Compact polyethylenimine-complexed mRNA vaccines.

Here we describe formulations comprising individual, polymer-complexed self-amplifying RNA (saRNA) molecules, designed for vaccination against infectious diseases and other prophylactic and therapeutic applications. When exposed to a large excess of the cationic polymer polyethylenimine (PEI), the single saRNA molecules in solution reorganize from an extended to a globular organization, characterized by a high packing density, low polymer mass fraction and, consequently, a very small size of the polyplex nanoparticles of about 30 nm. This format of PEI-complexed saRNA exhibits enhanced biological activity in comparison with previously described saRNA/PEI formulations, both in vitro and in vivo.

A globular protein exhibits rare phase behavior and forms chemically regulated orthogonal condensates in cells.

Proteins with chemically regulatable phase separation are of great interest in the fields of biomolecular condensates and synthetic biology. Intrinsically disordered proteins (IDPs) are the dominating building blocks of biomolecular condensates which often lack orthogonality and small-molecule regulation desired to create synthetic biomolecular condensates or membraneless organelles (MLOs). Here, we discover a well-folded globular protein, lipoate-protein ligase A (LplA) from E.

Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy.

Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary.

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.

A versatile chemical vapor synthesis reactor for in situ x-ray scattering and spectroscopy.

We describe a versatile reactor system for chemical vapor synthesis of nanoparticles, which enables in situ investigations of high temperature gas phase particle formation and transformation processes by x-ray scattering and x-ray absorption spectroscopy. The system employs an inductively heated hot wall reactor as the energy source to start nanoparticle formation from a mixture of precursor vapor and oxygen. By use of a modular set of susceptor segments, it is especially possible to change solely the residence time of the gas mixture while keeping all other process parameters (temperature, gas flow, pressure) constant.

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.

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, PPOx--PPrOx, features a relatively long poly(2-isopropenyl-2-oxazoline) (PPOx) backbone and short poly(2--propyl-2-oxazoline) (PPrOx) side chains. As a solvent, ethanol is used.

Clustering in ferronematics-The effect of magnetic collective ordering.

Clustering of magnetic nanoparticles can dramatically change their collective magnetic properties, and it consequently may influence their performance in biomedical and technological applications. Owing to tailored surface modification of magnetic particles such composites represent stable systems. Here, we report ferronematic mixtures that contain anisotropic clusters of mesogen-hybridized cobalt ferrite nanoparticles dispersed in liquid crystal host studied by different experimental methods-magnetization measurements, small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), and capacitance measurements.

Unraveling agglomeration and deagglomeration in aqueous colloidal dispersions of very small tin dioxide nanoparticles.

Hypothesis: Understanding deagglomeration, agglomerate formation and structure for very small nanoparticles (NPs), due to their more facile agglomeration, is critical for processing or tailoring agglomerates for nanostructured materials. We propose that by controlling and fine-tuning the interplay of agglomeration (colloidal interaction) and deagglomeration (hydrodynamic forces), the design of agglomerate size, microstructure and morphology is possible even for very small NPs.

Experiments: Here, we investigate very small SnO NPs (10 nm) generated in the gas phase as model system.

Probing the existence of non-thermal Terahertz radiation induced changes of the protein solution structure.

During the last decades discussions were taking place on the existence of global, non-thermal structural changes in biological macromolecules induced by Terahertz (THz) radiation. Despite numerous studies, a clear experimental proof of this effect for biological particles in solution is still missing. We developed a setup combining THz-irradiation with small angle X-ray scattering (SAXS), which is a sensitive method for detecting the expected structural changes.

Dependence of the Nanoscale Composite Morphology of FeO 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 FeO NPs before and after fibrillization at nanometer resolution.

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis.

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP that constitutes the anchor to the plasma membrane.

Cotton Textile/Iron Oxide Nanozyme Composites with Peroxidase-like Activity: Preparation, Characterization, and Application.

At present, both native and immobilized nanoparticles are of great importance in many areas of science and technology. In this paper, we have studied magnetic iron oxide nanoparticles and their aggregates bound on woven cotton textiles employing two simple modification procedures. One modification was based on the treatment of textiles with perchloric-acid-stabilized magnetic fluid diluted with methanol followed by drying.

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.

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production.

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.

Slowing down of dynamics and orientational order preceding crystallization in hard-sphere systems.

Despite intensive studies in the past decades, the local structure of disordered matter remains widely unknown. We show the results of a coherent x-ray scattering study revealing higher-order correlations in dense colloidal hard-sphere systems in the vicinity of their crystallization and glass transition. With increasing volume fraction, we observe a strong increase in correlations at both medium-range and next-neighbor distances in the supercooled state, both invisible to conventional scattering techniques.

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA.

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM).

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.

Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering.

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs.

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

We present colloidal nanocomposites formed by incorporating magnetite Fe O 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.

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.

Dynamics of soft nanoparticle suspensions at hard X-ray FEL sources below the radiation-damage threshold.

The application of X-ray photon correlation spectroscopy (XPCS) at free-electron laser (FEL) facilities enables, for the first time, the study of dynamics on a (sub-)nanometre scale in an unreached time range between femtoseconds and seconds. For soft-matter materials, radiation damage is a major limitation when going beyond single-shot applications. Here, an XPCS study is presented at a hard X-ray FEL on radiation-sensitive polymeric poly(-isopropylacrylamide) (PNIPAM) nanoparticles.

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.