silicon and 1-2-oleoylphosphatidylcholine

Supported lipid bilayers are model membranes formed at solid substrate surfaces. This architecture renders the membrane experimentally accessible to surface-sensitive techniques used to study their properties, including atomic force microscopy, optical fluorescence microscopy, quartz crystal microbalance, and X-ray/neutron reflectometry, and allows integration with technology for potential biotechnological applications such as drug screening devices. The experimental technique often dictates substrate choice or treatment, and it is anecdotally recognized that certain substrates are suitable for a particular experiment, but the exact influence of the substrate has not been comprehensively investigated. Here, we study the behavior of a simple model bilayer, phase-separating on a variety of commonly used substrates, including glass, mica, silicon, and quartz, with drastically different results. The distinct micron-scale domains observed on mica, identical to those seen in free-floating giant unilamellar vesicles, are reduced to nanometer-scale domains on glass and quartz. The mechanism for the arrest of domain formation is investigated, and the most likely candidate is nanoscale surface roughness, acting as a drag on the hydrodynamic motion of small domains during phase separation. Evidence was found that the physicochemical properties of the surface have a mediating effect, most likely because of the changes in the lubricating interstitial water layer between the surface and bilayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Aluminum Silicates; Diffusion; Glass; Lipid Bilayers; Membrane Microdomains; Phosphatidylcholines; Phosphatidylethanolamines; Quartz; Silicon; Surface Properties

A novel surface functionalization technique is presented for large-scale selective molecule deposition onto whispering gallery mode microgoblet cavities. The parallel technique allows damage-free individual functionalization of the cavities, arranged on-chip in densely packaged arrays. As the stamp pad a glass slide is utilized, bearing phospholipids with different functional head groups. Coated microcavities are characterized and demonstrated as biosensors.

Topics: Biosensing Techniques; Fluorescent Dyes; Glass; Microscopy; Microscopy, Fluorescence; Phosphatidylcholines; Phospholipids; Silicon; Surface Properties

The architecture of the plasma membrane is not only determined by the lipid and protein composition, but is also influenced by its attachment to the underlying cytoskeleton. Herein, we show that microscopic phase separation of "raft-like" lipid mixtures in pore-spanning bilayers is strongly determined by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb(3) were prepared on ordered pore arrays in silicon with pore diameters of 0.8, 1.2 and 2 μm, respectively, by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol-bearing cholesterol derivative rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liquid-ordered- (l(o)) and liquid-disordered (l(d)) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb(3) (40:35:20:5) membranes. The size of the l(o)-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl-β-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb(3)-doped membranes increased the l(o)-phase considerably and even induced l(o)-phase domains in non-phase separated bilayers composed of DOPC/sphingomyelin/cholesterol/Gb(3) (65:10:20:5).

Topics: beta-Cyclodextrins; Cholesterol; Gold; Lipid Bilayers; Microscopy, Fluorescence; Phase Transition; Phosphatidylcholines; Porosity; Protein Binding; Receptors, Cell Surface; Shiga Toxin 2; Silicon; Sphingomyelins; Temperature; Unilamellar Liposomes

Cationic amphiphile DDAB (dimethyl-dioctadecyl-ammonium-bromide) can spontaneously form water-dispersed and solid supported mimicking biomembrane structures as well as valuable DNA delivery vehicles whose shape, stability and transfection efficiency can be easily optimized on varying temperature, water content and chemical composition. In this framework, disclosing the thermotropic behavior of DDAB assemblies can be considered as an essential step in conceiving and developing new non-viral vector systems. Our work has been focused primarily on understanding the mesophase structure of silicon supported DDAB thin film on varying temperature at constant relative humidity by energy dispersive X-ray diffraction (EDXD). Diffraction results have then been employed in providing a more comprehensive dynamic light scattering (DLS) analysis of corresponding thermotropic water dispersed vesicles made up of DDAB alone and in combination with helper lecithin DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) liposomes. We found that above 55 °C silicon-supported DDAB films undergo a significant thinning effect, whilst DDAB-water vesicles exhibit a reduction in size polydispersity. Upon cooling to 25 °C a distinct silicon supported DDAB mesophase, exhibiting a relative humidity-dependent spacing, has been pointed out, and modeled in terms of a lyotropic metastable gel-crystalline phase.DDAB/DOPC-water vesicles show a temperature-dependent switching in size distribution, leading to promising biomedical applications.

