Formation Of Supported Lipid Bilayers Research Articles

Exploiting Conjugated Polyelectrolyte Photophysics toward Monitoring Real-Time Lipid Membrane-Surface Interaction Dynamics at the Single-Particle Level.

Herein we report the real-time observation of the interaction dynamics between cationic liposomes flowing in solution and a surface-immobilized charged scaffolding formed by the deposition of conjugated polyanion poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene (MPS-PPV) onto 100-nm-diameter SiO2 nanoparticles (NPs). Contact of the freely floating liposomes with the polymer-coated surfaces led to the formation of supported lipid bilayers (SLBs). The interaction of the incoming liposomes with MPS-PPV adsorbed on individual SiO2 nanoparticles promoted the deaggregation of the polymer conformation and led to large emission intensity enhancements. Single-particle total internal reflection fluorescence microscopy studies exploited this phenomenon as a way to monitor the deformation dynamics of liposomes on surface-immobilized NPs. The MPS-PPV emission enhancement (up to 25-fold) reflected on the extent of membrane contact with the surface of the NP and was correlated with the size of the incoming liposome. The time required for the MPS-PPV emission to reach a maximum (ranging from 400 to 1000 ms) revealed the dynamics of membrane deformation and was also correlated with the liposome size. Cryo-TEM experiments complemented these results by yielding a structural view of the process. Immediately following the mixing of liposomes and NPs the majority of NPs had one or more adsorbed liposomes, yet the presence of a fully formed SLB was rare. Prolonged incubation of liposomes and NPs showed completely formed SLBs on all of the NPs, confirming that the liposomes eventually ruptured to form SLBs. We foresee that the single-particle studies we report herein may be readily extended to study membrane dynamics of other lipids including cellular membranes in live cell studies and to monitor the formation of polymer-cushioned SLBs.

Tethered Lipid Bilayers within Porous Si Nanostructures: A Platform for (Optical) Real-Time Monitoring of Membrane-Associated Processes

The importance of cell membranes in biological systems has prompted the development of artificial lipid bilayers, which can mimic the cellular membrane structure. Supported lipid bilayers (SLBs) have emerged as a promising avenue for studying basic membrane processes and for possible biotechnological applications. Conventional methods for SLB formation involve the spreading of lipid vesicles on hydrophilic solid supports. Herein, a facile approach for the construction of tethered SLB within an oxidized porous Si (pSiO2) nanostructure, avoiding liposome preparation, is presented. We employ a two-step lipid self-assembly process, in which a first lipid layer is tethered to the pore walls resulting in a highly stable monolayer. A subsequent solvent exchange step induces the self-assembly of the unbound lipids into a robust SLB. Formation of pSiO2-SLB is confirmed by fluorescence resonance energy transfer (FRET), and the properties of the confined SLB are characterized by environment-sensitive fluorophores. The unique optical properties of the pSiO2 support are employed to monitor in real time the partitioning of a model amphiphilic molecule within the SLB via reflective interferometric Fourier transform spectroscopy (RIFTS) method. These self-reporting SLB platforms provide a highly generic approach for bottom-up construction of complex lipid architectures for performing biological assays at the micro- and nanoscale.

Facile Formation of Highly Mobile Supported Lipid Bilayers on Surface-Quaternized pH-Responsive Polymer Brushes

Poly(2-dimethylamino)ethyl methacrylate) (PDMA) brushes are grown from planar substrates via surface atom transfer radical polymerization (ATRP). Quaternization of these brushes is conducted using 1-iodooctadecane in n-hexane, which is a non-solvent for PDMA. Ellipsometry, AFM, and water contact angle measurements show that surface-confined quaternization occurs under these conditions, producing pH-responsive brushes that have a hydrophobic upper surface. Systematic variation of the 1-iodooctadecane concentration and reaction time enables the mean degree of surface quaternization to be optimized. Relatively low degrees of surface quaternization (ca. 10 mol % as judged by XPS) produce brushes that enable the formation of supported lipid bilayers, with the hydrophobic pendent octadecyl groups promoting in situ rupture of lipid vesicles. Control experiments confirm that quaternized PDMA brushes prepared in a good brush solvent (THF) produce non-pH-responsive brushes, presumably because the pendent octadecyl groups form micelle-like physical cross-links throughout the brush layer. Supported lipid bilayers (SLBs) can also be formed on the non-quaternized PDMA precursor brushes, but such structures proved to be unstable to small changes in pH. Thus, surface quaternization of PDMA brushes using 1-iodooctadecane in n-hexane provides the best protocol for the formation of robust SLBs. Fluorescence recovery after photobleaching (FRAP) studies of such SLBs indicate diffusion coefficients (2.8 ± 0.3 μm s–1) and mobile fractions (98 ± 2%) that are comparable to the literature data reported for SLBs prepared directly on planar glass substrates.

