Liquid-expanded Phase Research Articles

L-Ascorbic acid alkyl esters action on stratum corneum model membranes: An insight into the mechanism for enhanced skin permeation

L-ascorbic acid alkyl esters (ASCn) are lipophilic forms of vitamin C, which act as skin permeation enhancers. We investigated the physical changes induced by incorporating ASCn into stratum corneum (SC) lipid membranes and correlated this with the mechanism proposed in the literature for skin permeation enhancement phenomena. We used lipid monolayers to explore the 2D structure and elasticity of the lipid-enhancer systems. As a comparison, the classic permeation enhancer, oleic acid (OA) and the non-enhancer analogue stearic acid (SA) were analysed. The incorporation of ASCn or OA into SC membranes resulted in more liquid-like films, with a dose-dependent lowering of the compressibility modulus. Brewster angle microscopy (BAM) evidenced partial miscibility of the enhancer with SC lipid components, stabilising the liquid-expanded phase. At the nanoscale, AFM showed that SC lipids form heterogeneous membranes, which underwent structural alterations after incorporating ASCn and fatty acids, such as SA and OA. The lower, cholesterol-enriched phase appears to concentrate the enhancers, whilst the higher ceramide-enriched phase concentrated the non-enhancer SA. Our results and previously reported pieces of evidence indicate a strong pattern in which the rheological properties of SC lipid films are determinant for skin permeation phenomena.

Comprehensive Study of the Liquid Expanded-Liquid Condensed Phase Transition in 1,2-Dimyristoyl-sn-glycero-3-phospho-l-serine Monolayers: Surface Pressure, Volta Potential, X-ray Reflectivity, and Molecular Dynamics Modeling.

An integrated approach is applied to reveal fine changes in the surface-normal structure of 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) monolayers at the air-lipid-water interface occurring in a liquid expanded (LE)-liquid condensed (LC) transition. The combination of the Langmuir monolayer technique, X-ray reflectometry, and molecular dynamics (MD) modeling provides new insight into the molecular nature of electrostatic phenomena in different stages of lipid compression. A homemade setup with a laboratory X-ray source (λ = 1.54 Å) offers a nondestructive way to reveal the structural difference between the LE and LC phases of the lipid. The electron density profile in the direction normal to the interface is recovered from the X-ray reflectivity data with the use of both model-independent and model-based approaches. MD simulations of the DMPS monolayer are performed for several areas per lipid using the all-atom force field. Using the conventional theory of capillary waves, a comparison is made between the electron density profiles reconstructed from the X-ray data and those calculated directly from MD modeling, which demonstrates remarkable agreement between the experiment and simulations for all selected lipid densities. This confirms the validity of the simulations and allows an analysis of the contributions of the hydrophobic tails and hydrated polar groups to the electron density profile and to the dipole component of the electric field at the interface. According to the MD data, the dependence of the Volta potential on the area per lipid in the monolayer has a different molecular nature below and above the phase transition. In the LE state of the monolayer, the potential is determined mostly by the oriented water molecules in the polar region of the lipid. In the LE-LC transition, these molecules are displaced to the bulk, and their effect on the Volta potential becomes insignificant compared with the contribution of the hydrophobic tails. The hydrophobic tails are highly ordered in the state of the liquid crystal so that their dipole moments entirely determine the growth of the potential upon compression up to the monolayer collapse.

