Simulations of lipid transfer between a supported lipid bilayer and adsorbing vesicles

Recent experiments demonstrate transfer of lipid molecules between a charged, supported lipid membrane (SLB) and vesicles of opposite charge when the latter adsorb on the SLB. A simple phenomenological bead model has been developed to simulate this process. Beads were defined to be of three types, ‘n’, ‘p’, and ‘0’, representing POPS (negatively charged), POEPC (positively charged), and POPC (neutral but zwitterionic) lipids, respectively. Phenomenological bead–bead interaction potentials and lipid transfer rate constants were used to account for the overall interaction and transfer kinetics. Using different bead mixtures in both the adsorbing vesicle and in the SLB (representing differently composed/charged vesicles and SLBs as in the reported experiments), we clarify under which circumstances a vesicle adsorbs to the SLB, and whether it, after lipid transfer and changed composition of the SLB and vesicle, desorbs back to the bulk again or not. With this model we can reproduce and provide a conceptual picture for the experimental findings.     

pH-driven assembly of various supported lipid platforms: a comparative study on silicon oxide and titanium oxide

Supported lipid platforms are versatile cell membrane mimics whose structural properties can be tailored to suit the application of interest. By identifying parameters that control the self-assembly of these platforms, there is potential to develop advanced biomimetic systems that overcome the surface specificity of lipid vesicle interactions under physiological conditions. In this work, we investigated the adsorption kinetics of vesicles onto silicon and titanium oxides as a function of pH. On each substrate, a planar bilayer and a layer of intact vesicles could be self-assembled in a pH-dependent manner, demonstrating the role of surface charge density in the self-assembly process. Under acidic pH conditions where both zwitterionic lipid vesicles and the oxide films possess near-neutral electric surface charges, vesicle rupture could occur, demonstrating that the process is driven by nonelectrostatic interactions. However, we observed that the initial rupturing process is insufficient for propagating bilayer formation. The role of electrostatic interactions for propagating bilayer formation differs for the two substrates; electrostatic attraction between vesicles and the substrate is necessary for complete bilayer formation on titanium oxide but is not necessary on silicon oxide. Conversely, in the high pH regime, repulsive electrostatic interactions can result in the irreversible adsorption of intact vesicles on silicon oxide and even a reversibly adsorbed vesicle layer on titanium oxide. Together, the results show that pH is an effective tool to modulate vesicle-substrate interactions in order to create various self-assembled lipid platforms on hydrophilic substrates.     

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle–vesicle and vesicle–substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.     

A thermodynamic study of protein-induced lipid lateral phase separation. Effect of lysozyme on mixed lipid vesicles

In this work we study by differential scanning calorimetry (DSC) the lateral phase separation induced by a globular protein (lysozyme) on vesicles built-up by charged (phosphatidic acid) and neutral (phosphatidylcholine) lipids.The adsorption of the positively charged protein onto the negative vesicle surface induces the formation of micro-domains richer in the charged lipid component. This phenomenon is revealed as a splitting of the excess heat capacity peak associated to the melting of the lipid hydrocarbon chains.Also, the peak associated to the protein denaturation is shifted, suggesting the presence of adsorbed proteins onto the vesicle surface. The surface electrostatic potentials, both of proteins and vesicles, have been modulated by pH and ionic strength variations, showing a deep influence of the electric charges in modifying protein adsorption, rate of denaturation (related to unfolding enthalpy variation), and lipid micro-domain formation.Some of the present results have been rationalized on the basis of a theoretical model recently developed by the authors.     

Patterning polymerized lipid vesicles with soft lithography

The applications of soft lithography in patterning polymerized lipid vesicles of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine on glass substrates are reported. We demonstrate that the polymerized vesicles can be used as a high molecular weight ink to be transferred from a PDMS stamp onto a glass substrate to form two-dimensional stripes with a controlled separation. By combining channel flow with dewetting within microfluidic networks, we assemble the polymerized vesicle into three-dimensional stripes and one-dimension lines on glass substrates. Atomic force microscopy shows that these patterned vesicle structures are stable on glass substrates. The simple, stable, and precise immobilization of lipid vesicles on solid substrates will open up the possibility of integrating them in biosensors and microelectronic devices.     

