Quantitative size-resolved characterization of mRNA nanoparticles by in-line coupling of asymmetrical-flow field-flow fractionation with small angle X-ray scattering.

We present a generically applicable approach to determine an extensive set of size-dependent critical quality attributes inside nanoparticulate pharmaceutical products. By coupling asymmetrical-flow field-flow fractionation (AF4) measurements directly in-line with solution small angle X-ray scattering (SAXS), vital information such as (i) quantitative, absolute size distribution profiles, (ii) drug loading, (iii) size-dependent internal structures, and (iv) quantitative information on free drug is obtained. Here the validity of the method was demonstrated by characterizing complex mRNA-based lipid nanoparticle products. The approach is particularly applicable to particles in the size range of 100 nm and below, which is highly relevant for pharmaceutical products-both biologics and nanoparticles. The method can be applied as well in other fields, including structural biology and environmental sciences.

Novel Benchtop Magnetic Particle Spectrometer for Process Monitoring of Magnetic Nanoparticle Synthesis.

Magnetic nanoparticles combine unique magnetic properties that can be used in a variety of biomedical applications for therapy and diagnostics. These applications place high demands on the magnetic properties of nanoparticles. Thus, research, development, and quality assurance of magnetic nanoparticles requires powerful analytical methods that are capable of detecting relevant structural and, above all, magnetic parameters. By directly coupling nanoparticle synthesis with magnetic detectors, relevant nanoparticle properties can be obtained and evaluated, and adjustments can be made to the manufacturing process in real time. This work presents a sensitive and fast magnetic detector for online characterization of magnetic nanoparticles during their continuous micromixer synthesis. The detector is based on the measurement of the nonlinear dynamic magnetic response of magnetic nanoparticles exposed to an oscillating excitation at a frequency of 25 kHz, a technique also known as magnetic particle spectroscopy. Our results underline the excellent suitability of the developed magnetic online detection for coupling with magnetic nanoparticle synthesis based on the micromixer approach. The proven practicability and reliability of the detector for process monitoring forms the basis for further application fields, e.g., as a monitoring tool for chromatographic separation processes.

Nanoplastic Analysis by Online Coupling of Raman Microscopy and Field-Flow Fractionation Enabled by Optical Tweezers.

Nanoplastic pollution is an emerging environmental concern, but current analytical approaches are facing limitations in this size range. However, the coupling of nanoparticle separation with chemical characterization bears potential to close this gap. Here, we realize the hyphenation of particle separation/characterization (field-flow fractionation (FFF), UV, and multiangle light scattering) with subsequent chemical identification by online Raman microspectroscopy (RM). The problem of low Raman scattering was overcome by trapping particles with 2D optical tweezers. This setup enabled RM to identify particles of different materials (polymers and inorganic) in the size range from 200 nm to 5 µm, with concentrations in the order of 1 mg/L (109 particles L-1). The hyphenation was realized for asymmetric flow FFF and centrifugal FFF, which separate particles on the basis of different properties. This technique shows potential for application in nanoplastic analysis, as well as many other fields of nanomaterial characterization.

Inverse supercritical fluid extraction as a sample preparation method for the analysis of the nanoparticle content in sunscreen agents.

We demonstrate the use of inverse supercritical carbon dioxide (scCO2) extraction as a novel method of sample preparation for the analysis of complex nanoparticle-containing samples, in our case a model sunscreen agent with titanium dioxide nanoparticles. The sample was prepared for analysis in a simplified process using a lab scale supercritical fluid extraction system. The residual material was easily dispersed in an aqueous solution and analyzed by Asymmetrical Flow Field-Flow Fractionation (AF4) hyphenated with UV- and Multi-Angle Light Scattering detection. The obtained results allowed an unambiguous determination of the presence of nanoparticles within the sample, with almost no background from the matrix itself, and showed that the size distribution of the nanoparticles is essentially maintained. These results are especially relevant in view of recently introduced regulatory requirements concerning the labeling of nanoparticle-containing products. The novel sample preparation method is potentially applicable to commercial sunscreens or other emulsion-based cosmetic products and has important ecological advantages over currently used sample preparation techniques involving organic solvents.

Nanoparticle separation with a miniaturized asymmetrical flow field-flow fractionation cartridge.

Asymmetrical Flow Field-Flow Fractionation (AF4) is a separation technique applicable to particles over a wide size range. Despite the many advantages of AF4, its adoption in routine particle analysis is somewhat limited by the large footprint of currently available separation cartridges, extended analysis times and significant solvent consumption. To address these issues, we describe the fabrication and characterization of miniaturized AF4 cartridges. Key features of the down-scaled platform include simplified cartridge and reagent handling, reduced analysis costs and higher throughput capacities. The separation performance of the miniaturized cartridge is assessed using certified gold and silver nanoparticle standards. Analysis of gold nanoparticle populations indicates shorter analysis times and increased sensitivity compared to conventional AF4 separation schemes. Moreover, nanoparticulate titanium dioxide populations exhibiting broad size distributions are analyzed in a rapid and efficient manner. Finally, the repeatability and reproducibility of the miniaturized platform are investigated with respect to analysis time and separation efficiency.

Molecular mechanisms underlying FOXP3 induction in human T cells.

FOXP3 is playing an essential role for T regulatory cells and is involved in the molecular mechanisms controlling immune tolerance. Although the biological relevance of this transcription factor is well documented, the pathways responsible for its induction are still unclear. The current study reveals structure and function of the human FOXP3 promoter, revealing essential molecular mechanisms of its induction. The FOXP3 promoter was defined by RACE, cloned, and functionally analyzed using reporter-gene constructs in primary human T cells. The analysis revealed the basal, T cell-specific promoter with a TATA and CAAT box 6000 bp upstream the translation start site. The basal promoter contains six NF-AT and AP-1 binding sites, which are positively regulating the trans activation of the FOXP3 promoter after triggering of the TCR. The chromatin region containing the FOXP3 promoter was bound by NF-ATc2 under these conditions. Furthermore, FOXP3 expression was observed following TCR engagement. Promoter activity, mRNA, and protein expression of T cells were suppressed by addition of cyclosporin A. Taken together, this study reveals the structure of the human FOXP3 promoter and provides new insights in mechanisms of addressing T regulatory cell-inducing signals useful for promoting immune tolerance. Furthermore, the study identifies essential, positive regulators of the FOXP3 gene and highlights cyclosporin A as an inhibitor of FOXP3 expression contrasting other immunosuppressants such as steroids or rapamycin.