Planar lipid bilayers on solid supports mimic the fundamental structure of biological membranes and can be investigated using a wide range of surface-sensitive techniques. Despite these advantages, ...planar bilayer fabrication is challenging, and there are no simple universal methods to form such bilayers on diverse material substrates. One of the novel methods recently proposed and proven to form a planar bilayer on silicon dioxide involves lipid deposition in organic solvent and solvent exchange to influence the phase of adsorbed lipids. To scrutinize the specifics of this solvent-assisted lipid bilayer (SALB) formation method and clarify the limits of its applicability, we have developed a simplified, continuous solvent-exchange version to form planar bilayers on silicon dioxide, gold, and alkanethiol-coated gold (in the latter case, a lipid monolayer is formed to yield a hybrid bilayer) and varied the type of organic solvent and rate of solvent exchange. By tracking the SALB formation process with simultaneous quartz crystal microbalance–dissipation (QCM-D) and ellipsometry, it was determined that the acoustic, optical, and hydration masses along with the acoustic and optical thicknesses, measured at the end of the process, are comparable to those observed by employing conventional fabrication methods (e.g., vesicle fusion). As shown by QCM-D measurements, the obtained planar bilayers are highly resistant to protein adsorption, and several, but not all, water-miscible organic solvents could be successfully used in the SALB procedure, with isopropanol yielding particularly high-quality bilayers. In addition, fluorescence recovery after photobleaching (FRAP) measurements demonstrated that the coefficient of lateral lipid diffusion in the fabricated bilayers corresponds to that measured earlier in the planar bilayers formed by vesicle fusion. With increasing rate of solvent exchange, it was also observed that the bilayer became incomplete and a phenomenological model was developed in order to explain this feature. The results obtained allowed us to clarify and discriminate likely steps of the SALB formation process as well as determine the corresponding influence of organic solvent type and flow conditions on these steps. Taken together, the findings demonstrate that the SALB formation method can be adapted to a continuous solvent-exchange procedure that is technically minimal, quick, and efficient to form planar bilayers on solid supports.
Widely used in catalysis and biosensing applications, aluminum oxide has become popular for surface functionalization with biological macromolecules, including lipid bilayer coatings. However, it is ...difficult to form supported lipid bilayers on aluminum oxide, and current methods require covalent surface modification, which masks the interfacial properties of aluminum oxide, and/or complex fabrication techniques with specific conditions. Herein, we addressed this issue by identifying simple and robust strategies to form fluidic lipid bilayers on aluminum oxide. The fabrication of a single lipid bilayer coating was achieved by two methods, vesicle fusion under acidic conditions and solvent-assisted lipid bilayer (SALB) formation under near-physiological pH conditions. Importantly, quartz crystal microbalance with dissipation (QCM-D) monitoring measurements determined that the hydration layer of a supported lipid bilayer on aluminum oxide is appreciably thicker than that of a bilayer on silicon oxide. Fluorescence recovery after photobleaching (FRAP) analysis indicated that the diffusion coefficient of lateral lipid mobility was up to 3-fold greater on silicon oxide than on aluminum oxide. In spite of this hydrodynamic coupling, the diffusion coefficient on aluminum oxide, but not silicon oxide, was sensitive to the ionic strength condition. Extended-DLVO model calculations estimated the thermodynamics of lipid–substrate interactions on aluminum oxide and silicon oxide, and predict that the range of the repulsive hydration force is greater on aluminum oxide, which in turn leads to an increased equilibrium separation distance. Hence, while a strong hydration force likely contributes to the difficulty of bilayer fabrication on aluminum oxide, it also confers advantages by stabilizing lipid bilayers with thicker hydration layers due to confined interfacial water. Such knowledge provides the basis for improved surface functionalization strategies on aluminum oxide, underscoring the practical importance of surface hydration.
