Stability of emulsions stabilised by two physiological surfactants: L-alpha-phosphatidylcholine and sodium taurocholate

The emulsion phase formed within the stomach and duodenum during digestion of a fatty meal has been modelled using two physiological surfactants, the phospholipid L-alpha-phosphatidylcholine (PC) and the bile salt sodium taurocholate (NaT). Upon dilution of the phospholipid stabilised emulsions with a solution of NaT the bile salt became incorporated into the oil/water interface imparting a negative charge to the droplet surface. The magnitude of the droplet microelectrophoretic mobility for the mixed PC and NaT system was 47% of that found for emulsion droplets stabilised by NaT alone. But the electrostatic repulsion between droplets was not sufficient to account for the observed improvement in emulsion stability to coalescence. It is suggested that a residual liquid crystalline phospholipid interface is present imparting a significant steric component to the stabilisation of the emulsions droplets.     

Dynamic behavior of lung surfactant

We have previously developed an adsorption-limited model to describe the exchange of lung surfactant and its fractions to and from an air-liquid interface in oscillatory surfactometers. Here we extend this model to allow for diffusion in the liquid phase. Use of the model in conjunction with experimental data in the literature shows that diffusion-limited transport i.s important for characterizing the transient period from the start of oscillations to the achievement of steady-state conditions. Matching previous data shows that upon high levels of film compression, large changes occur in adsorption rate, desorption rate, and diffusion constant, consistent with what one might expect if the subsurface region was greatly enriched in DPPC. Collapse of the surfactant film that occurs during compression leads to a .significant elevation of surfactant concentration immediately heneath the interface, consistent with the subsurface depot of surfactant that has heen postulated by other investigators. Modeling studies also uncovered a phenomenon of surfactant behavior in which the interfacial tension remains constant at its minimum equilibrium value while the film is compressed, hut without collapse of the film. The phenomenon was due to desorption of surfactant from the interface and termed "pseudo-film collapse.' The new model also gave improved agreement with steady-state oscillatory cycling in a pulsating bubble surfactometer.     

Protein adsorption at the oil/water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements.

The dynamics of protein adsorption at an oil/water interface are examined over time scales ranging from seconds to several hours. The pendant drop technique is used to determine the dynamic interfacial tension of several proteins at the heptane/aqueous buffer interface. The kinetics of adsorption of these proteins are interpreted from tension/log time plots, which often display three distinct regimes. (I) Diffusion and protein interfacial affinity determine the duration of an initial induction period of minimal tension reduction. A comparison of surface pressure profiles at the oil/water and air/water interface reveals the role of interfacial conformational changes in the early stages of adsorption. (II) Continued rearrangement defines the second regime, where the resulting number of interfacial contacts per protein molecule causes a steep tension decline. (III) The final regime occurs upon monolayer coverage, and is attributed to continued relaxation of the adsorbed layer and possible build-up of multilayers. Denaturation of proteins by urea in the bulk phase is shown to affect early regimes.     

Phospholipid monolayers at the triolein-saline interface: production of microemulsion particles and conversion of monolayers to bilayers

Interfacial tensions of phospholipid monolayer at the triolein (TO)-saline interface were measured. The adsorption isotherms and the interfacial pressure-molecular area curves were evaluated on the basis of the measurements. Phosphatidylcholine (PC) forms a highly condensed monolayer, with a large lateral attractive interaction; phosphatidylethanolamine (PE) and phosphatidylserine (PS) form expanded monolayers with smaller lateral interaction energies. At the lowest interfacial tension (the highest interfacial pressure), the mole fractions of PC, PE, and PS in the monolayers are estimated as 0.95, 0.73, and 0.88, respectively. Therefore, PC forms the most stable monolayer at the interface. These results are consistent with the finding that the stable TO particles in aqueous solution were produced by using PC as an emulsifier, and PE and PS did not stabilize the particles. The phase diagram of TO and PC mixtures in saline obtained from theoretical considerations predicts the equilibrium conversion of the monolayers on TO particles to bilayers. This process may be closely related to the transformations of very low density lipoproteins and chylomicrons to high-density lipoproteins in plasma. The particle sizes of the emulsion are calculated theoretically as a function of PC mole fraction in the TO-PC mixture and compared with the experimental values obtained from quasi-elastic light scattering (QLS) measurements.     

