Floret-shaped solid domains on giant fluid lipid vesicles induced by pH

Lateral lipid phase separation of titratable PS or PA lipids and their assembly in domains induced by changes in pH are significant in liposome-based drug delivery: environmentally responsive lipid heterogeneities can be tuned to alter collective membrane properties such as permeability (altering drug release) and surface topography (altering drug carrier reactivity) impacting, therefore, the therapeutic outcomes. At the micrometer scale fluorescence microscopy on giant unilamellar fluid vesicles (GUVs) shows that lowering pH (from 7.0 to 5.0) promotes condensation of titratable PS or PA lipids into beautiful floret-shaped domains in which lipids are tightly packed via hydrogen-bonding and van der Waals interactions. The order of lipid packing within domains increases radially toward the domain center. Lowering pH enhances the lipid packing order, and at pH 5.0 domains appear to be entirely in the solid (gel) phase. Domains phenomenologically comprise a circular "core" cap beyond which interfacial instabilities emerge resembling leaf-like stripes. At pH 5.0 stripes are of almost vanishing Gaussian curvature independent of GUVs' preparation path and in agreement with a general condensation mechanism. Increasing incompressibility of domains is strongly correlated with a larger number of thinner stripes per domain and increasing relative rigidity of domains with smaller core cap areas. Line tension drives domain ripening; however, the final domain shape is a result of enhanced incompressibility and rigidity maximized by domain coupling across the bilayer. Introduction of a transmembrane osmotic gradient (hyperosmotic on the outer lipid leaflet) allows the domain condensation process to reach its maximum extent which, however, is limited by the minimal expansivity of the continuous fluid membrane.     

Crystalline protein domains and lipid bilayer vesicle shape transformations

Cellular membranes can take on a variety of shapes to assist biological processes including endocytosis. Membrane-associated protein domains provide a possible mechanism for determining membrane curvature. We study the effect of tethered streptavidin protein crystals on the curvature of giant unilamellar vesicles (GUVs) using confocal, fluorescence, and differential interference contrast microscopy. Above a critical protein concentration, streptavidin domains align and percolate as they form, deforming GUVs into prolate spheroidal shapes in a size-dependent fashion. We propose a mechanism for this shape transformation based on domain growth and jamming. Osmotic deflation of streptavidin-coated GUVs reveals that the relatively rigid streptavidin protein domains resist membrane bending. Moreover, in contrast to highly curved protein domains that facilitate membrane budding, the relatively flat streptavidin domains prevent membrane budding under high osmotic stress. Thus, crystalline streptavidin domains are shown to have a stabilizing effect on lipid membranes. Our study gives insight into the mechanism for protein-mediated stabilization of cellular membranes.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Two-component membrane material properties and domain formation from dissipative particle dynamics

The material parameters (area stretch modulus and bending rigidity) of two-component amphiphilic membranes are determined from dissipative particle dynamics simulations. The preferred area per molecule for each species is varied so as to produce homogeneous mixtures or nonhomogeneous mixtures that form domains. If the latter mixtures are composed of amphiphiles with the same tail length, but different preferred areas per molecule, their material parameters increase monotonically as a function of composition. By contrast, mixtures of amphiphiles that differ in both tail length and preferred area per molecule form both homogeneous and nonhomogeneous mixtures that both exhibit smaller values of their material properties compared to the corresponding pure systems. When the same nonhomogeneous mixtures of amphiphiles are assembled into planar membrane patches and vesicles, the resulting domain shapes are different when the bending rigidities of the domains are sufficiently different. Additionally, both bilayer and monolayer domains are observed in vesicles. We conclude that the evolution of the domain shapes is influenced by the high curvature of the vesicles in the simulation, a result that may be relevant for biological vesicle membranes.     

Substrate effects on interactions of lipid bilayer assemblies with bound nanoparticles

Understanding the interactions of nanoparticles with lipid membranes is crucial in establishing the mechanisms that govern assembly of membrane-based nanocomposites, nanotoxicology, and biomimetic inspired self-assembly. In this study, we explore binding of charged nanoparticles to lipid bilayers, both as liposomes and substrate supported assemblies. We find that the presence of a solid-support, regardless of curvature, eliminates the ability of zwitterionic fluid phase lipids to bind charged nanoparticles.     

