Substrate-supported Lipid Bilayers Research Articles

Substrate-supported Model Membrane as a Versatile Analytical/Biosensing Platform.

Biological membranes composed of a lipid bilayer and associated proteins work as a platform for highly selective and sensitive detection in nature. Substrate-supported lipid bilayers (SLBs) are a model system of the biological membrane that are mechanically stable, accessible to highly sensitive analytical techniques, and amenable to micro-fabrication, such as patterning. The surface of SLBs can effectively suppress the non-specific binding of proteins, and enhance selective detection by specific interactions. These features render SLBs highly attractive for the development of devices that utilize artificially mimicked cellular functions. Furthermore, SLBs can be combined with nanoscopic spaces, such as nano-channels and nano-pores, that can reduce the detection volume and suppress the non-specific background noise, enhancing the signal-to-background noise (S/B) ratio. SLBs therefore provide promising platforms for a wide range of biomedical and environmental analyses.

Lipid flip-flop and desorption from supported lipid bilayers is independent of curvature.

Flip-flop of lipids of the lipid bilayer (LBL) constituting the plasma membrane (PM) plays a crucial role in a myriad of events ranging from cellular signaling and regulation of cell shapes to cell homeostasis, membrane asymmetry, phagocytosis, and cell apoptosis. While extensive research has been conducted to probe the lipid flip flop of planar lipid bilayers (LBLs), less is known regarding lipid flip-flop for highly curved, nanoscopic LBL systems despite the vast importance of membrane curvature in defining the morphology of cells and organelles and in maintaining a variety of cellular functions, enabling trafficking, and recruiting and localizing shape-responsive proteins. In this paper, we conduct molecular dynamics (MD) simulations to study the energetics, structure, and configuration of a lipid molecule undergoing flip-flop and desorption in a highly curved LBL, represented as a nanoparticle-supported lipid bilayer (NPSLBL) system. We compare our findings against those of a planar substrate supported lipid bilayer (PSSLBL). Our MD simulation results reveal that despite the vast differences in the curvature and other curvature-dictated properties (e.g., lipid packing fraction, difference in the number of lipids between inner and outer leaflets, etc.) between the NPSLBL and the PSSLBL, the energetics of lipid flip-flop and lipid desorption as well as the configuration of the lipid molecule undergoing lipid flip-flop are very similar for the NPSLBL and the PSSLBL. In other words, our results establish that the curvature of the LBL plays an insignificant role in lipid flip-flop and desorption.

Molecular Interactions Between Silver Nanoparticles and Model Cell Membranes

Silver (Ag) nanoparticles (NPs) are well known for their antibacterial properties. However, concerns have been raised on their possible toxicity to humans. This work is aimed to understand molecular interactions between Ag NPs and model mammalian cell membranes. Sum frequency generation (SFG) vibrational spectroscopy was used to study such interactions, supplemented by attenuated total reflectance–Fourier transform infrared spectroscopy (ATR–FTIR). Based on the SFG and ATR–FTIR results, it was found that Ag NPs could induce flip-flop of substrate supported lipid bilayers serving as model mammalian cell membranes. The Ag NPs could accumulate onto the model cell membrane and may aggregate. The Ag NP–model cell membrane interactions depend on the Ag NP solution concentration. At low Ag NP solution concentration, lipid flip-flop was observed. At higher Ag NP concentrations, Ag NPs caused lipid flip-flop faster and might aggregate. Therefore, the lipid flip-flop rates and Ag NP accumulation/aggregation rates are directly related to the Ag NP concentration of the subphase in contact with the lipid bilayer.

Real-time investigation of interactions between nanoparticles and cell membrane model.

Understanding the internal cellular processes of micron/nanoparticles will be important for particles toxicity studies or drug-delivery systems designing. The details and mechanisms of the cellular up-take process of micron/nano particles can hardly be described in real-time. In this study, cellular internalization of micron/nanoparticles was investigated by fluorescence spectroscopy, flow cytometry and sum frequency generation vibrational spectroscopy (SFG). Model cell membranes such as substrate supported lipid bilayers and lipid vesicles were used in this research. Especially, SFG was used to examine the behavior of each leaflet of the lipid bilayer while interacting with micron/nanoparticles. Experiments of SFG show direct evidences that micron/nanoparticles attachment lead to the lipid orientation. Vesicle dye-leakage model were used to study long-term interactions on model membrane. Results from this study provide in-depth insight into the molecular interactions between micron/nanoparticles and cell membranes, which will help to understand the particles toxicity and will be useful for the designing of micron/nanoparticles for applications.

