**Acknowledgements**

**7. Relation between TiNSs-caused vacuoles and endosomal pathway**

of vacuoles is related with some part of the endosomal pathway.

**8. Discussion**

170 Cytotoxicity

area of 8400 nm2

20 nm have a volume of 4189 nm3

lent to the volume of one nanosphere (4189 nm3

Previous studies have reported formation of vacuoles originating from endocytic organelles, where the vacuolization of late endosomes was induced by the inhibition of kinases for regulation of vesicular transport and sorting [45–47], and several bacterial toxins induced vacuoles having an endosomal/lysosomal origin [48–50]. To examine the relation between TiNS-induced vacuoles and the endosomal pathway, monocyte-derived adherent cells were incubated with fluorescence-dye-labeled dextran to visualize endosome structures. The adherent cells were prepared using a pre-culture of PBMCs. TiNSs were then added to the culture of endosome-visualized adherent cells at 10 μg/ml, and observations were then made of vacuoles using the fluorescence derived from dextran. In the control culture without TiNSs, a diffuse fluorescence was observed. However, exposure to TiNSs induced vacuoles in the adherent cells, where most of the vacuoles showed co-localization with the fluorescence derived from endocytosed dextran. These observations indicate that TiNS-induced formation

Our study revealed the unique toxicity of TiNSs. This 2D nano-scaled material has the harmful potential to cause caspase-dependent apoptosis of immune cells to the same degree as asbestos. In particular, monocytes showed formation of marked vacuoles prior to cell death upon exposure to TiNSs, which were later found in the vacuoles and suggest the actual engulfment of TiNSs by monocytes. A consideration of these findings with the observation regarding co-localization of vacuoles with endosomal dextran indicates that engulfed TiNSs entered the endosomal pathway, leading to the formation of vacuoles in monocytes and subsequent cell death. As mentioned previously, TiNSs have a very large surface area per gram. **Figure 3** represents an illustration showing the large surface area of 2D nano-materials. Nano-scaled spheres with a diameter of

and surface area of 1257 nm2

the nanosphere. Thus, nanosheets have an extremely large surface that probably enhances the chemical activities of titanium oxide, which might contribute to the toxicity of TiNSs. In addition, TiNSs are negatively charged on the surface due to the presence of the oxygen atom, which might cause cationic interference in endo-lysosomes that leads to the formation of vacuoles. Various kinds of stimulation such as oxidative stress disrupt the integrity of the lysosomal membrane and cause lysosomal-membrane permeabilization (LMP), which triggers cell death including caspase-dependent apoptosis [51, 52]. Autophagy is the machinery of the intracellular degradation process, which is also the part of the intracellular membrane system and is linked to the endolysosomal pathway [51, 53]. Furthermore, recent studies have been accumulating concerning a new type of cell death associated with large vacuoles, named methuosis, although it is thought

as the sum of both sides, which is 6.68 times as large as the surface area of

with diagonals of 20 and 30 nm, a depth of 1 nm and resulting volume of 300 nm3

same density as a nanosphere. The total volume of 14 nanosheets (4200 nm3

. Diamond-shaped nanosheets

), but those nanosheets result in a total surface

have the

) is almost equiva-

The authors thank Ms. Tamayo Hatayama, Shoko Yamamoto and Miho Ikeda for their technical help. The authors declare that there is no conflict of interests regarding the publication of this chapter.
