**6. Identification of intra-vacuolar TiNSs in monocytes**

**5. TiNSs-caused cell death and vacuole formation**

**Figure 1.** TEM images of TiNSs used in the present study and illustrations of a single TiNS and TiO6

octahedron [34].

the culture. The effects of exposure to bulk TiO<sup>2</sup>

TiNSs or asbestos, but not to TiO2

images. (C) Illustration of a TiO6

168 Cytotoxicity

depth of one and a half of a sideways TiO6

Before the culture experiments, the diameters of TiNSs in water and in culture media supplemented with 10% of FBS were measured using a Zetasizer. The results showed that the diameter of about 25 nm in water is consistent with the size estimated by TEM images, while that in the media was about 422 nm, indicating induced agglomeration of TiNSs in the culture media. In addition, TiNSs in water showed a zeta potential of −22.1 mV. To examine the effects of exposure to TiNSs on human immune cells, peripheral blood mononuclear cells (PBMCs) were prepared and cultured with various doses of TiNSs, or with asbestos as a control cytotoxic material, for 7 days. Exposure to asbestos caused an increase in annexin V (Anx)-positive apoptosis of cells when exposed to more than a dose of 1 μg/ml at day 2 after culture, whereas apoptosis was not observed in the culture exposed to TiNSs even at the maximum dose of 10 μg/ml. However, exposure to TiNSs caused dose-dependent apoptosis to the same degree as asbestos at day 7 of

were synthesized by Dr. Yoshioka and observed by TEM. These images show the uniform diamond shapes of TiNSs in our present study. Some TiNSs appear to be piled up showing darker diamonds. Scale bars of 100 (left) and 50 nm (right) are shown. (B) Illustration of single TiNSs having a diamond shape with almost 20- and 30-nm diagonals, based on TEM

octahedron in the orthostatic position, of which TiNSs are composed. TiNSs have the

with TiNSs, but they did not cause apoptosis of PBMCs. The cell death induced by exposure to

lation, apoptotic cells with a low DNA content, using flow cytometry. Interestingly, marked formation of vacuoles was observed in the culture of PBMCs exposed to TiNSs but not the other materials. It was confirmed by staining with fluorescence-labeled antibodies to CD14 that

particles and crystalline silica were compared

octahedron. (A) TiNSs

or silica, was also confirmed by measuring the sub-G1 popu-

The results obtained from the cell cultures demonstrated the characteristic toxicity of TiNSs for monocytes, comprising apoptosis associated with the striking formation of vacuoles. Therefore, we investigated the presence of intracellular microstructures in monocytes exposed to TiNSs. Monocytes were isolated from human PBMCs, cultured with TiNSs at 10 μg/ml and then harvested at day 1 or 2 after the culture for subsequent TEM observations. The TEM images showed rapid formation of vacuoles in monocytes even at day 1, and the number and size of vacuoles increased at day 2. It is noteworthy that nano-scaled materials with TiNS-like shapes were found within the vacuoles of the monocytes and that most of the material was located near the inner surface of the vacuolar membrane (**Figure 2**). In order to confirm whether these intra-vacuolar nano-scaled materials were TiNSs, we observed the inner surface of the vacuolar membrane in monocytes using scanning electron microscopy (SEM), followed by energy dispersion X-ray (EDX) analysis for titanium. SEM observations showed that there was a rough area in the inner surface of the vacuolar membrane in monocytes harvested at day 1 after the culture with TiNSs. The rough area of the vacuolar membrane was also seen in other monocytes exposed to TiNSs. Analysis of the rough area by EDX confirmed the presence of titanium, in contrast to results for the cytosolic region in TiNS-exposed or control monocytes. These overall findings indicate that TiNSs were actually engulfed by monocytes and included in the vacuoles.

**Figure 2.** Observation of microstructures in TiNS-exposed monocytes by transmission electron microscope (TEM). The images are taken at day 1 after culture with TiNSs. It can be seen that monocytes have obvious vacuoles even at this early time. Additionally, nano-scaled materials with TiNS-like shapes can be seen inside the vacuoles, and most of the nano-scaled material is located near the vacuolar membrane. Finally, it was confirmed by scanning electron microscopy (SEM) with energy dispersion X-ray (EDX) analysis that these materials included titanium, indicating that the material contained TiNSs. Scale bars of 5 μm (left), 1 μm (upper right) and 100 nm (bottom right) are shown.
