Barium Titanate (BaTiO3) Nanoparticles Exert Cytotoxicity through Oxidative Stress in Human Lung Carcinoma (A549) Cells

Barium titanate (BaTiO3) nanoparticles (BT NPs) have shown exceptional characteristics such as high dielectric constant and suitable ferro-, piezo-, and pyro-electric properties. Thus, BT NPs have shown potential to be applied in various fields including electro-optical devices and biomedicine. However, very limited knowledge is available on the interaction of BT NPs with human cells. This work was planned to study the interaction of BT NPs with human lung carcinoma (A549) cells. Results showed that BT NPs decreased cell viability in a dose- and time-dependent manner. Depletion of mitochondrial membrane potential and induction of caspase-3 and -9 enzyme activity were also observed following BT NP exposure. BT NPs further induced oxidative stress indicated by induction of pro-oxidants (reactive oxygen species and hydrogen peroxide) and reduction of antioxidants (glutathione and several antioxidant enzymes). Moreover, BT NP-induced cytotoxicity and oxidative stress were effectively abrogated by N-acetyl-cysteine (an ROS scavenger), suggesting that BT NP-induced cytotoxicity was mediated through oxidative stress. Intriguingly, the underlying mechanism of cytotoxicity of BT NPs was similar to the mode of action of ZnO NPs. At the end, we found that BT NPs did not affect the non-cancerous human lung fibroblasts (IMR-90). Altogether, BT NPs selectively induced cytotoxicity in A549 cells via oxidative stress. This work warrants further research on selective cytotoxicity mechanisms of BT NPs in different types of cancer cells and their normal counterparts.


S1.1. Endpoint chromogenic limulus amebocyte lysate (LAL) assay
The endpoint chromogenic limulus amebocyte lysate (LAL) assay kit (Lonza, Basel, Switzerland) was used to examine endotoxin contamination in BT NPs. This assay has a sensitivity range of 0.1 EU/ml -1.0 EU/ml. Briefly, BT NPs at a concentration of 50 μg/ml was were mixed with the LAL supplied in the test kit and incubated at 37 °C for 10 min. A peptide substrate solution was then mixed with the LAL-sample mixture and incubated at 37 °C for next 6 min. The reaction was then stopped by addition of stop reagent supplied with the kit. In addition to the complete reaction mixture (i.e. BT NPs+LAL+substrate), two additional mixtures were prepared to check the possible interference of BT NPs in the assay, namely BT NPs+LAL and BT NPs+substrate. If endotoxin is present in the sample, a yellow color should develop only in the complete reaction mixture, not in other two mixtures. The absorbance of the enzymatically cleaved p-nitroaniline part of the substrate peptide was measured at 405 nm in a microplate reader (Synergy-HT, BioTek, Vinnoski, VT, USA). Since this absorbance is in direct proportion to the amount of endotoxin present, the concentration of endotoxin can be calculated from a standard curve using LPS.

S1.2. MTT assay
Cell viability was measured by MTT assay [1] with some specific changes [2]. This assay measures the mitochondrial function by determining the ability of living cells to reduce MTT into blue formazon product. Briefly, 20,000 cells/well seeded in a 96-well plate and allowed for 24 h to attach on the surface of the plate. Then, cells were treated to different concentrations of BT NPs (0-200 μg/ml) for different exposure times (24, 48, and 72 h). After the completion of exposure time, culture medium was removed from each well to avoid interference of BT NPs and replaced with new medium containing MTT solution in an amount equal to 10% of culture volume. The 96-well plate is now incubated for 3 h at 37 °C until a purple colour formazan product was appeared. The resulting formazan product was dissolved in acidified isopropanol. Further, 96-well plate was centrifuged at 2300×g for 5 min to settle down the BT NPs, if present in the solution. Then, a 100 μl supernatant was transferred to other fresh wells of 96-well plate and absorbance was measured at 570 nm using a microplate reader (Synergy-HT, BioTek, Vinnoski, VT, USA).

S1.3. NRU assay
Neutral red uptake (NRU) assay was performed following the procedure as described by Borenfreund and Puerner [3] with some specific changes [2]. Briefly, 20,000 cells/well were seeded in 96-well plates and allowed for 24 h to attach on the surface of the plate. Then, cells were treated to different concentrations of BT NPs (0-200 μg/ml) for 24, 48, and 72 h time intervals. After the completion of exposure time, culture medium was aspirated and cells were washed with phosphate buffer saline (PBS) before being incubated for 3 h in culture medium supplemented with neutral red (50 μg/ml). Then, medium was washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. Cells were further incubated for 20 min at 37 °C in a mixture of acetic acid (1%) and ethanol (50%) to extract the dye. The 96-well plates were then centrifuged at 2300×g for 5 min to settle the remaining NPs, if present in the solution. After this, 100 μl of supernatant was transferred to new wells of 96well plate and the absorbance was recorded at 540 nm using a microplate reader (Synergy-HT, BioTek).

