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.

Determination of Internal Density Profiles of Smart Acrylamide-Based Microgels by Small-Angle Neutron Scattering: A Multishell Reverse Monte Carlo Approach.

The internal structure of nanometric microgels in water has been studied as a function of temperature, cross-linker content, and level of deuteration. Small-angle neutron scattering from poly( N-isopropylmethacrylamide) (volume phase transition ≈ 44 °C) microgel particles of radius well below 100 nm in D2O has been measured. The intensities have been analyzed with a combination of polymer chain scattering and form-free radial monomer volume fraction profiles defined over spherical shells, taking polydispersity in size of the particles determined by atomic force microscopy into account. A reverse Monte Carlo optimization using a limited number of parameters was developed to obtain smoothly decaying profiles in agreement with the experimentally scattered intensities. The results are compared to the swelling curve of microgel particles in the temperature range from 15 to 55 °C obtained from photon correlation spectroscopy (PCS). In addition to hydrodynamic radii measured by PCS, our analysis provides direct information about the internal water content and gradients, the strongly varying steepness of the density profile at the particle-water interface, the total spatial extension of the particles, and the visibility of chains. The model has also been applied to a variation of the cross-linker content, N, N'-methylenebisacrylamide, from 5 to 15 mol %, providing insight on the impact of chain architecture and cross-linking on water uptake and on the definition of the polymer-water interface. The model can easily be generalized to arbitrary monomer contents and types, in particular mixtures of hydrogenated and deuterated species, paving the way to detailed studies of monomer distributions inside more complex microgels, in particular core-shell particles.

Smart Starch-Poly( N-isopropylacrylamide) Hybrid Microgels: Synthesis, Structure, and Swelling Behavior.

In this study, we present hybrid microgels made of starch nanoparticles (SNPs) and poly( N-isopropylacrylamide) [p(NIPAM)]. SNPs were formed through nanoprecipitation. Hybrid microgels were prepared by surfactant-free precipitation polymerization (SFPP) or in the presence of surfactant precipitation polymerization (PP) at different NIPAM/SNP ratios. Dynamic light scattering results of hybrid microgels synthesized by SFPP revealed changes in volume phase transition temperature according to SNP amount, where the increase in the hydrophilic content caused small shifts in the lower critical solution temperature (LCST), reaching nearly 35 °C. Colloidal stability was improved with the SNP content, leading to increased stability because of the hydroxyl groups. Small-angle X-ray scattering indicates a core-shell structure above the LCST, where SNPs chains cover a p(NIPAM) core. Swelling curves experimentally obtained were analyzed using the Flory-Rehner model, where the interaction parameter (χ) has been modeled either by a series expansion of the swelling ratio or by a Hill-like equation for a cooperative thermotropic transition.

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.

Polyacrylates in the presence of an extraordinary monovalent cation-Solution behavior and metal nanoparticle formation.

Sodium polyacrylate (NaPA) in dilute aqueous solution at an ionic strength of [NaNO3] = 0.01M establishes a rich phase behavior in the presence of low amounts of silver cations, which were introduced at a few millimoles or less by replacing the corresponding amount of Na+ cations. Beyond an extremely low level of Ag+ cations, anionic PA chains aggregate. By increasing the concentration of Ag+, the aggregates become denser and keep on growing without limit. Once a certain range of [Ag+] is reached, the instantaneously formed dense aggregates remain stable. Irradiation of the PA aggregate solutions with UV-light induces formation of silver nanoparticles (Ag-Nps). Based on a combination of UV-vis spectroscopy, light scattering, transmission electron microscopy, and small angle neutron scattering, the mechanism of this NaPA assisted formation of Ag-Nps is studied. One focus of the study is lying on the effect of the two different solution states of dense aggregates, corresponding to the unstable growing AgPA aggregates and to the stable AgPA aggregates and another focus is aiming at the characterisation of the morphology of the generated hybrid particles composed of Ag-Nps and hosting PA chains.

Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate.

l-Amino acid oxidases (l-AAO) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS) stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

Redox-Dependent Conformational Dynamics of Decameric 2-Cysteine Peroxiredoxin and its Interaction with Cyclophilin 20-3.

