The role of the hydrophobic force in bilayer adhesion and fusion

The Surface Forces Apparatus technique was used for measuring the adhesion, deformation, and fusion of bilayers supported on mica. The technique allows the molecular rearrangements to be followed in real time during the fusion process, and the most important forces involved to be identified. The adhesion between two bilayers can be increased by two orders of magnitude if they are thinned so as to expose more hydrophobic groups. For all the bilayer systems studied a single basic fusion mechanism was found in which the bilayers do not “overcome” the short-range repulsive steric-hydration forces; instead, local bilayer deformations allow these repulsive forces to be “bypassed”. The results further indicate that the most important force leading to the direct fusion of bilayers is the hydrophobic attraction acting between the hydrophobic interiors of bilayers (1, 2).     

Tip penetration through lipid bilayers in atomic force microscopy

In studies of solid supported lipid bilayers with atomic force microscopes (AFM) the force between tip and bilayer is routinely measured. During the approach of the AFM tip in aqueous electrolyte first a short-range repulsive force is observed. For many solid-like and some liquid-like lipid bilayers a subsequent break-through is observed. We observe such a break-through also for dioleoyloxypropyl-trimethylammonium chloride (DOTAP) which is expected to be liquid-like. Here we describe a model which assumes that the jump reflects the penetration of the AFM tip through the lipid bilayer. The model predicts a logarithmical dependence of the break-through force on the approaching velocity of the AFM tip. Two parameters are introduced: The ratio A/αV, α being a geometric factor, A being the area over which pressure is exerted on the bilayer, V the activation volume, and k₀, the rate of spontaneous formation of a hole in the lipid bilayer that is big enough to allow the break-through of the tip. Experiments with bilayers consisting of DOTAP and dioleoylphosphatidylserine (DOPS) show that the break-through forces behave in the predicted way. For DOTAP we obtain ratios A/αV of about 58 nm⁻¹ and rates k₀ ranging from 1.9×10⁻⁸ to 2.5×10⁻⁴ s⁻¹. For DOPS the corresponding values are 162 nm⁻¹ and 2.0 s⁻¹.     

Role of nanomechanical properties in the tribological performance of phospholipid biomimetic surfaces

The role of phospholipid bilayers in controlling and reducing frictional forces between biological surfaces is investigated by three complementary experiments: friction forces are measured using a homemade tribometer, mechanical resistance to indentation is measured by AFM, and lipid bilayer degradation is controlled in situ during friction testing using fluorescence microscopy. DPPC lipid bilayers in the solid phase generate friction coefficients as low as 0.002 (comparable to that found for cartilage) that are stable through time. DOPC bilayers formed by the vesicle fusion method or the adsorption of mixed micelles generate higher friction coefficients. These coefficients increased through time, during which the bilayers degraded. The friction coefficient is correlated with the force needed to penetrate the bilayer with the AFM tip. With only one bilayer in the contact region, the friction increased to a similar value of about 0.08 for the DPPC and DOPC. Our study therefore shows that good mechanical stability of the bilayers is essential and suggests that the low friction coefficient is ensured by the hydration layers between adjacent lipid bilayers.     

非支撑磷脂双层膜制备及温度对其力学性质的影响

结合聚苯乙烯球刻蚀和微机电系统技术加工氮化硅纳米多孔膜, 并在其上用囊泡法制备非支撑磷脂双层膜, 通过温控原子力显微术(AFM)的成像模式和力曲线模式对非支撑磷脂双层膜的形貌和力学性质进行研究. 实验结果表明, 该方法制备的非支撑磷脂双层膜具有流动性, 能进行自我修复, 该特点有利于提供足够的非支撑磷脂双层膜区域用于其性质研究; 非支撑磷脂双层膜的膜破力和粘滞力均随着温度的升高而减小, 即膜的机械稳定性随着温度的升高而降低. 非支撑磷脂双层膜膜破力小于支撑磷脂双层膜的膜破力, 并且非支撑磷脂双层膜粘滞力随温度的变化趋势与支撑磷脂双层膜的变化趋势相反.     