S1.4. Mitochondrial membrane potential assay
Mitochondrial membrane potential (MMP) was quantified using rhodamine-plate and allowed for 24 h to attach on the surface. Then, cells were treated to different concentrations of BT NPs (25-100 μg/ml) for 24 h. ZnO NPs (25 μg/ml for 24 h) was used as a positive control. At the end of exposure, cells were harvested and washed twice with PBS. Cells were further exposed with 10 μg/ml of Rh-123 probe for 1 h at 37 °C in the dark. Cells were washed with PBS and fluorescent intensity of Rh-123 was measured at a microplate reader (Synergy-HT, BioTek). A parallel set of experiment in 24-well plate (1×10 5 cells/well) was also prepared to capture the fluorescent intensity of Rh-123 probe in control, BT NPs (50 μg/ml for 24 h) or ZnO NPs (25 μg/ml for 24 h) treated groups using a DMi8 fluorescent microscope (Leica Microsystems, GmbH, Wetzlar, Germany). Cell images were captured at 20× magnification.

S1.5. Reactive oxygen species generation assay
Intracellular ROS generation was assayed using 2,7-dichlorofluorescin diacetate (DCFH-DA) as reported in our previous work [4]. ROS level was estimated through two distinc methods; quantitative analysis and microscopic fluorescence imaging. For quantitative assay, 20,000 cells/well were seeded in a 96-well blackbottomed plates and allowed to adhere for 24 h in a CO2 incubator at 37 °C. Cells were further exposed to different concentrations of BT NPs (25-100 μg/ml) for 24 h. ZnO NPs (25 μg/ml for 24 h) was used as a positive control. In some set of experiments, cells were co-exposed with 2 mM of N-acetylcycteine (NAC) (Sigma-Aldrich, St.

S1.6. Preparation of cell extract
In brief, cells were cultured in 6-well plate (1×10 6 cells/well) and exposed to different concentrations of BT NPs (25-100 μg/ml) for 24 h. ZnO NPs (25 μg/ml for 24 h) was used as a positive control. In some set of experiments, cells were co-exposed

S1.7. Caspase-3 and caspase-9 enzymes assay
Caspase-3 and caspase-9 enzymes activity was measured using BioVision colorimetric kits (Milpitas, CA, USA). This assay is based on the principle that activated caspases in apoptotic cells cleave the synthetic substrates to release free chromophore p-nitroanilide (pNA), which was recorded at 405 nm. The pNA produced after specific action of caspase-3 and caspase-9 on tertrapeptide substrates were DEVD-pNA and LEHD-pNA, respectively. In brief, reaction mixture consisted of 50 μl of control and treated cell extract protein (50 μg), 50 μl of 2X reaction buffer (containing 10 mM dithiothreitol) and 5 μl of 4 mM DEVD-pNA (for caspase-3) or LEHD-pNA (for caspase-9) substrate in a total volume of 105 μl. The reaction mixture was incubated at 37 °C for1 h and absorbance of the product was recorded at 405 nm by a microplate reader (Synergy-HT, BioTek).

S1.8. Glutathione assay
The glutathione (GSH) level was quantified using Ellman's method [5]. Briefly, a mixture of 0.1 ml of cell extract and 0.9 ml of 5% TCA was centrifuged (2500×g) for 15 min at 4 °C. Then, 0.5 ml of the supernatant was added into 1.5 ml of 0.01% 5,5'dithiobis-(2-nitrobenzoic acid (DTNB) and the reaction was monitored at 412 nm. The amount of GSH was expressed in terms of nanomole/mg protein.

S1.9. Glutathione peroxidase enzyme assay
Activity of glutathione peroxidase (GPx) enzyme was measured using protocol of Rotruck and co-workers [6]. Briefly, a reaction mixture contained 20 μl of 0.1 M GSH, 10 μl of cell extract, 100 μl of 2 mM NADPH, 100 μl of 10 U/ml glutathione reductase, 800 μl of 0.2 M Tris-HCl, and 10 μl of 5 mM t-butylhydroperoxide. The oxidation rate of NADPH was monitored at 320 nm. S1.10. Glutathione reductase enzyme assay Glutathione reductase (GR) enzyme activity was measured using method of Carlberg and Mannervik [7]. The method is based on the oxidation of NADPH to NADP + catalysed by GR. Activity of GR was estimated by following reduction in absorbance at 340 nm due to oxidation of NADPH. The reaction mixture contained 25 mM phosphate buffer (pH 7.8), 0.5 mM oxidized glutathione, 0.12 mM NADPH and an aliquot of the cell extract. Correction was made for oxidation of NADPH in the absence of oxidized glutathione.