Molecular interactions between amantadine and model cell membranes.

Sum frequency generation (SFG) vibrational spectroscopy was applied to study molecular interactions between amantadine and substrate supported lipid bilayers serving as model cell membranes. Both isotopically asymmetric and symmetric lipid bilayers were used in the research. SFG results elucidated how the water-soluble drug, amantadine, influenced the packing state of each leaflet of a lipid bilayer and how the drugs affected the lipid flip-flop process. It is difficult to achieve such detailed molecular-level information using other analytical techniques. Especially, from the flip-flop rate change of isotopically asymmetric lipid bilayer induced by amantadine, important information on the drug-membrane interaction mechanism can be derived. The results show that amantadine can be associated with zwitterionic PC bilayers but has a negligible influence on the flip-flop behavior of PC molecules unless at high concentrations. Different effects of amantadine on the lipid bilayer were observed for the negatively charged DPPG bilayer; low concentration amantadine (e.g., 0.20 mM) in the subphase could immediately disturb the outer lipid leaflet and then the lipid associated amantadine molecules gradually reorganize to cause the outer leaflet to return to the original orderly packed state. Higher concentration amantadine (e.g., 5.0 mM) immediately disordered the packing state of the outer lipid leaflet. For both the high and low concentration cases, amantadine molecules only bind to the outer PG leaflet and cannot translocate to the inner layer. The presence of amantadine within the negatively charged lipid layers has certain implications for using liposomes as drug delivery carriers for amantadine. Besides, by using PC or PG bilayers with both leaflets deuterated, we were able to examine how amantadine is distributed and/or oriented within the lipid bilayer. The present work demonstrates that SFG results can provide an in-depth understanding of the molecular mechanisms of interactions between water-soluble drugs and model cell membranes.

Lipid Reassembly in Asymmetric Langmuir–Blodgett/Langmuir–Schaeffer Bilayers

Molecular-reorganization-induced morphology alteration in asymmetric substrate-supported lipid bilayers (SLBs) was directly visualized by means of atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF) microscopy. SLB samples were fabricated on mica-on-glass and glass substrates by Langmuir-Blodgett (LB)/Langmuir-Schaeffer (LS) using binary lipid mixtures, namely, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and ternary mixtures DOPC/DPPC/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), labeled with 0.2 mol % Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (TR-DHPE) dye. Phase segregations were characterized by TIRF imaging, and DPPC-enriched domain structures were also observed. Interestingly for ∼40% (n = 6) of the samples with binary mixtures in the LB leaflet and a single component in the LS leaflet, that is, (DOPC/DPPC)(LB)+DOPC(LS), the contrast of the DPPC domains changed from the original dark (without dye) to bright (more TR dye partitioning) on TIRF images, returning to dark again. This contrast reverse was also correlated to AFM height images, where a DPPC-DPPC gel phase was spotted after the TIRF image contrast returned to dark. The rupture force mapping results measured on these binary mixture samples also confirmed unambiguously the formation of DPPC-DPPC gel domain components during the contrast change. The samples were tracked over 48 h to investigate the lipid molecule movements in both the DPPC domains and the DOPC fluid phase. The fluorescence contrast changes from bright to dark in SLBs indicate that the movement of dye molecules was independent of the movement of lipid molecules. In addition, correlated multimodal imaging using AFM, force mapping, and fluorescence provides a novel route to uncover the reorganization of lipid molecules at the solid-liquid interface, suggesting that the dynamics of dye molecules is highly structure dependent.

