A new reverse wormlike micellar system: mixtures of bile salt and lecithin in organic liquids

We report a new route for forming reverse wormlike micelles (i.e., long, flexible micellar chains) in nonpolar organic liquids such as cyclohexane and n-decane. This route involves the addition of a bile salt (e.g., sodium deoxycholate) in trace amounts to solutions of the phospholipid lecithin. Previous recipes for reverse wormlike micelles have usually required the addition of water to induce reverse micellar growth; here, we show that bile salts, due to their unique "facially amphiphilic" structure, can play a role analogous to that of water and promote the longitudinal aggregation of lecithin molecules into reverse micellar chains. The formation of transient entangled networks of these reverse micelles transforms low-viscosity lecithin organosols into strongly viscoelastic fluids. The zero-shear viscosity increases by more than 5 orders of magnitude, and it is the molar ratio of bile salt to lecithin that controls the viscosity enhancement. The growth of reverse wormlike micelles is also confirmed by small-angle neutron scattering (SANS) experiments on these fluids.     

Computer Simulations of the Formation of Bile Salt Micelles and Bile Salt/DPPC Mixed Micelles in Aqueous Solutions

Brownian dynamics simulations for a coarse-grained model have been performed to study the formation of micelles from bile salts and mixed micelles with dipalmitoyl-phosphatidylcholine (DPPC) in aqueous solutions. The particular association behavior of bile salts as facial surfactants was shown to be caused by their special molecular architecture with a hydrophilic and a hydrophobic side. The experimentally observed smooth transition into the micellar region with increasing concentration is reproduced. Micelle size distributions have been evaluated at different bile salt concentrations. Typical structures of pure bile salt micelles could be identified. The composition and the structure of mixed micelles have been studied in their dependence on the bile salt/lipid concentration ratio in the aqueous solution. We have found that the bile salt fraction in the mixed micelles increases considerably with increasing bile salt/lipid concentration ratio and decreasing micelle size. The structural and thermodynamic features of micelle formation in the aqueous bile salt solutions with DPPC, which we have studied with the coarse-grained model, are in good qualitative agreement with experimental findings.     

Cholesterol Binding to Simple Micelles in Aqueous Bile-Salt–Cholesterol Solutions

The true thermodynamic activity (A_T) of cholesterol (Ch) in aqueous solutions containing taurocholate (TC)–Ch was determined by employing a direct assay of a 1 × 2-cm silicone polymer film with 0.025 cm thickness. Using theA_Tdata, information on the nature of micellar species present in the TC–Ch system, and employing a binding-site model previously developed for tauroursodeoxycholate (TUDC)–Ch and taurochenodeoxycholate (TCDC)–Ch systems, it appeared that the Ch-binding affinity for simple bile-salt micelles corresponds precisely with the order of hydrophobicity, TUDC < TC < TCDC. Further, although simple TC micelles and simple TCDC micelles have similar binding capacities, the first Ch binding to a simple TC micelle may not significantly facilitate the second Ch binding, as occurs in simple TCDC micelles. For TUDC–Ch, TC–Ch, and TCDC–Ch systems, the concentration of bound simple micelles increased with increasingA_Tvalues, whereas the unbound simple micelle concentration decreased proportionally. These results provide insights into the possible influence of bile-salt species on Ch-binding to simple micelles in bile-salt–Ch solutions.     

Micellar bile salt mobile phases for the liquid chromatographic separation of routine compounds and optical, geometrical, and structural isomers

Aqueous solutions of bile salts, i.e. sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium taurocholate (NaTC), are characterized and evaluated as reversed-phase liquid chromatographic (RPLC) mobile phases. The separation of the ASTM-recommended RPLC test mix in addition to more than 50 other compounds on a C18 column demonstrates the viability of these bile salts as HPLC mobile phases. The Armstrong-Nome theory was applied and found to adequately describe the partitioning behavior of solutes eluted with these bile salts at low surfactant concentrations. The effect of alcohol additives on chromatographic retention and efficiency was also assessed. Not only are the bile salt molecules rigid and chiral, but they form helical micellar aggregates as well. Consequently, many isomeric compounds can be easily resolved with this mobile phase additive. The base-line resolution of some binaphthyl-type enantiomers with a standard C18 column and the bile salt micellar mobile phases is also demonstrated. In addition, these bile salt mobile phases may be preferable to conventional hydroorganic mobile phase systems for the separation of many classes of routine compounds. A brief prospectus on the future utilization of bile salts in liquid chromatography is presented.     

