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Questo lavoro fa parte del progetto Europeo H2020 ASINA, il quale mira a promuovere delle soluzioni Safe-by-design (SbD) per nuovi nanomateriali (NMs) lungo tutto il loro ciclo vitale. L’adozione di innovativi approcci SbD è di grande importanza nel quadro del Green Deal della Commissione Europea. Negli ultimi decenni, la produzione e l’uso di NMs ha subito una sostanziale crescita, sollevando preoccupazioni a proposito del potenziale rischio associato al loro rilascio e all’esposizione della popolazione. Le nanoparticelle (NPs) di argento (Ag) e di biossido di titanio (TiO2) sono tra i NMs più ampiamente utilizzati e le loro applicazioni in vari prodotti, a causa delle proprietà antibatteriche delle prime e dell’attività fotocatalitica delle seconde, stanno aumentando. Lo scopo di questo lavoro è stato di identificare il pericolo posto da NMs metallici di nuova sintesi, disegnati secondo un approccio SbD, verso la salute umana durante la loro produzione e il loro utilizzo. Il primo passo ha previsto il test delle nuove NPs di Ag e TiO2 con diversi rivestimenti per confrontare i loro effetti tossicologici con NPs di riferimento senza alcun rivestimento. Gli endpoint sono stati selezionati secondo un approccio Adverse Outcome Pathway (AOP), il quale ha consentito l’identificazione dei principali eventi connessi alla tossicità di queste NPs. La vitalità cellulare, la risposta infiammatoria, i livelli di specie reattive dell’ossigeno, il danno ossidativo al DNA, e le interazioni tra cellule e NPs sono state analizzate nel modello alveolare in vitro A549. In seguito, un New Approach Methodology (NAM) è stato sviluppato per identificare e valutare il rischio delle NPs identificate come più sicure nella prima parte dello studio considerando la potenziale esposizione umana durante la loro produzione. Lo scopo è stato quello di definire meglio il pericolo di queste NPs. La selezione di dosi di esposizione appropriate per la definizione del pericolo ha un’importanz, This work is part of the H2020 European project ASINA which aims to promote Safe-by-Design (SbD) solutions for new nanomaterials (NMs) across all life cycle stages. The adoption of SbD novel approaches for new NMs is of pivotal importance in the framework of the European Commission’s Green Deal. Over the past decades, the production and use of NMs have seen substantial growth, raising concerns regarding the potential risk associated with their release and population exposure. Silver (Ag) and titania (TiO2) nanoparticles (NPs) are among the most widely used NMs and their applications in various products, due to the antibacterial properties of the first and photocatalytic activity of the latter, are increasing. The aim of this work is to identify the potential hazard posed by newly developed metal based NMs, designed according to a SbD approach, toward human health during their production and use. The initial step involved testing the newly developed Ag and TiO2 NPs coated with different agents to compare their toxicological effects to reference uncoated NPs. The endpoints were selected according to an Adverse Outcome Pathway (AOP) approach that enabled the identification of the main events linked to the toxicity of these NPs. Cell viability, inflammatory response, reactive oxygen species levels, oxidative DNA damage, and cell-NPs interactions were analyzed in the alveolar in vitro model, A549. Furthermore, a New Approach Methodology (NAM) was developed to identify and evaluate the hazard of the safer NPs (based on the results of the first part of this study) considering the potential human exposure during NMs production. The goal was to improve their hazard and risk definition. The selection of appropriate exposure doses for hazard assessment has gained increasing importance in the risk assessment framework. To this end, starting from monitoring campaign data and applying the MPPD model to determine the lung retained dose of NPs, it was possible to estimate the doses

A substantial increase in engineered nanoparticles in consumer products has been observed, heightening human and environmental exposure. Inhalation represents the primary route of human exposure, necessitating a focus on lung toxicity studies. However, to avoid ethical concerns the use of in vitro models is an efficient alternative to in vivo models. This study utilized an in vitro human alveolar barrier model at air-liquid-interface with four cell lines, for evaluating the biological effects of different gold nanoparticles. Exposure to PEGylated gold nanospheres, nanorods, and nanostars did not significantly impact viability after 24 h, yet all AuNPs induced cytotoxicity in the form of membrane integrity impairment. Gold quantification revealed cellular uptake and transport. Transcriptomic analysis identified gene expression changes, particularly related to the enhancement of immune cells. Despite limited impact, distinct effects were observed, emphasizing the influence of nanoparticles physicochemical parameters while demonstrating the model's efficacy in investigating particle biological effects.

The growing demand for effective antimicrobials determined a significant increase of new nanoformulated materials to fight against bacteria and viruses, to be used in many industrial sectors including textiles, cosmetics, pharmaceuticals, and air and water filtering. Nano silver (Ag)-based materials, well-known for their antibacterial properties, are among the most exploited ones. However, the toxicity mechanisms of Ag nanoparticles (NPs) are still debated, requiring more evidence to support a safe-by-design (SbD) strategy for these materials. This study used zebrafish (D. rerio) to assess and compare the toxic and adverse effects (AEs) of commercial Ag-NPs (naked and PVP-coated) and newly synthesized hydroxyethylcellulose-coated Ag-NPs in solution (Ag-HECs) or the powder (Ag-HECp) form. Statistical correlation analysis between AEs and NP physico-chemical (p-chem) properties, such as size, surface charge, and solubility, was also performed. The results ranked the materials as follows in terms of acute lethality (LC50) and malformation (EC50) effects: Ag-HECp > Ag-HECs > Ag-PVP > Ag-NKD. Notable AEs included axial defects, pericardial edema, and reduced lipid yolk consumption, impacting embryo growth and hatching time. Correlation analyses showed that the stabilizing agent HEC, though a safe polymer, played a significant role in modulating Ag-NPs' reactivity toward embryonic structures. The study discusses the biological mode of action and potential molecular events underlying the observed effects. These findings contribute to understanding the biological targets and AEs modulated by tuning Ag-NPs' properties and will additionally feed the SbD frameworks under development for implementing safe and sustainable Ag-based nano-enabled antimicrobial materials.

