A large number of reported studies give some insights regarding cytotoxicity induced by several nanomaterials [4–9]. However, because these data are, for the most part, not obtained in the context of the same experimental set-up, it is difficult to compare with other cytotoxicity results, thus presenting an issue in the interpretation of the results. Therefore, our study was designed to evaluate and compare the toxicity induced by 24 nanoparticles, in the same experimental set-up. As expected, our results demonstrate toxicity of some, but not all, of the nanoparticles tested. Moreover, our study clearly highlights the difference of sensitivity between cell types and cytotoxicity assays that has to be carefully taken into account when assessing nanoparticle toxicity.
We found that in most cases MTT was more sensitive than Neutral Red assay to assess nanoparticle toxicity, as shown by the higher number of calculable TC50 values with MTT assay than with the Neutral Red one. Moreover, TC50 values were almost every time lower for MTT assay as compared to Neutral Red (additional file 1, additional file 2, additional file 3, additional file 4, additional file 5, additional file 6 and additional file 7). Such results are in accordance with data from literature where many examples can be found of different degrees of toxicity that could be determined for the same particle, depending on the toxicity test used [9, 12–14]. This observation could be explained by the interference between the assay and the nanomaterial tested . However, as described in the method section, we performed both assays carefully, (trying to avoid) making sure that no nanomaterial was present in the supernatant when reading the optical density (Neutral Red assay) or that it didn't modify the measurement (MTT assay). Another explanation probably lies in the nature of each assay, one based on the uptake and subsequent lysosomal accumulation of a supravital dye (Neutral Red assay), and the other mainly based on the metabolic activity of the mitochondria (MTT assay). As the cellular targets are not the same, one can expect the cellular answer not to be identical, depending on the cell death mechanism . Such reasoning can also be used when comparing toxicity data obtained with A549 and THP-1 cells, where, in our experimental setting, A549 cells showed less sensitivity than THP-1 cells; TC50 values obtained with A549 cells were higher than those obtained with THP-1 cells. If such a difference in cell sensitivity is expected, those results appear in slight contradiction with those of Soto et al.  who analyzed the cytotoxic effects of several aggregated nanomaterials and, although finding a similar trend in both cell lines, A549 cells were shown to be more sensitive as compared to the THP-1 cells. However, they used naïve THP-1 cells (not PMA-activated) and evaluated cytotoxicity at only one time point (48 hours) after exposure to the different nanomaterials. Indeed, our results clearly showed that, whatever the cell type, there is an increase in the observed cytotoxicity, not only dose-dependently, but also time-dependently. Chang et al. , in a study comparing normal human fibroblasts to human epithelial tumour cells, proposed that the cytotoxicity induced by silica nanoparticles depends on the metabolic activity of the cell line. In that study, fibroblasts cells, with long doubling times, were more susceptible than epithelial tumor cells, which present shorter doubling times. In our study, we used two cell lines with similar doubling time (22.9 and 26 hours for A549 and THP-1 cells respectively, [ATCC product data sheet]). However, we used PMA-activated THP-1 cells, and it has been shown that PMA not only differentiates the monocytic THP-1 cells into macrophages, but also inhibits their proliferation . Therefore, the paradigm proposed by Chang et al.  could apply to our study and explain the better sensitivity of THP-1 as compared to that of A549 cells. Another possibility to explain the difference of sensitivity observed between the two cell types is the function of phagocytosis that characterizes macrophages (THP-1 cells), but not alveolar epithelial cells (A549 cells). As such, PMA-differentiated THP-1 macrophages have a greater ability to take in particle aggregates through phagocytic mechanisms that would likely increase macrophage response to nanomaterials. Such higher sensitivity for macrophages has been shown in response to metals from combustion-derived particulate matter, after the evaluation of both cell metabolism and cell death . The authors showed that rat alveolar macrophages (NR8383 cell line) were most sensitive to metals by nearly one order of magnitude in metal concentration, followed by the two alveolar epithelial cell lines studies (rat RLE-6TN and human A549). Further studies would be needed to clarify this point.
