Evaluation of asymmetric liposomal nanoparticles for encapsulation of polynucleotides

Conventional lipid bilayer liposomes have similar inner and outer leaflet compositions; asymmetric liposomes have different lipid leaflet compositions. The goal of this work is to place cationic lipids in the inner leaflet to encapsulate negatively charged polynucleotides and to place neutral/anionic lipids on the outer leaflet to decrease nonspecific cellular uptake/toxicity. Inverse emulsion particles have been developed with a single lipid leaflet of cationic and neutral lipids surrounding an aqueous core containing a negatively charged 21-mer DNA oligo. The particles are accelerated through an oil-water interface, entrapping a second neutral lipid to form oligo encapsulated unilamellar liposome nanoparticles. Inverse emulsion particles can be consistently produced to encapsulate an aqueous environment containing negatively charged oligo. The efficiency of encapsulated liposome formation is low and depends on the hydrocarbon used as the oil phase. Dodecane, mineral oil, and squalene were tested, and squalene, a branched hydrocarbon, yielded the highest efficiency.     

Formation of artificial lipid bilayers using droplet dielectrophoresis

We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be extended to create programmable BLM arrays and networks.     

Voltage control of droplet interface bilayer lipid membrane dimensions

A novel approach to control the area of anchor-free droplet interface bilayer (DIB) lipid membranes is presented. Unsupported DIB lipid membranes are formed at the interface of phospholipid-coated aqueous droplets dispensed in dodecane oil. Using electrodes inserted into the droplets, an external voltage is applied which modulates the effective DIB area. Electrical (capacitance or current) and optical (imaging of DIB lateral length) recordings were simultaneously performed. Alpha-hemolysin (αHL) single channel insertions into the DIB were recorded. Currents across the DIB were measured as a function of voltage and αHL concentration in the droplets. Nonlinear response is observed for current, DIB lateral length and area, and capacitance with respect to voltage. Voltage induced changes in interfacial tension modulated the DIB-oil contact angle and the membrane contact length, which provided control of membrane dimensions. Comparison of these results is made to the electrowetting effect, which is also governed by effect of voltage on the interfacial tension. This approach provides active control of the number of ion channels inserted into the DIB.     

Functional bionetworks from nanoliter water droplets

We form networks from aqueous droplets by submerging them in an oil/lipid mixture. When the droplets are joined together, the lipid monolayers surrounding them combine at the interface to form a robust lipid bilayer. Various protein channels and pores can incorporate into the droplet-interface bilayer (DIB), and the application of a potential with electrodes embedded within the droplets allows ionic currents to be driven across the interface and measured. By joining droplets in linear or branched geometries, functional bionetworks can be created. Although the interfaces between neighboring droplets comprise only single lipid bilayers, the structures of the networks are long-lived and robust. Indeed, a single droplet can be "surgically" excised from a network and replaced with a new droplet without rupturing adjacent DIBs. Networks of droplets can be powered with internal "biobatteries" that use ion gradients or the light-driven proton pump bacteriorhodopsin. Besides their interest as coupled protocells, the droplets can be used as devices for ultrastable bilayer recording with greatly reduced electrolyte volume, which will permit their use in rapid screening applications.     

Triggered release of molecules across droplet interface bilayer lipid membranes using photopolymerizable lipids

A combination of nonpolymerizable phospholipids (DPPC or DPhPC) and a smaller amount of cross-linking photopolymerizable phospholipids (23:2 DiynePC) is incorporated in an unsupported artificial lipid bilayer formed using the droplet interface bilayer (DIB) approach. The DIB is formed by contacting lipid monolayer-coated aqueous droplets against each other in a dodecane-lipid medium. Cross-linking of the photopolymerizable lipids incorporated in the DIB was obtained by exposure to UV-C radiation (254 nm), resulting in pore formation. The effect of cross-linking on the DIB properties was characterized optically by measuring the diffusion of selectively encapsulated dye molecules (calcein) from one droplet of the DIB to the other droplet. Changes in DIB conductivity due to UV-C exposure were investigated using current-voltage (I-V) measurements. The leakage of dye molecules across the DIB and the increase in DIB conductivity after UV-C exposure indicates the formation of membrane pores. The results indicate that the DIB approach offers a simple and flexible platform for studying phototriggered drug delivery systems in vitro.     

Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Calcium adsorption and displacement: characterization of lipid monolayers and their interaction with membrane-active peptides/proteins

Background  

The first target of antimicrobial peptides (AMPs) is the bacterial membrane. In the case of Gram-negative bacteria this is the outer membrane (OM), the lipid composition of which is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is made up solely from lipopolysaccharides (LPSs). LPS, therefore, represents the first target of AMPs. The binding and intercalation of polycationic AMPs is driven by the number and position of negatively charged groups of the LPS. Also, proteins other than cationic AMPs can interact with LPS, e.g. leading eventually to a neutralization of the endotoxic effects of LPS. We compared different biophysical techniques to gain insight into the properties of the electrical surface potentials of lipid monolayers and aggregates composed of LPSs and various phospholipids and their interaction with peptides and proteins.     

Electrical behavior of droplet interface bilayer networks: experimental analysis and modeling

Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. Such DIB networks might be used as model systems for the study of membrane-based biological phenomena. In this study, we develop and experimentally validate an electrical modeling approach for DIB networks by applying it to describe the current flow through a simple network containing protein pores and blocking molecules. We demonstrate the use of SPICE (Simulation Program with Integrated Circuit Emphasis) for simulating the electrical behavior of DIB networks. The modular and scalable nature of DIB networks should enable a straightforward extension of the analysis presented in this paper to large, complex networks.     

Direct quantitation of peptide-mediated protein transport across a droplet-interface bilayer

We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof-of-concept, the cell-penetrating peptide, Pep-1, was used to carry horseradish peroxidase (HRP) across droplet-interface bilayers (DIBs). Two submicroliter, lipid-encased aqueous droplets form a membrane at the contacting interface, through which enzyme-peptide complexes pass during transport. Following transport, the droplets are separated and the captured enzymes are assayed by a fluorogenic reaction. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that leaflet charge symmetry may play a role in Pep-1-mediated protein translocation. We anticipate that the DIB method may be useful for a variety of transport-based studies.     

Asymmetric insertion of membrane proteins in lipid bilayers by solid-state NMR paramagnetic relaxation enhancement: a cell-penetrating Peptide example

A novel solid-state NMR technique for identifying the asymmetric insertion depths of membrane proteins in lipid bilayers is introduced. By applying Mn (2+) ions on the outer but not the inner leaflet of lipid bilayers, the sidedness of protein residues in the lipid bilayer can be determined through paramagnetic relaxation enhancement (PRE) effects. Protein-free lipid membranes with one-side Mn (2+)-bound surfaces exhibit significant residual (31)P and lipid headgroup (13)C intensities, in contrast to two-side Mn (2+)-bound membranes, where lipid headgroup signals are mostly suppressed. Applying this method to a cell-penetrating peptide, penetratin, we found that at low peptide concentrations, penetratin is distributed in both leaflets of the bilayer, in contrast to the prediction of the electroporation model, which predicts that penetratin binds to only the outer lipid leaflet at low peptide concentrations to cause an electric field that drives subsequent peptide translocation. The invalidation of the electroporation model suggests an alternative mechanism for intracellular import of penetratin, which may involve guanidinium-phosphate complexation between the peptide and the lipids.     

Model of an asymmetric DPPC/DPPS membrane: effect of asymmetry on the lipid properties. A molecular dynamics simulation study

The study of asymmetric lipid bilayers is of a crucial importance due to the great number of biological process in which they are involved such as exocytosis, intracellular fusion processes, phospholipid-protein interactions, and signal transduction pathway. In addition, the loss of this asymmetry is a hallmark of the early stages of apoptosis. In this regard, a model of an asymmetric lipid bilayer composed of DPPC and DPPS was simulated by molecular dynamics simulation. Thus, the asymmetric membrane was modeled by 264 lipids, of which 48 corresponded to DPPS- randomly distributed in the same leaflet with 96 DPPC. In the other leaflet, 120 DPPC were placed without DPPS-. Due to the presence of a net charge of -1 for the DPPS- in physiological conditions, 48 Na+ were introduced into the system to balance the charge. To ascertain whether the presence of the DPPS- in only one of the two leaflets perturbs the properties of the DPPC in the other leaflet composed only of DPPC, different properties were studied, such as the atomic density of the different components across the membrane, the electrostatic potential across the membrane, the translational diffusion of DPPC and DPPS, the deuterium order parameters, lipid hydration, and lipid-lipid charge bridges. Thus, we obtained that certain properties such as the surface area lipid molecule, lipid head orientation, order parameter, translational diffusion coefficient, or lipid hydration of DPPC in the leaflet without DPPS remain unperturbed by the presence of DPPS in the other leaflet, compared with a DPPC bilayer. On the other hand, in the leaflet containing DPPS, some of the DPPC properties were strongly affected by the presence of DPPS such as the order parameter or electrostatic potential.     

Oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel KcsA

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.     

Preparation and characterization of asymmetric planar supported bilayers composed of poly(bis-sorbylphosphatidylcholine) on n-octadecyltrichlorosilane SAMs

Planar supported lipid bilayers (PSLBs) have been widely studied as biomembrane models and biosensor scaffolds. For technological applications, a major limitation of PSLBs composed of fluid lipids is that the bilayer structure is readily disrupted when exposed to chemical, mechanical, and thermal stresses. A number of asymmetric supported bilayer structures, such as the hybrid bilayer membrane (HBM) and the tethered bilayer lipid membrane (tBLM), have been created as an alternative to symmetric PSLBs. In both HBMs and tBLMs, the inner monolayer is covalently attached to the substrate while the outer monolayer is typically composed of a fluid lipid. Here we address if cross-linking polymerization of the lipids in the outer monolayer of an asymmetric supported bilayer can achieve the high degree of stability observed previously for symmetric PSLBs in which both monolayers are cross-linked [E.E. Ross, L.J. Rozanski, T. Spratt, S.C. Liu, D.F. O'Brien, S.S. Saavedra, Langmuir 19 (2003) 1752]. To explore this issue, HBMs composed of an outer monolayer of a cross-linkable lipid, bis-sorbylphosphatidylcholine (bis-SorbPC), and an inner SAM were prepared and characterized. Several experimental conditions were varied: vesicle fusion time, polymerization method, and polymerization time and temperature. Under most conditions, bis-SorbPC cross-linking stabilized the HBM such that its bilayer structure was largely preserved after drying; however these films invariably contained sub-micron scale defects that exposed the hydrophobic core of the HBM. The defects appear to be caused by desorption of low molecular weight oligomers when the film is removed from water, rinsed, and dried. In contrast, poly(bis-SorbPC) PSLBs prepared under similar conditions by Ross et al. were nearly defect free. This comparison shows that formation of a cross-linked network in the outer leaflet of an asymmetric supported bilayer is insufficient to prevent lipid desorption; inter-leaflet covalent linking appears to be necessary to create supported poly(lipid) assemblies that are impervious to repeated drying and rehydration. The difference in stability is attributed to inter-leaflet cross-linking between monolayers which can form in symmetric bis-SorbPC PSLBs.     

Direct detection of membrane channels from gels using water-in-oil droplet bilayers

We form planar lipid bilayers between an aqueous droplet and a hydrogel support immersed in a lipid-oil solution. By scanning the bilayer over the surface of an SDS-PAGE gel, we are able to directly detect membrane proteins from gels using single-channel recording. Using this technique, we are able to examine low levels of endogenous protein from cell extracts without the need for over-expression. We also use droplet bilayers to detect small molecules from hydrogels. The bilayers show enhanced stability compared to conventional planar lipid bilayers, and both bilayer size and position can be controlled during an experiment. Hydrogel scanning with droplet bilayers provides a new method for the discovery and characterization of ion channels with the potential for high-throughput screening.     

Tethered bilayer lipid micromembranes for single-channel recording: the role of adsorbed and partially fused lipid vesicles

A mercury-supported bilayer lipid micromembrane was prepared by anchoring a thiolipid monolayer to a mercury cap electrodeposited on a platinum microdisc about 20 μm in diameter; a lipid monolayer was then self-assembled on top of the thiolipid monolayer either by vesicle fusion or by spilling a few drops of a lipid solution in chloroform on the cap and allowing the solvent to evaporate. Single-channel recording following incorporation of the alamethicin channel-forming peptide exhibits quite different features, depending on the procedure followed to form the distal lipid monolayer. The "spilling" procedure, which avoids the formation of adsorbed or partially fused vesicles, yields very sharp single-channel currents lasting only one or two milliseconds. These are ascribed to ionic flux into the hydrophilic spacer moiety of the thiolipid. Conversely, the vesicle-fusion procedure yields much longer single-channel openings analogous to those obtained with conventional bilayer lipid membranes, albeit smaller. This difference in behavior is explained by ascribing the latter single-channel currents to ionic flux into vesicles adsorbed and/or partially fused onto the tethered lipid bilayer, via capacitive coupling.     