Entropic stabilization and equilibrium size of lipid vesicles

We have studied the phase behavior of zwitterionic phospholipid dioleoylphosphatidylcholine (DOPC) vesicles (membranes) and interpreted our results using scaling arguments in combination with molecular realistic self-consistent field (SCF) calculations. DOPC membranes acquire a partial negative charge per lipid molecule at intermediate NaBr concentrations. As a result of this, dilute DOPC solutions form stable unilamellar vesicles. Both at low and high salt concentrations phase separation into a lamellar and a vesicular phase is observed. The vesicle radius decreases as a power law with decreasing lipid concentration. This power-law concentration dependence indicates that the vesicle phase is entropically stabilized; the size of the DOPC vesicles result from a competition between the bending energy and translation and undulation entropy. This scaling behavior breaks down for very small vesicles. This appears to be consistent with SCF predictions that point to the fact that in this regime the mean bending modulus kc increases with curvature. The SCF theory predicts that, at low ionic strength, the membrane stability improves when there is more charge on the lipids. Upon a decrease of the ionic strength, lipids with a full negative charge form vesicles that grow exponentially in size because the mean bending modulus increases with decreasing ionic strength. At the same time the Gaussian bending modulus becomes increasingly negative such that the overall bending energy tends to zero. This indicates that small micelles become the dominant species. The SCF theory thus predicts a catastrophic break down of giant vesicles in favor of small micelles at sufficiently low ionic strength and high charge density on the lipids.     

Supported lipid bilayers on biocompatible polysaccharide multilayers

Formation of supported lipid bilayers on soft polymer cushions is a useful approach to decouple the membrane from the substrate for applications involving membrane proteins. We prepared biocompatible polymer cushions by the layer-by-layer assembly of two polysaccharide polyelectrolytes, chitosan (CHI) and hyaluronic acid, on glass and silicon substrates. (CHI/HA)(5) films were characterized by atomic force microscopy, giving an average thickness of 57 nm and roughness of 25 nm in aqueous solution at pH 6.5. Formation of zwitterionic lipid bilayers by the vesicle fusion method was attempted using DOPC vesicles at pH 4 and 6.5 on (CHI/HA)(5) films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; a combination of fluorescence and AFM indicated that this was attributable to formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. By contrast, more uniform bilayers with mobile lipids were produced at pH 4. Fluorescence recovery after photobleaching gave diffusion coefficients that were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. These results demonstrate that polysaccharides provide a useful alternative to other polymer cushions, particularly for applications where biocompatibility is important.     

Simulations of lipid adsorption on TiO2 surfaces in solution

Molecular dynamics simulations are carried out to study the adsorption of three lipids, namely, DOPC, DOPS, and DMTAP, on TiO2(110) rutile surfaces and the influence of the interface on their conformational properties. Three types of rutile (110) surfaces, characterized by a different degree of hydroxylation (the neutral nonhydroxylated and hydroxylated surfaces and a partially hydroxylated surface with charge density corresponding to physiological pH) are investigated using force fields derived from ab initio calculations and experimental data. It is found that the stability of the adsorbate and the strength of the attachment are strictly connected with the nature of both the lipid and the surface. Direct coordination of the phosphate or carbonyl oxygens of the lipids with available titanium sites, observed in the case of partially or nonhydroxylated layers, determines stronger adsorption and, as a consequence, reduced dynamics. For a given hydration state of the surface, the adsorption strengths are in the order DOPS > DOPC > DMTAP, in agreement with experimental data according to which the presence of DOPS units inside lipid bilayers favors stronger adsorption and lower mobility. The adsorption geometry, the hydration state of the lipid headgroups, and the dynamical processes (detachment, diffusion, etc.) occurring at the lipid/oxide interface are analyzed in detail, putting on a roughly quantitative basis time scales and energy barriers of the latter processes.     

Simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption

Recent experimental investigations of the kinetics of vesicle adsorption in solution on SiO2 demonstrate a thermally activated transition from adsorbed intact vesicles to a supported lipid bilayer. Our Monte Carlo simulations clarify the mechanism of this process. The model employed is an extension of the model used earlier to describe vesicle adsorption at room temperature. Specifically, it includes limitations of the adsorption rate by vesicle diffusion in the solution, and adsorption- and lipid-membrane-induced rupture of arriving and already adsorbed vesicles. Vesicles and lipid molecules, formed after rupture of vesicles, are considered immobile. With these ingredients, the model is able to quantitatively reproduce the temperature-dependent adsorption kinetics, including a higher critical surface concentration of intact vesicles for lower temperatures, and the apparent activation energy for the vesicle-to-bilayer transition E(a) approximately 5 kcal/mol.     

Dynamic interaction between oppositely charged vesicles: aggregation, lipid mixing, and disaggregation

We investigated dynamic interactions between oppositely charged small unilamellar vesicles using positively charged vesicles containing 1,2-dioleoyl-3-trimethylammonium-propane or 3beta-[N-(N('),N(')-dimethylaminoethane)-carbamoyl] cholesterol and negatively charged vesicles containing L-alpha-phosphatidyl-DL-glycerol. Aggregation, lipid bilayer mixing, contents mixing and contents leakage were systematically examined using optical density measurements, fluorescence resonance energy transfer assays, fluorescence quenching assays, light-scattering analyses, and freeze-fracture transmission electron microscopy. The oppositely charged vesicles aggregated immediately. Lipid mixing was observed, but there was no mixing of the contents. The vesicle aggregates disaggregated spontaneously after several minutes. The surface potential of the disaggregated vesicles was neutralized. From these results, we infer that the lipids in the external monolayers were exchanged between the oppositely charged vesicles while the internal monolayers remained intact. The two types of cationic lipids used exhibited different speeds of disaggregation.     

Real-time QCM-D monitoring of electrostatically driven lipid transfer between two lipid bilayer membranes

The lipid exchange/transfer between lipid membranes is important for many biological functions. To learn more about how the dynamics of such processes can be studied, we have investigated the interaction of positively and negatively charged lipid vesicles with supported lipid bilayers (SLBs) of opposite charge. The vesicle-SLB interaction leads initially to adsorption of lipid vesicles on the SLB, as deduced from the mass uptake kinetics and the concerted increase in dissipation, monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Eventually, however, vesicles (and possibly other lipid structures) desorb from the SLB surface, as judged from the mass loss and the dissipation decrease. The mass loss is approximately as large as the initial mass increase; i.e., at the end of the process the mass load is that of a SLB. We interpret this interesting kinetics in terms of initial strong electrostatic attraction between the added vesicles and the SLB, forming a structure where lipid transfer between the two bilayers occurs on a time scale of 10-40 min. We suggest that this lipid transfer causes a charge equilibration with an accompanying weakening of the attraction, and eventually repulsion, between the SLB and vesicles, leading to desorption of vesicles from the SLB. The composition of the latter has thus been modified compared to the initial one, although no net mass increase or decrease has occurred. Direct evidence for the lipid exchange was obtained by sequential experiments with alternating positive and negative vesicles, as well as by using fluorescently labeled lipids and FRAP. The above interpretation was further strengthened by combined QCM-D and optical reflectometry measurements.     

Dissipation-enhanced quartz crystal microbalance studies on the experimental parameters controlling the formation of supported lipid bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.     

A silicate gel for promoting deposition of lipid bilayers

A simple method for modifying a polymer surface to induce lipid bilayer formation by vesicle fusion is described. A silicate gel was prepared by condensation of tetraethyl orthosilicate (TEOS) in the presence of acid. When applied to a poly(methylmethacrylate) substrate, either a rough or a smooth layer could be produced, depending on the method used for the application. The smooth surface induced formation of a supported lipid bilayer by fusion of lipid vesicles; the rough silicate surface induced adsorption of a vesicle layer. A high-frequency acoustic waveguide device was used to follow the initial adsorption of vesicles, the transition from a vesicle layer to a bilayer, and the formation of a complete bilayer; the time required to form a bilayer was determined as a function of lipid concentration in suspension. The presence of a bilayer on the smooth silicate surface was confirmed by fluorescence recovery after photobleaching. An additional procedure is described to modify a gold surface to induce bilayer formation.     

