Aescin-Cholesterol Complexes in DMPC Model Membranes: A DSC and Temperature-Dependent Scattering Study.

The saponin aescin, a mixture of triterpenoid saponins, is obtained from the seeds of the horse chestnut tree Aesculus hippocastanum. The ß-form employed in this study is haemolytically active. The haemolytic activity results from the ability of aescin to form strong complexes with cholesterol in the red blood cell membrane. In this study, we provide a structural analysis on the complex formation of aescin and cholesterol when embedded in a phospholipid model membrane formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In this work, the temperatures investigated extend from DMPC's Lß' to its Lα phase in dependence of different amounts of the saponin (0-6 mol% for calorimetric and 0-1 mol% for structural analyses) and the steroid (1-10 mol%). At these aescin contents model membranes are conserved in the form of small unilamellar vesicles (SUVs) and major overall structural modifications are avoided. Additionally, interactions between aescin and cholesterol can be studied for both phase states of the lipid, the gel and the fluid state. From calorimetric experiments by differential scanning calorimetry (DSC), it could be shown that both, the steroid and the saponin content, have a significant impact on the cooperative phase transition behaviour of the DMPC molecules. In addition, it becomes clearly visible that the entire phase behaviour is dominated by phase separation which indeed also depends on the complexes formed between aescin and cholesterol. We show by various methods that the addition of cholesterol alters the impact of aescin on structural parameters ranging from the acyl chain correlation to vesicle-vesicle interactions. While the specific saponin-phospholipid interaction is reduced, addition of cholesterol leads to deformation of SUVs. The analyses of the structures formed were performed by wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS).

Temperature dependent self-organization of DMPC membranes promoted by intermediate amounts of the saponin aescin.

The plant-derived biosurfactant aescin is naturally present in many plants and is used for treatment of disorders such as varicose veins and inflammation of veins. The hemolytic activity of this saponin is attributed to its interaction with cholesterol in the red blood cell membrane. This work investigates the phase and aggregation behavior of saponin-containing model membranes consisting of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The aescin concentrations studied range from 1 mol% to 7 mol% with respect to the total lipid content. The methods of choice to elucidate the structural picture are small-angle scattering of X-rays (SAXS) and neutrons (SANS) and cryogenic transmission electron microscopy (cryo-TEM). SANS and SAXS revealed that at lower aescin contents vesicular structures are conserved and vesicles tend to aggregate already at aescin contents of around 1 mol%. Aggregation and vesicle deformation effects are found to be stronger when the phospholipids are in the L [Formula: see text] phase. With increasing aescin content, mixed structures, i.e. aggregated and deformed vesicles and solubilized bilayer fragments, are present. This was proven for a sample with 4 mol% aescin by cryo-TEM. An increasing aescin amount leads to membrane decomposition and free standing bilayers which tend to build stacks at high temperature. These stacks are characterized by SAXS using the modified Caillé theory. Analyses and model dependent fitting reveal formation of well-defined structures beginning at 7 mol% aescin.

Interaction of the Saponin Aescin with Ibuprofen in DMPC Model Membranes.

In the present work, we study the interaction of the saponin aescin with the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen at concentrations of 1.2-2.5 mM. These amounts are higher than those usually used for medication (10-300 µM) to show possible structures and formulations for orally absorbed drug delivery systems. It is shown how the interaction of both substances, separately or together, alters the thermotropic phase behavior of the 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayer in the presence of different amounts of aescin, ranging from 20 µM to 1 mM. The methods of choice are differential scanning calorimetry (DSC), and additionally wide-angle (WAXS) and small-angle X-ray scattering (SAXS). We found that these two additives, aescin and ibuprofen, alter the temperature-dependent structural appearance of the DMPC membrane depending on the aescin and drug content. The presence of the saponin and the drug become visible on different length scales, i.e., ranging from a global structural change to inner-membrane interactions. DSC reveals that the drug and saponin alter the cooperativity of the DMPC phase transition in a concentration-dependent manner. Furthermore, there is a significant difference between the drug-containing compared to the drug-free systems. By WAXS, we could resolve that aescin reverses the strong impact of ibuprofen on the diffraction peak of DMPC. Both molecules interact strongly with the phospholipid headgroups. This becomes visible in a changing area per lipid and shifting phase transition to higher temperatures. SAXS experiments reveal that the addition of ibuprofen leads to major morphological changes in the phospholipid bilayer. SAXS experiments performed on representative samples do not only cover the drug-saponin interaction within the bilayer from the structural perspective but also confirm the visually observed macroscopic concentration and temperature-dependent phase behavior. Vesicular shape of extruded samples is conserved at low aescin contents. At intermediate aescin content, aggregation between vesicles occurs, whereby the strength of aggregation is reduced by ibuprofen. At high aescin contents, DMPC bilayers are solubilized. The kind of formed structures depends on temperature and drug content. At low temperature, separated bilayer sheets are formed. Their size increases with ibuprofen in a concentration-dependent manner. At high temperature, the drug-free system reorganizes into stacked sheets. Whereas sheets at 5 mol % ibuprofen close to vesicles, the ones with 10 mol % of the drug increase massively in size. Altogether, ibuprofen was found to rather enhance than inhibit structural and thermotropic membrane modifications induced by the aescin on the DMPC model membrane.