Probing the properties of lipopolysaccharide monolayers and their interaction with the antimicrobial peptide polymyxin B by atomic force microscopy

In contrast to the majority of all known cell types, Gram-negative bacteria have a second membrane, the outer membrane, which is an asymmetric bilayer composed of a phospholipid inner leaflet and a glycolipid outer leaflet. The glycolipid layer, in most cases being composed of a lipopolysaccharide (LPS), is the first target for antimicrobial agents. To get a basic understanding of the membrane-forming properties of LPS, we reconstituted monolayers of deep rough mutant LPS from Salmonella enterica serova Minnesota (R595 LPS), its lipid A moiety, and of the synthetic tetraacyl compound 406 (resembling the biosynthetic lipid A precursor IVa) at the air-water interface of a film balance. The liquid-expanded (LE) and liquid-condensed (LC) domains in the coexisting region were investigated with epifluorescence and, after transferring the monolayer onto mica, as a Langmuir-Blodgett film, with atomic force microscopy (AFM). The fluorescence and the AFM images showed identical domain structure. The higher resolution of the AFM images, however, contained more topographic details. Different heights and adhesion forces between the LE and LC domains could be observed. Differences in the adhesion forces between the AFM tip and the sample were determined in the repulsive and the attractive dynamic scanning modes, demonstrating the importance of a careful interpretation of height images. We propose that an increase in the lateral pressure causing the LE-LC transition of the monolayers leads to a reorientation of the molecules due to a tilt angle between the alkyl chains and the diglucosamine backbone. LPS monolayers have been utilized as a simplified reconstitution model of the outer membrane to study the interaction with antimicrobial agents. We investigated the action of the polycationic peptide polymyxin B (PMB) and found dramatic influences on the domain structures.     

Direct force measurement of the stability of poly(ethylene glycol)-polyethylenimine graft films

The stability and passivity of poly(ethylene glycol)-polyethylenimine (PEG-PEI) graft films are important for their use as antifouling coatings in a variety of biotechnology applications. We have used AFM colloidal-probe force measurements combined with optical reflectometry to characterize the surface properties and stability of PEI and dense PEG-PEI graft films on silica. Initial contact between bare silica probes and PEI-modified surfaces yields force curves that exhibit a long-range electrostatic repulsion and short-range attraction between the surfaces, indicating spontaneous desorption of PEI in the aqueous medium. Further transfer of PEI molecules to the probe occurs with subsequent application of forces between FR = 300 and 500 microN/m. The presence of PEG reduces the adhesive properties of the PEI surface and prevents transfer of PEI molecules to the probe with continuous contact, though an initial desorption of PEI still occurs. Glutaraldehyde crosslinking of the graft films prevents both the initial desorption and subsequent transfer of the PEI, resulting in sustained attractive interaction forces of electrostatic origin between the negatively charged probe and the positively charged copolymer graft films.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

Ultrathin spin-coated dioleoylphosphatidylcholine lipid layers in dry conditions: a combined atomic force microscopy and nanomechanical study

Atomic force microscopy (AFM) has been used to study the structural and mechanical properties of low concentrated spin-coated dioleoylphosphatidylcholine (DOPC) layers in dry environment (RH ≈ 0%) at the nanoscale. It is shown that for concentrations in the 0.1-1 mM range the structure of the DOPC spin-coated samples consists of an homogeneous lipid monolayer ～1.3 nm thick covering the whole substrate on top of which lipid bilayer (or multilayer) micro- and nanometric patches and rims are formed. The thickness of the bilayer structures is found to be ～4.5 nm (or multiples of this value for multilayer structures), while the lateral dimensions range from micrometers to tens of nanometer depending on the lipid concentration. The force required to break a bilayer (breakthrough force) is found to be ～0.24 nN. No dependence of the mechanical values on the lateral dimensions of the bilayer structures is evidenced. Remarkably, the thickness and breakthrough force values of the bilayers measured in dry environment are very similar to values reported in the literature for supported DOPC bilayers in pure water.     

