From molecular modelling to photophysics of neutral oligo- and polyfluorenes incorporated into phospholipid bilayers.

The combination of various experimental techniques with theoretical simulations has allowed elucidation of the mode of incorporation of fluorene based derivatives into phospholipid bilayers. Molecular dynamics (MD) simulations on a fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayer, with benzene (B), biphenyl (BP), fluorene (F) and tri-(9,9-di-n-octylfluorenyl-2,7-diyl), TF, have provided insights into the topography of these molecules when they are present in the phospholipid bilayer, and suggest marked differences between the behavior of the small molecules and the oligomer. Further information on the interaction of neutral fluorenes within the phospholipid bilayer was obtained by an infrared (IR) spectroscopic study of films of DMPC and of the phospholipid with PFO deuterated specifically on its alkyl chains (DMPC-PFO-d34). This was complemented by measurements of the effect of F, TF and two neutral polymers: polyfluorene poly(9,9-di-n-octylfluorenyl-2,7-diyl), PFO, and poly(9,9-di-n-dodecylfluorenyl-2,7-diyl), PFD, on the phospholipid phase transition temperature using differential scanning calorimetry (DSC). Changes in liposome size upon addition of F and PFO were followed by dynamic light scattering. In addition, the spectroscopic properties of F, TF, PFO and PFD solubilised in DMPC liposomes (absorption, steady-state and time-resolved fluorescence) were compared with those of the same probes in typical organic solvents (chloroform, cyclohexane and ethanol). Combining the insight from MD simulations with the results at the molecular level from the various experimental techniques suggests that while the small molecules have a tendency to be located in the phospholipid head group region, the polymers are incorporated within the lipid bilayers, with the backbone predominantly orthogonal to the phospholipid alkyl chains and with interdigitation of them and the PFO alkyl chains.

Role of the electrostatic depletion attraction on the structure of charged liposome-polymer mixtures.

The effect of adding charged nonadsorbing polymers to electrostatically structured suspensions of charged liposomes has been experimentally studied by means of light scattering techniques. The static structure factor of the mixtures is analyzed using two polymers of different sizes. As the polymer concentration increases, the main peak of the structure factor decreases and shows an important shift to larger values of the scattering vector. Such displacement is the consequence of the electrostatic-enhanced depletion attraction induced by the polymers that counteracts the electrostatic repulsion. For the shorter polymer, the system remains stable for all studied polymer concentrations. However, for the long polymer chains, the effective attraction induced at the highest polymer density studied is strong enough to destabilize the mixture. In this case, the aggregation of the liposomes leads to clusters of nearly linear morphology. The PRISM theory is employed to calculate the effective pair potential between liposomes. The theoretical predictions are able to support the experimental observations, and provide an explanation of the interplay between the electrostatic repulsive interaction and the depletion attraction. In particular, they show that the depletion attraction is especially long ranged, and is dominated by electrostatic effects rather than entropic.

Use of high-pressure freeze fixation and freeze fracture electron microscopy to study the influence of the phospholipid molar ratio in the morphology and alignment of bicelles.

The high-pressure freeze fixation and freeze fracture electron microscopy techniques were combined with the (31)P nuclear magnetic resonance to study the morphological transitions of two different dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine aggregates by the effect of temperature. Through these techniques, the relationship between magnetic alignment and the morphology of alignable and non-alignable aggregates was evaluated. The micrographs related to the non-alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented rounded objects at a temperature below the dimyristoyl-phosphatidilcholine phase transition (T(m)) and, above this temperature an increase of viscosity was followed by the appearance of large elongated aggregates. The micrographs related to the alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented discoidal objects below T(m). Above T(m), when the best alignment was achieved, the images showed large areas of lamellar stacked bilayers and the presence of some multilamellar vesicles. Our results reveal that the composition of the aggregates is a key factor determining the morphological transitions of the bicellar systems. Understanding of the rules governing these transitions is crucial to modulate characteristics of these systems and to adequate them for different applications.

