Mechanical properties and stability measurement of cholesterol-containing liposome on mica by atomic force microscopy

The micromechanical properties of pure and cholesterol modified egg yolk phosphatidylcholine (EggPC) vesicles prepared by sonication were studied by atomic force microscopy (AFM) on mica surface. The force curves between an AFM tip and an unruptured vesicle were obtained by contact mode. During approach, two repulsion regions with two breaks were observed. The slopes of the two repulsive force regimes for the pure EggPC vesicles are determined to be several times lower than that of EggPC/cholesterol vesicles. The elastic properties from force plot analysis based on the Hertzian model showed that Young's modulus (E) and the bending modulus (kc) of cholesterol-modified vesicles increased several-fold compared with pure EggPC vesicles. The significant difference is attributed to the enhanced rigidity of the EggPC vesicles as a result of the incorporation of cholesterol molecules. The behavior of cholesterol-modified vesicles upon adsorption is different from that in solution as revealed by mechanical properties. The results indicate that AFM can provide a direct method to measure the mechanical properties of adsorbed small liposomes and to detect the stability change of liposomes.     

A facile route for creating "reverse" vesicles: insights into "reverse" self-assembly in organic liquids

Reverse vesicles are spherical containers in organic liquids (oils) consisting of an oily core surrounded by a reverse bilayer. They are the organic counterparts to vesicles in aqueous solution and could potentially find analogous uses in encapsulation and controlled release. However, few examples of robust reverse vesicles have been reported, and general guidelines for their formation do not exist. We present a new route for forming stable unilamellar reverse vesicles in nonpolar organic liquids, such as cyclohexane and n-hexane. The recipe involves mixing short- and long-chain lipids (lecithins) with a trace of a salt such as sodium chloride. The ratio of short- to long-chain lecithin controls the type and size of self-assembled structure. As this ratio is increased, a spontaneous transition from reverse micelles to reverse vesicles occurs. Small-angle neutron scattering (SANS) and transmission electron microscopy (TEM) confirm the presence of unilamellar vesicles in the corresponding solutions. Average vesicle diameters can be tuned from 60 to 250 nm depending on the sample composition.     

New vesicle formation upon oleate addition to preformed vesicles

The size distribution of new vesicles formed after addition of oleate in different forms to preformed egg yolk phosphatidylcholine (EggPC) vesicles was studied by gel exclusion chromatography. The addition of oleate to preformed vesicles resulted in the formation of new small vesicles. Fission of preformed vesicles incorporated by oleate and partial solubilization of the vesicles by addition of oleate in micellar form were involved in the process of the new small vesicle formation.     

Size control of mixed egg yolk phosphatidylcholine (EggPC)/oleate vesicles

The size distribution after addition of oleate surfactant to preformed Egg yolk phosphatidylcholine (EggPC) vesicles was investigated by gel filtration chromatography combining with dynamic light scattering. Phospholipid and oleate concentration, fluorescence intensity and size of the vesicles were measured for each elution fraction. The spontaneous vesiculation of oleate at pH 8.5 was accelerated in the presence of preformed EggPC vesicles. The size distribution of newly formed vesicles was dependent on the preformed vesicular size. For example, oleate addition to large preformed vesicles (230 nm) resulted in altering of vesicles to both larger and smaller than preformed ones, while addition of oleate to small preformed vesicles (50 nm) led to the formation of only larger vesicles without exhibition of newly small vesicles. The combinations of gel filtration chromatography and dynamic light scattering could provide more detailed insight into the size change of newly formed vesicles.     

Effect of chain lengths of PEO-PPO-PEO on small unilamellar liposome morphology and stability: an AFM investigation