Combining colloidal probe atomic force and reflection interference contrast microscopy to study the compressive mechanics of hyaluronan brushes

We describe a method that combines colloidal probe atomic force microscopy (AFM) and reflection interference contrast microscopy (RICM) to characterize the mechanical properties of thin and solvated polymer films. When analyzing polymer films, a fundamental problem in colloidal probe AFM experiments is to determine the distance at closest approach between the probe and the substrate on which the film is deposited. By combining AFM and RICM in situ, forces and absolute distances can be measured simultaneously. Using the combined setup, we quantify the compressive mechanics of films of the polysaccharide hyaluronan that is end-grafted to a supported lipid bilayer. The experimental data, and comparison with polymer theory, show that hyaluronan films are well-described as elastic, very soft and highly solvated polymer brushes. The data on these well-defined films should be a useful reference for the investigation of the more complex hyaluronan-rich coats that surround many living cells.     

Long-Range Interaction Forces between Polymer-Supported Lipid Bilayer Membranes

Much of the short-range forces and structures of softly supported DMPC bilayers has been described previously. However, one interesting feature of the measured force-distance profile that remained unexplained is the presence of a long-range exponentially decaying repulsive force that is not observed between rigidly supported bilayers on solid mica substrate surfaces. This observation is discussed in detail here based on recent static and dynamic surface force experiments. The repulsive forces in the intermediate distance regime (mica-mica separations from 15 to 40 nm) are shown to be due not to an electrostatic force between the bilayers but to compression (deswelling) of the underlying soft polyelectrolyte layer, which may be thought of as a model cytoskeleton. The experimental data can be fit by simple theoretical models of polymer interactions from which the elastic properties of the polymer layer can be deduced.     

Compressibility study of quaternary phospholipid blend monolayers

The mechanical properties of liposome membranes are strongly dependent on type and ratio of lipid compounds, which can have important role in drug targeting and release processes when liposome is used as drug carrier. In this work we have used Brewster's angle microscopy to monitor the lateral compression process of lipid monolayers containing as helper lipids either distearoyl phosphatidylethanolamine (DSPE) or dioleoyl phophatidylethanolamine (DOPE) molecules on the Langmuir trough. The compressibility coefficient was determined for lipid blend monolayers containing the helper lipids above, cholesterol, distearoyl phosphatidylcholine (DSPC) and pegylated-DSPE at room temperature. Two variables, the cholesterol fraction and the ratio ρ between the helper lipid (either DSPE or DOPE) and the reference lipid DSPC, were studied by multivariate analysis to evaluate their impact on the compressibility coefficient of the monolayers. The cholesterol level was found to be the most significant variable for DSPE blends while the ratio ρ was the most significant one for DOPE blend monolayers. It was also found that these two variables can exhibit positive interaction and the same compressibility value can be obtained with different blend compositions.     

DOPC,DOPE和神经酰胺对鞘磷脂/胆固醇双层膜结构的影响

用LB技术和原子力显微镜(AFM)研究了1,2-二油酸甘油-3-磷脂酰胆碱(DOPC)、1,2-二油酸甘油-3-磷脂酰乙醇胺(DOPE)和神经酰胺(Ceramide)对鞘磷脂(SM)/胆固醇(Chol)结构的影响. 实验结果表明, 在表面压力较低时, 每种混合脂双层膜都呈现均匀分布的脂双层结构. 随着表面压力的增加, 形态发生了明显的变化: (1) SM/Chol二元组分双层膜形成均一的液态有序相微区结构, 衬底覆盖率达到80%; (2) DOPC的加入促使SM/Chol双层膜出现相分离现象, SM/Chol形成的液态有序相 “岛状” 微区结构漂浮在液态无序相的DOPC上部, 约占总面积的30%; (3) DOPE与SM/Chol形成的双层膜明显不同于DOPC/SM/Chol, 呈现出液态无序相、液态有序相及凝胶相3相共存的结构; (4) Ceramide诱导了SM/Chol双层膜结构发生重排, 两层脂分子间发生翻转形成囊泡结构, 部分神经酰胺从液态有序相中分离形成小颗粒结构. 在较高膜压下, 各系统都呈现出具有特定形态的双层膜结构. 分子官能团的成键能力决定了双层膜形态结构.     

