Solid-Supported Assembly Composed of n-Octyl-ß-D-glucopyranoside and 1,2-Dioleoyl-sn-glycero-3-phosphocholine in Equilibrium with Its Ambient Aqueous Solution System Including Dispersed Assembly.

In the last few decades, the preparation of solid-supported lipid bilayers by immersing a solid substrate in an aqueous solution where the lipid is dissolved with the aid of a surfactant, followed by dilution of the solution, has been reported. In this study, we attempted to interpret the evolution of supported surfactant/lipid assemblies towards the supported lipid bilayer in terms of a phase equilibrium between the supported assembly phase and its ambient solution system consisting of the dispersed surfactant/lipid assembly phase and the bulk solution phase comprising monomeric surfactant and lipid. We characterized the supported assembly formed on hydrophilized Ge or mica substrates in equilibrium with aqueous solutions containing various concentrations of the nonionic surfactant, n-octyl-ß-D-glucopyranoside (OG) and the amphoteric phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), using interaction-force-profile measurements by atomic force microscopy (AFM), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). We also investigated the ambient solution system using equilibrium dialysis to obtain the partition equilibrium profile of OG between the bulk solution and dispersed assembly phases in the micellar or vesicular states. These studies indicate that the properties of the supported assembly depend on the composition of the dispersed assembly and concentration of monomerically dissolved OG. Further, a type of micellar-bilayer state transition occurs in the supported assembly, roughly synchronized with that in the dispersed assembly.

Layer-by-layer assembly of multi-layered droplet interface bilayers (multi-DIBs).

Droplet interface bilayers (DIBs) have tremendous promise as platforms for fundamental biomembrane studies and in biotechnology. Being composed of a single bilayer however limits their biomimetic potential, as many cell membrane motifs are composed of multiple aligned bilayers. We describe a technology to manufacture cell-sized multi-layered DIBs (multi-DIBs) by coating giant unilamellar vesicles with a further monolayer, and allowing such structures to make contact with themselves or a monolayer coated droplet. This easily customisable strategy will pave the way for an expanded repertoire of DIB functionality, for example by facilitating the incorporation of multiple-bilayer spanning protein complexes.

Chitosan-based melatonin bilayer coating for maintaining quality of fresh-cut products.

This work was designed to develop the chitosan-based melatonin layer-by-layer assembly (CMLLA) via the inclusion method. The structural characterizations and interaction present in CMLLA were investigated by the scanning electron microscope (SEM), X-ray diffraction (XRD) and Fourier Transform-Infrared spectroscopy (FTIR). The ratio of chitosan (CH) to carboxymethylcellulose (CMC) greatly influenced the mechanical properties, including the tensile strength, moisture content and color performance. Results showed that both antioxidant and antimicrobial properties of CMLLA were enhanced with the addition of melatonin (MLT). Furthermore, it was demonstrated that the CMLLA with 1.2 % (w/v) CH, 0.8 % (w/v) CMC and 50 mg/L MLT better contributed to the delay of chlorophyll degradation and the maintenance of shelf-life quality. Results from this study might open up new insights into the approaches of quality improvement of postharvest fresh products by incorporating the natural antioxidant compounds into natural polymers.

Biogenic supported lipid bilayers as a tool to investigate nano-bio interfaces.

HYPOTHESIS: Extracellular Vesicles (EVs) are natural nanosized lipid vesicles involved in most intercellular communication pathways. Given their nature, they represent natural cell membrane models, with intermediate complexity between real and synthetic lipid membranes. Here we compare EVs-derived (EVSLB) and synthetic Supported Lipid Bilayers (SLBs) in the interaction with cationic superparamagnetic iron oxide nanoparticles (SPIONs). The aim is twofold: (i) exploit SPIONs as nanometric probes to investigate the features of EVSLBs as novel biogenic platforms; (ii) contribute at improving the knowledge on the behavior of SPIONs with biological interfaces. EXPERIMENTS: Quartz Crystal Microbalance, X-ray Reflectivity, Grazing-incidence Small-angle X-ray Scattering, Atomic Force Microscopy, Confocal Microscopy data on SPIONs-EVSLB were systematically compared to those on SPIONs challenging synthetic SLBs, taken as references. FINDINGS: The ensemble of experimental results highlights the much stronger interaction of SPIONs with EVSLBs with respect to synthetic SLBs. This evidence strongly supports the hypotheses on the peculiar structure of EVSLBs, with cushioned non-flat areas and extended exposed surface; in addition, it suggests that these features are relevant in the response of biogenic membranes to nano-objects. These findings contribute to the fundamental knowledge on EVSLBs, key for their development both as biomimetic membranes, or as platforms for biomedical applications.

