Passive Macromolecular Translocation Mechanism through Lipid Membranes.

The translocation of biologically active macromolecules through cell membranes is of vital importance for cells and is a key process for drug delivery. Proteins exploit specific conformational changes in their secondary structure to facilitate membrane translocation. For the large class of biological and synthetic macromolecules, where such conformational adaptions are not possible, guidelines to tailor the structure of monomers and macromolecules to aid membrane translocation and cross-membrane drug delivery would be highly desirable. Here, we use alternating amphiphilic macromolecules to systematically investigate the relation between polarity, polymer chain length, lipid chain length, polymer concentration, and temperature on membrane partition and translocation rate. We employed pulse field gradient NMR and confocal fluorescence microscopy to determine membrane adsorption and desorption rate constants and partitioning coefficients. We find that translocation is a two-step process involving a fast adsorption and membrane insertion process and a slower desorption process. Membrane insertion is a key step that determines the molecular weight, concentration, and temperature dependences. Passive translocation is possible on time scales from minutes to hours. Macromolecules with different adapted hydrophilic/hydrophobic comonomer sequences show the same translocation rate, indicating that common optimized translocation conditions can be realized with a variety of monomer chemical structures. The investigated copolymers are biocompatible, biodegradable, and capable of transporting a hydrophobic payload through the lipid membrane. This detailed understanding of the macromolecular translocation mechanism enables to better tailor the delivery of active agents using macromolecular carriers.

Structure formation of PNIPAM microgels in foams and foam films.

Responsive aqueous foams are very interesting from a fundamental point of view and for various applications like foam flooding or foam flotation. In this study thermoresponsive microgels (MGs) made from poly(N-isopropyl-acrylamide) (PNIPAM) with varying cross-linker content, are used as foam stabilisers. The foams obtained are thermoresponsive and can be destabilised by increasing the temperature. The structuring of MGs inside the foam films is investigated with small-angle neutron scattering and in a thin film pressure balance. The foam films are inhomogeneous and form a network-like structure, in which thin and MG depleted zones with a thickness of ca. 30 nm are interspersed in a continuous network of thick MG containing areas with a thickness of several 100 nm. The thickness of this continuous network is related to the elastic modulus of the individual MGs, which was determined by atomic force microscopy indentation experiments. Both, the elastic moduli and foam film thicknesses, indicate a correlation to the network elasticity of the MGs predicted by the affine network model.

Inelastic neutron scattering analysis with time-dependent Gaussian-field models.

Converting neutron scattering data to real-space time-dependent structures can only be achieved through suitable models, which is particularly challenging for geometrically disordered structures. We address this problem by introducing time-dependent clipped Gaussian field models. General expressions are derived for all space- and time-correlation functions relevant to coherent inelastic neutron scattering for multiphase systems and arbitrary scattering contrasts. Various dynamic models are introduced that enable one to add time-dependence to any given spatial statistics, as captured, e.g., by small-angle scattering. In a first approach, the Gaussian field is decomposed into localized waves that are allowed to fluctuate in time or to move either ballistically or diffusively. In a second approach, a dispersion relation is used to make the spectral components of the field time-dependent. The various models lead to qualitatively different dynamics, which can be discriminated by neutron scattering. The methods of this paper are illustrated with oil/water microemulsion studied by small-angle scattering and neutron spin-echo. All available data-in both film and bulk contrasts, over the entire range of q and τ-are analyzed jointly with a single model. The analysis points to the static large-scale structure of the oil and water domains while the interfaces are subject to thermal fluctuations. The fluctuations have an amplitude of around 60 Å and contribute to 30% of the total interface area.

Stabilizing the Solid-Electrolyte Interphase with Polyacrylamide for High-Voltage Aqueous Lithium-Ion Batteries.

The introduction of "water-in-salt" electrolyte (WiSE) concept opens a new horizon to aqueous electrochemistry that is benefited from the formation of a solid-electrolyte interphase (SEI). However, such SEI still faces multiple challenges, including dissolution, mechanical damaging, and incessant reforming, which result in poor cycling stability. Here, we report a polymeric additive, polyacrylamide (PAM) that effectively stabilizes the interphase in WiSE. With the addition of 5 molar % PAM to 21 mol kg-1 LiTFSI electrolyte, a LiMn2 O4 ∥L-TiO2 full cell exhibits enhanced cycling stability with 86 % capacity retention after 100 cycles at 1 C. The formation mechanism and evolution of PAM-assisted SEI was investigated using operando small angle neutron scattering and density functional theory (DFT) calculations, which reveal that PAM minimizes the presence of free water molecules at the anode/electrolyte interface, accelerates the TFSI- anion decomposition, and densifies the SEI.