The morphology and stability of small unilamellar egg yolk phosphatidylcholine (EggPC) liposomes modified with the Pluronic copolymer (poly (oxyethylene)-poly (oxypropylene)-poly (oxyethylene) (PEO-PPO-PEO)) with different compositions on mica surface have been investigated using atomic force microscopy. Morphology studies reveal significant morphological changes of liposomes upon incorporating the Pluronic copolymer. Bilayers are observed for Pluronic with small hydrophilic (PEO) chain lengths such as L81 [(PEO)2(PPO)40(PEO)2] and L121 [(PEO)4(PPO)60(PEO)4]; bilayer and vesicle coexistence is observed for P85 [(PEO)26(PPO)39.5(PEO)26] and F87 [(PEO)61.1(PPO)39.7(PEO)61.1]; and stable vesicles are observed for F88 [(PEO)103.5(PPO)39.2(PEO)103.5], F127 [(PEO)100(PPO)65(PEO)100], and F108 [(PEO)132.6(PPO)50.3(PEO)132.6]. The micromechanical properties of Pluronic-modified EggPC vesicles were studied by analyzing AFM approaching force curve. The bending modulus (k(c)) of the Pluronic-modified EggPC vesicles increased several-fold compared with that of the pure EggPC vesicles. The significant difference is due to the enhanced rigidity of the EggPC vesicles as a result of the incorporation of PPO molecules and PEO chains. Based on the analysis of onset point by AFM and diameters of vesicles by light scattering, it was concluded that the favorable model to describe the polymer-bilayer interaction is the membrane-spanning model.     

Effect of preformed egg phosphatidylcholine vesicles on spontaneous vesiculation of oleate micelles

An addition of oleate micellar solution to two original sizes (180 nm and 50 nm) of preformed vesicles was studied using gel exclusion chromatography, dynamic light scattering and freeze fracture electron microscopy. The effect of molar ratios of phospholipid and oleate on size distribution of newly formed vesicles was investigated by varying molar concentrations of these two components. After adding an equiamount of oleate to 180 nm-preformed vesicles or 50 nm-preformed vesicles, a relatively monodisperse population of newly formed vesicles was detected. For the high amount of oleate addition to two original sizes of preformed vesicles, the results were quite different. New large vesicles and a number of new small vesicles were observed in samples of mixed EggPC/oleate suspension in the presence of preformed vesicles with 180 nm of size, whereas, only some new large vesicles were detected in samples of mixed EggPC/oleate suspension in the presence of preformed vesicles with 50 nm of size. We assumed that the number of new small vesicles, with size close to preformed vesicles, increased in the latter case. The transformation of mixed EggPC/oleate micelles to mixed vesicles was investigated. The results showed that transformation of mixed EggPC/oleate micelles to vesicles was remarkably faster than transformation of mere oleate micelles to vesicles. The above findings suggested that new mixed EggPC/oleate vesicles with small size were presumably formed by partial solubilization.     

Probing small unilamellar EggPC vesicles on mica surface by atomic force microscopy

Sonicated small unilamellar egg yolk phosphatidylcholine (EggPC) vesicles were investigated using atomic force microscopy (AFM) imaging and force measurements. Three different topographies (convex, planar, and concave shape) of the EggPC vesicles on the mica surface were observed by tapping mode in fluid, respectively. It was found that the topography change of the vesicles could be attributed to the interaction force between the AFM tip and vesicles. Force curves between an AFM tip and an unruptured vesicle were obtained in contact mode. During approach, two breaks corresponding to the abrupt penetration of upper and lower bilayer of vesicle were exhibited in the force curve. Both breaks spanned a distance of around 4 nm close to the EggPC bilayer thickness. Based on Hertz analysis of AFM approach force curves, the Young's modulus (E) and the bending modulus (kc) for pure EggPC vesicles were measured to be (1.97 +/- 0.75) x 10(6)Pa and (0.21 +/- 0.08) x 10(-19)J, respectively. The results show that the AFM can be used to obtain good images of intact and deformed vesicles by tapping mode, as well as to probe the integrity and bilayer structure of the vesicles. AFM force curve compare favorably with other methods to measure mechanical properties of soft samples with higher spatial resolution.     