DMPC vesicle structure and dynamics in the presence of low amounts of the saponin aescin.

Vesicle shape and bilayer parameters are studied by small-angle X-ray (SAXS) and small-angle neutron (SANS) scattering in the presence of the saponin aescin. We confirm successful incorporation of aescin molecules by analysis of the radii of gyration RG and study furthermore the impact of aescin incorporation on bilayer thickness parameters from the neutron and X-ray perspective. Additionally, the bending elasticity (κ) of these 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicle bilayers is studied in the presence of aescin. Neutron spin-echo spectroscopy (NSE) allows to detect subtle changes in the dynamics and κ of lipid membranes. Changes of κ are detectable at temperatures below and above the main phase transition temperature Tm of the lipid. The impact of aescin is much more significant below Tm. It has been found that below Tm the addition of aescin to the vesicles decreases the value of κ and softens the bilayer. Above Tm the value of κ increases with increasing aescin content and the bilayer becomes more rigid. Altogether, we demonstrate by analysis of the structure and dynamics of the vesicles that the impact of aescin strongly depends on the lipid state. Below Tm the membrane becomes fluidized and softer, above Tm solidified and stiffer compared to a DMPC membrane without additive at similar conditions.

Formation of a transient amorphous solid in low density aqueous charged sphere suspensions.

Colloidal glasses formed from hard spheres, nearly hard spheres, ellipsoids and platelets or their attractive variants, have been studied in great detail. Complementing and constraining theoretical approaches and simulations, the many different types of model systems have significantly advanced our understanding of the glass transition in general. Despite their early prediction, however, no experimental charged sphere glasses have been found at low density, where the competing process of crystallization prevails. We here report the formation of a transient amorphous solid formed from charged polymer spheres suspended in thoroughly deionized water at volume fractions of 0.0002-0.01. From optical experiments, we observe the presence of short-range order and an enhanced shear rigidity as compared to the stable polycrystalline solid of body centred cubic structure. On a density dependent time scale of hours to days, the amorphous solid transforms into this stable structure. We further present preliminary dynamic light scattering data showing the evolution of a second slow relaxation process possibly pointing to a dynamic heterogeneity known from other colloidal glasses and gels. We compare our findings to the predicted phase behaviour of charged sphere suspensions and discuss possible mechanisms for the formation of this peculiar type of colloidal glass.

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.

Sugar Surfactant Based Microemulsions at Solid Surfaces: Influence of the Oil Type and Surface Polarity.

The structure of sugar-surfactant-based bicontinuous microemulsions in the bulk and at hydrophilic and hydrophobic solid planar surfaces was studied by means of neutron scattering techniques (SANS, NR, and GISANS). In particular, the influence of the type of oil (tetradecane and methyl oleate) on the structural properties in the vicinity of surfaces was investigated at different oil-to-water ratios. In the case of hydrophilic surfaces, the analysis of the scattering length density profiles reveals an induced ordering of the oil and water domains perpendicular to the solid-liquid interface in both sets of microemulsions. At hydrophobic surfaces, differences in the near-surface ordering between microemulsions containing polar and nonpolar oils are observed.