Mobile lipid bilayers on gold surfaces through structure-induced lipid vesicle rupture.

Forming fluid supported lipid bilayers (SLBs) on a gold surface can enable various lipid-membrane-associated biomolecular interactions to be investigated by several surface sensing techniques, such as surface plasmon resonance and scanning tunneling microscopy. However, forming fluid SLBs on a gold surface through lipid vesicle deposition continues to pose a challenge. In this study, we constructed nanograting structures on a gold surface to induce lipid vesicle rupture for forming a mobile layer of SLBs. Observations based on fluorescence recovery after photobleaching showed that SLBs on the prepared grating supports had some fluidity, while SLBs on the planar support had no fluidity. The anisotropic fluorescence intensity recovery shape changes observed in the SLBs on the grating support suggested that a second layer of SLBs partially formed on top of the first layer in contact with the gold surface and extended along the grating structure. Comparisons of the relative amounts of second bilayer and the fluorescence recovery fractions on supports with various grating edge densities suggested that the second layer formed at the edge regions and that the coverage ratio was directly proportional to the grating edge density. All of these results showed that the grating edges could serve as vesicle-rupture-inducing sites for the formation of a mobile second SLB on a gold surface. The formation of the second layer of SLBs at the edge regions but not in the flat regions enabled us to determine the second layer locations and provided us with an opportunity to pattern mobile lipid bilayers on gold surfaces by controlling the edge locations.

Electroformation from patterned single-layered supported lipid bilayers for formation of giant vesicles with narrow size distribution

We describe a method of producing solvent-free giant vesicles (GVs) with a narrow size distribution from patterned supported lipid bilayers (SLBs). The SLBs were prepared on a patterned SiO2 surface by vesicle fusion, and the GVs were formed from the SLBs by electroformation. Fluorescence observation showed the formation of single-layered SLBs on circular patterns of SiO2 with a diameter of 20 µm, the area of which corresponded to that of a unilamellar vesicle of 10 µm diameter. The electroformation from the patterned SLBs produced the GVs with a median diameter of 8.53 µm and a coefficient of variation of 10.9%.

Mechanics of membrane fusion/pore formation

Lipid bilayers play a fundamental role in many biological processes, and a considerable effort has been invested in understanding their behavior and the mechanism of topological changes like fusion and pore formation. Due to the time- and length-scale on which these processes occur, computational methods have proven to be an especially useful tool in their study. With their help, a number of interesting findings about the shape of fusion intermediates could be obtained, and novel hypotheses about the mechanism of topological changes and the involvement of peptides therein were suggested. In this work, we try to present a summary of these developments together with some hitherto unpublished results, featuring, among others, the shape of stalks and fusion pores, possible modes of action of the influenza HA fusion peptide and the SNARE protein complex, the mechanism of supported lipid bilayer formation by vesicle spreading, and the free energy and transition pathway of the fusion process.

Controllable occurrence of free-standing lipid membranes on nanograting structured supports.

Supported lipid bilayers (SLBs) have been widely used to study protein-lipid membrane interactions because their planar geometry is suitable for many surface analysis tools. However, the friction coupling between the support and the membrane can influence the properties of biomolecules in the membrane. Many studies have attempted to span SLBs over nanostructured supports to create free-standing regions in SLBs for biosensor applications. However, membranes following the support surface contour are more frequently observed than are free-standing membranes on structured supports, indicating that the parameter range suitable for formation of free-standing SLBs might be narrow and more information is necessary to understand the required conditions. The objective of this study was to estimate the system energies of free-standing and contour-following membrane states and determine which state is the most energetically favorable under various conditions. For a lipid membrane preferring to stay close to the support, an energy reward occurs when they are in close proximity; however, increasing the contact area on a structured surface can result in an energy penalty because of the bending of the lipid bilayer. Whether the energy reward or the energy penalty dominates could determine the membrane state. We used the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and the Helfrich bending theory to relate the energy sizes to experimentally controllable parameters. We experimentally examined whether the membrane state followed the model prediction when we used various buffer ionic strengths, various lipid types, and nanograting supports with three different geometries. Because it is difficult to observe the experimental membrane state directly at the nanoscale, we developed a method to use the fluorescence recovery shape change after photobleaching to distinguish experimental membrane states at the micrometer scale. Our experimental results closely matched the theoretical predictions, suggesting that the developed model can be used to predict suitable conditions for formation of free-standing bilayers on nanostructured solid supports.

