ZnO nanostructures have attracted global interest because of their excellent optoelectronic, piezoelectric, ferromagnetic, and optical properties. Therefore, evaluating the biocompatibility of ZnO nanostructures is important. However, contradictory results on the biocompatibility of ZnO nanostructures were reported in numerous studies 123456. For example, no or low cytotoxic effect was observed on rat L2 lung epithelial cells and primary rat lung alveolar macrophage treated with ZnO nanoparticles (NPs) 2. ZnO nanowires were shown to be completely biocompatible on HeLa cells but cytotoxic on L929 cells at a dose of 100 μg/mL 3. In vitro experiments indicated that ZnO NPs have potential applications in cancer diagnosis and therapy 789. However, significant cytotoxic effects were observed when cells were treated with ZnO nanostructures 10111213141516. For example, the treatment of different cells (such as epithelial A549, A431, BEAS-2B cells, and macrophage RAW 264.7 cells) with ZnO NPs induces remarkable intracellular oxidative stress and DNA damage 171819. To date, only a few investigations have evaluated the cytotoxicity of complex ZnO nanostructures assembled by nanoscale building blocks.

Zn²⁺ is a vital component of enzymes and proteins and an ionic signal among various intracellular organelles and storage depots 20. It modulates protein function by binding to and detaching from intracellular zinc-dependent proteins. Nevertheless, excess free Zn²⁺ is cytotoxic and can induce serious neuronal injury 212223. Considerable evidence shows that free Zn²⁺ in the extracellular fluid results in amyloid deposition, one of the pathological hallmarks of Alzheimer’s disease 2425. To date, little is known about the neurotoxic effects of ZnO nanostructures. In the present study, the neurotoxic effects of ZnO hierarchical architectures (including NPs and hollow microspheres consisting of NPs, the prism-like and flower-like structures) were evaluated using RSC96 Schwann cells as the model. RSC96 Schwann cells are the main supportive cells of the peripheral nervous system and are responsible for the myelination of axons. Cell viability was measured through the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and flow cytometry was employed to analyze cell apoptosis and cell cycle. The decomposition of ZnO hierarchical architectures in cell culture media was measured using atomic absorption spectrometry.

The morphologies of ZnO hierarchical architectures were characterized through scanning electron microscopy (SEM) and TEM observation. As shown in Figures 1a and 2a, monodispersed spherical ZnO NPs with an average diameter of ca. 35 nm were obtained. Hollow ZnO microspheres with an average diameter of ca. 2.7 μm were also obtained (Figure 1b). Numerous ZnO NPs were adsorbed on the rough surface of the microspheres. As shown in the inset of Figure 1b, the ZnO microspheres consisted of numerous NPs that are slightly larger than those in Figure 1a. The TEM image in Figure 2b further confirms the hollow structure of the microspheres consisting of NPs that are ca. 45 nm in diameter (Figure 2b). A hexahedral, prism-like ZnO was synthesized with a sharp but irregular tip (Figures 1c and 2c). The prism-like structure was ca. 2.5 to 6.0 μm in diameter and ca. 18.0 to 60.0 μm in length. Figures 1d and 2d show SEM and TEM images of the flower-like structure which consisted of sword-like leaves and prism-like pistils. Both the leaves and the prism-like pistils were ca. 500 to 600 nm in diameter (or in width) and several microns in length.