Many cell membrane functions emerge from the lateral presentation of membrane receptors. The link between the nanoscale organization of the receptors and ligand binding remains, however, mostly ...unclear. In this work, we applied surface molecular imprinting and utilized the phase behavior of lipid bilayers to create platforms that recapitulate the lateral organization of membrane receptors at the nanoscale. We used liposomes decorated with amphiphilic boronic acids that commonly serve as synthetic saccharide receptors and generated three lateral modes of receptor presentationrandom distribution, nanoclustering, and receptor crowdingand studied their interaction with saccharides. In comparison to liposomes with randomly dispersed receptors, surface-imprinted liposomes resulted in more than a 5-fold increase in avidity. Quantifying the binding affinity and cooperativity proved that the boost was mediated by the formation of the nanoclusters rather than a local increase in the receptor concentration. In contrast, receptor crowding, despite the presence of increased local receptor concentrations, prevented multivalent oligosaccharide binding due to steric effects. The findings demonstrate the significance of nanometric aspects of receptor presentation and generation of multivalent ligands including artificial lectins for the sensitive and specific detection of glycans.
As a simple and efficient technique, the solvent-assisted lipid bilayer (SALB) formation method offers a versatile approach to fabricating a planar lipid bilayer on solid supports. Corresponding ...mechanistic aspects and the role of various governing parameters remain, however, to be better understood. Herein, we first scrutinized the effect of lipid concentration (0.01 to 5 mg/mL) and solvent type (isopropanol, n-propanol, or ethanol) on SALB formation on silicon oxide in order to identify optimal conditions for this process. The obtained fluid-phase lipid layers on silicon oxide were investigated by using the quartz crystal microbalance with dissipation monitoring, epifluorescence microscopy, and atomic force microscopy. The experimental results indicate that, in alcohol, lipid attachment to the substrate is reversible and reaches equilibrium in accordance with the bulk lipid concentration. During the solvent-exchange step, the water fraction increases and the deposited lipids are converted into planar bilayer fragments, along with the concurrent adsorption and rupture of micelles within an optimal lipid concentration range. In addition, fluid-phase lipid bilayers were successfully formed on other substrates (e.g., chrome, indium tin oxide, and titanium oxide) that are largely intractable to conventional methods (e.g., vesicle fusion). Moreover, gel-phase lipid bilayers were fabricated as well. Depending on the phase state of the lipid bilayer during fabrication, the corresponding adlayer mass varied by approximately 20% between the fluid- and gel-phase states in a manner which is consistent with the molecular packing of lipids in the two arrangements. Taken together, our findings help to explain the mechanistic details of SALB formation, optimize the corresponding procedure, and demonstrate the general utility for fabricating gel- and fluid-phase planar lipid bilayers.
The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of ...Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 μm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells.
Artificial organelles are envisioned as nanosized assemblies with intracellular biocatalytic activity to provide the host cells with non-native or missing/lost function. Hybrid vesicles loaded with ...glucose oxidase (NRGOx) or β-galactosidase (NRβ-Gal) and equipped with lysosomal escape ability are assembled using phospholipids and the block copolymer poly(cholesteryl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate). The co-localization of the building blocks and the catalytic activity of NRGOx and NRβ-Gal are illustrated. The intracellular activity of the nanoreactors in RAW 264.7 macrophages is confirmed by an enhanced reduction in viability for cells pre-incubated with NRGOx in the presence of glucose due to the generation of cytotoxic hydrogen peroxide compared to the controls. In addition, RAW 264.7 macrophages and primary human macrophages equipped with NRβ-Gal are able to intracellularly convert β-Gal-NONOate into nitric oxide. The successful use of these hybrid vesicles to equip host macrophages with additional catalytic activity diversifies the available toolbox of nanocarriers with envisioned application in cell mimicry.
The accurate determination of the maximum turnover number and Michaelis constant for membrane enzymes remains challenging. Here, this problem has been solved by observing in parallel the hydrolysis ...of thousands of individual fluorescently labeled immobilized liposomes each processed by a single phospholipase A2 molecule. The release of the reaction product was tracked using total internal reflection fluorescence microscopy. A statistical analysis of the hydrolysis kinetics was shown to provide the Michaelis–Menten parameters with an accuracy better than 20 % without variation of the initial substrate concentration. The combined single‐liposome and single‐enzyme mode of operation made it also possible to unravel a significant nanoscale dependence of these parameters on membrane curvature.
