Freeze-fracture transmission electron microscopy studies on the self-assemblies of amphiphilic solutions

Self-assemblies of amphiphiles in solutions were investigated by using freeze-fracture transmission electron microscopy (FF-TEM). Especially, vesicles were characterized by FF-TEM and the transition of self-assemblies was determined. The stacked lamellar La-phase was prepared without shear forces by a chemical reaction. The stacked lamellar La-phase can be transformed into multilamellar vesicles by the shearing forces that occur when the stacked lamellar La-phase sample is turned upside down a few times. The multilamellar vesicles can also be transformed into unilamellar vesicles by high shearing forces. These transitions were demonstrated by FF-TEM measurements. 2n2+-induced vesicle formation in the single-chain surfactant solutions was first achieved.     

Hydrogels from phospholipid vesicles

It is shown that phospholipid dispersions with a few percent of diacylphosphocholine PC in water can be swollen to single-phase lyotropic liquid crystalline L_α-phases by the addition of co-solvents like glycerol, 1,3-butyleneglycol BG or 1,2-propyleneglycol PG. The birefringent L_α-phases contain small unilamellar and multilamellar vesicles if the temperature of the samples is above the Krafft-Temperature T_m of the phospholipid. When such transparent birefringent viscous samples are cooled down below T_m the samples are transformed into birefringent gels. Cryo-TEM and FF-TEM measurements show that the bilayers of the vesicles are transformed from the liquid to the crystalline state during the transformation while the vesicle structure remains. The bilayers of the crystalline vesicles form adhesive contacts in the gel. Pulsed-field gradient NMR measurements show that two different kinds of water or co-solvent can be distinguished in the gels. One type of solvent molecules can diffuse like normal solvent in a continuous bulk phase. A second type of water diffuses much more slowly. This type of solvent is obviously trapped in the vesicles. The permeability of the crystalline vesicles for water and solvent molecules is much lower in the crystalline state than in the fluid state.     

Photochemical control of molecular assembly formation in a catanionic surfactant system

Photochemical control of vesicle disintegration and reformation in aqueous solution was examined using a mixture of 4-butylazobenzene-4'-(oxyethyl)trimethylammonium bromide (AZTMA) as the photoresponsive cationic surfactant and sodium dodecylbenzenesulfonate (SDBS) as the anionic surfactant. Spontaneous vesicle formation was found in a wide-ranging composition of the trans-AZTMA/SDBS system. AZTMA molecules constituting vesicles underwent reversible trans-cis photoisomerization when irradiated with ultraviolet and visible light. Transmission electron microscopy observations using the freeze-fracture technique (FF-TEM) showed that UV light irradiation caused the vesicles to disintegrate into coarse aggregates and visible light irradiation stimulated the reformation of vesicles (normal control). A detailed investigation of the phase state and the effects of UV and visible light irradiation on the AZTMA/SDBS system with the use of electroconductivity, dynamic/static light scattering, and surface tension measurements and FF-TEM observations revealed that in the AZTMA-rich composition (AZTMA/SDBS 9:1) a micellar solution before light irradiation became a vesicular solution after UV light irradiation and visible light irradiation allowed the return to a micellar solution (reverse control). Thus, we could photochemically control the disintegration (normal control) and reformation (reverse control) of vesicles in the same system.     

Swelling of Lalpha-phases by matching the refractive index of the water-glycerol mixed solvent and that of the bilayers in the block copolymer system of (EO)15-(PDMS)15-(EO)15

The swelling of Lalpha-phases from the block copolymer polyethylenoxide-b-polydimethylsiloxane-polyethylenoxide (EO)15-(PDMS)15-(EO)15 in water/glycerol mixtures is reported. At low and medium polymer concentrations (<60%), the block copolymer forms a turbid vesicular dispersion in water. With time, the small unilamellar vesicles (SUV) and the large multilamellar vesicles (MLV) separate into a two phase L1/Lalpha-system. The turbid dispersions of the Lalpha-phase below 60% of the compound become more and more transparent with increasing glycerol and at 60% of glycerol become completely clear. Replacement of water by the solvent glycerol thus lowers the turbidity of the dispersion and swells the interlamellar distance between the bilayers. A 20% aqueous L1/Lalpha-dispersion can thus be transformed into a single birefringent transparent Lalpha-phase. The swelling of the Lalpha-phase in water and the decrease of the turbidity of the dispersion by the addition of glycerol is explained by the matching of the refractive index of the solvent to the refractive index of the bilayers of the block copolymer. The matching of a refractive index lowers the Hamaker constant in the DLVO theory between the bilayers and therefore decreases the attraction between the bilayers what allows them to swell to a larger separation. The microstructures in the phases were determined by cryo- and FFR-TEM. The interlamellar distance between the bilayers was determined by SAXS measurements. The viscous properties of the Lalpha-phases were determined by oscillatory rheological measurements. In comparison to other Lalpha-phases from normal surfactants, the Lalpha-phases from the block copolymer (EO)15-(PDMS)15-(EO)15 have low shear moduli. This is probably due to the high flexibility of the poly dimethylsiloxane block in the bilayers what can be recognized on the non-spherical shapes of the SUV's.     

Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous

This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy. Upon solution exchange and acidification of the extravesicular solution (from pH 9 to 3.5), we observed for each vesicle a biphasic decay in fluorescence. Through single-vesicle analysis, we found that rate constants for the first decay followed a Poisson distribution, whereas rate constants for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the solubility-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate constant and vesicle intensity (dependent on size) correlated to give an average permeability coefficient; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our experiments.     

Influence of hydrocarbon surfactant on the aggregation behavior of silicone surfactant: observation of intermediate structures in the vesicle-micelle transition

Intermediate structures of the aggregates in the aqueous solution of an ABA-type silicone surfactant and in the process of an SDS-induced vesicle-micelle transition are reported. In single ABA silicone surfactant aqueous solutions, large multilamellar vesicles (MLV), small single lamellar vesicles (SLV), threadlike micelles (TLM), and spheroidal micelles were observed. Interestingly, a large amount of TLMs were found entrapped into the large MLVs, but not in SLVs. Disintegration of the small vesicles inside the MLVs indicates that the entrapped TLM are from the disintegrated membrane of the entrapped small vesicles. Addition of SDS induced a transition from vesicles or threadlike micelles to spheroidal micelles. The intermediate structures, such as the appearance of small holes in the vesicle membrane, the budding of threadlike micelles from the membrane fracture, and the clusters of spheroidal micelles, were observed with increase of the SDS concentration. The electrical conductivity measurements indicated that complex micelles of SDS and silicone surfactant were formed in the solution due to the interaction between the SDS and PEO part of the silicone surfactant.     

Structural Changes Induced in the Surfactant System C(12)E(4)/Benzyl Alcohol/Water by the Admixture of the Cationic Surfactant Cetylpyridinium Chloride

The influence of the addition of the cationic surfactant cetylpyridinium chloride (CPyCl) on the structure of the different phases of the ternary surfactant system C(12)E(4)/benzyl alcohol/water in the dilute region has been studied by means of small angle neutron scattering (SANS) and freeze-fracture microscopy (FF-TEM). In the ternary system various different subregions of the L(alpha)-phase were identified as a function of the concentration of the cosurfactant, benzyl alcohol. Addition of small amounts of CPyCl suppresses these different L(alpha)-phases in favor of the one composed of multilamellar vesicles. Addition of somewhat larger amounts (up to 2 mol% relative to the total surfactant concentration) destabilizes the formation of bilayer structures completely and leads to the formation of micellar solutions. This demonstrates that in this surfactant system the incorporation of very small amounts of cationic surfactant has a pronounced and systematic fluence on its phase behavior and its structures. Copyright 2001 Academic Press.     

Characterizing assembly morphology changes during solubilization process of dimyristoyl phosphocholine vesicles by n-dodecyl triethylammonium bromide

In the present work, the assembly morphology changes during the solubilization process of the sonicated unilamellar vesicles from dimyristoyl phosphocholine (DMPC) by a cationic surfactant, n-dodecyl triethylammonium bromide (DTEAB) were well characterized with DSC, FF-TEM and DLS and fluorescence probes technique. Based on an analysis on the above results, a primary multi-stage model was brought forward to sketch the assembly morphology changes during the DMPC vesicle solubilization by DTEAB. In comparison with classical models, vesicles division, tubule-like structure formation and fission to vesicle were found in the middle stages of this model. Additionally, it is the first time that the transversally-cut profiles of tubule-like structures were observed during vesicle solubilization process.     

