Toxicity of functional nano-micro zinc oxide tetrapods: impact of cell culture conditions, cellular age and material properties

Abstract

With increasing production and applications of nanostructured zinc oxide, e.g., for biomedical and consumer products, the question of safety is getting more and more important. Different morphologies of zinc oxide structures have been synthesized and accordingly investigated. In this study, we have particularly focused on nano-micro ZnO tetrapods (ZnO-T), because their large scale fabrication has been made possible by a newly introduced flame transport synthesis approach which will probably lead to several new applications. Moreover, ZnO-T provide a completely different morphology then classical spherical ZnO nanoparticles. To get a better understanding of parameters that affect the interactions between ZnO-T and mammalian cells, and thus their biocompatibility, we have examined the impact of cell culture conditions as well as of material properties on cytotoxicity. Our results demonstrate that the cell density of fibroblasts in culture along with their age, i.e., the number of preceding cell divisions, strongly affect the cytotoxic potency of ZnO-T. Concerning the material properties, the toxic potency of ZnO-T is found to be significantly lower than that of spherical ZnO nanoparticles. Furthermore, the morphology of the ZnO-T influenced cellular toxicity in contrast to surface charges modified by UV illumination or O2 treatment and to the material age. Finally, we have observed that direct contact between tetrapods and cells increases their toxicity compared to transwell culture models which allow only an indirect effect via released zinc ions. The results reveal several parameters that can be of importance for the assessment of ZnO-T toxicity in cell cultures and for particle development.

Figure 1. Morphology (A–D) and cytotoxicity (E) of ZnO-T structures synthesized at different dates.

Ages of ZnO-T used for cell treatment were: A = 16–20 weeks, B = 13–17 weeks, C = 8–12 weeks and D = 3–7 weeks. Passage number of normal human dermal fibroblasts (NHDF): P7–P11. Seeded cell number: 50000 cells/cm²; ZnO-T concentration: 0.05–10 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. [Each symbol represents the mean ± SE of n = 3–5 independent experiments with fourfold determinations. CTRL is short abbreviation for control].

Figure 2. Cytotoxic effect of ZnO-T with different surface charges.

Cytotoxicity of untreated or either pre-treated ZnO-T with O₂ or UV-light. Cell viability of normal human dermal fibroblasts (NHDF) was determined by the MTT assay. Passage number of fibroblasts: P8–P20. Seeded cell number: 50000 cells/cm² ZnO-T concentration: 0.05–10 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of at least n = 3–5 independent experiments with fourfold determinations.

Figure 3. Effect of ZnO-T morphology on cytotoxicity: Phase contrast microscopy (A–D) and scanning electron microscopy (SEM) (E–H).

A, E: ZnO-T “thick”; B, F: ZnO-T “fine”; C, G: ZnO-T reference “crushed”; D, H: ZnO-T “reference”, (I–K) Viability (MTT-assay) of treated human dermal fibroblasts (NHDF). Passage number of NHDF: P10–P14. Seeded cell number: 50000 cells/cm²; ZnO-T concentration: 0.05–10 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3–5 independent experiments with fourfold determinations.

Figure 4. Percentage increase or decrease of cytotoxicity depending on size/shape of ZnO-T compared to the reference probe (ZnO-T reference) at each concentration [% viable cells of probe/% viable cells of reference * 100].

Each value shown is the mean of three independent experiments (± SE. Statistical significance determined by one way analysis of variance: thick vs. crushed 2 mg/ml (** = p<0.01); thick vs. fine 5 mg/ml (* = p<0.05); thick vs. crushed 5 mg/ml (*** = p<0.001) (post hoc test: Bonferroni).

Figure 5. Effect of ZnO-T on cell viability determined by the MTT-assay depending on passage number of normal human dermal fibroblasts (NHDF).

Seeded cell number: 50000 cells/cm²; ZnO-T concentration: 0.2–5 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments. Statistical significance determined by one way analysis of variance: P7–P12 vs. P14–P19 2 mg/ml (** = p<0.01) and 5 mg/ml (* = p<0.05).

Figure 6. Effect of cell density on ZnO-T cytotoxicity.

