Miscibility and Langmuir Studies of the Interaction of E2 (279-298) Peptide Sequence of Hepatitis G Virus/GB Virus-C with Dipalmitoylphosphatidyl Choline and Dimiristoylphosphatidyl Choline Phospholipids.

Mixed monolayers of E2(279-298), a synthetic peptide belonging to the structural protein E2 of the GB virus C (GBV-C), formerly know as hepatitis G virus (HGV), and the phospholipids dipalmitoylphosphatidyl choline (DPPC) and dimiristoylphosphatidyl choline (DMPC),which differ in acyl chains length, were obtained at the A/W interface (monolayers of extension) in order to provide new insights on E2/phospholipids interaction. Analysis of the surface pressure-area isotherms, Brewster angle microscopy images, relative thickness, and mean areas per molecule has allowed us to establish the conditions under which the mixed components of the monolayer are miscible or immiscible and know how the level of the E2/phospholipid interaction varies with the composition of the mixed films, the surface pressure, and the hydrocarbon chains length of the phospholipids. The steric hindrance caused by the penetration of the polymer strands into the more or less ordered hydrocarbon chains of the phospholipids was suggested to explain the differences in the peptide interaction with the phospholipids studied. Therefore, the novelty of results obtained with the Langmuir film balance technique, supplemented with BAM images allow us to achieve a deeper understanding of the interaction.

Characterization of poly(2-hydroxyethyl methacrylate) (PHEMA) contact lens using the Langmuir monolayer technique.

The behavior of poly(2-hydroxyethyl methacrylate) (PHEMA) polymer monolayer spread on water was studied under various experimental conditions. The influence of subphase pH and temperature, compression speed, elapsed time from the deposit of the monolayer and the recording of the surface pressure-area (π-A) isotherms, as well as the number of polymer molecules deposited at the air/water surface (surface concentration) was studied. The obtained results show that PHEMA exhibits a very stable monolayer given that it is unaffected by modifications in the majority of these variables. Only the elapsed time between the spreading of the monolayer and the beginning of compression causes a small change in the π-A isotherms that consists in an increase in the area occupied by the film. This is attributed to the greater unfolding with time of the polymer's monomers at the air/water interface. The plateau that appears on π-A curves of the PHEMA monolayer is attributed to the reorientation of their hydroxyethyl polar groups through their C-O-C bonds, as well as to the reorientation of the ethylene (CH(2)) groups that link the monomers, which provokes a folding of the polymer's chains causing an accordion configuration. The existence of this structure is confirmed by the presence of numerous noise peaks in the relative thickness versus time curve corresponding to this region. In the same fashion, the images observed from Brewster angle microscopy (BAM) reveal the existence of light-dark "bands" relative to the different regions of this particular structure.

How to obtain a well-spread monolayer of lysozyme at the air/water interfaces.

The purpose of this study is to define the conditions required to obtain a complete spreading of the lysozyme monolayer at the A/W interface. To this end, using Trurnit's method, the influence of the ionic strength of the substrate, the elapsed time between the spreading of the monolayer and the beginning of its compression, and the number of lysozyme molecules spread at the interface was studied. The results obtained show that the lysozyme spreading is conditioned by the unfolding of amino acid chains which form part of its structure, so that such unfolding is hindered, either because of an excessive accumulation of lysozyme molecules on the substrate surface or because the waiting time necessary to get this unfolding is not long enough, regardless of the number of spread molecules. The complete unfolding of lysozyme, which involves the loss of tertiary and secondary structures, has been obtained in this work under certain conditions: (1) using a substrate with a high saline concentration (NaCl 3-3.5 M) and at a pH value close to the isoelectric point of lysozyme (pI=11) in order to reduce the solubility and the electrical potential of the protein, (2) increasing the elapsed time between the spreading of the protein and the beginning of its compression to 3h in order to get a complete unfolding of lysozyme, which is a very slow process, (3) using an initial spreading area of 2.1m(2)/mg or larger (equivalent to a protein surface concentration of less than 2×10(12) molec/cm(2)) to reduce the accumulation of protein molecules at the surface, thus facilitating their unfolding, and (4) using Trurnit's spreading method, although for the spreading of this protein was found to be insufficiently significant.

