In vitro methods to study the cytotoxicity, cell transformation capacity and genotoxicity of nanoparticles—application to cobalt ferrite and silver nanoparticles

Abstract

The objective of the work was to investigate in vitro cytotoxic, genotoxic and carcinogenic effects induced by nanoparticles (NPs) of industrial interest on a range of cell cultures. The cytotoxicity of two sizes of CoFe2O4 NPs, paramagnetic particles interesting for different biomedical applications, was investigated by Neutral Red uptake (NR) and Colony Forming Efficiency (CFE) assays, using six mammalian cell lines at concentrations between 10 and 120 μM for 72 hours of exposure. The cytotoxic effect exhibited a dose-effect relationship for Balb3T3 cells as assessed by the CFE assay. CoFe2O4 NPs were also studied for carcinogenicity and genotoxicity using the Balb3T3 model assessed by the Cell Transformation Assay (CTA, at concentrations between 1 and 60 μM for 72 hours of treatment) and Cytokinesis-Block Micronucleus test (CBMN, at concentrations between 1 and 60 μM for 24 hours of treatment). No effect was observed for both the particles at the doses and time points investigated. Four sizes of Ag NPs, chosen due to their antimicrobial properties, were investigated for cytotoxicity (by CFE, at concentrations between 0.01 and 10 μM for 24 and 72 hours of treatment), carcinogenicity (by CTA, at concentrations between 0.5 and 5 μM for 72 hours of treatment) and genotoxicity (by CBMN, at concentrations between 1 and 10 μM for 24 hours of treatment) to Balb3T3 mouse fibroblasts. Ag NPs resulted in a significant cytotoxic effect but no carcinogenic and genotoxic effect at the doses and time points investigated. Physicochemical characterization was carried out for the NPs tested, measuring the size distribution and surface charge by Dynamic Light Scattering (DLS), imaging by Scanning Electron Microscopy (SEM), the purity and ions’ leakage by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and the sedimentation by UV-Visible spectrometry.