Solubilization of negatively charged DPPC/DPPG liposomes by bile salts

The interactions of the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC) in 0.1 M NaCl (pH 7.4) with membranes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) and mixtures of DPPC and DPPG at molar ratios of 3:1 and 1:1 were studied by means of high-sensitivity isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and differential scanning calorimetry (DSC). The partition coefficients and the transfer enthalpies for the incorporation of bile salt molecules into the phospholipid membranes were determined by ITC. The vesicle-to-micelle transition was investigated by ITC, DLS, and DSC. The phase boundaries for the saturation of the vesicles and their complete solubilization established by ITC were in general agreement with DLS data, but systematic differences could be seen due to the difference in detected physical quantities. Electrostatic repulsion effects between the negatively charged bile salt molecules and the negatively charged membrane surfaces are not limiting factors for the vesicle-to-micelle transition. The membrane packing constraints of the phospholipid molecules and the associated spontaneous curvature of the vesicles play the dominant role. DPPG vesicles are transformed by the bile salts into mixed micelles more easily or similarly compared to DPPC vesicles. The saturation of mixed DPPC/DPPG vesicles requires less bile salt, but to induce the solubilization of the liposomes, significantly higher amounts of bile salt are needed compared to the concentrations required for the solubilization of the pure phospholipid systems. The different solubilization behavior of DPPC/DPPG liposomes compared to the pure liposomes could be due to a specific "extraction" of DPPG into the mixed micelles in the coexistence region.     

Cholesterol Monohydrate Dissolution in Bile Salt‐Lecithin Solutions

A modified dissolution rate equation was used to quantitatively investigate the contribution of simple bile salt (BS) micelles and mixed BS‐lecithin (L) micelles to a cholesterol monohydrate (ChM) dissolution. Using a least‐squares technique to assess the relationship between the ChM dissolution rate and BS concentration at a constant L concentration, good curve‐fittings were obtained when the BS monomer concentration was set to equal the critical micellar concentration (CMC). For taurochenodeoxycholate (TCDC), a dihydroxy BS, the resulting values of parameters show that the simple TCDC micelle rate constant (ks) increases, but the mixed TCDC‐L micelle rate constant (kM ) decreases with increasing L concentrations. As for taurocholate (TC), a tri‐hydroxy BS, a ChM dissolution study was conducted over the initial 2 hour period in different TC‐L solutions. A similar curve‐fitting analysis revealed that the simple TC micelle ks is independent of L concentration and is much higher than the kM of mixed TC‐L micelles. Moreover, the outcome of the analysis supports previously reported equilibrium dialysis study results concerning the BS to L ratio of mixed BS‐L micelles. According to the collision theory, the resulting ks and kM values are interpreted.     

Effect of sodium deoxycholate and sodium cholate on DPPC vesicles: A fluorescence anisotropy study with diphenylhexatriene

Effects of two bile salts, namely sodium deoxycholate (NaDC) and sodium cholate (NaC), on DPPC small unilamellar vesicles have been investigated using the steady-state fluorescence anisotropy (r _ss ) of diphenylhexatriene (DPH) as a tool. It was found that the variation of r _ss is sensitive enough to monitor different stages of interaction of bile salts with DPPC vesicles. NaDC induced significant changes in the membrane well below its CMC (6 mM). Even at 4 mM, which is still lower than the CMC, the phospholipids were completely solubilised by the NaDC micelles. The effect of NaC on DPPC vesicles, however, was much less significant, especially in the sub-micellar concentration regime. Being more hydrophilic NaC does not interact with the membrane efficiently. Complete solubilisation of phospholipids took place only when the concentration of NaC was above its CMC (16 mM). The experiments also showed that the bile salt-induced changes of vesicle structure were strongly dependent on the concentration of the bile salt and not on the molar ratio of lipid and bile salt.     