Interactions in monolayers: a study of the behavior of poly(methyl methacrylate)-lysozyme mixed films from surface pressure-area and ellipsometric measurements.

Mixed monolayers of PMMA-lysozyme show the existence of negative deviations from the additivity of the molecular areas (A(m)) when the composition of polymer mixtures is less than X(PMMA) 0.6, regardless of the surface pressure of the monolayers. The maximum deviation occurs in the mixed monolayer with composition X(PMMA) 0.25, which is attributed to the formation of a complex consisting of one polymer molecule and three protein molecules (1:3 stoichiometry), stabilized by hydrogen bonds between the NH groups of the protein and the CO groups of the polymer as well as by van der Waals attractive forces between the hydrocarbon chains of both components. When the relative proportion of the components in the mixed films significantly differs from the value corresponding to the stoichiometry of the complex (as in mixtures with X(PMMA) > 0.6), this complex cannot be formed, causing an immiscible system where the values of the experimental molecular areas coincide with those corresponding to ideal behavior. Measurements of monolayer thickness and BAM images allow confirmation on the microscopic level of the structural characteristics deduced from the π-A isotherms.

Approach to knowledge of the interaction between the constituents of contact lenses and ocular tears: mixed monolayers of poly(methyl methacrylate) and dipalmitoyl phosphatidyl choline.

Mixed monolayers of poly(methyl methacrylate) (PMMA), the main component of hard contact lenses, and dipalmitoyl phosphatidyl choline (DPPC), a characteristic phospholipidic constituent of ocular tear films, were selected as an in vitro model in order to observe the behavior of contact lenses on the eye. Using Langmuir monolayer and Brewster angle microscopy (BAM) techniques, the interaction between both components was analyzed from the data of surface pressure-area isotherms, compressional modulus-surface pressure, and relative film thickness versus time elapsed from the beginning of compression, together with BAM images. Regardless of the surface pressure at which the molecular/monomer areas (A(m)) were recorded, the A(m) mole fractions of PMMA (X(PMMA)) plots show that the experimental results match the theoretical values calculated from additivity rule A(m) = X(PMMA)A(PMMA) + X(DPPC)A(DPPC). The application of the Crisp phase rule to the phase diagram of the PMMA-DPPC system can explain the existence of a mixed monolayer made up of miscible components with ideal behavior at surface pressures below 25 mN/m. However, at very high surface pressures, when collapse is reached (at 60 mN/m), the single collapsed components are segregated into two independent phases. These results allows us to argue that PMMA hard contact lenses in the eye do not alter the structural characteristics of the phospholipid (DPPC) in tears.

Interactions between polymers and lipid monolayers at the air/water interface: surface behavior of poly(methyl methacrylate)-cholesterol mixed films.

The behavior of mixed monolayers of cholesterol and poly(methyl methacrylate) (PMMA) with molecular weights of M(w) = 120,000 g/mol and M(w) = 15,000 g/mol was investigated at the air/water interface using Langmuir and Brewster angle microscopy techniques. From the data of surface pressure (pi)-area (A) isotherms, compressional modulus-surface pressure (C(s)(-1)-pi) curves, and film thickness, complemented with Brewster angle microscopy images, the interaction between the components was analyzed. Regardless of the surface pressure (pi = 10, 20, or 30 mN/m) at which the mean molecular/monomer areas (Am) were calculated, the Am-mole fraction plots (corresponding to X(PMMA) = 0.1, 0.3, 0.5, 0.7, and 0.9) show that all the experimental points obtained are placed on the theoretical straight line calculated according to the additivity rule. This fact, together with the existence of two collapses in the mixed monolayers and with the fact that the surface pressure of the liquid-expanded LE-L'E phase transition of PMMA does not change with the monolayer composition, demonstrates the immiscibility of the film components at the interface. The application of the Crisp phase rule to the phase diagram of PMMA-cholesterol mixed monolayers helps to explain the existence of a biphasic system, regardless of their composition and surface pressure. Besides, Brewster angle microscopy (BAM) images showed the existence of heterogeneous cholesterol domains with high reflectivity immersed in a homogeneous polymer separate phase with low reflectivity.