Effect of mild nanoscopic confinement on the dynamics of ionic liquids.

Ionic liquids are molten salts without an additional solvent and are discussed as innovative solvents and electrolytes in chemical processing and electrochemistry. A thorough microscopic understanding of the structure and ionic transport processes is essential for tailored applications. Here, we study the influence of "mild" nanoscopic confinement on the structure and diffusion properties of an ionic liquid, 1-ethyl-3-methylimidazolium acetate, using scattering techniques. The structure is analyzed by X-ray diffraction, while neutron backscattering spectroscopy is used for the study of the diffusion processes in these systems. Interpreting the diffusion processes in terms of a jump-diffusion model allowed us to deduce the confinement effects on the jump length and residence time, both increased at elevated temperatures in confinement. The applied "mild" confinement, which leaves room for 10-25 times the domain spacing, allows us to observe in great detail how the onset of domain distortion decelerates the dynamics.

Structure and Dynamics of the Central Lipid Pool and Proteins of the Bacterial Holo-Translocon.

The bacterial Sec translocon, SecYEG, associates with accessory proteins YidC and the SecDF-YajC subcomplex to form the bacterial holo-translocon (HTL). The HTL is a dynamic and flexible protein transport machine capable of coordinating protein secretion across the membrane and efficient lateral insertion of nascent membrane proteins. It has been hypothesized that a central lipid core facilitates the controlled passage of membrane proteins into the bilayer, ensuring the efficient formation of their native state. By performing small-angle neutron scattering on protein solubilized in "match-out" deuterated detergent, we have been able to interrogate a "naked" HTL complex, with the scattering contribution of the surrounding detergent micelle rendered invisible. Such an approach has allowed the confirmation of a lipid core within the HTL, which accommodates between 8 and 29 lipids. Coarse-grained molecular dynamics simulations of the HTL also demonstrate a dynamic, central pool of lipids. An opening at this lipid-rich region between YidC and the SecY lateral gate may provide an exit gateway for newly synthesized, correctly oriented, membrane protein helices, or even small bundles of helices, to emerge from the HTL.

Deformation of Mesoporous Titania Nanostructures in Contact with D2 O Vapor.

For many applications, mesoporous titania nanostructures are exposed to water or need to be backfilled via infiltration with an aqueous solution, which can cause deformations of the nanostructure by capillary forces. In this work, the degree of deformation caused by water infiltration in two types of mesoporous, nanostructured titania films exposed to water vapor is compared. The different types of nanostructured titania films are prepared via a polymer template assisted sol-gel synthesis in conjunction with a polymer-template removal at high-temperatures under ambient conditions versus nitrogen atmosphere. Information about surface and inner morphology is extracted by scanning electron microscopy and grazing incidence small-angle neutron scattering (GISANS) measurements, respectively. Furthermore, complementary information on thin film composition and porosity are probed via X-ray reflectivity. The backfilling induced deformation of near surface structures and structures inside the mesoporous titania films is determined by GISANS before and after D2 O infiltration. The respective atmosphere used for template removal influences the details of the titania nanostructure and strongly impacts the degree of water induced deformation. Drying of the films shows reversibility of the deformation.

Stealth carriers for low-resolution structure determination of membrane proteins in solution.

Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial nanoscale bilayer disc carriers that mimic the native bilayer environment allows the handling of membrane proteins in solution. This enables the use of small-angle scattering techniques for fast and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly nontrivial fashion, making subsequent data analysis challenging. Here, an elegant solution to circumvent the intrinsic complexity brought about by the presence of the carrier disc is presented. In combination with small-angle neutron scattering (SANS) and the D2O/H2O-based solvent contrast-variation method, it is demonstrated that it is possible to prepare specifically deuterated carriers that become invisible to neutrons in 100% D2O at the length scales relevant to SANS. These `stealth' carrier discs may be used as a general platform for low-resolution structural studies of membrane proteins using well established data-analysis tools originally developed for soluble proteins.

Anchoring vs bridging: new findings on polymer additives in bicontinuous microemulsions.

