The formation of supported lipid bilayers on silica nanoparticles revealed by cryoelectron microscopy

The controlled fabrication of biocompatible devices made of lipid bilayers deposited onto flat solid supports presents interest as models of cell membranes as well as for their biotechnological applications. We report here on the formation of supported lipid bilayers on silica nanoparticles (nanoSLBs). The successive steps of the adsorption of lipid vesicles on nanoparticles and the formation of nanoSLBs are revealed in detail by cryotransmission electron microscopy (cryo-EM). The formation of nanoSLBs was achieved for liposomes with positive, neutral, and low net negative charge, while liposomes with a high net negative charge adsorbed to silica nanoparticles but did not rupture. The nanoSLBs were found to follow faithfully the surface contours of the particles, information yet unavailable for SLB formation on planar solid substrates.     

Titration force microscopy on supported lipid bilayers

The use of chemically modified atomic force microscopy (AFM) probes allows us to measure the surface charges of supported planar lipid bilayers with high sensitivity through the force spectroscopy operation mode. By controlling the chemistry of the tip, we can perform a classical analytical chemistry titration where the titration agent is a weak acid (attached to the AFM tip) with the particularity of being performed in surface rather than in solution and, especially, at the nanometric scale. Thus, the AFM tip acts as a real "nanosensor". The approaching curves of the force plots reveal that electrostatic interactions between the tip and the supported membrane play a key role. Besides, the plot of the adhesion force (measured from the retracting curve of the force plots) versus pH displays a nonsigmoidal shape with a peak in the adhesion force attributed to high-energy hydrogen bonds. One of these peaks corresponds to the pKa of the surface under study and the other to the pKa of the titrating probe attached to the tip.     

Non-Brownian diffusion of membrane molecules in nanopatterned supported lipid bilayers

Molecules associated with the outer surface of living cells exhibit complex, non-Brownian patterns of diffusion. In this report, supported lipid bilayers were patterned with nanoscale barriers to capture key aspects of this anomalous diffusion in a controllable format. First, long-range diffusion coefficients of membrane-associated molecules were significantly reduced by the presence of the barriers, while short-range diffusion was unaffected. Second, this modulation was more pronounced for large molecular complexes than for individual lipids. Surprisingly, the quantitative effect of these barriers on long-range lipid diffusion could be accurately simulated using a simple, continuum-based model of diffusion on a nanostructured surface; we thus describe a metamaterial that captures the properties of the outer membrane of living cells.     

Effect of C60 on solid supported lipid bilayers

We show that water-soluble fullerenes accumulate on the surface of zwitterionic and cationic supported bilayers to different extents. We propose on the basis of bilayer thicknesses, phase-transition temperatures, and fullerene movement that the water-soluble fullerenes do not penetrate into the hydrocarbon tails of supported bilayers. These findings are important to toxicity issues concerning fullerene materials and the development of decorated lipid bilayers for future drug delivery or sensor application.     

Protein separation by electrophoretic-electroosmotic focusing on supported lipid bilayers

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field, and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch.     

Probing insertion and solubilization effects of lysolipids on supported lipid bilayers using microcantilevers

The interaction of surfactants with lipid membranes can result in composition change, area expansion, solubilization, or the formation of protrusion features of the membranes. Amphipathic surfactant molecules are simplified analogues to membrane-active drugs and peptides which are known for inserting into lipid bilayers; however, the effect of these amphipathic molecules on supported membranes is not well characterized. In this paper we explore the use of microcantilever sensors to quantify surfactants' effects on lipid membranes. We use microcantilevers which are coated with lipid membranes to probe the interactions between lysolipids and supported lipid bilayers (SLBs). In particular, we investigate the effects of four zwitterionic surfactants similar to phospholipids: lysolipids of different aliphatic chain lengths (lysophosphocholines, lysoPCs, 12:0, 14:0, 16:0, and 18:0) on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine-supported lipid bilayers. By monitoring the deflection of the microcantilevers, real-time free energy changes in the SLBs upon the addition of lysolipids can be detected. Additionally, the bending direction reveals whether the lysoPCs incorporate into or solubilize the SLB. When the bulk lysoPC concentration is less than its critical micelle concentration (CMC), we observe a compressive bending of the microcantilever, indicating adsorption to the SLB. Additionally, the change in surface stress is found to be proportional to the amount of membrane-bound lysoPCs. For bulk concentrations greater than the CMC, lysoPCs 12:0 and 14:0, there is tensile bending, indicating that the lysoPCs begin to solubilize and destroy the SLBs. Interestingly, this is not observed for lysoPCs with longer chain lengths. This new method of using microcantilevers for detecting and quantifying the surfactant insertion and solubilization of SLBs offers additional insights into the interactions between small amphipathic molecules and lipid membranes.     