Interactions between membrane sterols and phospholipids in model mammalian and fungi cellular membranes--a Langmuir monolayer study.

This paper is aimed at investigating sterol/phospholipid interactions in the exact proportion that occurs in fungi/mammalian cells. We have performed a thorough analysis of surface pressure (pi)-area (A) isotherms with the Langmuir monolayer technique, complemented with Brewster angle microscopy (BAM) images. The following mixtures were analysed: cholesterol (Chol)-dipalmitoyl phosphatidylcholine (DPPC), Chol-dioleoyl phosphatidylcholine (DOPC), ergosterol (Erg)-DPPC, and Erg-DOPC. For each system, two different concentrations of the sterols were used, 13 and 30%, corresponding to the range of concentration found in various natural membranes. The obtained results show the existence of attractive interactions between phospholipids and cholesterol. Mixtures with ergosterol behave quite differently, i.e. either the interactions are repulsive (mixtures with DPPC) or the system is ideal (mixtures with DOPC). The obtained results have implications in the polyene antibiotics mode of action, i.e. the polyenes may interact easier with ergosterol, present in fungi cells, as compared to cholesterol--the main sterol of the mammalian cellular membranes.

Study of adsorption and penetration of E2(279-298) peptide into Langmuir phospholipid monolayers.

The peptide corresponding to the sequence (279-298) of the Hepatitis G virus (HGV/GBV-C) E2 protein was synthesized, and surface activity measurements, pi-A compression isotherms, and penetration of E2(279-298) into phospholipid monolayers spread at the air-water interface were carried out on water and phosphate buffer subphases. The results obtained indicated that the pure E2(279-298) Langmuir monolayer exhibited a looser packing on saline-buffered than on pure water subphase and suggest that the increase in subphase ionic strength stabilizes the peptide monolayer. To better understand the topography of the monolayer, Brewster angle microscopy (BAM) images of pure peptide monolayers were obtained. Penetration of the peptide into the pure lipid monolayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) and into mixtures of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) at various initial surface pressures was investigated to determine the ability of these lipid monolayers to host the peptide. The higher penetration of peptide into phospholipids is attained when the monolayers are in the liquid expanded state, and the greater interaction is observed with DMPC. Furthermore, the penetration of the peptide dissolved in the subphase into these various lipid monolayers was investigated to understand the interactions between the peptide and the lipid at the air-water interface. The results obtained showed that the lipid acyl chain length is an important parameter to be taken into consideration in the study of peptide-lipid interactions.

The influence of subphase temperature on miltefosine-cholesterol mixed monolayers.

Effects of the subphase temperature on the surface pressure (pi)-area (A) isotherms of mixed monolayers of miltefosine (hexadecylphosphocholine), a potential anticancer drug, and cholesterol were investigated at the air/water interface, which were supplemented with Brewster angle microscopy (BAM) observations. Comparison of the collapse pressure values, mean molecular areas, excess areas and excess free energy of mixing between the mixed monolayer at various molar ratios and the pure component monolayers showed that, regardless of the subphase temperature, the investigated miltefosine-cholesterol system is much more stable than that the pure component monolayers, suggesting strong attractive interactions between miltefosine and cholesterol in mixed monolayers. As a consequence, it was postulated that stable "complexes" of the two components could form at the interface, for which stoichiometry may vary with the subphase temperature. Such "surface complexes" should be responsible for the contraction of the mean molecular area and thus the high stability of the mixed monolayer.

Influence of the saturation chain and head group charge of phospholipids in the interaction of hepatitis G virus synthetic peptides.

