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In this work the interaction between human serum albumin (HSA) and a monofluorinated phospholipid, 1-palmitoyl-2-[16-fluoropalmitoyl-phosphatidylcholine] (F-DPPC), was studied by using Langmuir monolayer and Brewster angle microscopy (BAM) techniques. Different amounts of F-DPPC were spread on a previously formed HSA monolayer located at the air/water interface at 25°C and the mixed monolayers thus obtained showed the existence of a liquid expanded-liquid condensed (LE-LC) phase transition (at 14 mN/m), attributed to the pure F-DPPC monolayer, coexisting with a second transition (at 22-24 mN/m) corresponding to the protein conformational change from an unfolded state to another in "loops" configuration. Relative thickness measurements recorded during the compression of the mixed monolayers showed the existence of an "exclusion" surface pressure (π(exc)), above which the protein is squeezed out the interface, but not totally. BAM images reveal that some protein molecules in a packed "loops" configuration remain at the interface at surface pressures higher than the "exclusion" surface pressure. The application of the Defay-Crisp phase rule to the phase diagram of the F-DPPC/HSA system can explain the existence of certain regions of surface pressure in which the mixed monolayer components are miscible, as well as those others that they are immiscible.

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The study of the interaction of a cationic polymer as PEI with phospholipids membranes is of special relevance for gene therapy because the PEI is a potential nonviral vector to transfer DNA in living cells. We used light scattering, zeta potential, and electron transmission microscopy to characterize the interaction between DMPG and DOPC liposomes with PEI as a function of the charge molar ratio, pH, temperature, initial size of the liposomes, and headgroup of the lipids. Unexpectedly, a double charge inversion and two different ranges of PEI-liposome concentrations where an aggregation occurs were found, when the proper pH and initial size of the liposomes were chosen. The interaction is analyzed in terms of the interaction potential proposed by Velegol and Thwar for colloidal particles with a nonuniform surface charge distribution. Results show a remarkable dependence of the stability on pH and the initial size of the liposomes, which explains the low reproducibility of the experiments if no special care is taken in preparing the samples. Comparatively small changes in the pH or in the liposomes size lead to a completely different stability behavior.

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The aim of this study is to deepen the understanding of the behavior of human serum albumin (HSA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed monolayers. For this purpose, different amounts of DPPC were spread at 25°C on the water surface containing a monolayer of HSA. Surface film balance and Brewster angle microscopy techniques have been used to analyze the structural and energetic characteristics (structure, topography, thickness, miscibility and interactions) of these mixtures. HSA/DPPC mixed monolayers exhibit two phase transitions evidenced by two discontinuities in the corresponding π-A isotherms and by two minimum values in the compressional modulus (C(s)(-1))-surface pressure (π) curves. The plot of the molecular areas occupied by the mixed monolayers as function of the mass fraction of DPPC shows the absence of deviations from linearity, a typical behavior for ideal or inmiscible system. This result was confirmed from the values calculated for the free energy of excess (ΔG(exc)), which are practically zero whatever the composition of the mixtures and the surface pressures at which ΔG(exc) values were calculated. In addition, relative thickness values of HSA/DPPC mixed monolayers showed the existence of an exclusion surface pressure (π(exc)), below which the monolayer is composed of a mixture of both components, while above π(exc) the HSA molecules are squeezed out the interface, but not totally. In fact, although in this region DPPC domains predominate at the interface, the existence of protein molecules in a packing "loops" configuration can be observed in BAM images. Moreover, relative thickness measurements confirm this hypothesis.

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The influence of La(3+) on the colloidal stability of liposomes made up by two zwitterionic phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-[16-fluoropalmitoyl-phosphatidylcholine (F-DPPC), in aqueous media has been investigated by dynamic light scattering and electrophoretic mobility. The critical aggregation concentration (c.a.c.) of La(3+) for F-DPPC and DPPC liposomes were experimentally obtained, and the results were compared with theoretical predictions using the Derjaguin-Landau-Verwey-Overbeek theory. In order to evaluate the influence of the state of the bilayer on the stability of liposomes, all experiments were performed at temperatures below and above the chain-melting phase-transition temperature of lipids (transition temperature of lipids). Changes in the size of both types of liposomes and high values of polydispersity in the presence of La(3+) showed that these ions induce aggregation of liposomes at 25 °C and at 60 °C. At 25 °C, when the bilayer of F-DPPC liposomes is interdigited, DPPC liposomes are more resistant to aggregation than the liposomes formed with F-DPPC. However, this difference disappears at 60 °C, when both bilayers have the same conformation. The experimental results also indicate that the c.a.c. is higher at 60 °C than at 25 °C for both types of liposomes. In fact, it has been observed by dynamic light scattering measurements that aggregation of liposomes at 25 °C can be prevented by increasing the solution temperature for La(3+) concentrations near to the c.a.c. Moreover, the behavior of these liposomes in the presence of the ion was studied at temperatures above and below the transition temperature of the phospholipids.