Kinetics of liposome disintegration from foam film studies: effect of the lipid bilayer phase state

The kinetics of interfacial liposome breakdown is investigated in the thin liquid film microinterferometric set up of Scheludko et al. Suspensions of small unilamellar vesicles of dimyristoylphosphatidylcholine are studied at temperatures above and below the temperature of the main gel-liquid crystal first order phase transition. The experimentally established time traces of the velocity of thinning of foam films are used to estimate the kinetic (rate) constants of interfacial liposome disintegration. New and previously established data for other lipids are summarized and compared with results from kinetic measurements of lipid monolayer formation. The thin film experiments confirm the existence of interfacial liposomal aggregates. A change in the kinetic behaviour is observed, due to the 'melting' of the hydrophobic tails in the lipid aggregates. This may have various consequences of biological and pharmacological importance.     

Molecular chain orientation of DNA films induced by both the magnetic field and the interfacial effect

DNA films showing highly homogeneous orientation of molecular chains were successfully prepared by drying a semidiluted solution in a horizontal magnetic field. Most of the molecular chain elements in the obtained film were found to be one-dimensionally oriented, as shown by X-ray diffraction, polarization microscopy, and linear dichroism spectroscopy. Because a DNA chain is theoretically expected to orientate only in divergent directions perpendicular to a magnetic field, this result suggests that the DNA chains were aligned not only by a magnetic field but also by the interfacial effect that induced the chains to fit along the air-liquid interface. The descent speed of an air-liquid interface by evaporation was faster than the estimated diffusion rate of DNA, suggesting an emergence of a concentrated layer near the surface. As proved by polarization microscopy, this emergence led to the transitional formation of a nematic-like liquid crystalline phase, which resulted in a DNA film with good chain alignment and unitary orientation. This mechanism underlying chain alignment was supported by molecular weight dependency, in which higher molecular weight DNA is more likely to evince chain alignment that exhibits a higher degree of birefringence. Low molecular weight components have such high thermal motility that it would be difficult to fit them along the air-liquid interface in the early stage of drying. For chain alignment, it was preferable to use an initial concentration of DNA lower than a critical concentration for liquid crystal formation so that the possible diffusion and assembly in a diluted solution would be essential for chain alignment. The DNA film exhibited obvious linear dichroism, indicating the potential for further applications.     

Effects of Lecithin Addition in Oil or Water Phase on the Stability of Emulsions Made with Whey Proteins

The effects of lecithin addition in oil or water phase on the stability of oil-in-water emulsions made with 0.1 wt% whey protein and 10 wt% n-tetradecane at neutral and acidic pH were studied by monitoring the gravitational creaming and phase separation. The effects of lecithin addition on the interfacial behavior of β-lactoglobulin were also studied to compare with the results of emulsion stability. At neutral pH, crude phosphatidylcholine (PC) from egg yolk or soybean increased the stability of the emulsion made with protein and lowered the interfacial tension of protein films more effectively than pure egg PC. A more remarkable effect on both the emulsion stability and the interfacial tension was found when crude PC was added in the oil phase rather than in the water phase. The purity of lecithins and the way to add them are suggested to be very important to make a stable emulsion with protein. On acidic pH (4.5 or 3.0), the increased creaming or phase separation in a whey protein-stabilized emulsion, but the lowered interfacial tension of β-lactoglobulin films, were found upon the addition of pure or crude PC in oil or water phase. These results suggest that in acidic pH, densely packed films may be formed on a planar oil–water interface, but not on adsorbed layers around oil droplets in an emulsion.     

A correlation between secondary structure and rheological properties of low-density lipoproteins at air/water interfaces

The secondary structure of apolipoprotein B-100 is studied within the bulk phase and at the air/water interface. In these “in viro” experiments, infrared reflection absorption spectroscopy (IRRAS) study was performed at the air/water interface while circular dichroism (CD) was conducted in the bulk phase. In the bulk phase, the conformational structure containing a significant amount of β–structure, whereas varying amount of α-helix, unordered structures, and β-sheet were observed at the air/water interface depending on the low-density lipoprotein (LDL) film interfacial pressure. The present IRRAS results demonstrate the importance of interfacial pressure-induced structural conformations on the apoB-100. A correlation between the secondary structure of the apoB-100 protein and the monomolecular film elasticity at the air/water interface was also established. The orientation of apoB-100 with respect to the LDL film-normal was found to depend on the interfacial pressure exhibited by the monomolecular film. These results may shed light on LDL’s pivotal role in the progression of atherosclerotic coronary artery disease as demonstrated previously by clinical trials.     

