Sedimentation of Nanoparticles in in vitro Toxicity Assays

Attached extended abstract published in the conference proceedingsJRC.DG.I.5-Nanobioscience

Interaction of magnetic nanoparticles with U87MG cells studied by synchrotron radiation X-ray fluorescence techniques

International audienceSynchrotron radiation (SR) X-ray microscopy combined with X-ray fluorescence (XRF) microspectroscopy provides unique information that have pushed the frontiers of biological research, particularly when investigating intracellular mechanisms. This work reports an SR-XRF microspectroscopy investigation on the distribution and the potential toxicity of Fe 2 O 3 and CoFe 2 O 4 nanoparticles (NPs) in U87MG glioblastoma-astrocytoma cells. The U87MG cells exposed to NPs concentrations ranging from 5 to 250 mg/ml for 24 h were analyzed in order to monitor both morphological and chemical changes. The SR-XRF maps complemented with XRM absorption and phase contrast images have revealed different intracellular distribution patterns for the two nanoparticles types allowing different mechanism of toxicity to be deduced

Changes in Caco-2 cells transcriptome profiles upon exposure to gold nanoparticles

Abstract Higher efficacy and safety of nano gold therapeutics require examination of cellular responses to gold nanoparticles (AuNPs). In this work we compared cellular uptake, cytotoxicity and RNA expression patterns induced in Caco-2 cells exposed to AuNP (5 and 30 nm). Cellular internalization was dose and time-dependent for both AuNPs. The toxicity was observed by colony forming efficiency (CFE) and not by Trypan blue assay, and exclusively for 5 nm AuNPs, starting at the concentration of 200 μM (24 and 72 h of exposure). The most pronounced changes in gene expression (Agilent microarrays) were detected at 72 h (300 μM) of exposure to AuNPs (5 nm). The biological processes affected by smaller AuNPs were: RNA/zinc ion/transition metal ion binding (decreased), cadmium/copper ion binding and glutathione metabolism (increased). Some Nrf2 responsive genes (several metallothioneins, HMOX, G6PD, OSGIN1 and GPX2) were highly up regulated. Members of the selenoproteins were also differentially expressed. Our findings indicate that exposure to high concentration of AuNPs (5 nm) induces metal exposure, oxidative stress signaling pathways, and might influence selenium homeostasis. Some of detected cellular responses might be explored as potential enhancers of anti-cancer properties of AuNPs based nanomedicines

Physicochemical characterisation of gold, silica and silver nanoparticles in water and in serum-containing cell culture media

This report presents the results from a study organised under the coordination of JRC as part of a project aiming at the adaptation of the in vitro micronucleus test (Test Guideline 487) for the assessment of manufactured NMs. The aim of the first step of the project was to evaluate the physicochemical characterisation of selected representative nanomaterials (5 nm gold, 30 nm gold, 22 nm silica, 30 nm citrate and 30 nm PVP stabilised silver nanoparticles) in pure water and in different complete culture media. The results of the study show that using a combination of different characterisation techniques is important to providing reliable information about the agglomeration behaviour of the tested nanoparticles in complete cell culture media (CCM). Most of the materials exhibited mild agglomeration in serum containing CCM. Only the PVP functionalised silver nanoparticles showed a size distribution change in all of the culture media that is so small that it could be attributed to solely protein adsorption without notable agglomeration. Silica nanoparticles were found to be the most sensitive to interaction with serum containing CCM, showing massive concentration and time dependent agglomeration strongly affected by the CCM composition. Extensive agglomeration might lead also to the accelerated sedimentation of the particles changing drastically the true, effective dose that the cells will receive under in vitro conditions1, 2. Thus, it has to be investigated in more detail and taken in account when designing in vitro experiments in the next phase of the project.JRC.F.2-Consumer Products Safet

Pro- and anti-oxidant properties of near-infrared (NIR) light responsive carbon nanoparticles