Emergence of a linear slope region of the isotherm in the first-order liquid-expanded–liquid-condensed phase transition in Langmuir monolayers

A nonhorizontal slope in the isotherm has been observed in the two-phase coexisting region of the first-order liquid-expanded (LE)-liquid-condensed (LC) phase transition in Langmuir monolayers for many decades. We show that the simple analysis of a phenomenological Landau free energy involving the coupling-energy contributions of molecular lateral density (ρ) with spontaneous collective chain tilt (θ) and two-dimensional strain (ɛ_{s}) inside the LC domain enables one to understand the origin of a nonhorizontal straight-line slope in the LE-LC phase coexistence region of the isotherm. The presence of ρ-ɛ_{s} coupling must be essential for the appearance of the straight-line shape of a nonhorizontal plateau in the isotherm. Moreover, it is found from the comparison of the two-dimensional contour plots of the free energy that an LE phase may persist significantly even at the later stage of the straight-line regime beyond a transition midpoint surface pressure in the presence of this coupling. The persistence of the LE phase may lead to the delay of transition progress as manifested more clearly by the appearance of a compressibility plateau in the coexistence region that indicates the existence of persistent equilibrium density fluctuations in the monolayer.

Effects of γ-zein peptides on lipid membrane organization: Quartz crystal microbalance with dissipation and Langmuir monolayer studies

γ-zein peptides (VHLPPP)n present great potential as efficient molecule carriers across the cell membrane. To further understand the binding behavior of peptides with cell membrane, simplified models consisting of peptides and mono/bi-layer membranes (Langmuir monolayer and liposome) were used to studied the resulting response in the structure of peptide and cell membrane in the present work. The results showed that (VHLPPP)n=1,3 bound to dipalmitoylphosphatidylcholine (DPPC) liposomes and was induced to self-assemble into polyproline II (PP II) structure. The incorporation of (VHLPPP)n=1,3 in the DPPC Langmuir monolayers resulted in an increase in the surface pressure and a decrease in the collapse pressure, suggesting the increase of lipid monolayer fluidity. Fluorescence images further confirmed that the presence of (VHLPPP)n=1,3 promoted the formation of liquid-expanded (LE, dis-ordered) phases in lipid monolayers. Atomic force microscopy images found that (VHLPPP)n=1,3 preferred to penetrate in LE phase domains. In addition, (VHLPPP)3 possesses higher binding affinity with DPPC membrane models compared to (VHLPPP)1.

Temperature-Responsive Hydrophobic Silica Nanoparticle Ultrasound Contrast Agents Directed by Phospholipid Phase Behavior.

In this paper, we report ultrasonically active nanoscale contrast agents that behave as thermometric sensors through phase change in their stabilizing phospholipid monolayer. Phospholipid-stabilized, hydrophobic mesoporous silica nanoparticles (P@hMSNs) are known to interact with high-intensity focused ultrasound (HIFU) to promote cavitation at their surfaces, which can be used for both imaging and therapy. We show that the lateral lipid phase behavior of the phosphocholine lipid dictates the acoustic contrast of the P@hMSNs. When the lipids are in the gel phase below their melting temperature, the P@hMSNs generate detectable microbubbles when exposed to HIFU. However, if the lipids exhibit a liquid expanded phase, the P@hMSNs cease to generate bubbles in response to HIFU insonation. We verify that the heating and subsequent transition of lipid coating the hMSN are associated with the loss of acoustic response by doping laurdan dye into the lipid monolayer and imaging lipid phase through red shifts in emission spectra. Similarly, cessation of cavitation was also induced by adding a fluidizing surfactant such as Triton X, which could be reversed upon washing away the excess surfactant. Finally, by controlling for the partial fluidization caused by the adsorption of protein, P@hMSNs may be used as thermometric sensors of the bulk fluid temperature. These findings not only impact the utilization of nanoscale agents as stimulus-responsive ultrasound contrast agents but also have broader implications for how cavitation may be initiated at surfaces coated by a surfactant.