Penetration and growth of DPPC/DHPC bicelles inside the stratum corneum of the skin.

The effect of dipalmitoyl phosphatidylcholine (DPPC)/dihexanoyl phosphatidylcholine (DHPC) bicelles on the microstructure of pig stratum corneum (SC) in vitro was evaluated. The physicochemical characterization of these nanoaggregates revealed small disks with diameters around 15 nm and a thickness of 5.4 nm. Upon dilution, the bicelles grow and transform into vesicles. Cryogenic scanning electron microscopy (cryo-SEM) images of the SC pieces treated with this system showed vesicles of about 200 nm and lamellar-like structures in the intercellular lipid areas. These vesicles probably resulted from the growth and molecular rearrangement of the DPPC/DHPC bicelles after penetrating the SC. The presence of lamellar-like structures is ascribed to the interaction of the lipids from bicelles with the SC lipids. The bicellar system used is suitable to penetrate the skin SC and to reinforce the intercellular lipid areas, constituting a promising tool for skin applications.

Morphological effects of ceramide on DMPC/DHPC bicelles.

Freeze fracture electron microscopy and dynamic light scattering were applied to characterize the morphological changes of DMPC/DHPC bicellar systems induced by the addition of ceramides. The results demonstrate a tendency of the DMPC/DHPC aggregates to evolve forming elongated or tubular structures with the increase of the temperature. At 20 degrees C, low concentrations of ceramide promote the appearance of elongated structures with twisted zones. Higher concentrations of this lipid lead to the formation of liposomes along the elongated structures. The increase of the temperature to 40 degrees C induces the growth of the structures containing low concentrations of ceramide forming branched aggregates. In samples with high amounts of ceramide, the increase of temperature causes phase separation and the formation of a mixed system composed by liposomes and multilamellar tubules. The morphological effects induced by ceramides in this new membrane model give new insights for the role played by this lipid in biological membranes.

Influence of the temperature in the adsorption of sodium dodecyl sulfate on phosphatidylcholine liposomes.

The influence of the temperature on the adsorption of monomeric and micellar solutions of the anionic surfactant sodium dodecyl sulfate (SDS) on phosphatidylcholine (PC) liposomes was investigated using the fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS). The number of adsorbed molecules was quantified by measuring changes in the electrostatic potential (Psi(o)) of the liposomes/probe during an incubation with SDS at varying temperatures. At low surfactant concentrations (from 0.05 to 0.25 mM), the increase in temperature reduced the number of surfactant molecules incorporated per vesicle regardless of the incubation time, whereas at high surfactant concentrations (from 0.50 to 1.0 mM) the incubation time has an opposite effect on this process. Thus, after 10s, the surfactant adsorption decreased with temperature, yet it increased progressively with time. The adsorption was linear with temperature below critical micellar concentration (CMC) of SDS and this linear tendency did not change above CMC. This suggests an adsorption of SDS monomers regardless of the surfactant concentration.

[Relation between the viscosity of enteral diets and mechanical complications in their administration according to the nasogastric catheters]. / Relación entre la viscosidad de las dietas enterales y las complicaciones mecánicas en su administración según el diámetro de la sonda nasogástrica.

OBJECTIVE: The use of enteral nutrition has increased enormously over the last few years and at the same time the standard indications for parenteral nutrition, whether in hospitals or at home, have gradually been defined. The most common way to administer enteral nutrition is intermittently and using gravity, through a nasogastric catheter or through gastrostomy. In our daily practice it has been observed that there was at times a considerable delay in the administration of the diet by means of a nasogastric catheter and the tube was sometimes blocked due to its small calibre and/or the excessive viscosity of the formula. It was therefore proposed to study the viscosities of polymeric enteral diets with and without fibre at this hospital to identify the relationship with the time taken for their intermittent, gravity-driven administration through different calibres of nasogastric catheter at the maximum flow rate. RESULTS: Some of the enteral diets under study exceeded the recommended time for intermittent administration by gravity (20-40 minutes). CONCLUSIONS: We feel it is essential for the product label to provide information on the optimal calibre of the nasogastric catheter required for administration of the product.