Spin and fluorescence label studies on bile salt micelles

The interior structure of micelles formed by bile salts, which differ in the number and location of the hydroxyl groups attached to the steroid nucleus, was studied by the spin label and fluorescence label methods. The results show that the interior structure of micelles formed by bile salts possessing two hydroxyl groups is more rigid than that of micelles formed by trihydroxy bile salts regardless of the terminal hydrophilic group. Even in the case of dihydroxy bile salts possessing two hydroxyl groups in the same location, the interior structures of their micelles are different from each other depending on the orientation of their hydroxyl groups. It is considered that hydroxyl groups as well as the terminal hydrophilic group play an important role in the micellar formation of bile salts.     

Phase behavior and rheological properties of lecithin/TTAOH/H<Subscript>2</Subscript>O mixtures

The phase behavior, structures, and rheological properties of lecithin/tetradecyltrimethylammonium hydroxide (TTAOH)/water system were investigated by cryogenic transmission electron microscopy (cryo-TEM), polarization optical microscope, ¹H and ³¹P nuclear magnetic resonance (NMR) spectra, surface tension, and rheological measurements. With the variation of mixing molar ratios and concentrations of lecithin and TTAOH, the system exhibits the phase transition from micelles (L₁ phase) to vesicles (L_α phase) through a phase separation region. The rod-like micelles, uni- and multilamellar vesicles were determined by means of cryo-TEM observations. The surface tension and rheological measurements were performed to follow the phase transition. The samples of L₁ phase region behave as Newton fluids at low concentration of lecithin. With the increase of the lecithin concentration, a shear-thinning L₁ phase at the shearing rate 100 s⁻¹ was found. The samples of \textL_a {{\text{L}}_{\alpha }} phase region show viscoelastic properties of the typical vesicles. The interactions between lecithin and TTAOH were monitored by ¹H and ³¹P NMR spectra. These results could contribute towards the understanding of the basic function of lecithin in biological membranes and membranous organelles.     

Activities and viscosities of aqueous bile acid salt-lecithin solutions

Isopiestic and viscosity measurements on aqueous sodium cholate-lecithin solutions are reported at high concentrations which include the physiological region as a lower limit. At low cholate concentration, the addition of lecithin increases the activity of water, consistent with earlier studies showing increases of micellar weights. At higher cholate concentrations, the activity of water is independent of lecithin concentrations. Studies of binary solutions of bile acid salts and detergents agree very closely with earlier work.     

Simple model for the growth behaviour of mixed lecithin-bile salt micelles

Mixed lecithin-bile salt micelles are known to have a cylindrical or worm-like structure. We investigated their shape, length, flexibility and cross-sectional structure using small-angle neutron scattering (SANS). A broad range of sample compositions was studied varying both the total amphiphile concentration and the molar ratio of bile salt (sodium taurochenodeoxycholate, NaTCDC) to lecithin (egg yolk phosphatidylcholine, EYL). The length of the micelles was quantitatively linked to the micellar composition by introducing a simple model. The model takes into account the partitioning of lecithin and bile salt between the bulk, cylindrical parts and the end caps of the micelles. The model also sheds light on the organization of the micelles, both in their cylindrical regions and end caps.     

Study of biologically relevant physical-chemical properties of bile salts by reverse-phase liquid chromatography

Summary The study of chromatographic behaviour of bile salts with reverse-phase HPLC columns (C18 and C8) and buffered water — methanol eluent systems indicated that the octanol-water partition coefficient and the micellar cholesterol-solubilizing capacity are linearly correlated with the chromatographic mobilities if eluents with pH>7 and 0.154 M saline concentration are used (correlation coefficients 0.98 and 0.86 respectively). The critical micellar concentration was poorly correlated with the chromatographic mobility under the various conditions tested. The pKa values of bile salts were determined by the changes of mobility with the pH of eluent. Values in the range 4.92–5.06 and 4.27–4.47 were found respectively for unconjugated and glycineconjugated bile salt, in agreement with previous estimates. Values in the range 0.74–1.02 represent the first direct evaluation for taurine-conjugates.     

Micellar electrokinetic chromatography of tri aza aromatic ligand compounds of iron (II): influence of bile salt type on enantiomeric separation.

