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Since their antimicrobial properties, Silver nanoparticles (AgNPs) are among the most exploited nanomaterials in consumer products. Size and surface reactivity and coating are critical physicochemical properties responsible for NP toxicity. In this study the reproductive and developmental toxicity of citrate- (Cit-) and branched polyethyleneimine- (BPEI-) coated 10 nm AgNPs was investigated on Xenopus laevis fertilization and embryos by the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The effects were compared also with the exposure to Ag ions. Light, confocal and electron microscopy analyses have been performed on sperms and embryos to characterize the NP-cell interactions and uptake, as well as the histological and ultrastructural lesions. The results suggest that the positively-charged BPEI-AgNPs are more effective in inducing adverse reproductive and developmental outcomes, likely as a consequence of the enhanced interactions with sperm membrane and embryonic barriers (e.g. fertilization envelope and intestine) in respect of the negatively-charged Cit-AgNPs. Although Ag + resulted strongly embryolethal, the BPEI-AgNPs showed the highest teratogenic index, pointing out the role of NP size and functional coating in determining the teratogenicity. No lethal and slight malformation effects were induced by Cit-AgNPs. We conclude that possible reduced fertility and teratogenic risks may be associated to BPEI-AgNPs exposure, but the modality of NP-tissue interactions and the teratogenic mechanism need further scrutiny to be defined. This work was supported by Fondazione Cariplo (OverNanotox 2013-0987).

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.

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.

International audience; 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.

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

The antibacterial activity of metal oxide (MeO) nanoparticles (NPs) has been extensively reported. Nevertheless, there is a general concern about MeO NPs toxicity, and much efforts are being devoted to improve a safe employment of these nanomaterials by surface functionalization. In this work, we propose to investigate if the coating of copper and zinc oxides (CuO and ZnO) NPs with the polymers poly(ethylene imine) (PEI) or poly(ethylene glycol) (PEG) is able protect non-target cells and organisms from the toxicity of antibacterial MeOs NPs. The overall results obtained exposing lung cells and Xenopus laevis embryos to CuO-PEG, CuO-PEI, ZnO-PEG and ZnO-PEI, indicate that PEG, but not PEI coating, is able to exert a protective function against MeOs toxicity. The ability of PEG shell to reduce the adverse impact of MeOs NPs is in part associated to a protection from the oxidative stress, even if in vitro results indicate that peghylation is not able to prevent pro-inflammatory responses. We suggest that the different intracellular availability of the metal oxides upon capping with PEG or PEI, together with the involvement of multiple mechanisms of action, may be responsible for the different toxicological outcome on lung cells viability and Xenopus mortality and malformations.

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.

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.