2-Cysteine peroxiredoxins (2-CysPrxs) switch between functions as a thiol peroxidase, chaperone, an interaction partner and possibly a proximity-based oxidase in a redox-dependent manner. In photosynthetic eukaryotes, 2-CysPrx localizes to the plastid, functions in the context of photosynthesis and enables an ascorbate peroxidase-independent water-water cycle for detoxifying H2O2 The high degree of evolutionary conservation of 2-CysPrx suggests that the switching is an essential characteristic and needed to transduce redox information to downstream pathways and regulation. The study aimed at exploring the dissociation behavior of 2-CysPrx and its interactions with cyclophilin depending on bulk phase conditions. Isothermal titration microcalorimetry (ITC), dynamic light scattering and size exclusion chromatography (SEC) proved the previously suggested model that reduced 2-CysPrx below a critical transition concentration (CTC) exists in its dimeric state, and above the CTC adopts the decameric state. The presence of cyclophilin 20-3 (Cyp20-3) affected the CTC of a 2-CysPrx decamer suggesting interaction which was further quantified by direct titration of 2-CysPrx with Cyp20-3, and in overlays. Finally catalytic inactivation assays showed the higher catalytic efficiency of 2-CysPrx at pH 8 compared with pH 7.2, but also revealed increased inactivation by hyperoxidation at pH 8. Interestingly, calculation of the average turnover number until inactivation gave rather similar values of 243 and 268 catalytic cycles at pH 8 and pH 7.2, respectively. These quantitative data support a model where 2-CysPrx and Cyp20-3, by interaction, form a redox-sensitive regulatory module in the chloroplast which is under control of the photosynthesis-linked stromal pH value, the redox state and additional stromal protein factor(s).

Inner structure of adsorbed ionic microgel particles.

Microgel particles of cross-linked poly(NIPAM-co-acrylic acid) with different acrylic acid contents are investigated in solution and in the adsorbed state. As a substrate, silicon with a poly(allylamine hydrochloride) (PAH) coating is used. The temperature dependence of the deswelling of the microgel particles was probed with atomic force microscopy (AFM). The inner structure of the adsorbed microgel particles was detected with grazing incidence small angle neutron scattering (GISANS). Small angle neutron scattering (SANS) on corresponding microgel suspensions was performed for comparison. Whereas the correlation length of the polymer network shows a divergence in the bulk samples, in the adsorbed microgel particles it remains unchanged over the entire temperature range. In addition, GISANS indicates changes in the particles along the surface normal. This suggests that the presence of a solid surface suppresses the divergence of internal fluctuations in the adsorbed microgels close to the volume phase transition.

Bending stiffness of biological membranes: what can be measured by neutron spin echo?

Large vesicles obtained by the extrusion method represent adequate membrane models to probe membrane dynamics with neutron radiation. Particularly, the shape fluctuations around the spherical average topology can be recorded by neutron spin echo (NSE). In this paper we report on the applicable theories describing the scattering contributions from bending-dominated shape fluctuations in diluted vesicle dispersions, with a focus on the relative relevance of the master translational mode with respect to the internal fluctuations. Different vesicle systems, including bilayer and non-bilayer membranes, have been scrutinized. We describe the practical ranges where the exact theory of bending fluctuations is applicable to obtain the values of the bending modulus from experiments, and we discuss about the possible internal modes that could be alternatively contributing to shape fluctuations.

Thermoresponsive Microgel Coatings as Versatile Functional Compounds for Novel Cell Manipulation Tools.

For the effective use of live cells in biomedicine as in vitro test systems or in biotechnology, non-invasive cell processing and characterisation are key elements. Thermoresponsive polymer coatings have been demonstrated to be highly beneficial for controlling the interaction of adherent cells through their cultivation support. However, the widespread application of these coatings is hampered by limitations in their adaptability to different cell types and because the full range of applications has not yet been fully explored. In the work presented here, we address these issues by focusing on three different aspects. With regard to the first aspect, by using well-defined laminar flow in a microchannel, a highly controllable and reproducible shear force can be applied to adherent cells. Employing this tool, we demonstrate that cells can be non-invasively detached from a support using a defined shear flow. The second aspect relates to the recent development of simple methods for patterning thermoresponsive coatings. Here, we show how such patterned coatings can be used for improving the handling and reliability of a wound-healing assay. Two pattern geometries are tested using mouse fibroblasts and CHO cells. In terms of the third aspect, the adhesiveness of cells depends on the cell type. Standard thermoresponsive coatings are not functional for all types of cells. By coadsorbing charged nanoparticles and thermoresponsive microgels, it is demonstrated that the adhesion and detachment behaviour of cells on such coatings can be modulated.

