In vitro cytotoxicity of SiO₂ or ZnO nanoparticles with different sizes and surface charges on U373MG human glioblastoma cells

Introduction

Nanoparticles (NPs) are objects with at least one dimension less than 100 nm.¹ Many NPs offer unique and beneficial properties, and they have been widely used in medical, pharmaceutical, food, cosmetics, electronics, and other industries (reviewed in Uskokovic).² Silicon dioxide (SiO₂) and zinc oxide (ZnO) NPs have photocatalytic activities and are commonly used in various consumer products (eg, cosmetics) and biomedical applications (eg, drug delivery and theranostics) (reviewed in Fan and Lu³ and Fine et al⁴).

Given their widespread use, humans are constantly exposed to SiO₂ and ZnO NPs, which can enter the body via ingestion, inhalation, and dermal absorption.⁵ NPs may interact with tissue macromolecules and adversely affect cellular physiology.^6–10 Numerous studies have investigated the cytotoxic effects of SiO₂ and ZnO NPs,^11–19 but many controversies remain due to the physical and chemical properties of these NPs.

Furthermore, we do not yet fully understand the underlying cell-death mechanisms induced by SiO₂ and ZnO NPs. For example, SiO₂ and ZnO NPs reportedly trigger the intrinsic apoptotic pathway by generating reactive oxygen species (ROS) and inducing the p53 pathway to activate the caspase cascade.^20–23 Both SiO₂ and ZnO NPs were shown to activate the initiator caspase-9 and the executioner caspase-3 in human lung epithelial cells and dermal fibroblasts.^20–23 However, a recent study found that ZnO NP-mediated apoptosis was not related to ROS generation or the p53 pathway.²⁴ Thus, the cell-death pathways mediated by SiO₂ and ZnO NPs are still the subject of some debate.

In this study, we investigated the cytotoxic effects of SiO₂ and ZnO NPs on the U373MG human glioblastoma cell line. Since cytotoxic potentials may be affected by the physical and chemical properties of NPs, such as their sizes and surface charges, we selected SiO₂ and ZnO NPs of different sizes (100 nm and 20 nm) and surface charges and examined their cytotoxic and apoptotic effects on U373MG cells.

Materials and methods

Cells, reagents, and preparation of NPs

The U373MG cells were maintained in Dulbecco’s Modified Eagle’s Medium (Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (Biowest), penicillin (100 U/mL), and streptomycin (100 μg/mL). Zinc chloride was purchased from Sigma-Aldrich (St Louis, MO, USA). The 20 and 100 nm ZnO NPs were purchased from Sumitomo Osaka Cement Co, Ltd (Lot number 141319) (Tokyo, Japan) and American Elements (Lot number 1871511079-673) (Los Angeles, CA, USA), respectively. The surface charge of the ZnO NPs was modified with citrate (for a negative charge) and L-serine (for a positive charge), as previously reported.²⁵ The 20 and 100 nm SiO₂ NPs were purchased from E&B Nanotech Co, Ltd (Ansan-si, South Korea). To reduce their negative charge, the SiO₂ NPs were treated with L-arginine (R). Detailed information regarding the characterizations and physicochemical properties of the SiO₂ and ZnO NPs can be found in Kim et al.²⁶

Western blot analysis

Cell lysates were harvested, fractionated, and transferred to nitrocellulose membranes as described previously.²⁷ Antibodies to poly-(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and alpha-tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA) and Sigma-Aldrich, respectively. Enhanced chemiluminescence detection reagents (Thermo Fisher Scientific, Waltham, MA, USA) and secondary peroxidase-labeled anti-mouse or anti-rabbit immunoglobulin G antibodies (Amersham Biosciences, Piscataway, NJ, USA) were used according to the manufacturer’s directions.

Cell viability and DNA fragmentation assays

Cell viability was determined by using the CellTiter-Glo luminescent cell viability assay (Promega Corporation, Madison, WI, USA) according to the manufacturer’s directions. DNA fragmentation was determined by using the DeadEnd™ Fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System (Promega Corporation) according to the manufacturer’s directions. Nuclei were stained using Vectasheild mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA).

