Mixed Langmuir monolayers of cholesterol and `essential' fatty acids

Langmuir monolayers of cholesterol and various fatty acids, such as stearic, oleic, linoleic, α-linolenic, and arachidonic acids, spread at the air/water interface are investigated. The system of cholesterol and stearic acid is found to be immiscible, with only one collapse, occurring at the same surface pressure for all composition range. However, surface pressure (π) – area (A) isotherms of cholesterol/unsaturated fatty acids show a characteristic course with two collapse states. The pressure of the first collapse varies with the proportion of the components in the mixture, while the second collapse, occurring at the surface pressure characteristic of cholesterol alone, is independent of mole fraction of the investigated fatty acid. The application of the surface phase rule indicates that the unsaturated fatty acids/cholesterol mixtures are miscible up to the surface pressure corresponding to the first collapse. Negative values of the excess free energy of mixing in all composition ranges prove that the mixtures are stable. The interactions existing in mixtures of cholesterol and unsaturated fatty acids possessing even numbers of double bonds are strongest in the lower region of fatty acid proportion, and the results are consistent with the minimum values of the excess free energy of mixing, indicating the most stable mixtures. For cholesterol and unsaturated fatty acids with odd numbers of double bonds the behavior is different, and the strongest interactions occur in both low and high regions of mole fraction of an acid. Received: 2 May 2000 Accepted: 26 October 2000     

Thermodynamic characteristics of mixed monolayers of amphotericin B and cholesterol

Surface-pressure (Pi) and surface-area isotherms as a function of surface area were measured for monolayers of amphotericin B (AmB) and cholesterol mixtures at the air/water interface at 10, 20, and 30 degrees C. When chloroform/methanol was used as a spreading solvent, the Pi-A isotherms of the mixed monolayers exhibited characteristic transitions from the gas to liquid-expanded, then liquid-condensed, and finally the solid state. The expanding effect in monolayers was accompanied by a large Pi-A hysteresis and a positive excess of free energy of mixing at high Pi. At low Pi, a condensing effect was observed with the most significant deviation from ideality occurring at a mole fraction of AmB (XAmB) of 0.67. Free energy calculations revealed a condensing effect at low Pi and an expanding effect at high Pi except at 30 degrees C, where a condensing effect was observed for XAmB around 0.33. In contrast, when 2-propanol/water was used as spreading solvent, the mixed monolayers at 20 degrees C exhibited Pi-A isotherms which obey van der Waals equation of state, with no visible transitions, low hysteresis, a condensing effect, and a negative free energy of mixing. The most stable monolayers were produced from mixtures of AmB and cholesterol with a 2:1 stoichiometry.     

The action of polysilicic acid on mixed monolayers of cholesterol and lecithins

Silicic acid produces marked expansion in dipalmitoyl lecithin films and in mixed cholesterol-dipalmitoyl lecithin films with more than 50 % phospholipid. In the presence of silicic acid, these films no longer exhibit a transition region between the liquid-expanded and liquid-condensed states. Mixed cholesterol-dilauroyl lecithin.films are also expanded by silicic acid. In the absence of silicic acid, the addition of cholesterol produces greater condensation in dilauroyl lecithin films than in films of dipalmitoyl lecithin, but the reverse is the case if the substrate contains silicic acid. These results have been interpreted in terms of a possible electrostatic interaction between negatively charged dissociated silicate ions and the positively charged trimethyl ammonium group of the choline fragment.     

A study of the miscibility of bile components in mixed monolayers at the air-liquid interface I. Cholesterol,lecithin, and lithocholic acid

Experimental isotherms, obtained by compressing monomolecular layers formed by a number of compounds involved in gallstone formation (cholesterol, L-α-phosphatidylcholine, and lithocholic acid), are studied. Mixed monolayers consisting of pairs of these substances are also analyzed. The additivity rule for molecular areas is used for characterizing the possible interactions between such components. Applying the phase rule to the surface systems studied in conditions of extended/condensed liquid equilibrium allows the miscibility of the components in both phases to be determined. Some hypotheses are given regarding the kind of molecular rearrangements that take place when each system suffers a phase transformation. The general conclusion reached is that, at the experimental conditions used (pH=6.00 and T =298 K), significant interactions occur between the molecules of the compounds studied.     

Lupane type triterpenes as structuring elements in the monolayers and films of lecithin and fullerene derivatives

Using compression and wetting isotherm analysis, it was shown that lupane triterpenes (betulinol, betulinol diacetate, betulinic acid) change crucially the state of monolayers and films of C₆₀ fullerene, nitroxide malonate C₆₀ methanofullerene, and lecithin and also the films of their mixtures at the water—air interface. The structuring action of triterpenes in the presence of lecithin and C₆₀ fullerene at high triterpene contents in the film gives rise to uniaxially oriented films similar to thin films of triterpenes (atomic force microscopy data). The formation of mixed bis-nitroxide malonate methanofullerene—triterpene films with excess of the latter affords structures shaped like crater-like bowls. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1369–1378, July, 2008.     