The cytotoxicity of the ZnO hierarchical architectures on RSC96 Schwann cells was evaluated via MTT assay. As shown in Figure 3a,b, no or very low cytotoxic effect was observed when RSC96 Schwann cells were treated with the prism-like and flower-like ZnO for 12 h at different doses (4, 8, 40, 80, and 400 μg/mL). Significant cytotoxic effects were observed when the treatment was prolonged up to 48 h at the high dose of 400 μg/mL. Cell viability sharply decreased to ca. 58.2% and 52.8% compared with that of the control. Dose- and time-dependent cytotoxic effects were observed when the cells were treated with ZnO NPs and microspheres. For example, a slight cytotoxic effect was observed when the cells were treated with ZnO NPs for 24 h at low doses (lower than 40 μg/mL). Cell viability was decreased to ca. 86.5% at an exposed dose of 40 μg/mL compared with that of the control. Acute cytotoxic effects were detected when the dose of the ZnO NPs was increased to 400 μg/mL, resulting in a sharp decrease in cell viability to ca. 25% after 12 h treatment. Almost complete death of cells occurred after 24 h of treatment. Remarkable time-dependent cytotoxic effects were observed at high doses (higher than 80 μg/mL). After being treated with ZnO NPs for 6 to 48 h, the cell viability was decreased from ca. 74.1% to 52.0% and from ca. 76.7% to 1.7% at doses of 80 and 400 μg/mL, respectively.

It is noteworthy that both dose- and time-dependent cytotoxic effects were more significant when RSC96 Schwann cells were treated with ZnO microspheres. For example, no cytotoxic effect was observed when the cells were treated at low doses (lower than 8 μg/mL) for 48 h. The significant cytotoxic effects were observed when the dose was higher than 80 μg/mL. For example, cell viability was decreased to ca. 57.8% when RSC96 Schwann cells were treated with ZnO microspheres at a dose of 400 μg/mL for 6 h. Time-dependent cytotoxic effects were observed when the cells were treated at high doses (higher than 40 μg/mL) for 48 h. For example, cell viability decreased to ca. 83.2%, 53.5%, 34.8%, and 6.8% when the cells were treated at a dose of 80 μg/mL for 6, 12, 24, and 48 h, respectively.

To investigate the cytotoxic effects of ZnO hierarchical architectures on RSC96 Schwann cells, cell apoptosis and necrosis were measured through a flow cytometry. As shown in Figure 4, RSC96 cells treated with ZnO hierarchical architectures at a dose of 8 μg/mL for 12 h grew normally and did not display apoptosis or necrosis, suggesting the absence of significant cytotoxic effects. When the dose was increased to 80 μg/mL, a low apoptosis rate of ca. 24.7% and 6.4% was observed corresponding to the treatment with the prism-like and flower-like ZnO, respectively. By contrast, a significant apoptosis rate of ca. 51.3% and 39.2% was detected when the cells were treated with ZnO NPs and microspheres at a dose of 80 μg/mL for 12 h, respectively. Furthermore, a significant necrosis rate of ca. 16.4% was observed in the cells treated with ZnO microspheres.

The cell cycle distribution was further analyzed to evaluate the cytotoxic effects of ZnO hierarchical architectures. Most cells are usually in the growth phase (G1), wherein various enzymes (especially those needed for DNA replication) are synthesized. In the subsequent S phase, DNA synthesis is completed to double the chromosomes for division. The G2 phase starts from the end of DNA synthesis until the beginning of mitosis, involving the synthesis of microtubules. The M phase lasts throughout the procedure during which the mother cell is divided into two separate but identical daughter cells 27. In the present study, the major cell population was found in the G1 and S phases in the control group. However, a significant increase of cell population in G2/M phase, accompanied by a decrease of cell population in the G1 and S phases, was observed when RSC96 Schwann cells were treated with ZnO hierarchical architectures at a dose of 8 μg/mL for 12 h (Table 1). These results suggest that the slight G2/M cell cycle arrest was induced by the low-dose treatment of ZnO hierarchical architectures, although no significant cytotoxic effect was detected according to the MTT assay. The population of cells in the G2/M phase was further increased when the cells were treated with ZnO hierarchical architectures at a dose of 80 μg/mL (Table 1), suggesting a more serious G2/M cell cycle arrest than that at the lower dose. These results show that cell cycle was significantly influenced by the treatment of ZnO hierarchical architectures because of serious G2/M cell cycle arrest.

Data were expressed as Mean ± SD (n = 5).