Use of a fluorescence spectroscopy technique to study the adsorption of sodium dodecylsulfonate on liposomes.

The fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(0)) of the bilayers. The abrupt decrease in the fluorescence intensity already detected 10 s after the surfactant addition and the slight fluorescence variations with time indicated that the surfactant adsorption was very fast and almost complete. For a given number of monomers adsorbed, a linear dependence between the lipid and C(12)-SO(3) concentrations was obtained, indicating a similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C(12)-SO(3) concentration above its critical micellar concentration (CMC). In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C(12)-SO(3) ability to be adsorbed on liposomes) and the bilayer/aqueous phase coefficients (K). The fact that the lowest values for Re were always reached after 10 s of incubation corroborates the rapid kinetic of the process. The decrease in the C(12)-SO(3) partitioning (K) when the number of surfactant molecules exceeded 15000 was possibly due to the electrostatic repulsion between the free and the adsorbed monomers, which could hinder the incorporation of new monomers on the charged surface of liposomes.

Relación entre la viscosidad de las dietas enterales y las complicaciones mecánicas en su administración según el diámetro de la sonda nasogástrica / Relationship between the viscosity of enteral diets and mechanical complications in their administration depending on the calibre of the nasogastric catheter

Objetivo: La utilización de la nutrición enteral se ha incrementado enormemente en los últimos años y al mismo tiempo se han ido normalizando las indicaciones de la nutrición parenteral, tanto a nivel hospitalario como domiciliario. La forma más habitual de administrar la nutrición enteral es mediante sonda nasogástrica (SNG) o por gastrostomía, de forma intermitente y por gravedad. En nuestra práctica diaria observamos que en algunas ocasiones existía un tiempo de retraso considerable en la administración de la dieta por SNG y a veces la sonda se obstruía debido a su pequeño diámetro o a la viscosidad excesiva de la fórmula. Así que nos planteamos estudiar las viscosidades de las dietas enterales poliméricas con y sin fibra de nuestro hospital y relacionarlas con el tiempo que se emplea en su administración intermitente por gravedad a través de SNG de diferentes diámetros a la máxima velocidad de flujo. Resultados: Algunas de las dietas enterales estudiadas sobrepasan el tiempo recomendado para su administración intermitente por gravedad (20-40 minutos).Conclusiones: Consideramos imprescindible que en la etiqueta del producto se aporte inforrnación sobre el calibre óptimo de la SNG necesaria para su administración (AU)

Objective: The use of enteral nutrition has increased enormously over the last few years and at the same time the standard indications for parenteral nutrition, whet-her in hospitals or at home, have gradually been defined. The most common way to administer enteral nutrition is intermittently and using gravity, through a nasogastric catheter or through gastrostomy. In our daily practice it has been observed that there was at times a considerable delay in the administration of the diet by means of a nasogastric catheter and the tube was sometimes blocked due to its small calibre and/or the excessive viscosity of the formula. It was therefore proposed to study the viscosities of polymeric enteral diets with and without fibre at this hospital to identify the relationship with the time taken for their intermittent, gravity-driven administration through different calibres of nasogastric catheter at the maximum flow rate. Results: Some of the enteral diets under study exceeded the recommended time for intermittent administration by gravity (20-40 minutes). Conclusions: We feel it is essential for the product la-bel to provide information on the optimal calibre of the nasogastric catheter required for administration of the product (AU)

Interaction of alpha-amylase with n-alkylammonium bromides.

