"In vitro methods to study the cytotoxicity, cell transformation capacity and genotoxicity of nanoparticles - application to cobalt ferrite and silver nanoparticles"
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The objective of this thesis was to investigate the in vitro cytotoxic, genotoxic and transforming effects induced by nanoparticles (NPs) of industrial interest on a range of cell cultures. The cytotoxicity of two sizes of CoFe2O4 NPs, paramagnetic particles of interest in different biomedical applications, was investigated using the Neutral Red uptake (NR) and Colony Forming Efficiency (CFE) assays using six mammalian cell lines at concentrations between 10 and 120 μM. More specifically, cytotoxicity was evaluated after 72-hour treatment of five cell lines (A549, CaCo2, HaCaT, HepG2, MDCK) with 10, 20, 40, 60, 80, 100 and 120 μM concentrations of NPs using NR, and 10, 20, 30, 40, 50, 60, 80, 100 and 120 μM concentrations of NPs using CFE. In parallel with these tests, cytotoxicity was also evaluated in mouse Balb3T3 fibroblasts using (i) NR after 72-hour treatment with 10, 20, 40, 60, 80, 100 and 120 μM concentrations of NPs, and (ii) CFE after both 72-hour treatment with 1, 5, 10, 20, 30, 40, 50, 60, 80, 100 and 120 μM concentrations of NPs, and 24-hour treatment with 1, 10, 60 and 120 μM concentrations of NPs. The cytotoxic effect exhibited a dose-effect relationship for Balb3T3 cells as assessed using the CFE assay. The testing of a more extensive concentration range of NPs in Balb3T3 cells (i.e., 1 and 5 μM in addition to the concentrations tested in the other five cell lines) over a 72-hour exposure time using CFE, together with the additional test using a 24-hour exposure time allowed appropriate concentration ranges to be determined for use in subsequent experiments using the Cell Transformation (CTA) and Cytokinesis-Block Micronucleus (CBMN) assays. The cell transformation capacity and genotoxicity of CoFe2O4 NPs were investigated using the Balb3T3 model, and assessed using the CTA (specifically at concentrations of 1, 5, 20 and 60 μM for 72 hours of treatment) and CBMN (specifically at concentrations of 1, 10 and 60 μM for 24 hours of treatment). The CoFe2O4 NPs induced neither effect at the doses and time points investigated. Four sizes of Ag NPs, chosen for their antimicrobial properties, were assessed for cytotoxicity (using CFE at concentrations of 0.1, 0.5, 1, 5 and 10 μM for 24 hours of treatment and of 0.01, 0.1, 0.5, 1, 2.5, 5 and 10 μM for 72 hours of treatment), cell transformation capacity (using CTA at concentrations of 0.5, 2.5 and 5 μM for 72 hours of treatment) and genotoxicity to Balb3T3 mouse fibroblasts (using CBMN at concentrations of 1, 5 and 10 μM for 24 hours of treatment). The Ag NPs had a significant cytotoxic effect, but no cell transformation or genotoxic effects at the doses and time points investigated. Physicochemical characterization of the chosen NPs was performed; size distribution and surface charge were measured by Dynamic Light Scattering (DLS), imaging by Scanning Electron Microscopy (SEM), the purity and ion leakage by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and the sedimentation by UV-Visible spectrometry.