The use of a complex tetra-culture alveolar model to study the biological effects induced by gold nanoparticles with different physicochemical properties.

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.

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.

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.

Silver 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.

Characterization of microparticles derived from waste plastics and their bio-interaction with human lung A549 cells.

Microplastics (MPs) represent a worldwide emerging relevant concern toward human and environmental health due to their intentional or unintentional release. Human exposure to MPs by inhalation is predicted to be among the most hazardous. MPs include both engineered, or primary MPs, and secondary MPs, materials obtained by fragmentation from any plastic good. The major part of the environmental MPs is constituted by the second ones that are irregular in size, shape and composition. These features make the study of the biological impact of heterogenous MPs of extremely high relevance to better estimate the real toxicological hazards of these materials on human and environmental organisms. The smallest fractions of plastic granules, relying on the micron-sized scale, can be considered as the most abundant component of the environmental MPs, and for this reason, they are typically used to perform toxicity tests using in vitro systems representative of an inhalation exposure scenario. In the present work, MPs obtained from industrial treatment of waste plastics (wMPs < 50 µm) were investigated, and after the physico-chemical characterization, the cytotoxic, inflammatory and genotoxic responses, as well as the modality of wMPs interactions with alveolar lung cells, were determined. Obtained results indicated that, at high concentrations (100 µg/ml) and prolonged exposure time (48 h), wMPs affect biological responses by inducing inflammation and genotoxicity, as a result of the cell-wMP interactions, also including the uptake of the smaller particles.

Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging.

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (INaL) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca2+ leak. Electrical and intracellular Ca2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.

Microplastics from miscellaneous plastic wastes: Physico-chemical characterization and impact on fish and amphibian development.

Microplastic pollution represents a global problem with negative impacts on aquatic environment and organisms' health. To date, most of the laboratory toxicological studies on microplastics (MPs) have made use of single commercial micro and nano-polymers, which do not reflect the heterogeneity of environmental MPs. To improve the relevance of the hazard assessment, micrometer-sized plastic particles of miscellaneous non-reusable waste plastics, with size <100 µm and <50 µm (waste microplastics, wMPs), were characterized by microscopic and spectroscopic techniques and tested on developing zebrafish and Xenopus laevis by FET and FETAX assays respectively. Moreover, the modalities of wMP interaction with the embryonic structures, as well as the histological lesions, were explored by light and electron microscopy. We have shown that wMPs had very heterogeneous shapes and sizes, were mainly composed of polyethylene and polypropylene and contained metal and organic impurities, as well as submicrometric particle fractions, features that resemble those of environmental occurring MPs. wMPs (0.1-100 mg/L) caused low rate of mortality and altered phenotypes in embryos, but established species-specific biointeractions. In zebrafish, wMPs by adhering to chorion were able to delay hatching in a size and concentration dependent manner. In Xenopus embryos, which open stomodeum earlier than zebrafish, wMPs were accumulated in intestinal tract, where produced mechanical stress and stimulated mucus overproduction, attesting an irritation response. Although wMP biointeractions did not interfere with morphogenesis processes, further studies are needed to understand the underlying mechanisms and long-term impact of these, or even smaller, wMPs.

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.

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 Xenopus laevis.

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 - Xenopus (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.

Do Nanoparticle Physico-Chemical Properties and Developmental Exposure Window Influence Nano ZnO Embryotoxicity in Xenopus laevis?

The growing global production of zinc oxide nanoparticles (ZnONPs) suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1-100 mg/L) specifically synthesized for industrial purposes with different sizes, shapes (round, rod) and surface coatings (PEG, PVP) was tested using the frog embryo teratogenesis assay-Xenopus (FETAX) to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.