The Pro-Apoptotic Effect of Silica Nanoparticles Depends on Their Size and Dose, as Well as the Type of Glioblastoma Cells

Abstract

Despite intensive investigations, nanoparticle-induced cellular damage is an important problem that has not been fully elucidated yet. Here, we report that silica nanoparticles (SiNPs) demonstrated anticancer influence on glioblastoma cells by the induction of apoptosis or necrosis. These effects are highly cell type-specific, as well as dependent on the size and dose of applied nanoparticles. Exposure of LN-18 and LBC3 cells to different sizes of SiNPs-7 nm, 5-15 nm, or 10-20 nm-at dosages, ranging from 12.5 to 1000 µg/mL, for 24 and 48 h reduced the viability of these cells. Treatment of LN-18 and LBC3 cells with 7 nm or 10-20 nm SiNPs at doses ≥50 µg/mL caused a strong induction of apoptosis, which is connected with an increase of intracellular reactive oxygen species (ROS) production. The 5-15 nm SiNPs exhibited distinct behavior comparing to silica nanoparticles of other studied sizes. In contrast to LBC3, in LN-18 cells exposed to 5-15 nm SiNPs we did not observe any effect on apoptosis. These nanoparticles exerted only strong necrosis, which was connected with a reduction in ROS generation. This suggests that SiNPs can trigger different cellular/molecular effects, depending on the exposure conditions, the size and dose of nanoparticles, and cell type of glioblastoma.

Keywords: apoptosis; glioblastoma; nanotoxicity; silica nanoparticles.

Figure 1

The viability of LBC3 cells (A–C) treated with different concentrations (12.5 to 1000 µg/mL) of silica nanoparticles in three different sizes—7 nm (A), 5–15 nm, (B) and 10–20 nm (C), for 24 and 48 h. Mean values from three independent experiments ± SD are presented. Note: Significant alterations are expressed relative to controls and marked with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001. Statistical significance was considered if p < 0.05.

Figure 2

The viability of LN-18 cells (A–C) treated with different concentrations (12.5 to 1000 µg/mL) of silica nanoparticles in three different sizes—7 nm (A), 5–15 nm (B), and 10–20 nm (C)—for 24 and 48 h. Mean values from three independent experiments ± SD are presented. Note: Significant alterations are expressed relative to controls and marked with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001. Statistical significance was considered if p < 0.05.

Figure 3

The effect of 7 nm silica nanoparticles on apoptosis and necrosis of LBC3 (A–D) and LN-18 (E–H) cells evaluated by annexin V assay. The cells were incubated for 24 and 48 h in Dulbecco’s Modified Eagle Medium (DMEM) with 25 μg/mL, 50 μg/mL, 100 μg/mL, 300 μg/mL, or 600 μg/mL of 7 nm silica nanoparticles. The cells were double-stained Fluorescein Isothiocyanate (FITC)-Annexin V and propidium iodide (PI). Representative Flow Cytometry (FACS) analysis via Annexin V-FITC/PI staining for 24 h and 48 h is presented. The bar graphs present the percentage of apoptotic cells as a sum of Q2 and Q4 quadrants (Figure 3C,G) and necrotic cells as a Q1 quadrant (Figure 3D,H) of the analyzed cell population. Mean values of the percentage of apoptotic and necrotic cells from three independent experiments ± SD are presented. Note: significant alterations are expressed relative to controls and marked with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001. Statistical significance was considered if p < 0.05.

Figure 4

The effect of 5–15 nm silica nanoparticles on apoptosis and necrosis of LBC3 (A–D) and LN-18 (E–H) cells evaluated by Annexin V assay. The cells were incubated for 24 and 48 h in DMEM with 25 μg/mL, 50 μg/mL, 100 μg/mL, 300 μg/mL, or 600 μg/mL of 5–15 nm silica nanoparticles. The cells were double-stained with FITC–Annexin V and PI. Representative FACS analysis via Annexin V-FITC/PI staining for 24 h and 48 h is presented. Bar graph presenting the percentage of apoptotic cells as a sum of Q2 and Q4 quadrants (Figure 4C,G) and necrotic cells as a Q1 quadrant (Figure 4D,H) of analyzed cells population. Mean values of the percentage of apoptotic and necrotic cells, from three independent experiments ± SD are presented. Note: significant alterations are expressed relative to controls and marked with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001. Statistical significance was considered if p < 0.05.

Figure 5

The effect of 10–20 nm silica nanoparticles on apoptosis and necrosis of LBC3 (A–D) and LN-18 (E–H) cells evaluated by Annexin V assay. The cells were incubated for 24 and 48 h in DMEM with 25 μg/mL, 50 μg/mL, 100 μg/mL, 300 μg/mL, or 600 μg/mL of 10–20 nm silica nanoparticles. The cells were double-stained with FITC-Annexin V and PI. Representative FACS analysis via Annexin V-FITC/PI staining for 24 h and 48 h is presented. Bar graphs present the percentage of apoptotic cells as a sum of Q2 and Q4 quadrants (C,G), necrotic cells as a Q1 quadrant (D,H) of the analyzed cell population. Mean values of the percentage of apoptotic and necrotic cells from three independent experiments ± SD are presented. Note: significant alterations are expressed relative to controls and marked with asterisks: ** p < 0.01; *** p < 0.001. Statistical significance was considered if p < 0.05.

Figure 6

Intracellular reactive oxygen species (ROS) production induced by three different sizes—7 nm (A), 5–15 nm (B), and 10–20 nm (C)—of silica nanoparticles (SiNPs) in LBC3 cells for 24 and 48 h. Note: significant alterations are expressed relative to controls and marked with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001. Statistical significance was considered if p < 0.05.

Figure 7

Intracellular reactive oxygen species (ROS) production induced by three different sizes—7 nm (A), 5–15 nm (B) and 10–20 nm (C)—of SiNPs in LN-18 cells for 24 and 48 h. Note: significant alterations are expressed relative to controls and marked with asterisks: * p < 0.05; *** p < 0.001. Statistical significance was considered if p < 0.05.