Using the Langmuir technique, we have studied the properties at the air/water interface and the interaction of the hepatitis G virus synthetic peptide E1(53-66) and its palmitoyl derivative with membrane phospholipids. These phospholipids had different characteristics referring to the net charge and saturation of the acyl chain. The palmitoyl derivative was more stable at the air/water interface and in the kinetic at constant area measurements showed higher incorporation to the monolayer. The interaction was higher for saturated phospholipids and those with a negative net charge. When the peptides were in the subphase, they produced changes in the miscibility of mixed monolayers composed of DPPC/DPPG or DOPC/DOPG. It can be deduced from the results obtained that electrostatic interactions play a major role, but when the peptide is derivatized with the palmitoyl chain, hydrophobic interactions are added to the former ones. The interaction is also influenced by the saturation of the acyl chain.

Surface behavior of oleoyl palmitoyl phosphatidyl ethanolamine (OPPE) and the characteristics of mixed OPPE-miltefosine monolayers.

Langmuir monolayers of oleoyl palmitoyl phosphatidyl ethanolamine (OPPE) were investigated at the air/water interface by means of surface pressure (pi)-area (A) isotherms complemented with Brewster angle microscopy images upon film compression/expansion. The characteristic phase transition appearing in the course of pi/A isotherms was attributed to the coexistence of two liquid-expanded phases of different molecular ordering. The interactions between OPPE and hexadecylphosphocholine (miltefosine) were studied at different subphase pHs (2, 6, and 10) at 20 degrees C and analyzed with mean molecular area (A12)-, excess area of mixing (Aexc)-, and excess free energy of mixing (DeltaGexc)-composition plots. The obtained results indicate that at pH 10, where both OPPE and miltefosine polar groups are negatively charged, attractive interactions are observed (reflected by negative deviations from ideality), contrary to expectation. This peculiar behavior is explained as being due both to water molecules, which surround negatively charged polar groups and increase the distance between them, weakening in this way the electrostatic repulsion forces; and to positively charged counterions present in the diffuse double layer, neutralizing their charge. In this way, the van der Waals attraction forces between hydrocarbon tails of both molecules predominate and are responsible for the observed negative deviations from ideal behavior. Similar explanations are given for the observed negative deviations at pH 2 where both polar groups are positively charged. At pH 6, the observed negative deviations at low surface pressures and positive deviations at high pressures are interpreted as being due to a change in orientation of polar groups upon monolayer compression.

Semifluorinated chains in 2D-(perfluorododecyl)-alkanes at the air/water interface.

Langmuir monolayers of a homologous series of perfluorododecyl-n-alkanes (general formula F(CF2)12(CH2)nH, abbreviated as F12Hn, where n = 6-20) are investigated through isotherms of surface pressure (pi) and electric surface potential (DeltaV) versus area (A) and quantitative Brewster angle microscopy. The investigated monolayers are found to be liquid in nature. The negative sign of the measured surface potential evidences the orientation of all the investigated molecules with their perfluorinated parts directed toward the air regardless of the length of the hydrogenated unit. Analysis of the direction of the molecular dipole moment with respect to the main axis indicates that the minimum effective dipole moment is achieved for a molecule oriented at an angle of about 35 degrees to the surface normal. The film thickness was evaluated from the relative intensity measurements. The results suggest that the F12Hn molecules are tilted to the interface in the vicinity of collapse, which is in accordance with the liquid character of their monolayers.

Miltefosine-cholesterol interactions: a monolayer study.

Mixed Langmuir monolayers of miltefosine (hexadecylphosphocholine) and cholesterol have been investigated by recording surface pressure-area (pi-A) isotherms at different subphase pHs (2, 6, and 10) and temperatures (10, 20, 25, and 30 degrees C). The change of both pH and temperature within the investigated range does not modify significantly the behavior of mixed films. The most pronounced effect involves condensation of the miltefosine monolayer by cholesterol, which diminishes in the following order: pH 6 > pH 2 > pH 10. The analyses of pi-A and compressibility modulus dependencies indicate the existence of interactions in the investigated system; at pH 2 and 6, the strongest were found to occur for the mixed film of miltefosine molar fraction (XM) between 0.6 and 0.7 (mean value, 0.66). Such a composition corresponds to the stable complex formation wherein 2 miltefosine molecules and 1 molecule of cholesterol are strongly bound together, mainly by attractive hydrophobic forces between their apolar tails. However, at pH 10 the highest stability occurs for mixtures containing a smaller proportion of miltefosine (XM = 0.5), which means that on alkaline subphases the ability to condense the miltefosine monolayer by cholesterol is less efficient as it requires a higher proportion of cholesterol (1:1 as compared to 1:2 at pH 2 and 6) to attain the maximum stability of the mixed film. The attractive forces between miltefosine and cholesterol are also weaker at pH 10 due to a greater solvatation of the miltefosine polar group. A similar trend is observed on increasing subphase temperature, when monolayers are more expanded.

