Spreading Of Liposomes Research Articles

Avoiding artifacts in liposome leakage measurements via cuvette- and liposome-surface modifications

The barrier properties of lipid membranes are often determined by investigating their solute permeability with the help of spectroscopic methods and the use of liposome-encapsulated self-quenching fluorescent dyes, for example, Carboxyfluorescein (CF). It was shown previously that liposome–surface interactions, and thus the choice of cuvette material, influence the result of such spectroscopic permeability/leakage experiments. In this work, we explore different methods to minimize the artifacts observed in spontaneous leakage measurements performed with cholesterol-containing liposomes. The spontaneous leakage of CF from liposomes with different composition and surface properties is monitored in cuvettes composed of quartz, polystyrene (PS), and Poly(methyl methacrylate) (PMMA). Our results show that significantly different leakage profiles are recorded for the exact same liposome batch depending on the cuvette material used. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) experiments indicate that these discrepancies likely arise from side processes occurring at the solution-cuvette interface, mainly, the attaching and spreading of liposomes. Further, we show that in some cases it is possible to minimize liposome–cuvette interactions, and reduce the experimental artifacts, by supplementing the liposomes with polyethylene glycol (PEG)-grafted lipids or gangliosides, and/or by pre-adsorbing free PEG to the cuvette walls. The collected data suggest that quartz cuvettes modified by adsorption of PEG8000 are suitable for spontaneous leakage experiments with POPC:cholesterol-based liposomes, while other cuvette materials perform poorly in the same experiments.

Spreading of a Unilamellar Liposome on Charged Substrates: A Coarse-Grained Molecular Simulation.

Supported lipid bilayers (SLBs) are able to accommodate membrane proteins useful for diverse biomimetic applications. Although liposome spreading represents a common procedure for preparation of SLBs, the underlying mechanism is not yet fully understood, particularly from a molecular perspective. The present study examines the effects of the substrate charge on unilamellar liposome spreading on the basis of molecular dynamics simulations for a coarse-grained model of the solvent and lipid molecules. Liposome transformation into a lipid bilayer of different microscopic structures suggests three types of kinetic pathways depending on the substrate charge density, that is, top-receding, parachute, and parachute with wormholes. Each pathway leads to a unique distribution of the lipid molecules and thereby distinctive properties of SLBs. An increase of the substrate charge density results in a magnified asymmetry of the SLBs in terms of the ratio of charged lipids, parallel surface movements, and the distribution of lipid molecules. While the lipid mobility in the proximal layer is strongly correlated with the substrate potential, the dynamics of lipid molecules in the distal monolayer is similar to that of a freestanding lipid bilayer. For liposome spreading on a highly charged surface, wormhole formation promotes lipid exchange between the SLB monolayers thus reduces the asymmetry on the number density of lipid molecules, the lipid order parameter, and the monolayer thickness. The simulation results reveal the important regulatory role of electrostatic interactions on liposome spreading and the properties of SLBs.

Liposome adhesion generates traction stress

Mechanical forces generated by cells modulate global shape changes required for essential life processes, such as polarization, division and spreading. Although the contribution of the cytoskeleton to cellular force generation is widely recognized, the role of the membrane is considered to be restricted to passively transmitting forces. Therefore, the mechanisms by which the membrane can directly contribute to cell tension are overlooked and poorly understood. To address this, we directly measure the stresses generated during liposome adhesion. We find that liposome spreading generates large traction stresses on compliant substrates. These stresses can be understood as the equilibration of internal, hydrostatic pressures generated by the enhanced membrane tension built up during adhesion. These results underscore the role of membranes in the generation of mechanical stresses on cellular length scales and that the modulation of hydrostatic pressure due to membrane tension and adhesion can be channelled to perform mechanical work on the environment.

Biomimetic surface modification with bolaamphiphilic archaeal tetraether lipids via liposome spreading.