We show for the first time the effect of telechelic polymers as additives in bicontinuous microemulsions. We combined macroscopic observations of the phase behavior with microscopic measurements of the structure by small-angle neutron scattering (SANS) to recover the two elastic moduli, κ and κ̅, namely the bending rigidity and saddle-splay modulus. On the basis of these results, we could classify the effect of telechelic polymers along with confinement, expressed as the ratio of the polymer end-to-end distance Ree and the oil-water domain size d. Their unique property to anchor at two points in the membrane (bridging) acts like a switch from antibooster to booster of the surfactant efficiency (between low and ultralow confinement). In the region of medium confinement, all telechelic polymers are in the bridging configuration and we have a maximum of the boosting effect, while at high confinement, the reversed behavior is found where anchoring and/or bridging do not play any role anymore.

Morphology and thermal transitions of self-assembled NIPAM-DMA copolymers in aqueous media depend on copolymer composition profile.

HYPOTHESIS: There is a lack of understanding of the interplay between the copolymer composition profile and thermal transition observed in aqueous solutions of N-isopropyl acrylamide (NIPAM) copolymers, as well as the correlation between this transition and the formation and structure of copolymer self-assemblies. EXPERIMENTS: For this purpose, we investigated the response of five copolymers with the same molar mass and chemical composition, but with different composition profile in aqueous solution against temperature. Using complementary analytical techniques, we probed structural properties at different length scales, from the molecular scale with Nuclear Magnetic Resonance (NMR) to the colloidal scale with Dynamic Light Scattering (DLS) and Small Angle Neutron Scattering (SANS). FINDINGS: NMR and SANS investigations strengthen each other and allow a clear picture of the change of copolymer solubility and related copolymer self-assembly as a function of temperature. At the molecular scale, dehydrating NIPAM units drag N,N-dimethyl acrylamide (DMA) moieties with them in a gradual collapse of the copolymer chain; this induces a morphological transition of the self-assemblies from star-like nanostructures to crew-cut micelles. Interestingly, the transition spans a temperature range which depends on the monomer distribution profile in the copolymer chain, with the asymmetric triblock copolymer specimen revealing the broadest one. We show that the broad morphological transitions associated with gradient copolymers can be mimicked and even surpassed by the use of stepwise gradient (asymmetric) copolymers, which can be more easily and reproducibly synthesized than linear gradient copolymers.

Multiscale Structural Insight into Dairy Products and Plant-Based Alternatives by Scattering and Imaging Techniques.

Dairy products and plant-based alternatives have a large range of structural features from atomic to macroscopic length scales. Scattering techniques with neutrons and X-rays provide a unique view into this fascinating world of interfaces and networks provided by, e.g., proteins and lipids. Combining these scattering techniques with a microscopic view into the emulsion and gel systems with environmental scanning electron microscopy (ESEM) assists in a thorough understanding of such systems. Different dairy products, such as milk, or plant-based alternatives, such as milk-imitating drinks, and their derived or even fermented products, including cheese and yogurt, are characterized in terms of their structure on nanometer- to micrometer-length scales. For dairy products, the identified structural features are milk fat globules, casein micelles, CCP nanoclusters, and milk fat crystals. With increasing dry matter content in dairy products, milk fat crystals are identified, whereas casein micelles are non-detectable due to the protein gel network in all types of cheese. For the more inhomogeneous plant-based alternatives, fat crystals, starch structures, and potentially protein structures are identified. These results may function as a base for improving the understanding of dairy products and plant-based alternatives, and may lead to enhanced plant-based alternatives in terms of structure and, thus, sensory aspects such as mouthfeel and texture.

Open-Bundle Structure as the Unfolding Intermediate of Cytochrome c' Revealed by Small Angle Neutron Scattering.

The dynamic structure changes, including the unfolding, dimerization, and transition from the compact to the open-bundle unfolding intermediate structure of Cyt c', were detected by a small-angle neutron scattering experiment (SANS). The structure of Cyt c' was changed into an unstructured random coil at pD = 1.7 (Rg = 25 Å for the Cyt c' monomer). The four-α-helix bundle structure of Cyt c' at neutral pH was transitioned to an open-bundle structure (at pD ~13), which is given by a numerical partial scattering function analysis as a joint-clubs model consisting of four clubs (α-helices) connected by short loops. The compactly folded structure of Cyt c' (radius of gyration, Rg = 18 Å for the Cyt c' dimer) at neutral or mildly alkaline pD transited to a remarkably larger open-bundle structure at pD ~13 (Rg = 25 Å for the Cyt c' monomer). The open-bundle structure was also supported by ab initio modeling.

