Extruded vesicles of dioctadecyldimethylammonium bromide and chloride investigated by light scattering and cryogenic transmission electron microscopy

Combined dynamic and static light scattering (DLS, SLS) and cryogenic transmission electron microscopy (cryo-TEM) were used to investigate extruded cationic vesicles of dioctadecyldimethylammonium chloride and bromide (DODAX, X being Cl(-) or Br(-)). In salt-free dispersions the mean hydrodynamic diameter, D(h), and the weight average molecular weight, M(w), are larger for DODAB than for DODAC vesicles, and both D(h) and M(w) increase with the diameter (varphi) of the extrusion filter. NaCl (NaBr) decreases (increases) the DODAB (DODAC) vesicle size, reflecting the general trend of DODAB to assemble as larger vesicles than DODAC. The polydispersity index is lower than 0.25, indicating the dispersions are rather polydisperse. Cryo-TEM micrographs show that the smaller vesicles are spherical while the larger ones are oblong or faceted, and the vesicle samples are fairly polydisperse in size and morphology. They also indicate that the vesicle size increases with phi and DODAB assembles as larger vesicles than DODAC. Lens-shaped vesicles were observed in the extruded preparations. Both light scattering and cryo-TEM indicate that the vesicle size is larger or smaller than phi when phi is smaller or larger than the optimal phi approximately 200 nm.     

Effects of synthetic lipids on solubilization and colloid stability of hydrophobic drugs

Aqueous miconazole (MCZ) aggregates were solubilized and/or colloidally stabilized by bilayer-forming synthetic amphiphiles such as dioctadecyldimethylammonium bromide (DODAB) or sodium dihexadecylphosphate (DHP) dispersions. Particle sizing, light absorption and scattering from drug particles, zeta-potential determination, and drug aggregation kinetics from turbidity changes in the presence or absence of lipid dispersions were obtained over a range of drug and lipid concentrations. The very low solubility of MCZ in water made possible the determination of size distributions for drug particles in water and comparison to those in the presence of DODAB or DHP nanosized bilayer fragments or entire and closed bilayer vesicles. Large drug aggregates disappeared upon incubation with nanosized bilayer fragments produced by ultrasonic dispersion with tip. Light-absorption spectra for MCZ in a poor solvent (water), in a good organic solvent (methanol), and in different lipid dispersions showed that solubilization depended on the presence of bilayer fragments. MCZ was poorly soluble in dispersions formed of closed bilayers (vesicles) of DODAB or DHP in the gel state and in phosphatidylcholine (PC) vesicles in the liquid-crystalline state. Increased hydrophobicity at the borders of bilayer fragments explained MCZ solubilization. At [MCZ]>0.4 mM, kinetics of drug aggregation, zeta-potential measurements, and size minimization were obtained upon addition of minute amounts of oppositely charged bilayer fragments ([DHP]=0.05 mM), making possible determination of a remarkable stabilizing effect of drug particles by coverage with anionic bilayer fragments. High drug colloid stability in the presence of charged bilayer fragments was achieved by two different means: (1). at large drug concentrations and small concentrations of bilayer fragments, coverage of large drug particles with bilayer fragments; (2). at large amounts of bilayer fragments, drug solubilization in its monomeric form at the borders of bilayer fragments. Inexpensive, synthetic bilayer fragments offered a large area of hydrophobic nanosurfaces dispersed and electrostatically stabilized in water, opening new prospects for drug solubilization and colloid stabilization of insoluble drug particles.     