Study on size growth in the vesicle formation upon detergent removal from phospholipid/ detergent mixed micelles

When 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) was removed from the mixed CHAPS/EggPC micelles, large vesicles were prepared by dialysis or by slow step-by-step dilution, but small vesicles were prepared by fast one-step dilution. When sodium cholate was removed from the sodium cholate/EggPC micelles, small vesicles formed either by dialysis or by dilution; however, in the presence of 5 mM Ca²⁺ large vesicles were produced by dialysis, while small vesicles were prepared by dilution. The size growth was related to a detergent-induced fusion of the vesicles containing a large amount of detergent. Using spectrophotometry, quasielastic light scattering and freeze–fracture electron microscopy the fusion events were investigated both through the process of vesicle solubilization by adding detergent and through the process of vesicle formation by diluting a mixed micelle. The results suggest that a rapid CHAPS-induced fusion of the vesicles led to the large resultant vesicles and that no fusion of vesicles containing sodium cholate is responsible for the formation of small vesicles. Furthermore, the ultimate vesicle size related to rapid or slow detergent removal is dependent on the kinetic aspects of the fusion. Received: 19 August 1999 Accepted: 18 February 2000     

Tethered bilayer lipid membranes studied by simultaneous attenuated total reflectance infrared spectroscopy and electrochemical impedance spectroscopy

The formation of tethered lipid bilayer membranes (tBLMs) from unilamelar vesicles of egg yolk phosphatidylcholine (EggPC) on mixed self-assembled monolayers (SAMs) from varying ratios of 6-mercaptohexanol and EO(3)Cholesteryl on gold has been monitored by simultaneous attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The influence of the lipid orientation (and hence the anisotropy) of lipids on a gold film on the dichroic ratio was studied by simulations of spectra with a matrix method for anisotropic layers. It is shown that for certain tilt angles of the dielectric tensor of the adsorbed anisotropic layer dispersive and negative absorption bands are possible. The experimental data indicate that the structure of the assemblies obtained varies with varying SAM composition. On SAMs with a high content of EO(3)Cholesteryl, tBLMs with reduced fluidity are formed. For SAMs with a high content of 6-mercaptohexanol, the results are consistent with the adsorption of flattened vesicles, and spherical vesicles have been found in a small range of surface compositions. The kinetics of the adsorption process is consistent with the assumption of spherical vesicles as long-living intermediates for surfaces of a high 6-mercaptohexanol content. No long-living spherical vesicles have been detected for surfaces with a large fraction of EO(3)Cholesteryl tethers. The observed differences between the surfaces suggest that for the formation of tBLMs (unlike supported BLMs) no critical surface coverage of vesicles is needed prior to lipid bilayer formation.     

Effect of a PEG lipid (DSPE-PEG2000) and freeze-thawing process on phospholipid vesicle size and lamellarity

Liposomes containing distearoylphosphatidylethanolamine with covalently linked polyethylene glycol of molecular weight 2,000 (DSPE-PEG2000) covering a range of 0–30 mol% were prepared by a mechanical dispersion or detergent-removal method. The effects of DSPE-PEG2000 on particle sizes and lamellarity of liposomes were investigated. The average diameters of vesicles prepared from both methods decreased when the concentration of DSPE-PEG2000 was increased. The decrease in vesicle size with increase in DSPE-PEG2000 was ascribed to the steric hindrance of strongly hydrated PEG. The significant decrease in the sizes of DSPE-PEG2000-containing EggPC vesicles prepared by the detergent-removal method could be explained by the postvesiculation size growth in the process of micelle–vesicle transition. For DMPC vesicles prepared by the detergent-removal method, electron micrographs showed that inclusion of DSPE-PEG2000 promoted vesicle formation. Based on the results of investigation of calcein entrapment efficiency, we concluded that the lamellarity of liposomes is reduced as PEG lipid concentration is increased. Fragmentation of multilamellar vesicles into smaller unilamellar vesicles occurred more readily when the liposome suspension was subjected to repetitive freeze-thawing. After five cycles of freezing and thawing, vesicles containing more than 0.5 mol% DSPE-PEG2000 were fragmented into unilamellar vesicles with diameters smaller than 300 nm.     

Electroformed Giant Vesicles from a Binary Mixture of Phospholipids and Quaternary Ammonium Salts

In this article, positively charged GVs were electroformed in a binary system of quaternary ammonium salts and egg phosphatidylcholine (EggPC) under an alternative current (AC) electric field. The diameter and charge density of the GVs is controlled by doping suitable cationic quaternary ammonium molecules into the EggPC bilayer. By developing positively charged GVs, there will be expanded the applications for phospholipids vesicles, especially the investigation of charge-induced interactions between cationic lipid membranes and macromolecules, such as colloidal particles or proteins.     