Investigations of the Interactions between Synthetic Antimicrobial Polymers and Substrate-Supported Lipid Bilayers Using Sum Frequency Generation Vibrational Spectroscopy

Sum frequency generation (SFG) vibrational spectroscopy was used to analyze interactions between solid-supported lipid bilayers acting as models for cellular membranes and several membrane-active random copolymers with different lipophilic side chains, named 0R (no group), 33Me (methyl group), 11Bz (benzyl group), and 33Bu (butyl group), according to both the identity and percentage of the side chains within the polymer. Biological tests of the minimum inhibitory concentration (MIC) and hemolytic concentration were performed. The inherent surface sensitivity of SFG allowed for independent monitoring of isotopically labeled lipid bilayer leaflets as a function of concentration to study polymer-bilayer interaction mechanisms. Concentrations at which each bilayer leaflet was disrupted were quantitatively determined for each copolymer. Spectroscopic evidence of interaction with the bilayer below the critical concentrations was observed for the 11Bz polymer. The lipophilic butyl side chain of the 33Bu polymer was found to be oriented parallel to the surface normal. This research shows that SFG is a useful analytical technique which provides unique details regarding the interaction mechanisms of these membrane-active copolymers and lipid bilayers.

Order at the Edge of the Bilayer: Membrane Remodeling at the Edge of a Planar Supported Bilayer Is Accompanied by a Localized Phase Change

We present experimental evidence for the existence of a unique molecular-level order in the vicinity of the bilayer's edge. Discrete patches of substrate-supported lipid bilayers exhibiting stable edge defects are prepared by confining vesicle fusion to hydrophilic patches of a chemically patterned substrate exhibiting hydrophilic patches in hydrophobic surrounding, and edge properties are characterized by fluorescence and vibrational spectroscopy based measurements. Specifically, wide-field fluorescence microscopy using phase-sensitive dyes, temperature-programmed fluorescence recovery measurements, and temperature-dependent attenuated total reflection Fourier transform infrared spectroscopy measurements are performed to characterize the local chain conformational properties, local diffusional characteristics, and phase discrimination afforded by phase-sensitive DiI fluorescent probes. We find that the bilayer structure near the edge is characterized by (1) an increase in intramolecular conformational order; (2) reduced effective lateral mobility; and (3) a distinctly higher local, effective gel-fluid transition temperature in comparison to their bulk counterpart. Together, these features signal the emergence of unique ordering presumably triggered by the hemimicellar configuration of the edge. These results are consistent with simulations of lyso-lipid micelles predicting the presence of dynamic clusters of ordered lipids in comparable micellar topology and disagrees with some recent interpretations of mobility near the edges of supported bilayers. Our results also offer the structural basis for the stability of defects and edges in fluid supported bilayers, and may be relevant in understanding the ordering and stabilization of pores, edges, and defects generated in membrane bilayers by proteins, curvature-sensitive lipids, antimicrobial peptides, and detergents.

Surface Electrostatics and Lipid-Substrate Interactions of Nanopore-Confined Lipid Bilayers

Substrate-supported lipid bilayers serve many purposes: from acting as versatile models of cellular membranes to biotechnological applications including substrate functionalization and stabilizing membrane proteins in functional conformations. While adsorption and subsequent reorganization of phospholipid vesicles on solid substrates were studied in the past, the exact nature of physicochemical interactions between the lipids and substrate surfaces remain largely unknown. Here we employed recently synthesized pH-sensitive spin-labeled phospholipids - derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE) with pH-reporting nitroxides that are covalently attached to the lipid's headgroup - to investigate surface electrostatics of nanotubular lipid bilayers confined in cylindrical nanopores. The lipid nanotubes were formed by self-assembling phospholipids inside ordered nanochannels of anodic aluminum oxide with pore diameters from 60 to 170 nm and diameter-to-pore length ratio of up to 1:1000. 31P NMR confirmed formation of macroscopically aligned lipid nanotubes with just 1-2° mosaic spread from zwitterionic DMPC, anionic DMPG, and their mixtures. Interfacial potentials were measured by carrying out titration experiments and observing the protonation state of the nitroxide tag by EPR. For nanopore-confined DMPC:DMPG (1:1) bilayers the protonation equilibrium was shifted to more acidic values: when the single lipid bilayer was deposited per nanopore the pKa of the nitroxide probe was shifted by (−0.91±0.05) pH units but only by (−0.34±0.05) when three bilayers per nanopore were present. Notably, the nitrogen hyperfine coupling constant for non-protonated nitroxides remained the same in all the samples indicating essentially the same interfacial dielectric environment. Thus, these shifts in pKa must come from changes in the lipid bialyer surface potential that was estimated to increase by 52±3 mV. EPR data on the lipid-substrate interface were combined with differential scanning calorimetry to elucidate effects of pore curvature, surface modification, and binding of antibacterial peptides on lipid-substrate interactions. Supported by DE-FG02-02ER15354.

