Sugar-based gemini surfactant with a vesicle-to-micelle transition at acidic pH and a reversible vesicle flocculation near neutral pH

A sugar-based (reduced glucose) gemini surfactant forms vesicles in dilute aqueous solution near neutral pH. At lower pH, there is a vesicle-to-micelle transition within a narrow pH region (pH 6.0-5.6). The vesicles are transformed into large cylindrical micelles that in turn are transformed into small globular micelles at even lower pH. In the vesicular pH region, the vesicles are positively charged at pH < 7 and exhibit a good colloidal stability. However, close to pH 7, the vesicles become unstable and rapidly flocculate and eventually sediment out from the solution. We find that the flocculation correlates with low vesicle zeta-potentials and the behavior is thus well predicted by the classical DLVO theory of colloidal stability. Surprisingly, we find that the vesicles are easily redispersed by increasing the pH to above pH 7.5. We show that this is due to a vesicle surface charge reversal resulting in negatively charged vesicles at pH > 7.1. Adsorption, or binding, of hydroxide ions to the vesicular surface is likely the cause for the charge reversal, and a hydroxide ion binding constant is calculated using a Poisson-Boltzmann model.     

Adsorption of gentamicin onto a bilayer lipid membrane

Addition of the aminoglycoside antibiotic, gentamicin (GM), to one side of a bilayer lipid membrane (BLM) results in a potential difference across the membrane. Evidence is presented that the membrane potential is caused by the adsorption of GM, bearing four positive charges, on the BLM surface. The experimental results are subjected to a quantitative analysis based on the double-layer theory and the Langmuir adsorption isotherm. The adsorption is saturated (i.e., the BLM is fully covered) at the bulk GM concentration of about 80 μmol/1. At this point, the calculated GM-induced increase in the BLM surface charge density is σ = 0.0054 C m⁻², which is equivalent to one positive charge per 50 lipids or one molecule of GM per 200 lipids.     

Protein binding onto surfactant-based synthetic vesicles

Synthetic vesicles were prepared by mixing anionic and cationic surfactants, aqueous sodium dodecylsulfate with didodecyltrimethylammonium or cetyltrimethylammonium bromide. The overall surfactant content and the (anionic/cationic) mole ratios allow one to obtain negatively charged vesicles. In the phase diagram, the vesicular region is located between a solution phase, a lamellar liquid crystalline dispersion, and a precipitate area. Characterization of the vesicles was performed by electrophoretic mobility, NMR, TEM, and DLS and we determined their uni-lamellar character, size, stability, and charge density. Negatively charged vesicular dispersions, made of sodium dodecylsulfate/didodecyltrimethylammonium bromide or sodium dodecylsulfate/cetyltrimethylammonium bromide, were mixed with lysozyme, to form lipoplexes. Depending on the protein/vesicle charge ratio, binding, surface saturation, and lipoplexes flocculation, or precipitation, occurs. The free protein in excess remains in solution, after binding saturation. The systems were investigated by thermodynamic (surface tension and solution calorimetry), DLS, CD, TEM, 1H NMR, transport properties, electrophoretic mobility, and dielectric relaxation. The latter two methods give information on the vesicle charge neutralization by adsorbed protein. Binding is concomitant to modifications in the double layer thickness of vesicles and in the surface charge density of the resulting lipoplexes. This is also confirmed by developing the electrophoretic mobility results in terms of a Langmuir-like adsorption isotherm. Charges in excess with respect to the amount required to neutralize the vesicle surface promote lipoplexes clustering and/or flocculation. Protein-vesicle interactions were observed by DLS, indicating changes in particle size (and in their distribution functions) upon addition of LYSO. According to CD, the bound protein retains its native conformation, at least in the SDS/CTAB vesicular system. In fact, changes in the alpha-helix and beta-sheet conformations are moderate, if any. Calorimetric methods indicate that the maximum heat effect for LYSO binding occurs at charge neutralization. They also indicate that enthalpic are by far the dominant contributions to the system stability. Accordingly, energy effects associated with charge neutralization and double-layer contributions are much higher than counterion exchange and dehydration terms.     

Characterization and Aggregation Behaviors of Mixed DDAB/SDS Solution With and Without Poly(4-styrenesulfonic Acid-Co-Maleic Acid) Sodium

Synthetic vesicles are formed by cationic and anionic surfactants, didodecyldimethylammonium bromide (DDAB), and sodium dodecylsulfate (SDS). The morphology, size, and aqueous properties of cationic/anionic mixtures are investigated at various molar ratios between cationic and anionic surfactants. The charged vesicular dispersions made of DDAB/SDS are contacted or mixed with negatively charged polyelectrolyte, poly(4-styrenesulfonic acid-co-maleic acid) sodium (PSSAMA), to form complexes. Depending on DDAB/SDS molar ratio or PSSAMA/vesicle charge ratio, complexes flocculation or precipitation occur. Characterization of the cationic/anionic vesicles or complexes formed by the catanionic vesicles and polyelectrolytes is performed by transmission electron microscope (TEM), dynamic light scattering (DLS), conductivity, turbidity, and zeta potential measurements. The size, stability, and the surface charge on the mixed cationic/anionic vesicles or complexes are determined.     

