Effects of titanium dioxide nanoparticle aggregate size on gene expression

Junko Okuda-Shimazaki, Saiko Takaku, Koki Kanehira, Shunji Sonezaki, Akiyohshi Taniguchi, Junko Okuda-Shimazaki, Saiko Takaku, Koki Kanehira, Shunji Sonezaki, Akiyohshi Taniguchi

Abstract

Titanium dioxide (titania) nanoparticle aggregation is an important factor in understanding cytotoxicity. However, the effect of the aggregate size of nanoparticles on cells is unclear. We prepared two sizes of titania aggregate particles and investigated their biological activity by analyzing biomarker expression based on mRNA expression analysis. The aggregate particle sizes of small and large aggregated titania were 166 nm (PDI = 0.291) and 596 nm (PDI = 0.417), respectively. These two size groups were separated by centrifugation from the same initial nanoparticle sample. We analyzed the gene expression of biomarkers focused on stress, inflammation, and cytotoxicity. Large titania aggregates show a larger effect on cell viability and gene expression when compared with the small aggregates. This suggests that particle aggregate size is related to cellular effects.

Keywords: cytotoxicity; gene expression; nanoparticles; titanium dioxide.

Figures

Figure 1.
Figure 1.
Particle size distribution of intensity measured, by dynamic light scattering analysis. Black circles show the size distribution of TPS, and open diamonds of TPL.
Figure 2.
Figure 2.
Microscopic images of TPS (A–D) and TPL (E–H)-exposed THP-1 cells. Titania particle-exposed cells were paraformaldehyde-fixed, and stained by Hoechst 33258 (nucleus marker; A, E) and rhodamine-phalloidin (F-actin marker, B, F). Titania particles were observed in differential interference images (C, G). (D, H) are merged images.
Figure 3.
Figure 3.
Microscopic images of TPS (A–D) and TPL (E–H)-exposed NCI-H292 cells. Titania particle-exposed cells were paraformaldehyde-fixed. Images represent the nucleus stained by Hoechst 33258 (A, E), F-actin stained by rhodamine-phalloidin (B, F), differential interference images to view titania particles (C, G), and merged images (D, H).
Figure 4.
Figure 4.
Cell viability test based on cytoplasmic ATP concentrations. THP-1 cells (A) or NCI-H292 cells (B) were exposed to each concentration of TPS (closed circle) or TPL (open circle). The results indicate mean ± SD, n ≥ 3 for each, *p < 0.01.
Figure 5.
Figure 5.
mRNA expression of stress- and toxicity- markers in TiO2 particle-exposed PMA-activated THP-1 cells (A) and NCI-H292 cells (B) (mean ± SD, n ≥ 3 for each). Cells were exposed to TPL (open bar) or TPS (solid bar) for 6 h or 24 h. mRNA expression was standardized by internal GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression and the relative expression level versus control (sterilized water added instead of TiO2 particles) is shown. Abbreviations: BTG2 (B-cell translocation gene 2), CCNG1 (cyclin G1), CHEK2 (CHK2 checkpoint homolog), HMOX1 (hemeoxigeanse 1), HSP (heat shock protein 70B′), IL6 (interleukin 6), IL8 (interleukin 8), TNF (tumor necrosis factor α). *p < 0.01, **p < 0.02.

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