A secondary aim of our study was to generate a generic experimental set-up for a cytotoxicity screening of nanoparticle toxicity. In order to validate our findings, the experiments were performed, for each material, in two independent laboratories. Data reported in Table 2 and additional file 1, additional file 2, additional file 3, additional file 4, additional file 5, additional file 6 and additional file 7 clearly show that, for highly toxic nanomaterials (Copper- or Zinc-based), there is a good reproducibility between the independent labs; TC50 values are very similar. The same is true for not toxic nanomaterials (Tungsten Carbide and Cobalt). The reproducibility of the results between the two independent labs performing the experiments can however be questioned for nanomaterials with intermediate toxicity (Nickel oxide, Nickel, Stainless steel for example). This discrepancy appears although we designed a strict experimental set-up with as much defined and fixed parameters as possible. One can't however exclude individual variables (temperature of the culture room, batch of culture medium, spectrophotometer sensitivity, ...) that could explain the discrepancies that we observed at least for nanomaterials with intermediate toxicity. We are conscious that although care was given to be as superposable as possible, the 3 labs implied in this study couldn't be exactly the same. From Figure 2, it is clear that a rather slight shift of the cytotoxicity curve, although presenting a similar slope, makes a huge difference in the final outcome (calculated TC50 value). It can therefore be considered as quite logical that materials with intermediate toxicity differ the most when analyzed by 2 separate labs. Interestingly, we also observed that each lab presents an individual sensitivity, assessed by the values of TC50 that could be calculated; values for Lab. A are mostly higher than the 2 other labs, and Lab. B gave the lowest TC50 values. Such discrepancies, although not explained, could play a part in the differences observed for nanomaterials with intermediate toxicity.
It is difficult to compare our results with data from literature, as, as stated before, the experimental set-up is critical and therefore, relative toxicity indexes can't be defined with results obtained from different studies. Our results indicate that, out of all nanoparticles studied, Copper- and Zinc-based nanomaterials present the highest toxicity, whatever their oxidation status. The high toxicity observed for Zn-based nanomaterials is concordant with results obtained in a recent study by Park et al.  on A549 cells exposed to various inhalable metal nanoparticles. Indeed, they found that, out of 6 different nanoparticles, 100 nm Zn nanoparticles were the most cytotoxic to A549 cells, as assess by DNA fragmentation and apoptosis experiments. Interestingly, there was no uptake of Zn particles, and no change in cell morphology, the mechanism of toxicity remaining unknown . In the same study, toxicity induced by Ni nanoparticles was also evaluated, and the authors demonstrated a similar increase in DNA fragmentation for Ni nanoparticles as compared to Zn nanoparticles. This is different from our results, where Zn-based nanoparticles showed higher cytotoxicity for both cell types. However, in the study by Park, there is no chemical analysis of the nanomaterial tested, and the equivalent spherical diameter is about twice that of the particles used in our study. Finally, as mentioned earlier, this discrepancy could be explained by the evaluation of different parameters (DNA fragmentation versus mitochondrial metabolism).
Physico-chemical characteristics of nanoparticles (such as size, chemical composition, crystalline structure, surface properties, ...) are proposed to be critical determinants of their toxic potential [9, 18]. In the present study, we failed to show any correlation between the cytotoxicity induced by each nanoparticle, assessed by TC50 values, and its equivalent spherical diameter or specific surface area. Surface area is the physico-chemical parameter usually proposed to represent at best the specific toxicity of nanoparticles, with a good correlation between the particle surface area and the inflammatory response of animal exposed to the nanoparticles [19–22]. However, several studies also failed to demonstrate such a relationship [4, 23], and care must be taken when trying to associate toxic potential of nanoparticles to only one single physico-chemical parameter, as it is probably the matter of the association of several parameters. Moreover, few of the particles we used were of similar chemical composition, therefore probably weakening a potential association between their induced cytotoxicity and their equivalent spherical diameter or specific surface area. Finally, primary particle size considerations may sometime be misleading, particularly when considering the aggregation propensity of nanomaterials, particularly in a biological medium containing salts and proteins [24–26]. The discrepancies we observed in nanoparticle-induced toxicity could be the result of differential penetration , generation of oxidative stress , inflammation , or a combination of several events that result in a particular toxicity mechanism. More studies are clearly needed to have a comprehensive understanding of nanoparticle-induced toxicity.