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The surface behavior of two zwitterionic phospholipids: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-[16-fluoropalmitoyl-phosphatidylcholine] (F-DPPC), has been investigated at the air-water interface at the temperature range from 10 to 30 °C. Surface pressure-area isotherms, BAM images and thickness-time curves were obtained for monolayers made from these pure phospholipids and from their mixtures.The comparative study of the behavior of both phospholipid monolayers with temperature showed some differences as the disappearance of the liquid expanded (LE)-liquid condensed (LC) phase transition at low temperatures for the DPPC but not for F-DPPC, because the F-DPPC monolayer is more expanded and more resistant to changes of temperature. On the other hand, film elasticity (C(s)(-1)) values calculated for both phospholipids show that the film condensation diminishes when the temperature increases, in accordance with the results obtained from surface pressure measurements.BAM images for F-DPPC monolayers recorded at different surface pressures and temperatures show the existence of numerous ovoid-like domains when the LE-LC phase transition is reached. However, in the LE and LC phases, homogeneous images were obtained. Time evolution of relative thickness along the compression of F-DPPC and DPPC monolayers shows similar behavior of both phospholipids, except at low temperatures.For DPPC/F-DPPC mixed systems, the plots of the mean molecular area as a function of F-DPPC mole fraction (X(F-DPPC)) indicated that, whatever the surface pressure, the experimental results match the theoretical values calculated from the additivity rule, a typical behavior for ideal mixed monolayers made of miscible components. This conclusion is confirmed from the values calculated for the free energy of excess (ΔG(exc)) of this system, which are practically zero, whatever the composition of the mixtures and the surface pressure at which ΔG(exc) values were calculated.

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Differential Scanning Calorimetry (DSC) was used to study the effect of the incorporation of a series of semifluorinated diblocks F(n)H(m) (F(6)H(10), F(6)H(16), F(8)H(14), F(8)H(16), F(8)H(18) and F(8)H(20)) on the gel and liquid states of the bilayer of large multilamellar DMPC and DPPC liposomes. The presence of the F(n)H(m) diblocks affects slightly the T(m) of the main gel-liquid transitions of DMPC and DPPC, but is accompanied by the appearance of a second transition in the calorimetric traces whose T(m) is mainly determined by the length of the F(n) segment. The DSC results are consistent with the previously established conclusion that the F(n) segments of the diblocks form a central layer in the core of the lipid bilayer, with the H(m) segments being interdigitated with the lipid chains. The DSC traces suggest that the structure of the fluorinated liposomes is a double bilayer at 3:4 and 1:2 and a trilayer at 2:1 lipid/F(n)H(m) molar ratios. At temperatures between the two phase transitions T(m)'s, the fluorinated liposomes are neither in a gel-like or a liquid-like state but rather possess both characteristics.

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Colloidal aggregation using liposomes has been studied in this chapter. As criteria of stability, the stability factor, an extension of the DLVO theory of colloidal stability, the fractal dimension of the liposome aggregates and the different regimes of aggregation (RLCA and DLCA) and the temperature have been used.

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The physicochemical and elastic properties of Langmuir mixed monolayers composed by dehydrocholic acid (HDHC) and didodecyldimethylammonium bromide (DDAB) were evaluated. The experiments were performed with a constant surface pressure penetration Langmuir balance based on Axisymmetric Drop Shape Analysis (ADSA). The behavior of such amphiphiles in monolayer was clearly non-ideal and would be seriously influenced by the amount of HDHC molecules present. The presence of bile acid type molecules caused the monolayer be more condensed (A(c) diminution) and the intermolecular attractive interactions be stronger (high epsilon(0) values). This fact would be related to H-bond formation between water and carboxilate and carbonile groups in the cholesteric ring and agreed with the existence of laterally structured microdomains at the monolayer (determined by the analysis of the first virial coefficient, b(0)<1, of the state equation). The miscibility of both surfactants in the monolayer, their high bulk hydrophobicity (pi(c)>35 mJ m(-2)) just with the obtained negative values of the free energy of mixing Delta G(mix), and the excess second virial coefficient (b(1))(E) obtained allows us to infer that net attractive interaction existed between HDHC and DDAB molecules at the monolayer and that mixed systems would be able to be used in the formulation of supramolecular assemblies.