Smart Homopolymer Microgels: Influence of the Monomer Structure on the Particle Properties.

In this work, we compare the properties of smart homopolymer microgels based on N-n-propylacrylamide (NNPAM), N-isopropylacrylamide (NIPAM) and N-isopropylmethacrylamide (NIPMAM) synthesized under identical conditions. The particles are studied with respect to size, morphology, and swelling behavior using scanning electron and scanning force microscopy. In addition, light scattering techniques and fluorescent probes are employed to follow the swelling/de-swelling of the particles. Significant differences are found and discussed. Poly(N-n-propylacrylamide) (PNNPAM) microgels stand out due to their very sharp volume phase transition, whereas Poly(N-isopropylmethacrylamide) (PNIPMAM) particles are found to exhibit a more homogeneous network structure compared to the other two systems.

Fluctuation dynamics of bilayer vesicles with intermonolayer sliding: experiment and theory.

The presence of coupled modes of membrane motion in closed shells is extensively predicted by theory. The bilayer structure inherent to lipid vesicles is suitable to support hybrid modes of curvature motion coupling membrane bending with the local reorganization of the bilayer material through relaxation of the dilatational stresses. Previous experiments evidenced the existence of such hybrid modes facilitating membrane bending at high curvatures in lipid vesicles [Rodríguez-García, R., Arriaga, L.R., Mell, M., Moleiro, L.H., López-Montero, I., Monroy, F., 2009. Phys. Rev. Lett. 102, 128201.]. For lipid bilayers that are able to undergo intermonolayer sliding, the experimental fluctuation spectra are found compatible with a bimodal schema. The usual tension/bending fluctuations couple with the hybrid modes in a mechanical interplay, which becomes progressively efficient with increasing vesicle radius, to saturate at infinity radius into the behavior expected for a flat membrane. Grounded on the theory of closed shells, we propose an approximated expression of the bimodal spectrum, which predicts the observed dependencies on the vesicle radius. The dynamical features obtained from the autocorrelation functions of the vesicle fluctuations are found in quantitative agreement with the proposed theory.

Thermoresponsive copolymer microgels.

Colloidal aqueous microgels have attracted notable attention due to their possible applications in both polymer chemistry and the (bio)material sector. Due to their soft internal network structure and their response to different external stimuli, they are suitable as carrier systems for different kinds of molecules (e.g. nanoparticles, drugs, diagnostic agents, and enzymes), as optically active materials or even as model systems for fundamental research on soft condensed matter. Within the class of microgels, a variety of molecular structures is possible. The aim of this manuscript is to review recent progress in the domain of copolymer microgels with different architectures. Although most of the work is based on microgels with poly(N-isopropyl acrylamide) (NIPAM) as one component, non-NIPAM based copolymer systems are also presented and discussed.

Kinetic and structural features of a dyestuff coaggregation studied by time-resolved static light scattering.

A binary dyestuff aggregate with a distinct stoichiometry is formed in dilute aqueous solution upon addition of Mg(2+) ions. The aggregation process was investigated with time-resolved multiangle static light scattering resulting in a sequence of static scattering curves. The scattering curves were analyzed with respect to the aggregation kinetics as well as the structure of the growing aggregates. The aggregation kinetics was based on the time evolution of the weight-averaged molar mass values extracted from the intercepts of the static scattering curves. A kinetic model that considers solely a nucleation step and monomer addition in its most simple form was developed in order to describe the evolution of time-dependent mass data. In addition, a kinetic model introduced by Lomakin et al. (Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1125) for the description of ß-amyloid aggregation was adapted to the same experimental data. Application of the two kinetic models offered significant information on the role of magnesium ions within the aggregation process and provided a deeper understanding of the aggregation mechanism. Correlation of the size parameters extracted from the initial slopes of the scattering curves with the respective mass data as well as direct fitting of the scattering curves with the wormlike chain model yield a consistent set of model parameters.