Milk membrane lipid vesicle structures studied with Cryo-TEM

Milk fat globule membrane (MFGM) lipids have been studied in the presence and absence of proteins β-lactoglobulin and β-casein. The aim of this study was to relate the self-assembly structure, e.g. vesicles, formed in aqueous dispersions of MFGM lipids to the lipid composition, electrolyte composition as well as the effect of added milk proteins, i.e. β-lactoglobulin and β-casein. For this purpose, vesicles of phospholipid mixtures, containing dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), dioleoylphosphatidylethanolamine (DOPE), phosphatidylinositol (PI) and dioleoylphosphatidylserine (DOPS) at composition corresponding to that of the MFGM, were prepared by extrusion. The morphology of the formed structures of different sample compositions was studied with cryogenic transmission electron microscopy (Cryo-TEM). Mixtures of membrane lipid with a composition (e.g. 80% DOPE, 12% DOPC and 8% SM) that at high lipid content give liquid crystalline phases at the boundary of lamellar to reversed hexagonal phase rather formed microtubular structures than vesicles at high water content. A large proportion of multilamellar vesicles is formed in buffer and divalent salts than in pure water. A small increase in the interlayer spacing of the multilamellar vesicle was observed in the presence of β-casein.     

Phase Behavior, Structure, and Physical Properties of the Quaternary System Tetradecyldimethylamine Oxide, HCl, 1-Hexanol, and Water

The phase diagram of the ternary surfactant system tetradecyldimethylamine oxide (TDMAO)/HCl/1-hexanol/water shows with increasing cosurfactant concentration an L(1) phase, two L(alpha) phases (a vesicle phase L(alpha1) and a stacked bilayer phase L(alphah)), and an L(3) phase, which are separated by the corresponding two-phase regions L(1)/L(alpha) and L(alpha)/L(3). In this investigation, the system was studied where some of the TDMAO was substituted by the protonated TDMAO. Under these conditions, one finds for constant surfactant concentration of 100 mM TDMAO a micellar L(1) phase, an L(alpha1) phase (consisting of multilamellar vesicles), and an interesting isotropic L(1)(*) phase in the middle of the L(1)/L(alpha) two-phase region. The L(1)(*) phase exists at intermediate degrees of charging of 30-60% and for 40-120 mM TDMAO and 70-140 mM hexanol concentration. At surfactant concentrations less than 80 mM the L(1)(*)-phase borders directly on the L(1) phase. The phase transition between the L(1) phase and the L(1)(*) phase was detected by electric conductivity and rheological measurements. The conductivity values show a sharp drop at the L(1)/L(1)(*) transition, and the zero shear viscosity of the L(1)(*) phase is much lower than in L(1) phase. The form and size of the aggregates in L(1)(*) were detected with FF-TEM and SANS. This phase contains small unilamellar vesicles (SUV) of about 10 nm and some large multilamellar vesicles with diameters up to 500 nm. The system exhibits another peculiarity. For 100 mM surfactant, the clear L(alpha1)-phase exists only at chargings below 30%. With oscillating rheological measurements a parallel development of the storage modulus G' and the loss modulus G" was observed. Both moduli are frequency independent and the system possesses a yield stress. The storage modulus is a magnitude larger than the loss modulus. Copyright 2000 Academic Press.     

Shear-induced permeation and fusion of lipid vesicles

This paper introduces a novel approach to controlling membrane permeability in free unilamellar vesicles using shearing in the presence of a detergent with a large head-group to tune pore formation. Such shear-induced permeation could offer a simple means of postencapsulating bioactive molecules to prepare vesicle vectors for drug delivery. Using UV absorption, fluorescence emission, dynamic light scattering, and electron microscopy, we investigated the membrane permeability and the morphology of unilamellar lipid vesicles (diameter in the range 50-400 nm) subjected to a shear stress in the presence of a small amount of nonionic surfactant (Brij 76). Shear-induced leakage and fusion events were observed. We analyzed the significance of the vesicle size, the shear rate, and the surfactant-to-lipid ratio for the observed phenomena. The present approach is evaluated for postloading of preformed vesicles.     