Vesicle-micelle transition in aqueous mixtures of the cationic dioctadecyldimethylammonium and octadecyltrimethylammonium bromide surfactants

The vesicle-micelle transition in aqueous mixtures of dioctadecyldimethylammonium and octadecyltrimethylammonium bromide (DODAB and C(18)TAB) cationic surfactants, having respectively double and single chain, was investigated by differential scanning calorimetry (DSC), steady-state fluorescence, dynamic light scattering (DLS) and surface tension. The experiments performed at constant total surfactant concentration, up to 1.0 mM, reveal that these homologous surfactants mix together to form mixed vesicles and/or micelles, depending on the relative amount of the surfactants. The melting temperature T(m) of the mixed DODAB-C(18)TAB vesicles is larger than that for the neat DODAB in water owing to the incorporation of C(18)TAB in the vesicle bilayer. The surface tension decreases sigmoidally with C(18)TAB concentration and the inflection point lies around x(DODAB) approximately 0.4, indicating the onset of micelle formation owing to saturation of DODAB vesicles by C(18)TAB molecules. When x(DODAB)>0.5 C(18)TAB molecules are mainly solubilised by the vesicles, but when x(DODAB)<0.25 micelles are dominant. Fluorescence data of the Nile Red probe incorporated in the system at different surfactant molar fractions indicate the formation of micelle and vesicle structures. These structures have apparent hydrodynamic radius R(H) of about 180 and 500-800 nm, respectively, as obtained by DLS measurements.     

Interaction of sodium cholate with dioctadecyldimethylammonium chloride vesicles in aqueous dispersion

Mixtures of dioctadecyldimethylammonium chloride (DODAC) cationic vesicle dispersions with aqueous micelle solutions of the anionic sodium cholate (NaC) were investigated by differential scanning calorimetry, DSC, turbidity and light scattering. Within the concentration range investigated (constant 1.0 mM DODAC and varying NaC concentration up to 4 mM), vesicle → micelle → aggregate transitions were observed. The turbidity of DODAC/NaC/water depends on time and NaC/DODAB molar concentration ratio R. At equilibrium, turbidity initially decreases smoothly with R to a low value (owing to the vesicle–micelle transition) when R = 0.5–0.8 and then increases steeply to a high value (owing to the micelle–aggregate transition) when R = 0.9–1.0. DSC thermograms exhibit a single and sharp endothermic peak at T_m ≈ 49 °C, characteristic of the melting temperature of neat DODAC vesicles in water. Upon addition of NaC, T_m initially decreases to vanish around R = 0.5, and the main transition peak broadens as R increases. For R > 1.0 two new (endo- and exothermic) peaks appear at lower temperatures indicating the formation of large aggregates since the dispersion is turbid. All samples are non-birefringent. Dynamic light scattering (DLS) data indicate that both DODAC and DODAC/NaC dispersions are highly polydisperse, and that the mean size of the aggregates tends to decrease as R increases.     

Kinetics of breakdown of vesicles from didodecyldimethylammonium bromide induced by single chain surfactants and by osmotic stress in aqueous solution

Didodecyldimethylammonium bromide (DDAB) forms vesicles spontaneously by simple solubilization of the solid into water at a concentration of ≈2.5 mM. Vesicles can be observed by the increase in turbidity of the aqueous solution of DDAB and by the increase in absorbance (at λ_max=490 nm) of a lipophilic dye (Sudan III) solubilized into the vesicular bilayer. This vesicle system has been perturbed by addition of single-chain surfactants in order to study the transition from a vesicle-stable region to a mixed-micelle region. Vesicle breakdown involves the initial incorporation of a single-chain surfactant into the vesicular bilayer, followed by subsequent disintegration of the vesicle. The progress of reaction has been observed by monitoring turbidity changes using a stopped-flow spectrophotometer. The rate of breakdown of vesicles depends on the concentration and hydrophobic properties of the added single-chain surfactant. In addition, hypertonic and hypotonic osmotic stresses have been investigated.     