Formation of supported lipid bilayers by vesicle fusion: effect of deposition temperature.

We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main phase-transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid bilayers formed below or above Tm by neutron reflection and investigated the effect of subsequent cooling to below the Tm. Our results clearly show that a continuous supported bilayer can be formed with high surface coverage below the lipid Tm. We also demonstrate that the high dissipation responses observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported.

Asymmetric supported lipid bilayer formation via methyl-β-cyclodextrin mediated lipid exchange: influence of asymmetry on lipid dynamics and phase behavior.

Supported lipid bilayers (SLBs) are broadly used as minimal membrane models and commonly produced by vesicle fusion (VF) on solid supports. Despite its advantages, VF does not allow the controlled formation of bilayers that mimic the leaflet asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick, and versatile method to produce SLBs with a desired asymmetric lipid composition which is stable for ca. 4 h. We apply methyl-β-cyclodextrin mediated lipid exchange to SLBs formed by VF to enrich the upper leaflet of the bilayer with sphingomyelin. The bilayer asymmetry is assessed by fluorescence correlation spectroscopy, measuring the lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we report the production of asymmetric SLBs (aSLBs) in the presence of a glycosylphosphatidylinositol-anchored protein, reconstituted in the bilayer both, via direct protein insertion, and via proteoliposomes fusion. We finally apply aSLBs to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. The observed differences in terms of phase separation in symmetric and asymmetric SLBs with the same overall lipid composition provide further experimental evidence that the transversal lipid distribution affects the overall lipid miscibility and allow to temporally investigate leaflet mixing.

Preparation of mica supported lipid bilayers for high resolution optical microscopy imaging.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

Control of the Morphology of Lipid Layers by Substrate Surface Chemistry

In this study, surface coatings were used to control the morphology of the deposited lipid layers during vesicle spreading, i.e., to control if liposomes self-assemble on a surface into a supported lipid bilayer or a supported vesicular layer. The influence of the properties of the surface coating on formation of the deposited lipid layer was studied with quartz crystal microbalance and two-wavelength multiparametric surface plasmon resonance techniques. Control of lipid self-assembly on the surface was achieved by two different types of soft substrate materials, i.e., dextran and thiolated polyethylene glycol, functionalized with hydrophobic linkers for capturing the lipid layer. The low-molecular-weight dextran-based surface promoted formation of supported lipid bilayers, while the thiolated polyethylene glycol-based surface promoted supported vesicular layer formation. A silicon dioxide surface was used as a reference surface in both measurement techniques. In addition to promoting supported lipid bilayer formation of known lipid mixtures, the dextran surface also promoted supported lipid bilayer formation of vesicles containing the cell membrane extract of human hepatoblastoma cells. The new dextran-based surface was also capable of protecting the supported lipid bilayer against dehydration when exposed to a constant flow of air. The well-established quartz crystal microbalance technique was effective in determining the morphology of the formed lipid layer, while the two-wavelength surface plasmon resonance analysis enabled further complementary characterization of the adsorbed supported lipid bilayers and supported vesicular layers.

Cell adhesion on supported lipid bilayers functionalized with RGD peptides monitored by using a quartz crystal microbalance with dissipation

Supported lipid bilayer (SLB) is one of the most widely used structures to mimic cell membranes. To study the cell–cell, cell–matrix and cell–material interactions, supported lipid bilayers (SLBs) functionalized with RGD peptides (SLBs–RGD) were prepared by vesicle fusion on a SiO2 quartz crystal, and subsequently bone mesenchymal stem cells (BMSCs) adhesion was analyzed. A quartz crystal microbalance with dissipation (QCM-D) was utilized to detect the dynamic adsorption behavior of lipid vesicles and BMSCs in real time. Observations obtained by QCM-D signals are confirmed by conducting fluorescence microscopy.QCM-D measurements showed the SLB formation starts at the critical concentration of the vesicles. More BMSCs adhered on SLBs–RGD than on SLBs. With the presence of SLBs, the adhesion cells on SLBs surfaces had a rounded morphology, and cells on SLBs–RGD will take long time to rearrange their cytoskeleton, which led to incomplete spreading compared with SiO2. Differences in adhesion density and adhesion properties of the cells on the different substrates could be traced at the dissipation versus frequency (ΔD/Δf) plots. These results indicate that RGD in/on SLBs could provide anchorage sites for more cells adhesion. QCM-D is demonstrated to be a useful tool for evaluating the interactions between various biological and non-biological systems in situ and in real-time.