Structures of pulmonary surfactant films adsorbed to an air-liquid interface in vitro

Phospholipid films can be preserved in vitro when adsorbed to a solidifiable hypophase. Suspensions of natural surfactant, lipid extract surfactants, and artificial surfactants were added to a sodium alginate solution and filled into a captive bubble surfactometer (CBS). Surfactant film was formed by adsorption to the bubble of the CBS for functional tests. There were no discernible differences in adsorption, film compressibility or minimal surface tension on quasi-static or dynamic compression for films formed in the presence or absence of alginate in the subphase of the bubble. The hypophase-film complex was solidified by adding calcium ions to the suspension with the alginate. The preparations were stained with osmium tetroxide and uranyl acetate for transmission electron microscopy. The most noteworthy findings are: (1) Surfactants do adsorb to the surface of the bubble and form osmiophilic lining layers. Pure DPPC films could not be visualized. (2) A distinct structure of a particular surfactant film depends on the composition and the concentration of surfactant in the bulk phase, and on whether or not the films are compressed after their formation. The films appear heterogeneous, and frequent vesicular and multi-lamellar film segments are seen associated with the interfacial films. These features are seen already upon film formation by adsorption, but multi-lamellar segments are more frequent after film compression. (3) The rate of film formation, its compressibility, and the minimum surface tension achieved on film compression appear to be related to the film structure formed on adsorption, which in turn is related to the concentration of the surfactant suspension from which the film is formed. The osmiophilic surface associated surfactant material seen is likely important for the surface properties and the mechanical stability of the surfactant film at the air-fluid interface.     

Surface behavior,microheterogeneity and adsorption equilibrium of myelin at the air-water interface

Interfacial films of whole myelin membrane adsorb at the air-water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy.     

Atomic force microscopy analysis of rat pulmonary surfactant films

Pulmonary surfactant facilitates breathing by forming a surface tension reducing film at the air-liquid interface of the alveoli. The objective was to characterize the structure of surfactant films using endogenous rat surfactant. Solid-support surfactant films, at different surface pressures, were obtained using a Langmuir balance and were analyzed using atomic force microscopy. The results showed a lipid film structure with three distinct phases: liquid expanded, liquid ordered and liquid condensed. The area covered by the liquid condensed domains increased as surface pressure increased. The presence of liquid ordered phase within these structures correlated with the cholesterol content. At a surface pressure of 50 mN/m, stacks of bilayers appeared. Several structural details of these films differ from previous observations made with goat and exogenous surfactants. Overall, the data indicate that surfactant films demonstrate phase separation at low surface pressures and multilayer formation at higher pressure, features likely important for normal surfactant function.     

Analysis of the diffusion of bile salt/phospholipid micelles in rat intestinal mucin

The interaction of sodium taurocholate/egg phosphatidylcholine (TC/PC) micelles with mucin was determined to investigate the exclusion of lipids by mucus in the absorption process. The distribution of TC/PC was assessed at two intermicellar and three phospholipid concentrations with isolated, rat intestinal mucin (RIM) by dialysis. The diffusion coefficients were measured by NMR spectroscopy. At high [PC], RIM had lower [PC] relative to the control, while the concentration of TC was largely independent of mucin concentration. The PC diffusion coefficients were reduced in the presence of RIM. The magnetization decay of TC was compared with simulations to provide estimates of the monomeric diffusivity and exchange rate constant. The rate constants increased with increasing micelle concentration, and the free TC diffusion coefficient was reduced in the presence of mucin. Mucin has an exclusive effect on TC/PC mixed micelles that has been quantitatively determined through the use of diffusion measurements of dialyzed samples.     