Through investigations of the self-assembly behavior of three different tetraether lipids, the authors successfully established a solid supported, biomimetic tetraether lipid membrane via liposome spreading. These bolaamphiphilic lipids are the main compound in membranes of archaea, extremophile microorganisms, which underwent an enormous adaptation to extreme conditions in their natural environment with regard to temperature, pH, and high salt concentrations. Starting from a mathematical point of view, the authors calculated hydrophilic-lipophilic balance values for each lipid and recognized a wide difference in self-assembly potentials relying on size and hydrophilic properties of the lipid head groups. These results were in good accordance with data generated by lipid experiments at the air-water interface applying a Langmuir-Blodgett film balance so that the self-assembly potential of two different tetraether lipids was found to be sufficient to form stable liposomes in aqueous media. Liposomes composed of the main phospholipid of the archaea strain Sulfolobus acidocaldarius fused covalently on silanized glass substrates and formed a monomolecular lipid layer with upright standing molecules at film consistent thicknesses of approximately 5 nm determined by ellipsometry and atomic force microscopy. This work can be considered as a basic strategy to find optimized lipid properties in terms of liposome formation and spreading in water, and it is the first report about archaeal liposome fusing on surfaces to establish a solid supported lipid monolayer.

Molecular Dynamics Interactions between Silicon Electrodes and Phospholipids

In experiments involving the application of an external electric field to a biological system, the field is assumed to be uniform and isotropic far from the electrode interface, but adsorption of ions and molecules directly at the interface can complicate the structure of the charged double-layer. Reaction-kinetic models of lipid vesicle adhesion1 have utilized continuum parameters such as the rate of lipid monolayer formation and corresponding charge displacement. Experiments have suggested2 that vesicle lipid composition strongly affects the rates of lipid monolayer formation, but no atomically detailed studies have been conducted. To fill this gap we have run molecular dynamics simulations on POPS, POPC, and octane at the charged silicon interface to investigate the conformation, orientation, kinetics, and energetics of charged and uncharged molecules, respectively. This allows for direct comparisons between rates cited in the literature to those obtained in simulations, and provides researchers with additional per-molecule energetics which will further characterize lipid adhesion mechanisms near a charged interface.1 Ružic, I, Pecar-Ilic, J, DeNardis, NI (2010). Mathematical model for kinetics of organic particle adhesion at an electrified interface. Journal of Electroanalytical Chemistry 642:120-1262 Hernandez, VA, Scholz, F (2008). The lipid composition determines the kinetics of adhesion and spreading of liposomes on mercury electrodes. Bioelectrochemistry 74:149-156

Intrinsic Heterogeneity in Liposome Suspensions Caused by the Dynamic Spontaneous Formation of Hydrophobic Active Sites in Lipid Membranes

The spontaneous, dynamic formation of hydrophobic active sites in lipid bilayer membranes is studied and characterized. It is shown that the rates of formation and consumption of these active sites control at least two important properties of liposomes: their affinity for hydrophobic surfaces and the rate by which they spontaneously release encapsulated molecules. The adhesion and spreading of liposomes onto hydrophobic polystyrene nanoparticles and the spontaneous leakage of an encapsulated fluorescent dye were monitored for different liposome compositions employing Cryo-TEM, DLS, and fluorescence measurements. It was observed that an apparently homogeneous, monodisperse liposome suspension behaves as if composed by two different populations: a fast leaking population that presents affinity for the hydrophobic substrate employed, and a slow leaking population that does not attach immediately to it. The results reported here suggest that the proportion of liposomes in each population changes over time until a dynamic equilibrium is reached. It is shown that this phenomenon can lead to irreproducibility in, for example, spontaneous leakage experiments, as extruded liposomes leak much faster just after preparation than 24 h afterward. Our findings account for discrepancies in several experimental results reported in the literature. To our knowledge, this is the first systematic study addressing the issue of an existing intrinsic heterogeneity of liposome suspensions.