Results

Effect of SiO₂ or ZnO NPs on the viability of U373MG human glioblastoma cells

The U373MG cells were treated with various concentrations of SiO₂ or ZnO NPs with different sizes and surface charges (ZnO^AE100(+) , ZnO^AE100(−), ZnO^SM20(+), ZnO^SM20(−), SiO₂^EN100(R), SiO₂^EN100(−), SiO₂^EN20(R), and SiO₂^EN20(−)). After 24 hours, cell viability was measured using the CellTiter-Glo assay, which determines the presence of live and metabolically active cells by measuring adenosine triphosphate (Figure 1). Treatment with 6 mg/mL of SiO₂^EN100(R) and SiO₂^EN100(−) reduced the viability of U373MG cells by 68% and 65%, respectively (Figure 1A and B). Interestingly, the 20 nm SiO₂ NPs were more toxic to U373MG cells than 100 nm SiO₂ NPs. Treatment with 0.6 and 0.9 mg/mL of SiO₂^EN20(R) reduced the viability of U373MG cells by 90% and 98%, respectively (Figure 1C). Similarly, treatment with 0.6 and 0.8 mg/mL of SiO₂^EN20(−) reduced the viability of U373MG cells by 23% and 96%, respectively (Figure 1D). The cytotoxicity of SiO₂ NPs was not cell-type specific as we observed similar levels of cytotoxicity against human dermal fibroblast and HCT116 human colorectal carcinoma cells (data not shown). The half-maximal inhibitory concentration values for the cytotoxicity of SiO₂^EN100(R), SiO₂^EN100(−), SiO₂^EN100(R), and SiO₂^EN20(−) against U373MG cells at 24 hours after treatment were 4.36, 4.93, 0.41, and 0.68 mg/mL, respectively (Table 1).

Figure 1 The effect of SiO2 NPs on the viability of U373MG cells. Notes: U373MG cells were treated with various concentration of (A) SiO2EN100(R), (B) SiO2EN100(−), (C) SiO2EN100(R), or (D) SiO2EN20(−) NPs. At 24 hours after treatment, cell viability was determined with the CellTiter-Glo assay. To calculate the relative luciferase activities, the luciferase activities at 0 hours after treatment were set to 100%. The data shown here represent the results from three independent experiments. Abbreviations: NPs, nanoparticles; RLU, relative luminescence units; SiO2, silicon dioxide.

Table 1 The IC50 values for the cytotoxicity of SiO2 or ZnO NPs against U373MG cells at 24 hours Abbreviations: IC50, half-maximal inhibitory concentration; NPs, nanoparticles; SiO2, silicon dioxide; ZnO, zinc oxide.

Compared to the SiO₂ NPs, the ZnO NPs were significantly more toxic to U373MG cells as treatment with 20 μg/mL of ZnO^AE100(+) , ZnO^AE100(−), ZnO^SM20(+), and ZnO^SM20(−) for 24 hours reduced the viability of U373MG cells by 53%, 47%, 74%, and 53%, respectively (Figure 2). The half-maximal inhibitory concentration values for the cytotoxicity of ZnO^AE100(+), ZnO^AE100(−), ZnO^SM20(+), and ZnO^SM20(−) on U373MG cells at 24 hours after treatment were 19.67, 20.47, 16.82, and 19.67 μg/mL, respectively (Table 1). Since treatment with 20 μg/mL of zinc chloride exhibited no cytotoxic effect against U373MG cells, the observed cytotoxicity appeared to have been due to the effect of the ZnO NPs (rather than Zn₂⁺) on U373MG cells (Figure 3).

Figure 2 The effect of ZnO NPs on the viability of U373MG cells. Notes: U373MG cells were treated with 0, 2, 5, 10, 15, or 20 μg/mL of (A) ZnOAE100(+), (B) ZnOAE100(−), (C) ZnOSM20(+), or (D) ZnOSM20(−) NPs. At 24 hours after treatment, cell viability was determined with the CellTiter-Glo assay. To calculate the relative luciferase activities, the luciferase activities at 0 hours after treatment were set to 100%. The data shown here represent the results from three independent experiments. Significant differences between samples were determined by the P-value of a two-sample t-test (P<0.05). Abbreviations: NPs, nanoparticles; RLU, relative luminescence units; ZnO, zinc oxide.

Figure 3 The effect of non-nano ZnCl2 on the viability of U373MG cells. Notes: U373MG cells were treated with or without 20 μg/mL ZnCl2. At 24 hours after treatment, cell viability was determined with the Celltiter-Glo assay. To calculate the relative luciferase activities, the luciferase activities of mock-treated cells were set to 100%. The data shown here represent the results from three independent experiments. Abbreviations: RLU, relative light unit; ZnCl2, zinc chloride.

Taken together, these results indicate that the ZnO NPs were more toxic than SiO₂ NPs against U373MG cells. Furthermore, the 20 nm SiO₂ NPs were more toxic than the 100 nm NPs, whereas the cytotoxicity of ZnO NPs was not affected by their size or surface charge in our experimental systems.