The decomposition of ZnO hierarchical architectures was measured by monitoring the change of Zn²⁺ concentration in the culture media at different time intervals via atomic absorption spectroscopy. As shown in Figure 5, a significant enhancement of Zn²⁺ concentration was observed with increasing incubation time. This result suggests that the decomposition process occurred during the incubation period. It is noteworthy that the release of Zn²⁺ in the ZnO NPs-treated group was lower than that in the other groups at a dose of 8 μg/mL, but higher than in the prism-like and flower-like ZnO-treated group at a dose of 80 μg/mL. The highest Zn²⁺ concentration was observed in the ZnO microspheres-treated group at both tested doses (e.g., ca. 3.7 to 9.6 μg/mL at time intervals from 6 to 48 h at a dose of 80 μg/mL). These results suggest that at a dose of 80 μg/mL, the release of Zn²⁺ from both ZnO NPs and microspheres was faster than from the flower-like and prism-like ZnO. This result is consistent with the cytotoxic effect observed via the MTT assay.

Compared with bulk counterparts, more atoms are located on the surfaces of smaller NPs, which can interact with biological systems more effectively 28. In the present work, ZnO NPs and hollow microspheres consisting of NPs displayed more significant cytotoxic effects in dose- and time-dependent manners on RSC96 Schwann cells than the bulk prism-like and flower-like structures. This implies that the cytotoxic effects of complex architectures are most likely predominated by the nanoscale building blocks. The precise cytotoxic mechanisms of ZnO nanostructures still remain indistinct. ZnO NPs can induce a significant accumulation of intracellular reactive oxygen species (ROS) in various cells (such as monocytes and lymphocytes and WIL2-NS human lymphoblastoid cells) in a size-dependent manner. This treatment of ZnO NPs results in the direct alteration of mitochondrial functionality, increase of intracellular Ca²⁺ level, and expression of genes involved in apoptosis and oxidative stress responses 293031. In the present work, ZnO NPs and microspheres induced significant cell apoptosis in a dose-dependent manner. The treatment of RSC96 cells with ZnO hierarchical architectures resulted in a remarkable G2/M cell cycle arrest, even at a sublethal dose (8 μg/mL), implying the early DNA damage. Furthermore, the release of Zn²⁺ in the cell culture media was consistent with the cytotoxic effect of ZnO hierarchical architectures on RSC96 Schwann cells. Alterations in Zn²⁺ homeostasis displayed powerful stimulatory effects on multiconductance cation channels in the inner mitochondrial membrane and ROS generation. A loss of Zn²⁺ homeostasis may result in cell apoptosis or necrosis 1632. Moreover, the effects of Zn²⁺ level on Ca²⁺ homeostasis were also reported which is another crucial signal pathway closely related to cell apoptosis and necrosis 3133. The effects of ZnO NPs on the cell cycle have rarely been studied. Wang et al. 34 reported that the exposure of human embryonic kidney HEK293 cells to SiO₂ NPs results in the accumulation of cells in the G2/M phase in a dose-dependent manner. AshaRani et al. 35 found that Ag NPs caused a concentration-dependent increase of cell population in the G2/M phase in both normal human lung fibroblast IMR90 cells and human glioblastoma U251cells.

In summary, the cytotoxic effects of ZnO hierarchical architectures, such as NPs and hollow microspheres consisting of NPs, the prism-like and flower-like structures, were evaluated using RSC96 Schwann cells as the model. The ZnO NPs and microspheres displayed significant cytotoxic effects on RSC96 Schwann cells in time- and concentration-dependent manners. The treatment of cells with ZnO NPs and microspheres induced remarkable cell apoptosis and G2/M cell cycle arrest which were associated with the decomposition of ZnO hierarchical architectures and the subsequent release of Zn²⁺ in the culture media. These results provide new insights into the cytotoxic effects of complex architectures that could be prominently dominated by nanoscale building blocks.

The authors declare that they have no competing interests.

YY and XC carried out MTT assay and cell cycle and apoptosis measurement, as well as data analysis. QL, HS, and DC were responsible for the synthesis and characterization of ZnO hierarchical architectures, as well as the measurement of Zn²⁺ concentration in culture media. QW designed the whole work and wrote the manuscript. All authors read and approved the final manuscript.