Milos Steinhart

Combining structure and dynamics: non-denaturing high-pressure effect on lysozyme in solution

Small-angle X-ray scattering (SAXS) and elastic and quasi-elastic neutron scattering techniques were used to investigate the high-pressure-induced changes on interactions, the low-resolution structure and the dynamics of lysozyme in solution. SAXS data, analysed using a global-fit procedure based on a new approach for hydrated protein form factor description, indicate that lysozyme completely maintains its globular structure up to 1500 bar, but significant modifications in the protein–protein interaction potential occur at approximately 600–1000 bar. Moreover, the mass density of the protein hydration water shows a clear discontinuity within this pressure range. Neutron scattering experiments indicate that the global and the local lysozyme dynamics change at a similar threshold pressure. A clear evolution of the internal protein dynamics from diffusing to more localized motions has also been probed. Protein structure and dynamics results have then been discussed in the context of protein–water interface and hydration water dynamics. According to SAXS results, the new configuration of water in the first hydration layer induced by pressure is suggested to be at the origin of the observed local mobility changes.

Green synthesis of hyaluronan fibers with silver nanoparticles

The application of green chemistry in the nano-science and technology is very important in the area of the preparation of various materials. In this work, an eco-friendly chemical method was successfully used for the preparation of hyaluronan fibers containing silver nanoparticles (AgNPs). Thus, hyaluronic acid (HA) was dissolved in an aqueous solution of sodium hydroxide to prepare a transparent solution, which was used for the preparation of fibers by a wet-spinning technique. Consequently, silver nanoparticles inside the fiber were prepared. Different parameters affecting the preparation of final product, such as concentration of silver nitrate, hyaluronan fiber concentration, time and temperature of the reaction, pH of the reaction mixture, were studied. AgNPs were confirmed by transmission electron microscopy (TEM), X-ray diffraction (XRD), two-dimensional X-ray scattering (2D SWAXS), UV/Vis spectroscopy, inductively coupled plasma optical emission spectrometry (ICP OES) and scan electron microscopy (SEM). Mechanical properties of prepared fibers were also measured.

Effects of Pressure and Temperature on the Self-Assembled Fully Hydrated Nanostructures of Monoolein−Oil Systems

Synchrotron small-angle X-ray scattering (SAXS) was applied for studying the effects of hydrostatic pressure and temperature on the structural behavior of fully hydrated tetradecane (TC)-loaded monoolein (MO) systems. Our main attention focused on investigating the impact of isobaric and isothermal changes on the stability of the inverted type discontinuous Fd3m cubic phase as compared to the inverted type hexagonal (H(2)) liquid crystalline phase. The present results show that compressing the TC-loaded Fd3m phase under isothermal conditions induces a significant increase of its lattice parameter: it approximately increases by 1 A per 75 bar. Further, the Fd3m phase is more pressure-sensitive as compared to the Pn3m and the H(2) phases. At ambient temperatures, we observed the following structural transitions as pressure increases: Fd3m --> H(2) --> Pn3m. Our findings under isobaric conditions reveal more complicated structural transitions. At high pressures, we recorded the interesting temperature-induced structural transition of (Pn3m + L(alpha)) --> (Pn3m + L(alpha) + H(2)) --> (L(alpha) + H(2)) --> H(2) --> Fd3m --> traces of Fd3m coexisting with L(2). At high pressures and low temperatures, the TC molecules partially crystallize as indicated by the appearance of an additional diffraction peak at q = 3.46 nm(-1). This crystallite disappears at high temperatures and also as the system gets decompressed. The appearance of the Pn3m and the L(alpha) phases during compressing the fully hydrated MO/TC samples at high pressures and low temperatures is generally related to a growing hydrocarbon chain condensation, which leads to membrane leaflets with less negative interfacial curvatures (decreasing the spontaneous curvatures |H(0)|). Both the effects of pressure and temperature are discussed in detail for all nonlamellar phases on the basis of molecular shape and packing concepts.

High pressure cell for small- and wide-angle x-ray scattering

A compact high pressure cell and its control environment designed for small- and wide-angle x-ray scattering experiments under hydrostatic pressure up to 3000 bar and temperatures between −20 °C and +80 °C are described. With this system x-ray scattering experiments can be performed at constant pressure and temperature with a conventional laboratory x-ray source and it can be easily modified to carry out time resolved pressure jump studies at synchrotron radiation sources. The computer-aided pressure adjustment has a calibrated accuracy of ±0.5%, and temperature is computer-controlled to a precision of ±0.05 °C. The instrument has been designed to investigate systems of biological interest, especially lipid-water dispersions, but it is equally possible to measure highly viscous or solid samples. The performance is illustrated by a pressure dependent small- and wide-angle x-ray scattering study of a phospholipid-water dispersion at constant temperature.

High-pressure instrument for small- and wide-angle x-ray scattering. II. Time-resolved experiments

An instrument to facilitate small- and wide-angle x-ray scattering measurements of samples under elevated hydrostatic pressures or exposed to pressure jumps is described. Pressure from atmospheric up to 0.35 GPa is produced by a motor-driven, piston-type generator and transferred through a network containing pressurizing liquid to a sample cell. The cell, with the optical path length of 1.6 mm, has two Beryllium windows with a total transmission of 0.4 (for x rays of λ=0.154 nm) and low background scattering. Scattering can be observed at angles up to 30°. Samples can be solid or liquid with a minimal volume less than 30 μl and the irradiated volume up to 3 μl. Separation of the samples from the pressurizing medium is accomplished by Teflon pistons. The high-pressure network has two sections separated by a pneumatic valve. The inner section is connected permanently to the cell and the outer one to the pressure generator. For pressure jumps, the outer section is brought to a different pressure level than t...

