Cytotoxic and molecular assessment with copper and iron nanocomposite, act as a soft eradicator against cancer cells

2.1 Experimental

2.2 Characterizations of the prepared powder material (CuFeO₂NCs)

The crystalline character, phases, size of the prepared powder were examined via X-ray diffraction pattern (XRD, PANalytical XPert Pro, Colorado, U.S.A.) with Cu_Kα radiation (λ = 1.54178 Å) in range from 20 to 80° with 6°/min scanning speed. The morphological observation was confirmed via SEM (JEOL, JED-2200 series, Tokyo, Japan) at room temperature. To analyse the processed powder morphology, sprinkled homogeneously on a carbon tape and pasted on sample holder. The sample holder was moved to sputtering chamber, where conducting layer of platinum (Pt) was coated for ∼3 s. The sample was fixed in a sample holder and analyzed the morphology at room temperature. Further the morphological investigation was again analyzed via TEM (JEOL, JSM, 2010, Tokyo, Japan). The functional chemical bonding in terms of asymmetric and symmetric stretching mode of vibrations between the metal and oxygen were confirmed via Fourier transform infrared (FTIR; Perkin Elmer-FTIR Spectrum-100, Shelton, C.T, U.S.A.) spectroscopy analyzed within the range of 4000–400 cm⁻¹.

2.3 Cells culture

The cells (C2C12) were purchased from American Type Culture and collection (ATCC, U.S.A). The procured cells vial were thawed at 37 °C for 2–3 min and then after transferred it to a 75 mm² flask and cultured with cell culture medium (Dulbecco’s Modified Eagle’s Medium, DMEM) containing 10–12% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin) in a humid environment incubated at 37 °C with 5% CO₂. The medium was refilled at an alternate day and were subcultured after reached 85–90% confluence.

2.4 MTT assay

The % cells viability was examined via using cell proliferation kit I (MTT, ROCHE, Ltd, U.S.A)) and utilized as per instructions. The cells were initially cultured in a 96-wells plates with concentration range ∼1 × 10⁴ and nurtured the plate for 24 h at 37 °C in an incubator with 5% CO₂. When the cells were reached to their optimal confluence, the different concentrations of NCs (0.5 μg/mL, 1 μg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL) were mixed it and to keep the solution in an incubator for 24 and 48 h. For the viability test of the cells with NCs, the MTT assay ((3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide, 10 µL/well)) was mixed with cell culture solution. Including the control solution, the MTT solution was added with NCs samples and again incubated the culture solution at 37 °C for 4 h. When the incubation period was accomplished the sample was moved from the incubator and to this sample a solubilizing buffer (100 µL/well) was mixed. In the culture flask, a purple color solution was appeared and it fainted over pipetting. Due to MTT assay as formazan precipitate was formed and it dissolved with DMSO in both control and treated samples. The % viability was tested with MTT assay through Elisa Reader (Bio Rad, 570 nm). The absorbance of purple color solution was measured through spectrophotometer at usually ∼570 nm wave length. The percentage (%) viability was calculated as follows:

% Viability = OD (optical densities) in sample well/OD in control well × 100

2.5 qPCR study

The RNA was extracted from the cultured cells with at low and high concentration of the treated material with selective protocol via Trizol as per according to the manufacturer’s protocol. The purity of RNA was examined via the optical density between 260 and 280 nm. The cDNA was formed with using M-MLV reverse transcriptase as acknowledged in the literature (Wahab et al., 2016). The intensities of genes bands were measured from quantity one program (Bio-Rad, USA) through gels electrophoresis.

2.6 Statistical analysis

The statistical assessments were expressed and authenticated the student T-test, mean ± SD and standard pool at significant value (P < 0.05).