DODAB and DODAC bilayer-like aggregates in the micromolar surfactant concentration domain

In the millimolar concentration domain (typically 1 mM), dioctadecyldimethylammonium bromide and chloride (DODAX, X representing Br⁻ or Cl⁻ counterions) molecules assemble in water as large unilamellar vesicles. Differential-scanning calorimetry (DSC) is a suitable technique to obtain the melting temperature (T _m) characteristic of surfactant bilayers, while fluorescence spectroscopy detects formation of surfactant aggregates, like bilayers. These two techniques were combined to investigate the assembly of DODAX molecules at micromolar concentrations, from 10 to 100 μM. At 1 mM surfactant, T _m ≈ 45 °C and 49 °C, respectively, for DODAB and DODAC. DSC and fluorescence of Nile Red were used to show the formation of DODAX aggregates, at the surfactant concentration as low as 10 μM, whose T _m decreases monotonically with increasing DODAX concentration to attain the value for the ordinary vesicles. The data indicate that these aggregates are organized as bilayer-like structures.     

Cationic liposomes in mixed didodecyldimethylammonium bromide and dioctadecyldimethylammonium bromide aqueous dispersions studied by differential scanning calorimetry, Nile Red fluorescence, and turbidity

The thermotropic phase behavior of cationic liposomes in mixtures of two of the most investigated liposome-forming double-chain lipids, dioctadecyldimethylammonium bromide (DODAB) and didodecyldimethylammonium bromide (DDAB), was investigated by differential scanning calorimetry (DSC), turbidity, and Nile Red fluorescence. The dispersions were investigated at 1.0 mM total surfactant concentration and varying DODAB and DDAB concentrations. The gel to liquid-crystalline phase transition temperatures (Tm) of neat DDAB and DODAB in aqueous dispersions are around 16 and 43 degrees C, respectively, and we aim to investigate the Tm behavior for mixtures of these cationic lipids. Overall, DDAB reduces the Tm of DODAB, the transition temperature depending on the DDAB content, but the Tm of DDAB is roughly independent of the DODAB concentration. Both DSC and fluorescence measurements show that, within the mixture, at room temperature (ca. 22 degrees C), the DDAB-rich liposomes are in the liquid-crystalline state, whereas the DODAB-rich liposomes are in the gel state. DSC results point to a higher affinity of DDAB for DODAB liposomes than the reverse, resulting in two populations of mixed DDAB/DODAB liposomes with distinctive phase behavior. Fluorescence measurements also show that the presence of a small amount of DODAB in DDAB-rich liposomes causes a pronounced effect in Nile Red emission, due to the increase in liposome size, as inferred from turbidity results.     

Temperature quenched DODAB dispersions: fluid and solid state coexistence and complex formation with oppositely charged surfactant

Dilute dispersions of the synthetic bilayer forming double-chained cationic lipid dioctadecyldimethylammonium bromide (DODAB) were investigated. In dispersions sonicated above the chain melting temperature Tm (approximately 45 degrees C) it was found by H NMR that about 50% of the surfactant chains remained fluid when the samples were cooled to room temperature, which is 20 degrees C below Tm. In contrast, there was no sign of a fluid fraction in unsonicated samples at room temperature. The addition of the anionic surfactant sodium dodecyl sulfate (SDS) to DODAB dispersions at room temperature resulted in the formation of an essentially stoichiometric DODA-DS complex with frozen chains, as seen by titration calorimetry and H NMR experiments. For sonicated samples, turbidity experiments demonstrated that, after a fast complexation reaction, the system remains colloidally stable unless the SDS-to-DODAB mixing ratio is too close to unity. H NMR experiments also showed that in the unreacted DODAB the fraction of fluid chains remained close to 50%, indicating either that SDS reacts equally fast with fluid and frozen DODAB or that there is a relaxation of the fluid fraction after the complexation. The melting enthalpy and the melting temperature of the alkyl chains rise gradually as the mixing ratio increases. We observed with cryo-TEM that the fraction of large unilamellar vesicles was significantly larger after addition of SDS. This indicates vesicle fusion. Based on both wide- and small-angle X-ray scattering patterns, the structure of the equimolar SDS-DODAB complex at 25 degress C was proposed to be lamellar.     

