Cytotoxicity and apoptosis induction of zinc ferrite nanoparticle through the oxidative stress pathway in human breast cancer cells

2.1 Experimental

The synthesis of zinc ferrite was performed with the use of zinc nitrate hexahydrate, iron nitrate nonahydrate and sodium hydroxide. The chemicals related to the formation of nanoparticles were purchased from Aldrich chemical corporation, U.S.A and used as received. In an experiment, the zinc nitrate hexahydrate (0.1 M), and iron nitrate nonahydrate (0.3 M) were mixed in double distilled water in a 250 mL capacity beaker, under constant stirring. Once the solution was dissolute completely, sodium hydroxide (0.03 M) was added to this mixture dropwise and stirred completely. The pH of the prepared mixture was noted which reached to 12.75 and then after transferred to the relaxed it for 6 hour (h) at 90^οC. The obtained precipitates were filtered and washed with distilled water/ethanol. The product dried at room temperature for 48 h and keep for further analysis.

2.2 Characterizations of the prepared product

The XRD (Cu_Kα radiation (λ = 1.54178 Å) with rotation angle ranging from 10° to 80° with 6°/min scanning speed, was used for the analysis of the prepared powder. Morphology of the prepared powder was analysed with using FESEM as described earlier (Ahmad et al., 2021). Also the FTIR spectroscopy was analysed in the range of 400–4000 cm⁻¹. The thermal stability test in was performed with terms of TGA instrument (Mettler Toledo AG, Analytical CH-8603, Schwerzenbach, Switzerland) was also conducted for the analysis of prepared product. For this experiment ∼ 6.8 mg of powder was loaded into alumina crucibles (Al₂O₃) and heated till to 800 °C with a heating ramp of 20 °C/min under nitrogen gas with a flow of 20 mL/min.

2.3 Reagents and consumables for cell culture and cancer cells study

Human breast cancer (MCF-7) cells (ATCC, US) were maintained in DMEM culture medium supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin as an antibiotic, and 100 lg/mL streptomycin at 37 °C in a humidified 5 % CO₂ incubator.

2.4 Cell viability assays (MTT and NRU)

The cell viability was determined MTT assay and carried out as according to (Mosmann, 1983) with some minor modifications. Cells were seeded in 96-well plates (∼1 × 10⁴ cells/ well, 200 μL/well). Cells were incubator at 37 °C with humid environment to grow the cells until 70–80 % confluency. After incubation of 24 h culture medium was removed and washed three to four times with PBS. Thereafter, the MTT solution (final MTT concentration 10 μg/mL) was added and incubated the cells at 37 °C for 2 h. After incubation, the MTT solution was removed without disturbing the cells and DMSO (200 μL/well) was added, the plates were shake gently for the mixing of formazan crystals. The plate absorbance was observed with microplate reader (Multiskan Ex, Thermo Scientific, China) at 550 nm. The (%) cells viability was calculated as described below formula. $$\%viability=[(totalcells-viablecells)/totalcells]\times 100$$

Another indicator of cytotoxity treated cells were also quantitated by NRU assay as described method (Borenfreund and Puerner, 1985). For the NRU assay, cells were seeded in 96-well plates and allowed to grow overnight at 37 °C in a CO₂ incubator (5 % CO₂ humidity). Once the incubation was completed, the media was discarded and fresh medium was added. The cells were allowed to grow further for 48 h at 37 °C in a CO₂ incubator. After 48 h, the cell culture medium was discarded and washed with PBS. The neutral red (NR) dye (50 μg/mL) freshly prepared in culture medium was added in all wells and incubated for 3 h at 37 °C in a CO₂ incubator. Then, NR dye was discarded, and cells were washed again with PBS to completely remove the dye. Finally, the absorbance of red colour developed in the wells was measured at 550 nm on a microplate reader.

2.5 Intracellular ROS detection

The ROS was measured with using 2, 7-dichloro di hydrofluoresce in diacetate (DCFH-DA, Sigma Aldrich, USA) dye, which is utilized as a fluorescent agent and followed the protocol as described earlier (Wang and Joseph, 1999). The DCFH-DA enters the cell and reacts with ROS to form fluorescent compound dichlorofluorescein (DCF). The cells were exposed with ZnFe₂O₄NPs for 24 h, and washed well with PBS then incubated for 30 min in DCFH-DA (20 μM) in dark medium at 37 °C. It was analysed with fluorescence microscope.

