Nanoparticles are defined as elementary particles with a size between 1 and 100 nm for at least 50% (in number). They can be made from natural materials, or manufactured. Due to their small sizes, novel toxicological issues are raised and thus determining the accurate size of these nanoparticles is a major challenge. In this study, we performed an intercomparison experiment with the goal to measure sizes of several nanoparticles, in a first step, calibrated beads and monodispersed SiO2 Ludox®, and, in a second step, nanoparticles (NPs) of toxicological interest, such as Silver NM-300 K and PVP-coated Ag NPs, Titanium dioxide A12, P25(Degussa), and E171(A), using commonly available laboratory techniques such as transmission electron microscopy, scanning electron microscopy, small-angle X-ray scattering, dynamic light scattering, wet scanning transmission electron microscopy (and its dry state, STEM) and atomic force microscopy. With monomodal distributed NPs (polystyrene beads and SiO2 Ludox®), all tested techniques provide a global size value amplitude within 25% from each other, whereas on multimodal distributed NPs (Ag and TiO2) the inter-technique variation in size values reaches 300%. Our results highlight several pitfalls of NP size measurements such as operational aspects, which are unexpected consequences in the choice of experimental protocols. It reinforces the idea that averaging the NP size from different biophysical techniques (and experimental protocols) is more robust than focusing on repetitions of a single technique. Besides, when characterizing a heterogeneous NP in size, a size distribution is more informative than a simple average value. This work emphasizes the need for nanotoxicologists (and regulatory agencies) to test a large panel of different techniques before making a choice for the most appropriate technique(s)/protocol(s) to characterize a peculiar NP.

Nanoparticles (NPs) can be toxic, depending on their physico-chemical characteristics. Macroautophagy/autophagy could represent a potential underlying mechanism of this toxicity. We therefore set up a study aimed to characterize in depth the effects, on autophagy, of macrophage exposure to NPs, with a particular attention paid to the role of NP physico-chemical characteristics (specifically chemical composition, shape, size, length, crystal phase, and/or surface properties). We demonstrate that exposure to carbon nanotubes (CNT) but not to spherical NPs leads to the blockage of the autophagic flux. We further identified lysosomal dysfunction, in association with the downregulation of SNAPIN expression, as the underlying mechanism responsible for the CNT-induced autophagy blockade. These results identify for the first time the shape as a major determinant of the interaction of NPs with the autophagy pathway. Moreover, identifying the lysosomes and SNAPIN as primary targets of MWCNT toxicity opens new directions in the interpretation and understanding of nanomaterial toxicity.

Silver nanoparticles (Ag-NPs) are used in a variety of consumers' goods. Their toxicological impact is currently intensely studied, mostly upon acute exposure, but their intracellular dissolution and fate is rather poorly documented. In this study, murine primary macrophages were exposed to a single high but non-lethal dose of Ag-NPs or to repeated, low doses of Ag-NPs. Cells were either collected immediately after acute exposure or after 72 h of recovery in the NP-free exposure medium. Ag intracellular content and distribution were analyzed by particle-induced X-ray emission, transmission electron microscopy coupled to energy-dispersive spectroscopy analysis and inductively coupled plasma mass spectrometry. In parallel, macrophage functionality as well as inflammatory and thiol-responses were assessed after Ag-NP exposure. We show that Ag accumulation in macrophages is similar upon acute and repeated exposure to Ag-NPs, and that Ag is partly expelled from cells during the 72 h recovery stage. However, acute exposure leads to a strong response of macrophages, characterized by reduced mitochondrial membrane potential, phagocytic capacity and nitric oxide (NO) production upon lipopolysaccharide (LPS) stimulation. Under this condition, we also show an increased release of proinflammatory cytokines as well as a decreased release of anti-inflammatory cytokines. This response is reversible since these biomarkers reach their basal level after the recovery phase; and is much less intense in repeatedly exposed cells. These results suggest that repeated exposure of macrophages to Ag-NPs, which is a more realistic exposure scenario than acute exposure, leads to significant Ag intracellular accumulation but a much less intense toxicological response.

