Egg Phosphatidylcholine Vesicles Research Articles

Native E. coli inner membrane incorporation in solid-supported lipid bilayer membranes

Solid-supported bilayer lipid membranes (SBLMs) containing membrane protein have been generated through a simple lipid dilution technique. SBLM formation from mixtures of native Escherichia coli bacterial inner membrane (IM) vesicles diluted with egg phosphatidylcholine (egg PC) vesicles has been explored with dissipation enhanced quartz crystal microbalance (QCM-D), atomic force microscopy (AFM), attenuated total internal-reflection Fourier-transform infrared spectroscopy (ATR-FTIR), and fluorescence recovery after photobleaching (FRAP). QCM-D studies reveal that SBLM formation from vesicle mixtures ranging between 0% and 100% IM can be divided into two regimes. Samples with < or = 40% IM form SBLMs, while samples of greater IM fractions are dominated by vesicle adsorption. FRAP experiments showed that the bilayers formed from mixed vesicles with < or = 40% IM were fluid, and comprised a mixture of both egg PC and IM. ATR-FTIR measurements on SBLMs membranes formed with 30% IM confirm that protein is present. SBLM formation was also explored as a function of temperature by QCM-D and FRAP. For samples of 30% IM, QCM-D data show a decreased mass and viscoelasticity at elevated temperatures, and an increased fluidity is observed by FRAP measurements. These results suggest improved biomimetic characteristics can be obtained by forming and maintaining the system at, or close to, 37 degrees C.

Novel Method for Obtaining Homogeneous Giant Vesicles from a Monodisperse Water-in-Oil Emulsion Prepared with a Microfluidic Device

A novel technique called the "lipid-coated ice droplet hydration method" is presented for the preparation of giant vesicles with a controlled size between 4 and 20 microm and entrapment yields for water-soluble molecules of up to about 30%. The method consists of three main steps. In the first step, a monodisperse water-in-oil emulsion with a predetermined average droplet diameter between 4 and 20 microm is prepared by microchannel emulsification, using sorbitan monooleate (Span 80) and stearylamine as emulsifiers and hexane as oil. In the second step, the water droplets of the emulsion are frozen and separated from the supernatant hexane solution by precipitation, followed by a removal of the supernatant and followed by the replacement of Span 80 by using a hexane solution containing egg yolk phosphatidylcholine, cholesterol, and stearylamine (5:5:1, molar ratio). This procedure is performed at -10 degrees C to keep the water droplets of the emulsion in a frozen state and thereby to avoid extensive water droplet coalescence. In the third step, hexane is evaporated at -4 to -7 degrees C and an external water phase is added to the remaining mixture of lipids and water droplets to form giant vesicles that have an average size in the range of that of the initial emulsion droplets (4-20 microm). The entrapment yield and the lamellarity of the vesicles obtained depend on the lipid/water droplet ratio and on the composition of the external water phase. At high lipid/water droplet ratio, the giant vesicles have a thicker membrane (indicating multilamellarity) and a higher entrapment yield than in the case of a low lipid/water droplet ratio. The highest entrapment yield ( approximately 35%) is obtained if the added external water phase contains preformed unilamellar egg phosphatidylcholine vesicles with an average diameter of 50 nm. The addition of these small vesicles minimizes the water droplet coalescence during the third step of the vesicle preparation, thereby decreasing the extent of release of water-soluble molecules originally present in the water droplets. The GVs prepared can be extruded through polycarbonate membranes to yield large unilamellar vesicles with about 100 nm diameter. This size reduction, however, leads to a decrease in the entrapment yield to about 12% due to solute leakage from the vesicles during the extrusion process.

The Amphiphilic Self‐Assembling Peptide EAK16‐I as a Potential Hydrophobic Drug Carrier

It is crucial for hydrophobic drugs to be dissolved and stabilized by carriers in aqueous systems and then to be delivered into target cells. An amphiphilic self‐assembling peptide EAK16‐I (Ac‐AEAKAEAKAEAKAEAK‐NH2) is reported here to be able to stabilize a model hydrophobic compound, pyrene, in aqueous solution, resulting in the formation of colloidal suspensions. Egg phosphatidylcholine (EPC) vesicles are used as plasma membranes mimic. Fluorescence data shows that the pyrene is presented in the crystalline form when stabilized by EAK16‐I and molecularly migrates from its peptide encapsulations into the membrane bilayers of EPC vesicles when the suspension is mixed with EPC vesicles. Furthermore, the release rate can be controlled by changing peptide‐to‐pyrene ratio, and the higher ratios lead to the slower release rates due to a thicker encapsulation on the pyrene microcrystals. This demonstrates that EAK16‐I, as a promising nanobiomaterial, has the potential to be a hydrophobic compounds carrier.

