Development of a multilevel approach for the evaluation of nanomaterials toxicity

Aim: To develop a multilevel approach that includes different toxicity tests and gene-expression studies for toxicity evaluation of engineered nanomaterials developed for biomedical applications. Materials & methods: K-562, MCF-7 and U-937 human-derived cell lines were used as models for in vitro toxicity tests. These tests included viability assays (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium [MTS] assay); evaluation of apoptosis/necrosis by propidium iodide staining and DNA laddering assay; evaluation of mitochondrial toxicity (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethyl-benzimidazolcarbocyanine iodide [JC-1] assay); transmission electron microscopy analysis and gene expression analysis by DNA microarray. For in vivo toxicity evaluation, Swiss mice were used for monitoring acute or chronic effects. Two superparamagnetic contrast agents approved for human use (Resovist® and Primovist®) and two new lanthanide-based luminescent nanoparticles were tested. Results & discussion: The nanomaterials approved for human use did not show significant toxicities in our assays. Toxicity studies performed on lanthanide-based nanoparticles (EDTA120 and EDTA120D) complexed with the chelating agent EDTA revealed that these nanomaterials induced necrosis in U-937 and K-562 cells while no toxicity was observed in MCF-7 cells. Moreover, no in vivo effects have been observed. The comparative analysis of the nanomaterials and their separated components showed that the toxicity in U-937 and K-562 cells was mainly due to the presence of EDTA. Conclusion: The multilevel approach proved to be useful for nanomaterial toxicity characterization. In particular, for the lanthanide-based nanoparticles tested in this work, the EDTA was identified as the main cause of the toxicity in vitro, suggesting a possible applicability of these nanoparticle suspensions for in vivo optical imaging