Anionic solid lipid nanoparticles supported on protamine/DNA complexes

The objective of this study was to design novel anionic ternary nanoparticles for gene delivery. These ternary nanoparticles were equipped with protamine/DNA binary complexes (150-200?nm) as the support, and the anionic formation was achieved by absorption of anionic solid lipid nanoparticles (≤20?nm) onto the surface of the binary complexes. The small solid lipid nanoparticles (SLNs) were prepared by a modified film dispersion-ultrasonication method, and adsorption of the anionic SLNs onto the binary complexes was typically carried out in water via electrostatic interaction. The formulated ternary nanoparticles were found to be relatively uniform in size (257.7 ± 10.6?nm) with a 'bumpy' surface, and the surface charge inversion from 19.28 ± 1.14?mV to -17.16 ± 1.92?mV could be considered as evidence of the formation of the ternary nanoparticles. The fluorescence intensity measurements from three batches of the ternary nanoparticles gave a mean adsorption efficiency of 96.75 ± 1.13%. Circular dichroism spectra analysis showed that the protamine/DNA complexes had been coated by small SLNs, and that the anionic ternary nanoparticles formed did not disturb the construction of the binary complexes. SYBR Green I analysis suggested that the ternary nanoparticles could protect the DNA from nuclease degradation, and cell viability assay results showed that they exhibit lower cytotoxicity to A549 cells compared with the binary complexes and lipofectamine. The transfection efficiency of the ternary nanoparticles was better than that of naked DNA and the binary complexes, and almost equal to that of lipofectamine/DNA complexes, as revealed by inversion fluorescence microscope observation. These results indicated that the anionic ternary nanoparticles could facilitate gene transfer in cultured cells, and might alleviate the drawbacks of the conventional cationic vector/DNA complexes for gene delivery in vivo.     

Evanescent wave cavity ringdown spectroscopy: a platform for the study of supported lipid bilayers

Evanescent wave cavity ringdown spectroscopy (EW-CRDS) is advocated as an approach for monitoring the formation of supported lipid bilayers (SLBs) on quartz substrates in situ and for the quantitative study of fast molecular adsorption kinetics at the resulting modified biomimetic surface. This approach is illustrated using SLBs of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Complementary atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) measurements confirm the formation of bilayers on quartz. The subsequent interaction of the porphyrin, 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p',p',p'-tetrasulfonic acid tetrasodium hydrate (TPPS) with the cationic bilayer-modified silica surface has been studied using EW-CRDS combined with an impinging-jet to deliver analyte to the surface in a well-defined manner. The adsorption of TPPS to the bilayer was kinetically controlled and the adsorption rate constant was found to be 1.7 (±0.6) × 10(-4) cm s(-1) from finite element modeling of the jet hydrodynamics and associated convective-diffusion equation, coupled to a first-order surface process describing adsorption. These proof-of-concept studies provide a platform for the investigation of molecular processes at biomembranes using EW-CRDS for chemical species showing optical absorbance in the visible and ultraviolet range.     

Formation of lipid sheaths around nanoparticle-supported lipid bilayers

High-surface-area nanoparticles often cluster, with unknown effects on their cellular uptake and environmental impact. In the presence of vesicles or cell membranes, lipid adsorption can occur on the nanoparticles, resulting in the formation of supported lipid bilayers (SLBs), which tend to resist cellular uptake. When the amount of lipid available is in excess compared with that required to form a single-SLB, large aggregates of SLBs enclosed by a close-fitting lipid bilayer sheath are shown to form. The proposed mechanism for this process is one where small unilamellar vesicles (SUVs) adsorb to aggregates of SLBs just above the gel-to-liquid phase transition temperature, T(m) , of the lipids (as observed by dynamic light scattering), and then fuse with each other (rather than to the underlying SLBs) upon cooling below T(m) . The sacks of SLB nanoparticles that are formed are encapsulated by the contiguous close-fitting lipid sheath, and precipitate below T(m) , due to reduced hydration repulsion and the absence of undulation/protrusion forces for the lipids attached to the solid support. The single-SLBs can be released above T(m) , where these forces are restored by the free lipid vesicles. This mechanism may be useful for encapsulation/release of drugs/DNA, and has implications for the toxic effects of nanoparticles, which may be mitigated by lipid sequestration.     