The widespread use of silver nanoparticles (Ag NPs) in food and consumer products suggests the relevance of human oral exposure to these nanomaterials (NMs) and raises the possibility of adverse effects in the gastrointestinal tract. The aim of this study was to investigate the toxicity of Ag NPs in a human intestinal cell line, either uncoated or coated with polyvinylpyrrolidone (Ag PVP) or hydroxyethylcellulose (Ag HEC) and digested in simulated gastrointestinal fluids. Physicochemical transformations of Ag NPs during the different stages of in vitro digestion were identified prior to toxicity assessment. The strategy for evaluating toxicity was constructed on the basis of adverse outcome pathways (AOPs) showing Ag NPs as stressors. It consisted of assessing Ag NP cytotoxicity, oxidative stress, genotoxicity, perturbation of the cell cycle and apoptosis. Ag NPs caused a concentration-dependent loss of cell viability and increased the intracellular level of reactive oxygen species as well as DNA damage and perturbation of the cell cycle. In vitro digestion of Ag NPs did not significantly modulate their toxicological impact, except for their genotoxicity. Taken together, these results indicate the potential toxicity of ingested Ag NPs, which varied depending on their coating but did not differ from that of non-digested NPs.

Incomplete combustion processes in diesel engines produce particulate matter (PM) that significantly contributes to air pollution. Currently, there remains a knowledge gap in relation to the physical and chemical characteristics and also the biological reactivity of the PM emitted from old- and new-generation diesel vehicles. In this study, the emissions from a Euro 3 diesel vehicle were compared to those from a Euro 6 car during the regeneration of a diesel particulate filter (DPF). Different driving cycles were used to collect two types of diesel exhaust particles (DEPs). The particle size distribution was monitored using an engine exhaust particle sizer spectrometer and an electrical low-pressure impactor. Although the Euro 6 vehicle emitted particulates only during DPF regeneration that primarily occurs for a few minutes at high speeds, such emissions are characterized by a higher number of ultrafine particles (<0.1 mu m) compared to those from the Euro 3 diesel vehicle. The emitted particles possess different characteristics. For example, Euro 6 DEPs exhibit a lower PAH content than do Euro 3 samples; however, they are enriched in metals that were poorly detected or undetected in Euro 3 emissions. The biological effects of the two DEPs were investigated in human bronchial BEAS-2B cells exposed to 50 mu g/mL of PM (corresponding to 5.2 mu g/cm(2)), and the results revealed that Euro 3 DEPs activated the typical inflammatory and procarcinogenic pathways induced by combustion-derived particles, while Euro 6 DEPs were less effective in regard to activating such biological responses. Although further investigations are required, it is evident that the different in vitro effects elicited by Euro 3 and Euro 6 DEPs can be correlated with the variable chemical compositions (metals and PAHs) of the emitted particles that play a pivotal role in the inflammatory and carcinogenic potential of airborne PM.

Zinc oxide (ZnO) is the most efficient curing activator employed in the industrial rubber production. However, ZnO and Zn(II) ions are largely recognized as an environmental hazard being toxic to aquatic organisms, especially considering Zn(II) release during tire lifecycle. In this context, aiming at reducing the amount of microcrystalline ZnO, a novel activator was recently synthetized, constituted by ZnO nanoparticles (NPs) anchored to silica NPs (ZnO-NP@SiO2-NP). The objective of this work is to define the possible hazards deriving from the use of ZnO-NP@SiO2-NP compared to ZnO and SiO2 NPs traditionally used in the tire industry. The safety of the novel activators was assessed by in vitro testing, using human lung epithelial (A549) and immune (THP-1) cells, and by the in vivo model zebrafish (Danio rerio). The novel manufactured nanomaterial was characterized morphologically and structurally, and its effects evaluated in vitro by the measurement of the cell viability and the release of inflammatory mediators, while in vivo by the Fish Embryo Acute Toxicity (FET) test. Resulting data demonstrated that ZnO-NP@SiO2-NP, despite presenting some subtoxic events, exhibits the lack of acute effects both in vitro and in vivo, supporting the safe-by-design development of this novel material for the rubber industry.

Multidrug antimicrobial resistance is a constantly growing health care issue associated with increased mortality and morbidity, and huge financial burden. Bacteria frequently form biofilm communities responsible for numerous persistent infections resistant to conventional antibiotics. Herein, novel nanoparticles (NPs) loaded with the natural bactericide farnesol (FSL NPs) are generated using high-intensity ultrasound. The nanoformulation of farnesol improved its antibacterial properties and demonstrated complete eradication of Staphylococcus aureus within less than 3 h, without inducing resistance development, and was able to 100% inhibit the establishment of a drug-resistant S. aureus biofilm. These antibiotic-free nano-antimicrobials also reduced the mature biofilm at a very low concentration of the active agent. In addition to the outstanding antibacterial properties, the engineered nano-entities demonstrated strong antiviral properties and inhibited the spike proteins of SARS-CoV-2 by up to 83%. The novel FSL NPs did not cause skin tissue irritation and did not induce the secretion of anti-inflammatory cytokines in a 3D skin tissue model. These results support the potential of these bio-based nano-actives to replace the existing antibiotics and they may be used for the development of topical pharmaceutic products for controlling microbial skin infections, without inducing resistance development., Peer Reviewed, Postprint (published version)