Selective adsorption of protein molecules on phase-separated sapphire surfaces

Site-selective adsorption of protein molecules was found on sapphire surfaces that exhibit a phase separation into two domains: weakly charged hydrophobic domain and negatively charged hydrophilic one. Ferritin and bovine serum albumin molecules, which are negatively charged in a buffer solution, are adsorbed to the hydrophobic domains. Avidin molecules, which are positively charged, are adsorbed to the other domain. Fibrinogen molecules, which consist of both negative and positive modules, are adsorbed to the whole sapphire surface. Hemoglobin molecules, whose net charge is almost zero, are also adsorbed to the whole surfaces. These results indicate that electrostatic double layer interaction is the primary origin of the observed selectivity. Dependence of protein adsorption or desorption behaviors on the pH value can also be interpreted by the proposed model.     

Photochemical growth of silver nanoparticles on c(-) and c(+) domains on lead zirconate titanate thin films

The photochemical growth of silver nanoparticles on the negative domains of lead zirconate titanate thin films is reported. A sample of highly [100] orientated lead zirconate titanate, with a ratio of 30:70, that was 65-70 nm thick grown on Pt-coated MgO was poled by use of piezoresponse force microscopy to produce defined regions of surface positive and negative polarization. A comparison between the growth of silver nanoparticles on the surface of the lead zirconate titanate when illuminated with two sources of super band gap UV is given. In both cases the wavelength of illumination leads to growth on the positive domains but only illumination with a Honle H lamp, with a high photon output over 250-200 nm, caused significant growth of silver nanoparticles on the negative domain. The deposition on the negative domain is explained in terms of changed band bending due to the excitation of electrons into the conduction band, the rate of decay to the ground state, and dimensions of the ferroelectric film. The rate of deposition of silver nanoparticles on the negative domains is approximately half that on the positive domains.     

Local concentration of gel phase domains in supported lipid bilayers under light irradiation in binary mixture of phospholipids doped with dyes for photoinduced activation

Recently, lipid bilayers supported on solid substrates are considered to offer potential as biological devices utilizing biological membranes and membrane proteins. In particular, artificially patterned supported bilayers hold great promise for the development of biological devices. In this study, we show control of the formation and location of phase-separated domain structures by light irradiation for gel phase and liquid-crystalline phase separation structures in a DMPC-DOPC binary lipid bilayer tagged with dye molecules on SiO2/Si substrates. Upon light irradiation, the gel phase domain structures disappeared from the phase-separated bilayers. This disappearance indicates that the light irradiation causes a local increase in the temperature of the lipid bilayer. In this disappearance phenomenon, the photoinduced activation of dye lipids, e.g. fluorescent lipids, is considered to play an important role, since the same phenomenon does not occur in lipid bilayers that have a low concentration of dye lipids. Thus, the local increase in temperature is propagated by light absorption of the dye lipid and subsequent photoinduced activation of nonradiative molecular vibrations. Subsequent interruption of the photoinduced activation for molecular motion allowed the gel phase domain structures to precipitate and grow again. Moreover, the domain area fraction remaining after the photoinduced activation was higher than that before the photoinduced activation. This result indicates that the local increase in temperature propagated by dye-excitation enhances formation of the gel phase domains. By utilizing this phenomenon, we could preferentially induce formation of domain structures within the light-irradiated regions. This technique could be the basis for a new patterning technique based on domain structures. Moreover, these domain structure patterns can be eliminated by increasing the temperature, allowing rewritable patterning.     

