Bilayer Morphologies Research Articles

Stimuli-Responsive Membrane Anchor Peptide Nanofoils for Tunable Membrane Association and Lipid Bilayer Fusion.

Self-assembled peptide nanostructures with stimuli-responsive features are promising as functional materials. Despite extensive research efforts, water-soluble supramolecular constructs that can interact with lipid membranes in a controllable way are still challenging to achieve. Here, we have employed a short membrane anchor protein motif (GLFD) and coupled it to a spiropyran photoswitch. Under physiological conditions, these conjugates assemble into ∼3.5 nm thick, foil-like peptide bilayer morphologies. Photoisomerization from the closed spiro (SP) form to the open merocyanine (MC) form of the photoswitch triggers rearrangements within the foils. This results in substantial changes in their membrane-binding properties, which also varies sensitively to lipid composition, ranging from reversible nanofoil reformation to stepwise membrane adsorption. The formed peptide layers in the assembly are also able to attach to various liposomes with different surface charges, enabling the fusion of their lipid bilayers. Here, SP-to-MC conversion can be used both to trigger and to modulate the liposome fusion efficiency.

Key Process and Factors Controlling the Direct Translocation of Cell-Penetrating Peptide through Bio-Membrane.

Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately. Also, the time-course analysis was introduced for the geometric calculation of adsorption and internalization amount per lipid molecule consisting of GUV. As a result, we found that adsorption and internalization assumed to occur successively by CPP molecule come into contact with membrane lipid. Adsorption is quick to saturate within 10 min, while cytolysis of each CPP on the membrane follows successively. After adsorption is saturated, cytolysis proceeds further linearly by time with a different rate constant that is dependent on the osmotic pressure. We also found that temperature and lipid composition influence cytolysis by modulating lipid mobility. The electrolyte in the outer media is also affected as a chemical mediator to control CPP cytolysis by following the Hoffmeister effect for membrane hydration. These results confirmed the mechanism of cytolysis as temporal and local phase transfer of membrane lipid from Lα to Mesh1, which has punctured bilayer morphologies.

Double-Layer Morphologies from a Silicon-Containing ABA Triblock Copolymer.

A combined experimental and self-consistent-field theoretical (SCFT) investigation of the phase behavior of poly(stryrene- b-dimethylsiloxane- b-styrene) (PS- b-PDMS- b-PS, or SDS32) thin films during solvent vapor annealing is presented. The morphology of the triblock copolymer is described as a function of the as-cast film thickness and the ratio of two different solvent vapors, toluene and heptane. SDS32 formed terraced bilayer morphologies even when the film thickness was much lower than the commensurate thickness. The morphology transitioned between bilayer cylinders, bilayer perforated lamellae, and bilayer lamellae, including mixed structures such as a perforated lamella on top of a layer of in-plane cylinders, as the heptane fraction during solvent annealing increased. SCFT modeling showed the same morphological trends as a function of the block volume fraction. In comparison with diblock PS- b-PDMS with the same molecular weight, the SDS32 offers a simple route to produce a diversity of well-ordered bilayer structures with smaller feature sizes, including the formation of bilayer perforated lamellae over a large process window.

Pearling Bifurcations in the Strong Functionalized Cahn--Hilliard Free Energy

The functionalized Cahn--Hilliard free energy supports phase-separated morphologies of distinct codimension, including codimension-one bilayer and codimension-two filament morphologies. We characte...

Molecular Mechanism of Ionic-Liquid-Induced Membrane Disruption: Morphological Changes to Bilayers, Multilayers, and Vesicles.

The application of ionic liquids (ILs) in many industrially relevant processes provides an urgent need to better understand their molecular interactions with biological systems. A detailed understanding of the cytotoxicity mechanism of ILs can be helpful in facilitating the molecular design of nontoxic ILs. Using coarse-grained molecular dynamics (MD) simulations, we investigate the effects of imidazolium-based ILs on several lipid bilayer morphologies. Our results demonstrate that the asymmetric insertion of IL cations into one side of a lipid bilayer leaflet enhances the leaflet strain, which upon reaching a critical value triggers a morphological disruption in the bilayer. Consistently, the bending modulus of the bilayer is reduced by 1 to 2 orders of magnitude relative to that of an IL-free planar bilayer prior to the disruption event. Our results suggest that ILs that can easily insert into the lipid bilayer without diffusing across or inducing lipid flip-flop can be more disruptive to a lipid biomembrane.