Organization of beta-cyclodextrin under pure cholesterol, DMPC, or DMPG and mixed cholesterol/phospholipid monolayers

The complexation of beta-cyclodextrin with monolayers of cholesterol, DMPC, DMPG, and mixtures of those lipids has been studied using Brewster microscopy, PMIRRAS, and ab initio calculations. An oriented channel-like structure of beta-cyclodextrin, perpendicular to the air/water interface, was observed when some cholesterol molecules were present at the interface. This channel structure formation is the first step in the cholesterol dissolution in the subphase. With pure DMPC and DMPG monolayers, weaker, less organized complexes are formed, but they disappear almost completely at high surface pressure, and only a small amount of phospholipid is dissolved in the subphase.     

Electron transfer studies on cholesterol LB films assembled on thiophenol and 2-naphthalenethiol self-assembled monolayers

We have formed the cholesterol monolayer and multilayer LB films on the self-assembled monolayers of 2-naphthalenethiol (2-NT) and thiophenol (TP) and studied the electrochemical barrier properties of these composite films using cyclic voltammetry and electrochemical impedance spectroscopy. We have also characterized the cholesterol monolayer film using grazing angle FTIR, scanning tunneling microscopy (STM) and atomic force microscopy (AFM). Cholesterol has a long hydrophobic steroid chain, which makes it a suitable candidate to assemble on the hydrophobic surfaces. We find that the highly hydrophobic surface formed by the self-assembled monolayers (SAM) of 2-NT and TP act as effective platforms for the fabrication of cholesterol monolayer and multilayer films. The STM studies show that the cholesterol monolayer films on 2-NT form striped patterns with a separation of 1.0 nm between them. The area per cholesterol molecule is observed to be 0.64 nm2 with a tilt angle of about 28.96 degrees from the surface normal. The electrochemical studies show a large increase in charge transfer resistance and lowering of interfacial capacitance due to the formation of the LB film of cholesterol. We have compared the behavior of this system with that of cholesterol monolayer and multilayers formed on the self-assembled monolayer of thiophenol.     

Simultaneous microanalysis of bile acids and cholesterol in bile by glass capillary column gas chromatography

Simultaneous quantitative microanalysis of bile acids and cholesterol was carried out by enzymatic hydrolysis, the formation of the ethyl ester dimethylethylsilyl ether derivatives and subsequent analysis by glass capillary gas chromatography. A complete separation and satisfactory recovery of cholesterol and the five major bile acids commonly occurring in human and hamster bile were obtained. The method is applicable to individual small animal models such as hamster from which only a small amount of bile is available.     

Glycoprotein adsorption into bilirubin/cholesterol mixed monolayers at the air-water interface

The adsorption of α₁-acid glycoprotein into bilirubin/cholesterol mixed monolayers with various component molar ratios is investigated using surface pressure-area (π-A) isotherms and (dπ/dA)-A curves. The results showed that the surface area per molecule increased after the adsorption/insertion of glycoprotein molecules into the monolayers. The compressibility of mixed monolayers increased as a result of hydrogen bonding between bilirubin and glycoprotein molecules, while the interactions between bilirubin and cholesterol are weakened. The adsorption of glycoprotein into a monolayer induced changes in molecular surface area depending on the surface pressure and molar fraction of bilirubin. The transmission electron microscopy of mixed monolayers confirmed the insertion of glycoprotein particles of spherical shape with an average diameter of about 80 nm into the monolayer. The text was submitted by the authors in English.     

Interfacial properties of Pluronics and the interactions between Pluronics and cholesterol/DPPC mixed monolayers

Pluronics are triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) with wide range of hydrophilic-lipophilic balance. In order to investigate the relationship between the chemical structures of Pluronics and the interfacial properties at the air-water interface by monolayer techniques, Pluronics L61, P65, F68, P84, P123, L35, and P105 were selected. Since cholesterol influenced substantially the molecular packing stage and the characteristics of cell membranes, the interactions between Pluronics and model cell membranes in the absence and presence of cholesterol were compared. The results of pi-A isotherms and surface elasticities of Pluronic monolayers indicated that the first and second transition like stage were mainly affected by the numbers of EO and PO monomers, respectively. Pluronics with higher hydrophobicities demonstrated larger surface activities and penetration abilities to dipalmitoylphosphatidylcholine (DPPC) monolayers, which might be due to hydrophobic interactions and van der Waals forces. In the presence of cholesterol, hydrogen bonding effects was supposed to exist between the 3beta-hydroxy group of cholesterol and ether oxygen of PEO chains, which led Pluronic F68, with the longest PEO chain herein, to exhibit significantly higher penetration ability. Our findings proposed a theoretical basis for selection of optimized drug carriers and the starting point for further investigations.     