Structuring of supported hybrid phospholipid bilayers on electrodes with phospholipase A2

The effect of a lipolytic enzyme, pork pancreatic phospholipase A(2), on hybrid bilayer membranes was monitored using voltammetry, impedance spectroscopy and surface plasmon resonance. The hybrid bilayers were prepared by Langmuir-Schaefer transfer of lipid monolayers onto gold electrodes modified with self-assembled alkanethiol monolayers, or by liposome spreading. The electrodes were immersed in the phospholipase aqueous solution to allow adsorption of the enzyme and cleavage of the ester bond in the sn-2 position of phospholipids in the outer leaflet of the hybrid layers. The action of phospholipase A(2) led to perforation of the lipid films. Impedance spectroscopy and surface plasmon resonance were used for monitoring enzyme adsorption, phospholipid hydrolysis and product desorption. The results obtained show that transport efficiency of an electroactive probe, ferrocyanate, and of an electroactive drug, doxorubicin, through the bilayer depends on the action of the enzyme; the state of the lipid layer covering the electrode surface depends on the latter as well. Cyclic voltammetry and electrochemical impedance spectroscopy were used to study this effect. The doxorubicin reduction/oxidation signals appearing at potentials close to those observed using a bare gold electrode indicated facilitated penetration of the drug into the layer. The results obtained were interpreted in terms of pore formation in the lipid matrix; phospholipase A(2) can be considered as a nano-device for high precision perforation of the lipid layer.

Study of the temporal distribution of the adhesion-spreading events of liposomes on a mercury electrode

The formal analysis of the mechanism of adhesion spreading of liposomes at mercury electrodes shares several characteristics with the mechanism of metal nucleation at electrodes. It is shown that the description of the temporal distribution of the adhesion-spreading events is similar to that of the temporal distribution of metal clusters. Both processes are stochastic in nature and can be described by the Poisson distribution. Using this approach, a previously proposed model for the overall adhesion-spreading mechanism, considering the formation of active sites on the liposome and the actual attachment of the liposomes to the mercury surface, is validated.

The Electrochemistry of Liposomes

AbstractThis review aims to summarize the current state of research concerning the interaction of electrodes with liposomes suspended in solutions. Main attention is given to the complex mechanism of adhesion and spreading of liposomes on mercury electrodes. That mechanism can be studied with the help of chronoamperometry, where each adhesion‐spreading event appears as a capacitive current spike. Integration of these spikes produces charge versus time transients that can be modeled and simulated, revealing the details of the multi‐step adhesion‐spreading process. Whereas the number of spikes per time mirrors the macro‐kinetics, the analysis of the time behavior of each spike mirrors the micro‐kinetics of each adhesion‐spreading event. The reviewed studies show that this approach provides a new tool to study the properties of liposome membranes. The adhesion‐spreading of liposomes on mercury electrodes has strong similarities to the process of vesicle fusion, which makes these studies a biomimetic model allowing one to deduce the effects of foreign molecules in bilayer membranes.

The lipid composition determines the kinetics of adhesion and spreading of liposomes on mercury electrodes

The dependence of membrane properties on their composition was studied by following the adhesion and spreading of unilamellar and multilamellar liposomes on static mercury electrodes with the help of chronoamperometry. The analysis of the peak-shaped signals allows determining the kinetic parameters of the three-step adhesion-spreading process. The presence of cholesterol in the membrane stabilizes the bilayer in the liquid-crystalline phase, and destabilizes the gel phase. The kinetic parameters also show the effect of superlattice formation in the DMPC–cholesterol system. The detergent triton X-100 is only incorporated in the liquid-crystalline DMPC membranes, and it is expelled to the solution when the membrane is transformed to the gel phase. In the liquid-crystalline membrane, it enhances the adhesion-spreading of liposomes on mercury. The lytic peptides mastoparan X and melittin affect the adhesion-spreading in a similar manner. For the rupture-spreading step, their effect is explained by pore formation. The results obtained with lecithins of different length suggest that the bilayer opening process has much in common with flip-flop translocations. For this process the activation energies were found to be independent of the chain length of the lecithin molecules, while the preexponential factor in the Arrhenius equation decreases drastically for longer chains.