Supported Bilayer Lipid Membrane Arrays on Photopatterned Self‐Assembled Monolayers

This work demonstrates the use of photocleavable cholesterol derivatives to create supported bilayer lipid membrane arrays on silica. The photocleavable cholesteryl tether is attached to the surface by using the reaction of an amine-functionalized self-assembled monolayer (SAM) and the N-hydroxysuccinimide-based reagent 9. The resultant SAM contains an ortho-nitrobenzyl residue that can be cleaved by photolysis by using soft (365 nm) UV light regenerating the original amine surface, and which can be patterned using a mask. The photoreaction yield was approximately 75 % which was significantly higher than previously found for related ortho-nitrobenzyl photochemistry on gold substrates. The SAMs were characterized by means of contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Patterned surfaces were characterized with SEM and AFM. After immersing the patterned surface into a solution containing small unilamellar vesicles of egg phosphatidylcholine (PC), supported lipid membranes were formed comprised of lipid bilayer over the amine functionalized "hydrophilic" regions and lipid monolayer over the cholesteryl "hydrophobic" regions. This was confirmed by fluorescence microscopy and AFM. FRAP studies yielded a lateral diffusion coefficient for the probe molecule of 0.14+/-0.05 microm(2) s(-1) in the bilayer regions and approximately 0.01 microm(2) s(-1) in the monolayer regions. This order of magnitude difference in diffusion coefficients effectively serves to isolate the bilayer regions from one another, thus creating a bilayer array.

Single GUV Method Reveals Interaction of Tea Catechin (−)-Epigallocatechin Gallate with Lipid Membranes

Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (−)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration (∼150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 μM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.

Liposome Size Analysis by Dynamic/Static Light Scattering upon Size Exclusion-/Field Flow-Fractionation

The aim of the current study was to analyse the particle size distribution of a liposome dispersion, which contained small egg phosphatidylcholine vesicles and had been prepared by high-pressure homogenisation, by various size analysis techniques. Such liposomes were chosen since they can be looked at as a prototype of drug nano-carriers. Three sub-micron particle size analysis techniques were employed: (1) fixed-angle quasi-elastic laser light scattering or photon correlation spectroscopy (PCS), (2) size exclusion chromatographic (SEC) fractionation with subsequent (off-line) PCS size-analysis and quantification of the amount of particles present in the sub-fractions, and (3) field-flow-fractionation coupled on-line with a static light scattering and a refractive index (RI)-detector. When designing liposome-based drug carrier systems, a reliable and reproducible analysis of their size and size distribution is of paramount importance: Not only does liposome size influence the nanocarrier's in-vitro characteristics such as drug loading capacity, aggregation and sedimentation but also it is generally acknowledged that the pharmacokinetic behaviour and biodistribution of the carrier is strongly size-dependent. All three approaches of liposome size analysis used here were found to yield useful results, although they were not fully congruent. PCS indicated either a broad, mono-modal, log-normal size distribution in the range of below 20 to over 200 nm in diameter, or alternatively, a bimodal distribution with two discrete peaks at 30 to 70 nm and 100 to over 200 nm. Which of the two distribution models represented the best fit depended primarily on the data collection times used. In contrast, both fractionating techniques revealed a size distribution with a large, narrow peak well below 50 nm and a minor, broad, overlapping peak or tail extending to over 100 nm in diameter. The observed differences in liposome size distribution may be explained by the inherent limitations of the different size analysis techniques, such as the detection limit and the fact that PCS is overemphasizing bigger particle sizes.