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Langmuir monolayers of the hydrogenated/fluorinated catanionic surfactant cetyltrimethylammonium perfluorooctanoate at the air/water interface are studied at room temperature. Excess Gibbs energies of mixing, DeltaG(E), as well as transition areas and pressures, were obtained from the surface pressure-area isotherm. The DeltaG(E) curve indicates that tail-tail interactions are more important than head-head interactions at low pressures and vice versa. Atomic force microscopy and molecular dynamics simulations allowed a fine characterization of the monolayer structure as a function of the area per molecule at mesoscopic and nanoscopic size scales, respectively. A combined analysis of the techniques allow us to conclude that electrostatic interactions between the ionic head groups are dominant in the monolayer while hydrophobic parts are of secondary importance. Overall, results obtained from the different techniques complement to each other, giving a comprehensive characterization of the monolayer.

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The interaction between two serum blood proteins, namely human serum albumin (HSA) and human immunoglobulin G (IgG), with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) liposomes has been studied in detail using dynamic light scattering, flow cytometry, enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility, differential scanning calorimetry (DSC), and surface tension measurements. HSA and IgG interact with liposomes forming molecular aggregates that remain stable at protein concentrations beyond those of total liposome coverage. Both HSA and IgG penetrate into the liposome bilayer. An ELISA assay indicates that the Fc region of IgG is the one that is immersed in the DMPC membrane. The liposome-protein interaction is mainly of electrostatic nature, but an important hydrophobic contribution is also present.

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Hexadecyltrimethylammonium bromide (C(16)TAB)-sodium perfluorooctanoate (C(8)FONa) and hexadecylpyridynium bromide (C(16)PyB)-C(8)FONa catanionic semifluorinated mixtures have been studied by conductivity, dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and polarizing microscopy. The regular solution theory, applicable for a limited fluorinated molar ratio, does not predict long-range electrostatic interactions. The results are consistent with the fact that in the hydrogenated-rich region the interaction is attractive in both catanionic mixtures. The systems containing pyridinium headgroups were of the stronger interaction. A transition from micelles was found in both mixtures as a function of fluorinated molar ratio. Special attention was devoted to the effect of the head group in the system properties. The information related with the mean vesicle radius measured by DLS was compared with the vesicle size distribution as well as the elastic properties of the bilayer measured with cryo-TEM.

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Using grazing incidence small-angle X-ray scattering (GISAXS), and atomic force microscopy (AFM) it has been recently demonstrated that linear fluorocarbon-hydrocarbon diblocks (FnHm) self-assemble in water/air interfaces forming elongated and circular hemimicelles. Those structures have been observed for diblocks with at least eight fluorinated carbons. Based on the lack of a collapse pressure for F6H16, and due to the fact that no stable surface pressure values are reached under compression, it has been concluded that these molecules do not form stable monolayers. It has been also suggested that F6H16 and shorter diblocks desorb from the water surface under compression. It is not easy to accept that a significant concentration of so hydrophobic molecules can be stable in aqueous solution even when the employed experimental techniques were not able to clearly detect a well defined structure on the interface. In the present work the adsorption and arrangement of F6H16 and F6H10 at the water surface are studied by molecular dynamics (MD) simulations as a function of the available area per molecule. Starting from a random mixture, the spontaneous formation of elongated hemimicelles is observed for both systems when the area per molecule is higher than approximately 50 A(2). For intermediate areas two pseudo-phases, one rich in hydrocarbons and the other with higher fluorocarbon concentration, are formed. For the systems with less than approximately 30 A(2) available per molecule the formation of multilayers is observed. This is the first time that the dynamics and structure of perfluoroalkane (PFA) films, and in particular of hemimicelles on a liquid surface, are observed and characterized at atomic level.

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The complexations between catalase and the sodium perfluorooctanoate/sodium octanoate and sodium perfluorooctanoate/sodium dodecanoate systems have been studied by a combination of electrophoresis and spectroscopy measurements. The numbers of adsorption sites on the protein were determined from the observed increases of the zeta-potential as a function of surfactant concentration in the regions where the adsorption was a consequence of the hydrophobic effect. The Gibbs energies of adsorption of the surfactants onto the protein were evaluated and the results show that for all systems, Gibbs energies are negative and larger, in absolute values, at low values of surfactant concentration where binding to the high energy sites takes place, and become less negative as more surfactant molecules bind, suggesting a saturation process. The role of hydrophobic interactions in these systems has been demonstrated to be the predominant. Spectroscopy measurements suggest conformational changes on catalase depending on the surfactant mixture as well as the mixed ratio. No isosbestic point or shifts have been found showing that catalase has spectrophotometrically one kind of binding site for these surfactant mixtures.