The power of statistical analysis: The kinetics of the phospholipase A2 digestion of individual liposomes composed of dye‐modified lipids was measured by total internal reflection fluorescence imaging (see picture). In this way both kcat and Km could be extracted without the need to vary the initial concentration of the phospholipid substrate. This approach based on single liposomes and single enzyme molecules may give information on the effect of membrane morphology on reaction kinetics.
This paper describes the application of a solvent-exchange method to prepare supported membranes containing high fractions of cholesterol (up to ∼57 mol %) in an apparent equilibrium. The method ...exploits the phenomenon of reverse-phase evaporation, in which the deposition of lipids in alcohol (e.g., isopropanol) is followed by the slow removal of the organic solvent from the water-alcohol mixture. This in turn induces a series of lyotropic phase transitions successively producing inverse-micelles, monomers, micelles, and vesicles in equilibrium with supported bilayers at the contacting solid surface. By using the standard cholesterol depletion by methyl-β-cyclodextrin treatment, a quartz crystal microbalance with dissipation monitoring assay confirms that the cholesterol concentration in the supported membranes is comparable to that in the surrounding bulk phase. A quantitative characterization of the biophysical properties of the resultant bilayer, including lateral diffusion constants and phase separation, using epifluorescence microscopy and atomic force microscopy establishes the formation of laterally contiguous supported lipid bilayers, which break into a characteristic domain-pattern of coexisting phases in a cholesterol concentration-dependent manner. With increasing cholesterol fraction in the supported bilayer, the size of the domains increases, ultimately yielding two-dimensional cholesterol bilayer domains near the solubility limit. A unique feature of the approach is that it enables preparation of supported membranes containing limiting concentrations of cholesterol near the solubility limit under equilibrium conditions, which cannot be obtained using conventional techniques (i.e., vesicle fusion).
The dynamic nature of micellar nanostructures is employed to form a self-assembled Förster resonance energy transfer (FRET) nanoplatform for enhanced sensing of DNA. The platform consists of lipid ...oligonucleotide FRET probes incorporated into micellar scaffolds, where single recognition events result in fusion and fission of DNA mixed micelles, triggering the fluorescence response of multiple rather than a single FRET pair. In comparison to conventional FRET substrates where a single donor interacts with a single acceptor, the micellar multiplex FRET system showed ∼20- and ∼3-fold enhancements in the limit of detection and FRET efficiency, respectively. This supramolecular signal amplification approach could potentially be used to improve FRET-based diagnostic assays of nucleic acid and non-DNA based targets.
In this study, we employed the solvent-assisted lipid bilayer (SALB) formation method to fabricate charged membranes on solid supports. The SALB formation method exploits a ternary mixture of ...lipid-alcohol-aqueous buffer to deposit lamellar phase structures on solid supports upon gradual increase of the buffer fraction. Using the quartz crystal microbalance with dissipation (QCM-D) technique, we investigated the formation of negatively and positively charged membranes via the SALB formation method and directly compared with the vesicle fusion method on two different oxide films. Bilayers containing an increasing fraction of negatively charged DOPS lipid molecules were successfully formed on both SiO2 and Al2O3 substrates using the SALB formation method at physiological pH (7.5). In contrast, the vesicle fusion method did not support bilayer formation on Al2O3 and those containing more than 10% DOPS ruptured on SiO2 only under acidic conditions (pH 5). Characterization of the fraction of negatively charge DOPS by in situ annexin 5A binding assay revealed that the fraction of DOPS lipid molecules in the bilayers formed on Al2O3 is significantly higher than that formed on SiO2. This suggests that the SALB self-assembly of charged membranes is predominantly governed by the electrostatic interaction. Furthermore, our findings indicate that when multicomponent lipid mixtures are used, the relative fraction of lipids in the bilayer may differ from the fraction of lipids in the precursor mixture.
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