Orientational changes in dipalmitoyl phosphatidyl glycerol Langmuir monolayers.

Dipalmitoyl phosphatidyl glycerol (DPPG) as Langmuir monolayers at the air/water interface was investigated by means of surface pressure measurements in addition to Brewster angle microscopy (BAM) during film compression/expansion. A characteristic phase transition region appeared in the course of surface pressure-area (pi-A) isotherms for monolayers spread on alkaline water or buffer subphase, while on neutral or acidic water the plateau region was absent. This phase transition region was attributed to the ionization of DPPG monolayer. It has been postulated that the ionization of the phosphatidyl glycerol group leads to its increased solvation, which probably provokes both a change in the orientation of the polar group and its deeper penetration into bulk phase. Film compression along the transition region provokes the dehydration of polar groups and subsequent change of their conformation, thus causing the DPPG molecules to emerge up to the interface. Quantitative Brewster angle microscopy (BAM) measurements revealed that along the liquid-expanded to liquid-condensed phase transition the thickness of the ionized DPPG monolayer increases by 4.2 A as a result of the conformational changes of the ionized polar groups, which tend to emerge from the bulk subphase up to the surface.

Structural and topographical characteristics of dipalmitoyl phosphatidic acid in Langmuir monolayers.

Dipalmitoyl phosphatidic acid (DPPA) monolayers at the air-water interface were studied from surface pressure (Pi)-area (A) isotherms and at the microscopic level with Brewster angle microscopy (BAM) under different conditions of temperature, pH, and ionic strength. BAM images were recorded simultaneously with Pi-A isotherms during the monolayer compression-expansion cycles. DPPA monolayers show a structural polymorphism from the liquid-expanded (LE)-liquid-condensed (LC) transition region at lower surface pressures toward liquid-condensed and solid (S) structures at higher surface pressures. An increase in temperature, pH, or ionic strength provokes an expansion in the monolayer structure. The results obtained from the Pi-A measurements are confirmed by the monolayer topography and relative reflectivity. The measurements of relative reflectivity upon monolayer compression showed an increase in relative monolayer thickness of 1.25 and 3.3 times throughout the full monolayer compression from the liquid-expanded to the liquid-condensed and solid states, respectively.

Desorption of Amphotericin B from Mixed Monolayers with Cholesterol at the Air/Water Interface.

The desorption of amphotericin B (AmB) from mixed monolayers with cholesterol was studied by monitoring temporal changes in the relative area (A(t)/A(0)) as a function of time at constant surface pressures, corresponding to three distinct regions of the AmB isotherm. The loss of AmB molecules from the interface was found to obey the Ter Minassian Saraga model [J. Colloid Interface Sci. 11, 398 (1956)]. In the low surface pressure region the overall process of desorption is controlled by a one-step dissolution (at small times) followed by diffusion. At high pressures, however, the process of dissolution is more complex and consists of two steps that differ in rate. The presence of cholesterol molecules in the monolayer prevents the desorption of AmB molecules as evidenced by the constant desorption values. The analysis of the kinetics parameters confirms our previous findings, based on the analysis of excess functions, that the highest interactions, due to the stable complex formation, appear for the mixture of X(AmB)=0.7. Copyright 2001 Academic Press.

5-Azidoimidacloprid and an acyclic analogue as candidate photoaffinity probes for mammalian and insect nicotinic acetylcholine receptors.

