Nabeshi et al. Nanoscale Research Letters 2011, 6:93 http://www.nanoscalereslett.com/content/6/1/93 C failed to display any detectable cytotoxicity up to con- centrations of 1000 μg/ml (Figure 1). These results demonstrate that the cytotoxic effect of nSP70 is reduced by surface modification. Some reports indicated that the exposure of cells to silica nanoparticles leads to membrane damage, caspase activation, and cell death via apoptosis. The precise trigger for these silica nano- particle-induced cellular effects is uncertain. One study concluded that lysosomal destabilization was the initia- tion factor [23], whereas other investigations suggest that mitochondrial membrane damage is the critical event [24,25]. However, it is possible that multiple fac- tors (i.e., membrane damage, caspase activation, lysoso- mal destabilization, and mitochondrial membrane damage) are involved in nSP70-mediated cytotoxicity. Unfortunately, it is difficult to establish a comprehensive mechanism of nSP70 cytotoxicity based on previous observations, which have been rather inconsistent. Some reports indicate that the cellular uptake and trafficking of nanoparticles is involved in cellular signal- ing, which then leads to cytotoxicity. However, the rela- tionship between the surface properties of nanoparticles and cellular uptake/trafficking is poorly understood. Page 3 of 6 To establish why a different biological effect was induced by surface modification, the intracellular locali- zation of fluorescent-labeled nanosilicas was investi- gated. RAW 264.7 cells treated with 100 μg/ml of nSP70, nSP70-N, and nSP70-C were observed by confo- cal laser scanning microscopy. After 3-h incubation, all nanosilicas were found to be localized in the cytoplasm as punctate fluorescent dots regardless of surface modi- fication (Figure 2). Interestingly, distinctive distributions were observed in individual cases. For the nSP70-treated cells, punctate fluorescence was observed in both the cytoplasm and nucleus. However, nSP70-N appeared to adsorb to the plasma membrane because bright fluores- cence was observed along the outline of the cell. In the nSP70-C-treated group, only intracellular punctate fluorescence was observed, suggesting that these parti- cles were efficiently incorporated into the cells. Unfortu- nately, detailed intracellular localization of the silica nanoparticles was not apparent from these images. Nonetheless, differences of intracellular localization of nanosilica might have an effect on cytotoxicity. It is reported that the entry of nanosilica into the nucleus induces dysfunction of the nucleus and genotoxicity via Figure 1 Effect of unmodified and modified nanosilica on cell proliferation. The proliferation of RAW 264.7 cells after incubation with nSP70 (circle), nSP70-N (square), or nSP70-C (triangle) for 24 h was evaluated using the 3H-thymidine incorporation assay. The percentage increase in cell proliferation was calculated relative to the negative control. Data are presented as means ± SD.