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Biocompatible coating based on bovine serum albumin (BSA) was applied on two different TiO 2 nanoparticles (aeroxide P25 and food grade E171) to investigate properties and stability of resulting TiO 2 @BSA composites, under the final perspective to create a "Safe-by-Design" coating, able to uniform, level off and mitigate surface chemistry related phenomena, as naturally occurring when nano-phases come in touch with proteins enriched biological fluids. The first step towards validating the proposed approach is a detailed characterization of surface chemistry with the quantification of amount and stability of BSA coating deposited on nanoparticles' surfaces. At this purpose, we implemented an orthogonal multi-techniques characterization platform, providing important information on colloidal behavior, particle size distribution and BSA-coating structure of investigated TiO 2 systems. Specifically, the proposed orthogonal approach enabled the quantitative determination of bound and free (not adsorbed) BSA, a key aspect for the design of intentionally BSA coated nano-structures, in nanomedicine and, overall, for the control of nano-surface reactivity. In fact, the BSA-coating strategy developed and the orthogonal characterisation performed can be extended to different designed nanomaterials in order to further investigate the protein-corona formation and promote the implementation of BSA engineered coating as a strategy to harmonize the surface reactivity and minimize the biological impact., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Asymmetric-flow field-flow fractionation (AF4) has been recognized as an invaluable tool for the characterisation of particle size, polydispersity, drug loading and stability of nanopharmaceuticals. However, the application of robust and high quality standard operating procedures (SOPs) is critical for accurate measurements, especially as these complex drug nanoformulations are most often inherently polydisperse. In this review we describe a unique international collaboration that lead to the development of a robust SOP for the measurement of physical-chemical properties of nanopharmaceuticals by multi-detector AF4 (MD-AF4) involving two state of the art infrastructures in the field of nanomedicine, the European Union Nanomedicine Characterization Laboratory (EUNCL) and the National Cancer Institute-Nanotechnology Characterisation Laboratory (NCI-NCL). We present examples of how MD-AF4 has been used for the analysis of key quality attributes, such as particle size, shape, drug loading and stability of complex nanomedicine formulations. The results highlight that MD-AF4 is a very versatile analytical technique to obtain critical information on a material particle size distribution, polydispersity and qualitative information on drug loading. The ability to conduct analysis in complex physiological matrices is an additional very important advantage of MD-AF4 over many other analytical techniques used in the field for stability studies. Overall, the joint NCI-NCL/EUNCL experience demonstrates the ability to implement a powerful and highly complex analytical technique such as MD-AF4 to the demanding quality standards set by the regulatory authorities for the pre-clinical safety characterization of nanomedicines., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

This work is dealing with the use of polystyrene (PS) nanoparticles as substrates for bioanalytical specific interactions. Different techniques were used for the accurate characterization of the PS nanoparticles of 100 nm and 196 nm before coating them with a layer of antibodies against immunoglobulins of type E (aIgE), giving to the particle a specific functionality. The formation of the aIgE adsorbed layer was monitored using centrifugal particle separation (CPS) and centrifugal field flow fractionation (CF3) experiments, which allowed to determine the size changes and the adsorbed mass. Particle sizes were also measured with DLS, used both as stand-alone instrument and coupled to CF3 (CF3-DLS). The complementary information obtained from the CPS and CF3-DLS measurements allowed the estimation of the density of the aIgE shell. The proteins immobilized at the surface fully retained their activity, as proven by the reactions between the functionalized PS-aIgE particles and immunoglobulins of type E (IgE) dispersed in suspensions prepared on purpose., (Copyright © 2019. Published by Elsevier B.V.)