Shear-induced phase transitions in sucrose ester surfactant

The behavior of a commercial sucrose stearate blend has been examined by means of various experimental techniques (differential scanning calorimetry, light polarization and electron microscopy, and rotational rheometry). A partial phase diagram in water has been established. It shows that the binary system forms a lamellar lyotropic mesophase and that the melting behavior is characterized by a lamellar gel-lamellar liquid crystalline phase transition. The identification of the liquid crystalline phase has been carried out from textural observation using polarization microscopy and freeze-fracture electron microscopy. At low surfactant concentrations, the phase transition has been followed through rheological experiments. Furthermore, a shear-induced transition, from the lamellar phase (sheets of surfactant bilayers including a few large multilamellar vesicles) to an onion phase, has been observed above a critical temperature of 43 degrees C. The vesicles so obtained did not relax over more than 3 weeks. The presence of a small ratio of distearate in the sugar ester blend seems to be the key to vesicle formation at low surface-active material concentration.     

Vesicle growth and deformation in a surfactant solution below the Krafft temperature

We have studied vesicle growth and deformation in aqueous solutions of nonionic surfactant C(16)E(7) below the Krafft temperature by means of an optical microscope. It has been found that vesicles become larger by fusing together, and that the growth rate is slower than that of the unilamellar vesicle or emulsion systems due to the multilamellar structures of shells in a vesicle. The deformation of the vesicles depends on the temperature quench depth, and we found the transformation from spherical vesicles to string-like domains at a certain quench-temperature. From the small angle X-ray scattering and confocal microscope experiments, it can be deduced that the deformation of vesicles would be induced by osmotic pressure due to the micellar concentration difference between inside and outside of vesicles.     

Highly efficient capture and long-term encapsulation of dye by catanionic surfactant vesicles

Vesicles formed from the cationic surfactant, cetyltrimethylammonium tosylate (CTAT) and the anionic surfactant, sodium dodecylbenzenesulfonate (SDBS), were used to sequester the anionic dye carboxyfluorescein. Carboxyfluorescein was efficiently sequestered in CTAT-rich vesicles via two mechanisms: encapsulation in the inner water pool and electrostatic adsorption to the charged bilayer. The apparent encapsulation efficiency (22%) includes both encapsulated and adsorbed fractions. Entrapment of carboxyfluorescein by SDBS-rich vesicles was not observed. Results show the permeability of the catanionic membrane is an order of magnitude lower than that of phosphatidylcholine vesicles and the loading capacity is more than 10 times greater.     

Spontaneous formation of vesicles of diblock copolymer EO6BO11 in water: a SANS study

Small angle neutron scattering (SANS) is used to study the structures formed in water by a diblock copolymer EO6BO11 (having 6 ethylene oxide, EO, and 11 butylene oxide, BO, units). The data show that polymer solutions over a broad concentration range (0.05-20 wt %) contain vesicular structures at room temperature. Interestingly, these vesicles could be formed without any external energy input, such as extrusion, which is commonly required for the formation of other block copolymer or lipid vesicles. The EO6BO11 vesicles are predominantly unilamellar at low polymer concentrations, whereas at higher polymer concentrations or temperatures there is a coexisting population of unilamellar and multilamellar vesicles. At a critical concentration and temperature, the vesicular structures fuse into lyotropic arrays of planar lamellar sheets. The findings from this study are in broad agreement with the work of Harris et al. (Langmuir, 2002, 18, 5337), who used electron microscopy to identify the vesicle phase in the same system.     

Fluorescence microscopic investigation on morphological changes of giant multilamellar vesicles induced by amphiphilic additives

Adding an artificial bolaamphiphile to a dispersion of giant multilamellar vesicles (GMVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) induced a cup-shaped deformation in GMVs accompanied by partial extrusion of the inner vesicle; thereafter, the deformed vesicles returned to their original shape. On the other hand, when the artificial bolaamphiphile together with a surfactant was added to the vesicular dispersion, these deformation and reformation dynamics were transmitted from the outer membranes in GMVs to the inner membranes until an intact inner vesicle was extruded out of the outer membrane. The microscopic aspects of these processes were investigated using amphiphiles tagged with individual fluorophores.     

Vesicles Formation Induced by Layered Double Hydroxides in Mixture of Lauryl Sulfonate Betaine and Sodium Dodecyl Benzenesulfonate

This paper reports that structurally positively charged layered double hydroxides (LDHs) nanoparticles induce the vesicle formation in a mixture of a zwitterionic surfactant, lauryl sulfonate betaine (LSB), and an anionic surfactant, sodium dodecyl benzenesulfonate (SDBS). The existence of vesicles was demonstrated by negative‐staining (NS‐TEM) and freeze‐fracture (FF‐TEM) transmission electron microscopy and confocal laser scanning microscopy (CLSM). The size of vesicles increased with the increase of volume ratio (Q) of Mg₃Al‐LDHs sol to the SDBS/LSB solution. A new composite of LDHs nanoparticles encapsulated in vesicles was formed. A possible mechanism of LDHs‐induced vesicle formation was suggested. The positive charged LDHs surface attracted negatively charged micelles or free amphiphilic molecules, which facilitated their aggregation into a bilayer membrane. The bilayer membranes could be closed to form vesicles that have LDHs particles encapsulated. It was also found that an adsorbed compound layer of LSB and SDBS micelles or molecules on the LDHs surface played a key role in the vesicle formation.     