In vitro ultrasound-mediated leakage from phospholipid vesicles

Interest in using ultrasound energy in wound management and intracellular drug delivery has been growing rapidly. Development and treatment optimization of such non-diagnostic applications requires a fundamental understanding of interactions between the acoustic wave and phospholipid membranes, be they cell membranes or liposome bilayers. This work investigates the changes in membrane permeation (leakage mimicking drug release) in vitro during exposure to ultrasound applied in two frequency ranges: “conventional” (1MHz and 1.6MHz) therapeutic ultrasound range and low (20kHz) frequency range. Phospholipids vesicles were used as controllable biological membrane models. The membrane properties were modified by changes in vesicle dimensions and incorporation of poly(ethylene glycol) i.e. PEGylated lipids. Egg phosphatidylcholine vesicles with 5mol% PEG were prepared with sizes ranging from 100nm to 1μm. Leakage was quantified in terms of temporal fluorescence intensity changes observed during carefully controlled ultrasound ON/OFF time intervals. Custom-built transducers operating at frequencies of 1.6MHz (focused) and 1.0MHz (unfocused) were used, the Ispta of which were 46.9W/cm2 and 3.0W/cm2, respectively. A commercial 20kHz, point-source, continuous wave transducer with an Ispta of 0.13W/cm2 was also used for comparative purposes. Whereas complete leakage was obtained for all vesicle sizes at 20kHz, no leakage was observed for vesicles smaller than 100nm in diameter at 1.6 or 1.0MHz. However, introducing leakage at the higher frequencies became feasible when larger (greater than 300nm) vesicles were used, and the extent of leakage correlated well with vesicle sizes between 100nm and 1μm. This observation suggests that physico-chemical membrane properties play a crucial role in ultrasound mediated membrane permeation and that low frequency (tens of kilohertz) ultrasound exposure is more effective in introducing permeability change than the “conventional” (1MHz) therapeutic one. The experimental data also indicate that the leakage level is controlled by the exposure time. The results of this work might be helpful to optimize acoustic field and membrane parameters for gene or drug delivery. The outcome of this work might also be useful in wound management.

Conformation and orientation of penetratin in phospholipid membranes

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.

Generation of Superparamagnetic Liposomes Revealed as Highly Efficient MRI Contrast Agents for in Vivo Imaging

Maghemite (gamma-Fe2O3) nanocrystals stable at neutral pH and in isotonic aqueous media were synthesized and encapsulated within large unilamellar vesicles of egg phosphatidylcholine (EPC) and distearoyl-SN-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG(2000), 5 mol %), formed by film hydration coupled with sequential extrusion. The nonentrapped particles were removed by flash gel exclusion chromatography. The magnetic-fluid-loaded liposomes (MFLs) were homogeneous in size (195 +/- 33 hydrodynamic diameters from quasi-elastic light scattering). Iron loading was varied from 35 up to 167 Fe(III)/lipid mol %. Physical and superparamagnetic characteristics of the iron oxide particles were preserved after liposome encapsulation as shown by cryogenic transmission electron microscopy and magnetization curve recording. In biological media, MFLs were highly stable and avoided ferrofluid flocculation while being nontoxic toward the J774 macrophage cell line. Moreover, steric stabilization ensured by PEG-surface-grafting significantly reduced liposome association with the macrophages. The ratios of the transversal (r2) and longitudinal (r1) magnetic resonance (MR) relaxivities of water protons in MFL dispersions (6 < r2/r1 < 18) ranked them among the best T2 contrast agents, the higher iron loading the better the T2 contrast enhancement. Magnetophoresis demonstrated the possible guidance of MFLs by applying a magnetic field gradient. Mouse MR imaging assessed MFLs efficiency as contrast agents in vivo: MR angiography performed 24 h after intravenous injection of the contrast agent provided the first direct evidence of the stealthiness of PEG-ylated magnetic-fluid-loaded liposomes.

Differential effects of uncharged aminoamide local anesthetics on phospholipid bilayers, as monitored by 1H-NMR measurements

We have collected evidences of a “transient site” for the local anesthetics (LA) lidocaine, etidocaine, bupivacaine and mepivacaine in sonicated egg phosphatidylcholine (EPC) vesicles. The effects of the uncharged anesthetic species at a fixed LA/EPC ratio inside the bilayer were measured by chemical shifts (C.S.) and longitudinal relaxation times (T 1) of the lipid hydrogens. Two sort of changes were detected: (I) decrease, indicating specific orientation of the LA aromatic ring (measured as up-field C.S. changes by the short-range ring-current effect) and less rotational freedom (smaller T 1 values) for EPC hydrogens such as the two glycerol-CH 2 and the choline-CH 2 bound to the PO 4− group, probably due to the nearby presence of the LA; (II) increase, indicating the aromatic ring is now perpendicular to the orientation observed before (causing down-field changes in C.S.) and larger T 1 values for all the choline and glycerol hydrogens, as a result of LA insertion behind these well-organized bilayer regions. The less hydrophobic, linear and nonlinear (lidocaine and mepivacaine, respectively) aminoamide analogs provide similar effects—described in I; their hydrophobic counterparts (etidocaine and bupivacaine) also produced comparable effects (depicted in II). The preferential positioning and orientation of each LA inside the bilayer is then determined by its hydrophobic and steric properties. We propose that this “transient site” in the lipid milieu exists also in biological membranes, where it can modulates the access of the uncharged LA species to its site(s) of action in the voltage-gated sodium channel.