Micellar electrokinetic chromatography is used with a variety of bile salt micelles to separate the enantiomers of bis(8-((pyridine-2-methylene)amino)quinoline)iron(II) hexafluorophosphate, Fe(PMAQ)2(PF6)2; bis(8-((pyridine-2-methylene)amino)lepidine iron(II) hexafluorophosphate, Fe(PMAL)2(PF6)2; and bis(1-(2-pyridinyl)ethylidine)-8-aminoquinoline iron(II) hexafluorophosphate, Fe(PEAQ)2(PF6)2. The influence of ten different bile salts on the resolution of each pair of enantiomers is investigated. Significant changes in resolution are seen depending upon the bile salt used. The dihydroxy bile salts are superior to the trihydroxy bile salts in terms of resolution, and the taurine or glycine conjugated bile salts yield better results than the unconjugated bile salts. Resolution for most enantiomers is maximized in a buffer solution containing 10-15% acetone and employing either taurochenodeoxycholic or glycochenodeoxycholic acid as the bile salt. Evidence for the separation of the corresponding Fe(III) complexes is presented.     

Micellar aggregates and hydrogels from phosphonobile salts

The aggregation properties of novel bile acid analogs-phosphonobile salts (PBS)-have been studied. The critical micellar concentration of 23 and 24-phosphonobile salts were measured using fluorescence and 31P NMR methods. All the ten synthesized phosphonobile salts formed gels at different pH ranges in water. The pH range at which individual PBSs could gelate water was narrow and influenced by the number and conformation of hydroxyl groups. A reversible thermochromic system has been developed (with 23-phosphonodeoxycholate at pH 3.3), which changes color upon gelation. The investigation of the first hydrogels derived from trihydroxy bile acid analogs 1 and 6 was made using fluorescence, 31P NMR, X-ray crystallography, circular dichroism and SEM. The present studies reveal that the gel network consists of a chiral, fibrous structure possessing hydrophobic interiors.     

A Fluorescence Probe Study on the Chiral Microenvironment of Bile Salt Organized Media

Bile salt aggregation in aqueous solution has been an active area of research, with primary emphasis on the physiological role of bile salts in lipid solubilization. More recently, bile salt media has attracted attention as an alternative to conventional micellar reagents for separations and luminescence analysis, especially for applications in chiral separations. In this study, pyrene and chiral molecular l,1'-bi-2-naphthol as fluorescence probe were used to study chiral microenvironment of bile salt organized media.     

Measurement of bilirubin partition coefficients in bile salt micelle/aqueous buffer solutions by micellar electrokinetic chromatography

The partition coefficients for the distribution of bilirubin between aqueous phosphateborate buffer and cholic, taurocholic, taurodeoxycholic, and taurochenodeoxycholic micelles have been measured by micellar electrokinetic chromatography at pH 8.5. Determination of the partition coefficients required that the critical micelle concentration and partial specific volumes be determined for each bile salt. Critical micelle concentrations were slightly higher for the trihydroxy bile salts. Partial specific volumes of the bile salt micelles differed very little from each other, and for each bile salt they were constant over the concentration range studied, which was typically from slightly above the critical micelle concentration to 35 mM. Capacity factors were corrected for the effects of applied voltage by extrapolation of the capacity factor to zero applied volts. The free solution mobility of bilirubin, determined in the absence of bile salt, was also corrected for the effects of applied voltage. Plots of extrapolated capacity factor versus phase ratio yield the partition coefficient as the slope of a linear fit to the data. Partition coefficients for bilirubin were significantly higher for dihydroxy bile salts than for trihydroxy bile salts.     