Seeking to quantify the ferromagnetic-to-antiferromagnetic interface coupling resulting in exchange bias with various thin-film conformations

Ni3Fe/(Ni, Fe)O thin films with bilayer and nanocrystallite dispersion morphologies are prepared with a dual ion beam deposition technique permitting precise control of nanocrystallite growth, composition, and admixtures. A bilayer morphology provides a Ni3Fe-to-NiO interface, while the dispersion films have different mixtures of Ni3Fe, NiO, and FeO nanocrystallites. Using detailed analyses of high resolution transmission electron microscopy images with Multislice simulations, the nanocrystallites' structures and phases are determined, and the intermixing between the Ni3Fe, NiO, and FeO interfaces is quantified. From field-cooled hysteresis loops, the exchange bias loop shift from spin interactions at the interfaces are determined. With similar interfacial molar ratios of FM-to-AF, we find the exchange bias field essentially unchanged. However, when the interfacial ratio of FM to AF was FM rich, the exchange bias field increases. Since the FM/AF interface ‘contact’ areas in the nanocrystallite dispersion films are larger than that of the bilayer film, and the nanocrystallite dispersions exhibit larger FM-to-AF interfacial contributions to the magnetism, we attribute the changes in the exchange bias to be from increases in the interfacial segments that suffer defects (such as vacancies and bond distortions), that also affects the coercive fields.

Sensitivity analysis of current generation in organic solar cells—comparing bilayer, sawtooth, and bulk heterojunction morphologies

Organic solar cells (OSC) show great potential as a low-cost energy source. In addition, their mechanical flexibility allows the added advantage of use on a wide variety of surfaces. In recent years, progress in experimental strategies and modeling approaches have enabled enhancing the power conversion efficiencies of OSC. In particular, simulation based analysis has played a significant role in improving our understanding of the charge transport phenomena in the active layer of these devices. The excitonic drift-diffusion (EDD) model has been used widely to simulate the generation and transport properties of bulk heterojunction (BHJ) solar cells. The EDD model – which is derived from the Boltzmann transport is dependent on a number of input parameters such as (1) material properties (mobility and permittivity), (2) operating conditions (illumination and device thickness), and (3) active layer morphology.A comprehensive sensitivity analysis of the short-circuit current, Jsc, to the input parameters is performed. This helps in rank ordering the input parameters and operating conditions – by strength and relevance – on their impact on Jsc. We particularly focus our investigations on understanding how the active layer morphology affects the sensitivity of Jsc. To accomplish this we analyze three classes of morphologies: bilayer, BHJ, and sawtooth. The results show significant differences in sensitivities between BHJ, sawtooth, and bilayer morphologies. Short-circuit current in BHJ structure shows higher sensitivity to material properties than either sawtooth and bilayer structure, suggesting that the necessity for finer control of material properties to counteract the increased disorder in the active layer morphology. The electrode current is found to be most sensitive to illumination intensity for all three morphologies. We report some interesting trends that may help choose the most sensitive parameters to vary for designing OSC's with better performance.

A Stepwise “Micellization–Crystallization” Route to Oblate Ellipsoidal, Cylindrical, and Bilayer Micelles with Polyethylene Cores in Water

Micellar polymorphism from block copolymers has been well documented, but most attention has focused on noncrystalline hydrophobic systems. We have investigated the micellization in water of model diblock copolymers with semicrystalline polyethylene (PE) as the core-forming component. Poly(N,N-dimethylacrylamide)–polyethylene (AE) diblock copolymers were synthesized by a combination of anionic and RAFT polymerizations. The bulk nanostructures were probed by small-angle X-ray scattering (SAXS) and AE diblock copolymers were found to be moderately segregated at 140 °C. Dispersions of AE amphiphiles in water were prepared by direct dissolution at 120 °C (i.e., above the melting transition of PE) followed by cooling to 25 °C. By manipulating the composition of AE diblock copolymers, discrete structures with oblate ellipsoidal, cylindrical, and bilayer morphologies were produced, as evidenced in cryogenic transmission electron microscopy (cryo-TEM). The self-assembled aggregates were also studied by small-angle neutron scattering (SANS) and dilute solution rheology. The semicrystalline nature of the nanostructures was further revealed by differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS). A stepwise “micellization–crystallization” process was proposed as the micelle formation mechanism, as supported by the existence of similar nanostructures at 120 °C using SANS. This strategy holds promise for a general protocol toward the production of giant wormlikemore » micelles and vesicles with semicrystalline polymeric cores.« less