Dependence of Antimicrobial Selectivity and Potency on Oligomer Structure Investigated Using Substrate Supported Lipid Bilayers and Sum Frequency Generation Vibrational Spectroscopy

Sum frequency generation (SFG) vibrational spectroscopy was used to study interactions between solid-supported lipid bilayers mimicking microbial and erythrocyte cellular membranes and synthetic antimicrobial arylamide oligomers named 2, 3, and 4, designed with the facial amphiphilicity common to naturally occurring antimicrobial peptides. The three compounds have the same backbone structure but varied side chains. The inherent interfacial sensitivity of SFG allowed for simultaneous monitoring of lipid ordering in the individual bilayer leaflets and orientation of 2, 3, and 4 upon interaction with the bilayer. Critical concentrations at which the inner leaflet is disrupted were determined for each oligomer. Spectral evidence of the oligomers' interaction with the bilayer below the critical concentrations was also found. Oligomers 2 and 3 tilted toward the bilayer surface normal, in agreement with previous experimental and simulation results. These oligomers selectively interact with microbial membrane models over erythrocyte membrane models, correlating well to previously published SFG studies on antimicrobial oligomer 1. It was shown that the oligomers interact with the lipid bilayers differently, indicating their different activity and selectivity. This research further shows that SFG is a particularly useful technique for the investigation of interaction mechanisms between cell membranes and membrane-active molecules. Additionally, SFG provides details of the specific interactions between these novel antimicrobials and lipid bilayers.

Structure from substrate supported lipid bilayers (Review)

Highly aligned, substrate supported membranes have made it possible for physical techniques to extract unambiguous structural information previously not accessible from commonly available membrane dispersions, or so-called powder samples. This review will highlight some of the major breakthroughs in model membrane research that have taken place as a result of substrate supported samples.

Multiple Orientation of Melittin inside a Single Lipid Bilayer Determined by Combined Vibrational Spectroscopic Studies

Despite the availability of several mature structure determination techniques for bulk proteins, determination of structural and orientational information of interfacial proteins, e.g., in cell membranes or on biomaterial surfaces, remains a difficult problem. We combine sum frequency generation (SFG) vibrational spectroscopy with attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) to investigate the orientation of alpha-helical peptides reconstituted in substrate supported lipid bilayers. Melittin was chosen as a model for alpha-helical peptides, and its orientation when interacting with a supported 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) bilayer has been examined. Through polarization analysis using amide I signals obtained from both SFG and ATR-FTIR measurements, the orientation distribution of melittin inside a DPPG bilayer was deduced using several trial distribution functions. Melittin was modeled as either an ideal helix or a helix with a bent structure. It was found that a simple distribution function such as a delta-distribution or a Gaussian distribution was not adequate to describe the melittin orientation distribution inside a DPPG bilayer. Instead, two populations of melittin, corresponding to two melittin-bilayer association states, could be used to interpret the experimentally observed result. The method employed in this study demonstrates the feasibility of acquiring a more accurate orientation distribution of peptides/proteins in situ using a combination of vibrational spectroscopic techniques without exogenous labeling.

Neutron and X-Ray Diffraction Studies of Substrate Supported Lipid Bilayers

not Available.

Observing a Molecular Knife at Work

Sum frequency generation (SFG) vibrational spectroscopy has been employed to study the molecular interactions between a single substrate supported lipid bilayer and an amphiphilic antibiotic compound 1, with a design based on the common structural motif of natural antimicrobial peptides. The interfacial sensitivity of SFG allows real-time in situ monitoring of ordering changes in both leaflets of the bilayer and orientation of 1 simultaneously. A critical concentration of about 0.8 microg/mL of 1 is found, above which the inner leaflet of the bilayer is significantly perturbed. This concentration corresponds well to the minimum inhibition concentration of 1 that is obtained from bacterial experiments. Orientation of 1 in the bilayer is shown to be perpendicular to the bilayer surface, in agreement with simulation results. SFG can be developed into a very informative technique for studying the cell membrane and the interactions of membrane-active molecules.