The interaction of alpha-amylase with n-alkylammonium bromides above and below their critical micellar concentrations (cmc) has been studied in buffer at pH 7 and 10 by UV spectrophotometry, photon correlation spectroscopy and Doppler microelectrophoresis. This interaction produces a complex that is dependent on pH of the medium. This complex appears at surfactant concentrations below the cmc, which means that individual surfactant molecules can bind tightly to native alpha-amylase. The complex maintains its aggregation state when the concentration of surfactants with a hydrocarbon chain of 16 carbons increases, but not for surfactants of 12 and 14 carbons. Measurements of zeta-potential indicate the influence of electrostatic and hydrophobic forces. When the size of the aggregate is maximal, proteins are at their point of zero charge. In such conditions, Van der Waals forces and contacts between the alkyl chain and the hydrophobic core of the protein favour the formation of a larger aggregate.

Use of a fluorescence spectroscopy technique to study the adsorption of sodium dodecylsulfonate on liposomes.

The fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS) was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine (PC) bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(o)) of the bilayers. The abrupt decrease in the fluorescence intensity detected even 10 s after the surfactant addition and the slight fluorescence variations with time indicated that the surfactant adsorption was very fast and almost complete. For a given number of monomers adsorbed a linear dependence between the lipid and C12-SO3 concentrations was obtained, indicating similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C12-SO3 concentration above its CMC. In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C12-SO3 ability to be adsorbed on liposomes) and the bilayer/aqueous phase coefficients (K). The fact that the lowest values for Re were always reached after 10 s of incubation corroborates the rapid kinetics of the process. The decrease in the C12-SO3 partitioning (K) when the number of surfactant molecules exceeded 15000 was possibly due to the electrostatic repulsion between the free and the adsorbed monomers, which could hinder the incorporation of new monomers on the charged surface of liposomes.

Location of Pinacyanol in Micellar Solutions of N-Alkyl Trimethylammonium Bromide Surfactants.

The interaction of pinacyanol (PIN), a cationic dye formed by monomer and dimer species, with three cationic surfactants (DTAB, TTAB, and HTAB) has been studied spectroscopically and by acid-base equilibrium in the micellar concentration range. In the presence of surfactants, the absorption maximum of the two main peaks undergoes bathochromic shifts. The spectral shifts suggest a hydrophobic environment of the chromophore. The presence of micelles favors the monomer species; i.e., it reduces the extent of dimerization. The pK(a) of PIN in micellar medium is similar to the value in pure water. When acid-base equilibrium was considered, the changes in the interfacial pK(a) allowed to us to determine the constant dielectric for the interfacial region (epsilon=69). This led to the conclusion that the dye must be solubilized between the solution and the hydrocarbon chain core, i.e., in the aqueous micellar interface. This location can be explained by a cation-pi interaction between the uncharged ring system of the dye and the cationic headgroups of the surfactants. Copyright 2001 Academic Press.

Influence of cholesterol on liposome fluidity by EPR. Relationship with percutaneous absorption.

The influence of liposome composition on bilayer fluidity and its effect on the percutaneous absorption into the skin were investigated. Liposomes formed with saturated or unsaturated phospholipids (H-PC or PC) with varying amounts of cholesterol were prepared and their penetration behaviour into the stratum corneum was followed up by means of the stripping method. The order and dynamics of the hydrophobic domain of the vesicles were studied using electron paramagnetic resonance (EPR) methodology. Phospholipid composition and the amount of cholesterol exert a considerable influence on the penetration behaviour of the probe encapsulated in the liposomes. This behaviour is closely related to the fluidity characteristics of these liposomes studied by EPR. Therefore, a penetration mechanism of the vesicles into the skin, based on the incorporation of lipids into the skin lipids and on fluidity behaviour, is suggested.

Influence of the fluidity of liposome compositions on percutaneous absorption.