Interaction between Pluronic F127 and dioctadecyldimethylammonium bromide (DODAB) vesicles studied by differential scanning calorimetry

A number of fundamental studies on the interactions between lipid bilayers and (ethylene oxide)-b-(propylene oxide)-b-(ethylene oxide) copolymers (PEO-PPO-PEO, Pluronics) have been carried out recently as model systems for the complex behavior of cell membranes with this class of polymers often employed in pharmaceutical formulations. We report here a study by differential scanning calorimetry (DSC) of the interactions in water between Pluronic F127 (F127), and the cationic vesicles of di-n-octadecyldimethylammonium bromide (DODAB), as a function of concentration of the two components (DODAB 0.1 and 1.0 mM; F127 0.1 to 5.0 mM) and of the sample preparation protocol. The DSC studies follow the critical micellization temperature (cmt ≈ 27 °C at 1.0 mM) of F127 and the gel-liquid crystal transition (T(m) ≈ 45 °C) of the DODAB bilayer and of F127/DODAB mixtures. Upon heating past T(m), vesicle/polymer mixtures undergo an irreversible conversion into mixed DODAB/F127 micelles and/or F127-bearing vesicles, depending on the relative amount of each component, together with, in some cases, residual intact F127 micelles or DODAB vesicles. Sample preparation protocol is shown to have little impact on the composition of mixed systems once they are heated above T(m).     

Adsorption behavior of DODAB/DPPC vesicles on silica

The interaction between composite dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) bilayer vesicles in the gel state and silica is investigated over the 0-20% DODAB range from determination of adsorption curves, silica sedimentation, particle sizing and zeta-potentials. At 1 mg/mL silica, 0% DODAB, pH 6.3, over the 0-150 mM NaCl range of ionic strengths, high affinity adsorption curves were barely affected by ionic strength and all of them exhibited limiting adsorption values above the level expected for single bilayer deposition. At 1 mg/mL silica, 2% DODAB, pH 6.3 and 1 mM NaCl, high affinity adsorption curves fortuitously presented limiting adsorption indicative of one bilayer deposition on each silica particle. At %DODAB<2% or %DODAB>2%, limiting adsorption was above and below the level expected for bilayer deposition, respectively. Increasing %DODAB in the vesicle composition negatively modulated the limiting adsorption on silica despite the increasing surface charge on vesicles and electrostatic attraction between vesicles and particles. The results point out the difficulty of closed vesicle disruption (required for bilayer deposition from vesicles) when the bilayer is tightly packed in the rigid gel state and might be of interest for analytical applications of immobilized vesicles on silica.     

Temperature-induced structural and permeability changes in dioctadecyldimethylammonium bromide (DODAB) vesicles: a fluorescence investigation using 2-[(p-methylamino)phenyl]-3,3-dimethyl-5-carboethoxy-3H-indole as a probe

An investigation of the temperature dependence of the fluorescence spectral characteristics of 2-[(p-methyl-amino)phenyl]-3,3-dimethyl-5-carboethoxy-3H indole (I) in aqueous micelles(sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB)) and surfactant vesicles (dioctadecyldimethylammonium bromide (DODAB)) is presented. The gel-to-liquid crystalline phase transition temperature T_c, determined to be approximately 309 K (36°C) for DODAB vesicles, is in close agreement with the value reported previously. A pretransition at 294 K (21°C) has also been obtained. The blue shift in the fluorescence maximum and the increase in bandwidth are accounted for by the displacement of molecule I towards the interior of the bilayer as a function of temperature. Arrhenius plots for the non-radiative decay processes competing with fluorescence as a function of temperature show relatively high values for the activation energy above the phase transition temperature, indicating the displacement of molecule I to a new, more viscous, less polar site compared with its initial location in DODAB. The highest value of the activation energy in water indicates that the decay dynamics of this molecule are different in water than in the organized media studied here.     