2.6 Mitochondrial membrane potentials (MMP)

The MMP were determined by JC-1 (5, 50, 6, 60-Tetrachloro-1, 10, 3, 30-tetraethyl benzimi- dazolylcarbocyanine iodide) (Smiley et al., 1991). In normal cells JC-1 forms complexes known as J-aggregates with intense fluorescence. On the other hand, in apoptotic cells with lower membrane potential, JC-1 remains in the monomeric. The cells were seeded at appropriate densities in plate reader. After the treatment, media was aspirated and labelled with 5 lM JC-1 in HEPES buffered HBSS for 20 min. Fluorescence of JC-1 aggregate (red in color) and JC-1 monomer (green in colour) in cells were taken by a fluorescence microscope using green filter cube.

2.7 Glutathione level

For Glutathione (GSH) level measurement, MCF-7 cells was cultured in flasks. The cells were exposed to 50, 100, and 200 μg/mL of ZnFe₂O₄NPs for 24 h. Subsequently, the cells were lysed in cell lysis buffer [1 × 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2.5 mM sodium pyrophos- phate, 1 mM Na₂EDTA, and 1 % Triton]. Centrifuge 15,000 × g for 10 min at 4 °C, then supernatant, obtained was kept on ice until following (Eiiman et al., 1959). Briefly, a mixture of 0.1 mL of cell extract (supernatant) and 0.9 mL of 5 % TCA was centrifuged (2300 × g for 15 min at 4 °C). Thereafter, 0.5 mL of supernatant was added to 1.5 mL of 0.01 % 5, 5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and measured spectrophometrically at 412 nm.

2.8 Lipid peroxidation (LPO)

Lipid peroxidation (LPO) was assayed by the following of (Ohkawa et al., 1979). MCF-7 cells were exposed to 50, 100, and 200 μg/mL of Zn-Fe NPs for 24 h. Briefly, 0.1 mL of cell extract and 1.9 mL of 0.1 M sodium phosphate buffer (pH 7.4) were incubated at 37 °C for 1 h. After incubation, cells were precipitated with 5 % TCA and then centrifuged to 2300 × g for 15 min, to collect the supernatant. After incubation, 1.0 mL of 1 % TBA was added to the supernatant and put in hot water for 15 min. After cooling, the absorbance was taken at 532 nm.

2.9 Real-time PCR (RT-PCR)

Total RNA was purified from MCF-7 cells from untreated and treated with ZnFe₂O₄NPs for 24 h using the RNeasy mini Kit (Qiagen). RNA concentration were determined by Nanodrop 8000 spectro photometer (Thermo Scientific, USA). The cDNA was prepared by the MLV reverse transcriptase (GE Health Care, UK). For the quantification of apoptotic and anti-apoptotic gene was determined by using Roche^@LightCycler^@480 (96-well block), following the cycling program. Reaction mixture of 20 µL taking 75 ng of the cDNA and 5 µM of each primer with 2 × of SYBR Green I dye Roche Diagnostics. The cycling conditions at 95 °C for 10 min denaturation step, followed by 40 cycles of 95 °C for 15 s denaturation, 60 °C for 20 s annealing, and 72 °C for 20 s elongation steps (Ahmad et al., 2023). Data were normalized by GAPDH, a house keeping control gene.

2.10 Data analysis

The statistical analysis data. Statistical significance was measured at p < 0.05. The experiments were carried out in triplicates manner. The images were captured at constant exposure time.

3.1 Morphological results (XRD, FESEM and TEM results

The crystallographic structure of ZnFe₂O₄NPs was recognised of powder XRD assessment. The powder XRD patterns is presented in Fig. 1. The peak positions are 29.98 〈2 2 0〉, 35.28 〈3 1 1〉, 36.92 〈2 2 2〉, 42.84 〈4 0 0〉, 53.2 〈4 2 2〉, 56.64 〈5 1 1〉 and 62.22 〈4 4 0〉 All appears diffraction peaks were indexed to the face cantered regular spinel cubic structure (JCPDS card No. 89–1009) (Rameshbabu et al., 2014). No other impurity peaks were detected, which endorse the purity of the NPs. The strong sharp peaks suggested that ZnFe₂O₄NPs were well crystallized. The average crystallite size of ZnFe₂O₄NPs was estimated using Scherrer’s equation. The calculated average crystallite size was ∼ 27 ± 1 nm.

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Fig. 1

X-ray diffraction pattern of prepared zinc ferrite nanoparticles (ZnFe₂O₄NPs).

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Morphology of ZnFe₂O₄NPs was further examined by FESEM and TEM. Fig. 2A and B display the typical FESEM and TEM micrographs of ZnFe₂O₄NPs, respectively. The micro graph indicate that ZnFe₂O₄NPs have spherical morphology with average particle size of around 27 ± 1 nm.