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Bile acids (deoxycholic and dehydrocholic acids) spread mixed monolayers behavior at the air/water interface were studied as a function of subphase pH using a constant surface pressure penetration Langmuir balance based on the Axisymmetric Drop Shape Analysis (ADSA). We examined the influence of electrostatic, hydrophobic and hydration forces on the interaction between amphiphilic molecules at the interface by the collapse area values, the thermodynamic parameters and equation of state virial coefficients analysis. The obtained results showed that at neutral (pH=6.7) or basic (pH=10) subphase conditions the collapse areas values are similar to that of cholanoic acid and consistent with the cross-sectional area of the steroid nucleus (approximately 40 A(2)). The Gibbs energy of mixing values (DeltaG(mix)<0) and the first virial coefficients of the equation of state (b(0)<1) indicated that a miscible monolayer with laterally structured microdomains existed. The aggregation number (1/b(0)) was estimated within the order of 6 (pH=6.7) and 3 (pH=10). At pH=3.2, acidic subphase conditions, no phase separation occurs (DeltaG(mix)<0) but a high expanded effect of the monolayer could be noted. The mixed monolayer behavior was no ideal and no aggregates were formed (b(0)> or =1). Such behavior indicates that the polar groups of the molecules interacts each other more strongly by repulsive electrostatic forces than with the more hydrophobic part of the molecule.

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The binding of sodium perfluorooctanoate (C8FONa), sodium octanoate (C8HONa), lithium perfluorooctanoate (C8FOLi), and sodium dodecanoate (C12HONa) onto myoglobin, ovalbumin, and catalase in water has been characterized using electrophoretic mobility. The tendency of the protein-surfactant complexes to change their charge in the order catalase < ovalbumin < myoglobin was observed which was related to the contents of alpha-helices in the proteins. alpha-Helices are more hydrophobic than beta-sheets. The effect of surfactant on the zeta potentials follows C8HONa < C8FONa < C8FOLi < C12HONa for catalase and ovalbumin; and C8HONa < C8FOLi < C8FONa < C12HONa for myoglobin. The numbers of binding sites on the proteins were determined from the observed increases of the zeta-potential as a function of surfactant concentration in the regions where the binding was a consequence of the hydrophobic effect. The Gibbs energies of binding of the surfactants onto the proteins were evaluated. For all systems, Gibbs energies are negative and large at low concentrations (where binding to the high energy sites takes place) and become less negative at higher ones. This fact suggests a saturation process. Changes in Gibbs energies with the different proteins and surfactants under study have been found to follow same sequence than that found for the charge. The role of hydrophobic interactions in these systems has been demonstrated to be the predominant.

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A study of the fractal dimension of the aggregation of three different types of large unilamellar vesicles, formed by egg yolk phosphatidylcholine (EYPC), dimyristoyl-phosphocholine (DMPC), and dipalmitoyl-phosphocholine (DPPC), in the presence of La3+, is presented. Aggregate liposome fractal dimensions were calculated by two methods, aggregation kinetics, using the approaches diffusion-limited cluster aggregation (DLCA) and reaction-limited cluster aggregation (RLCA) and angle-scattering light dispersion. Electrophoretic measurements show a similar variation of the zeta potential (zeta potential) for EYPC and DPPC, with a small increase of initial positive values. However, the zeta potential of DMPC changes from a initial negative value to near zero with increasing La3+ concentration. The evolution of the aggregate sizes was followed by light scattering. DPPC and DMPC show a RLCA regimen growth at low La3+ concentrations and a DLCA regimen at higher concentrations. In the case of EYPC, the final size of aggregation strongly depends on La3+ concentration. The calculated fractal dimension is in the range 1.8 to 2.1.