Micellization of dissymmetric cationic gemini surfactants and their interaction with dimyristoylphosphatidylcholine vesicles

The micellization process of a series of dissymmetric cationic gemini surfactants [CmH2m+1(CH3)2N(CH2)6N(CH3)2C6H13]Br2 (designated as m-6-6 with m = 12, 14, and 16) and their interaction with dimyristoylphosphatidylcholine (DMPC) vesicles have been investigated. In the micellization process of these gemini surfactants themselves, critical micelle concentration (cmc), micelle ionization degree, and enthalpies of micellization (DeltaHmic) were determined, from which Gibbs free energies of micellization (DeltaGmic) and entropy of micellization (DeltaSmic) were derived. These properties were found to be influenced significantly by the dissymmetry in the surfactant structures. The phase diagrams for the solubilization of DMPC vesicles by the gemini surfactants were constructed from calorimetric results combining with the results of turbidity and dynamic light scattering. The effective surfactant to lipid ratios in the mixed aggregates at saturation (Resat) and solubilization (Resol) were derived. For the solubilization of DMPC vesicles, symmetric 12-6-12 is more effective than corresponding single-chain surfactant DTAB, whereas the dissymmetric m-6-6 series are more effective than symmetric 12-6-12, and 16-6-6 is the most effective. The chain length mismatch between DMPC and the gemini surfactants may be responsible for the different Re values. The transfer enthalpy per mole of surfactant within the coexistence range may be associated with the total hydrophobicity of the alkyl chains of gemini surfactants. The transfer enthalpies of surfactant from micelles to bilayers are always endothermic due to the dehydration of headgroups and the disordering of lipid acyl chain packing during the vesicle solubilization.     

A COMPARISON OF THERMALLY INDUCED CHANGES IN SONICATED PHOSPHOLIPID AND SURFACTANT VESICLES BY MEANS OF INTRA-MOLECULAR EXCIMER-FORMING PROBE

Abstract The molecule (1,l'-dipyrenyl)-methyl ether (dipyme) was used for monitoring the bilayer fluidity of surfactant and sonicated phospholipid vesicles. In the latter systems, the observed transition temperatures ( Tc ) are identical with those found by different methods. Surfactant vesicles prepared from dioctadecyldimethylammonium bromide (DODAB) and dihexadecylphosphate (DHP) molecules manifest a similar fluidity of their bilayers as those of sonicated phospholipid vesicles below their Tc. However, unlike in phospholipid vesicles, there was no significant change of the bilayer structure above Tc observed in surfactant vesicles. DHP vesicles formed in pure water provide a different solubilization site for dipyme than those prepared in a buffer solution. Such sites are characterized by a relatively high local concentration of the probe and the appearance of the blue shifted spectrum of the excimer.     

Effect of coating electrolytes on two-tailed surfactant bilayer coatings in capillary electrophoresis

Surfactants such as dioctadecyldimethylammonium bromide (DODAB) form semi-permanent coatings that effectively prevent adsorption of cationic proteins onto the fused silica capillary in capillary electrophoresis (CE). The bilayer coating is generated by flushing the capillary with a 0.1 mM surfactant solution. However, formation of the bilayer is strongly dependent on the coating electrolyte. The effect of counter-ions, electrolyte concentrations and buffer co-ions were monitored based on: the separation of basic model proteins; the adsorption kinetics of DODA⁺ onto fused silica; and dynamic light scattering (DLS) to determine vesicle size. Low concentrations (≤10.0 mM) and/or weakly associating buffers such as phosphate (pH 3.0), acetate (pH 4.0) and chloride should be used for DODAB coating solutions. Dissolving the surfactant in strongly associating electrolyte, such as phosphate at pH 7.0, results in poor coating of the capillary surface. Effective cationic bilayer coatings are formed if the buffer conditions favor formation of vesicles with diameters < 300 nm. Monitoring turbidity at 400 nm provides a convenient means of verifying vesicle diameter variation of <5 nm; that is, that the coating solution is effective.     