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Fig. 2

(A) and (B) shows the FESEM and TEM image of the prepared zinc ferrite nanoparticles (ZnFe₂O₄NPs) respectively.

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3.2 Chemical functional analysis (FTIR results)

The functional groups in the prepared powder was examined by the FTIR spectroscopy and represent as Fig. 3A. The peak at 570 cm⁻¹ is related to ZnFe₂O₄ whereas the bands at 3200–3600 cm⁻¹ correspond to H-O–H mode of vibration. The small asymmetric stretching mode of vibration of O–H was observed between 1627 cm⁻¹. The symmetric and asymmetric stretching vibrational peak occurs between 1383 cm⁻¹ which designates the vibration of NO₃^-1 ions (Dina et al., 2020, Kumar et al., 2019).

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Fig. 3

(A) FTIR spectroscopy of zinc ferrite nanoparticles (ZnFe₂O₄NPs), (B) Thermogravimetric analysis (TGA) of zinc ferrite nanoparticles (ZnFe₂O₄NPs).

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3.3 Thermogravimetric analysis (TGA results)

Thermogravimetric analysis (TGA) provides the information related to the % mass loss with increasing the temperature. Fig. 3B. shows two-step weight loss for the prepared sample, the first one is related to the used solvent elimination whereas other one is the stabilization phase respectively. From the obtained data reveals that the initial step phase starts at 311 °C and completed at 430 °C with a weight loss of 1.60 %. The secondary weight loss starts at 530 °C, which is also knows as chemical decomposition phase, completed at 900 °C with a total weight loss was 3.10 %. The secondary phase shows the stabilization of the prepared material and from with this information it shows that a very minute quantity was loosed. From the experiment it reveals that the prepared material is highly stable at high temperature and are in consistent with other studies (XRD and FTIR).

3.4 Microscopic results of cultured and treated cells

The morphology of the cultured cells and treated cells (ZnFe₂O₄NPs) were examined via microscopic study at 24 h incubation periods in a range of different concentrations of ZnFe₂O₄ NPs (50, 100 and 200 µg/mL) (Fig. 4). The initially grown cells was used as a control (Fig. 4A) to compare the treated cells. The image shows that the long and oval shaped structure with nucleated positions initially but once the incubation time increases, the cells size were further increased. Fig. 4B, At initial treated sample at low concentration (50 µg/mL) of cells sizes were decreased and higher concentration (100 to 200 µg/mL) of ZnFe₂O₄NPs, the growth of cells was much affected, which are very clear from the obtained images (Fig. 4C and D).

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Fig. 4

ZnFe₂O₄NPs-induced morphological changes in MCF-7 cells. Cells were exposed to ZnFe₂O₄NPs at various concentrations for 24 h. Images were taken using phase contrast inverted microscope at 20x magnifications.

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3.5 MTT assay

The toxicity in cells was very widely observed via MTT assay as detailed described above with the incorporation of ZnFe₂O₄NPs. The obtained data reveals that the viability of cancer cells were diminished with the incorporation of ZnFe₂O₄NPs and these data’s were concentration/ dose-dependent. The cancer cells viability was decreases at 24 h 100 %, 101 %, 100 %, 93 %, 86 %, 67 % and 55 % (Fig. 5) for the concentrations of 2, 5, 10, 25, 50, 100 and 200 μg/mL respectively (p < 0.05 for each). The MTT calculation shows that at initial concentrations (2, 5, 10 μg/mL) of material doesn’t shows any change in the cells viability, whereas at higher concentration of ZnFe₂O₄NPs shows a sequential change in the viability of cells and cytotoxicity was much influenced.

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Fig. 5

Cytotoxic assessment of ZnFe₂O₄NPs in MCF-7 cells by MTT assay. Cells were exposed to various concentrations of ZnFe₂O₄NPs for 24 h.

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3.6 NRU assay

The cytotoxicity was further examined via NRU assay treated with ZnFe₂O₄NPs in MCF-7 cells. The assay was calculated as described in the material and method with using control and treated cancer cells, exposed in a range of different concentrations from 2 to 200 µg/ml for 24 h incubation. The experiment shows that viability of cells were affected with the incorporation of ZnFe₂O₄NPs and affected the cells growth. The NRU assay was decreases at 24 h 107 %, 107 %, 102 %, 97 %, 88 %, 71 % and 53 % (Fig. 6) for the concentrations of 2, 5, 10, 25, 50, 100 and 200 μg/mL correspondingly (p < 0.05 for each). As MTT assay, the NRU data’s also in consistent and express that the cells viability initially not affected much whereas as the concentrations of ZnFe₂O₄NPs exceeded the cytotoxicity was much influenced.

