Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

Development and Characterization of a 96-Well Exposure System for Safety Assessment of Nanomaterials.

In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air-liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air-liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cell-based assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivo-like and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.

Physicochemical characterization and quantification of nanoplastics: applicability, limitations and complementarity of batch and fractionation methods.

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)-multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC-MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)-based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC-MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation.

Fast and Purification-Free Characterization of Bio-Nanoparticles in Biological Media by Electrical Asymmetrical Flow Field-Flow Fractionation Hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis Detection.

Accurate physico-chemical characterization of exosomes and liposomes in biological media is challenging due to the inherent complexity of the sample matrix. An appropriate purification step can significantly reduce matrix interferences, and thus facilitate analysis of such demanding samples. Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4) provides online sample purification while simultaneously enabling access to size and Zeta potential of sample constituents in the size range of approx. 1-1000 nm. Hyphenation of EAF4 with Multi-Angle Light Scattering (MALS) and Nanoparticle Tracking Analysis (NTA) detection adds high resolution size and number concentration information turning this setup into a powerful analytical platform for the comprehensive physico-chemical characterization of such challenging samples. We here present EAF4-MALS hyphenated with NTA for the analysis of liposomes and exosomes in complex, biological media. Coupling of the two systems was realized using a flow splitter to deliver the sample at an appropriate flow speed for the NTA measurement. After a proof-of-concept study using polystyrene nanoparticles, the combined setup was successfully applied to analyze liposomes and exosomes spiked into cell culture medium and rabbit serum, respectively. Obtained results highlight the benefits of the EAF4-MALS-NTA platform to study the behavior of these promising drug delivery vesicles under in vivo like conditions.

Integration of Inverse Supercritical Fluid Extraction and Miniaturized Asymmetrical Flow Field-Flow Fractionation for the Rapid Analysis of Nanoparticles in Sunscreens.

We report the use of inverse supercritical fluid extraction (SFE) and miniaturized asymmetrical flow field-flow fractionation (mAF4) for the preparation and subsequent analysis of titanium dioxide nanoparticles in model and commercial sunscreens. The approach allows for the fast and reliable fractionation and sizing of TiO2 nanoparticles and their quantitation in commercial products. This new method represents a powerful and efficient tool for the verification of nanoparticle content in a wide range of matrixes, as demanded by recently introduced regulatory requirements. Furthermore, the use of carbon dioxide as an environmentally friendly solvent is in line with the increasing need for ecologically compatible analytical techniques.

Effect of Protein Corona on Silver Nanoparticle Stabilization and Ion Release Kinetics in Artificial Seawater.

In parallel with the growing use of nanoparticle-containing products, their release into the environment over the coming years is expected to increase significantly. With many large population centers located in near-coastal areas, and increasing evidence that various nanoparticles may be toxic to a range of organisms, biota in estuarine and coastal waters may be particularly vulnerable. While size effects may be important in cases, silver nanoparticles have been found to be toxic in large part due to their release of silver ions. However, there is relatively little data available on how nanoparticle coatings can affect silver ion release in estuarine or marine waters. We have found that albumin, as a model for biocorona-forming macromolecules which nanoparticles may encounter in wastewater streams, stabilizes silver colloids from agglomeration in high salinity marine waters by electrosteric repulsion for long time periods. A minimum mass ratio of about 130 for albumin:silver nanoparticles (40 nm) was required for stable dispersion in seawater. Increasing albumin concentration was also found to reduce dissolution of nanoparticles in seawater with up to 3.3 times lower concentrations of silver ions noted. Persistent colloids and slow sustained ion release may have important consequences for biota in these environmental compartments.

Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.

The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques. The characterization strategy and the analytical measurements described here will have an important role during formulation development and in determining robust quality attributes ultimately supporting the quality assessment of these innovative RNA therapeutics.

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.

Fluorescence Labeling of Cellulose Nanocrystals-A Facile and Green Synthesis Route.

Efficient chemical modification of cellulose nanocrystals (CNCs) by grafting commonly involves aprotic solvents, toxic reactants, harsh reaction conditions, or catalysts, which have negative effects on the particle character, reduced dispersibility and requires further purification, if products are intended for biomedical applications. This work, in contrast, presents a robust, facile, and green synthesis protocol for the grafting of an amino-reactive fluorophore like fluorescein isothiocyanate (FITC) on aqueous CNCs, combining and modifying existent approaches in a two-step procedure. Comparably high grafting yields were achieved, which were confirmed by thermogravimetry, FTIR, and photometry. The dispersive properties were confirmed by DLS, AF4-MALS, and TEM studies. The presented route is highly suitable for the introduction of silane-bound organic groups and offers a versatile platform for further modification routes of cellulose-based substrates.