Solubilization of dodac small unilamellar vesicles by sucrose esters: A fluorescence study

A fluorescence method was employed to study the solubilizing interactions of several sucrose esters with Dioctadecyldimethylammonium chloride small unilamellar vesicles. In this paper we studied four different alkyl esters of sucrose, saturation and solubilization concentrations (C_sat and C_sol), the ester–DODAC molar ratio (R_e) and bilayer/aqueous partition coefficients (K) were measured by monitoring changes in laurdan generalized polarization values. A new critical surfactant concentration at lower values than saturation concentration was observed. All critical concentrations showed linear dependence with DODAC concentration. The decrease in the length of surfactant alkyl chain (upper cmc) led to an increase in its ability to saturate and solubilize vesicles and to a decrease in its bilayer affinity. Consequently the shorter alkyl chain (lauryl sucrose ester), the higher ability to saturate and solubilize the vesicles, whereas the longer chain (stearyl sucrose ester), exhibited the highest degree of partitioning into the vesicles.     

Solubilization mechanism of vesicles by surfactants: effect of hydrophobicity

Simulations based on dissipative particle dynamics are performed to investigate the solubilization mechanism of vesicles by surfactants. Surfactants tend to partition themselves between vesicle and the bulk solution. It is found that only surfactants with suitable hydrophobicity are able to solubilize vesicles by forming small mixed micelles. Surfactants with inadequate hydrophobicity tend to stay in the bulk solution and only a few of them enter into the vesicle. Consequently, the vesicle structure remains intact for all surfactant concentrations studied. On the contrary, surfactants with excessive hydrophobicity are inclined to incorporate with the vesicle and thus the vesicle size continues to grow as the surfactant concentration increases. Instead of forming discrete mixed micelles, lipid and surfactant are associated into large aggregates taking the shapes of cylinders, donuts, bilayers, etc. For addition of surfactant with moderate hydrophobicity, perforated vesicles are observed before the formation of mixed micelles and thus the solubilization mechanism is more intricate than the well-known three-stage hypothesis. As the apparent critical micellar concentration (φ(s,v)(a,CMC)) is attained, pure surfactant micelles form and the vesicle deforms because the distribution of surfactant within the bilayer is no longer uniform. When the surfactant concentration reaches φ(s,v)(p), the vesicle perforates. The extent of perforation grows with increasing surfactant concentration. The solubilization process begins at φ(s,v) (sol), and lipids leave the vesicle and join surfactant micelles to form mixed micelles. Eventually, total collapse of the vesicle is observed. In general, one has φ(s,v)(a,CMC)≤φ(s,v)(p)≤φ(s,v)(sol).     

Vesicles accelerate proton transfer from carbon up to 850-fold

[reaction: see text] We have analyzed the different catalytic effects of surfactant aggregates upon the rate-determining hydroxide ion induced deprotonation reaction of 1. Vesicles are more effective catalysts than micelles, most likely providing a more apolar microenvironment at the substrate binding sites. We suggest that this leads to a catalytic reaction involving less strongly hydrated hydroxide ions. In the case of DODAB and DODAC vesicles, binding of cholesterol to the bilayer further increases the catalytic efficiency.     

Surfactant effects of chlorpromazine and imipramine on lipid bilayers containing sphingomyelin and cholesterol

The surface-active drugs chlorpromazine (CPZ) and imipramine (IP) have been tested on large unilamellar vesicles composed of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (Ch) in different proportions. The well-characterized nonionic detergent Triton X-100 (TX) has also been used in parallel experiments. Leakage of vesicular aqueous contents and bilayer solubilization have been measured for each surfactant molecule and vesicle composition. All three surface-active molecules behave in a qualitatively similar way, irrespective of bilayer composition: they induce leakage at concentrations well below their critical micellar concentrations (cmc) and solubilization near the cmc. In these events, the potency of the three surfactants under study increases with decreasing cmc, in the order IP相似文献   