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval. The workshop participants came up with recommendations highlighting methodological gaps that should be addressed in ongoing projects addressing the regulatory science of nanomedicines. In addition, individual opinions of experts relevant to progress of the regulatory science in the field of nanomedicine were summarised in the format of a survey., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Asymmetric Flow Field-Flow Fractionation (AF4) combined with multidetector analysis form a promising technique in the field of nanoparticle characterization. This system is able to measure the dimensions and physicochemical properties of nanoparticles with unprecedented accuracy and precision. Here, for the first time, this technique is optimized to characterize the interaction between an archetypal antimicrobial peptide and synthetic membranes. By using charged and neutral liposomes it is possible to mimic some of the charge characteristics of biological membranes. The use of AF4 system allows determining, in a single analysis, information regarding the selectivity of the peptides, the quantity of peptides bound to each liposome, the induced change in the size distribution and morphology of the liposomes. The results obtained provide relevant information for the study of structure-activity relationships in the context of membrane-induced antimicrobial action. This information will contribute to the rational design of potent antimicrobial agents in the future. Moreover, the application of this method to other liposome systems is straightforward and would be extremely useful for a comprehensive characterization with regard to size distribution and protein interaction in the nanomedicine field., (Copyright © 2015. Published by Elsevier B.V.)

With the ever growing use of nanoparticles in a broad range of industrial and consumer applications there is increasing likelihood that such nanoparticles will enter the aquatic environment and be transported through freshwater systems, eventually reaching estuarine or marine waters. Due to silver's known antimicrobial properties and widespread use of silver nanoparticles (AgNP), their environmental fate and impact is therefore of particular concern. In this context we have investigated the species-specific effects of low concentrations of 60 nm AgNP on embryonal development in Mediterranean sea urchins Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. The sensitivity of urchin embryos was tested by exposing embryos to nanoparticle concentrations in the 1-100 μg L(-1) range, with times of exposure varying from 30 min to 24 h (1 h-48 h for S. granularis) post-fertilisation which corresponded with fertilized egg, 4 cell, blastula and gastrula development phases. The most sensitive species to AgNP was A. lixula with significant modulation of embryonal development at the lowest AgNP concentrations of 1-10 μg L(-1) with high numbers of malformed embryos or arrested development. The greatest impact on development was noted for those embryos first exposed to nanoparticles at 6 and 24 h post fertilisation. For P. lividus, similar effects were noted at higher concentrations of 50 μg L(-1) and 100 μg L(-1) for all times of first exposure. The S. granularis embryos indicated a moderate AgNP impact, and significant developmental abnormalities were recorded in the concentration range of 10-50 μg L(-1). As later post-fertilisation exposure times to AgNP caused greater developmental changes in spite of a shorter total exposure time led us to postulate on additional mechanisms of AgNP toxicity. The results herein indicate that toxic effects of AgNP are species-specific. The moment at which embryos first encounter AgNP is also shown to be an important factor in the development of abnormalities, and future applications of the sea urchin embryo development test for nanoparticle toxicity testing should carefully address the specific phase of development of embryos when nanoparticles are first introduced., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The use of nanotechnology-based products is constantly increasing and there are concerns about the fate and effect on the aquatic environment of antimicrobial products such as silver nanoparticles. By combining different characterization techniques (asymmetric flow field-flow fractionation, single particle ICP-MS, UV-Vis) we show that it is possible to assess in detail the agglomeration process of silver nanoparticles in artificial seawater. In particular we show that the presence of alginate or humic acid differentially affects the kinetic of the agglomeration process. This study provides an experimental methodology for the in-depth analysis of the fate and behaviour of silver nanoparticles in the aquatic environment., (Copyright © 2015. Published by Elsevier Ltd.)