Topics: Humidity; Light; Membranes, Artificial; Molecular Structure; Phosphatidylcholines; Quaternary Ammonium Compounds; Scattering, Radiation; Silicon; Surface-Active Agents; Temperature; Water; X-Ray Diffraction

Ethanol-lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Alcohols; Biophysics; Ethanol; Gels; Lipid Bilayers; Membrane Fluidity; Membrane Microdomains; Microscopy, Atomic Force; Phosphatidylcholines; Phospholipids; Protein Structure, Tertiary; Silicon

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms.

Topics: Animals; Cell Line, Tumor; Female; Gene Silencing; Genetic Therapy; Humans; Liposomes; Mice; Mice, Nude; Nanoparticles; Ovarian Neoplasms; Phosphatidylcholines; Receptor, EphA2; RNA, Small Interfering; Silicon; Xenograft Model Antitumor Assays

Specular neutron reflectivity was used to study the time course and nature of the interaction of the positively charged, peripheral membrane protein cytochrome c with supported bilayers of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) containing the anionic lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylserine (POPS). The supported bilayers were prepared by deposition on silicon blocks of two monolayers of DOPC, the second of which contained either 10 or 20 mol % POPS at surface pressures of either 15 or 20 mN/m using a combination of Langmuir-Blodgett and Schaefer deposition techniques. Each supported bilayer was initially characterized by specular neutron reflectivity using subphases of 10 mM NaCl aqueous solutions. Regardless of POPS content and bilayer deposition pressure, the molecular architecture of the bilayers was similar. The addition of cytochrome c resulted in an almost immediate change in reflectivity, which was well modeled by assuming that an additional layer was present next to the outer leaflet of the bilayer. The thickness of this layer, which contained the volume fraction of approximately 15% protein, was approximately 30 A (consistent with the cross-section of a single cytochrome c molecule). The addition of cytochrome c to the subphase also resulted in a change in the structure of the phospholipid bilayer, suggesting some penetration of cytochrome c into the bilayer. Specular neutron reflectivity studies after careful washing with solvent showed that although most of the protein was washed off by flushing 10 mM NaCl D2O through the cell a small amount remained both within the bilayer and bound to the membrane surface.

Topics: Cell Membrane; Cytochromes c; Lipid Bilayers; Neutron Diffraction; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Silicon; Sodium Chloride

We conducted time-dependent, in situ x-ray reflectivity measurements on the formation of substrate-supported lipid monolayers and bilayers at solid-liquid interfaces, buried under an aqueous buffer with various concentrations (5, 10, 20, 40, and 50 microg/ml ) of lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The DOPC bilayer is formed on the hydrophilic surface of a bare Si substrate, while the DOPC monolayer is formed on a hydrophobic octadecylthricholorsilane (OTS) monolayer-coated Si substrate. The evolution of the reflectivity curves from the lipid bilayers is well described by lateral growth of bilayer islands, consistent with the rupture and fusion model for the adsorption of lipid vesicles to solid-liquid interfaces. By contrast, the formation of the lipid monolayer on OTS-coated Si occurs through a relatively fast coverage of the entire interfacial area, followed by an increase in the monolayer thickness. For both monolayers and bilayers, the rate of lipid layer growth increases with increasing lipid concentration in the buffer solution.

Topics: Adsorption; Biophysics; Dose-Response Relationship, Drug; Electrons; Lipid Bilayers; Lipids; Membrane Lipids; Molecular Conformation; Phosphatidylcholines; Phospholipids; Silanes; Silicon; Surface Properties; Time Factors; X-Rays

Curved lipid membranes are ubiquitous in living systems and play an important role in many biological processes. To understand how curvature and lipid composition affect membrane formation and fluidity, we have assembled and studied mixed 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) supported lipid bilayers on amorphous silicon nanowires grown around carbon nanotube cores with controlled wire diameters ranging from 20 to 200 nm. We found that lipid vesicles fused onto nanowire substrates and formed continuous bilayers for all DOPC-DOPE mixtures tested (with the DOPE content of up to 30%). Our measurements demonstrate that nanowire-supported bilayers are mobile, exhibit fast recovery after photobleaching, and have a low concentration of defects. Lipid diffusion coefficients in these high-curvature tubular membranes are comparable to the values reported for flat supported bilayers and increase slightly with decreasing nanowire diameter. A free space diffusion model adequately describes the effect of bilayer curvature on the lipid mobility for nanowire substrates with diameters greater than 50 nm, but shows significant deviations from the experimental values for smaller diameter nanowires.