Lateral mobility of phospholipid molecules in thin liquid films

The Fluorescence Recovery After Photobleaching (FRAP) method was applied to measure the lateral mobility of the fluorescent lipid analog, dioctadecylindocarbocyanine perchlorate (Dil-C18), in microscopic thin liquid films (Foam Films (FFs)). The foam film structures were comprised of two phosphatidylcholine monolayers adsorbed at air/water interfaces which sandwiched a thin liquid core. Lateral diffusion of the DiI molecules in the plane of the monolayers was determined as a function of the thickness of the thin liquid core of the film between the FF monolayers. The results obtained indicated that the diffusion coefficient was strongly dependent both on the distance between the FF monolayers in the range 4 nm to 85 nm (corresponding to the FF thickness) and on the film type. The applicability of the FRAP method for studying the molecular mobility in phospholipid FFs was demonstrated. Considerable differences in the surface diffusion coefficient of Dil were observed, ranging between 2 × 10^–8 cm²/s and 22 × 10^–8 cm²/s in so called yellow, gray, common black and Newton black FFs. The effect of the presence of polyethylene glycol (PEG-400) in the liquid core of lecithin FFs on surface diffusion was also studied. The surface diffusion results from the FF studies were compared with data from black lipid membranes (BLMs). These structures are related in thickness terms but the molecular orientation in FFs is the reverse of that in BLMs. Correspondence to: Z. Lalchev     

X-ray diffraction and foam film investigations of PC head group interaction in water/ethanol mixtures

The influence of ethanol on single phospholipid monolayers at the water/air interface and in foam films has been investigated. Grazing incidence X-ray diffraction investigations (GIXD) of Langmuir monolayers from 1,2-distearoyl-phosphatidylcholine (DSPC) spread on water subphases with different amounts of ethanol were performed. The thickness and free specific energy of formation of foam films stabilized by 1,2-dimyristoyl-phosphatidylcholine (DMPC) at different concentrations of ethanol in the film forming dispersions were measured. The GIXD investigations show that the tilt angle of the alkyl chains in the PC lipid monolayer decreases with increasing concentration of ethanol caused by a decrease of the diameter of the head groups. With increasing ethanol content of the solution also the thickness of the aqueous core of PC lipid foam films decreases. We assume that ethanol causes a decreasing probability for the formation of hydrogen bonds of water molecules to the PC head groups. The distinct difference between the effects of ethanol on lipid bilayers as described in the literature and on monolayers and foam films found in this study is discussed. Whereas PC monolayers at the water/air interface become unstable above 25 vol.% ethanol, the PC foam films are stable up to 50 vol.% ethanol. This is related to the decrease of the surface excess energy per lipid molecule by the interaction between the two film surfaces.     

Thin liquid films and monolayers of DMPC mixed with PEG and phospholipid linked PEG

In this work thin liquid films (TLFs) and monolayers at the air/water interface formed by dimyristoylphosphatidylcholine (DMPC) and by DMPC mixed with poly ethylene glycols (PEGs) and dimyristoylphosphatidylethanolamine (DMPE) linked PEGs were studied. Film forming dispersions were composed of two types of particles: liposomes and micelles. TLFs stability, threshold concentration C _t (i.e., the minimum one for stable film formation), and hydrodynamic behavior were measured. At equivalent conditions, DMPC films were Newton black films (real bilayers), while DMPE-PEGs films were much thicker with free water between the monolayers. DMPE-PEG addition to DMPC films caused both C _t decrease (depending on PEG moiety length and Mw) and change of TLF formation mechanism. TLFs’ hydrodynamic behavior also strongly depended on DMPE-PEG content and Mw. It was observed that thinning of the DMPC and DMPE-PEGs films continued to different film types and thickness, being much thicker for the latter films. Addition of free PEGs (PEG-200/6000) did not alter TLF type or stability, but changed TLF thinning time, confirming that free PEGs with Mw<8000 could not penetrate in the membrane and alter “near-membrane” water layer viscosity. Monolayer studies showed improved formation kinetics of both adsorbed and spread films, decrease of surface tension (equilibrium and dynamic), and of film compression/decompression histeresis area in DMPE-PEGs monolayers compared with DMPC pure films. Our study shows that combining the models of phospholipid TLFs and monolayers provide the opportunity to investigate the properties of membrane surface and to clarify some mechanisms of its interactions with membrane-active agents.     

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir-Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.     