The 5-azido analogue of the major insecticide imidacloprid, 1-(5-azido-6-chloropyridin-3-ylmethyl)-2-nitroiminoimidaz olidine (1), and an acyclic analogue, N-(5-azido-6-chloropyridin-3-ylmethyl)-N'-methyl-N' '-nitroguanidine (2), were prepared in good yields as candidate photoaffinity probes for mammalian and insect nicotinic acetylcholine receptors (nAChRs). The essential intermediate was 5-azido-6-chloropyridin-3-ylmethyl chloride (3) prepared in two ways: from 6-chloro-5-nitronicotinic acid by selective reduction and then diazotization, and from N-(6-chloropyridin-3-ylmethyl)morpholine by an electrophilic azide introduction with lithium diisopropylamide followed by chlorine substitution of morpholine with ethyl chloroformate. Coupling of 3 with 2-nitroiminoimidazolidine gave 1. Conversion of 3 to 2 was achieved in good yields via the hexahydrotriazine intermediate 14. Fortuitously, the azido substituent in 1 and 2 increases the affinity 7-79-fold for rat brain and recombinant alpha4beta2 nAChRs (K(i)s 4.4-60 nM competing with [(3)H](-)-nicotine) while maintaining high potency on both insect nAChRs (Drosophila and Myzus) (K(i)s 1-15 nM competing with [(3)H]imidacloprid). Azidopyridinyl compounds 1 and 2 are therefore candidate photoaffinity probes for characterization of both mammalian and insect receptors.

Molecular organisation of amphotericin B at the air-water interface in the presence of sterols: a monolayer study.

Using the monolayer technique to study the surface behaviour of systems consisting of amphotericin B (AmB) and various sterols, the components were found to interact with each other. The interactions observed are accounted for by postulating that, at low surface pressures, AmB and different sterols form mixed films where the former lies parallel and the latter normal to the air-water interface in such a way that the polar groups in both components establish hydrogen bonds that lead to the formation of an AmB-sterol 'complex' of 2:1 stoichiometry at the interface. At high surface pressures, AmB molecules rearrange themselves normal to the interface; this gives rise to the Van der Waals interactions between non-polar chains of both components that vary with the nature and composition of the system. The occurrence of these hydrophobic interactions prevents the desorption of AmB into the subphase, which is consistent with the positive excess areas of mixing obtained under these surface pressure conditions. Among the four sterols studied, ergosterol exhibits the strongest interaction with AmB and beta-sitosterol the weakest. Cholesterol and stigmasterol show intermediate behaviour.

Surface pressure-area isotherms of mixed cyclosporin-poly(isobutylcyanoacrylate) monolayers spread at the air/water interface.

The pi-A isotherms of mixed monolayers of cyclosporin and poly(isobutylcyanoacrylate) (PIBCA) show that the molecular areas of cyclosporin and PIBCA are additive regardless of the pH of the substrate or the physical state of the monolayers. All these mixed films collapse at the same surface pressure; therefore, application of the two-dimensional phase rule implies that cyclosporin and PIBCA are immiscible at the interface. This conclusion may have important implications with regard to the formulation of PIBCA-cyclosporin nanoparticles for cyclosporin administration, though further research in this direction will require consideration of the role played by the coadjuvants used for PIBCA polymerization during nanoparticle formation.

Spectrofluorimetric study of the binding of 1-anilinonaphthalene-8-sulfonate to bovine serum albumin.

We studied the binding of the fluorescent probe 1-anilinonaphthalene-8- sulfonate to bovine serum albumin by spectrofluorimetric techniques. The binding of this probe to bovine serum albumin was via two types of sites: one with a high affinity constant but relatively fewer in number and the other with a lower affinity constant but greater in number. The number of binding sites of each type of site and their respective association constants varied with the features of the moiety and the temperature. The results indicate that the simplistic interpretation involving a single type of interaction of hydrophobic nature can no longer be supported and that additional involvement of other, nonhydrophobic types of binding should be considered. This thermodynamic study allowed calculation of changes in enthalpy, Gibbs free energy, and entropy values that are consistent with this hypothesis.