Preliminary Toxicological Analysis in a Safe-by-Design and Adverse Outcome Pathway-Driven Approach on Different Silver Nanoparticles: Assessment of Acute Responses in A549 Cells

International audienceSilver nanoparticles (Ag NPs) are among the most widely used metal-based nanomaterials (NMs) and their applications in different products, also as antibacterial additives, are increasing. In the present manuscript, according to an adverse outcome pathway (AOP) approach, we tested two safe-by-design (SbD) newly developed Ag NPs coated with hydroxyethyl cellulose (HEC), namely AgHEC powder and AgHEC solution. These novel Ag NPs were compared to two reference Ag NPs (naked and coated with polyvinylpyrrolidone—PVP). Cell viability, inflammatory response, reactive oxygen species, oxidative DNA damage, cell cycle, and cell–particle interactions were analyzed in the alveolar in vitro model, A549 cells. The results show a different toxicity pattern of the novel Ag NPs compared to reference NPs and that between the two novel NPs, the AgHEC solution is the one with the lower toxicity and to be further developed within the SbD framework

Antibacterial and In Vivo Studies of a Green, One-Pot Preparation of Copper/Zinc Oxide Nanoparticle-Coated Bandages

Simultaneous water and ethanol-based synthesis and coating of copper and zinc oxide (CuO/ZnO) nanoparticles (NPs) on bandages was carried out by ultrasound irradiation. High resolution-transmission electron microscopy demonstrated the effects of the solvent on the particle size and shape of metal oxide NPs. An antibacterial activity study of metal-oxide-coated bandages was carried out against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). CuO NP-coated bandages made from both water and ethanol demonstrated complete killing of S. aureus and E. coli bacteria within 30 min., whereas ZnO NP-coated bandages demonstrated five-log reductions in viability for both kinds of bacteria after 60 min of interaction. Further, the antibacterial mechanism of CuO/ZnO NP-coated bandages is proposed here based on electron spin resonance studies. Nanotoxicology investigations were conducted via in vivo examinations of the effect of the metal-oxide bandages on frog embryos (teratogenesis assay—Xenopus). The results show that water-based coatings resulted in lesser impacts on embryo development than the ethanol-based ones. These bandages should therefore be considered safer than the ethanol-based ones. The comparison between the toxicity of the metal oxide NPs prepared in water and ethanol is of great importance, because water will replace ethanol for bulk scale synthesis of metal oxide NPs in commercial companies to avoid further ignition problems. The novelty and importance of this manuscript is avoiding the ethanol in the typical water:ethanol mixture as the solvent for the preparation of metal oxide NPs. Ethanol is ignitable, and commercial companies are trying the evade its use. This is especially important these days, as the face mask produced by sonochemistry (SONOMASK) is being sold all over the world by SONOVIA, and it is coated with ZnO

An integrated new approach methodology for inhalation risk assessment of safe and sustainable by design nanomaterials

The production and use of nanomaterials (NMs) has increased over the last decades posing relevant questions on their risk after release and exposure of the population or sub-populations. In this context, the safe and sustainable by design (SSbD) approach framework requires to assess the potential hazard connected with intrinsic properties of the material along the whole life cycle of the NM and/or of the nano enabled products. Moreover, in the last years, the use of new advanced methodologies (NAMs) has increasingly gained attention for the use of alternative methods in obtaining relevant information on NMs hazard and risk. Considering the SSbD and the NAMs frameworks, within the ASINA H2020 project, we developed new NAMs devoted at improving the hazard and risk definition of different Ag and TiO2 NPs. The NAMs are developed considering two air liquid interface exposure systems, the Vitrocell Cloud-α and the Cultex Compact module and the relevant steps to obtain reproducible exposures are described. The new NAMs build on the integration of environmental monitoring campaigns at nano-coating production sites, allowing the quantification by the multiple-path particle dosimetry (MPPD) model of the expected lung deposited dose in occupational settings. Starting from this information, laboratory exposures to the aerosolized NPs are performed by using air liquid interface exposure equipment and human alveolar cells (epithelial cells and macrophages), replicating the doses of exposure estimated in workers by MPPD. Preliminary results on cell viability and inflammatory responses are reported. The proposed NAMs may represent possible future reference procedures for assessing the NPs inhalation toxicology, supporting risk assessment at real exposure doses

Iron nanoparticle bio-interactions evaluated in Xenopus laevis embryos, a model for studying the safety of ingested nanoparticles

Iron nanoparticles (NPs) have been proposed as a tool in very different fields such as environmental remediation and biomedical applications, including food fortification against iron deficiency, even if there is still concern about their safety. Here, we propose Xenopus laevis embryos as a suitable model to investigate the toxicity and the bio-interactions at the intestinal barrier of Fe3O4 and zerovalent iron (ZVI) NPs compared to Fe(II) and (III) salts in the 5 to 100 mg Fe/L concentration range using the Frog Embryo Teratogenesis Assay in Xenopus (FETAX). Our results demonstrated that, at concentrations at which iron salts induce adverse effects, both iron NPs do not cause acute toxicity or teratogenicity even if they accumulate massively in the embryo gut. Prussian blue staining, confocal and electron microscopy allowed mapping of iron NPs in enterocytes, along the paracellular spaces and at the level of the basement membrane of a well-preserved intestinal epithelium. Furthermore, the high bioaccumulation factor and the increase in embryo length after exposure to iron NPs suggest greater iron intake, an essential element for organisms. Together, these results improve the knowledge on the safety of orally ingested iron NPs and their interaction with the intestinal barrier, useful for defining the potential risks associated with their use in food/feed fortification

Teratogenic hazard of BPEI-coated silver nanoparticles to <i>Xenopus laevis</i>

<p>Silver nanoparticles (AgNPs) are among the most exploited antimicrobial agents and are used in many consumer products. Size and surface reactivity are critical physico-chemical properties responsible for NPs toxicity, and surface coatings, often used to functionalize or stabilize AgNPs, can influence their toxic profile and biocompatibility. In the current study the developmental toxicity of (1) negatively charged citrate-coated AgNPs (Cit-AgNPs), (2) positively charged branched polyethylenimine-coated AgNPs (BPEI-AgNPs), and (3) Ag⁺ (from 0.0625 to 0.75 mg Ag/L) was investigated by the standard Frog Embryo Teratogenesis Assay – <i>Xenopus</i> (FETAX). In order to identify the most sensitive developmental phase, embryos were also exposed during different embryonic stages. Morphological and bio-physical studies were performed to characterize tissue lesions and NP uptake. The results suggest that Ag⁺ was strongly embryo-lethal. Contrary to Cit-AgNPs, the positively charged BPEI-AgNPs exert a concentration-dependent effect on lethality and malformations of embryos. The BPEI-AgNPs showed the highest teratogenic index (TI = 1.6), pointing out the role of functional coating in determining the developmental hazard. The highest susceptibility to BPEI-AgNPs was during early embryogenesis, when embryos are still enclosed in the fertilization envelope, and the post-stomodeum opening stages, when NPs ingestion occurs. In BPEI-AgNPs treated larvae, the histological examination revealed irregular intestinal diverticula coupled with edematous connective tissue. Small NPs aggregates are mapped throughout the intestinal mucosa and secondary target organs by two-photon excitation microscopy. We conclude that a teratogenic risk may be associated with BPEI-AgNPs exposure, but the modality of NP-tissue interactions and the teratogenic mechanism need further investigations to be better defined.</p