Photocatalytic behavior of TiO2 nanoparticles supported on porous aluminosilicate surface modified by cationic surfactant

In order to utilize the photocatalytic function of TiO₂ nanoparticles in materials manufactured from organic polymeric compounds, such as paper, resins, and textiles, TiO₂ nanoparticles supported on aluminosilicate, which contained 1, 5, and 10 wt% of TiO₂ were prepared by mixing commercial TiO₂ nanoparticles and porous aluminosilicate at pH 7 in a cationic surfactant aqueous solution. Most of the supported TiO₂ nanoparticles on the aluminosilicate surface were observed by TEM–EDS (energy depressive X-ray spectroscopy) analysis. TiO₂ nanoparticles supported on aluminosilicate reduced the formaldehyde concentration from 20 to 0 ppm after UV irradiation for 20 h; the reduction of formaldehyde concentration under UV irradiation was obviously different from that in the dark. Moreover, a paper mixed with 20 wt% of TiO₂ nanoparticles supported on aluminosilicate bleached the stains colored with cigarette tar after UV irradiation for 6 h. However, the paper maintained its initial tensile strength even after UV irradiation for 1 year; in contrast, the paper mixed with a simple dry mixture of TiO₂ powder and aluminosilicate lost approximately half of its initial tensile strength after a year. TiO₂ nanoparticles supported on aluminosilicate could exhibit photocatalytic activity without decomposing the organic polymeric compounds.     

Poly(aniline) nanowires in sol-gel coated ITO: a pH-responsive substrate for planar supported lipid bilayers

Facilitated ion transport across an artificial lipid bilayer coupled to a solid substrate is a function common to several types of bioelectronic devices based on supported membranes, including biomimetic fuel cells and ion channel biosensors. Described here is fabrication of a pH-sensitive transducer composed of a porous sol-gel layer derivatized with poly(aniline) (PANI) nanowires grown from an underlying planar indium-tin oxide (ITO) electrode. The upper sol-gel surface is hydrophilic, smooth, and compatible with deposition of a planar supported lipid bilayer (PSLB) formed via vesicle fusion. Conducting tip AFM was used to show that the PANI wires are connected to the ITO, which convert this electrode into a potentiometric pH sensor. The response to changes in the pH of the buffer contacting the PANI nanowire/sol-gel/ITO electrode is blocked by the very low ion permeability of the overlying fluid PSLB. The feasibility of using this assembly to monitor facilitated proton transport across the PSLB was demonstrated by doping the membrane with lipophilic ionophores that respond to a transmembrane pH gradient, which produced an apparent proton permeability several orders of magnitude greater than values measured for undoped lipid bilayers.     

Infrared spectroscopy of fluid lipid bilayers

Infrared spectroscopy is a powerful technique for examining lipid bilayers; however, it says little about the fluidity of the bilayer-a key physical aspect. It is shown here that it is possible to both acquire spectroscopic data of supported lipid bilayer samples and make measurements of the membrane fluidity. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR) is used to obtain the spectroscopic information and fluorescence recovery after photobleaching (FRAP) is used to determine the fluidity of the samples. In the infrared spectra of lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the following major peaks were observed; nu(as)(CH3) 2954 cm(-1), nu(s)(CH3) 2870 cm(-1), nu(as)(CH2) 2924 cm(-1), nu(s)(CH2) 2852 cm(-1), nu(C=O) 1734 cm(-1), delta(CH2) 1463-1473 cm(-1), nu(as)(PO2-) 1226 cm(-1), nu(s)(PO2-) 1084 cm(-1), and nu(as)(N+(CH3)3) 973 cm(-1). The diffusion coefficient of the same lipid bilayer was measured to be 3.5 +/- 0.5 micom(2)/s with visual recovery also noted through use of epifluorescence microscopy. FRAP and visual data confirm the formation of a uniform, mobile supported lipid bilayer. The combination of ATR-FT-IR and FRAP provides complementary data giving a more complete picture of fully hydrated model membrane systems.     

Degradative transport of cationic amphiphilic drugs across phospholipid bilayers

Drug molecules must cross multiple cell membrane barriers to reach their site of action. We present evidence that one of the largest classes of pharmaceutical drug molecules, the cationic amphiphilic drugs (CADs), does so via a catalytic reaction that degrades the phospholipid fabric of the membrane. We find that CADs partition rapidly to the polar-apolar region of the membrane. At physiological pH, the protonated groups on the CAD catalyse the acid hydrolysis of the ester linkage present in the phospholipid chains, producing a fatty acid and a single-chain lipid. The single-chain lipids rapidly destabilize the membrane, causing membranous fragments to separate and diffuse away from the host. These membrane fragments carry the drug molecules with them. The entire process, from drug adsorption to drug release within micelles, occurs on a time-scale of seconds, compatible with in vivo drug diffusion rates. Given the rate at which the reaction occurs, it is probable that this process is a significant mechanism for drug transport.     