Atomic force microscopy assisted immobilization of lipid vesicles.

We report on a new approach to direct the immobilization of unilamellar lipid vesicles on substrate-supported lipid bilayers in a spatially confined manner. The adsorption of vesicles from solution is limited to areas of disorder in the bilayers, which is induced by scanning a pattern in situ with an atomic force microscopy (AFM) tip using high imaging forces. Lines of vesicles with a length exceeding 25 microm and a width corresponding to that of a single surface-immobilized vesicle have been fabricated. The adsorbed vesicles are effectively immobilized and do not desorb spontaneously. However, AFM with forces of several nanoNewtons allows one to displace vesicles selectively. The novel methodology described, which may serve as a platform for research on proteins incorporated in the lipid bilayers comprising the vesicles, does not require chemical labeling of the vesicles to guide their deposition.

Planar supported bilayer polymers formed from bis-diene lipids by Langmuir-Blodgett deposition and UV irradiation.

Substrate-supported lipid bilayers have been prepared from bis-diene functionalized phosphorylcholine (PC) lipids and polymerized by UV irradiation. The overall bilayer structure is largely preserved upon removal from water, although significant loss of material occurs from the upper leaflet of the bilayer, likely due to desorption at the air/water interface. The morphology and surface structure of the bilayer, as observed by AFM, indicate a substantially different arrangement of the lipids in the hydrated and dehydrated states, presumably due to the loss of water from the near surface region. These changes have been correlated with infrared spectral shifts sensitive to the conformation of the hydrocarbon chains. Protein adsorption studies show that rehydrated, polymerized bilayers retain a degree of resistance to BSA adsorption intermediate between model hydrophobic and fluid PC lipid bilayer surfaces. The degree of protein adsorption is correlated with desorption of material from the upper leaflet of the bilayer upon drying, which produces voids at which hydrophobically driven protein adsorption occurs.

光リソグラフィーを用いた脂質二分子膜の微細パターン化技術

光リソグラフィーを用いた脂質二分子膜の微細パターン化技術

Part 1. Direct Measurement of Depletion Attraction and Thin Film Viscosity between Lipid Bilayers in Aqueous Polyethylene Glycol Solutions

Using the surface forces apparatus, we have measured the static and hydrodynamic forces between two substrate supported lipid bilayers in water and semidilute aqueous solutions of 8000 MW poly(ethylene glycol) (PEG), a neutral polymer for which water is a good solvent. At small separations (D < RF, Flory radius), an enhanced adhesion due to depletion attraction was measured. Conversely, at larger separations we found a repulsive barrier, which is not predicted by mean-field theories. Dynamic measurements of the viscosity as a function of film thickness reflected the same pattern of behavior as exhibited in the static force experiments: Thus, in the absence of polymer, the shear plane was at the lipid-water interface, while in the 8000 MW polymer solution the viscosity decreased as a function of decreasing surface separation due to a polymer-depleted layer of lower viscosity at the bilayer surface. We discuss our results in terms of depletion attraction, depletion stabilization, thin film lubrication, the...

Part 2. Crossover from Depletion Attraction to Adsorption: Polyethylene Glycol Induced Electrostatic Repulsion between Lipid Bilayers

Using the surface forces apparatus, we have measured the static and hydrodynamic forces between two substrate-supported lipid bilayers in water and semidilute, aqueous solutions of poly(ethylene glycol) (PEG), a neutral polymer for which water is a good solvent. In part 1 (previous paper in this issue), we presented detailed measurements of depletion attraction in the presence of 8000 molecular weight PEG. In part 2 (this work), we address the behavior of higher molecular weight 20 000 PEG. Specifically, the higher molecular weight PEG adsorbs to the surfaces and both a steric as well as an unexpected electrostatic repulsion were measured. We determined that higher MW PEG chelates metal cations and thereby induces an apparent electrostatic charge on the bilayer surfaces upon adsorption.