聚对苯甲酰胺液晶态球晶生长机理

本文用热台偏光显微镜和透射电子显微镜研究了聚对苯甲酰胺(PBA)/H_2SO_4液晶态生长球晶的形态结构和结晶机理。结果表明,PBA/H_2SO_4向列型液晶相具有过冷状态,可达30—40℃左右,并可生长球晶结构,一般直径可达5毫米左右。20Wt％溶液在不同的过冷态结晶时,可以生成三种形态的球晶结构,这是由于构成球晶的基本结构单元——有序微区结构在不同过冷条件下堆砌排列的规整程度不同的缘故。球晶的形态结构只与过冷程度有关,而与溶液的浓度无关。同时还研究了变温和剪切应力下结晶的球晶形态。     

Phase separation and fractal domain formation in phospholipid/diacetylene-supported lipid bilayers

Phase separation in lipid bilayers is a phenomenon dependent on many environmental parameters such as pH, temperature, ionic strength, and pressure. Its importance in biological systems is reflected by the fact that it has been implicated in the spatial reorganization of plasma membranes, which leads to signaling and stimulation. Here, we present the study of phase separation, domain formation, and domain morphology of supported lipid bilayers composed of mixtures of diacetylene lipids and phospholipids. We have used high-resolution fluorescence and atomic force microscopy to characterize the phase separation between these lipids, and have found that at temperatures below 40 degrees C diacetylene molecules form fractal-like domains. These molecules aggregate in tetralayer stacks with an average monolayer thickness of 3 nm. Boundary and area fractal dimensions were calculated to quantify the domain growth and morphology. A transition from dendritic to dense branching growth was observed as the relative diacetylene concentration was increased. The ability to tailor the growth pattern by changing the relative amount of diacetylene molecules makes this a useful model system for the study of nonequilibrium growth phenomena. In addition, we have explored the possibility of promoting diacetylene domain nucleation through the use of nanostructured surfaces. We found that nanoscale perturbations acted as nucleation sites and modified the growth pattern of diacetylene domains. Phase separation induced by nanometer-scale perturbations could prove useful in selectively positioning lipid patches with specific compositions.     

Curvature modulates the self-assembly of amphiphilic molecules

In this work, we used lattice Monte Carlo simulations and theoretical model calculations to show how the self-assembly of adsorbed amphiphilic molecules is affected by the local curvature of solid surfaces. It is found that, beyond a critical curvature value, solid surface geometry governs the spatial ordering of aggregates and may induce the morphological transitions. The simulation results show how the curvature of solid surfaces modulates the distribution of aggregates: the anisotropy in local curvature along and perpendicular to the cylindrical surfaces tends to generate orientationally ordered cylindrical micelles. To account for the morphological transitions induced by the local curvature of solid surfaces, we constructed a theoretical model which includes the Helfrich bending energy, the deformation energy of aggregates induced by solid surfaces, and the adsorption energy. The model calculations indicate that on highly curved solid surfaces the bending energy for bilayer structure sharply increases with surface curvature, which in turn induces the morphological transition from bilayer to cylindrical structure. Our results suggest that the local curvature provides a means of controlling the spatial organization of amphiphilic molecules.     

Lipid fluorination enables phase separation from fluid phospholipid bilayers

To probe the effect of lipid fluorination on the formation of lipid domains in phospholipid bilayers, several new fluorinated and non-fluorinated synthetic lipids were synthesised, and the extent of phase separation of these lipids from phospholipid bilayers of different compositions was determined. At membrane concentrations as low as 1% mol/mol, both fluorinated and non-fluorinated lipids were observed to phase separate from a gel-phase (solid ordered) phospholipid matrix, but bilayers in a liquid disordered state caused no phase separation; if the gel-phase samples were heated above the transition temperature, then phase separation was lost. We found incorporation of perfluoroalkyl groups into the lipid enhanced phase separation, to such an extent that phase separation was observed from cholesterol containing bilayers in the liquid ordered phase.     

Inclusion-mediated lipid organization in supported membranes on a patterned substrate

We theoretically investigate the effects of inclusions on the domain formation in mixed lipid bilayers supported on a geometrically patterned substrate. It is found that the inclusions may distribute quite differently with varying volume fraction and size of inclusions. The distribution of inclusions will effectively change the spontaneous curvature of the inclusion-rich lipid domains, and consequently can sort the lipid domains in the supported bilayers. By varying the volume fraction and size of inclusions, we obtain a rich variety of laterally organized lipid bilayers and reveal some interesting transitions between these structures. The present model provides a possible strategy to control the domain formation in the supported membranes, and may yield some theoretical insight into the design of biosensors by the reorganization of lipids and inclusions.     