Performance of PEGylated chitosan and poly (L-lactic acid-co-ε-caprolactone) bilayer vascular grafts in a canine femoral artery model.

The fabrication of a functional small-diameter vascular graft with good biocompatibility, in particular hemocompatibility, has become an urgent clinical necessity. We fabricated a native bilayer, small-diameter vascular graft using PEGylated chitosan (PEG-CS) and poly (L-lactic acid-co-ε-caprolactone; PLCL). To stabilize the inner layer, a PEG-CS blend with PLCL at ratio of 1:6 was casted on a round metal bar by a drip feed, and the outer layer, a PLCL blend with water-soluble PEG that acted as a sacrificial part to enhance pore size, was fabricated by electrospinning. The results showed excellent hemocompatibility and strong mechanical properties. In vitro, the degradation of the graft was evaluated by measuring the graft structure, mass loss rate, and changes in molecular weight. The results indicated that the graft had adequate support for the regeneration of blood vessels before collapse. An in vivo study was performed in a canine femoral artery model for up to 24 weeks, which demonstrated that the PEGylated bilayer grafts possessed excellent structural integrity, high compatibility with blood, good endothelial cell (EC) and smooth muscle cell (SMC) growth, and high expression levels of angiogenesis-related proteins, features that are highly similar to autologous blood vessels. Moreover, the results showed almost negligible calcification within 24 weeks. These findings confirm that the bilayer graft mimics native cells, thereby effectively improving vascular remodeling.

Mechanical and transport properties of chitosan-zwitterionic phospholipid vesicles.

Chitosan is a polysaccharide that has shown promise in liposomal drug delivery because of certain desirable properties such as muco-adhesivity, biodegradability and low toxicity. In this study, chitosan-bearing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles were prepared using inverse phase precursor method to measure their mechanical and transport properties. We show that while an increase in chitosan: lipid molar ratio in the vesicle bilayer at pH 7 led to a substantial increase in its bending modulus, chitosan-mediated change in bending modulus was diminished at pH 4.5. Water permeability across the vesicle bilayer, as well as phospholipid diffusivity within supported lipid bilayers, were also found to decrease with increasing chitosan: lipid molar ratio. Together, these findings demonstrate that incorporation of chitosan in phospholipid bilayers modulates the mechanical and transport properties of liposomes which may affect their in vivo circulation time and drug release rate.

Incorporation of PEGylated δ-decalactone into lipid bilayers: thermodynamic study and chimeric liposomes development.

Liposomes have been on the market as drug delivery systems for over 25 years. Their success comes from the ability to carry toxic drug molecules to the appropriate site of action through passive accumulation, thus reducing their severe side effects. However, the need for enhanced circulation time and site and time-specific drug delivery turned research focus on other systems, such as polymers. In this context, novel composites that combine the flexibility of polymeric nanosystems with the properties of liposomes gained a lot of interest. In the present work a mixed/chimeric liposomal system, composed of phospholipids and block copolymers, was developed and evaluated in regards with its feasibility as a drug delivery system. These innovative nano-platforms combine advantages from both classes of biomaterials. Thermal analysis was performed in order to offers an insight into the interactions between these materials and consequently into their physicochemical characteristics. In addition, colloidal stability was assessed by monitoring z-potential and size distribution over time. Finally, their suitability as carriers for biomedical applications was evaluated by carrying out in vitro toxicity studies.