Analysis of complex particle mixtures by asymmetrical flow field-flow fractionation coupled to inductively coupled plasma time-of-flight mass spectrometry.

Asymmetrical flow field-flow fractionation (AF4) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS) has been widely used to characterize metal containing particles. This study demonstrates the advantages of coupling AF4 with ICP-time-of-flight mass spectrometry (ICP-TOFMS) in standard and single particle modes to determine size distribution, elemental composition, and number concentration of composite particles. The coupled system was used to characterize two complex particle mixtures. The first mixture consisted of particles extracted from micro-alloyed steels with two size populations of different elemental composition. The second mixture consisted of particles extracted from soil spiked with various engineered nanoparticles (ENPs). The equivalent hydrodynamic sizes of individual micro-alloyed steel particles were up to 6 times larger than the sizes determined by single particle (sp)-ICP-TOFMS. The larger AF4 sizes were attributed to the presence of a surface coating, which is not reflected in the core size determined by sp-ICP-TOFMS. Two particle populations could not be separated by AF4 due to their broad size distributions but were resolved by sp-ICP-TOFMS using their unique elemental signatures. Multi-angle light scattering and ICP-TOFMS signals of soil suspensions increased with the spiked ENP concentrations. However, only after conducting full element screening and single particle fingerprinting by ICP-TOFMS could this increase be attributed to enhanced extraction efficiency of natural particles and the risk for false conclusions be eliminated. In this study, we describe how AF4 coupled to ICP-TOFMS can be applied to study complex samples of inorganic particles which contain organic compounds.

Influence of Physicochemical Characteristics and Stability of Gold and Silver Nanoparticles on Biological Effects and Translocation across an Intestinal Barrier-A Case Study from In Vitro to In Silico.

A better understanding of their interaction with cell-based tissue is a fundamental prerequisite towards the safe production and application of engineered nanomaterials. Quantitative experimental data on the correlation between physicochemical characteristics and the interaction and transport of engineered nanomaterials across biological barriers, in particular, is still scarce, thus hampering the development of effective predictive non-testing strategies. Against this background, the presented study investigated the translocation of gold and silver nanoparticles across the gastrointestinal barrier along with related biological effects using an in vitro 3D-triple co-culture cell model. Standardized in vitro assays and quantitative polymerase chain reaction showed no significant influence of the applied nanoparticles on both cell viability and generation of reactive oxygen species. Transmission electron microscopy indicated an intact cell barrier during the translocation study. Single particle ICP-MS revealed a time-dependent increase of translocated nanoparticles independent of their size, shape, surface charge, and stability in cell culture medium. This quantitative data provided the experimental basis for the successful mathematical description of the nanoparticle transport kinetics using a non-linear mixed effects modeling approach. The results of this study may serve as a basis for the development of predictive tools for improved risk assessment of engineered nanomaterials in the future.

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension.

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most "active" component of a nanoparticle dose. Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1-1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration). We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20-100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

Multi-endpoint toxicological assessment of polystyrene nano- and microparticles in different biological models in vitro.

Nanoplastics (NP) and microplastics (MP) accumulate in our environment as a consequence of the massive consumption of plastics. Huge knowledge-gaps exist regarding uptake and fate of plastic particles in micro- and nano-dimensions in humans as well as on their impact on human health. This study investigated the transport and effects of 50 nm and 0.5 µm COOH-modified polystyrene (PS) particles, as representatives for NP and MP, in different biological models in vitro. Acute toxicity and potential translocation of the particles were studied at the human intestinal and placental barrier using advanced in vitro co-culture models. Furthermore, embryotoxicity and genotoxicity were investigated as highly sensitive endpoints. Polystyrene was not acutely toxic in both sizes (nano- and microparticles). No transport across the intestinal and placental barrier but a cellular uptake and intracellular accumulation of PS nano- and microparticles were determined. The particles were identified as weak embryotoxic and non-genotoxic. In contrast to single-organ studies, this multi-endpoint study is providing a data-set with the exact same type of particles to compare organ-specific outcomes. Our study clearly shows the need to investigate other types of plastics as well as towards long-term or chronic effects of plastic particles in different biological models in vitro.