A study of liposome formation using a solution (isoperibol) calorimeter

A solution (isoperibol) calorimeter has been employed to study the process of formation of phospholipid vesicles from natural and synthetic phospholipid films. Phospholipid films were hydrated in the solution calorimeter at temperatures exceeding the main phospholipid phase transition temperature, with continuous agitation to ensure conversion of the hydrating bilayers into multilamellar liposomes. It was seen that retention of chloroform in phospholipid films altered the apparent enthalpy change of vesicle formation to a far greater extent than would be expected from the contribution of the enthalpy of solution of chloroform; this indicates that chloroform alters the hydration process of the lipid. The overall measured enthalpy change for the formation of egg phosphatidylcholine vesicles was exothermic, whilst that for dimyristoylphosphatidylcholine was endothermic. This difference, it is suggested, results from the influence of the hydrocarbon chains mostly on the hydration process and also on the process of vesicle formation.

Influence of cetyltrimethylammonium bromide on phosphatidylcholine-coated capillaries.

Large unilamellar vesicles of egg-phosphatidylcholine (eggPC), a naturally occurring phospholipid, were used in capillary electrophoresis (CE) for semi-permanent coating of fused silica capillaries. The stability of the phospholipid coating was tested at different cetyltrimethylammonium bromide (CTAB) concentrations with and without CaCl(2) present in the coating solution. The effect of physical factors influencing the coating stability (e.g. duration of the coating time, storage temperature of the coating solution) were also studied. Standing overnight in background electrolyte (BGE) solution did not alter the eggPC phospholipid coating noticeably. The performance of the coating was tested with a mixture of basic proteins (lysozyme, ribonuclease A and alpha-chymotrypsinogen A). Highest efficiencies (over 200,000 plates m(-1)) were achieved when the capillary was filled for 15 h with a liposome solution containing both CTAB and CaCl(2).

Insertion and partition of sodium taurocholate into egg phosphatidylcholine vesicles.

To get a continuous description of the insertion and partition processes of sodium taurocholate (TC) into the lipid bilayers of vesicles that can serve as a model for understanding the mechanism of destabilization by the bile salts of liposomes used as drug carriers for oral administration. The progressive solubilization of egg phosphatidylcholine vesicles during TC addition at controlled rates was followed by continuous turbidity (OD) and resonance energy transfer (RET) between two fluorescent probes. The influence of the lipid and TC concentrations as well as the rate of TC addition on the processes were examined. Continuous turbidity recordings allowed following of the size and composition evolutions of the mixed TC/lipid aggregates formed at different steps of the vesicle-micelle transition. The solubilization mechanism is governed by complex kinetics that depend on the surfactant concentration and its addition rate. A two-step process characterizes the evolution of the vesicular state: interaction of TC molecules with the external monolayer of the vesicles first occurs. The homogeneous distribution of TC within the lipid matrix after its insertion is a very slow process. A micellar structural reorganization is observed when TC is added rapidly. This work provides detailed information on the slow insertion and diffusion kinetics of TC in liposomal bilayers by using a dynamic study which mimics physiological phenomena of digestion.

Self-assembling peptide as a potential carrier of hydrophobic compounds.

Microcrystals of a hydrophobic cargo were stabilized by EAK16 II, a self-assembling oligopeptide, and suspended in aqueous solution. Pyrene was used as a model hydrophobic compound. Egg phosphatidylcholine (EPC) vesicles were prepared to mimic a cell membrane. Pyrene was released from its EAK16 II coating into EPC vesicles. The excimer decay profiles were acquired. They showed that pyrene is present in the crystalline form when stabilized by EAK16 II, it is molecularly dispersed in EPC vesicles, and it is completely released from its EAK16 II coating into the membrane bilayers. The release of pyrene from the microcrystals coated with EAK16 II into the EPC membrane was followed by fluorescence as a function of time. The amount of pyrene released into the EPC vesicles at a given time was quantified using a calibration curve. The concentration of pyrene released was determined as a function of time, and the concentration-versus-time profile was fitted with one exponential. The rate of pyrene release was found to depend on the peptide-to-pyrene molecular ratio. Higher peptide-to-pyrene ratios lead to slower transfer of pyrene to the lipophilic environment. Scanning electron micrographs demonstrated that a thicker coating on the pyrene crystals results in a slower release. The data presented in this work demonstrate that the self-assembling EAK16 II can stabilize a hydrophobic cargo in aqueous solution and deliver it into a lipophilic environment, and that the rate of transfer can be adjusted by tuning the peptide-to-pyrene ratio.