Hydration forces between silica surfaces: experimental data and predictions from different theories

Silica is a very interesting system that has been thoroughly studied in the last decades. One of the most outstanding characteristics of silica suspensions is their stability in solutions at high salt concentrations. In addition to that, measurements of direct-interaction forces between silica surfaces, obtained by different authors by means of surface force apparatus or atomic force microscope (AFM), reveal the existence of a strong repulsive interaction at short distances (below 2 nm) that decays exponentially. These results cannot be explained in terms of the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory, which only considers two types of forces: the electrical double-layer repulsion and the London-van der Waals attraction. Although there is a controversy about the origin of the short-range repulsive force, the existence of a structured layer of water molecules at the silica surface is the most accepted explanation for it. The overlap of structured water layers of different surfaces leads to repulsive forces, which are known as hydration forces. This assumption is based on the very hydrophilic nature of silica. Different theories have been developed in order to reproduce the exponentially decaying behavior (as a function of the separation distance) of the hydration forces. Different mechanisms for the formation of the structured water layer around the silica surfaces are considered by each theory. By the aid of an AFM and the colloid probe technique, the interaction forces between silica surfaces have been measured directly at different pH values and salt concentrations. The results confirm the presence of the short-range repulsion at any experimental condition (even at high salt concentration). A comparison between the experimental data and theoretical fits obtained from different theories has been performed in order to elucidate the nature of this non-DLVO repulsive force.     

Direct submolecular scale imaging of mesoscale molecular order in supported dipalmitoylphosphatidylcholine bilayers

Supported dipalmitoylphosphatidylcholine (DPPC) bilayers are widely used membrane systems in biophysical and biochemical studies. Previously, short-range positional and orientational order of lipid headgroups of supported DPPC bilayers was observed at room temperature using low deflection noise frequency modulation atomic force microscopy (FM-AFM). While this ordering was supported by X-ray diffraction studies, it conflicted with diffusion coefficient measurements of gel-phase bilayers determined from fluorescence photobleaching experiments. In this work, we have directly imaged mica-supported DPPC bilayers with submolecular resolution over scan ranges up to 146 nm using low deflection noise FM-AFM. Both orientational and positional molecular ordering were observed in the mesoscale, indicative of crystalline order. We discuss these results in relation to previous biophysical studies and propose that the mica support induces mesoscopic crystalline order of the DPPC bilayer at room temperature. This study also demonstrates the recent advance in the scan range of submolecular scale AFM imaging.     

Surfactant boundary lubricant film modified by an amphiphilic diblock copolymer

The effect of the uptake of a low-molecular-weight amphiphilic diblock copolymer on the morphology of didodecyldimethylammonium bromide (DDAB) adsorbed layers on mica, the interactions between two coated surfaces, and the frictional properties of the boundary film have been studied using an atomic force microscope and a dynamic surface forces apparatus nanotribometer. When DDAB-coated surfaces in aqueous solution were compressed, hemifusion or removal of the adsorbed surfactant bilayers could not be induced, and no frictional force could be measured between the surfaces, which display superior lateral cohesion and lubricant properties. Coadsorbing octadecyl end modified poly(ethylene oxide) chains at low density facilitates hemifusion, generating significant shear stress and leading to stick-slip instabilities. The mixed films regain their lateral cohesion at higher adsorbed copolymer densities, but an extra short-range attraction brings the adsorbed layers into adhesive contact without causing bilayer hemifusion. Here, noticeable frictional forces are also measured.     

