Formation Of Supported Phospholipid Bilayers Research Articles

Neutron reflectivity study of substrate surface chemistry effects on supported phospholipid bilayer formation on [formula omitted] sapphire

Oxide-supported phospholipid bilayers (SPBs) used as biomimetic membranes are significant for a broad range of applications including improvement of biomedical devices and biosensors, and in understanding biomineralization processes and the possible role of mineral surfaces in the evolution of pre-biotic membranes. Continuous-coverage and/or stacked SPBs retain properties (e.g., fluidity) more similar to native biological membranes, which is desirable for most applications. Using neutron reflectivity, we examined the role of oxide surface charge (by varying pH and ionic strength) and of divalent Ca2+ in controlling surface coverage and potential stacking of dipalmitoylphosphatidylcholine (DPPC) bilayers on the (112¯0) face of sapphire (α-Al2O3). Nearly full bilayers were formed at low to neutral pH, when the sapphire surface is positively charged, and at low ionic strength (I=15mM NaCl). Coverage decreased at higher pH, close to the isoelectric point of sapphire, and also at high I⩾210mM, or with addition of 2mM Ca2+. The latter two effects are not additive, suggesting that Ca2+ mitigates the effect of higher I. These trends agree with previous results for phospholipid adsorption on α-Al2O3 particles determined by adsorption isotherms and on single-crystal (101¯0) sapphire by atomic force microscopy, suggesting consistency of oxide surface chemistry-dependent effects across experimental techniques.

1,2-Dielaidoylphosphocholine/1,2-dimyristoylphosphoglycerol supported phospholipid bilayer formation in calcium and calcium-free buffer

Phospholipid membranes are useful in the field of biocatalysis because a supported phospholipid membrane can create a biomimetic platform where biocatalytic processes can readily occur. In this work, supported bilayer formation from sonicated phospholipid vesicles containing 1,2-dielaidoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] was studied using a quartz crystal microbalance with dissipation monitoring and an atomic force microscope. The molar percentages of DEPC and DMPG were varied to determine the effect of overall lipid composition on supported bilayer formation. This work also explored the effect that calcium ion concentration had on supported bilayer formation. Results show that vesicles with up to 50mol% dimyristoylphosphoglycerol can form a supported bilayer without the presence of calcium ions; however, supported bilayer formation in calcium buffer was inhibited as the anionic (negatively charged) lipid concentration increased. Data suggest that supported phospholipid bilayer formation in the absence of Ca2+ from vesicles containing negatively charged lipids is specific to phosphatidylglycerol.

Electrostatic effects on deposition of multiple phospholipid bilayers at oxide surfaces

We investigated electrostatic effects on the formation of multiple supported phospholipid bilayers (SPB) by varying the oxide substrate, ionic strength, the presence of divalent Ca(2+), and phospholipid (PL) headgroup charge. Whereas the current understanding of processes and forces controlling SPB formation is based primarily on studies involving planar substrates, we report results from experiments using aqueous suspensions of quartz (α-SiO(2)) and corundum (α-Al(2)O(3)) particles. Using fluorescent dye-loaded dipalmitoylphosphatidylcholine (DPPC) vesicles, we determined that the vesicles underwent oxide particle-induced rupture and formed supported planar bilayers rather than a supported vesicle layer. Adsorption isotherms of DPPC at pH 7.2 in solutions of varying ionic strength set by NaCl, and with or without 2 mM Ca(2+), support our hypotheses that van der Waals forces predominantly account for two DPPC bilayers, and that adsorption beyond the second bilayer occurs at low ionic strength due to extension of the electric double-layer near the oxide surface. In contrast, adsorption isotherms of anionic dipalmitoylphosphatidylserine (DPPS) and cationic dipalmitoylethylphosphatidylcholine (DPEPC) show that adsorption of highly charged bilayers is decreased or prevented altogether due to bilayer-oxide and/or bilayer-bilayer repulsion. Results have potential implications for biomedical, industrial, and environmental remediation applications involving SPBs and for proto-cell stability in origin-of-life hypotheses.

Influence of Nanotopography on Phospholipid Bilayer Formation on Silicon Dioxide

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.

Supported phospholipid bilayer formation on hydrophilicity-controlled silicon dioxide surfaces

We investigated the influence of surface hydroxyl groups (-OHs) on the supported planar phospholipid bilayer (SPB) formation and characteristics. We prepared SiO2 surfaces with different hydrophilicity degree by annealing the SiO2 layer on Si(100) formed by wet chemical treatments. The hydrophilicity reduced with irreversible thermal desorption of -OHs. We formed SPB of dimyristoylphosphatidylcholine on the SiO2 surfaces by incubation at a 100-nm-filtered vesicle suspension. The formation rate was faster on less hydrophilic surfaces. We proposed that a stable hydrogen-bonded water layer on the SiO2 surface worked as a barrier to prevent vesicle adhesion on the surface. Theoretical calculation indicates that water molecules on vicinal surface -OHs take a stable surface-unique geometry, which disappears on an isolated -OH. The surface -OH density, however, affected little the fluidity of once formed SPBs, which was measured by the fluorescence recovery after the photobleaching method. We also describe the area-selective SPB deposition using surface patterning by the focused ion beam.

Dissipation-Enhanced Quartz Crystal Microbalance Studies on the Experimental Parameters Controlling the Formation of Supported Lipid Bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.