Research on the vesicle-micelle transition by 1H NMR relaxation measurement

We have investigated how the dynamics of surfactant molecules changes with the vesicle-micelle transition by (1)H NMR relaxation studies on the sodium decyl sulfate (SDeS)-decyltrimethylammonium bromide (DeTAB)-deuterium oxide system. The study has been planned with reference to the phase diagram of the SDeS-DeTAB-water system deduced from thermodynamic analysis of the surface tension data. The spin-lattice relaxation time (T(1)) and the spin-spin relaxation time (T(2)) are measured at 90 and 400 MHz at various total molalities, m, and compositions, X(2), of the surfactants. The data were analyzed according to the "two-step" model developed by Wennerstr?m et al. and molecular dynamics of the surfactant is discussed from the viewpoint of correlation time tau(f) associated with the local fast motion of the surfactant molecule, correlation time tau(s) associated with the slow overall motions of the aggregate and surfactant molecules within it, and local order parameter S. We find tau(s) of vesicles is an order of magnitude larger than that of micelles signifying that the tumbling of vesicle particles and surfactant diffusion over the vesicle are much slower than those for micelle. Tau(f) and S for vesicles are also larger than those for micelles. Molecular environments of the surfactant are also discussed from the dependence of the chemical shifts on m at constant X(2) or from that on X(2) at constant m. When the chemical shifts in vesicle and micelle are compared at constant m, the chemical shifts in vesicle are displaced to a lower magnetic field than those in micelle, which implies that the surfactant molecules are arranged more closely to each other in the vesicle than in the micelle.     

Polymer-induced structural changes in lecithin/sodium dodecyl sulfate-based multilamellar vesicles

Aqueous concentrated lecithin mixtures (asolectin from soybean) show typical lamellar liquid crystalline behavior and the individual lamellae tend to form spherical supramolecular structures, i.e., multilamellar vesicles. When part of the lecithin is replaced by the anionic surfactant sodium dodecyl sulfate (SDS), the compact multilamellar vesicles disappear and the viscosity decreases. By adding poly(diallyldimethylammonium chloride) (PDADMAC) to the lecithin/SDS system, the formation of multilamellar vesicles can be induced again and the viscosity increases. However, one characteristic feature of these polymer-modified systems is a temperature-dependent phase transition from a compact multilamellar vesicle phase to a more swollen liquid crystalline phase. The polymer-modified multilamellar compact vesicles are of interest for utilization as new thermosensitive drug delivery systems.     

Interaction between zero-charged catanionic vesicles and PEO–PPO–PEO triblock copolymers

We investigate the interaction between zero-charged catanionic vesicles and PEO–PPO–PEO (poly(ethylene oxide–poly(propylene oxide)–poly(ethylene oxide)) triblock copolymers. The 25-mg mL^?1 aqueous solution of tetradecyltrimethylammonium laurate (TTAL) contains closely packed uni- and multi-lamellar vesicles and shows viscoelastic properties with a dominant elastic modulus (G′) over a viscous modulus (G″). When a small amount of F127 ((EO)₉₇(PO)₆₉(EO)₉₇) or F68 ((EO)₇₆(PO)₂₉(EO)₇₆) was added, an improvement of the viscoelasticity was observed at suitable polymer concentrations. Freeze–fracture transmission electron microscopy (FF-TEM) observations on an F68-containing system revealed interesting aggregate transition from vesicles to flexible tubules and back to vesicles. The improvement of the viscoelasticity of the vesicular solution containing F68 or F127 can be explained by the formation of tubule and polymer–vesicle associates, while no such phenomenon was noticed for P123 ((EO)₁₉(PO)₆₉(EO)₁₉) which has the highest propylene oxide (PO) content and the strongest ability to self-associate in aqueous solution. In all the cases, vesicles will be destroyed and phase separation can be observed at high polymer contents (>5-mg mL^?1).