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Development of an integrated platform for nanoparticle analysis to verify their possible toxicity and the eco-toxicity

Final Report Summary - DIPNA (Development of an integrated platform for nanoparticle analysis to verify their possible toxicity and the eco-toxicity)

The aim of the DIPNA project was to provide knowledge about the impact of four different NPs (cobalt, gold, cerium and iron oxide) applied in wet suspension and in dry state on different types of cells (human immortalised THP-1, HepG2, CaCo-2 and A549 cell lines, murine 3T3, human CD34-derived Dendritic cells (DC), and human monocytes) to design assays and identify biomarkers of nanotoxicity. The project developed also technological solutions concerning the set-up of singlet NP / cell interaction with sensors to evaluate the effects, a device for an automatic evaluation of the toxicological impact of nanoparticles, and instruments for field analysis of nanopollution in occupational sites.

The results of the biological tests, carried out following the conditions of toxicological tests to assess the risks of molecules and ions, indicate that none of the NPs tested, either in dry or in wet conditions, affected the viability and capacity of proliferation of various cell types in culture. No signs of apoptosis were detected. The immunological biomarkers selected, such as cytokines, chemokines and TLR expression, were not affected in a dose-dependent fashion in the time frame of the in vitro tests.

A dose-dependent increase in the production of reactive oxygen species could be observed only for cobalt NPs. Also the expression of inflammation-related genes, such as IL-18Ra, TIR8 and caspase-1, was not modulated by NPs, either upon acute administration or when given in chronic or cumulative fashion over 15 days of cell culture. In the same conditions, silver NPs (used as control) likewise did not induce any relevant toxic effect nor could affect innate / inflammatory parameters.

The finding that only cobalt NPs seem to have toxic effects is explained as follows. Cobalt NPs can corrode in the culture medium and release cobalt ions. The toxicity of cobalt ions is well known. They are not in an equilibrium state and can readily combine with other ionic species present in the medium or in cells, and affect cell functions. Thus, toxicity of cobalt NPs can be indirectly due to the release of cobalt ions, rather than the consequence of NP-cell interaction. The identification of cobalt phosphate particles after chronic exposure of cells to cobalt NPs suggests that NP corrosion and ion release is indeed occurring.

Different systems were constructed to simulate singlet NP / cell interaction. Chips were built to monitor membrane changes by single cell impedance spectroscopy. A nanodispenser was devised for depositing NPs deposition in array format on cell culture supports. Microinjection techniques and Raman spectroscopy have been successfully combined with Principal component analysis (PCA) to detect the single cell / (few) NPs interaction.

The singlet NP / cell interaction did not cause any measurable effect except for the appearance of signals in Raman spectroscopy. Such signals are due to phosphate groups present in NP-microinjected cells (but not in solvent-injected control cells), suggesting that microinjection of few NPs in a single cell affected the cellular phosphate metabolism.

An interesting system was developed for the repeated spraying of dry NPs in air and in a liquid medium containing cells. The system works very well in air but not in the medium.

An automated system was designed and constructed to measure the possible toxicity of NPs. The breadboard system consists of a small incubator with in-built controllers for CO2, temperature and humidity, a fluidic system with pump and valves to transport reagents and samples, and two optical detection units, for optical absorption measurement and for refractive index measurement respectively. The system detects the proliferation of A549 cells (untreated versus exposed to NPs) based on Alamar blue staining.

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