MembraneDyn: simulating the dynamics of supported membrane stacks on the nanosecond timescale.

The static structure factor and the undulation dynamics of a solid-supported membrane stack have previously been calculated by Romanov and Ul'yanov [Romanov & Ul'yanov (2002). Phys. Rev. E, 66, 061701]. Based on this prior work, the calculation has been extended to cover the membrane dynamics, i.e. the intermediate scattering function as a Fourier transform of the van Hove correlation function of the membrane stack. Fortran code which calculates the intermediate scattering function for a membrane stack on a solid support is presented. It allows the static and dynamic scattering functions to be calculated according to the derivation of Romanov and Ul'yanov. The physical properties of supported phospholipid bilayers can be examined in this way and the results can be directly compared with results obtained from grazing-incidence neutron spin-echo spectroscopy experiments.

Multiple scattering and resolution effects in small-angle neutron scattering experiments calculated and corrected by the software package MuScatt.

This article deals with multiple scattering effects that are important for the method of small-angle neutron scattering (SANS). It considers three channels for the coherent elastic, the incoherent elastic and the incoherent inelastic scattering processes. The first channel contains the desired information on the experiment. Its multiple scattering effects can be desmeared, as shown in the later sections of the article. The other two channels display a nearly constant background as a function of the scattering angle. The incoherent elastic scattering is treated by the theory of Chandrasekhar, allowing for multiple scattering even at large scattering angles. The transfer to a single representative thermalized wavelength by the inelastic scattering - as a simplification - is assumed to happen by a single scattering event. Once the transition to this altered wavelength has happened, further incoherent multiple scattering is considered. The first part of the paper deals with the multiple scattering effects of light water. In the later part of the article, deconvolution algorithms for multiple scattering and instrumental resolution of the elastic coherent signal as implemented in the program MuScatt are described. All of these considerations are interesting for both reactor-based instruments with velocity selectors and time-of-flight SANS instruments and may improve the reliability of the data treatment.

Small-angle scattering for beginners.

Many experimental methods are available for the characterization of nanostructures, but most of them are limited by stringent experimental conditions. When it comes to analysing nanostructures in the bulk or in their natural environment - even as ordinary as water at room temperature - small-angle scattering (SAS) of X-rays or neutrons is often the only option. The rapid worldwide development of synchrotron and neutron facilities over recent decades has opened unprecedented possibilities for using SAS in situ and in a time-resolved way. But, in spite of its huge potential in the field of nanomaterials in general, SAS is covered far less than other characterization methods in non-specialized curricula. Presented here is a rigorous discussion of small-angle scattering, at a technical level comparable to the classical undergraduate coverage of X-ray diffraction by crystals and which contains diffraction as a particular case.

Distribution and orientation of nerve fibers and myelin assembly in a brain section retrieved by small-angle neutron scattering.

The structural connectivity of the brain has been addressed by various imaging techniques such as diffusion weighted magnetic resonance imaging (DWMRI) or specific microscopic approaches based on histological staining or label-free using polarized light (e.g., three-dimensional Polarized Light Imaging (3D-PLI), Optical Coherence Tomography (OCT)). These methods are sensitive to different properties of the fiber enwrapping myelin sheaths i.e. the distribution of myelin basic protein (histology), the apparent diffusion coefficient of water molecules restricted in their movements by the myelin sheath (DWMRI), and the birefringence of the oriented myelin lipid bilayers (3D-PLI, OCT). We show that the orientation and distribution of nerve fibers as well as myelin in thin brain sections can be determined using scanning small angle neutron scattering (sSANS). Neutrons are scattered from the fiber assembly causing anisotropic diffuse small-angle scattering and Bragg peaks related to the highly ordered periodic myelin multilayer structure. The scattering anisotropy, intensity, and angular position of the Bragg peaks can be mapped across the entire brain section. This enables mapping of the fiber and myelin distribution and their orientation in a thin brain section, which was validated by 3D-PLI. The experiments became possible by optimizing the neutron beam collimation to highest flux and enhancing the myelin contrast by deuteration. This method is very sensitive to small microstructures of biological tissue and can directly extract information on the average fiber orientation and even myelin membrane thickness. The present results pave the way toward bio-imaging for detecting structural aberrations causing neurological diseases in future.

Mutually Beneficial Combination of Molecular Dynamics Computer Simulations and Scattering Experiments.