Adsorption of oil into surfactant monolayers and structure of mixed surfactant+oil films

We describe a tensiometric method for determining the adsorption isotherm of an oil on a surfactant monolayer adsorbed at the air–water surface. The method involves measuring the surface pressure of oil, π_oil, as a function of its activity, a_oil, varied by changing the relative vapour pressure. We compare the isotherm of dodecane adsorption onto a C₁₂E₅ monolayer determined in this way with that measured directly using neutron reflectivity. The agreement between the two allows us to conclude that, at least for this system, addition of oil does not result in a change in the chemical potential of the surfactant. Structural analysis of the dodecane+C₁₂E₅ mixed film has been performed with neutron reflectivity using two contrasts. In one, only the surfactant chain region is highlighted, whilst in the other only the oil film is visible. We document, for the first time, changes in thickness and packing density of both the oil and surfactant chains in the mixed surfactant+oil layer upon increasing oil content. For a diverse range of other oil+surfactant systems, we have determined the initial (S_i) and equilibrium (S_e) spreading coefficients of oil by measuring π_oil following addition of liquid oil (at unit activity) to surfactant solution surfaces. Those systems in which S_e is close to zero display a repulsive van der Waals component of the disjoining pressure-oil film thickness isotherm, whilst with toluene as oil the calculated isotherm is attractive, consistent with non-spreading observed for this oil.     

Polypeptides mixed monolayers: collapse mechanism

The mechanism of the collapse process of monolayers of poly-L-alanine and of its mixtures with poly--methyl-L-glutamate was studied at the water/air interface at temperatures of 15°, 20°, 25°, and 30 °C.From measurements of the collapse surface pressure as a function of molar ratios and from the determination of the collapse kinetics, as well as from ellipsometrical measurements of the thickness of the film, the complete solubility of the components, even in the collapsed phase, was deduced.Furthermore, activation energies and values ofG*,H*, andS* in relationship to the kinetics of this process were deduced; it was shown that this process is constituted of a first phase of nucleation and of a second phase of growth both for the poly-L-alanine alone and for its mixtures with poly--methyl-L-glutamate.     

Retention and separation studies of cholesterol and bile acids using thermostated thin-layer chromatography.

The influence of temperature on retention and separation of cholesterol and bile acids, using reversed-phase thin-layer chromatography, was studied. As mobile phases methanol-water mixtures of various compositions were used. Chromatographic experiments were performed using vapor-saturated chambers at temperatures ranging from 5 to 60 degrees C. A linear relationship between R(M) values and temperature (1/T) as well as mobile phase composition was observed. The elution order of steroids under the conditions investigated was discussed. Each chromatogram was evaluated using simple optimization parameters and the best chromatographic conditions for the separation of multicomponent samples were chosen.     

Thermodynamic behavior and relaxation processes of mixed DPPC/cholesterol monolayers at the air/water interface

This study investigated the thermodynamic behavior and relaxation processes of mixed DPPC/cholesterol monolayers at the air/water interface at 37°C. Surface pressure–area isotherms and relaxation curves for the mixed monolayers were obtained by using a computer-controlled film balance. In the thermodynamic analysis of the mixed monolayers, the areas of monolayers exhibited negative deviations from the ideal values at all compositions for lower surface pressures. However, at higher surface pressures, distinctively positive deviations from ideality were observed at lower DPPC contents. Excess free energies of mixing had been calculated and the most stable state of the mixed monolayer with x_DPPC=0.5 or 0.6 was found. Moreover, the relaxation kinetics of the mixed monolayers was investigated by measuring the surface area as a function of time at a constant surface pressure of 40 mN m⁻¹. It was shown that the relaxation processes could be described by the models considering nucleation and growth mechanisms.     

Penetration of beta-lactoglobulin into monoglyceride monolayers. dynamics, interactions, and topography of mixed films

In this work we have analyzed the penetration of betalactoglobulin into a monoglyceride monolayer (monopalmitin or monoolein) spread at the air-water interface and its effects on the structural, dilatational, and topographical characteristics of mixed films. Dynamic tensiometry, surface film balance, Brewster angle microscopy (BAM), and surface dilatational rheology have been used, maintaining the temperature constant at 20 degrees C and the pH and ionic strength at 7 and 0.05 M, respectively. The initial surface pressure (mN/m) of the spread monoglyceride monolayer (pii(MONOGLYCERIDE)) at 10, 20, and the collapse point is the variable studied. Beta-lactoglobulin can penetrate into a spread monoglyceride monolayer at every surface pressure. The penetration of beta-lactoglobulin into the monoglyceride monolayer with a more condensed structure, at the collapse point of the monoglyceride, requires monoglyceride molecular loss by collapse and/or desorption. However, the structural, topographical, and dilatational characteristics of monoglyceride penetrated by beta-lactoglobulin mixed monolayers are essentially dominated by the presence of monoglyceride (either monopalmitin or monoolein) in the mixed film. In fact, monoglyceride molecules have the capacity to re-enter the monolayer after expansion and recompression of the mixed monolayer. Thus, monoglyceride molecular loss by collapse and/or desorption is reversible. The topography of the monolayer under dynamic conditions corroborates these conclusions.     

Stereoselective interactions of a specialized antibody with cholesterol and epicholesterol monolayers

The stereoselective recognition by monoclonal antibodies of two-dimensional monolayers of cholesterol spread at the air-water interface is presented. Using immunofluorescence, we show that one antibody, raised and selected against crystals of cholesterol monohydrate, specifically recognizes monolayers of cholesterol, but not monolayers of epicholesterol--its epimeric form. This demonstrates that stereoselective recognition also applies to protein-surface interactions.