Fabio Franchini

Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts

Gold nanoparticles (AuNPs) are currently used in several fields including biomedical applications, although no conclusive information on their cytotoxicity is available. For this reason this work has investigated the effects of AuNPs in vitro on Balb/3T3 mouse fibroblasts. Results obtained exposing cells for 72 h to AuNPs 5 and 15 nm citrate stabilized, revealed cytotoxic effects only for AuNPs 5 nm at concentration ≥ 50 μM if measured by colony forming efficiency (CFE). To understand the differences in cytotoxicity observed for the two AuNPs sizes, we investigated the uptake and the intracellular distribution of the nanoparticles. By TEM it was observed that 5 and 15 nm AuNPs are internalized by Balb/3T3 cells and located within intracellular endosomal compartments. Quantification of the uptake by ICP-MS showed that AuNPs internalization enhanced even up to 72 h. Disruption of the actin cytoskeleton was evident, with cell footprints narrow and contracted; effects more remarkable in cells exposed to 5 nm AuNP. The mechanism of NPs cell internalization was investigated using immunocytochemistry and western blot. No significant effect was observed in the expression level of caveolin, while reduction of the expression and degradation of the clathrin heavy chain was observed in cells exposed for 72 h to AuNPs.

Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse fibroblasts

Nanotechnology is an emerging field that involves the development, manufacture and measurement of materials and systems in the submicron to nanometer range. Its development is expected to have a large socio-economical impact in practically all fields of industrial activity. However, there is still a lack of information about the potential risks of manufactured nanoparticles for the environment and for human health. In this work, we studied the cytotoxicity, genotoxicity and morphological transforming activity of cobalt nanoparticles (Co-nano) and cobalt ions (Co(2+)) in Balb/3T3 cells. We also evaluated Co-nano dissolution in culture medium and cellular uptake of both Co-nano and Co(2+). Our results indicated dose-dependent cytotoxicity, assessed by colony-forming efficiency test, for both compounds. The toxicity was higher for Co-nano than for Co(2) after 2 and 24 h of exposure, while dose-effect relationships were overlapping after 72 h. Statistically significant results were observed for Co-nano with the micronucleus test and the comet assay, while for Co(2+) positive results were observed only with the latter. In addition, even when Co-nano was genotoxic (at >1 microM), no evident dose-dependent effect was observed. Concerning morphological transformation, we found a statistically significant increase in the formation of type III foci (morphologically transformed colonies) only for Co-nano. Furthermore, we observed a higher cellular uptake of Co-nano compared with Co(2+).

Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells

Abstract Despite human gastrointestinal exposure to nanoparticles (NPs), data on NPs toxicity in intestinal cells are quite scanty. In this study we evaluated the toxicity induced by zinc oxide (ZnO) and titanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPs produced significant cytotoxicity, evaluated by two different assays. The presence of foetal calf serum in culture medium significantly reduced ZnO NPs toxicity as well as ion leakage and NP-cell interaction. The two NPs increased the intracellular amount of reactive oxygen species (ROS) after 6 h treatment. However, only ZnO NPs increased ROS and induced IL-8 release both after 6 and 24 h. Experimental data indicate a main role of chemical composition and solubility in ZnO NPs toxicity. Moreover our results suggest a key role of oxidative stress in ZnO NPs cytotoxicity induction related both to ion leakage and to cell interaction with NPs in serum-free medium.

Size and mass determination of silver nanoparticles in an aqueous matrix using asymmetric flow field flow fractionation coupled to inductively coupled plasma mass spectrometer and ultraviolet–visible detectors

The powerful antibacterial properties of engineered silver nanoparticles (AgNPs) have, in recent years, led to a great increase in their use in consumer products such as textiles and personal care products offers. This widespread and often indiscriminate use of nano-silver is inevitably increasing the probability that such materials be accidentally or deliberately lost into the environment. Once present in the environment the normally useful antibacterial properties of the silver may instead become a potential hazard to both man and the environment. In the face of such concerns it therefore desirable to develop easy, reliable and sensitive analytical methods for the determination of nano-sized silver in various matrices. This paper describes a method for the simultaneous determination of particles-size and mass-concentration of citrate-stabilized silver nano-particles in aqueous matrices by asymmetric flow field flow fractionation coupled to an ICP-mass spectrometer and UV/vis detector. In particular, this work has evaluated the use of pre-channel injections of mono-dispersed silver nano-particles as a means of accurate size and mass-calibration. The suitability of the method as a means to generate accurate and reliable results was verified by determination of parameters such as precision under repeatability conditions, linearity, accuracy, recovery and analytical sensitivity.

A quantitative in vitro approach to study the intracellular fate of gold nanoparticles: from synthesis to cytotoxicity

Abstract Due to their physico-chemical characteristics, gold nanoparticles (AuNPs) seem to be suitable for biomedical and therapeutic applications even if conflicting data on their toxicological profiles are present in literature. In order to better understand if AuNPs could be safe we must consider different biological endpoints such as cytotoxicity, genotoxicity, inflammation and biopersistence. Starting from these considerations, one of the first issues to be assessed is to better understand if AuNPs can be internalized by cells. In this work, we propose a methodological approach to radioactivate AuNPs by neutron activation and the quantification of their internalization by two in vitro cell systems such as MDCK and HepG2 after 24 h of exposure. Despite a dose-dependent internalization, no evidence of cytotoxicity, determined by two different standard in vitro methods such as Neutral Red Uptake and Colony Forming Efficiency, was observed.