The penetration into the stratum corneum of fluorescein, as the acid form or as a sodium salt, encapsulated in liposomes formed by liquid- or gel-state phospholipids, with or without cholesterol, was investigated in humans by the stripping method. Liposomes prepared by extrusion were applied to the forearms of healthy human volunteers and 30 min later, strippings were performed. Fluorescein was extracted and determined by spectrofluorimetry. The skin penetration of sodium fluorescein was higher from fluid liposomes (phosphatidylcholine) than from rigid liposomes (hydrogenated phosphatidylcholine), but it was independent of the content of cholesterol. It seems that the liquid-crystalline state of the lipids is the main aspect involved in the fluidity of the liposome bilayer itself as well as in the interaction with the lipids of the stratum corneum. The similar enhanced penetration behavior obtained for unsaturated liposomes containing sodium or acid fluorescein seems to support the hypothesis of a previous destruction of the vesicles during its passage through the lipid intercellular pathway in the stratum corneum.

Electrophoretic properties of dodecyltrimethylammonium bromide micelles in KBr solution.

Charge in ionic micelles determines the trends of their stability and their practical applications. Charge can be calculated from zeta potential (zeta) measurements, which, in turn, can be obtained by Doppler microelectrophoresis. In this study, the electrophoretic properties of dodecyltrimethylammonium bromide (DTAB) in KBr aqueous solution (0-6 mM) were determined by Doppler microelectrophoresis. At very low surfactant concentrations (up to 6 mM), zeta potential was quite constant and due to the ionized monomers (DTA+). Above 6 mM, zeta potential increased to a maximum at surfactant concentrations still below the critical micellar concentration (CMC). This increase could be explained by a formation of nonmicellar aggregates of DTAB. Then, above the CMC, zeta potential underwent an abrupt reduction, which was dependent qualitatively and quantitatively on KBr concentration, and which could be due to an increase of the number of counterions adsorbed on the micelle surface. Calculation of effective micellar charge from zeta potential gave the surface charge density. Comparing this value with the theoretical, obtained from geometrical considerations, a fraction of 0.29 of charged micellar headgroups was obtained when DTAB was in aqueous solution, which is consistent with the value obtained by conductivity measurements.

Chemical degradation of liposomes by serum components detected by NMR.

Interaction between serum components and liposomes is an oxygen-dependent exothermic process. We studied the interaction of 100 nm extruded liposomes (bearing positive, negative or no charge) with foetal calf serum by 1H NMR and 13C NMR, in order to further our understanding of these reactions. Studies of aqueous or organic extracts obtained after 2 h, 1 day or 1 week, showed hydrolysis to be a degradation process concomitant with the interaction with serum. Oxidation was identified as additional to hydrolysis in the process of degradation. Oxidation produced aldehydes, acids and alcohols, although aldehydes and alcohols were prone to further decomposition and only appeared transiently. Alkenes and other oxidized compounds predominated in those products derived from oxidation. In stearylamine-containing liposomes some aldehydes and a nitroderivative were found as degradation products. Such metabolites are apolar and their presence might explain the intrinsic toxicity of this kind of liposome in cell cultures. The work described in the present study revealed the chemical degradation of liposomes in the serum used. In all cases the results obtained were compared with liposomes not incubated with serum.

The effect of liposomes on skin barrier structure.

The present work deals with the 'in vivo' stripping technique to evaluate the percutaneous absorption of sodium fluorescein (NaFl) vehiculized in two different liposome preparations formed by phosphatidylcholine (PC) and lipids mimicking the stratum corneum (SC; ceramides, cholesterol, palmitic acid and cholesteryl sulphate), respectively. Furthermore, the possible effect of these vesicles on the SC lipid alkyl chain conformational order were evaluated at different depths of SC by non-invasive biophysical techniques: Corneometer, Tewameter and especially ATR-FTIR. The results of NaFl percutaneous absorption indicate the highest penetration in the case of incorporation in PC liposomes, which could be related to the increase in SC lipid disorder detected by ATR-FTIR, i.e. a decrease in skin barrier function. On the other hand, SC lipid liposomes have been shown to have a higher affinity for SC owing to the high amount of NaFl found in this layer, suggesting a greater reservoir capacity of SC when similar lipid composition formulation is applied. A lipid order increase is observed by infrared spectroscopy, when these types of liposomes are topically applied, resulting in a strong barrier effect. These results could be useful in designing specific liposomal topical applications.