Bile salt-induced disintegration of egg phosphatidylcholine liposomes: a kinetic study based on turbidity changes

The disintegration kinetics of egg phosphatidylcholine small unilamellar liposomes in various bile salts (nine species) were investigated by monitoring turbidity changes with a stopped-flow apparatus. The pseudo-first-order rate constants obtained as a function of bile salt concentration (up to 25 mM) were analyzed based on a two-step model in which a penetration-saturation step of bile salt into the bilayer and a lamellar-micellar transition step were assumed for the disintegration mechanism of the bilayer. The order of the rate of the penetration-saturation step, which is assumed to be a measure of the disintegration ability, was as follows: SCDOC greater than SDOC greater than STCDOC greater than STDOC greater than STC greater than SC greater than SGCDOC greater than SGDOC greater than SGC. The results indicated that (1) the dihydroxy bile salts have a greater disintegration ability than the corresponding trihydroxy bile salts, (2) the chenodeoxy bile salts have a greater ability than the corresponding deoxy-bile salts regardless of non-conjugated or conjugated form, (3) the taurine conjugates always have a greater ability than the glycine conjugates. The penetration-saturation rate of the bile salts against the lipid bilayer depends considerably on the chemical nature of each bile salt, varying by a factor of about 10(5). In the conjugated bile salts alone, they were in a narrower range of a factor of 10(3). The physical integrity of liposomes can hardly be maintained in the bile salt-rich intestinal tract but the resulting mixed micelles may contribute substantially to solubilization and enhanced delivery of drugs.     

Sodium and Calcium Laurylamidomethylsulfate: Aqueous Micellar Phases, Their Properties, and a Precipitate of Vesicles

A new anionic surfactant (M-LAMS) that is capable of forming intermolecular hydrogen bonds was investigated. Inverse solubilities of Na and Ca salts were found. Critical micelle concentration and aggregation behavior were determined by surface tension, light scattering, electric birefringence, and SANS measurements. It is found that the Na salt forms globular micelles while the Ca salt forms rodlike micelles. The phase behavior of the micellar solutions with increasing cosurfactant concentration was also studied. It is observed that 100 mM Na-LAMS solutions in the presence of 100 mM CaCl(2) undergo several phase transformations with increasing n-hexanol concentration. We found not only the expected micellar L(1) phase and a lamellar phase at concentrations quite low for this kind of system, but also a novel phase: At a cosurfactant/surfactant ratio x(C) of 1.2 a white precipitate is formed at the bottom of the sample. With increasing ratio x(C) the precipitate dissolves into a liquid crystalline L(alpha) phase that at x(C)=3.2 is transformed into an L(3) or sponge phase. Investigation by FF-TEM, light microscopy, and SANS shows that the precipitate consists of agglomerated polydisperse multilamellar vesicles. The vesicles consist of densely packed bilayers that contain little water. The bilayer thickness is about 20 ? and independent of its composition whereas the interlamellar distance is strikingly linked to concentrations of cosurfactant (surfactant/cosurfactant ratio) and electrolyte. With increasing cosurfactant content, the bilayers become less rigid and resulting thermal undulations force the membranes apart and weaken their interactions until a common L(alpha) phase is formed. This transition is an example of a bonding-nonbonding transition of membranes. Copyright 2001 Academic Press.     

Imaging Cholesterol Vesicles Aggregation in Bile Models

Investigation of aggregation of cholesterol vesicles is essential for the understanding of gallstones prior to the appearance of cholesterol monohydrate crystals. In this article, according to existing data based on various microscopic techniques and present data on various microstructural pathways leading to cholesterol crystal formation in bile models, we considered the bile models composed of cholesterol, bile salts and lecithin and distilled water and employed light polarization microscopy and microelectrophoresis apparatus. Concentrated isotropic solutions of bile models were diluted to induce cholesterol supersaturation and start an evolution of microstructures, leading to cholesterol crystallization.     

Self-assembly in aqueous bile salt solutions

The salts of bile acids (“bile salts”) self-assemble in aqueous solution, similar to classical amphiphiles. The micellization is not only driven by the hydrophobic effect, but also hydrogen binding. Moreover, instead of a small, hydrophilic head and a flexible, hydrophobic tail, bile salts are rigid, almost flat molecules with weakly separated hydrophobic and hydrophilic faces. This results in a complex self-assembly behaviour with very distinct aggregate properties. Some characteristics resemble the behaviour of classical amphiphiles, while others are very different and reminiscent of other classes of molecules, for example low-molecular weight gelators or chromonic materials. We review the peculiar properties of bile salt aggregates, concentrating on general trends rather than specific values and comparing them to classical amphiphiles.