We showcase the combination of experimental neutron scattering data and molecular dynamics (MD) simulations for exemplary phospholipid membrane systems. Neutron and X-ray reflectometry and small-angle scattering measurements are determined by the scattering length density profile in real space, but it is not usually possible to retrieve this profile unambiguously from the data alone. MD simulations predict these density profiles, but they require experimental control. Both issues can be addressed simultaneously by cross-validating scattering data and MD results. The strengths and weaknesses of each technique are discussed in detail with the aim of optimizing the opportunities provided by this combination.

Diatom-inspired self-assembly for silica thin sheets of perpendicular nanochannels.

HYPOTHESIS: Multistage silicate self-organization into light-weight, high-strength, hierarchically patterned diatom frustules carries hints for innovative silica-based nanomaterials. With sodium silicate in a biomimetic sol-gel system templated by a tri-surfactant system of hexadecyltrimethylammonium bromide, sodium dodecylsulfate, and poly(oxyethylene-b-oxypropylene-b-oxyethylene) (P123), mesoporous silica nanochannel plates with perpendicular channel orientation are synthesized. The formation process, analogous to that of diatom frustules, is postulated to be directed by an oriented self-assembly of the block copolymer micelles shelled with charged catanionic surfactants upon silication. EXPERIMENTS: The postulated formation process for the oriented silica nanochannel plates was investigated using time-resolved small-angle X-ray and neutron scattering (SAXS/SANS) and freeze fracture replication transmission electron microscopy (FFR-TEM). FINDINGS: With fine-tuned molar ratios of the anionic, cationic, and nonionic surfactants, the catanionic combination and the nonionic copolymer form charged, prolate ternary micelles in aqueous solutions, which further develop into prototype monolayered micellar plates. The prolate shape and maximized surfactant adsorption of the complex micelles, revealed from combined SAXS/SANS analysis, are of critical importance in the subsequent micellar self-assembly upon silicate deposition. Time-resolved SAXS and FFR-TEM indicate that the silicate complex micelles coalesce laterally into the prototype micellar nanoplates, which further fuse with one another into large sheets of monolayered silicate micelles of in-plane lamellar packing. Upon silica polymerization, the in-plane lamellar packing of the micelles further transforms to 2D hexagonal packing of vertically oriented silicate channels. The unveiled structural features and their evolution not only elucidate the previously unresolved self-assembly process of through-thickness silica nanochannels but also open a new line of research mimicking free-standing frustules of diatoms.

Confined polymer dynamics on clay platelets.

The structure and dynamics of poly(ethylene oxide) adsorbed on dispersed clay platelets are investigated by small-angle neutron scattering and neutron spin-echo spectroscopy. The intermediate scattering function has a mobile contribution described by the Zimm theory and an immobile contribution that is constant within the time window. The immobile fraction as a function of the scattering vector Q is described by a Lorentz function, from which a localization length is determined. The relaxation rates grow with polymer concentration in agreement with dielectric measurements but contrary to pure polymer gels.

3D printed spherical environmental chamber for neutron reflectometry and grazing-incidence small-angle neutron scattering experiments.

In neutron scattering on soft matter, an important concern is the control and stability of environmental conditions surrounding the sample. Complex sample environment setups are often expensive to fabricate or simply not achievable by conventional workshop manufacturing. We make use of state-of-the-art 3D metal-printing technology to realize a sample environment for large sample sizes, optimized for investigations on thin film samples with neutron reflectometry (NR) and grazing-incidence small-angle neutron scattering (GISANS). With the flexibility and freedom of design given by 3D metal-printing, a spherical chamber with fluidic channels inside its walls is printed from an AlSi10Mg powder via selective laser melting (SLM). The thin channels ensure a homogeneous heating of the sample environment from all directions and allow for quick temperature switches in well-equilibrated atmospheres. In order to optimize the channel layout, flow simulations were carried out and verified in temperature switching tests. The spherical, edgeless design aids the prevention of condensation inside the chamber in case of high humidity conditions. The large volume of the sample chamber allows for high flexibility in sample size and geometry. While a small-angle neutron scattering (SANS) measurement through the chamber walls reveals a strong isotropic scattering signal resulting from the evenly orientated granular structure introduced by SLM, a second SANS measurement through the windows shows no additional background originating from the chamber. Exemplary GISANS and NR measurements in time-of-flight mode are shown to prove that the chamber provides a stable, background free sample environment for the investigation of thin films.