Understanding and improving assays for cytotoxicity of nanoparticles: what really matters?

Despite years of excellent individual studies, the impact of nanoparticle (NP) cytotoxicity studies remains limited by inconsistent data collection and analysis. It is often unclear how exposure conditions can be used to determine cytotoxicity quantitatively. Discrepancies due to using different measurement conditions, readouts and controls to characterize NP interactions with cells lead to further challenges. To examine which parameters are critical in NP cytotoxicity studies, we have chosen to examine two NP types (liposomes and quantum dots) at different concentrations incubated with two primary vascular endothelial cells, HUVEC and HMVEC-C for a standard time of 24 h. We paid close attention to the effects of positive controls and cell association on interpretation of cytotoxicity data. Various cellular responses (ATP content, oxidative stress, mitochondrial toxicity, and phospholipidosis) were measured in parallel. Interestingly, cell association data varied significantly with the different image analyses. However, cytotoxicity responses could all be correlated with exposure concentration. Cell type did have an effect on cytotoxicity reports. Most significantly, NP cytotoxicity results varied with the inclusion or exclusion of positive controls. In the absence of positive controls, one tends to emphasize small changes in cell responses to NPs.