Tuning Nanobodies' Bioactivity: Coupling to Ultrasmall Gold Nanoparticles Allows the Intracellular Interference with Survivin.

Nanobodies are highly affine binders, often used to track disease-relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease-relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin's multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein-protein interactions. However, as Survivin harbors no classical "druggable" binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X-ray scattering of the Survivin-SuN complex locates the proposed interaction interface between the C-terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2 nm) gold nanoparticles (SuN-N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability.

Rigid-to-Flexible Transition in a Molecular Brush in a Good Solvent at a Semidilute Concentration.

The structures of a molecular brush in a good solvent are investigated using synchrotron small-angle X-ray scattering in a wide range of concentrations. The brush under study, PiPOx239-g-PnPrOx14, features a relatively long poly(2-isopropenyl-2-oxazoline) (PiPOx) backbone and short poly(2-n-propyl-2-oxazoline) (PnPrOx) side chains. As a solvent, ethanol is used. By model fitting, the overall size and the persistence length as well as the interaction length and interaction strength are determined. At this, the interplay between form and structure factor is taken into account. The conformation of the molecular brush is traced upon increasing the solution concentration, and a rigid-to-flexible transition is found near the overlap concentration. Finally, the results of computer simulations of the molecular brush solutions confirm the experimental results.

Hydration in aqueous NaCl.

Atomistic details about the hydration of ions in aqueous solutions are still debated due to the disordered and statistical nature of the hydration process. However, many processes from biology, physical chemistry to materials sciences rely on the complex interplay between solute and solvent. Oxygen K-edge X-ray excitation spectra provide a sensitive probe of the local atomic and electronic surrounding of the excited sites. We used ab initio molecular dynamics simulations together with extensive spectrum calculations to relate the features found in experimental oxygen K-edge spectra of a concentration series of aqueous NaCl with the induced structural changes upon solvation of the salt and distill the spectral fingerprints of the first hydration shells around the Na+- and Cl--ions. By this combined experimental and theoretical approach, we find the strongest spectral changes to indeed result from the first hydration shells of both ions and relate the observed shift of spectral weight from the post- to the main-edge to the origin of the post-edge as a shape resonance.

Anomalous SAXS at P12 beamline EMBL Hamburg: instrumentation and applications.

Small-angle X-ray scattering (SAXS) is an established method for studying nanostructured systems and in particular biological macromolecules in solution. To obtain element-specific information about the sample, anomalous SAXS (ASAXS) exploits changes of the scattering properties of selected atoms when the energy of the incident X-rays is close to the binding energy of their electrons. While ASAXS is widely applied to condensed matter and inorganic systems, its use for biological macromolecules is challenging because of the weak anomalous effect. Biological objects are often only available in small quantities and are prone to radiation damage, which makes biological ASAXS measurements very challenging. The BioSAXS beamline P12 operated by the European Molecular Biology Laboratory (EMBL) at the PETRA III storage ring (DESY, Hamburg) is dedicated to studies of weakly scattering objects. Here, recent developments at P12 allowing for ASAXS measurements are presented. The beamline control, data acquisition and data reduction pipeline of the beamline were adapted to conduct ASAXS experiments. Modelling tools were developed to compute ASAXS patterns from atomic models, which can be used to analyze the data and to help designing appropriate data collection strategies. These developments are illustrated with ASAXS experiments on different model systems performed at the P12 beamline.

Dependence of the Nanoscale Composite Morphology of Fe3O4 Nanoparticle-Infused Lysozyme Amyloid Fibrils on Timing of Infusion: A Combined SAXS and AFM Study.