Solid-supported block copolymer membranes through interfacial adsorption of charged block copolymer vesicles

The properties of amphiphilic block copolymer membranes can be tailored within a wide range of physical parameters. This makes them promising candidates for the development of new (bio)sensors based on solid-supported biomimetic membranes. Here we investigated the interfacial adsorption of polyelectrolyte vesicles on three different model substrates to find the optimum conditions for formation of planar membranes. The polymer vesicles were made from amphiphilic ABA triblock copolymers with short, positively charged poly(2,2-dimethylaminoethyl methacrylate) (PDMAEMA) end blocks and a hydrophobic poly( n-butyl methacrylate) (PBMA) middle block. We observed reorganization of the amphiphilic copolymer chains from vesicular structures into a 1.5+/-0.04 nm thick layer on the hydrophobic HOPG surface. However, this film starts disrupting and dewetting upon drying. In contrast, adsorption of the vesicles on the negatively charged SiO2 and mica substrates induced vesicle fusion and formation of planar, supported block copolymer films. This process seems to be controlled by the surface charge density of the substrate and concentration of the block copolymers in solution. The thickness of the copolymer membrane on mica was comparable to the thickness of phospholipid bilayers.     

Covalently anchored lipid structures on amine-enriched polystyrene

The study of the adhesion of lipid vesicles on surfaces is of increasing interest in the field of medical implants and tissue engineering (protein-resistant surfaces), drug delivery, biosensors, and biochips. In this work, lipid coverage was developed from PEG-coated vesicles (with sizes from 100 to 300 nm) by covalently binding poly(ethylene glycol)-alpha-disteroylphosphatidylethanolamine-omega-benzotriazole carbonate (DSPE-PEG-BTC) molecules onto the surface amine groups by carbamate chemistry. Lipid surface density and the surface structure of multilamellar (MLVs) and extruded unilamellar (LUVs) vesicles deposited on three types of polystyrene (PS) well-plates were probed by fluorescence and atomic force microscopy (AFM) imaging. A significant difference in the vesicle surface coverage of PS substrates was observed with a substantial increase in lipid multilayers on the amine-enriched PS surface using both unilamellar and multilamellar vesicles.     

Properties of mixed lipid monolayers assembled on hydrophobic surfaces through vesicle adsorption

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.     

Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels

We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively.     

Migration of poly-L-lysine through a lipid bilayer

When a giant vesicle, composed of neutral and anionic lipid (90:10 mol %), comes into contact with various poly-l-lysines (MW 500-29 300), ropelike structures form within the vesicle interior. By using fluorescence lipids and epi-fluorescence microscopy, we have shown that both neutral and anionic lipids are constituents of the ropes. Evidence that the ropes are also comprised of poly-l-lysine comes from two experiments: (a) direct microinjection of poly(acrylic acid) into rope-containing vesicles causes the ropes to contract into small particles, an observation consistent with a polycation/polyanion interaction; and (b) direct microinjection of fluorescein isothiocyanate (a compound that covalently labels poly-l-lysine with a fluorescent moiety) into rope-containing vesicles leads to fluorescent ropes. The results may be explained by a model in which poly-l-lysine binds to the vesicle exterior, forms a domain, and enters the vesicle through defects or at the domain boundary. The model helps explain the ability of poly-l-lysine to mediate the permeation of a cancer drug, doxorubicine, into the vesicle interior.     

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.     

AFM characterization of spin-coated multilayered dry lipid films prepared from aqueous vesicle suspensions

We present a detailed AFM study on multilayered dry lipid films prepared from aqueous vesicle suspensions. Different preparation techniques were applied in order to optimize the preparation of homogeneous lipid films of various film thicknesses. Suspensions of preformed DOPC/DPPC vesicles were adsorbed onto indium tin oxide-coated glass coverslips, a substrate also commonly employed for the formation of giant liposomes. We found that the homogeneity of the lipid films could substantially be improved when applying a spin-coating step during the film preparation. These films were much more homogeneous than those prepared by conventional drop-casting and in addition the film thickness could be controlled. When using a combination of vesicle adsorption and spin-coating the quality and thickness of the films depended crucially on the lipid concentration of the vesicle suspension, the adsorption temperature and the adsorption time. For lipid films prepared by direct spin-coating the lipid concentration and the applied spin-coating sequence were critical parameters for the quality and thickness of the deposited lipid films.