Electrostatic and Structural Effects at the Adsorption of Polylysine on the Surface of the DMPS Monolayer

The effect of the adsorption of a polypeptide on the lateral interaction of dimyristoylphosphatidylserine molecules in different phase states on the surface of a 10 mM KCl aqueous solution has been studied. Changes in the surface pressure and Volta potential induced by the adsorption of large poly-D-lysine molecules (about 200 links in a chain) have been determined at different areas per lipid molecule in a monolayer. The adsorption of macromolecules noticeably increases the elasticity of the monolayer under lateral compression in the liquid expanded state of lipid and reduces the effective dipole moment from 0.48 to 0.38 D. These properties are in qualitative agreement with X-ray reflectometry data for the lipid monolayer obtained with synchrotron radiation with a photon energy of ≈70 keV. The electron density profiles perpendicular to the surface of the aqueous subphase have been reconstructed from reflectometry data within a model approach to the structure of an interface with two and three layers. These profiles indicate the existence of a wide diffuse polymer layer (150 ± 40) A in width at the interface of the monolayer in both the liquid expanded and liquid condensed states. A decrease in the area per molecule in the monolayer by a factor of 2 results in the doubling of the surface density of the macromolecule film. The adsorption of the polymer also affects the integral density of the layer of polar phospholipid groups, which decreases by a factor of ≈2 in the liquid expanded phase and by ∼30% in the liquid condensed phase.

Interaction of inhalable volatile organic compounds and pulmonary surfactant: Potential hazards of VOCs exposure to lung.

Exposure of volatile organic compounds (VOCs) towards lung leads to pulmonary dysfunctions and various lung diseases. However, the interaction of VOCs with pulmonary surfactant (PS) that directly comes into contact with inhaled VOCs is unknown. Here, simulated PS extracted from porcine lungs (EPS) was used to study the interaction with BTEX (i.e., benzene, toluene, ethylbenzene, and p-xylene) as representatives of VOCs. Surface pressure-area (π-A) isotherms showed that in the presence of individual BTEX, EPS monolayer's phase conversion from gas to liquid expanded phase was dramatically influenced and its collapse pressure decreased greatly compared to those of EPS alone, which was attributed to the alteration of EPS monolayer's microstructure characterized by atomic force microscopy and Brewster angle microscopy. Solubilization experiments manifested that EPS and its major components (dipalmitoyl phosphatidylcholine, DPPC; bovine serum albumin, BSA) exhibited obvious solubilization effects on individual BTEX. The solubilization capacity followed an order: EPS > DPPC > BSA, which was positively correlated with hydrophobicity of individual BTEX. Synergistic solubilization test unveiled that the mixed phospholipid components were largely responsible for the solubilization capacity of EPS. These findings indicate that VOCs exposure may induce potential pulmonary health risk due to the alteration of gas-liquid interfacial properties of PS.

Influence of volatile organic compounds (VOCs) on pulmonary surfactant monolayers at air-water interface: Implication for the pulmonary health

Understanding the interaction of pulmonary surfactant (PS) and volatile organic compounds (VOCs) at air-water interface is crucial for the assessment of pulmonary health. In this study, dipalmitoyl phosphatidylcholine (DPPC), the mainly effective components of PS, was used as a mimic of PS employed to explore its surface activity in the exposure of VOCs. The results showed that the surface tension of DPPC decreased significantly with the addition of VOCs. On a basis of structure-activity assessment, hydrogen-bond basicity and dispersion/cavity interaction of VOCs are responsible for the decreasing capacity of VOCs for DPPC’s surface tension. Surface pressure-molecular area (π-A) isotherms demonstrate that the phase conversion of DPPC monolayer from gas to liquid expanded phase was greatly impacted by VOCs. The collapse pressure of DPPC monolayer in VOCs exposure are lower compared to that of DPPC alone, which can be attributed to the lowest surface tension of DPPC solutions mixed with VOCs. Atomic force microscopy analysis elucidates that the presence of VOCs led to bigger apertures area of DPPC monolayer than that of DPPC alone, which is indicative of microstructure changes of DPPC monolayer in the exposure of VOCs. This study provides new evidences that the exposure of VOCs on DPPC may alter the air-water interface activity of PS.