Optical Scattering of Liposomes Suspended above a Surface

Lipid bilayers exhibit a different refractive index parallel and perpendicular to their surface. This optical birefringence is characteristic for the size, shape and orientation of the lipid molecules in the membrane [1].A new optical model to investigate the optical birefringence of liposomes suspended above a surface is presented. The solution to Maxwell's equation for particles suspended above a surface, as presented by Bobbert and Vlieger [2], is extended to investigate the optical scattering properties of liposomes suspended above a surface. Numerical simulations demonstrate that ellipsometry is highly sensitive to the distance of liposomes above a surface and that ellipsometry can be applied to determine the optical anisotropy of the liposome lipid bilayer, creating opportunities for the use of ellipsometry in the investigation of the supported lipid bilayer formation process.The optical model also allows for the analysis of more complex structures such as nanoparticles coated with a lipid bilayer, or liposomes whose lumen contains a spherical nanoparticle. The influence of the presence of a lipid bilayer and its optical anisotropy on ellipsometry measurements is presented. Also, it is investigated how model parameters such as lipid bilayer anisotropy can be experimentally separated from other model parameters such as e.g. the overall particle radius.[1] Z. Salamon and G. Tollin, Biophysical Journal, 80, 1557-1569 (2001); [2] P. Bobbert and J. Vlieger, Physica A: Statistical Mechanics and its Applications, 137A, 209-242 (1986)

Direct Observation and Control of Supported Lipid Bilayer Formation with Interferometric Scattering Microscopy

Supported lipid bilayers (SLB) are frequently used to study processes associated with or mediated by lipid membranes. The mechanism by which SLBs form is a matter of debate, largely due to the experimental difficulty associated with observing the adsorption and rupture of individual vesicles. Here, we used interferometric scattering microscopy (iSCAT) to directly visualize membrane formation from nanoscopic vesicles in real time. We observed a number of previously proposed phenomena such as vesicle adsorption, rupture, movement, and a wave-like bilayer spreading. By varying the vesicle size and the lipid-surface interaction strength, we rationalized and tuned the relative contributions of these phenomena to bilayer formation. Our results support a model where the interplay between bilayer edge tension and the overall interaction energy with the surface determine the mechanism of SLB formation. The unique combination of sensitivity, speed, and label-free imaging capability of iSCAT provides exciting prospects not only for investigations of SLB formation, but also for studies of assembly and disassembly processes on the nanoscale with previously unattainable accuracy and sensitivity.

Nanoscale Departures: Excess Lipid Leaving the Surface during Supported Lipid Bilayer Formation

The behavior of small liposomes on surfaces of inorganic oxides remains enigmatic. Under appropriate conditions it results in the formation of supported lipid bilayers (SLBs). During this process, some lipids leave the surface (desorb). We were able to visualize this by a combination of time-resolved fluorescence microscopy and fluorescence recovery after photobleaching studies. Our observations also allowed us to analyze the kinetics of bilayer patch growth during the late stages of SLB formation. We found that it entails a balance between desorption of excess lipids and further adsorption of liposomes from solution. These studies were performed with liposomes containing zwitterionic phospholipids (dioleoylphosphatidylcholine alone or a mixture of dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and cholesterol) on TiO2 in the presence of Ca(2+) but in the absence of other salts.

Lipid bilayers supported on bare and modified gold – Formation, characterization and relevance of lipid rafts

Supported lipid bilayers (SLB) comprise a very important set of model systems of biomembranes. In particular, they can be prepared on metallic surfaces such as gold electrodes, which allow a number of electrochemical studies and applications that could not be undertaken with other types of model systems, such as liposome suspensions. Also of special relevance is lipid bilayer composition, especially those combinations of lipids which permit the formation of biologically relevant membrane domains such as lipid rafts. Indeed, membrane domain organization is a crucial feature concerning not only the biophysical properties of the bilayer itself, but also the behavior and bioactivity of membrane-interacting biomolecules. In spite of its relevance, the presence of bilayer domains has not been central in many investigations involving lipid bilayers supported on conductive surfaces. Moreover, air exposed gold surface is hydrophobic, which is not ideally suitable to establish a proper interaction with the lipids polar head group. To overcome such limitation different strategies may be adopted, encompassing the fine tuning of the buffer conditions and previous surface modification by hydrophilic self-assembled monolayer (SAM) or thiolipids. This review will be focused on the formation and characterization of SLB and lipid rafts on gold substrates, illustrating the range of applications of such platforms, with examples of the study of electroactive molecules and the development of new biosensing interfaces.