Bursting and spreading of liposomes on the surface of a static mercury drop electrode

When liposomes suspended in an aqueous electrolyte solution hit the surface of a static mercury drop electrode (SMDE) they burst and spread. These events lead to capacitive signals and allow counting of liposomes as well as the determination of the number of molecules in each liposome.

IgG 1-glycolipidic LB films obtained by vertical deposition of an interfacial film formed through proteo-liposome spreading at the air/water interface

This study focuses on the formation of an immunoglobulin–glycolipid interfacial film through the disintegration of proteo-glycolipid liposomes at the air/buffer interface. The IgG-glycolipid liposomes were formed by mechanical dispersion and were spread on the buffer surface. The time-dependent increase in surface pressure following the liposome spreading was attributed to the formation of a surface film due to the interfacial reorganisation of the proteo-glycolipid vesicles. The presence of immunoglobulin in the glycolipid matrix was evidenced by the modification of the elastic properties of the interfacial film during the film compression. The transfer efficiency of the immunoglobulin with the glycolipid matrix by vertical Langmuir–Blodgett (LB) deposition procedure has been characterised through ATR–FTIR and fluorescence spectroscopies. Observations performed with Epi-fluorescence microscopy gives a direct evidence of the presence of immunoglobulin in the LB film.

Immunological proteo-glycolipidic interfacial film obtained from spreading of liposomes including ascitic fluid

This study focuses on the formation of an immunological proteo-glycolipidic interfacial film obtained from spreading of synthetic glycolipid liposomes including ascitic fluid. It is shown that the lipid–protein interactions created in liposomes before the monolayer formation are optimal to carry a large amount of proteinaceous material to the air–water interface. The strong amphipathic balance and the high fluidity of the hydrocarbon chains of the synthetic glycolipid used in this study are the two main properties enabling both retention of immunological proteins in the lipidic matrix and preservation of the structural organisation of the proteo-glycolipidic interfacial film during the transfer through vertical Langmuir–Blodgett deposition.

Ellipsometry Studies of Interfacial Film Formation in Emulsion Systems

The possibility of using in situ ellipsometry for studies of the formation of interfacial films in emulsion systems was investigated. It was found that ellipsometry studies on dilute emulsions can provide information on the adsorption kinetics, the adsorbed layer structure, and the mechanisms of formation of the adsorbed layer. At high electrolyte concentrations, the adsorption from a model o/w emulsion stabilized by egg lecithin was found to be more pronounced at hydrophilic and negatively charged silica than at hydrophobic methylated silica. Furthermore, the adsorbed layer thickness at both surfaces is smaller than the average droplet size, corresponding to either small or "flattened" droplets or liposomes. The adsorbed layer thickness is smaller at methylated silica than at silica, presumably due to a larger degree of emulsion droplet or liposome spreading at this surface. In both cases, the adsorbed layer thickness is independent of the adsorbed amount, i.e., the adsorbed layer formation proceeds by attachment of droplets, which pack more densely with an increasing adsorbed amount.

Spreading of liposomes at the air/water interface

Two types of film structure are formed when liposomes are spread at the air/water interface. At zero surface pressure, there is a slow transformation of the closed bilayered structure into a lipid monolayer. The internal content of the liposomes is released into the aqueous subphase. In contrast, when multilamellar liposomes are spread against a surface pressure, they retain their internal content at the air/water interface by forming multilayered structures. Among the liposomes which dipped through the interface an important fraction loses its internal content. During the spreading process at zero surface pressure, it seems that the outer layer of the liposome spreads with a better yield as compared with the inner layer. It is possible to use this spreading technique to determine the asymmetrical distribution of lipids across bilayers.