Understanding the formation process and the spatial distribution of nanoparticle (NP) clusters on amyloid fibrils is an essential step for the development of NP-based methods to inhibit aggregation of amyloidal proteins or reverse the assembling trend of the proto-fibrillary complexes that prompts pathogenesis of neuro degeneration. For this, a detailed structural determination of the diverse hybrid assemblies that are forming is needed, which can be achieved by advanced X-ray scattering techniques. Using a combined solution small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) approach, this study investigates the intrinsic trends of the interaction between lysozyme amyloid fibrils (LAFs) and Fe3O4 NPs before and after fibrillization at nanometer resolution. AFM images reveal that the number of NP clusters interacting with the lysozyme fibers does not increase significantly with NP volume concentration, suggesting a saturation in NP aggregation on the fibrillary surface. The data indicate that the number of non-adsorbed Fe3O4 NPs is highly dependent on the timing of NP infusion within the synthesis process. SAXS data yield access to the spatial distribution, aggregation manner and density of NP clusters on the fibrillary surfaces. Employing modern data analysis approaches, the shape and internal structural morphology of the so formed nanocomposites are revealed. The combined experimental approach suggests that while Fe3O4 NPs infusion does not prevent the fibril-formation, the variation of NP concentration and size at different stages of the fibrillization process can impose a pronounced impact on the superficial and internal structural morphologies of these nanocomposites. These findings may be applicable in devising advanced therapeutic treatments for neurodegenerative diseases and designing novel bio-inorganic magnetic devices. Our results further demonstrate that modern X-ray methods give access to the structure of-and insight into the formation process of-biological-inorganic hybrid structures in solution.

Effect of the concentration of protein and nanoparticles on the structure of biohybrid nanocomposites.

We present colloidal nanocomposites formed by incorporating magnetite Fe3 O4 nanoparticles (MNPs) with lysozyme amyloid fibrils (LAFs). Preparation of two types of solutions, with and without addition of salt, was carried out to elucidate the structure of MNPs-incorporated fibrillary nanocomposites and to study the effect of the presence of salt on the stability of the nanocomposites. The structural morphology of the LAFs and their interaction with MNPs were analyzed by atomic force microscopy and small-angle x-ray scattering measurements. The results indicate that conformational properties of the fibrils are dependent on the concentration of protein, and the precise ratio of the concentration of the protein and MNPs is crucially important for the stability of the fibrillary nanocomposites. Our results confirm that despite the change in fibrillary morphology induced by the varying concentration of the protein, the adsorption of MNPs on the surface of LAF is morphologically independent. Moreover, most importantly, the samples containing salt have excellent stability for up to 1 year of shelf-life.

Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering.

Messenger ribonucleic acid (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clinical practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clinical efficacy. In this study, pH-induced changes in internal molecular organization and overall physicochemical characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were determined for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate determination of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ determination of the structural response of the lipoplex systems to changes of the environmental pH similar to that observed for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.

Hydration in aqueous osmolyte solutions: the case of TMAO and urea.

The hydration and hydrogen-bond topology of small water solvated molecules such as the naturally occurring organic osmolytes trimethylamine N-oxide (TMAO) and urea are under intense investigation. We aim at furthering the understanding of this complex hydration by combining experimental oxygen K-edge excitation spectra with results from spectra calculated via the Bethe-Salpeter equation based on structures obtained from ab initio molecular dynamics simulations. Comparison of experimental and calculated spectra allows us to extract detailed information about the immediate surrounding of the solute molecules in the solvated state. We quantify and localize the influence of the solute on the hydrogen bond network of the water solvent and find spectroscopic fingerprints of a clear directional asymmetry around TMAO with strong and local kosmotropic influence around TMAO's NO head group and slight chaotropic influence around the hydrophobic methyl groups. The influence of urea on the local water network is qualitatively similar to that of TMAO but weaker in magnitude. The strongest influence of both molecules on the shape of the oxygen K-edge spectra is found in the first hydration shells.

Kinetics of pressure-induced nanocrystal superlattice formation.