Nanoparticles belong to the class of nanomaterial that are natural, incidental, or manufactured materials containing elementary particles where at least one external dimension is in the range of 1 nm to 100 nm for 50% or more of the particles in the number size distribution. Therefore, size criteria is a key control parameter for defining nanoparticles. Moreover, nanoparticles have the property of assembling, agglomerating, or aggregating elementary particles into larger entities. But, the definition clearly refers to elementary particle and thus fine methodology must be used to obtain the critical size of nanoparticles. Besides, nanotoxicology which is the field of study of toxicological effect of nanoparticle in health aims to relate putative toxicological effect to the size of nanoparticles as the current dogma in the field suggest a greatest impact/reactivity with smaller particles than larger ones. Thus, obtaining accurate size measurements of nanoparticles is of great scientific significance. In this work, we have combined several biophysical and microscopic methods to characterize the size of several nanoparticles of main interest in toxicological studies. Although we have worked on more than 50 different metallic nanoparticles, we only focused our interest on two families: Silver (Ag) and titane oxide (TiO 2 ). We have used atomic force microscopy, wet scanning transmission electron microscopy, dynamic light scattering, small‐angle X‐ray scattering. First, controls on well‐behaved nanosize samples were performed and maximum dispersion between low‐bound and high‐bound sizes was about 30%. The striking results of this work is the relatively large dispersion of results obtained on Ag and TiO 2 (several hundreds of percent) depending on the method used. Several hypotheses can explain these results: nanoparticle solutions are highly heterogeneous and some methods may capture different forms, measurement methods are biased toward a certain size, methodologies used to extract sizes are inaccurate. All these hypotheses are plausible and will be discussed in this work.

Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.

The development and the increasing production of manufactured nanoparticles (NP) are raising safety concerns because of the potential effects of NP on human health, particularly at the respiratory level. Several studies have shown that exposure to manufactured NP can induce pathogenic biological effects, with lung remodelling (fibrosis, emphysema…), depending on the physicochemical characteristics of the NP. Currently, oxidative stress and inflammation are the most widely accepted paradigms of NP toxicity; however, the exact underlying mechanisms in the biological effects of NP still remain unknown. Autophagy is a physiological process that allows the autodigestion of the subcellar components and which is also involved in the elimination of intracellular pathogens. It has been shown that this process can negatively regulate inflammation and oxidative stress. Thus, the hypothesis of this study is that a defective autophagy could be a new mechanism explaining, at least in part, NP effects. We choose to focus on TiO2 NP since it is one of the most abundantly produced and widely used NP. We used seven TiO2 NPs presenting different physicochemicals properties (size, crystal phase, surface embedding) and compared their effects to those of micron-size TiO2 and carbon black (CB - for chemical composition effects). NPs were characterized by electron microscopy, dynamic light scattering and X-ray diffraction. Murine macrophages (RAW cell line) were exposed to 50 μg/mL TiO2 and CB NP for 6 hours. Effects of NP on the autophagy process were analysed by looking at the expression of autophagy markers (LC3-II and p62) and lysosomal proteins (LAMPs and cathepsins). We also analysed the cytoskeleton network by fluorescence microscopy. Moreover, expression of inflammatory cytokines was determined in macrophages exposed to NP. All particles, except the micrometric one, induced an increase of LC3-II protein expression, indicating an accumulation of autophagosomes. These particles also increased p62 protein level, suggesting an autophagy blockade. This perturbation of the autophagy process by NP does not seem to result from a disruption of the cytoskeleton but from a defect in the lysosome function as suggested by a decrease of mature cathepsins expression. Moreover, preliminary results showed that exposure to TiO2 NP induce inflammation, at different levels depending on their physicochemicals characteristics. These results suggest that some TiO2 NP, depending on their physicochemical properties, can block the autophagy process. The future work will be to understand the mechanisms explaining this autophagy dysfunction and to determine its consequence on the toxicity induced by these NP.

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.