Lysozyme binding onto cat-anionic vesicles

Mixing aqueous sodium dodecylsulfate with cetyltrimethylammonium bromide solutions in mole ratios close to (1.7/1.0) allows the formation of cat-anionic vesicles with an excess of negative charges on the outer surface. The vesicular dispersions are mixed with lysozyme, and interact electrostatically with the positive charges on the protein, forming lipo-plexes. Dielectric relaxation, zeta-potential, and light scattering indicate the occurrence of interactions between vesicles and the protein. According to CD, the vesicle-adsorbed protein retains its native conformation. Binding and surface saturation, inferred by dielectric relaxation and zeta-potential, fulfil a charge neutralisation stoichiometry. Adsorbed lysozyme promotes the vesicle clustering and is concomitant with the lipo-plexes flocculation. Above the charge neutralisation threshold, lysozyme in excess remains dispersed in molecular form. Attempts were made to determine in what conditions protein release from the vesicles occurs. Accordingly, the full neutralisation of sodium dodecylsulfate in excess by cetyltrimethylammonium bromide ensures the lipo-plexes break-up, the precipitation of the mixed surfactants and the protein release in native form.     

Changes of conductance and compressibility of bilayer lipid membranes induced by oligonucleotide-cationic polyene antibiotic complexes

The positively charged polyene molecule amphotericin B 3-dimethylaminopropylamide (AMA) is an efficient agent for the delivery of antisense oligodeoxyribonucleotides (ODN) into target cells. In the present study, bilayer lipid membrane (BLM) conductance, elasticity modulus perpendicular to the membrane plane, surface potential and electrical capacitance were measured by conductance and electrostriction methods in the presence of AMA, pure or complexed to 20-mer single stranded ODN at different ratios. Pure AMA did not induce changes in conductance of cholesterol-containing BLM, but did induce an increase in elasticity modulus and surface potential. ODN/AMA complexes changed BLM properties depending on the charge ratio. The most pronounced effect on membrane conductance was observed for positively charged ODN/AMA complexes (charge ratio rho-/+=0.1), while for negatively charged complexes these changes were less marked/apparent, correlating to substantially lower binding constants. The effect of ODN/AMA complexes on elasticity modulus and charge potential was biphasic. After an increase in both values, a decrease was observed for higher incubation times and ODN/AMA concentrations. These results are interpreted as indicating that the membrane property changes result from the large AMA aggregates induced by the presence of the negatively charged ODN, which condensate on these aggregates. It is suggested that the decrease of elasticity modulus and surface potential in the presence of increasing incubation time and AMA concentration result from desorption of the complexes in the complex-free compartment of the BLM cell, or appearance of a non-linear conductance of the lipid bilayer. The first alternative would explain the AMA-induced transmembrane transfer of ODN.     

Control of lipid microstructures by molecular design and its implications

An overview of the current trends in the lipid design for specific applications has been presented. Lipids with different surface charge and hydrophobic backbone undergo aggregation to produce lamellae or bilayer and multilayer vesicles in aqueous media. Various aspects of present development of chiral superstructures and enzyme-mimics have been discussed. Utility of these molecules for potential applications in immunomodulation and sustained drug-delivery systems is also summarized.     

Analytical application of surface-affinity polymerized vesicular membranes to trace metal analysis by electrothermal atomic absorption spectrometry

The affinity of anionic polymerized vesicular membranes for metal cations in aqueous solutions is explored in terms of metal ion extraction and preconcentration. The method is based on the coordination of metal ions on the surface of charged polymerized vesicles via intra-vesicular complexes. These are causing changes in the selectivity, reactivity and inter-vesicular bridges which facilitate the aggregation of polymerized vesicles promoting phase separation. An analytical demonstration is shown by the optimization of the experimental conditions that enable the determination of antimony (III) in natural waters. The analytical features of the method including detection limits, precision and analytical recoveries from spiked natural water samples suggest that polymerized vesicular membranes can be a promising alternative to surfactant-mediated extractions of metal ions from aqueous matrices.     

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.     

Distribution of local anesthetics in lipid membranes

Absorption of local anesthetics into lipid membranes and adsorption onto their surfaces were studied as a function of the pH of aqueous bulk solutions by measuring lipid vesicle electrophoretic mobility, the partition of the anesthetics between the aqueous and membrane phases by the use of fluorescence and radioactive tracer methods, and the effect of the anesthetics on interfacial tension of lipid monolayers formed at the oil/aqueous interface.