Optical detection of ion-channel-induced proton transport in supported phospholipid bilayers

The integration of ion-channel transport functions with responses derived from nanostructured and nanoporous silica mesophase materials is demonstrated. Patterned thin-film mesophases consisting of alternating hydrophilic nanoporous regions and hydrophobic nanostructured regions allow for spatially localized proton transport via selective dimerization of gramicidin in lipid bilayers formed on the hydrophilic regions. The adjoining hydrophobic mesostructure doped with a pH sensitive dye reports the transport. The ease of integrating functional membranes and reporters through the use of patterned mesophases should enable high throughput studies of membrane transport.     

Neutron reflectometry of supported hybrid bilayers with inserted peptide

The insertion of a synthetic amphiphilic, α-helical peptide into a supported hybrid bilayer membrane (HBM) was studied by neutron reflectometry to elucidate the resulting nanostructure. The HBM consisted of a self-assembled monolayer of perdeuterated octadecanethiol on gold and an overlying leaflet of acyl-deuterated phosphatidylcholine (d-DMPC). Using contrast variation, several reflectivity spectra were recorded for each step of film fabrication, and simultaneously modeled. This analysis indicated that peptide insertion into the DMPC lipid leaflet is the likeliest mode of incorporation.     

Simple reconstitution of protein pores in nano lipid bilayers

We developed a new, simple and robust approach for rapid screening of single molecule interactions with protein channels. Our glass nanopipets can be fabricated simply by drawing glass capillaries in a standard pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ～140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by electrical and mechanical forces. We demonstrate the functional reconstitution of the E. coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-molecule level, demonstrating the ability of this method to detect single molecule interactions with protein channels. High-resolution conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-molecule studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.     

Surface properties of encapsulating hydrophobic nanoparticles regulate the main phase transition temperature of lipid bilayers: A simulation study

The main phase transition temperature of a lipid membrane, which is vital for its biomedical applications such as controllable drug release, can be regulated by encapsulating hydrophobic nanoparticles into the membrane. However, the exact relationship between surface properties of the encapsulating nanoparticles and the main phase transition temperature of a lipid membrane is far from clear. In the present work we performed coarse-grained molecular dynamics simulations to meet this end. The results show the surface roughness of nanoparticles and the density of surface-modifying molecules on the nanoparticles are responsible for the regulation. Increasing the surface roughness of the nanoparticles increases the main phase transition temperature of the lipid membrane, whereas it can be decreased in a nonlinear way via increasing the density of surface-modifying molecules on the nanoparticles. The results may provide insights for understanding recent experimental studies and promote the applications of nanoparticles in controllable drug release by regulating the main phase transition temperature of lipid vesicles.     

Simulation of fusion-mediated nanoemulsion interactions with model lipid bilayers

Perfluorocarbon-based nanoemulsion particles have become promising platforms for the delivery of therapeutic and diagnostic agents to specific target cells in a non-invasive manner. A "contact-facilitated" delivery mechanism has been proposed wherein the emulsifying phospholipid monolayer on the nanoemulsion surface contacts and forms a lipid complex with the outer monolayer of target cell plasma membrane, allowing cargo to diffuse to the surface of target cell. While this mechanism is supported by experimental evidence, its molecular details are unknown. The present study develops a coarse-grained model of nanoemulsion particles that are compatible with the MARTINI force field. Simulations using this coarse-grained model have demonstrated multiple fusion events between the particles and a model vesicular lipid bilayer. The fusion proceeds in the following sequence: dehydration at the interface, close apposition of the particles, protrusion of hydrophobic molecules to the particle surface, transient lipid complex formation, absorption of nanoemulsion into the liposome. The initial monolayer disruption acts as a rate-limiting step and is strongly influenced by particle size as well as by the presence of phospholipids supporting negative spontaneous curvature. The core-forming perfluorocarbons play critical roles in initiating the fusion process by facilitating protrusion of hydrophobic moieties into the interface between the two particles. This study directly supports the hypothesized nanoemulsion delivery mechanism and provides the underlying molecular details that enable engineering of nanoemulsions for a variety of medical applications.     

Excitation of Cy5 in self-assembled lipid bilayers using optical microresonators

Due to their sensitivity and temporal response, optical microresonators are used extensively in the biosensor arena, particularly in the development of label-free diagnostics and measurement of protein kinetics. In the present letter, we investigate using microcavities to probe molecules within biomimetic membranes. Specifically, a method for self-assembling lipid bilayers on spherical microresonators is developed and the bilayer-nature is verified. Subsequently, the microcavity is used to excite a Cy5-conjugated lipid located within the bilayer while the optical performance of the microcavity is characterized. The emission wavelength of the dye and the optical behavior of the microcavity agree with theoretical predictions.