Polymerization of diacetylene phospholipid bilayers on solid substrate: influence of the film deposition temperature

Micropatterned phospholipid bilayers on solid substrates offer an attractive platform for various applications, such as high throughput drug screening. We have previously developed a photopolymerization-based methodology for generating micropatterned bilayers composed of polymerized and fluid lipid bilayers. Lithographic photopolymerization of a diacetylene-containing phospholipid (DiynePC) allowed facile fabrication of compartmentalized arrays of fluid lipid membranes. Herein, we report on a key experimental parameter that significantly influences the homogeneity and quality of the fabricated polymeric bilayers, namely the temperature at which monolayers of monomeric DiynePC were formed on the water surface and transferred onto solid substrates by the Langmuir-Blodgett/Langmuir-Schaefer (LB/LS) technique. Using fluorescence microscopy and atomic force microscopy, it was found that polymerized bilayers were homogeneous, if bilayers of DiynePC were prepared below the triple point temperature (ca. 20 degrees C) of the monolayer, where a direct transition from the gaseous state to the liquid condensed state occurred. Bilayers prepared above this temperature had a markedly increased number of crack-like line defects. The differences were attributed to the domain structures in the monolayer that were transferred from the water surface to the substrate. Domain size, rather than the molecular packing in each domain, was concluded to play a critical role in the formation of defects. The spontaneous curvature and area changes of bilayers were postulated to cause destabilization and detachment of the films from the substrate upon polymerization. Our present results highlight the importance of controlling the domain structures for the homogeneity of polymerized bilayers required in technological applications.     

Counterion-mediated pattern formation in membranes containing anionic lipids

Most lipid components of cell membranes are either neutral, like cholesterol, or zwitterionic, like phosphatidylcholine and sphingomyelin. Very few lipids, such as sphingosine, are cationic at physiological pH. These generally interact only transiently with the lipid bilayer, and their synthetic analogs are often designed to destabilize the membrane for drug or DNA delivery. However, anionic lipids are common in both eukaryotic and prokaryotic cell membranes. The net charge per anionic phospholipid ranges from − 1 for the most abundant anionic lipids such as phosphatidylserine, to near − 7 for phosphatidylinositol 3,4,5 trisphosphate, although the effective charge depends on many environmental factors. Anionic phospholipids and other negatively charged lipids such as lipopolysaccharides are not randomly distributed in the lipid bilayer, but are highly restricted to specific leaflets of the bilayer and to regions near transmembrane proteins or other organized structures within the plane of the membrane. This review highlights some recent evidence that counterions, in the form of monovalent or divalent metal ions, polyamines, or cationic protein domains, have a large influence on the lateral distribution of anionic lipids within the membrane, and that lateral demixing of anionic lipids has effects on membrane curvature and protein function that are important for biological control.     

Adsorption and spontaneous rupture of vesicles composed of two types of lipids

To analyze the adsorption of single vesicles composed of two types of lipids (e.g., zwitterionic and positively charged lipids or zwitterionic and negatively charged lipids), we propose a statistical model taking into account lipid-surface interactions, lipid-lipid lateral interactions, and vesicle bending energy. Our treatment specifies how these parameters govern vesicle adsorption, shows how the radius of the vesicle-surface contact area may depend on the vesicle composition, and clarifies the conditions for vesicle rupture.     

Surface phase behavior and microstructure of lipid/PEG-emulsifier monolayer-coated microbubbles