We show that using asymmetric flow-field-flow fractionation and UV-vis detector it is possible to separate, characterize, and quantify the correct number size distribution of gold nanoparticle (AuNP) mixtures of various sizes in the 5-60 nm range for which simple dynamic light scattering measurements give misleading information. The size of the collected nanoparticles fractions can be determined both in solution and in the solid state, and their surface chemistry characterized by NMR. This method will find widespread applications both in the process of "size purification" after the synthesis of AuNP and in the identification and characterization of gold-based nanomaterials in consumer products., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mitochondria, known to share many common features with prokaryotic cells, accumulate several endogenous ligands of the pattern-recognition Toll-like receptor 4 (TLR4), such as the heat shock proteins (Hsp) 70 and 60. TLR4 specifically recognises and responds to LPS of Gram-negative bacteria and participates in both autoimmune reactions and tissue regeneration due to its ability to recognise endogenous ligands. In the present study we show that mitochondria extracts obtained from hydrogen peroxide-dysfunctionalised cells induce a pro-inflammatory response in human THP-1 myeloid leukaemia cells. This inflammatory response was similar to that caused by LPS and much stronger than that induced by the extracts of normal mitochondria. Such reactions include activation of stress-adaptation hypoxia-inducible factor 1 alpha (HIF-1α) and expression/release of the pro-inflammatory cytokines IL-6 and TNF-α. Pre-treatment of THP-1 myeloid macrophages with TLR4-neutralising antibody before exposure to mitochondria extracts or LPS attenuated the inflammatory responses. Signalling pathways recruited by TLR4 in response to LPS and mitochondria-derived ligands were found to be the same. An in vitro ELISA-based TLR4-ligand binding assay, in which the ligand-binding domain of human TLR4 was immobilised, showed that mitochondria extracts contain endogenous TLR4 ligands. These results were verified in surface plasmon resonance experiments in which the affinity of the ligands derived from dysfunctional mitochondria was comparable with that of LPS and was much higher than that observed for normal mitochondria., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition. To obtain and compare the three-dimensional structure of a large number of different complexes, however, we need a faster method than traditional experimental techniques. While biocomputational docking is fast, its results might not be accurate. Combining experimental validation with computational prediction may be a solution. As a proof of concept, here we isolated a monoclonal Ab from the blood of a human donor recovered from dengue virus infection, characterized its immunological properties, and identified its epitope on domain III of dengue virus E protein through simple and rapid NMR chemical shift mapping experiments. We then obtained the three-dimensional structure of the Ab/Ag complex by computational docking, using the NMR data to drive and validate the results. In an attempt to represent the multiple conformations available to flexible Ab loops, we docked several different starting models and present the result as an ensemble of models equally agreeing with the experimental data. The Ab was shown to bind a region accessible only in part on the viral surface, explaining why it cannot effectively neutralize the virus., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Researchers from Europe and the USA met at the Joint Research Center (JRC) of the European Commission to discuss how to integrate gene and protein expression analyses with bioinformatic tools in the field of ecotoxicology and how this new approach could be translated in improved risk assessment procedures. The measurements of gene and/or protein expression levels, upon exposure to a chemical or a stressor, can be used to develop robust molecular biomarkers that will allow the early detection of environmental stress, study long-term exposure and infer the mechanism of action. These molecular biomarkers should be linked to phenotypic end points of exposure such as adverse effects in growth and reproduction in single organisms and populations. At environmentally realistic exposure levels there could be "non-linear" dose-response curves, which should be accounted for in the experimental design and in the analyses of microarray and proteomic data. The application of gene and protein expression profiling in ecotoxicology will have a significant impact on the ecotoxicology field in the near future and international collaborations will play an important role in accelerating the application of those techniques.

The conformation in solution of three different metal(III)-transferrins, namely aluminum(III), gallium(III) and indium(III) transferrin, was investigated by absorption, CD, 1H NMR and 13C NMR spectroscopies. The formation of the respective metal-transferrin complexes and the characteristic 2:1 metal-to-protein binding stoichiometry were unambiguously demonstrated, in all cases, through UV difference studies. The 13C NMR spectra of these metallotransferrins in the carbonyl region are very similar to one another pointing out that the arrangement of the synergistic anion in the binding site must be essentially the same. However, the CD spectra in the near UV (aromatic region) reveal the occurrence of significant differences between indium transferrin, on one side, and the other two derivatives, on the other. Also, the 1H NMR spectra exhibit a number of different features suggesting the occurrence of metal-induced conformational heterogeneity around the metal sites. Such metal-induced conformational heterogeneity probably affects the transferrin-receptor recognition process, resulting in a different metabolic fate of these metals in the organisms.