At a pH much lower than the pK_a value of the local anesthetic, the charged form of the local anesthetic was only adsorbed onto the membrane surface, as determined from vesicle electrophoretic mobility, radioisotope tracer and the monolayer surface tension studies. Surface partition coefficients of the charged form of the local anesthetics on phosphatidylcholine and phosphatidylserine membranes were obtained from the data of electrophoretic mobilities for lipid vesicles. The surface partition coefficients of various local anesthetics paralleled those of the bulk partition coefficients.

As the pH of the solutions increased, the adsorbed amount of the charged form of the anesthetic at the membrane interface decreased, while the absorption of the uncharged form of the local anesthetic into the membrane increased. The total amount of local anesthetic adsorbed per unit area of the membrane generally increased as the pH of the solution increased. This was also observed from the measurements of the fluorescence of local anesthetics adsorbed into the membranes. At lower pH than that corresponding to the pK_a value of the local anesthetic, the amount of anesthetic adsorbed depended greatly upon the membrane surface charge. At a higher pH than its pK_a, it did not depend appreciably on the surface charge density of the membrane but did depend on the bulk partition coefficients between the aqueous and oil phases.     

Influence of inhalation anesthetics on ion transport across a planar bilayer lipid membrane

Ion transport from one aqueous phase (W1) to another (W2) across a planar bilayer lipid membrane (BLM) in the presence of inhalation anesthetics was electrochemically investigated. In the absence of inhalation anesthetics in the BLM system, no ion transport current flowed between W1 and W2 across the BLM. When inhalation anesthetics such as halothane, chloroform, diethyl ether and trichloroethylene were added to the two aqueous phases or the BLM, the ion transport current quite clearly appeared. When the ratio of the concentration of KCl or NaCl in W1 to that in W2 was varied, the zero current potential across the BLM was shifted. By considering the magnitude of the potential shift, we concluded that the ion transport current can be predominantly ascribed to the transport of Cl(-) across the BLM. Since the dielectric constants of these anesthetics are larger than that of the inner hydrophobic domain of the BLM, the concentration of hydrophilic electrolyte ions in the BLM increases with the increase in the dielectric constant of the inner hydrophobic domain caused by addition of these anesthetics. These situations lead to an increase in the ion permeability coefficient.     

Dynamics of DNA adsorption on and release from SDS-DDAB cat-anionic vesicles: a multitechnique study

DNA adsorption and release from cat-anionic vesicles made of sodium dodecylsulfate-dodecyldimethylammonium bromide (SDS-DDAB) in nonstoichiometric amounts was investigated by different electrochemical, spectroscopic, and biomolecular strategies. The characterization of the vesicular system was performed by dynamic light scattering, which allowed estimating both its size and distribution function(s). The interaction dynamics was followed by dielectric spectroscopy and zeta-potential, as well as by agarose gel electrophoresis, AGE. Also, circular dichroism, CD, measurements were carried out, to ascertain possible structural rearrangements of DNA, consequent to the interactions with the cat-anionic vesicles. CD demonstrates that vesicle-bound DNA retains its native conformation. The results obtained by the aforementioned techniques are consistent and indicate that binding saturation is obtained at a [DNA/vesicles] charge ratio close to 0.8, considering only the excess surface charges on the vesicles. This result is apparently in contradiction with a purely electrostatic approach and is tentatively ascribed to the distance between charges on the biopolymer and the vesicle surface, respectively. A possible interpretation is discussed. The nucleic acid can be completely retrieved from the vesicles upon addition of adequate amounts of SDS, which is the defective surfactant in the vesicular system. Precipitation of the poorly soluble SD-DDA salt results in an almost complete release of DNA.     

Effects of zwitterionic vesicles on the reactivity of benzoyl chlorides

A systematic study on the solvolysis reaction of substituted benzoyl chlorides in the presence of zwitterionic vesicles of dipalmitoyl phosphatidylcholine (DPPC) has been performed. Size, shape, surface charge, and polarity of the interface of the vesicular aggregates were determined using various techniques. The application of the pseudophase formalism allowed us to obtain the thermodynamic and kinetic coefficients characteristic of the reaction. The effects of vesicular aggregates on the solvolysis of benzoyl chlorides, which are known to be sensitive to the physical properties of the medium, depend on the nature of the substrate. For benzoyl chlorides with electron-donating groups, which react predominantly through a dissociative mechanism which is strongly affected by medium properties, the rate constant decreases as the vesicle concentration increases. On the other hand, for benzoyl chlorides with electron-withdrawing groups, which react mainly via an associative pathway, DPPC vesicles catalyze the solvolysis reaction.     