Cell viability of normal human dermal fibroblasts (NHDF) was determined by the MTT assay. Passage number: P11–P15. Seeded cell numbers: 25000 cells/cm², 50000 ells/cm² and 100000 cells/cm²; ZnO-T concentration: 0.05–5 mg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Statistical significance determined by one way analysis of variance: 25000 vs. 50000 cells/cm² 2 mg/ml (*** = p<0.001) and 5 mg/ml (*** = p<0.001); 100,000 vs. 50000 cells/cm² and 5 mg/ml (*** = p<0.001).

Figure 7. Phase contrast images of normal human dermal fibroblasts (NHDF) depending on passage numbers and seeding cell density after 24 h treatment with ZnO-T and ZnCl₂.

A, D, G and J: untreated NHDF; B, E, H and K: 0.5 mg/ml ZnO-T; C, F, I and L: 30 µg/ml ZnCl₂. Passage numbers: P11–P15 (A–C and G–I) vs. P22–P26 (D–F and J–L). Seeded cell number: 25000 (A–F) vs. 100000 cells/cm² (G–L); time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Additionally experiments were done with three different frozen stocks of NHDF to verify the result.

Figure 8. Effect of passage number and seeding cell density of NHDF on ZnO-T and ZnCl₂ cytotoxicity.

A and D: cell protein concentrations of controls determined by the Lowry-assay. Passage numbers: P11–P15 vs. P22–P26. Seeded cell numbers: 25000 (A–C) vs. 100000 cells/cm² (D–F); ZnO-T concentration: 0.05–5 mg/ml; ZnCl₂ concentration: 25–45 µg/ml; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Additionally experiments were done with three different frozen stocks of NHDF to verify the result. Statistical significance determined by one way analysis of variance: cell density: 100000 cells/cm², ZnO-T: P11–P15 vs. P22–P26 1 mg/ml (** = p<0.01), 2 mg/ml (*** = p<0.001) and 5 mg/ml (*** = p<0.001). Cell density: 100000 cells/cm², ZnCl₂: P11–P15 vs. P22–P26 35 µg/ml (* = p<0.05).

Figure 9. Comparing the cytotoxic effect of ZnCl₂, spherical ZnO NP and ZnO-T on normal human dermal fibroblasts (NHDF) determined by the MTT-assay.

Zn content is calculated in [mM] according to the used probe concentration. Passage number: P6–P9. Seeded cell number: 50000 cells/cm²; time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Statistical significance determined by one way analysis of variance: EC₅₀ values are highly significant (p<0,001) different (Table 7). The typical SEM images of spherical ZnO NP (average diameter is ∼ 150 nm) and ZnO are shown as inset images which were used for investigation in present case.

Figure 10. Role of direct cell contact for ZnO-T toxicity shown by phase contrast microscopy and changes in cell viability (MTT-Assay).

Column diagram shows the cytotoxicological effects of different probes on normal human dermal fibroblasts (NHDF). CTRL (Image: A) = cell culture medium as control; CTRL (S) (Image: B) = supernatant of cell culture medium 24 h pre-incubated on NHDF as control; ZnO-T = ZnO-T in dispersion directly on NHDF (10 mg/ml or 20 mg/ml (Images: not shown due to overlapping of ZnO tetrapods layer)); ZnO-T (S) – disp. + cells = supernatant of ZnO-T in culture medium pre-incubated on NHDF for 24 h (10 mg/ml (Image: C) or 20 mg/ml (Image: D)); ZnO-T (S) – disp. = supernatant of ZnO-T in culture medium pre-incubated for 24 h without NHDF (10 mg/ml (Image: G) or 20 mg/ml (Image H); ZnO-T in Transwells = ZnO-T in dispersion (10 mg/ml (Image: E) or 20 mg/ml (Image: F) into transwells to prevent cell contact. Each value shown is the mean of three independent experiments (± SE). Passage number: P12–P14. Seeded cell number: 50000 cells/cm²; time prior treatment: 48 h; duration of treatment: 24 h. Each column represent the mean ± SE of n = 3 independent experiments. Statistical significance determined by one way analysis of variance: ZnO-T vs. C/D, G/H and E/F (* = p<0.05, ns = not significant).