Detection, quantification and derivation of number size distribution of silver nanoparticles in antimicrobial consumer products

In 2011 the European Commission published its recommendation for a definition for the term nanomaterial which requires the materials to be characterized in terms of the number size distribution of their constituent particles. More recently, the definition has begun to be applied to the labelling of food and cosmetic products where any components present in the form of engineered nanomaterials must now be clearly indicated in the list of ingredients. The implementation of this definition requires that methods be developed and validated to accurately size particles with at least one external dimension in the range of 1–100 nm, and to quantify them on a ‘number-based’ particle size distribution. An in-house developed method based on Asymmetric Flow Field Flow Fractionation-Inductively Coupled Plasma Mass Spectrometry (AF4-ICP-MS) for the simultaneous detection and quantification of citrate-stabilised silver nanoparticles (AgNPs) in water, has been applied to real-world liquid antimicrobial consumer products based on colloidal silver. This transfer of the method from ideal model systems to real products was assessed in light of other techniques including Centrifugal Liquid Sedimentation (CLS), Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). Five out of six analysed products were found to contain AgNPs in the nano-range by means of a number of techniques including AF4-ICP-MS. Comparative analysis shows that CLS has sufficient size resolution to size AgNPs in the consumer products while DLS was unsuccessful probably due to sample polydispersivity. Despite the silver nanoparticles having unknown surface properties and stabilisation agents which could have influenced the sizing with AF4, a relatively good agreement between TEM and AF4-ICP-MS was observed. The AF4-ICP-MS data could be converted from mass-based to number-based distributions; this transformation, despite the possibility of experimental artefacts being mathematically amplified, has shown promising results.

Cyclotron Production of Radioactive

Nowadays, a wide variety of nanoparticles (NPs) are applied in different fields such as medical science and industry. Due to their large commercial volume, the OECD Working Party on Manufactured Nanomaterials (NMs) has proposed to study a set of 14 nanomaterials, one of which being cerium oxide (CeO2). In particular, CeO2 based NPs are widely used in automotive industry, healthcare, and cosmetics. In this paper, we propose a method for the production of radioactive CeO2 NPs. We demonstrate that they maintain the same physicochemical characteristics as the “cold” ones in terms of size distribution and Zeta potential; we develop a new protocol to assess their cellular interaction in immortalized mouse fibroblast cell line Balb/3T3, a model for the study of basal cytotoxicity and carcinogenic potential induced by chemicals and in the present case by NPs. Experimental result of this work, which shows a quasi-linear concentration-uptake response of cells, can be useful as a reference dose-uptake curve for explaining effects following biological uptake after exposure to CeO2 NPs.

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In this work, we present a complete physicochemical characterization of multi-wall carbon nanotubes (mwCNTs) in order to assess their potential toxicological effects in in vitro cell models using Colony Forming Efficiency (CFE) assay. We verified that Dimethyl Sulfoxide (DMSO) was a more suitable solvent to disperse mwCNTs compared to culture medium guaranteeing reproducibility in the preparation of testing dilutions. The CFE assay was carried out on five mammalian cell lines representing the potentially exposed and/or target organs for nanomaterials (lung, liver, kidney, intestine, skin), as well as on mouse fibroblasts cell line, which usually is considered a sensitive model to verify in vitro cytotoxicity of test compounds. A statistically significant toxic effect was found only in human alveolar basal epithelial cells and immortalized mouse fibroblasts, for which the interaction between mwCNTs and cells was additionally studied by Atomic Force and Scanning Electron Microscopy. In this study, we considered and suggested the CFE assay as a promising test for screening studies of cytotoxicity. In addition, combining in vitro tests with physicochemical analysis, this work underlines basic points to be considered when research on nanomaterials has to be carried out, to set up, in our opinion, well-defined and suitable experimental planning and procedures.

Nanoparticles and Their Application for In Vitro Uptake Studies

A simple method for the synthesis of lipophilic Ag NPs have been developed. The coated Ag NPs have been entrapped into a FDA-approved and targetable PEG-based polymeric nanoparticles, and this nanocarrier has been conjugated with the peptide chlorotoxin. Uptake experiments have shown a cell-specific recognition of the Ag-1-PNPs-Cltx on U87MG cell lines in comparison to Balb/3T3. The uptake of Ag into the cells was quantified and an interesting cytotoxic effect (IC50 = 45 μM) has been found on glioblastoma cell lines.

Colony Forming Efficiency and Microscopy Analysis of Multi-Wall Carbon Nanotubes Cell Interaction

Abstract Different in vitro assays are successfully used to determine the relative cytotoxicity of a broad range of compounds. Nevertheless, different research groups have pointed out the difficulty in using the same tests to assess the toxicity of nanoparticles (NPs). In this study, we evaluated the possible use of a microphysiometer, Bionas 2500 analyzing system Bionas GmbH®, to detect in real time changes in cell metabolisms linked to NPs exposure. We focused our work on response changes of fibroblast cultures linked to exposure by cobalt ferrite NPs and compared the results to conventional in vitro assays. The measurements with the microphysiometer showed a cobalt ferrite cytotoxic effect, confirmed by the Colony Forming Efficiency assay. In conclusion, this work demonstrated that the measurement of metabolic parameters with a microphysiometer is a promising method to assess the toxicity of NPs and offers the advantage to follow on-line the cell metabolic changes.