Effect of SiO₂ or ZnO NPs on caspase-3 activation

To determine whether SiO₂ or ZnO NPs induce apoptosis, U373MG cells were treated with the above-described SiO₂ or ZnO NPs, and caspase-3 activation was assessed by determining the proteolytic cleavage of PARP (from the native 116 kDa to 89 kDa) at 0, 1, 3, 6, and 9 hours after treatment. The treatment of U373MG cells with SiO₂ NPs at the concentrations shown to reduce cell viability by 85% to 90% was found to rapidly induce PARP cleavage at 1 hour after treatment (Figure 4; compare lane 2 with lane 1). ZnO^AE100(−) and ZnO^SM20(−) also rapidly induced PARP cleavage in U373MG cells at 1 hour after treatment (Figure 5A and B; compare lane 2 with lane 1), whereas ZnO^AE100(+) and ZnO^SM20(+) induced PARP cleavage at later time points at 9 and 6 hour, respectively (Figure 5C and D; compare lane 2 with lane 1). These data indicate that both SiO₂ and ZnO NPs induce caspase-3 activation, further suggesting that they both induce apoptosis.

Figure 4 The effect of SiO2 NPs on caspase activation. Notes: U373MG cells were treated with 9 mg/mL of (A) SiO2EN100(R) or (B) SiO2EN100(−) NPs, or 0.8 mg/mL of (C) SiO2EN100(R) or (D) SiO2EN20(−) NPs. At 0, 1, 3, 6 and 9 hours after treatment, PARP cleavage was determined by Western blot analysis. Abbreviations: NPs, nanoparticles; PARP, poly-(adenosine diphosphate-ribose) polymerase; SiO2, silicon dioxide.

Figure 5 The effect of ZnO nanoparticles on caspase activation. Notes: U373MG cells were treated with 20 μg/mL of (A) ZnOAE100(−), (B) ZnOSM20(−), (C) ZnOAE100(+), or (D) ZnOSM20(+) NPs. At 0, 1, 3, 6, 9 hours after treatment, PARP cleavage was determined by Western blot analysis. Abbreviations: NPs, nanoparticles; PARP, poly-(adenosine diphosphate-ribose) polymerase; ZnO, zinc oxide.

Effect of SiO₂ or ZnO NPs on chromosomal DNA fragmentation and damage

To further assess the apoptotic effects of SiO₂ or ZnO NPs, U373MG cells were treated with the above-described NPs for 6 hours, and chromosomal DNA fragmentation was determined using a TUNEL assay. As expected, SiO₂ NPs and ZnO NPs induced chromosomal DNA fragmentation in U373MG cells (Figure 6). At 6 hours after treatment with SiO₂^EN100(R), SiO₂^EN100(−), SiO₂^EN100(R), and SiO₂^EN20(−), 11.7%, 12.9%, 10.8%, and 10.1% of the cells were found to be TUNEL positive (Figure 6A). Compared to the SiO₂ NPs, the ZnO NPs were more potent in inducing chromosomal DNA fragmentation as 42.7%, 60.4%, 42.8%, and 19.5% of the cells treated with ZnO^AE100(+), ZnO^AE100(−), ZnO^SM20(+), and ZnO^SM20(−), respectively, were found to be TUNEL positive (Figure 6B). These data suggest that the ZnO NPs may be more effective at inducing chromosomal DNA fragmentation in these cells (Figure 6B). In addition to the TUNEL assay, the comet assay was employed to examine DNA damage in SiO₂ or ZnO NP-treated cells. Consistent with the TUNEL data, both SiO₂ and ZnO NPs induced DNA damage in U373MG cells (data not shown). Taken together, these data indicate that both SiO₂ and ZnO NPs reduce the viability of U373MG cells by inducing apoptosis, and further suggest that ZnO NPs may be more effective than SiO₂ NPs for inducing apoptosis in U373MG cells.

Figure 6 The effect of SiO2 or ZnO NPs on DNA fragmentation. Notes: U373MG cells were treated with (A) SiO2 or (B) ZnO NPs with different sizes and surface charges at the concentrations described above. At 6 hours after treatment, fragmented DNA was labeled with fluorescein-12-UTP (green) and visualized under fluorescence microscopy. Nuclei were visualized by DAPI staining (blue). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; NPs, nanoparticles; SiO2, silicon dioxide; ZnO, zinc oxide; UTP, uridine triphosphate.