Preparation of Lipid Nanodiscs with Lipid Mixtures.

Lipid nanodiscs provide a native-like lipid environment for membrane proteins, and they have become a valuable platform for the study of membrane biophysics. A range of biophysical and biochemical analyses are enabled when membrane proteins are captured in lipid nanodiscs. Two parameters that can be controlled when capturing membrane proteins in lipid nanodiscs are the radius, and hence the surface area of the lipid surface, and the composition of the lipid bilayer. Despite their emergence as a versatile tool, most studies with lipid nanodiscs in the literature have focused on nanodiscs of a single radius with a single lipid. In light of the complexity of biological membranes, it is likely that nanodiscs with multiple membrane components would be more sophisticated models for membrane research. It is possible to prepare nanodiscs with more complex lipid mixtures to probe the effects of lipid composition on several aspects of membrane biochemistry. Detailed protocols are described here for the preparation of nanodiscs with mixtures of phospholipids, incorporation of cholesterol, and incorporation of a spectroscopic lipid probe. These protocols provide starting points for the construction of nanodiscs with more physiological membrane compositions or with useful biophysical probes. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Assembly of mixed lipid nanodiscs Basic Protocol 2: Assembly of nanodiscs with cholesterol Basic Protocol 3: Incorporation of laurdan into nanodiscs for membrane fluidity measurements.

Elastic property of membranes self-assembled from diblock and triblock copolymers.

The elastic property of membranes self-assembled from AB diblock and ABA triblock copolymers, as coarse-grained model of lipids and the bolalipids, are studied using the self-consistent field theory (SCFT). Specifically, solutions of the SCFT equations, corresponding to membranes in different geometries (planar, cylindrical, spherical, and pore) have been obtained for a model system composed of amphiphilic AB diblock copolymers and ABA triblock copolymers dissolved in A homopolymers. The free energy of the membranes with different geometries is then used to extract the bending modulus, Gaussian modulus, and line tension of the membranes. The results reveal that the bending modulus of the triblock membrane is greater than that of the diblock membrane. Furthermore, the Gaussian modulus and line tension of the triblock membrane indicate that the triblock membranes have higher pore formation energy than that of the diblock membranes. The equilibrium bridging and looping fractions of the triblock copolymers are also obtained. Implications of the theoretical results on the elastic properties of biologically equivalent lipid bilayers and the bolalipid membranes are discussed.

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers.

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

Highly Stable Artificial Cells from Galactopyranose-Derived Single-Chain Amphiphiles.

Single-chain amphiphiles (SCAs) that self-assemble into large vesicular structures are attractive components of synthetic cells because of the simplicity of bilayer formation and increased membrane permeability. However, SCAs commonly used for vesicle formation suffer from restricted working pH ranges, instability to divalent cations, and the inhibition of biocatalysts. Construction of more robust biocompatible membranes from SCAs would have significant benefits. We describe the formation of highly stable vesicles from alkyl galactopyranose thioesters. The compatibility of these uncharged SCAs with biomolecules makes possible the encapsulation of functional enzymes and nucleic acids during the vesicle generation process, enabling membrane protein reconstitution and compartmentalized nucleic acid amplification, even when charged precursors are supplied externally.

Efavirenz oral delivery via lipid nanocapsules: formulation, optimisation, and ex-vivo gut permeation study.

Present investigation aimed to prepare, optimise, and characterise lipid nanocapsules (LNCs) for improving the solubility and bioavailability of efavirenz (EFV). EFV-loaded LNCs were prepared by the phase-inversion temperature method and the influence of various formulation variables was assessed using Box-Behnken design. The prepared formulations were characterised for particle size, polydispersity index (PdI), zeta potential, encapsulation efficiency (EE), and release efficiency (RE). The biocompatibility of optimised formulation on Caco-2 cells was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Then, it was subjected to ex-vivo permeation using rat intestine. EFV-loaded LNCs were found to be spherical shape in the range of 20-100 nm with EE of 82-97%. The best results obtained from LNCs prepared by 17.5% labrafac and 10% solutol HS15 when the volume ratio of the diluting aqueous phase to the initial emulsion was 3.5. The mean particle size, zeta potential, PdI, EE, drug loading%, and RE during 144 h of optimised formulation were confirmed to 60.71 nm, -35.93 mV, 0.09, 92.60, 7.39 and 55.96%, respectively. Optimised LNCs increased the ex vivo intestinal permeation of EFV when compared with drug suspension. Thus, LNCs could be promising for improved oral delivery of EFV.