Colloidal nanocrystals (NC) are known to self-organize into superlattices that promise many applications ranging from medicine to optoelectronics. Recently, the formation of high-quality PEGylated gold NC was reported at high hydrostatic pressure and high salt concentrations. Here, we study the formation kinetics of these superlattices after pressure jumps beyond their crystallisation pressure by means of small-angle X-ray scattering with few ms experimental resolution. The timescale of NC formation was found to be reduced the larger the width of the pressure jump. This is connected to an increase of crystal quality, i.e., the faster the NC superlattice forms, the better the crystal quality. In contrast to the formation kinetics, the melting of the NC superlattice is approximately one order of magnitude slower and shows linear kinetics.

A Multiperspective Approach to Solvent Regulation of Enzymatic Activity: HMG-CoA Reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions.

Smaller capillaries improve the small-angle X-ray scattering signal and sample consumption for biomacromolecular solutions.

Radiation damage by intense X-ray beams at modern synchrotron facilities is one of the major complications for biological small-angle X-ray scattering (SAXS) investigations of macromolecules in solution. To limit the damage, samples are typically measured under a laminar flow through a cell (typically a capillary) such that fresh solution is continuously exposed to the beam during measurement. The diameter of the capillary that optimizes the scattering-to-absorption ratio at a given X-ray wavelength can be calculated a priori based on fundamental physical properties. However, these well established scattering and absorption principles do not take into account the radiation susceptibility of the sample or the often very limited amounts of precious biological material available for an experiment. Here it is shown that, for biological solution SAXS, capillaries with smaller diameters than those calculated from simple scattering/absorption criteria allow for a better utilization of the available volumes of radiation-sensitive samples. This is demonstrated by comparing two capillary diameters di (di = 1.7 mm, close to optimal for 10 keV; and di = 0.9 mm, which is nominally sub-optimal) applied to study different protein solutions at various flow rates. The use of the smaller capillaries ultimately allows one to collect higher-quality SAXS data from the limited amounts of purified biological macromolecules.

Water-Mediated Protein-Protein Interactions at High Pressures are Controlled by a Deep-Sea Osmolyte.

The influence of natural cosolvent mixtures on the pressure-dependent structure and protein-protein interaction potential of dense protein solutions is studied and analyzed using small-angle X-ray scattering in combination with a liquid-state theoretical approach. The deep-sea osmolyte trimethylamine-N-oxide is shown to play a crucial and singular role in its ability to not only guarantee sustainability of the native protein's folded state under harsh environmental conditions, but it also controls water-mediated intermolecular interactions at high pressure, thereby preventing contact formation and hence aggregation of proteins.

Hydration in aqueous solutions of ectoine and hydroxyectoine.

We explore the influence of the two osmolytes ectoine and hydroxyectoine on the structure of pure water and aqueous NaCl solutions using non-resonant X-ray Raman scattering spectroscopy at the oxygen K-edge. Both ectoine and hydroxyectoine are naturally occurring organic osmolytes synthesized by halophilic organisms that live in high-salt and other extreme environments. We find that X-ray spectroscopic data at the oxygen K-edge are consistent with a scenario where both osmolytes affect the hydrogen bonding network of water on a local scale to in effect increase tetrahedral order. This supports the proposed stabilizing mechanism of the osmolytes for proteins: preferential exclusion of the osmolytes from the proteins' surface and preferential hydration of the macromolecules instead of complex alterations to the structure of water on a bulk scale. The effect of NaCl on water, a disruption of hydrogen bonds and tetrahedral order, acts in opposition to the localized water-binding effects of ectoine and hydroxyectoine. For ternary mixtures of osmolyte in the presence of NaCl, the effects seen in the spectra are found to be additive such that the mixed solutes generate a level of oppositional frustration in the water network.

Structuralization of magnetic nanoparticles in 5CB liquid crystals.

This work is devoted to the study of highly stable composite systems of the liquid crystal 4-n-pentyl-4'-cyanobiphenyl (5CB) doped with CoFe2O4 magnetic nanoparticles. Ferronematic samples were prepared with two different weight concentrations: sample A 0.085 wt% and sample B 0.062 wt%. The interaction of CoFe2O4 nanoparticles with the liquid crystal was investigated by small-angle X-ray-scattering and magnetization measurements. The obtained results reveal aggregates formed by magnetic nanoparticles that are oriented in the nematic phase. Moreover, the prepared samples show unexpected behaviour of a sudden change in magnetization, which is unusual for such ferronematics.