Negli ultimi decenni abbiamo assistito all’esplosione delle nanotecnologie, sviluppatesi con lo scopo di manipolare la materia a livello nanometrico. Secondo l’International Standard Organization (ISO) per Nanoparticelle si intendono nano-oggetti con dimensioni comprese tra 1 e 100 nm (ISO TS27687/2008). Questi innovative e sconosciuti nanomateriali (NMs) sono stati portati continuamente sul mercato, causando un’esposizione massiva per l’Uomo e l’Ambiente. La principale via di esposizione per l’uomo è l’inalazione. Le NPs, caratterizzate dale loro piccolo dimensioni (meno di 100 nm) sono in grado di raggiungere gli alveoli, ultima parte del nostro tratto respiratorio. La capacità dei NMs di depositarsi a livello alveolare è la ragione per la quale è d’obbligo considerare prioritari gli studi tossicologici a livello polmonare. Per quanto riguarda l’esposizione ambientale, le NPs vengono considerate come una classe di nuovi contaminanti soprattutto per gli habitat acquatici. Alla luce di ciò, studi nanotossicologici diventano fondamentale per comprendere gli effetti avversi delle NPs sugli organismi acquatici. L’utilizzo di vari modelli con diversi livelli di complessità permette di ottenere una caratterizzazione del comportamento delle NPs sempre più complete e dettagliato. Tre diversi moelli in vitro con un crescente livello di complessità sono proposti in questa tesi: da una semplice monocoltura di cellule alveolari, a un innovative modello 3D alveolare di tetracoltura e un organismo completo in via di sviluppo (Xenopus laevis). Le monocolture sono I sistemi più comunemente usati per uno screening ppeliminare degli effetti di composti chimici e particelle. In particolare, la linea cellular A549 (Human Alveolar Adenocarcinoma Cell Line) è un modello largamente utilizzato in esperimenti tossicologici. Negli ultimi anni, numeri modelli 3D in vitro sono stati proposti per studi predittivi di tossicologia. Questi modelli, più o meno complessi, mimano l’organozzazione tissutale cercando in questo modo di modulare la risposta del tessuto polmonare umano in modo più preciso rispetto a una monocoltura. Xenopus laevis è un classic modello di studi di embriologia, valido anche per studi di tossicologia acquatica. Nella procedura standardizzata, chiamata Frog Embryo Teratogenenis Assay – FETAX (ASTM 1998) gli embrioni vengono esposti durante il periodo di organogenesis. La tesi si struttura in una breve introduzione riguardante le nanotecnologia, le proprietà fisico-chimiche delle NPs e I modelli in vitro utilizzati nei diversi studi. Dopodiché, il manoscritto si compone di quatttro diversi capitol riguardanti altrettanti casi di studio. Inizialmente vengono affrontati gli effetti tossici di tre diverse forme di nZnO su una semplice monocoltura di cellule A549 (Capitolo 1); poi verranno proposti gli effetti di AuNPs su un complesso sistema 3D alveolare (Capitolo 2). La complessità dei modelli cresce passsando poi a un intero organism quali sono gli embrioni di Xenopus laevis; su questo modello sono state testate diversi nZnO (Capitolo 3) e due diverse AgNPs (Capitolo 4). Infine tutti i risultati ottenuti durante i tre anni di dottorato saranno ripresi e discussi. During the last decades, we are witnessing the explosion of the nanotechnologies, which have developed with the aim of manipulating matter in the nanoscale dimension. According to The International Standard Organization (ISO) “Nanoparticles indicate nano-objects with the three external dimensions ranging from 1 to 100 nm” (ISO TS27687/2008). The main route of human exposure is represented by inhalation. NPs characterized by their small size (less than 100 nm) are able to reach the alveoli site, the latest part of our air way tract. The capability of NMs to deposit into alveoli, is the reason why considerer human lung toxicological studies mandatory. About the environmental exposure, NPs are considered as a new class of pollutants especially for the aquatic habitats always considered as a sink for wastes. In the light of this, nanotoxicology studies have become fundamental for assessing the adverse effects of NPs on the aquatic organisms. The main physico-chemical properties that characterize NMs are ascribable to their size, shape, surface charge and coating agents. NM size and shape contribute significantly to their interaction with cells, however, functional groups on the NP surface are the primary dictators of many important nanomaterial properties, such as solubility and macromolecule and cell surface interactions. The knowledge of how cells interact with NPs and how their physico-chemical properties are relevant to toxic effects is of great importance in the perspective of proposing and supporting the development a Safe-by-Design (SbD) approach for the production of safer NPs. The use of several models with different level of complexity allows to obtain a more complete and well-described characterization of NP behavior. Different in vitro models with increasing complexity have been proposed in this thesis for a complete assessment: a simple alveolar monoculture, a novel 3D tetraculture alveolar model and a whole developing organism, the Xenopus laevis early embryos. Monoculture systems are the most commonly models used for the first screening of chemical and particle effects. In particular, the A549 Human Alveolar Adenocarcinoma Cell Line is an established model of Type II alveolar epithelium widely used to performe lung toxicity experiments. In the last years, numerous 3D in vitro lung models have been proposed to perform predictive toxicological inhalation studies. These models, more or less complex, mimic the tissue organization trying to modulate the response of the human lung tissue better than a monoculture. Xenopus laevis is a classical model for embryological studies, also suitable for aquatic toxicology assessment in the standardized procedure called Frog Embryo Teratogenenis Assay – FETAX (ASTM 1998). FETAX is conducted on fertilized Xenopus mid-blastula stage over the organogenesis period, representing an excellent development toxicity alert test, alternative to Mammals to predict the teratogenic potential of a compound with an accuracy of about 80%. The thesis starts with a brief introduction regarding the Nanotechnologies, the Nanoparticle physico-chemical properties and the in vitro models used in the different studies. After that, the manuscript proposes four different chapters regarding different cases of study. First, it will address the toxicological effects of three different shaped nZnO on a simple monoculture of A549 cells (Chapter 1 – paper in preparation); then the focus will place on the effects of AuNPs on a complex 3D in vitro alveolar model (Chapter 2 – paper in preparation). The complexity of the models will increase passing on a whole developing organism, Xenopus laevis; on this model the effects on embryos of several nZnOs (Chapter 3 – Bonfanti et al., 2015) and differently coated AgNPs (Chapter 4 – Colombo et al., 2017) will be discuss. Finally, all the results obtain during the three years of PhD will be gather together and discussed.

Glyphosate is the active ingredient in broad-spectrum herbicide formulations used in agriculture, domestic area and aquatic weed control worldwide. Its market is growing steadily concurrently with the cultivation of glyphosate-tolerant transgenic crops and emergence of weeds less sensitive to glyphosate. Ephemeral and lentic waters near to agricultural lands, representing favorite habitats for amphibian reproduction and early life-stage development, may thus be contaminated by glyphosate based herbicides (GBHs) residues. Previous studies on larval anuran species highlighted increased mortality and growth effects after exposure to different GBHs in comparison to glyphosate itself, mainly because of the surfactants such as polyethoxylated tallow amine present in the formulations. Nevertheless, these conclusions are not completely fulfilled when the early development, characterized by primary organogenesis events, is considered. In this study, we compare the embryotoxicity of Roundup® Power 2.0, a new GBH formulation currently authorized in Italy, with that of technical grade glyphosate using the Frog Embryo Teratogenesis Assay–Xenopus (FETAX). Our results evidenced that glyphosate was not embryolethal and only at the highest concentration (50 mg a.e./L) caused edemas. Conversely, Roundup® Power 2.0 exhibited a 96 h LC50 of 24.78 mg a.e./L and a 96 h EC50 of 7.8 mg a.e./L. A Teratogenic Index of 3.4 was derived, pointing out the high teratogenic potential of the Roundup® Power 2.0. Specific concentration-dependent abnormal phenotypes, such as craniofacial alterations, microphthalmia, narrow eyes and forebrain regionalization defects were evidenced by gross malformation screening and histopathological analysis. These phenotypes are coherent with those evidenced in Xenopus laevis embryos injected with glyphosate, allowing us to hypothesize that the teratogenicity observed for Roundup® Power 2.0 may be related to the improved efficacy in delivering glyphosate to cells, guaranteed by the specific surfactant formulation. In conclusion, the differences in GBH formulations should be carefully considered by the authorities, since sub-lethal and/or long-term effects (e.g. teratogenicity) can be significantly modulated by the active ingredient salt type and concentration of the adjuvants. Finally, the mechanistic toxicity of glyphosate and GBHs are worthy of further research.

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.

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.