High-Density Reconstitution of Functional Water Channels into Vesicular and Planar Block Copolymer Membranes

The exquisite selectivity and unique transport properties of membrane proteins can be harnessed for a variety of engineering and biomedical applications if suitable membranes can be produced. Amphiphilic block copolymers (BCPs), developed as stable lipid analogs, form membranes that functionally incorporate membrane proteins and are ideal for such applications. While high protein density and planar membrane morphology are most desirable, BCP–membrane protein aggregates have so far been limited to low protein densities in either vesicular or bilayer morphologies. Here, we used dialysis to reproducibly form planar and vesicular BCP membranes with a high density of reconstituted aquaporin-0 (AQP0) water channels. We show that AQP0 retains its biological activity when incorporated at high density in BCP membranes, and that the morphology of the BCP–protein aggregates can be controlled by adjusting the amount of incorporated AQP0. We also show that BCPs can be used to form two-dimensional crystals of AQP0.

Formation of Planar Bilayer Membranes on Solid Supports Using Peptide Gemini Surfactants

Abstract Peptide-based gemini surfactants, called PG-surfactants, consisting of a tri- or tetra-Asp peptide core and acetylated dodecylamine-conjugated Cys residues at both the N- and C-terminal ends (D3C12 and D4C12), form a stable planar bilayer membrane on a solid support. From the DLS and TEM analyses, D3C12 and D4C12 have a tendency to form bilayer morphologies in a buffer solution, which reflects their tendency to form a stable planar bilayer membrane.

PTEN binding properties for different model membrane morphologies

PTEN acts as an agonist to PI 3‐kinase signaling by removing the phosphate group in the 3 position of PI(3,4,5)P3. PTEN membrane association and activation requires the interaction with PI(4,5)P2 and PS. In biological membranes, these lipids may form upon interaction with other molecular entities domains that exhibit distinct compositional and physicochemical characteristics. It is the objective of this study to elucidate the relationship between lipid bilayer morphologies and the extent of PTEN binding and PTEN membrane association dynamics.MethodsTo characterize lipid morphologies in the absence and presence of PTEN, we used fluorescence microscopy measurements of mixed lipid giant unilamellar vesicles (GUVs). PTEN binding extent is evaluated using SPR and Trp fluorescence measurements. The binding kinetics of PTEN bilayer interaction is elucidated using stopped‐flow experiments.ResultsThe interaction of PTEN with mixed PC/PI(4,5)P2 model membranes results in the formation of PI(4,5)P2 enriched domains. Domain formation in the absence of PTEN is also observed when cholesterol is added when making mixed PC/PI(4,5)P2 vesicles. We find an increased PTEN binding in the presence of PI(4,5)P2 domains, which is probably due to increased local PI(4,5)P2 concentration. This work was supported by NIH R01 NS021716 and NSF CHEM 0922848 and 1058719.

Thermal Stress of Supported Lipid Bilayer Induces Formation and Collapse of Uniform Radius Tubules

Lipid bilayer morphologies and the transitions between them are important to many cellular processes. Supported lipid bilayer (SLB) provides a model system in which to quantitatively investigate transitions from planar to tubular and tubular to spherical morphologies. Following a small increase in temperature (∼5-10°C) flexible filaments extrude from a fluid SLB. Individual filaments can reach hundreds of microns in length before spontaneously collapsing into discs. We demonstrate that the filaments are tubular by decreasing the external buffer concentration, which causes them to swell, first into resolvable tubules with capped ends and then into giant vesicles. At high ionic strength, the sub-resolution tubules are adsorbed to the SLB, enabling the measurement of their radius to within ±5 nm using conventional fluorescence microscopy. The radius depends on the lipid tail composition and varies <10% along the tubule length. Under tension, tubules are even more uniform, having no measurable variation in radius.