Observation by optical second harmonic generation of the mean tilt angle of cyanine dyes during compression with a phase transition in a Langmuir-Blodgett trough

Optical second harmonic generation (SHG) signals from a cyanine dye during compression of a monolayer in a Langmuir-Blodgett trough were measured to investigate the mean tilt angle of the dye corresponding to the formation of an H-band. The pressure-surface area (π-A) isotherm for 3,3′-dioctadecyloxacarbocyanine molecules spread on an aqueous subphase showed a plateau region after a kink point in the isotherm characteristic, which suggests a phase transition from the liquid expanded (LE) phase to the liquid condensed (LC) phase. SHG signals from the monolayer were obtained as the surface area decreased for the input and output polarization combinations PP, MS, SP, PS, and SS, where the first and second letters denote the input and output polarizations, respectively, and M refers to the polarization midway between S and P. For polarization combination PP, the SHG signal increased steeply during compression, whereas for polarization combination MS, the signal increased gently. The SH field from the monolayer for polarization combination SP in the LC phase was almost inverted to the field in the LE phase. We estimated the mean polar angles from the SHG responses during compression. The angle decreased continuously by almost 5° from 28° with the growth of domains in the LC phase. Visible absorption spectra of monolayers transferred to glass substrates were also obtained. The area intensity of the peak in the spectra attributed to H-aggregates increased compared with that of the monomer as the surface area decreased in the LC phase. The results were consistent with the polar angle reduction in the LC phase observed by in situ SHG. The decrease of the angle in the LC phase probably allowed the cyanine dyes to pack together tightly and align side by side, promoting H-aggregation.

Quantification of PEG40St squeeze out from DSPC/PEG40St monolayers at higher molar ratios

Mixtures of 1,2 Distearoyl-sn-glycero-3-phosphocholine (DSPC) and polyoxyethylene-40-stearate (PEG40St) were prepared at different molar ratios and their miscibility were investigated using Langmuir isotherms. Pure DSPC monolayer exhibited a liquid-condensed (LC) phase whereas PEG40St monolayer exhibited liquid-expanded (LE) phase at the air-water interface at 22 ± 2 °C. At the collapse pressure of 33 mN/m, the PEG40St mean molecular area was calculated to be 28 Å2/molecule for 9:1 composition and 50 Å2/molecule for 5:5 composition, showing an increasing trend with the emulsifier content. A quantification method was developed to estimate the squeeze out amount of PEG40St from Langmuir isotherms of the DSPC/PEG40St mixtures at different molar ratios. Almost 93%, 82%, and 53% of PEG40St displaced for the 9:1, 7:3, and 5:5 mixtures, respectively, at the end of the first collapse plateau and showed a decreasing trend with the PEG40St content. Remaining PEG40St squeezed out at the end of the second collapse plateau, where 20% of PEG40St still contained within the 5:5 composition. It was concluded that increasing PEG40St content would be advantageous to design more stable lipid based microbubbles.

Interactions between model cell membranes and the neuroactive drug propofol

Vibrational sum frequency spectroscopy (VSFS) complemented by surface pressure isotherm and neutron reflectometry (NR) experiments were employed to investigate the interactions between propofol, a small amphiphilic molecule that currently is the most common general anaesthetic drug, and phospholipid monolayers. A series of biologically relevant saturated phospholipids of varying chain length from C18 to C14 were spread on either pure water or propofol (2,6-bis(1-methylethyl)phenol) solution in a Langmuir trough, and the change in the molecular structure of the film, induced by the interaction with propofol, was studied with respect to the surface pressure. The results from the surface pressure isotherm experiments revealed that propofol, as long as it remains at the interface, enhances the fluidity of the phospholipid monolayer. The VSF spectra demonstrate that for each phospholipid the amount of propofol in the monolayer region decreases with increasing surface pressure. Such squeeze out is in contrast to the enhanced interactions that can be exhibited by more complex amphiphilic molecules such as peptides. At surface pressures of 22–25 mN m−1, which are relevant for biological cell membranes, most of the propofol has been expelled from the monolayer, especially in the case of the C16 and C18 phospholipids that adopt a liquid condensed phase packing of its alkyl tails. At lower surface pressures of 5 mN m−1, the effect of propofol on the structure of the alkyl tails is enhanced when the phospholipids are present in a liquid expanded phase. Specifically, for the C16 phospholipid, NR data reveal that propofol is located exclusively in the head group region, which is rationalized in the context of previous studies. The results imply a non-homogeneous distribution of propofol in the plane of real cell membranes, which is an inference that requires urgent testing and may help to explain why such low concentration of the drug are required to induce general anaesthesia.

