In vitro multi-assay cytotoxicity assessment of iron oxide nanoparticles on neural cells and addressing spectrophotometric interference

Iron-rich nanoparticulate matter air pollution exposure is implicated in the pathophysiology of Alzheimers disease. The use of colorimetric cell viability assays to ascertain the potential cytotoxicity of iron oxide nanoparticles have highlighted conflicting results in various in vitro models, because of spectrophotometric interference. Attempts to resolve this confounding factor to reveal accurate measurements have been insufficient. To address this interference issue, we tested three control methods (readout correction, plate transfer, and above-plate reading) on in vitro cytotoxicity measurements via Neutral Red Uptake (NRU), Resazurin, and MTT assays, of iron oxide and magnetite nanoparticulate exposure on SK-N-SH and IMR-32 human neural cell lines. We observed varying degrees of interference based on cell line, nanoparticle type, exposure time, assay type, and the readout method (colorimetric or fluorometric). The readout correction method did not remove interference completely, the plate transfer method tended to cause overestimation of cell viability beyond the original interference, and fluorescence readings from above the plate were less sensitive than the conventional bottom reading. The Resazurin assay with fluorometric readout presented no interference in most conditions investigated in this work, therefore presents advantages over both colorimetric assays and interference control methods. It is recommended to forego colorimetric assays in future studies of nanoparticle cytotoxicity as they are more susceptible to interference.