Lipid bilayers on topochemically structured planar colloidal crystals: a versatile platform for optical recording of membrane-mediated ion transport

Phospholipid membranes assembled on a planar colloidal crystal functionalized with a proton sensitive fluorescence probe offers a useful model system for a convenient, on-chip, parallel, and fluorescence based assay of membrane-mediated ion transport. The fusion of small unilamellar vesicles on surface derivatized hydrophilic/hydrophobic planar colloidal crystals results in a single lipid bilayer on the hydrophilic region and a monolayer on the hydrophobic region. We show that this composite structure exhibits a laterally continuous outer leaflet spanning the two mono- and bilayer morphologies, thereby providing an effective seal against ionic permeability. This is in contrast to the structure of lipids assembled on a patterned wettability planar coverslip where there a lipophobic “moat” formed at the junction between the mono- and the bilayer configurations prevents forming well-sealed membrane. By embedding a pH sensitive fluorescent probe, namely, fluorescein, within the colloidal crystal interstices, we find that the colloidal crystal supported membrane configuration provides a practical assay for optical characterization of membrane mediated ionic transport. We demonstrate the ability of our platform for the case of passive transport of protons across a fluid phospholipid bilayer. The membrane permeability coefficients derived, including those associated with the biphasic permeation mechanism, match well with those reported for vesicle based assays and confirm the quantitative optical transduction. We also illustrate the use of this experimental platform for parallel measurements such as may be useful for the characterization of stochastic transport and high throughput measurements.

Retinol fluorescence: a simple method to differentiate different bilayer morphologies

The fluorescence spectra of retinol obtained in bilayer structures of two different systems with dodecyl tetraethylenglycol ether are shown. A correlation between the fluorescence intensity of retinol and the different topologies of bilayers has been found. We have tested this correlation with the C12E4/benzyl alcohol/water system, and we have also applied this idea to the study of the lamellar phases of the C12E4/PEG/water system. The highest fluorescence intensity of retinol corresponds to unilamellar vesicles, while the lowest is observed for multilamellar vesicles. The kinetic study of the degradation of vitamin A in these media is also related to the different microstructures of the bilayers.

A Class of Supported Membranes: Formation of Fluid Phospholipid Bilayers on Photonic Band Gap Colloidal Crystals

We report the formation of a new class of supported membranes consisting of a fluid phospholipid bilayer coupled directly to a broadly tunable colloidal crystal with a well-defined photonic band gap. For nanoscale colloidal crystals exhibiting a band gap at the optical frequencies, substrate-induced vesicle fusion gives rise to a surface bilayer riding onto the crystal surface. The bilayer is two-dimensionally continuous, spanning multiple beads with lateral mobilities which reflect the coupling between the bilayer topography and the curvature of the supporting colloidal surface. In contrast, the spreading of vesicles on micrometer scale colloidal crystals results in the formation of bilayers wrapping individual colloidal beads. We show that simple UV photolithography of colloidal crystals produces binary patterns of crystal wettabilities, photonic stopbands, and corresponding patterns of lipid mono- and bilayer morphologies. We envisage that these approaches will be exploitable for the development of optical transduction assays and microarrays for many membrane-mediated processes, including transport and receptor-ligand interactions.

Phospholipid Morphologies on Photochemically Patterned Silane Monolayers

We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.

Self-assembling phospholipid filaments

Aqueous dispersions of double-chain phospholipids spontaneously assemble into closed bilayers called vesicles (or liposomes). Although the vesicles are in general topologically spherical, cylindrical and helical liposomes have sometimes been observed. We present here video-enhanced microscopic studies of a diacetylenic phospholipid dispersed in ethanol/water, which reveal the existence of unusual bilayer morphologies. On cooling the dispersion from the isotropic phase, we have observed the formation of long (of the order of hundreds of micrometres), thin (0.2-2 microns) filaments, which fluctuate strongly. When the temperature is decreased further, the filaments rapidly retract into a mass of lipid. At constant temperature, on the other hand, the filaments transform into torus or ring-like vesicles. Such non-spherical structures have been predicted theoretically but not previously observed experimentally.