Ligand Layer Engineering To Control Stability and Interfacial Properties of Nanoparticles.

The use of mixed ligand layers including poly(ethylene glycol)-based ligands for the functionalization of nanoparticles is a very popular strategy in the context of nanomedicine. However, it is challenging to control the composition of the ligand layer and maintain high colloidal and chemical stability of the conjugates. A high level of control and stability are crucial for reproducibility, upscaling, and safe application. In this study, gold nanoparticles with well-defined mixed ligand layers of α-methoxypoly(ethylene glycol)-ω-(11-mercaptoundecanoate) (PEGMUA) and 11-mercaptoundecanoic acid (MUA) were synthesized and characterized by ATR-FTIR spectroscopy and gel electrophoresis. The colloidal and chemical stability of the conjugates was tested by dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and UV/vis spectroscopy based experiments, and their interactions with cells were analyzed by elemental analysis. We demonstrate that the alkylene spacer in PEGMUA is the key feature for the controlled synthesis of mixed layer conjugates with very high colloidal and chemical stability and that a controlled synthesis is not possible using regular PEG ligands without the alkylene spacer. With the results of our stability tests, the molecular structure of the ligands can be clearly linked to the colloidal and chemical stabilization. We expect that the underlying design principle can be generalized to improve the level of control in nanoparticle surface chemistry.

Influence of TMAO and urea on the structure of water studied by inelastic X-ray scattering.

We present a study on the influence of the naturally occurring organic osmolytes tri-methylamine N-oxide (TMAO) and urea on the bulk structure of water using X-ray Raman scattering spectroscopy. Addition of TMAO is known to stabilize proteins in otherwise destabilizing aqueous urea solutions. The experimental X-ray Raman scattering spectra change systematically with increasing solute concentration revealing different effects on the structure of water due to the presence of the two osmolytes. Although these effects are distinct for both molecular species, they have mutually compensating influences on the spectra of the ternary water-TMAO-urea mixtures. This compensation effect seen in the spectra vanishes only at the highest studied ternary concentration of 4 M : 4 M (TMAO : urea). Our experiment shows that the hydrogen-bonding structure of water remains rather intact in the presence of the aforementioned osmolytes if both of them are present.

Stabilizing effect of TMAO on globular PNIPAM states: preferential attraction induces preferential hydration.

We study the effect of the organic co-solute trimethylamine N-oxide (TMAO) on the volume phase transition of microgel particles made from poly(N-isopropylacrylamide) (PNIPAM) using dynamic light scattering (DLS) and all-atom molecular dynamics (MD) simulations. The DLS measurements reveal a continuous TMAO-induced shrinking process from a coil to a globular state of PNIPAM microgel particles. Analyzing the DLS data by the phenomenological Flory-Rehner theory verifies the stabilization of the globular state of the particles in the presence of TMAO. Complementary atomistic MD simulations highlight a pronounced accumulation of TMAO molecules around PNIPAM chains. We observe a significant preferential attraction between TMAO and the globular state of PNIPAM, which is additionally stabilized by a larger number of hydrating water molecules compared to pure aqueous solutions. Further DLS measurements were also conducted on PNIPAM suspensions with the co-solute urea added. The observed differences compared with the results obtained for TMAO support the proposed mechanism.

Concentration dependent effects of urea binding to poly(N-isopropylacrylamide) brushes: a combined experimental and numerical study.

