an ordered bilayer structure on a surface, such that fluorescence detection in surface-supported bilayers is not always trivial.     

Protecting, patterning, and scaffolding supported lipid membranes using carbohydrate glasses

Disaccharides are known to protect sensitive biomolecules against stresses caused by dehydration, both in vivo and in vitro. Here we demonstrate how interfacial accumulation of trehalose can be used to (1) produce rugged supported lipid bilayers capable of near total dehydration; (2) enable spatial patterning of membrane micro-arrays; and (3) form stable bilayers on otherwise lipophobic substrates (e.g., metal transducers) thus affording protecting, patterning, and scaffolding of lipid bilayers.     

Lipid bilayer disruption by polycationic polymers: the roles of size and chemical functional group

Polycationic polymers are used extensively in biology to disrupt cell membranes and thus enhance the transport of materials into the cell. The highly polydisperse nature of many of these materials makes obtaining a mechanistic understanding of the disruption processes difficult. To design an effective mechanistic study, a monodisperse class of polycationic polymers, poly(amidoamine) (PAMAM) dendrimers, has been studied in the context of supported dimyristoylphosphatidylcholine (DMPC) lipid bilayers using atomic force microscopy (AFM). Aqueous solutions of amine-terminated generation 7 (G7) PAMAM dendrimers caused the formation of 15-40-nm-diameter holes in lipid bilayers. This effect was significantly reduced for smaller G5 dendrimers. For G3, no hole formation was observed. In addition to dendrimer size, surface chemistry had a strong influence on dendrimer-lipid bilayer interactions. In particular, acetamide-terminated G5 did not cause hole formation in bilayers. In all instances, the edges of bilayer defects proved to be points of highest dendrimer activity. A proposed mechanism for the removal of lipids by dendrimers involves the formation of dendrimer-filled lipid vesicles. By considering the thermodynamics, interaction free energy, and geometry of these self-assembled vesicles, a model that explains the influence of polymer particle size and surface chemistry on the interactions with lipid membranes was developed. These results are of general significance for understanding the physical and chemical properties of polycationic polymer interactions with membranes that lead to the transport of materials across cell membranes.     

Absence of ethanol-induced interdigitation in supported phospholipid bilayers on silica surfaces

Membranes prepared by the adsorption of phospholipid vesicles on solid supports are much-used model systems in biomedical research. However, there is accumulating evidence that such membranes may not always be equivalent to the free-standing cellular membranes that they are modeling. In the present study, sonicated DOPC/DOPS (80/20 mol %) vesicles were adsorbed on hydrophilic silica surfaces, a system that has been demonstrated to produce confluent bilayers. In addition, pure DOPC and DLPC membranes were studied. It is demonstrated that ethanol-induced membrane interdigitation, as demonstrated for free-standing bilayers, does not occur in these supported membranes.     

Phospholipid bilayer formation on hydroxyapatite sol-gel synthesized films

Lipid bilayers supported by porous biomaterials are being explored as models for cell membranes. Hydroxyapatite is a relevant material currently being used extensively for biomedical applications. In this study, hydroxyapatite films produced via a sol-gel chemistry route have been characterized and explored as a scaffolding material for lipid membranes. The hydroxyapatite has been characterized using XRD, SEM, and AFM, followed by vesicle-fusion of lipids characterized by fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) to determine the diffusion coefficient of this system. The HA films produced in this work were found to produce slow lateral diffusion and, in the two-phase lipid systems, some domains were observed. The low lateral diffusion coefficients were believed to be a result of the large undulations present on the hydroxyapatite film surface.     

Fluid and air-stable lipopolymer membranes for biosensor applications

The behavior of poly(ethylene glycol) (PEG) conjugated lipids was investigated in planar supported egg phosphatidylcholine bilayers as a function of lipopolymer density, chain length of the PEG moiety, and type of alkyl chains on the PEG lipid. Fluorescence recovery after photobleaching measurements verified that dye-labeled lipids in the membrane as well as the lipopolymer itself maintained a substantial degree of fluidity under most conditions that were investigated. PEG densities exceeding the onset of the mushroom-to-brush phase transition were found to confer air stability to the supported membrane. On the other hand, substantial damage or complete delamination of the lipid bilayer was observed at lower polymer densities. The presence of PEG in the membrane did not substantially hinder the binding of streptavidin to biotinylated lipids present in the bilayer. Furthermore, above the onset of the transition into the brush phase, the protein binding properties of these membranes were found to be very resilient upon removal of the system from water, rigorous drying, and rehydration. These results indicate that supported phospholipid bilayers containing lipopolymers show promise as rugged sensor platforms for ligand-receptor binding.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.