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In the field of bioscience, the study of the interactions between blood proteins and fluorinated materials is very important from both theoretical and applied points of view. Fluorinated materials have potential use in drug delivery, as blood substitutes, and in biotechnology. Using a combination of ultraviolet-visible (UV-vis) and ultraviolet-circular dichroism (UV-CD) spectroscopies and ion-selective electrodes, the complete interaction of sodium perfluorooctanoate (SPFO) and the most important immunoglobulin (on a quantitative basis) in human serum, immunoglobulin G (IgG), has been evaluated. The study has been focused on bulk solution. By the application of an SPFO selective electrode, it was determined that there were true specific unions between surfactant molecules and IgG structure. The experimental data were presented as Koltz and Scatchard plots and analyzed on the basis of an empirical Hill equation. The conformational changes at the bulk solution were well characterized by UV-vis and UV-CD spectroscopies. As a consequence of these changes, the protein structure was affected.

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The interaction between sodium perfluorooctanoate (SPFO) and dodecyltrimethylammonium bromide (DTAB) was studied by several methods and it was found strongly synergistic. Above a mole fraction of SPFO in the surfactant mixture (alpha(SPFO))=0.38, the interaction is repulsive and increases with the content of SPFO in both, the overall mixture and micelles, whereas the interaction is attractive if DTAB is in excess. At alpha(SPFO)=0.38 the low miscibility between hydrocarbon and fluorocarbon is counterbalanced by the electrostatic attraction between the opposite charged head groups, and the micelle composition is ideal (i.e., the mole fraction of SPFO in micelles X(SPFO)=alpha(SPFO)=0.38). The solubility of fluorocarbon in hydrocarbon is lower than that of hydrocarbon in fluorocarbon. Micelles of DTAB act as a solvent for SPFO without important structural changes, whilst micelles of SPFO undergo important changes when dissolve DTAB. This asymmetry may be interpreted as caused by the difference in chain length that favors the inclusion of the shorter chain in micelles of the longer surfactant, but disfavors the opposite process. Above X(SPFO)=0.5 there is an excess adsorption of bromide ions on the mixed micelles surface, giving rise to a high zeta potential. Micelles of pure SPFO or pure DTAB show an important energy barrier which prevents micelle flocculation. The inclusion of SPFO in DTAB micelles produces a reduction of the energy barrier, which disappeared when alpha(SPFO)=0.5. This produces the flocculation of micelles giving rise to the formation of a non-birefringent coacervate, which is probably formed by unordered isometric clusters of micelles.

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In this paper we have corroborated the usefulness of spectroscopic techniques, such as UV-visible, in the study and thermodynamic characterization of the thermal unfolding of catalase as a function of the concentration and alkyl chain length of n-alkyltrimethylammonium bromides (CnTAB, n = 8, 10, and 12). For this reason, a thermodynamic model was used which included experimental data corresponding to the pre- and posttransition into the observable transition. It has been found that n-alkyltrimethylammonium bromides play two opposite roles in the folding and stability of catalase. They act as a structure stabilizer at a low molar concentration and as a destabilizer at a higher concentration. The maximum of the unfolding temperature has been found to decrease with the alkyl chain. The reason for this difference has been suggested to be the side chains involved. In the presence of C8TAB and C10TAB, Gibbs energies of unfolding (DeltaG(T)) decrease with concentration, whereas for C12TAB an increase has been observed. These findings can be explained by the fact that when differences in the hydrophobic nature of the surfactants exist, different pathways of unfolding may occur. Also, the presence of surfactants has been observed to affect the cold denaturation of catalase. Thermodynamic results suggest that the thermal denaturation of catalase in the presence of n-alkyltrimethylammonium bromides is a perfect transition between two states.

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Lanthanide ions such as La3+ and Gd3+ are well known to have large effects on the structure of phospholipid membranes. Unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) were prepared by sonication method and confirmed by transmission electron microscopy. The effects of concentration of gadolinium ions Gd3+ on DPPC unilamellar vesicles in aqueous media were studied by different techniques. As physical techniques, photon correlation spectroscopy, electrophoretic mobility, and differential scanning calorimetry were used. The theoretical predictions of the colloidal stability of liposomes were followed using the Derjaguin-Landau-Verwey-Overbeek theory. Changes in the size of liposomes and high polydispersities values were observed as Gd3+ concentration increases, suggesting that this cation induces the aggregation of vesicles. Electrophoretic mobility measurements on unilamellar vesicles as a function of Gd3+ ion concentration show that the vesicles adsorb Gd3+ ions. Above Gd3+ concentrations of 0.1 mol dm-3, the zeta potential and light scattering measurements indicate the beginning of aggregation process. For comparison with similar phospholipids, the zeta potential of phosphatidylcholine interacting with Gd3+ was measured, showing an analogous behavior. Differential scanning calorimetry has been used to determine the effect of Gd3+ on the transition temperature (Tc) and on the enthalpy (DeltaHc) associated with the process.

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