Temperature-Dependent Phase Behavior of Langmuir Films of 10,12-Pentacosadiynoic Acid at the Air/Water Interface and Its Effects on Chromatic Stability of the Polymerized Langmuir-Schaefer Films

The effect of temperature on the two-dimensional phase of 10,12-pentacosadiynoic acid (PCDA) Langmuir films at the air/water interface was investigated. The temperature of the Langmuir films was precisely controlled from 5 to 50 °C and their surface isotherms and in-situ visible absorption spectra were acquired. Depending on the temperature, the PCDA Langmuir films were found to be classified into a liquid-condensed (low temperatures, 5 and 25 °C) and a liquid-expanded phase (high temperatures, 40 and 50 °C). After polymerizing the PCDA Langmuir films with 254 nm UV light at the specific temperatures, the films were transferred to hydrophobic glass using the Langumir-Schaefer (LS) method. Upon thermal and pH stimuli, their chromatic transition characteristics were analyzed by visible spectroscopy. The liquid-condensed films were found to be more susceptible to thermal stimulus than the liquid-expanded films. The latter also showed remarkable chromatic stability against the pH in the region from 2 to 11, compared to the former. Thus, when sturdy films are required, the multilayered PCDA LS films prepared at the liquid-expanded phase are more suitable than the liquid-condensed films, and vice versa. This result is expected to be very useful for controlling the sensitivity and stability of polydiacetylene-based sensory systems simply by changing the polymerization temperature, even without synthesizing new monomers.

The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane.

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.

Aescin Incorporation and Nanodomain Formation in DMPC Model Membranes.

The saponin aescin from the horse chestnut tree is a natural surfactant well-known to self-assemble as oriented-aggregates at fluid interfaces. Using model membranes in the form of lipid vesicles and Langmuir monolayers, we study the mixing properties of aescin with the phase-segregating phospholipid 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC). The binary membranes are experimentally studied on different length scales ranging from the lipid headgroup area to the macroscopic scale using small-angle X-ray scattering (SAXS), photon correlation spectroscopy (PCS), and differential scanning calorimetry (DSC) with binary bilayer vesicles and Langmuir tensiometry (LT) with lipid monolayers spread on the surface of aescin solutions. The binary interaction was found to strongly depend on aescin concentration in two well differentiated concentration regimes. Below 7 mol %, the results reveal phase segregation of nanometer-sized aescin-rich domains in an aescin-poor continuous bilayer. Above this concentration, aescin-aescin interactions dominate, which inhibit vesicle formation but lead to the formation of new membrane aggregates of smaller sizes. From LT studies in monolayers, the interaction of aescin with DMPC was shown to be stronger in the condensed phase than in the liquid expanded phase. Furthermore, a destructuring role was revealed for aescin on phospholipid membranes, similar to the fluidizing effect of cholesterol and nonsteroidal anti-inflammatory drugs (NSAIDs) on lipid bilayers.

