Anodically Grown Titania Nanotube Induced Cytotoxicity has Genotoxic Origins

Abstract

Nanoarchitectures of titania (TiO₂) have been widely investigated for a number of medical applications including implants and drug delivery. Although titania is extensively used in the food, drug and cosmetic industries, biocompatibility of nanoscale titania is still under careful scrutiny due to the conflicting reports on its interaction with cellular matter. For an accurate insight, we performed in vitro studies on the response of human dermal fibroblast cells toward pristine titania nanotubes fabricated by anodic oxidation. The nanotubes at low concentrations were seen to induce toxicity to the cells, whereas at higher concentrations the cell vitality remained on par with controls. Further investigations revealed an increase in the G₀ phase cell population depicting that majority of cells were in the resting rather than active phase. Though the mitochondrial set-up did not exhibit any signs of stress, significantly enhanced reactive oxygen species production in the nuclear compartment was noted. The TiO₂ nanotubes were believed to have gained access to the nuclear machinery and caused increased stress leading to genotoxicity. This interesting property of the nanotubes could be utilized to kill cancer cells, especially if the nanotubes are functionalized for a specific target, thus eliminating the need for any chemotherapeutic agents.

Figure 1. SEM images of TiO₂ nanotube array films separated from substrates.

(a) Top and (b) lateral views of the nanotubes fabricated using the EG/NH₄F electrolyte. (c) and (d) show the images of the corresponding regions in films grown in the water/HF electrolyte.

Figure 2. TEM micrographs of TiO₂ nanotubes.

(a) Low magnification and (b) high magnification images of ethylene glycol (organic) electrolyte fabricated TiO₂ nanotubes. (c) Low magnification and (d) high magnification images of HF/H₂O (aqueous) electrolyte fabricated TiO₂ nanotubes.

Figure 3. Cellular toxicity analysis for the effects of TiO₂ nanotubes on human dermal fibroblasts.

The alamar blue assisted assay revealed a dose independent viability chart, with viability decreasing with decrease in concentration (a). Though an overall maximum viability remained at approximately 60%. (Columns, means of three independent experiments; bars ± SD). The live dead analysis (b–g) revealed a generally live cell population in the NTs treated group (e–g) which could be correlated to the residual esterase even in the cells undergoing apoptosis (f) (scale bar represents 500 μm). In the cell cycle analysis a predominant synthetic and mitotic phase was observed with the control cells (h), however in the NTs group a prominent resting phase was observed (i). The LDH release was found to be insignificant in treatment group when compared to control (j); The TCP expression were found to be unaffected (k) whereas the number of proliferating cells in the treatment group dropped by half when compared to control (l).

Figure 4. Cytoskeletal integrity analysis of the control and NTs treated group were performed with emphasis on actin, micotubulins and vinculin.

With respect to the actin filaments, a slight reduction in the network of the fibres was observed with the NT group (d) when compared with the control (b). In the case of microtubulins the control (f) and test groups (h) exhibited comparable tubular network. The vinculin expression was found to be undisturbed in the treatment group (l) when compared to control (j). (Scale bars represent 10 μm). Total FAK proteins were quantified with ELISA and their expression in control and treatment groups remained comparable (m). Overall, the cytoskeletal components, which form the basic backbone of a cell and are responsible for their structural integrity and cell-cell/cell-matrix interactions, remained unaffected by the TiO₂ NTs, with the exception of actin where a slight rearrangement of the fibers was observed.

Figure 5. Mitochondrial integrity and condition were assayed with the ROS generation and transition pore formation tests.

It was clearly evident that the NTs induced ROS production, especially nuclear ROS (d). The nuclear ROS in the treatment group was found to be increased by 7 fold than the control (e). The mitochondrial pore induction investigation revealed the absence of any distorted signals from the mitochondrial region (l) with discrete calcein signaling (k), indicative of a properly functioning mitochondrial system. (Scale bars represent 10 μm). It is evident from this assay that though the mitochondrial apparatus seems to be healthy and functioning properly, nuclear ROS is significantly enhanced on TiO₂ NTs exposure, which may be the precursor for numerous distortions in nuclear functioning.

Figure 6. Chromatin condensation and DNA break studies were carried out to assess the condition of the nuclear set up of the control and NT treated cells.

Prominent chromatin condensation and plasma membrane permeability (c,d) was recorded with the NTs treated group. (Scale bars represent 10 μm). The NF-κB expression was elevated in the treatment group when compared to control (e). Comet analysis confirmed significant increase in number of comet tails in treatment group (f). Consistent DNA breaks could also be observed with the H2AX test in the NTs group (k) when compared to the controls (h). (Scale bars represent 500 μm). This nuclear condensation and DNA alteration study is correlative of the ROS induction previously observed.