Size-dependent properties of small unilamellar vesicles formed by model lipids

The size-dependent behavior of small unilamellar vesicles is explored by dissipative particle dynamics, including the membrane characteristics and mechanical properties. The spontaneously formed vesicles are in the metastable state and the vesicle size is controlled by the concentration of model lipids. As the vesicle size decreases, the bilayer gets thinner and the area density of heads declines. Nonetheless, the area density in the inner leaflet is higher than that in the outer. The packing parameters are calculated for both leaflets. The result indicates that the shape of lipid in the outer leaflet is like a truncated cone but that in the inner leaflet resembles an inverted truncated cone. Based on a local order parameter, our simulations indication that the orientation order of lipid molecules decreases as the size of the vesicle reduces and this fact reveals that the bilayer becoming thinner for smaller vesicle is mainly attributed to the orientation disorder of the lipids. The membrane tension can be obtained through the Young-Laplace equation. The tension is found to grow with reducing vesicle size. Therefore, small vesicles are less stable against fusion. Using the inflation method, the area stretching and bending moduli can be determined and those moduli are found to grow with reducing size. Nonetheless, a general equation with a single numerical constant can relate bending modulus, area stretching modulus, and bilayer thickness irrespective of the vesicle size. Finally, a simple metastable model is proposed to explain the size-dependent behavior of bilayer thickness, orientation, and tension.     

Bias-dependent admittance in hybrid bilayer membranes

Artificial bilayer membranes provide a platform for bioelectronic devices based on their structural, sensing, and transport functions. In this letter, we report on the impedance response of an engineered membrane with a lower leaflet of octadecanethiol on gold and an outer leaflet of dioleoylphosphatidylcholine with the monomeric channel protein gramicidin. This hybrid bilayer exhibits an electrical response analogous to a solid-state diode: the admittance is very low (<10(-)(7) Omega(-)(1) cm(-)(2)) over a wide potential range but increases exponentially at negative potentials.     

Rectification of the current in alpha-hemolysin pore depends on the cation type: the alkali series probed by MD simulations and experiments

A striking feature of the alpha-hemolysin channel-a prime candidate for biotechnological applications-is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li(+) to Cs(+). Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel's end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.     

Asymmetry of lipid bilayers induced by monovalent salt: atomistic molecular-dynamics study

Interactions between salt ions and lipid components of biological membranes are essential for the structure, stability, and functions of the membranes. The specific ionic composition of aqueous buffers inside and outside of the cell is known to differ considerably. To model such a situation we perform atomistic molecular-dynamics (MD) simulations of a single-component phosphatidylcholine lipid bilayer which separates two aqueous reservoirs with and without NaCl salt. To implement the difference in electrolyte composition near two membrane sides, a double bilayer setup (i.e., two bilayers in a simulation box) is employed. It turns out that monovalent salt, being in contact with one leaflet only, induces a pronounced asymmetry in the structural, electrostatic, and dynamical properties of bilayer leaflets after 50 ns of MD simulations. Binding of sodium ions to the carbonyl region of the leaflet which is in contact with salt results in the formation of "Na-lipids" complexes and, correspondingly, reduces mobility of lipids of this leaflet. In turn, attractive interactions of chloride ions (mainly located in the aqueous phase close to the water-lipid interface) with choline lipid groups lead to a substantial (more vertical) reorientation of postphatidylcholine headgroups of the leaflet adjoined to salt. The difference in headgroup orientation on two sides of a bilayer, being coupled with salt-induced reorientation of water dipoles, leads to a notable asymmetry in the charge-density profiles and electrostatic potentials of bilayer constitutes of the two leaflets. Although the overall charge density of the bilayer is found to be almost insensitive to the presence of salt, a slight asymmetry in the charge distribution between the two bilayer leaflets results in a nonzero potential difference of about 85 mV between the two water phases. Thus, a transmembrane potential of the order of the membrane potential in a cell can arise without ionic charge imbalance between two aqueous compartments.     

Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.