Elemental carbon nanomaterials (ECNMs) are redox active agents that can be exploited to purposely modify the redox balance of cells. Both pro- or antioxidant properties have been reported. However, to the best of our knowledge, there are not comprehensive studies exploring both properties on the same material in view of a potential application in medicine. At the same time, the effect of the bulk structure on the pro/antioxidant properties is poorly known. Here, carbon nanoparticles (CNPs) derived by glucose with definite size and shape have been prepared, and their redox properties evaluated in cell free systems in the dark or following activation with a Near Infrared (NIR) laser beam (945 nm, 1.3 W/cm2). We found that, when irradiated with NIR, CNPs efficiently generate heat and singlet oxygen (1O2), a property that can be exploited for dual photo-thermal (PT)/photodynamic (PD) therapy in cancer. On the other hand, in the absence of photo-activation, CNPs react with both oxidant (hydroxyl radicals) and antioxidant (glutathione) species. When tested on a murine macrophages cell line (RAW 264.7) CNPs were clearly antioxidant. Furthermore, albeit efficiently internalized, CNPs do not exert cytotoxic effect up to 80 µg/ml and do not exacerbate TNF-α-mediated inflammation. Overall, the results reported herein suggest that CNPs may represent a new class of safe nanomaterials with potential applications in medicine

Evidence of SARS-CoV-2 bacteriophage potential in human gut microbiota

Background: In previous studies we have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth, and it is influenced by the administration of specific antibiotics. These observations are compatible with a 'bacteriophage-like' behaviour of SARS-CoV-2. Methods: We have further elaborated on these unusual findings and here we present the results of three different supplementary experiments: (1) an electron-microscope analysis of samples of bacteria obtained from a faecal sample of a subject positive to SARS-CoV-2; (2) mass spectrometric analysis of these cultures to assess the eventual de novo synthesis of SARS-CoV-2 spike protein; (3) sequencing of SARS-CoV-2 collected from plaques obtained from two different gut microbial bacteria inoculated with supernatant from faecal microbiota of an individual positive to SARS-CoV-2. Results: Immuno-labelling with Anti-SARS-CoV-2 nucleocapsid protein antibody confirmed presence of SARS-CoV-2 both outside and inside bacteria. De novo synthesis of SARS-CoV-2 spike protein was observed, as evidence that SARS-CoV-2 RNA is translated in the bacterial cultures. In addition, phage-like plaques were spotted on faecal bacteria cultures after inoculation with supernatant from faecal microbiota of an individual positive to SARS-CoV-2. Bioinformatic analyses on the reads obtained by sequencing RNA extracted from the plaques revealed nucleic acid polymorphisms, suggesting different replication environment in the two bacterial cultures. Conclusions: Based on these results we conclude that, in addition to its well-documented interactions with eukaryotic cells, SARS-CoV-2 may act as a bacteriophage when interacting with at least two bacterial species known to be present in the human microbiota. If the hypothesis proposed, i.e., that under certain conditions SARS-CoV-2 may multiply at the expense of human gut bacteria, is further substantiated, it would drastically change the model of acting and infecting of SARS-CoV-2, and most likely that of other human pathogenic viruses

"In vitro" comparative immune effects of different titanium compounds.

Exposure to Ti compounds is today an occupational and environmental health hazard. Object of this study was to determine "in vitro" effects of different Ti salts on cultured human peripheral blood mononuclear cells (PBMC) proliferation and cytokine release. 10−4 and 10−7 M Ti compounds did not modify spontaneous PBMC proliferation. Ti dioxide (a biocompatible material and sunscreen component) did not exert effects on phytoemagglutinin (PHA) stimulated PBMC proliferation and on PHA stimulated IFN-γ and TNF-α release from PBMC. On the other hand, 10−4 M Ti oxalate (with wide industrial applications) and Ti ascorbate (used mainly in agriculture) inhibited about 70 % the PHA stimulated PBMC proliferation; both these Ti compounds at 10−4 and 10−7 M concentrations significantly inhibited TNF-α release, while only Ti oxalate inhibited that of IFN-γ. Titanocene (used in chemotherapy) did not exert effects on PBMC proliferation but markedly inhibited IFN-γ and TNF-α release. On the whole, this study demonstrates that Ti dioxide is not immunotoxic; Ti oxalate shows marked immunotoxicity; titanocene exerts selective toxicity on cytokine release but not on PBMC proliferation, while Ti ascorbate affects TNF-α release from PBMC but not IFN-γ release. In conclusion, these data show that immunotoxicity of Ti depends on speciation

Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts

AbstractThe wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials.In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size