Preparations of synthetic phospholipids promote procoagulant activity on damaged vessels: studies under flow conditions.

BACKGROUND: The possibility of developing synthetic platelet substitutes that could promote hemostasis with prolonged shelf-life and increased safety is an appealing one. STUDY DESIGN AND METHODS: Preparations containing synthetic phospholipids were incorporated into blood samples (1.15 mg/mL) in which platelets and white cell counts had been experimentally reduced by a filtration procedure. Vesicles containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), or combinations of PC and PE and of PC and PS were tested in this system. Blood was recirculated (10 min; shear rate, 250/sec) through a perfusion chamber containing vascular segments. The ability of the various phospholipid preparations to promote fibrin formation on the damaged subendothelium was evaluated morphometrically and expressed as the percentage of fibrin coverage. Generation of thrombin in the system was monitored through the measurement of prothrombin fragments 1 and 2. RESULTS: Vesicles containing PC, PI, PE:PC (1:1), or PS:PC (1:3) increased fibrin deposition on the subendothelium (64.5 +/- 9.8%, 32.7 +/- 6.3%, 58.3 +/- 6.5%, and 46.6 +/- 15.2%, respectively; p < 0.01 vs. 11.5 +/- 1.2% in thrombocytopenic blood). Vesicles containing PE, PS, or PS:PC (3:1) did not show procoagulant effect. CONCLUSION: Synthetic phospholipid preparations promote a local procoagulant activity at sites of vascular damage when they are incorporated into thrombocytopenic blood maintained under flow conditions.

Influence of Size on Electrokinetic Behavior of Phosphatidylserine and Phosphatidylethanolamine Lipid Vesicles.

The aim of this study was to examine the electrokinetic properties of liposomes containing either phosphatidylethanolamine (PE) or phosphatidylserine (PS). Zeta potential was determined by laser-Doppler microelectrophoresis. The presence of PE enhanced the slight negative charge on the phosphate group of phosphatidylcholine. A linear relation between zeta potential and PE concentration was found. Absolute values of zeta potential were higher in PS liposomes and showed an exponential dependence on the PS content. The electrophoretic mobility depended not only on the percentage of aminophospholipid but also on the size of vesicles. This behavior can be explained by the relaxation effect. From geometrical considerations, it was possible to calculate the theoretical surface charge densities. Comparing experimental with theoretical values, an underestimation of zeta potential was found by the electrokinetic classic theory in the experimental conditions used. Copyright 1998 Academic Press.

Bilayer distribution of phosphatidylserine and phosphatidylethanolamine in lipid vesicles.

The distribution of phosphatidylethanolamine (PE) and phosphatydilserine (PS) in liposomes was studied as a function of aminophospholipid concentration using fluorescamine as labeling reagent. The method is suitable for such determination since, in the assay conditions, fluorescamine does not penetrate the vesicles nor does it disrupt them. The liposomes were obtained by sonication, extrusion, or mechanical dispersion (MLV). For any kind of vesicle, the percentage of PS in the external monolayer is higher than that obtained for PE in the corresponding vesicles. In extruded PS liposomes, this aminophospholipid is located preferentially in the outer layer, while for PE liposomes the localization depends on the size of vesicle. Sonicated liposomes present an asymmetrical distribution of both aminophospholipids, and the external location of PS or PE always predominates. In contrast, in MLV, aminophospholipids are mainly found in the inner layers of the vesicles, except for liposomes formed by the lowest PS proportion. A remarkable feature of PS liposomes is the reduction of vesicle size, especially in MLV liposomes, in comparison with neutral liposomes.