The insertion of Polybia-MP1 peptide into phospholipid monolayers is regulated by its anionic nature and phase state

Polybia-MP1 or simply MP1 (IDWKKLLDAAKQIL-NH2) is a peptide with broad-spectrum bactericidal activity and a strong inhibitory effect against cancer cells. The aim of this work was to evaluate the effect of biophysical properties such as membrane texture and film thickness on MP1 interaction with neutral and anionic lipid membranes. For this purpose, we first explored the peptide’s surface behavior. MP1 showed high surface activity, adsorbing onto bare air/aqueous interfaces up to higher surface pressures than the collapse pressure of MP1 Langmuir films. The MP1-lipid membrane interaction was studied using Langmuir phosphatidylcholine and phosphatidylserine (PS) monolayers as model membrane systems. PS was chosen since this negatively charged lipid was found predominantly on the outer leaflet of tumor cells, and it enhances MP1 activity for PS-containing membranes to a greater extent than for other negatively charged lipids. MP1 incorporated into anionic PS monolayers, which show a liquid-expanded (LE) phase or LE-liquid-condensed (LC) phase coexistence, up to lipid-packing densities higher than those of cell membranes. The mixed lipid/MP1 films were explored by Brewster angle microscopy and atomic force microscopy. MP1 partitioned preferentially into the LE phase state of PS films, and were thus excluded from the coexisting LC phase. This interaction had strong electrostatic bases: in pure water, the lipid-peptide interaction was strong enough to induce formation of reversible lipid-peptide 3D structures associated with the interface. MP1 incorporation into the LE phase was accompanied by a shift of the phase transition pressure to higher values and a thinning of the lipid film. These results showed a clear correlation between peptide penetration capacity and the presence or induction of the thin LE phase. This capacity to regulate membrane physical properties may be of relevance in the binding, incorporation and membrane selectivity of this promising antitumor peptide.

Energetic Basis for Inhibition of Calcium Phosphate Biomineralization by Osteopontin.

Calcium oxalate kidney stones form attached to Randall's plaques (RP), calcium phosphate (Ca-P) deposits on the renal papillary surface. Osteopontin (OPN) suppresses crystal growth in the complex process of urinary stone formation, but the inhibitory role of active domains of OPN involved in the initial formation of the RPs attached to epithelial cells has yet to be clarified. Here we demonstrate the thermodynamic basis for how OPN sequences regulate the onset of Ca-P mineral formation on lipid rafts as a model membrane. We first quantify the kinetics of hydroxyapatite (HAP) nucleation on membrane substrates having liquid-condensed (LC) and liquid-expanded (LE) phases using in situ atomic force microscopy (AFM). We find that rates are sequence-dependent, and the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy γ. Combined with single-molecule determination of the binding energy (ΔGB) of the OPN peptide segments adsorbed to the HAP (100) face, we show a linear relationship of γ and ΔGB, suggesting that the increase in the nucleation barriers correlates with strong peptide-crystal nuclei binding. These findings reveal fundamental energetic clues for inhibition of membrane-mediated nucleation by sequence motifs and subdomains within the OPN protein through spatial location of charged moieties and provide insight connecting peripheral cell membranes to pathological mineralization.

The miscibility of milk sphingomyelin and cholesterol is affected by temperature and surface pressure in mixed Langmuir monolayers.

The miscibility of milk sphingomyelin (milk-SM) and cholesterol was investigated in this study. The effect of different physical states of milk-SM on its interactions with cholesterol was determined by the recording of isotherms of compression of Langmuir films for temperatures above and below the gel to Lα phase transition of milk-SM (Tm∼34°C). For T=15°C<Tm, the liquid expanded (LE) to liquid condensed (LC) phase transition of milk-SM monolayers was observed at surface pressures of 10-15mN/m. For T=43°C>Tm, the milk-SM molecules were in a LE phase regardless of the surface pressure applied. A phase diagram pressure - milk-SM/cholesterol composition was established. This study demonstrated that both temperature and surface pressure affected the miscibility between the milk-SM and cholesterol. The strongest attractive forces (i.e. condensing effect) were identified for 30mol% cholesterol when the milk-SM was in the LE phase state.

