Neuron Compatibility and Antioxidant Activity of Barium Titanate and Lithium Niobate Nanoparticles

Abstract

The biocompatibility and the antioxidant activity of barium titanate (BaTiO₃) and lithium niobate (LiNbO₃) were investigated on a neuronal cell line, the PC12, to explore the possibility of using piezoelectric nanoparticles in the treatment of inner ear diseases, avoiding damage to neurons, the most delicate and sensitive human cells. The cytocompatibility of the compounds was verified by analysing cell viability, cell morphology, apoptotic markers, oxidative stress and neurite outgrowth. The results showed that BaTiO₃ and LiNbO₃ nanoparticles do not affect the viability, morphological features, cytochrome c distribution and production of reactive oxygen species (ROS) by PC12 cells, and stimulate neurite branching. These data suggest the biocompatibility of BaTiO₃ and LiNbO₃ nanoparticles, and that they could be suitable candidates to improve the efficiency of new implantable hearing devices without damaging the neuronal cells.

Keywords: PC12; ROS; apoptosis; neuronal cell line; oxidative stress; piezoelectric material.

Figure 1

Results of MTS assay of differentiated PC12 cells treated with three different concentrations of barium titanate (BaTiO₃) (a) or lithium niobate (LiNbO₃) (b) for 24 h, 48 h and 72 h. Cell viability was expressed as mean value percent ± SEM vs. control cells (NT). Cisplatin (Cpt) 13 µM and 25 µM were used as positive control. Asterisks indicate significant differences in comparison to negative control (NT). * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 2

Morphological analysis of differentiated PC12 cells treated with BaTiO₃ (d–l) or LiNbO₃ (m–u) for 24 h, 48 h and 72 h, respectively. Untreated cells were used as control (a–c) and 13 µM cisplatin was used as positive control (v–x). The nuclei were stained in blue by DAPI, the cytoskeleton was stained in red by phalloidin-TRITC. Scale bar 100μm. Arrows indicate apoptotic cells.

Figure 3

Immunocytochemical analysis of differentiated PC12 cells treated with BaTiO₃ (d–l) or LiNbO₃ (m–u) for 24 h, 48 h and 72 h. Untreated cells were used as control (a–c) and 13 µM cisplatin was used as positive control (v–x). Cytochrome c was stained in red by TRITC-conjugated secondary antibody; the nuclei were stained in blue by DAPI. Healthy cells show homogeneous cytochrome c staining while apoptotic cells show diffuse staining distribution. Scale bars 100 μm. Arrows indicate apoptotic cells.

Figure 4

Results of H₂DCFDA assay of differentiated PC12 treated with BaTiO₃ (a) or LiNbO₃ (b) for 24 h, 48 h and 72 h. Samples treated with 25 µM cisplatin (Cpt) for 24 h were used as positive control. The results are expressed as mean value percent ± SEM vs. control cells (NT). Asterisks indicate significant differences in comparison to NT. * = p < 0.05, *** = p < 0.001.

Figure 5

Evaluation of neurite network produced by differentiated PC12 cells treated with BaTiO₃ or LiNbO₃ for 24 h, 48 h, and 72 h. Cells treated with 13 µM cisplatin were used as positive control (Cpt). After incubation, 30 fields per treatment were acquired at magnification 10X and analysed measuring the differentiation grade (a), the number of neurites per cell (neurite number, (b)), the average length of neurites expressed in µm (neurite length, (c) and the number of branch points (d)). Data were expressed as mean value percent ± SEM vs. control cells (NT). Asterisks indicate significant differences in comparison to NT. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.