Characterization of self-assembled hybrid siloxane-phosphocholine bilayers.

We have synthesized six new hybrid siloxane phosphocholines (SiPCs) and examined their self-assembly behaviour in aqueous dispersions. Employing small angle X-ray scattering we have characterized SiPC bilayers. SiPCs exhibit differential self-assembly behaviour that results from the interplay between the siloxane fatty acid in the sn-2 position and the differing chain length fatty acids in the sn-1 position. SiPCs that possess a fatty acid chain of a C8-C14 chain length in the sn-1 position form unilamellar vesicles. Extending the fatty acid chain length to C16 and C18 allows for the formation of both unilamellar and multilamellar vesicles. We propose that the preferential formation of unilamellar vesicles is the result of an enhanced hydrophobic effect imparted by siloxane chains at the termini of lipid tails.

Spatiotemporal Kinetics of Supported Lipid Bilayer Formation on Glass via Vesicle Adsorption and Rupture.

Supported lipid bilayers (SLBs) represent one of the most popular mimics of the cell membrane. Herein, we have used total internal reflection fluorescence microscopy for in-depth characterization of the vesicle-mediated SLB formation mechanism on a common silica-rich substrate, borosilicate glass. Fluorescently labeling a subset of vesicles allowed us to monitor the adsorption of individual labeled vesicles, resolve the onset of SLB formation from small seeds of SLB patches, and track their growth via SLB-edge-induced autocatalytic rupture of adsorbed vesicles. This made it possible to perform the first quantitative measurement of the SLB front velocity, which is shown to increase up to 1 order of magnitude with time. This effect can be classified as dramatic because in many other physical, chemical, or biological kinetic processes the front velocity is either constant or decreasing with time. The observation was successfully described with a theoretical model and Monte Carlo simulations implying rapid local diffusion of lipids upon vesicle rupture.

Creating cross-linked lamellar block copolymer supporting layers for biomimetic membranes.

The long-standing goal in membrane development is creating materials with superior transport properties, including both high flux and high selectivity. These properties are common in biological membranes, and thus mimicking nature is a promising strategy towards improved membrane design. In previous studies, we have shown that artificial water channels can have excellent water transport abilities that are comparable to biological water channel proteins, aquaporins. In this study, we propose a strategy for incorporation of artificial channels that mimic biological channels into stable polymeric membranes. Specifically, we synthesized an amphiphilic triblock copolymer, poly(isoprene)-block-poly(ethylene oxide)-block-poly(isoprene), which is a high molecular weight synthetic analog of naturally occurring lipids in terms of its self-assembled structure. This polymer was used to build stacked membranes composed of self-assembled lamellae. The resulting membranes resemble layers of natural lipid bilayers in living systems, but with superior mechanical properties suitable for real-world applications. The procedures used to synthesize the triblock copolymer resulted in membranes with increased stability due to the crosslinkability of the hydrophobic domains. Furthermore, the introduction of bridging hydrophilic domains leads to the preservation of the stacked membrane structure when the membrane is in contact with water, something that is challenging for diblock lamellae that tend to swell, and delaminate in aqueous solutions. This new method of membrane fabrication offers a practical model for making channel-based biomimetic membranes, which may lead to technological applications in reverse osmosis, nanofiltration, and ultrafiltration membranes.

Tunable cell-surface mimetics as engineered cell substrates.

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed.

Headgroup-Inversed Liposomes: Biointerfaces, Supported Bilayers and Applications.