Discussion

In the present study, we investigated the cytotoxic effects of SiO₂ and ZnO NPs with two different sizes (20 and 100 nm) and charges (positive and negative) against the U373MG human glioblastoma cell line. The SiO₂ and ZnO NPs both reduced the viability of U373MG cells at 24 hours after treatment. The ZnO^SM20(+), ZnO^SM20(−), ZnO^AE100(+), and ZnO^AE100(−) NPs were 24-, 34-, 222-, and 241-fold more toxic to U373MG cells than their corresponding SiO₂ NP counterparts. These differences in the cytotoxicities of the SiO₂ and ZnO NPs may reflect differences in solubility, dissolution rate in the media, protein interactions, ROS generation, and/or the ability to activate the intrinsic apoptotic and/or necrotic pathways.

Other studies have indicated that both size and surface charge can influence the cytotoxicity of SiO₂ and ZnO NPs.^28–31 Consistent with these studies, we observed that particle size affected the cytotoxicity of SiO₂^EN100(R) and SiO₂^EN20(−) NPs, which were, respectively, eleven- and sevenfold more toxic than 100 nm counterparts against U373MG cells. Smaller NPs may be more effective at entering cells and organelles (eg, mitochondria), allowing them more opportunity to induce oxidative stress and apoptosis.^14,31–33 In contrast to the previous reports, however, we found that the surface charge of SiO₂ NPs had almost no effect on their cytotoxicity against U373MG cells. Although SiO₂^EN100(R) and SiO₂^EN100(R) NPs were slightly more toxic than SiO₂^EN100(−) and SiO₂^EN20(−), respectively, these differences were not significant. Also, inconsistent with the previous reports, we found that the cytotoxicity of ZnO NPs against U373MG cells was unaffected by their size and surface charge. Future work will be needed to examine these apparent discrepancies.

Treatment of U373MG cells with SiO₂ NPs was found to rapidly activate caspase-3 and induce apoptosis within 1 hour. Treatment with ZnO^AE100(−) and ZnO^SM20(−) NPs also activated caspase-3 by 1 hour after treatment, whereas ZnO^AE100(+) and ZnO^SM20(+) activated caspase-3 later (9 and 6 hours after treatment, respectively). Previous reports showed that SiO₂ and ZnO NPs may induce the intrinsic pathway for apoptosis via ROS-mediated p53 activation.^20–23 ROS-induced DNA damage activates p53, which triggers apoptosis by transactivating proapoptotic genes and activating other transcription-independent mechanisms.³⁴ However, Wilhelmi et al reported that ZnO NPs induce necrosis and apoptosis in macrophages via ROS- and p53-independent pathway.²⁴ Consistent with the latter study, we found that both SiO₂ and ZnO NPs induced apoptosis in U373MG cells, which express mutant p53. Other authors have suggested that, in addition to the ROS-mediated p53 activation pathway, SiO₂ and ZnO NPs may activate the p38 mitogen-activated protein kinase and/or c-Jun N-terminal kinase pathways to transactivate proapoptotic genes and induce apoptosis.^23,35

Since DNA damage induces the G₂/M DNA damage checkpoint to arrest the cell cycle,³⁶ it is not surprising that silica NPs induce cell cycle arrest at the G₂/M phase.³⁷ More specifically, they induce the G₂/M DNA damage checkpoint via the activation of Chk1, which phosphorylates p53.^37,38 Since Cdk2 has been reported to play an important role in p53-independent G₂/M checkpoint control,³⁹ we speculate that SiO₂ and ZnO NPs may arrest the cell cycle at the G₂/M phase in U373MG cells, possibly via a Cdk2-dependent pathway. In addition to apoptosis, SiO₂ and ZnO NPs may induce necrotic cell death in U373MG cells. Thus, further studies are needed to examine how SiO₂ and ZnO NPs induce apoptotic and/or necrotic cell death in human cell lines.

Conclusion

We herein investigated the cytotoxic effects of SiO₂ and ZnO NPs with different sizes and surface charges on the human glioblastoma cell line, U373MG. The overall cytotoxicity of the ZnO NPs was significantly higher than that of the SiO₂ NPs against U373MG cells. The cytotoxicity of the SiO₂ NPs was affected by the particle size, but not the surface charge, in our system, with the smaller SiO₂ NPs showing a higher cytotoxicity. In contrast, changes in the size and surface charge of the ZnO NPs had little or no effect on their cytotoxicity against U373MG cells. Both SiO₂ and ZnO NPs were found to activate caspase-3 and induce DNA fragmentation in our system. Thus, we report that the tested SiO₂ and ZnO NPs exhibited cytotoxic effects against U373MG cells, at least partly via the induction of apoptosis.

Acknowledgment

This research was supported by a grant (10182MFDS991) from the Ministry of Food and Drug Safety in 2010–2011.

Disclosure

The authors report no conflict of interest in this work.

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