Study of frictional properties of a phospholipid bilayer in a liquid environment with lateral force microscopy as a function of NaCl concentration

Friction properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-supported planar bilayers deposited on mica were tested in a liquid environment by lateral force microscopy. The presence of these bilayers was detected by imaging and force measurements with atomic force microscopy. To test how the presence of NaCl affects the frictional properties of the phospholipid bilayers, four DMPC bilayers were prepared on mica in saline media ranging from 0 to 0.1 M NaCl. Changes in the lateral vs vertical force curves were recorded as a function of NaCl concentration and related to structural changes induced in the DMPC bilayer by electrolyte ions. Three friction regimes were observed as the vertical force exerted by the tip on the bilayer increased. To relate the friction response to the structure of the DMPC bilayer, topographic images were recorded at the same time as friction data. Ions in solution screened charges present in DMPC polar heads, leading to more compact bilayers. As a consequence, the vertical force at which the bilayer broke during friction experiments increased with NaCl concentration. In addition, the topographic images showed that low-NaCl-concentration bilayers recover more easily due to the low cohesion between phospholipid molecules.     

Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Quantitative lateral force microscopy study of the dolomite (104)-water interface

The friction and lateral stiffness of the contact between an atomic force microscopy (AFM) probe tip and an atomically flat dolomite (104) surface were investigated in contact with two aqueous solutions that were in equilibrium and supersaturated with respect to dolomite, respectively. The two aqueous solutions yielded negligible differences in friction at the native dolomite-water interface. However, the growth of a Ca-rich film from the supersaturated solution, revealed by X-ray reflectivity measurements, altered the probe-dolomite contact region sufficiently to observe distinct friction forces on the native dolomite and the film-covered surface regions. Quantitative friction-load relationships demonstrated three physically distinct load regimes for applied loads up to 200 nN. Similar friction forces were observed on both surfaces below 50 nN load and above 100 nN load. The friction forces on the two surfaces diverged at intermediate loads. Quantitative measurements of dynamic friction forces at low load were consistent with the estimated energy necessary to dehydrate the surface ions, whereas differences in mechanical properties of the Ca-rich film and dolomite surfaces were evidently important above 50 nN load. Attempts to fit the quantitative stiffness-load data using a Hertzian contact mechanical model based on bulk material properties yielded physically unrealistic fitting coefficients, suggesting that the interfacial contact region must be explicitly considered in describing the static and dynamic contact mechanics of this and similar systems.     

Influence of cholesterol on the phase transition of lipid bilayers: a temperature-controlled force spectroscopy study

Cholesterol (Chol) plays the essential function of regulating the physical properties of the cell membrane by controlling the lipid organization and phase behavior and, thus, managing the membrane fluidity and its mechanical strength. Here, we explore the model system DPPC:Chol by means of temperature-controlled atomic force microscopy (AFM) imaging and AFM-based force spectroscopy (AFM-FS) to assess the influence of Chol on the membrane ordering and stability. We analyze the system in a representative range of compositions up to 50 mol % Chol studying the phase evolution upon temperature increase (from room temperature to temperatures high above the T(m) of the DPPC bilayer) and the corresponding (nano)mechanical stability. By this means, we correlate the mechanical behavior and composition with the lateral order of each phase present in the bilayers. We prove that low Chol contents lead to a phase-segregated system, whereas high contents of Chol can give a homogeneous bilayer. In both cases, Chol enhances the mechanical stability of the membrane, and an extraordinarily stable system is observed for equimolar fractions (50 mol % Chol). In addition, even when no thermal transition is detected by the traditional bulk analysis techniques for liposomes with high Chol content (40 and 50 mol %), we demonstrate that temperature-controlled AFM-FS is capable of identifying a thermal transition for the supported lipid bilayers. Finally, our results validate the AFM-FS technique as an ideal platform to differentiate phase coexistence and transitions in lipid bilayers and bridge the gap between the results obtained by traditional methods for bulk analysis, the theoretical predictions, and the behavior of these systems at the nanoscale.