Cytotoxic Effects of Zinc Oxide Nanoparticles on Human Glial Cells

: Despite being one of the most studied nanomaterials, much remains unknown about the mechanism of action of zinc oxide (ZnO) nanoparticles (NP). The aim of this work was to evaluate the effects on cell viability caused by exposure of glial cells to ZnO NP, and the role of Zn 2+ in the observed effects. The impact of ZnO NP or Zn 2+ ions on cell viability was assessed by MTT assay. Exposure to ZnO NP induced a significant decrease in cell viability. The presence Zn 2+ ions released from ZnO NP was not entirely responsible for the observed cytotoxic effects.


Introduction
One of the most widely used nanomaterials at present is zinc oxide (ZnO) nanoparticles (NP), with multiple applications in cosmetic, technological industry and, recently, also in biomedicine [1][2][3][4]. This widespread use means that humans are increasingly being exposed to these NP, raising concerns about how this could affect health. Due to welldemonstrated ability of these NP to easily reach the brain [3,5], their effects on the nervous system deserve particular attention. Despite being one of the most studied NP from a toxicological point of view, much remains unknown about how they may affect specific cell types, such as glial cells, and their mechanism of action. On this basis, the aim of this work was to evaluate the effects on cell viability caused by exposure of human glioblastoma A172 cells to ZnO NP, and the role of Zn 2+ in the observed effects.

Materials and Methods
A stock suspension of ZnO NP (CAS No. 1314-13-2, Sigma-Aldrich Co., final concentration 100 µg/mL) was prepared prior treatments in A172 cell culture medium and ultrasonicated (Branson Sonifier, USA), as previously described [6]. Average hydrodynamic size and zeta potential of particles were determined by dynamic light scattering (DLS) and  A flow cytometry approach (FACSCalibur, Becton Dickinson) was employed to assess the potential of the ZnO NP to enter the cells [7], whereas NP effects on viability were evaluated by MTT assay using a SPECTROstar Nano (BMG Labtech) microplate reader [8]. For these experiments, cells were incubated at different ZnO NP or ZnSO4 concentrations, or the control solutions (cell culture medium), for different exposures times.
Differences among groups were analysed by Kruskal-Wallis test, with Mann-Whitney U-test for two-by-two comparisons, by employing SPSS for Windows statistical package (version 20.0). The associations between two variables were analysed by Pearson's correlation. All experiments were run at least in triplicate. Experimental data were expressed as mean ± standard error and a p-value of <0.05 was considered significant.

Nanoparticle Characterization and Cellular Uptake
The ZnO NP employed in the present study were less than 100 nm spherical NP (data provided by the manufacturer). Results obtained from the analysis of hydrodynamic size and zeta potential are collected in Table 1. Despite the variable range obtained for zeta potential, dispersion of ZnO NP resulted quite stable, with slight variations in the hydrodynamic size, ranging between 273.97 nm and 315.01 nm.  Table 2 shows the results obtained from the flow cytometry analysis of cellular uptake of ZnO NP at different concentrations and exposure times.
According to flow cytometry results, NP were effectively internalized by the cells at all exposure times in a dose-dependent manner, although uptake rate was notably much lower at 3 h. These results agree with some previous studies that observed a similar doseand time-dependent cellular internalization of ZnO NP, evaluated by diverse methods, in other different cell types [9][10][11][12][13]. Nevertheless, current uptake results should be confirmed by means of a suitable microscopy technique.

Zn +2 Ion Release
Results obtained from the FAAS experiments to quantify the release of zinc ions from the ZnO NP are shown in Figure 1. Increased levels of dissolved Zn +2 ions from the ZnO NP surface were observed (from 2.5 µg/mL on) at all treatment periods (3, 24 and 48 h), but notably higher at the shortest time. The release of Zn 2+ ions from ZnO NP is well documented in the literature, and demonstrated that the solubility of these NP can be highly variable and dependent on both the NP physicochemical properties and the matrix in which they are suspended [14][15][16][17].

ZnO NP Effects on Glial Cells
After discarding any potential interference of the NP with MTT assay, the effects of ZnO NP on viability of glial A172 cells were evaluated. Results from these experiments are shown in Figure 2. Effects on cellular viability were moderate after 3 h of exposure, reaching 60% viability only at the highest concentrations. However, after 24 and 48 h of treatments, the decreases in viability were much marked, reaching values close to 90% mortality from 50 or 25 µg/mL on, respectively. Similarly, several authors previously described a dose-dependent decrease in cell viability of various cell types exposed to ZnO NP (reviewed in [18]).

Role of Zn +2 Ions Released from ZnO NP
In order to test the role of the Zn 2+ released from ZnO NP in the cytotoxic effects observed, as previously pointed out [19,20], in the present work A172 cells were treated with zinc sulfate (ZnSO4) at several concentrations chosen accordingly to FAAS analysis. Results obtained from these analyses showed that a significant decrease in cell viability was just observed at Zn 2+ ion concentrations greater than 0.2 and 0.3 mM for 24 and 48 h, respectively ( Figure 3). According to this, the release of Zn 2+ ions from of ZnO NP does not explain, at least completely, their effects on glial cells viability, and hence other different action mechanism, e.g., production of ROS or induction of oxidative stress, previously reported for these NP [21][22][23] should be considered to explain the observed effects.

Conclusions
ZnO NP were effectively internalized by A172 cells and the exposure induced a significant decrease in cell viability, depending on the dose and treatment duration. The ability of ZnO NP to release Zn 2+ ions into the medium in a concentration-dependent manner was confirmed; however, their presence was not entirely responsible for the observed cytotoxic effects. These results increase the knowledge about the toxic potential of ZnO NP in human glial cells and the involvement of released Zn 2+ ions in the observed effects.