Small‐angle X‐ray scattering and light scattering study of hybrid nanoparticles composed of thermoresponsive triblock copolymer F127 and thermoresponsive statistical polyoxazolines with hydrophobic moieties

A combination of new thermoresponsive statistical polyoxazolines, poly[(2-butyl-2-oxazoline)-stat-(2-isopropyl-2-oxazoline)] [pBuOx-co-piPrOx], with different hydrophobic moieties and F127 surfactant as a template system for the creation of thermosensitive nanoparticles for radionuclide delivery has recently been tested [Panek, Filippov, Hrubý, Rabyk, Bogomolova, Kucka & Stěpanek (2012). Macromol. Rapid Commun. 33, 1683–1689]. It was shown that the presence of the thermosensitive F127 triblock copolymer in solution reduces nanoparticle size and polydispersity. This article focuses on a determination of the internal structure and solution properties of the nanoparticles in the temperature range from 288 to 312 K. Here, it is demonstrated that below the cloud point temperature (CPT) the polyoxazolines and F127 form complexes that co-exist in solution with single F127 molecules and large aggregates. When the temperature is raised above the CPT, nanoparticles composed of polyoxazolines and F127 are predominant in solution. These nanoparticles could be described by a spherical shell model. It was found that the molar weight and hydrophobicity of the polymer do not influence the size of the outer radius and only slightly change the inner radius of the nanoparticles. At the same time, molar weight and hydrophobicity did affect the process of nanoparticle formation. In conclusion, poly(2-oxazoline) molecules are fully incorporated inside of F127 micelles, and this result is very promising for the successful application of such systems in radionuclide delivery.

A combined small-angle neutron and X-ray scattering study of block copolymers micellisation in supercritical carbon dioxide

Small angle neutron and X-ray scattering (SANS and SAXS) are used to investigate the monomer-aggregate transition of fluorocarbon-hydrocarbon diblock copolymers in supercritical carbon dioxide. SANS data are analyzed using a polydisperse sphere core-shell model. Synchrotron SAXS data have been collected by profiling the pressure at different temperatures, and critical micellization densities have been obtained for a series of diblock solutions. Finally pressure jump experiments, combined with synchrotron SAXS, have revealed two steps in the dynamics of the formation of the aggregates.

Kinetics of block-copolymer aggregation in super critical CO2

Small angle X-ray and neutron scattering (SAXS and SANS) are used to obtain structural information on the aggregation behavior of block-copolymers dissolved in supercritical CO2. The SANS technique is used to provide a detailed structural model for the micellar aggregates, which form below the critical micellization density (CMD), that we defined in our previous work. The SAXS technique (with a synchrotron source) is used to provide the first experimental information concerning the kinetic features of both formation and decomposition of such aggregates as soon as pressure jumps are applied to the solutions across the CMD. 2002 Elsevier Science B.V. All rights reserved.

Morphology and Kinetics of Aggregation of Silver Nanoparticles Induced with Regioregular Cationic Polythiophene

The aggregation kinetics of negatively charged borate-stabilized silver nanoparticles (NPs) induced by the cationic regioregular polythiophene polyelectrolyte poly{3-[6-(1-methylimidazolium-3-yl)hexyl]thiophene-2,5-diyl bromide} (PMHT-Br) and the morphology of formed aggregates have been investigated via ultraviolet-visible light (UV-vis) spectroscopy, transmission electron microscopy (TEM), zeta (ζ) potential measurements, dynamic light scattering (DLS), and time-resolved small-angle X-ray scattering (SAXS). Two or three populations of NPs are formed within milliseconds upon mixing the components, which differ in the mean size, extent of polymer coating, and time stability. These characteristics are primarily controlled by the PMHT-Br to Ag-NPs ratio. Population of single NPs of a mean size of ∼5 nm is present in every system and is mostly stable for a long time. At low ratios, the single NPs are most probably almost free of polymer chains and the second population includes slow, but in a limited extent, growing NPs in which single NPs might be interconnected by polymer chains. At the ratios corresponding to the charge balance in the system (ca. zero ζ-potential of NPs), the NPs aggregate, forming a second population that continuously grows in size, and finally undergo sedimentation. At the high ratios, three long-time stable populations of NPs are observed, having mean sizes of ca. 5, 13, and 35 nm; all NPs should be fully coated with PMHT-Br, giving them a positively charged stabilizing shell.

TIME-RESOLVED SAXS/WAXS STUDY OF PHASE BEHAVIOR AND CRYSTALLIZATION IN POLYMER BLENDS

Real-time SAXS and WAXS patterns have been simultaneously obtained during isothermal melt-crystallization of blends of low-molecular-weight poly(ethylene oxide) (PEO) and poly(methyl methacrylate) (PMMA). The analysis of results shows that the originally homogeneous, single-phase polymer blend separates into two phases. The PMMA molecules diffuse from the blend and form completely segregated regions while PEO starts to crystallize. The first and dominating effect at the beginning of crystallization is the formation of unstable lamellae of nonintegrally folded chains (NIF). The real-time crystallinity and density of the PEO crystalline phase in absolute units were obtained from the time-resolved SAXS/WAXS results. The structure development proceeds in two steps. A very fast evolution of PEO crystals from the melt starts to crystallize in disordered NIF lamellae with thick amorphous interlayers and with a lower density of crystalline phase. The steep growth of crystallinity and crystalline density mean quick thickening of crystalline part of lamellae and improvement of their crystalline structure. In the second step, the structure of the crystalline phase gradually improves and crystallinity grows very slowly. The recrystallization of NIF lamellae into extended chain lamellae (EC) and lamellae with once folded chains (1F) proceeds during both stages of crystallization.