Supercritical carbon dioxide as solvent in the lipase-catalyzed ethanolysis of fish oil: Kinetic study

Supercritical carbon dioxide (SC-CO2) has been used as green solvent in the lipase-catalyzed ethanolysis of fish oil by Lipozyme RM IM at mild, non-oxidative conditions and with no solvent residues. The effect of experimental conditions, initial substrate ethanol/oil molar ratio (2–38), pressure (7.5–30MPa), and temperature (323.15–353.15K) on equilibrium conversion, reaction rate and oxidative status of the products has been studied. No ethanol inhibition has been observed at high concentrations of ethanol, when putting in contact first the fish oil with the enzyme avoiding direct contact between the biocatalyst and ethanol. Operating pressure affected positively the reaction performance in the range investigated. Visual observation of the phase behaviour of the initial reaction mixture showed an “expanded liquid phase” that helped enhancing reaction rate, and a gas phase. Raising temperature accelerated the reaction up to a limit (343.15K), observing higher enzyme thermal stability than in other reaction media (313.15K). However, lipid oxidation increases with temperature. Up to 86±1% FAEE yield has been found at MR=6:1, 30MPa and 323.15K. Kinetic data have been correlated by using a mathematical model based on the elementary reactions of the 3-step transesterification. Kinetic rate constants, apparent activation volumes and energies are reported for the first time for a lipase-catalyzed ethanolysis reaction in SC-CO2.

Studying Gastric Lipase Adsorption Onto Phospholipid Monolayers by Surface Tensiometry, Ellipsometry, and Atomic Force Microscopy.

The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of the overall catalytic process carried out by these interfacial enzymes. Gastric lipase, for instance, shows an apparent optimum activity on triglycerides (TAG) at acidic pH, which is controlled by its pH-dependent adsorption at lipid-water interfaces. Since gastric lipase acts on TAG droplets covered by phospholipids, but does not hydrolyze these lipids, phospholipid monolayers spread at the air-water interfaces can be used as biomimetic interfaces to study lipase adsorption and penetration through the phospholipid layer, independently from the catalytic activity. The adsorption of recombinant dog gastric lipase (rDGL) onto 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monolayers can be monitored by surface tensiometry at various enzyme concentrations, pHs, and surface pressures (Π). These experimental data and the use of Langmuir adsorption isotherm and Verger-de Haas' lipase kinetics models further allow estimating various parameters including the adsorption equilibrium constant (KAds), the interfacial concentration [Formula: see text] , the molar fraction [Formula: see text] (ΦE*(%), mol%), and the molecular area [Formula: see text] of rDGL adsorbed onto the DLPC monolayer under various conditions. Additional insight into rDGL adsorption/insertion on phospholipid monolayers can be obtained by combining ellipsometry, Langmuir-Blodgett film transfer, and atomic force microscopy. When using multicomponent phospholipid monolayers with phase separation, these techniques allow to visualizing how rDGL preferentially partitions toward liquid expanded phase and at phase boundaries, gets adsorbed at various levels of insertion and impacts on the lateral organization of lipids.

Model Lung Surfactant Films: Why Composition Matters.

Lung surfactant replacement therapies, Survanta and Infasurf, and two lipid-only systems both containing saturated and unsaturated phospholipids and one containing additional palmitic acid were used to study the impact of buffered saline on the surface activity, morphology, rheology, and structure of Langmuir monolayer model membranes. Isotherms and Brewster angle microscopy show that buffered saline subphases induce a film expansion, except when the cationic protein, SP-B, is present in sufficient quantities to already screen electrostatic repulsion, thus limiting the effect of changing pH and adding counterions. Grazing incidence X-ray diffraction results indicate an expansion not only of the liquid expanded phase but also an expansion of the lattice of the condensed phase. The film expansion corresponded in all cases with a significant reduction in the viscosity and elasticity of the films. The viscoelastic parameters are dominated by liquid expanded phase properties and do not appear to be dependent on the structure of the condensed phase domains in a phase separated film. The results highlight that the choice of subphase and film composition is important for meaningful interpretations of measurements using model systems.