Controlling the pathway of formation of supported lipid bilayers of DMPC by varying the sodium chloride concentration

Supported lipid bilayers (SLBs) are synthetic ultrathin organic membranes which serve as model systems for cell membrane and are promising for future applications in diagnostic devices or for biomimetics. The pathway of SLB formation is yet partially understood. In the present study, the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO 2 surfaces was studied. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) on SiO 2 surfaces were investigated using dissipation enhanced quartz crystal microbalance (QCM-D) as a function of buffer composition, especially sodium chloride concentration. The lipid used here possesses a phase transition temperature (T m) of 24 °C which is close to the ambient and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris•HCl solutions containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density (Θ c) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. This pathway of SPB formation is referred to as pathway 1. In absence of CaCl 2, the kinetics of the SPB formation process are slowed down, but pathway 1 is still observed. In absence of sodium chloride, the passage through Θ c disappears and this behavior is referred to as pathway 2. The transition from pathway 1 to pathway 2 occurs at a sodium chloride concentration of about 75–90 mM. The role of sodium chloride in vesicle-substrate and vesicle–vesicle interactions is discussed.

Localized Surface Plasmon Resonance Sensing of Lipid-Membrane-Mediated Biorecognition Events

Supported phospholipid bilayers (SPBs) have emerged as important model systems for studies of the natural cell membrane and its components, which are essential for the integrity and function of cells in all living organisms, and also constitute common targets for therapeutic drugs and in disease diagnosis. However, the preferential occurrence of spontaneous SPB formation on silicon-based substrates, but not on bare noble-metal surfaces, has so far excluded the use of the localized surface plasmon resonance (LSPR) sensing principle for studies of lipid-membrane-mediated biorecognition reactions. This is because the LSPR phenomenon is associated with, and strongly confined to, the interfacial region of nanometric noble-metal particles. This problem has been overcome in this study by a self-assembly process utilizing localized rupture of phospholipid vesicles on silicon dioxide in the bottom of nanometric holes in a thin gold film. The hole-induced localization of the LSPR field to the voids of the holes is demonstrated to provide an extension of the LSPR sensing concept to studies of reactions confined exclusively to SPB-patches supported on SiO2. In particular, we emphasize the possibility of performing label-free studies of lipid-membrane-mediated reaction kinetics, including the compatibility of the assay with array-based reading (approximately 7 x 7 microm2) and detection of signals originating from bound protein in the zeptomole regime.

Cell adhesion on supported lipid bilayers.

The cell and protein repellent properties of supported phospholipid bilayer (SPB) membranes were investigated. The SPBs were prepared by vesicle adsorption on SiO(2) surfaces. The vesicles of phosphatidylcholine fuse and rupture, and form a supported bilayer covering the surface. We carried out cell culture experiments on several surfaces, including SPBs, using two types of epithelial cells to address the cell adhesional properties. The Quartz Crystal Microbalance Dissipation (QCM-D) technique was used to monitor the SPB formation and subsequent protein adsorption. Neither cell type adhered or proliferated on SiO(2) surfaces coated with SPBs, whereas both cell types adhered and proliferated on the three control surfaces of SiO(2), tissue culture glass, and TiO(2). The QCM-D measurements showed that about two orders of magnitude less mass adsorbed on a SPB surface compared to a TiO(2) surface, from serum-containing media (10% fetal bovine serum). The reduced adsorption on the SPB is a likely explanation for the nondetectable epithelial cell adhesion on the SPB surface. Biomembranes are therefore attractive candidate systems to achieve alternating cell-resistant and cell-interacting regions on surfaces, by including specific cell-binding proteins in the latter regions.

Formation of Supported Lipid Bilayer Membranes on SiO2 from Proteoliposomes Containing Transmembrane Proteins

We report the preparation of protein-containing supported phospholipid bilayers (SPBs) on silica (SiO2). The bilayers are formed from small proteoliposomes, which convert to an SPB when the liposomes adsorb on the surface. The kinetics of this conversion process was followed in real time, using the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. The proteoliposomes were prepared by reconstitution of two different proteins into small unilamellar liposomes (diameter ∼ 26 nm), creating proteoliposomes with diameters ranging from ca. 50 to 85 nm, depending on protein concentration. The two proteins were proton translocating nicotinamide nucleotide transhydrogenase (TH) from Escherichia coli and gramicidin A (GrA) from Bacillus brevis. The SPB formation process was measured and compared for different protein situations in the liposomes: (i) with the intact TH in the proteoliposomes, (ii) after removal of the water-exposed, hydrophilic domains of ...

Formation of Supported Phospholipid Bilayers from Unilamellar Vesicles Investigated by Atomic Force Microscopy

Since their introduction through the work of McConnell et al. in the early 80s, supported phospholipid bilayers (SPBs) have proven to be a versatile model system for investigating a wide variety of phenomena. Despite their continuous application in fundamental as well as applied research fields, the mechanism by which SPBs are formed from suspensions of unilamellar vesicles remains poorly understood. Utilizing the ability of atomic force microscopy (AFM) to investigate processes in situ and in real time, we have studied the early stages of SPB formation on mica. Unilamellar vesicles of various sizes, composed of zwitterionic phospholipids, were prepared by sonication or extrusion. Vesicles of all sizes investigated were found to adsorb to mica. Unruptured vesicles forming supported vesicular layers (SVLs), as well as disks, formed as a result of vesicle rupture, could be visualized by AFM. The behavior of the SVLs was found to depend on the vesicle size, the lipid concentration, and the presence or absence of Ca2+. The picture of the mechanism of SPB formation, which emerges from the results presented in this report, is critically compared with theoretical predictions and experimental results reported to date.