The binding effects of osmolytes on the conformational behavior of grafted polymers are studied in this work. In particular, we focus on the interactions between urea and poly(N-isopropylacrylamide) (PNIPAM) brushes by monitoring the ellipsometric brush thickness for varying urea concentrations over a broad temperature range. The interpretation of the obtained data is supported by atomistic molecular dynamics simulations, which provide detailed insights into the experimentally observed concentration-dependent effects on PNIPAM-urea interaction. In particular, in the low concentration regime (cu ≤ 0.5 mol L(-1)) a preferential exclusion of urea from PNIPAM chains is observed, while in the high concentration regime (2 ≤ cu ≤ 7 mol L(-1)) a preferential binding of the osmolyte to the polymer surface is found. In both regimes, the volume phase transition temperature (Ttr) decreases with increasing urea concentration. This phenomenon derives from two different effects depending on urea concentration: (i) for cu ≤ 0.5 mol L(-1), the decrease of Ttr is explained by a decrease of the chemical potential of bulk water in the surrounding aqueous phase; (ii) for cu ≥ 2 mol L(-1), the lower Ttr is explained by the favorable replacement of water molecules by urea, which can be regarded as a cross-linker between adjacent PNIPAM chains. Significant effects of the concentration-dependent urea binding on the brush conformation are noticed: at cu = 0.5 mol L(-1), although urea is loosely embedded between the hydrated polymer chains, it enhances the brush swelling by excluded volume effects. Beyond 0.5 mol L(-1), the stronger interaction between PNIPAM and urea reduces the chain hydration, which in combination with cross-linking of monomer units induces the shrinkage of the polymer brush.

Nano-beam X-ray microscopy of dried colloidal films.

We report on a nano-beam small angle X-ray scattering study on densely-packed, dried binary films made out of spherical silica particles with radii of 11.2 and 19.3 nm. For these three-dimensional thin films prepared by drop casting, only a finite number of colloidal particles contributes to the scattering signal due to the small beam size of 400 × 400 nm(2). By scanning the samples, the structure and composition of the silica particle films are determined spatially resolved revealing spatial heterogeneities in the films. Three different types of domains were identified: regions containing mainly large particles, regions containing mainly small particles, and regions where both particle species are mixed. Using the new angular X-ray cross-correlations analysis (XCCA) approach, spatial maps of the local type and degree of orientational order within the silica particle films are obtained. Whereas the mixed regions have dominant two-fold order, weaker four-fold and marginal six-fold order, regions made out of large particles are characterized by an overall reduced orientational order. Regions of small particles are highly ordered showing actually crystalline order. Distinct differences in the local particle order are observed by analyzing sections through the intensity and XCCA maps. The different degree of order can be understood by the different particle size polydispersities. Moreover, we show that preferential orientations of the particle domains can be studied by cross-correlation analysis yielding information on particle film formation. We find patches of preferential order with an average size of 8-10 µm. Thus, by this combined X-ray cross-correlation microscopy (XCCM) approach the structure and orientational order of films made out of nanometer sized colloids can be determined. This method will allow to reveal the local structure and order of self-assembled structures with different degree of order in general.

Efficient Narrowband Photoconductivity of the Excitonic Resonance in Two-Dimensional Ruddlesden-Popper Perovskites Due to Exciton Polarons.

Filter-less, wavelength-selective photodetectors made of perovskite usually rely on the charge collection narrowing mechanism, which intrinsically limits the response times. Using the narrow excitonic peak of, e.g., two-dimensional (2D) Ruddlesden-Popper perovskites as direct absorbers to realize color-selective photodetectivity promises faster responses. However, one major challenge in realizing such devices remains the separation and charge carrier extraction of the tightly bound excitons. Here, we report on filter-less color-selective photoconductivity in 2D perovskite butylammonium lead iodide thin film devices, exhibiting a distinct resonance in the photocurrent spectrum with a full width at half-maximum of 16.5 nm that correlates to the excitonic absorption. Our devices exhibit unexpectedly efficient charge carrier separation with an external quantum efficiency of ≤8.9% at the excitonic resonance, which we trace back to the involvement of exciton polarons. Our photodetector achieves response times of 150 µs and a maximum specific detectivity of 2.5 × 1010 Jones at the excitonic peak.