Micelles of Lipid−Oligonucleotide Conjugates: Implications for Membrane Anchoring and Base Pairing

This report examines the organization properties of new fluorescent DNA-lipids, either alone in water or in interaction with 1-octyl-beta-d-glucopyranoside micelles or egg phosphatidylcholine vesicles. We first describe the design and the syntheses of the conjugates. Then, we use UV-Vis absorption, steady-state fluorescence emission, electron microscopy, and fluorescence correlation spectroscopy after two-photon excitation to show that these DNA-lipids form spherical micelles in aqueous solution and incorporate much better in micelles than in vesicles. We also investigate the significance of the lipophilic chains of these DNA-lipids on the melting behavior of the double-stranded hybrids: in water melting curves are broadened whereas in amphiphilic assemblies duplexes melt as the unconjugated controls. This work is expected to be useful for improving the rational design of antisense medicines.

Interaction of Antiinflammatory Drugs with EPC Liposomes: Calorimetric Study in a Broad Concentration Range

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient ( K p), and/or the equilibrium constant ( K b). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, Δ H, and indirectly, Δ G and Δ S. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction—possibly the consequence of a modification of the membrane’s physical properties as it becomes saturated with the drug.

Electron Cryo‐Microscopy Reveals Mechanism of Action of Propranolol on Artificial Membranes

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective β‐blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time‐resolved electron cryo‐microscopy (cryo‐EM) under different experimental conditions. The liposome–drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin‐film vitrification. The time‐series cryo‐EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread‐like micelles, also called worm‐like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid–surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid‐to‐micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

Sulfonylureas rapidly cross phospholipid bilayer membranes by a free-diffusion mechanism.

Because sulfonylureas directly activate the exocytotic machinery, we were interested in the extent to which these compounds penetrate the beta-cell plasma membrane and the underlying molecular mechanism(s). We now provide evidence that sulfonylureas cross phospholipid bilayer membranes rapidly and effectively by a free-diffusion mechanism. Two sulfonylurea compounds investigated by 1H nuclear magnetic resonance spectroscopy, glibenclamide and tolbutamide, were found to incorporate into phospholipid bilayers, with the ionizable sulfonamide exposed to the aqueous interface and its apparent dissociation constant (pKa) increased to approximately 7.0. Diffusion of weak amphiphilic acids across membranes is associated with a measurable change in pH. Thus, by using a fluorescence-based pH assay, we could investigate the diffusion of sulfonylurea compounds across phospholipid bilayer membranes. A fluorescent pH indicator (pyranin or [2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein] [BCECF]) was trapped in egg phosphatidylcholine vesicles. Addition of glibenclamide decreased internal pH (pHin), and addition of albumin reversed this drop by 50%. With the same amount of tolbutamide, the decrease in pHin was much smaller, primarily because of the lower partitioning of tolbutamide into phospholipid bilayers. Using similar protocols, we also demonstrated diffusion by the same mechanism across the beta-cell plasma membrane. Thus, we now provide a molecular mechanism by which sulfonylureas can penetrate the plasma membrane and reach intracellular sites regulating exocytosis.

Temperature dependence of formation of a supported phospholipid bilayer from vesicles on SiO2.

Adsorption of egg-phosphatidylcholine vesicles and bilayer formation on a SiO2 surface was investigated in the temperature range 278-303 K using the quartz crystal microbalance-dissipation technique. The critical coverage for the vesicle-->bilayer transition is found to decrease with increasing temperature. The temperature dependence of the time-scale characterizing this transition can be represented in the Arrhenius form. Higher temperatures produce a bilayer with fewer trapped, nonruptured vesicles.

Calibration of Membrane Viscosity of the ReconstitutedVesicles by Measurement of the Rotational Diffusion of Bacteriorhodopsin

Membrane viscosity of the reconstituted vesicles was calibrated byrotational diffusion of bacteriorhodopsin (BR) indimyristoylphosphatidylcholine (DMPC) and egg phosphatidylcholine (PC)vesicles. Rotational diffusion of BR in the vesicles was measured byflash-induced absorption anisotropy decay. BR was, for the first time,reconstituted successfully into DMPC and egg PC vesicles. From themeasurement of flash-induced absorption anisotropy decay of BR, thevalue of rotational diffusion coefficient D was obtained from each curvefitting by a global fitting procedure and, in turn, membrane viscosityη was estimated from D. The results have shown that membrane viscosityis temperature-dependent. It was decreased as temperature increased, buta transition occurred in the region of the respective phase transitionof DMPC and egg PC, respectively. The decrease of η was fast near the phasetransition for DMPC and egg PC. Few effects of lipid/BR ratio andglycerol or sucrose in suspension medium on membrane viscosity were found.