Solubilization of phosphatidylcholine vesicles by hydrophobically modified poly(acrylamide)-co-(acrylic acid): effects of acrylic acid fraction and polymer concentration

The interaction of hydrophobically modified copolymers of acrylamide and acrylic acid, designated as PAM-C12-AA (X%) (X% indicates the percentage of acrylic acid unit and X = 5, 10, 20), with dimyristoylphosphatidylcholine (DMPC) vesicles has been studied. Complementary techniques including isothermal titration microcalorimetry (ITC), differential scanning calorimetry (DSC), turbidity measurement, calcein leakage measurement, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were used to get comprehensive information. The results show that PAM-C12-AA leads to solubilization of DMPC vesicles. There is a critical concentration (C(s)) for PAM-C12-AA to induce obvious vesicle disruption. This concentration is very close to the critical aggregation concentration (CAC) for the polymer self-aggregation. The Cs values are found to be similar for the three polymers. However, the disruption of DMPC vesicles induced by the polymers increases to a greater degree at higher AA fraction, owing to the increasing strength of interaction between the polymer and the lipid bilayer.     

The effect of electrolyte on the encapsulation efficiency of vesicles formed by the nonionic surfactant, 2C18E12

Encapsulation efficiencies of vesicles formed by the nonionic surfactant 1,2-dioctadecyl-rac-glycerol-3-omega-methoxydodecylethylene glycol (abbreviated as 2C18E12) and its phospholipid counterpart, distearoylphosphatidylcholine (DSPC) at 298 K, were determined by the entrapment of the water-soluble dye, carboxyfluorescein (CF) to be 0.045+/-0.001 and 0.03+/-0.04 L mol(-1) for 2C18E12 vesicles prepared using low osmolarity (270 m Osm) Krebs-Henseleit (K-H) buffer and a modified 'high salt' (1600 m Osm) variant of K-H buffer, respectively, and 0.64+/-0.01 and 0.31+/-0.04 Lmol(-1) for DSPC vesicles prepared under the same conditions and in the same buffers. Freeze fracture electron microscopy studies confirmed the presence of vesicles when 2C18E12 and DSPC were dispersed in water and both buffer solutions. Small angle neutron scattering (SANS) studies, using D2O in place of H2O, showed that when 2C18E12 vesicles were prepared in the 'high salt' variant of K-H buffer as opposed to K-H buffer or water, a higher proportion of multilamellar vesicles (MLV) were formed. Furthermore when prepared in the 'high salt' variant of K-H buffer, the 2C18E12 bilayers were thinner, and when present in the form of MLV exhibited a smaller layer of water separating the bilayers. However, even in the absence of electrolyte, 2C18E12 formed surprisingly thin bilayers due to the penetration of the polyoxyethylene chains into the hydrophobic chain region of the bilayer. Due to the dehydrating effect of the high concentration of electrolyte present in the 'high salt' variant of K-H, the polyoxyethylene head groups penetrated further into the hydrophobic region of the bilayer making the bilayer even thinner. In the case of the DSPC vesicles, although the SANS study showed an increase in the relative proportion of multilamellar to unilamellar vesicles when samples were prepared in the 'high salt' variant of K-H buffer, no differences were observed in the thickness and the d-spacing of the vesicle bilayers. Variable temperature turbidity measurements of 2C18E12, and DSPC vesicles prepared in water indicated phase changes at 320+/-0.5 and 327+/-0.5 K, respectively, and were unchanged when the 'high salt' variant of K-H buffer was used as hydrating medium. Taken together, these results suggest that a low phase transition temperature was not the reason for the poor entrapment efficiency of 2C18E12 vesicles but rather the very 'thin' hydrophobic barrier formed by the penetration of the polyoxyethylene chains into the hydrophobic region of the bilayer.