Determination of the optical interference of iron oxide nanoparticles in fluorometric cytotoxicity assays

Nanomaterials are known to exhibit unique interactions with light. Iron oxide nanoparticles (IONPs), composed of magnetite (black iron oxide) specifically, are known to be highly absorptive throughout the visible portion of the spectrum. We sought to investigate and overcome optical interference of IONPs in colorimetric, fluorometric and luminescence assays by introducing additional controls and determining the concentration-dependent contribution to optical artifacts which could confound, skew, or invalidate results. We tested the in vitro cytotoxicity of ∼8 nm spherical magnetite nanoparticles capped with alginate on a human lung carcinoma (A549) cell line for different exposure periods and at various concentrations. We observed significant interference with both the MTT reagent and the absorption at 590 nm, a concentration-dependent reduction in the luminescence, fluorescence at ∼490 nm (viability marker), and fluorescence at 530 nm (cytotoxicity marker). After introducing an additional correction, we obtained more accurate results, including a clear decrease in viability at 12-h post-treatment, with apparent near complete recovery after 24-h in addition to a dose-independent, time-dependent alteration in the cell proliferation rate. A small increase in cytotoxicity was noted at the 24-h timepoint at the two highest concentrations. According to our results, the MTT reagents appear to interact substantially with IONPs at concentrations above 0.1 mg/mL, therefore, this assay is not recommended for IONP cytotoxicity assessment at higher concentrations.