Phospholipids are a major component of the cell membrane. In most natural phospholipids, the phosphate acts as a bridge, connecting the other portion of the polar headgroup with the hydrophobic tails. Such bridging phosphate is chemically quite inert. Synthetic lipids inversing the headgroup polarity of phosphocholine (PC) have been recently reported, and these are named CP lipids with a terminal phosphate, or CPe with the terminal phosphate capped by an ethyl group. This Feature Article summarizes the properties and applications of such inversed lipids. First, CPe liposomes were found to be highly resistant to protein adsorption with an even longer blood circulation time than PC liposomes, allowing for enhanced accumulation in tumor sites. CPe liposomes do not interact with PC liposomes either, and this observation was different from that reported using CP polymers, which adhere strongly to cells. Second, CP liposomes interact strongly with many metal oxide nanoparticles (but not silica) forming supported lipid bilayers, while PC liposomes only form supported bilayers on silica. Finally, CP liposomes are good metal ligands based on their exposed terminal phosphate. Zn2+ binds to CP liposomes so strongly that Zn2+ sandwiched multilayered lipid structures were observed. Aside from these fundamental aspects, the potential applications of these headgroup-inversed lipids in drug delivery and biosensor development have also been described, which in turn has promoted fundamental biointerface insights.

Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels.

The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-a-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.

Effects of Sterol-Like Additives on Phase Transition Behavior of Ion-Pair Amphiphile Bilayers.

The incorporation of additive in lipid bilayers is one of the ordinary approaches for modulating their properties. Additive effect on phase transition of ion-pair amphiphile (IPA) bilayers, however, is not known. In this work, four double-chained IPAs with different hydrocarbon chain lengths and symmetry were designed and synthesized from single-chained cationic and anionic surfactants by the precipitation method. By using differential scanning calorimetry (DSC), the thermotropic transition behavior from gel phase (Lß) through rippled phase (Pß') if any to liquid-crystalline phase (Lα) was studied for bilayers of these lipid-like IPAs in excess water. The effects of three sterol-like additives (cholesterol, α-tocopherol, and α-tocopheryl acetate) in IPA bilayers on thermal phase behavior were then systematically investigated. The experimental results revealed that with increasing concentration of additive, the phase transition temperatures were unaffected on the one hand and the enthalpies of phase transition were decreased on the other hand. When the addition of additive exceeded a specific amount, the phase transition disappeared. More hasty disappearance of phase transition was found for IPAs with lower total number of carbon atoms in the hydrocarbon chains. For IPAs with the same total number of carbon atoms in the hydrocarbon chains, the disappearance of phase transition is more hasty for the asymmetric one than for the symmetric one. Similar effects on thermal phase behavior of four IPA bilayers were exhibited by the three additives with similar chemical structures. Possible mechanism of additive effects on phase transition of IPA bilayers was then proposed in line with that of lipid bilayers.

Thermodynamics of Indomethacin Adsorption to Phospholipid Membranes.

Using second-harmonic generation, we directly monitored adsorption of indomethacin, a nonsteroidal anti-inflammatory drug, to supported lipid bilayers composed of phospholipids of varying phase, cholesterol content, and head group charge without the use of extrinsic labels at therapeutically relevant aqueous concentrations. Indomethacin adsorbed to gel-phase lipids with a high binding affinity, suggesting that like other arylacetic acid-containing drugs, it preferentially interacts with ordered lipid domains. We discovered that adsorption of indomethacin to gel-phase phospholipids was endothermic and entropically driven, whereas adsorption to fluid-phase phospholipids was exothermic and enthalpically driven. As temperature increased from 19 to 34 °C, binding affinities to gel-phase lipids increased by 7-fold but relative surface concentration decreased to one-fifth of the original value. We also compared our results to the entropies reported for indomethacin adsorbed to surfactant micelles, which are used in drug delivery systems, and assert that adsorbed water molecules in the phospholipid bilayer may be buried deeper into the acyl chains and less accessible for disruption. The thermodynamic studies reported here provide mechanistic insight into indomethacin interactions with mammalian plasma membranes in the gastrointestinal tract and inform studies of drug delivery, where indomethacin is commonly used as a prototypical, hydrophobic small-molecule drug.