Topics: Biotechnology; Diffusion; Equipment Design; Lipid Bilayers; Lipids; Microscopy, Confocal; Nanotechnology; Nanowires; Phosphatidylcholines; Phosphatidylethanolamines; Silicon; Time Factors

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.

Topics: Aluminum Silicates; Biophysics; Biotechnology; Calcium; Cell Membrane; Edetic Acid; Fatty Acids, Monounsaturated; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylserines; Polyethylene Glycols; Quaternary Ammonium Compounds; Silicon; Silicon Dioxide; Time Factors

Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir-Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater. We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur. The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.

Topics: Biophysical Phenomena; Biophysics; Fatty Acids, Monounsaturated; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Phosphatidylcholines; Quaternary Ammonium Compounds; Silicon; Surface Properties

Annexin A5 is a protein that binds to membranes containing negatively charged phospholipids in a calcium-dependent manner. We previously found that annexin A5 self-assembles into two-dimensional (2D) crystals on supported lipid bilayers (SLBs) formed on mica while a monolayer of disordered trimers is formed on SLBs on silica. Here, we investigated in detail and correlated the adsorption kinetics of annexin A5 on SLBs, supported on silica and on mica, with the protein's 2D self-assembly behavior. For this study, quartz crystal microbalance with dissipation monitoring and ellipsometry were combined with atomic force microscopy. We find, in agreement with previous studies, that the adsorption behavior is strongly dependent on the concentration of dioleoylphosphatidylserine (DOPS) in the SLB and the calcium concentration in solution. The adsorption kinetics of annexin A5 are similar on silica-SLBs and on mica-SLBs, when taking into account the difference in accessible DOPS between silica-SLBs and mica-SLBs. In contrast, 2D crystals of annexin A5 form readily on mica-SLBs, even at low protein coverage (< or =10%), whereas they are not found on silica-SLBs, except in a narrow range close to maximal coverage. These results enable us to construct the phase diagram for the membrane binding and the states of 2D organization of annexin A5. The protein binds to the membrane in two different fractions, one reversible and the other irreversible, at a given calcium concentration. The adsorption is determined by the interaction of protein monomers with the membrane. We propose that the local membrane environment, as defined by the presence of DOPS, DOPC, and calcium ions, controls the adsorption and reversibility of protein binding.

Topics: Adsorption; Animals; Annexin A5; Calcium; Crystallization; Crystallography, X-Ray; Dimerization; Kinetics; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Rats; Recombinant Proteins; Silicon; Time Factors

We report on the normal and lateral forces between controlled-density mono- and bilayers of phospholipid co-adsorbed onto hydrophobic and hydrophilic solid supports, respectively. Interactions between 1,2-dioleoyl-sn-glycero-3-phosphocholine layers were measured using an atomic force microscope. Notable features of the normal force curves (barrier heights and widths) were found to correlate with the thickness and density of the supported lipid layers. The friction and normal force curves were also found interrelated. Thus, very low friction values were measured as long as the supported layer(s) resisted the normal pressure of the tip. However, as the applied load exceeded the critical value needed for puncturing the layers, the friction jumped to values close to those recorded between bare surfaces. The lipid layers were self-healing between measurements, but a significant hysteresis was observed in the force curves measured on approach and retraction, respectively. The study shows the potential of using atomic force microscopy for lipid layer characterization both with respect to structure and interactions. It further shows the strong lubricating effect of adsorbed lipid layers and how this varies with surface density of lipids. The findings may have important implications for the issue of joint lubrication.

Topics: Adsorption; Biophysical Phenomena; Biophysics; Detergents; Glucosides; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Silicon; Time Factors