Influence of liquid-layer thickness on pulmonary surfactant spreading and collapse

Pulmonary surfactant spreads on the thin ( approximately 0.1 microm) liquid layer that lines the alveoli, forming a film that reduces surface tension and allows normal respiration. Pulmonary surfactant deposited in vitro on liquid layers that are several orders of magnitude thicker, however, does not reach the low surface tensions ( approximately 0.001 N/m) achieved in the lungs during exhalation when the surfactant film compresses. This is due to collapse, a surface phase transition during which the surfactant film, rather than decreasing surface tension by increasing its surface density, becomes thicker at constant surface tension ( approximately 0.024 N/m). Formation of the collapse phase requires transport of surfactant to collapse sites, and this transport can be hindered in thinner liquid layers by viscous resistance to motion. Our objective is to determine the effect of the liquid-layer thickness on surfactant transport, which might affect surfactant collapse. To this end, we developed a mathematical model that accounts for the effect of the liquid-layer thickness on surfactant transport, and focused on surfactant spreading and collapse. Model simulations showed a marked decrease in collapse rates for thinner liquid layers, but this decrease was not enough to completely explain differences in surfactant film behavior between in vitro and in situ experiments.     

On liquid-liquid mass transfer in two-liquid-phase fermentations

Almost all two-liquid phase bioprocesses are characterized by the presence of surface active materials (biosurfactants), which significantly influence the interaction between the phases. In order to predict mass transfer rates during cultivations of Pseudomonas oleovorans biosurfactant was isolated from the biosuspension and added in defined amounts to n-octane/water model-dispersions. Effects of biosurfactant concentration on interfacial tension, mean Sauter-diameter, drop size distribution, dispersion stability and liquid-liquid mass transfer coefficients were studied. A comparison was made between calculated solvent transfer rates (STR) and measured solvent uptake rates (SUR) of P. oleovorans cultures. With increasing interfacial surfactant concentration interfacial tension and mean Sauter-diameter decreased until a minimum for both, interfacial tension and mean Sauter-diameter, were reached. Interfacial tension measurements indicate that these minima have to be attributed to a maximum monomolecular surfactant concentration and the formation of polymolecular adsorption layers. Drop size distributions showed that, coalescence and droplet break-up disappear because droplets are stabilized by the biosurfactant adsorption layers at the interface. Mass transfer regime shifted from forced convection and surface renewal to diffusion. Comparison of solvent uptake rates (SUR) and solvent transfer rates (STR) showed that n-octane transfer usually will not be limiting P. oleovorans cultures, however, can become dominant in cultures where solvents with very low miscibilities like n-decane are used.     

Interaction of gentamicin with phosphatidylserine/phosphatidylcholine mixtures in adsorption monolayers and thin liquid films: morphology and thermodynamic properties

Gentamicin possesses strong adverse actions like oto and nephrotoxicity. The latter is a result of strong gentamicin–acid phospholipid interactions, resulting in cell fusion, fission, etc., ions as calcium interact with gentamicin and effectively deter its toxicity. In this work, the interactions of gentamicin and Ca²⁺ with phosphatidylserine/phosphatidylcholine (PS/PC) mixtures of different ratio are experimentally characterized. Special attention is paid to bridge thermodynamic and morphological properties of adsorption monolayers and thin liquid films (TLFs) composed of these lipid mixtures. Our results show that gentamicin decreases the stability of common black TLFs formed of pure PS coupled with suppression of lipid surface adsorption to the monolayers at the air–water interface; also, gentamicin reveals effects of lowering of lipid spreading on the interface and significant loss of material during monolayer cycling, increase of condensed phase, and organization of dense net-like domain monolayer texture. Gentamicin addition results in opposite effects for films formed of DPPC/PS (95:5) mixture. It increases the stability of Newton black TLFs formed by DPPC/PS correlated with faster and stronger surface adsorption and better surface spreading; also, gentamicin lowers the amount of condensed phase and organization of domains of smaller size. We also showed that Ca²⁺ itself decreases the stability of common black TLFs formed of PS accompanied with weaker surface adsorption, formation of higher amounts of condensed phase and organization of domains. In our experiments, Ca²⁺ softens, even deters, the effects of gentamicin on both PS and DPPC/PS films.