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Fig. 6

Cytotoxic assessment of ZnFe₂O₄NPs in MCF-7 cells by NRU assay. Cells were exposed to various concentrations of ZnFe₂O₄NPs for 24 h.

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3.7 ROS generation in breast cancer cells (MCF-7) with ZnFe₂O₄NPs

A sequential change in ROS data was observed in MCF-7 cells after the exposure of ZnFe₂O₄NPs at 50, 100 and 200 μg/mL concentrations for 24 h (Fig. 7) with control. From the observed ROS with captured image and presented as Fig. 7A) and bar graph (Fig. 7B) shows that the ROS is increases with the increase of ZnFe₂O₄NPs concentrations as compared to control cells. The ROS were increase at 50 μg/mL, 100 μg/mL and 200 μg/mL were 120, 141, 171 % as compared to control (100 %) (Fig. 7B).

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Fig. 7

ROS generation induced by ZnFe₂O₄NPs in MCF-7 cells. (A) Fluorescence images showing intensity of DCF-DA dye after the ZnFe₂O₄NPs exposure at 0, 50, 100, and 200 μg/ml for 24 h. (B) The graph shows the percent ROS generation in MCF-7 cells. Data are presented as the mean ± SD of three different experiments.

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3.8 Mitochondrial membrane potential (MMP) with ZnFe₂O₄NPs

The MMP induction was observed with JC-1 red, JC-1 green and channel merged imaging and of monomer/aggregate both IC₅₀ and control MCF-7 cells treated with ZnFe₂O₄NPs (Fig. 8A). The level of MMP with control was determined at different concentration of ZnFe₂O₄NPs in MCF-7 cells exposed to 24 h at 25, 50, 100 and 200 µg/ml are 89 %, 75 %, 49 % and 43 % as shown in Fig. 8B. The decrease in MMP level was recorded in terms of fluorescence intensity of mitochondrial-specific dye JC-1. It is clearly presented that ZnFe₂O₄NPs decreased the fluorescence intensity.

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Fig. 8

MMP was detected by JC-1 in control and treated cells. Images taken in tandem for JC-1 aggregate (red) and JC-1 monomer (green) and merged Fig. 8 A. MMP level of percent control was determined at different concentration of ZnFe₂O₄NPs. Fig. 8 B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.9 Glutathione level

Depletion in GSH level in cultured MCF-7 cells exposed to 25, 50, 100, 200 µg/mL concentrations of ZnFe₂O₄NPs for 24 h and summarized in Fig. 9A. The result indicates that decreased the GSH levels in a dose-dependent manner. Fig. 9A shows a significant decrease in the GSH activity at higher concentrations as compared to the untreated control. A concentrationdependent significant decrease of 90 %, 82 %, 73 % and 62 % were observed in GSH activity at 25, 50, 100, and 200 µg/mL of ZnFe₂O₄NPs, respectively (Fig. 9A).

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Fig. 9

ZnFe₂O₄NPs -induced oxidative stress in MCF-7 cells exposed for 24 h. A Glutathione depletion, B Lipid peroxidation, Values are mean ± SE of three independent experiments.

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3.10 Lipid peroxidation (LPO)

The ZnFe₂O₄NPs-induced LPO in MCF-7 cells are summarized in Fig. 9B. A concentration-dependent significant increase in LPO was observed. An increase of 112, 124, 135 and 153 % at 25, 50, 100, and 200 µg/mL respectively were found in MCF-7 cells exposed for 24 h with control.

3.11 Gene expression

The quantitative RT-PCR study was performed to check the mRNA level in MCF-7 cancer cells treated with ZnFe₂O₄NPs for 24 h at a concentration of 100 μg/ml. A number of marker genes were selected such as genes (p53, bax, casp3 and bcl2) of the evaluation of mRNA level in cancer cells. The data shows a change with marker genes and upregulation of apoptotic genes in MCF-7 cells (p < 0.05 for each gene). The mRNA levels of tumour suppresser of p53 gene (fold change-2.6), the pro-apoptotic gene bax (fold change-1.6) and casp-3 (fold change-3.8) were upregulated with ZnFe₂O₄NPs, whereas expression of bcl-2 (fold change-0.7), which is an anti-apoptotic gene, was down regulated in cells treated with ZnFe₂O₄NPs (Fig. 10).

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Fig. 10

Quantification of ZnFe₂O₄NPs induced mRNA fold change, apoptotic genes (bax, p53, and casp3) and anti-apoptotic gene (bcl-2) was calculated. MCF-7 cells induced 200 µg/mL of ZnFe₂O₄NPs for 24 h. For the internal control GAPDH was used. The values are mean ± SE of three independent experiments.

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