Stability and Size Control of Reverse Vesicles

The stability and size control of reverse vesicles have been investigated for a sucrose monoalkanoate/hexaethylene glycol hexadecyl ether/decane/water system. The stability is highly dependent on the surfactant mixing ratio, amount of added water, and vesicle size. The size distribution of reverse vesicles produced by simple mixing is very large, but larger vesicles can be removed by means of the extrusion method and reasonably homogeneously size-distributed reverse vesicles can be obtained. If a probe-type ultrasonicator is used, the reverse vesicles obtained are homogeneous and of very small size (50-70 nm in diameter) and they are considered to be of the unilamellar type.     

High Yield of Supergiant Vesicles on Glyoxylic Acid Modified Aluminum Electrode

Supergiant vesicles are useful for investigating the interactions between lipid memebranes and collodial particles or macromolecules. Producing supergiant vesicles using the electroformation method lead to extraordinary findings in this work. The glyoxylic acid modified aluminum (GA-Al) electrode was prepared by the electro-reduction in an oxalic acid solution. High yield of supergiant vesicles with diameters >100 µm are successfully formed on a GA-Al electrode under an alternative current (AC) electric field. In contrast to platinum and indium tin oxide, which have been mainly used so far, this GA-Al electrode is preferable for the electroformation of egg phosphatidylcholine (EggPC) supergiant vesicles.     

Effect of freeze-thawing and polyethylene glycol (PEG) lipid on fusion and fission of phospholipid vesicles

The effect of freeze-thawing on the size of egg yolk phosphatidylcholine (EggPC) vesicles in the presence of 0-40 mol% distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG) was studied. Mean diameters of the vesicles fell into a range of 80-150 nm after 10 times freeze-thawing in spite of their original size. In the process of freeze-thawing, two opponent events, one is fission and the other is fusion, occurred at the same time. DSPE-PEG accelerated the fusion event.     

Physicochemical properties of PEG-grafted liposomes

Egg phosphatidylcholine (EggPC) or dimyristoylphosphatidylcholine (DMPC) liposomes containing polyethylene glycol (PEG)-lipids covering a range of 0-30 mol% have been prepared by Extrusion method. The physicochemical properties including size evolution and calcein permeation were evaluated to investigate the effect of PEG-lipids on bilayer structure. The results from quasielasetic light scattering (QELS), freeze-fracture microscopy, and gel exclusion chromatography revealed that presence of low concentration of PEG-lipid results in decreasing of vesicle size and further increase in the PEG-lipid concentrations lead to a transition from the lamellar membranes to micelles. The permeability for calcein increased with increase in concentration of distearoylphosphatidylethanolamine (DSPE)-PEG. On the other hand, the permeability decreased with low amount of cholesterol-PEG (blow 20% cholesterol-PEG) and increased with high amount of it. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the composition of the bilayer. The concentration of DSPE-PEG2000 incorporated into vesicles without damaging vesicle structures were <20 mol% for EggPC and <10% for DMPC.     

Formation of reverse vesicles in silicone surfactant systems

This study deals with the formation of reverse vesicles based on the phase behavior of silicone surfactants. The surfactants, polyoxyethylene–polydimethylsiloxane and polyoxyethylene–polyoxypropylene–polydimethylsiloxane copolymer, were found to form lamellar liquid crystal phases in three different types of silicone oil upon the addition of a certain amount of water. A conventional method in which reverse vesicles are prepared by physically dispersing this lamellar liquid crystal phase in oil was employed in addition to a technique based on a temperature-induced phase transition. The particle sizes and stabilities of the resulting reverse vesicles were evaluated.     

Reverse micelles dissolved in supercritical xenon: an NMR spectroscopic study

Reverse micelles currently gain increasing interest in chemical technology. They also become important in biomolecular NMR due to their ability to host biomolecules such as proteins. In the present paper, a procedure for the preparation of high-pressure NMR samples containing reverse micelles dissolved in supercritical xenon is presented. These reverse micelles are formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT). For the first time, NMR spectroscopy could be applied to reverse micelles in supercritical xenon. The AOT/H(2)O/Xe system was studied as a function of experimental parameters such as xenon pressure, water content, and salt concentration. Optimum conditions for reverse micelle formation in supercritical xenon could be determined. It is, furthermore, demonstrated that biomolecules such as amino acids and proteins can be incorporated into the reverse micelles dissolved in supercritical xenon.     