Langmuir trough methods and fluorescence microscopy were combined to investigate the phase behavior and microstructure of monolayer shells coating micron-scale bubbles (microbubbles) typically used in biomedical applications. The monolayer shell consisted of a homologous series of saturated acyl chain phospholipids and an emulsifier containing a single hydrophobic stearate chain and polyethylene glycol (PEG) head group. PEG-emulsifier was fully miscible with expanded phase lipids and phase separated from condensed phase lipids. Phase coexistence was observed in the form of dark condensed phase lipid domains surrounded by a sea of bright, emulsifier-rich expanded phase. A rich assortment of condensed phase area fractions and domain morphologies, including networks and other novel structures, were observed in each batch of microbubbles. Network domains were reproduced in Langmuir monolayers under conditions of heating–cooling followed by compression–expansion, as well as in microbubble shells that underwent surface flow with slight compression. Domain size decreased with increased cooling rate through the phase transition temperature, and domain branching increased with lipid acyl chain length at high cooling rates. Squeeze-out of the emulsifier at a surface pressure near 35 mN/m was indicated by a plateau in Langmuir isotherms and directly visualized with fluorescence microscopy, although collapse of the solid lipid domains occurred at much higher surface pressures. Compression of the monolayer past the PEG-emulsifier squeeze-out surface pressure resulted in a dark shell composed entirely of lipid. Under certain conditions, the PEG-emulsifier was reincorporated upon subsequent expansion. Factors that affect shell formation and evolution, as well as implications for the rational design of microbubbles in medical applications, are discussed.     

Modulation of the spontaneous curvature and bending rigidity of lipid membranes by interfacially adsorbed amphipathic peptides

Amphipathic alpha-helical peptides are often ascribed an ability to induce curvature stress in lipid membranes. This may lead directly to a bending deformation of the host membrane, or it may promote the formation of defects that involve highly curved lipid layers present in membrane pores, fusion intermediates, and solubilized peptide-micelle complexes. The driving force is the same in all cases: peptides induce a spontaneous curvature in the host lipid layer, the sign of which depends sensitively on the peptide's structural properties. We provide a quantitative account for this observation on the basis of a molecular-level method. To this end, we consider a lipid membrane with peptides interfacially adsorbed onto one leaflet at high peptide-to-lipid ratio. The peptides are modeled generically as rigid cylinders that interact with the host membrane through a perturbation of the conformational properties of the lipid chains. Through the use of a molecular-level chain packing theory, we calculate the elastic properties, that is, the spontaneous curvature and bending stiffness, of the peptide-decorated lipid membrane as a function of the peptide's insertion depth. We find a positive spontaneous curvature (preferred bending of the membrane away from the peptide) for small penetration depths of the peptide. At a penetration depth roughly equal to half-insertion into the hydrocarbon core, the spontaneous curvature changes sign, implying negative spontaneous curvature (preferred bending of the membrane toward the peptide) for large penetration depths. Despite thinning of the membrane upon peptide insertion, we find an increase in the bending stiffness. We discuss these findings in terms of how the peptide induces elastic stress.     

Polymorphic domains in monolayers of isomeric triple-chain phospholipids

Monolayers of two isomeric branched chain phosphatidyl cholines at the air/water interface have been studied by means of fluorescence microscopy. The lipids differ in the position of the branched chain at the glycerol backbone and carry three chains per headgroup of almost equal length. Most qualitative features of the compression isotherms are similar except a difference of 4 Ų/molecule in the minimum molecular area at high lateral pressures. This indicates a more condensed solid phase of compound C2 and is also reflected in the shapes of domains observed in the LE/LC phase coexistence range: domains with sharp edges and a mostly hexagonal shape are formed. On the other hand, the compound C1 with a larger limiting molecular area exhibits a smooth domain boundary and a shape instability as theoretically predicted.     

Line tension and coalescence in heterogeneous membranes

Membrane inhomogeneity gives rise to a perturbation energy that may be manifested as line tension between regions of different compositions. Here we calculate the perturbation energy of a phase-separated fluid membrane composed of domains embedded in a continuous phase and relate it to the line tension in the system. We find that the effective line tension due to the thickness mismatch between the phases varies nonmonotonically as a function of the domain size and spacing when those decrease below about 20 nm. Although we find that the characteristic time scale for domain coalescence increases with domain size, the coalescence time for domains on the order of 25 to 50 nm may be as long as several days. The time scale for domain coalescence is found, quite surprisingly, to be maximal when the bending and area moduli of the domain and continuous phase are similar and to decrease when one phase is stiffer than the other. Domain stability is also found to vary as a function of spacing; as a result, small domains on the order of 5 nm become unstable and tend to coalesce when the domain-phase area fraction increases above 0.5, whereas larger domains on the order of 50 nm become unstable only when the area fraction increases above 0.8.