Unraveling Supported Lipid Bilayer Formation Kinetics: Osmotic Effects

Solid-supported lipid bilayers are used as cell membrane models and form the basis of biomimetic and biosensor platforms. The mechanism of their formation from adsorbed liposomes is not well-understood. Using membrane-permeable solute glycerol, impermeable solutes sucrose and dextran, and a pore forming peptide melittin, we studied experimentally how osmotic effects affect the kinetics of the adsorbed liposome-to-bilayer transition. We find that its rate is enhanced if adsorbed liposomes are made permeable but is not significantly retarded by impermeable solutes. The results are explained in terms of adsorbed liposome deformation and formation of transmembrane pores.

Insight into the Lytic Mechanism of Antimicrobial Piscidin 1 and 3 using QCM-D

In this work, we focused on piscidin 1 (P1) and 3 (P3), which are twenty-two-residue-long amphipathic, cationic antimicrobial peptides (AMPs) isolated from hybrid striped bass. Extensive work has been reported on characterizing the atomic-level structure of both P1 and P3 in magnetically and mechanically aligned bilayers using solid-state NMR. Here we investigated the mechanism of action of both P1 and P3 with supported lipid bilayers (SLB), using a quartz crystal microbalance with dissipation monitoring (QCM-D). In order to mimic bacterial cell membranes, we selected a 3:1 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Liposomes were deposited onto a QCM-D silicon oxide crystal until SLB formation, then P1 and P3 were introduced into the system and the interaction between the peptide and the lipid system was monitored by changes in frequency (Δf) and energy dissipation (ΔD). Different concentrations (2-20 μM) of the peptide and buffer conditions (pH 6.0 and 7.4) were tested. In particular we observed a Δf increase (∼ mass removal) as we increased the concentration of P1 at pH 7.4, suggesting lipid removal, membrane thinning, or pore formation; while at pH 6.0 the Δf decrease (∼ mass addition) was probably due to the electrostatic interaction between the more cationic peptide and the anionic POPG. For a quantitative assessment of the effects on the SLB, we have obtained the shear viscosity and shear modulus as a function of piscidin type and concentration via a fit of the well-known Voigt model to the data. In all cases, ΔD values were a clear indication of the destabilization of the SLB. Overall, this study, which demonstrates quantitative P1 and P3 disruption of a bacterial membrane mimic, assists in better understanding the mode of action of piscidins and related AMPs.

Vesicle deposition on hydrophilic solid surfaces

Supported lipid bilayers (SLBs) are popular as model systems for cell membranes and are promising for future applications in diagnostic devices and biomimetics. However, the mechanism of SLB formation is not fully understood. In this study, the deposition process of a single small unilamellar vesicle on hydrophilic supports is explored by dissipative particle dynamics. Depending on the lipid tail hydrophobicity and vesicle size, there exist three distinctive pathways of vesicle deposition, involving non-disintegrated, partially disintegrated, and completely disintegrated vesicle. A morphological phase diagram is presented and it reveals that a vesicle with weak tail hydrophobicity and small size tends to be completely disintegrated upon deposition. In contrast, the adsorption of an intact vesicle is observed as it possesses strong tail hydrophobicity and large size. Moreover, our simulation results clearly indicate that the deposition process of a vesicle onto the solid surfaces can be adjusted by altering the vesicular membrane tension and the interaction strength between the lipid head and the solid support. Finally, the deposition process of multiple vesicles on the hydrophilic solid surface is also investigated. The result shows that the fusion of vesicles has a tendency to hinder the formation of a SLB.

Simulation of edge facilitated adsorption and critical concentration induced rupture of vesicles at a surface

We investigate the kinetics of supported lipid bilayer formation by the adsorption and rupture of uncharged phosphatidylcholine lipid vesicles on to a solid substrate. We model the adsorption process taking into account the distinct vesicle rupture events and growth processes. This includes (i) the initial adhesion and vesicle rupture that nucleates bilayer islands, (ii) the growth and merger of bilayer islands, (iii) enhanced adhesion of vesicles to the bilayer edge, and (iv) the final desorption of excess vesicles from the substrate. These simulation studies give insight into prior experimental observations of adsorption in which an overloading of lipid on the solid substrate occurs before formation of the final supported lipid bilayer. Our model provides an explanation for the features of the interesting universal master curve that was observed for the surface fluorescence intensity in the experimental investigations of Weirich et al.