Reversible self-organization of a UV-responsive PEG-terminated malachite green derivative: vesicle formation and photoinduced disassembly

This paper describes the assembly and disassembly of vesicles formed by a UV-responsive poly(ethylene glycol) terminated malachite green derivative. The UV-responsive amphiphile with both a hydrophobic malachite green group and a hydrophilic PEG group can self-organize into vesicles in water before UV irradiation. However, upon UV irradiation, the photochromic moiety can be ionized to its corresponding cation, leading to the disassembly of these vesicles. In addition, the cation can thermally recover its electrically neutral form, and the disassembled species can form vesicles reversibly on the basis of a thermal reverse reaction. The reverse reaction is temperature-controlled and can be speeded up by thermal treatment. By using various characterization techniques, e.g., transmission electron microscopy, dynamic light scattering, UV-visible spectroscopy, and NMR spectroscopy, we have confirmed that the vesicle structures can be formed, disassembled, and recovered by the above-mentioned treatments. It is anticipated greatly that this line of research may provide new insights into the mechanism behind stimuli-responsive formation and rupture of molecular assemblies, facilitating the design and synthesis of new surface active molecules for the fabrication of stimuli-responsive materials with designed functions.     

Probing the lateral organization of membranes: fluorescence repercussions of pyrene probe distribution

Phospholipids pyrene labeled are widely used to investigate dynamics and organizations of membranes. We studied pyrene probe lateral distribution by analyzing the variations of the molar absorption coefficient (epsilon) versus probe concentrations, in small unilamellar vesicles (SUV) made of phospholipids and/or glycolipids, with pyrene labeled phosphatidylcholine (PyPC) or phosphatidylglycerol (PyPG). The results were interpreted according to an infinite associative model. They indicated that an effective self-association process corresponding to K ranging from 30 to 100 M(-1) occurred with those probes incorporated in dimannosyl diacylglycerol (DMDG). In contrast, after SUV labeling of egg yolk phosphatidylcholine (EggPC) or phosphatidylglycerol (EggPG), K values < 1 M(-1) were determined. The corresponding percentages of various stacked forms of pyrene probes were calculated. They indicated that, for a 3% PyPG labeling, the monomer represented 21% of n-mers in DMDG and 94% in EggPC. The analysis of fluorescence experiments carried out on the same samples indicated that: (i) the fluorescence process of pyrene probes was generated by the monomers: and (ii) the excimer forming resulted from a diffusional encounter between one excited and one non-excited monomer. A correction of fluorescence data allowing a more correct interpretation of fluorescence measurements was proposed.     

Characterization of aggregate structures of phospholipid in the process of vesicle solubilization with sodium cholate using laser light scattering method

The aggregate structures formed during vesicle solubilization by sodium cholate, and their properties, were characterized by static laser light scattering (SLS) and electrophoretic light scattering (ELS) methods. The change in dissymmetry value Z₄₅ was observed by examining the regions of vesicles and micelles. The angular light scattering intensity data could be fitted with a modified shell model for the vesicles and a hollow cylinder model for the mixed micelles. In the case of the vesicles, the scattering curves were fitted with a spherical shell model by introducing the interparticle scattering factor S(q) and taking into account the intervesicle positional correlations, which is a function of the fractal dimension (D) and the interparticle correlation length (L). On the basis of the physical meanings of the fractal dimension and interparticle correlation length, the molecular packings of the membrane and the repulsive interaction between the vesicles were analyzed. Furthermore, using electrophoretic light scattering (ELS) the zeta potentials on the mixed vesicles were found to increase with the molar ratio (Re) of sodium cholate to egg yolk phosphatidylcholine (EggPC) in the membrane. It is suggested that the electrostatic properties of the vesicles result in repulsive interaction which is responsible for no fusion of the mixed vesicles. In addition, in the transition from vesicles to micelles, a cylinder-like micelle appeared as an intermediate structure.