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 vesicle adsorption for different lipid mixtures

Numerous experimental studies of lipid vesicle adsorption on solid surfaces show that electrostatic interactions play an important role for the kinetics and end result. The latter can, e.g., be intact vesicles or supported lipid bilayers (SLB). Despite an accumulated quite large experimental data base, the understanding of the underlying processes is still poor, and mathematical models are scarce. We have developed a phenomenological model of a vesicle adsorbing on a substrate, where the charge of the surface and the charge and polar state of the lipid headgroup can be varied. With physically reasonable assumptions and input parameters, we reproduce many key experimental observations, clarify the details of some experiments, and give predictions and suggestions for future experiments. Specifically, we have investigated the influence of different lipid mixtures (different charges of the headgroups) in the vesicle on the outcome of a vesicle adsorption event. For different mixtures of zwitterionic lipids with positive and negative lipids, we investigated whether the vesicle adsorbs or not, and--if it adsorbs--to what extent it gets deformed and when it ruptures spontaneously. Diffusion of neutral vesicles on different types of negatively charged substrates was also simulated. The mean surface charge density of the substrate was varied, including or excluding local fluctuations in the surface charge density. The simulations are compared to available experiments. A consistent picture of the influence of different lipid mixtures in the vesicle on adsorption, and the influence of different types of substrates on vesicle diffusion, appear as a result of the simulation data.     

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.     

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.     

Voltammetric study on ion transport across a bilayer lipid membrane in the presence of a hydrophobic ion or an ionophore

This review describes voltammetric studies on ion transport from one aqueous phase (W1) to another (W2) across a bilayer lipid membrane (BLM) containing a hydrophobic ion, valinomycin (Val) or gramicidin A (GA). In particular, the ion transport mechanisms are discussed in terms of the distribution of a pair of ions between aqueous and BLM phases. By addition of a small amount of hydrophobic ion into W1 and/or W2 containing a hydrophilic salt as a supporting electrolyte, the hydrophobic ion was distributed into the BLM with the counter ion to maintain electroneutrality within the BLM phase. It was found that the counter ion was transferred between W1 and W2 across the BLM by applying a membrane potential. Facilitated transport of alkali ions across a BLM containing Val as an ion carrier compound, could be interpreted by considering not only the formation of the alkali metal ion–Val complex but also the distribution of both the objective cation and the counter ion. In the case of addition of GA as a channel-forming compound into the BLM, the facilitated transport of alkali ions across the BLM depended on the ionic species of the counter ions. It was discovered that the influence of the counter ion on the facilitated transport of alkali ions across the BLM could be explained in terms of the hydrophobicity and the ionic radius of the counter ion.     

Opposing effects of cation binding and hydration on the bending rigidity of anionic lipid bilayers

We correlate the molecularly realistic self-consistent field predictions for the mean bending modulus kc of charged lipid vesicles with experimental observations of the size R of corresponding vesicles that are produced by the freeze-thaw method. We elaborate on the Ansatz that the bending modulus is related to the membrane persistence length and that this length scale sets the radius of the vesicles. Alkali cations have a remarkable effect on the mean bending modulus and thus on the equilibrium radius of negatively charged entropically stabilized dioleoylphosphatidylglycerol (DOPG) vesicles. Where cation hydration typically results in thicker and thus stiffer membranes, specific adsorption to the bilayer surface results in a decrease of the surface charge density and the thickness of the membrane-associated electric double layer. As a result of these opposing effects on kc and R, the largest DOPG vesicles are found in the presence of K+, which combines an intermediate hydration enthalpy and PG-binding affinity.     

Ion transport across a bilayer lipid membrane facilitated by valinomycin

The facilitated ion transport from one aqueous phase, W1, to another, W2, across a bilayer lipid membrane, BLM, containing valinomycin, Val, as an ionophore was investigated by voltammetry. Cyclic voltammograms for the ion transfer were symmetrical about the origin (0 V, 0 A) and the magnitude of the ion transfer current increased with an increase in the absolute value of the applied potential. The magnitude of the ion transfer current at a definite potential in the voltammograms depended on the cation species added to W1 and W2 and was proportional to the concentration of Val in the BLM. The magnitude of the ion transfer current at a definite potential also varied in proportion to the hydrophobicity of the counter anion in W1 and W2. Taking into account the conjugated ion transfers at the W1|BLM and BLM|W2 interfaces, the positive current that flowed from W1 to W2 across the BLM was attributable to both the transfer of the complex-forming cation from W1 to the BLM and the transfer of the anion, which was distributed in the BLM as the counter ion from W2 to W1. The transfer from the BLM to W1 occurred at the W1|BLM interface and both the transfer of the cation from the BLM to W2 and the transfer of the anion from W2 to the BLM at the BLM|W2 interface. The negative current was then attributed to the opposite reaction. The voltammograms were asymmetrical with the origin when the ion components in W1 and W2 were different.