3.1 X-ray diffraction (XRD) results

XRD provide the information related to the processed powder crystallite size, phases and crystalline of the material. In this experiment, the analysis was performed in range of 20-80° with 6°/min scanning speed with 40 kV and 30 mA current. The obtained spectroscopic data reveals that all peaks and their corresponding positions are very clear such as (〈0 0 6〉 (30.13), 〈0 1 2〉 (35.33), 〈0 1 5〉 (43.21), 〈0 1 8〉 (57.18) and 〈1 1 0〉 (62.73) are with powder diffraction data analogous with Joint Committee on Powder Diffraction Standards (JCPDS) with card number 039-0246. The acquired crystal geometry of rhombohedral space group R₃m with lattice constant lattice constant were a_h = 53.03 Å and c_h = 517.09 Å respectively (Fig. 1) (Galakhov et al., 1997). In all the defined peaks show that the material is highly crystalline with defined facts. The diffraction pattern also shows that there is no other peaks related to impurity was formed except the processed material NCs, which again validates that the material is pure oxide nanomaterials. Average crystallite size and other parameters such as phases, peaks positions, FWHM of the processed powder was calculated to ∼44 ± 1 nm through well-known Scherer equation (Cullity, 1978).

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Fig. 1

XRD pattern of NCs via solution process.

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3.2 Morphological evaluation of the processed powder

The powder morphology was examined through SEM and obtained data is presented as Fig. 2. The low (Fig. 2a) and high (Fig. 2b) magnification scales shows that in a larger amount of NCsNPs are seen in a wide captured areas. Several particles are seen in the images, some are combined with other one and some are seems to be separate and single. The powder was also checked at high magnification scale and found that the entire surface was full of spherical shaped nanostructures, with combined to other nanospheres (Fig. 2c & d). The grown crystallite surfaces exhibit a defined shape with full array bunches of nanostructures with an organized in manner.

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Fig. 2

SEM images of NCs: at low magnification (a) and (b) high magnification.

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Further for more detailed observation related to the structural assessment of the prepared powder, sample was again checked via TEM and the obtained result is presented as Fig. 3. From the obtained images it illustrates that NCs are in a grouped with other particles (Fig. 3) with spherical in shape. The average diameter of each nanoparticle is in the range of 45–50 nm in size (Fig. 3). The data’s are in full consistent with SEM and XRD observations (Figs. 1 & 2).

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Fig. 3

Displayed the low magnification TEM images of NCs.

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3.3 FTIR spectroscopy results of NCs

Fig.4. Shows the FITR results of the prepared powder, which shows a wide and shallow band observed in the range of 3200–3600 cm⁻¹ designates the hydroxyl (H–O–H, 3340 cm⁻¹) group vibration mode (Barnabé et al., 2013). The small peak at 2341 cm⁻¹ designates the atmospheric CO₂ whereas the sharp peak observed at 1641 cm⁻¹ shows the vibrational mode of hydroxyl group (–OH) molecule. The vibrations of nitro group (NO₃^2–), was observed at 1053 cm⁻¹ respectively. The oxide peaks was observed at 602 cm⁻¹ and it demonstrates the formation of nanocomposite (CuFeO₂). From the obtained data and their peaks observations reveals that the –OH group changes to the oxides and shows the formation of NCs (Fig. 4) (Schmachtenberg et al., 2019).

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Fig. 4

Shows the FTIR spectra of grown NCs.

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3.4 Morphology of cultured cells visualized via microscopy

The cells were cultured in a culture medium DMEM and incubated at defined time intervals 24 (Panel A) and 48 h (Panel B). When the cells were incubated and reached to their optimal confluence, the cells morphologies were captured as treated (with NCs) and control (without NCs) cells and presented as Fig. 5. The cells were exposed with different concentration of NCs (0.5 μg/mL, 1 μg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, and 100 µg/mL) at different incubations (24 and 48 h) period. Initially, single cells were seen at many places were as it’s nucleated upon their confluence. The nucleated/confluence cells were exposed with NCs at an above mentioned condition. From the recovered images, it’s very clear that as the doses of NCs increases with the cells, the cancer cells growth were much affected and high density of cells were reduced (Fig. 5) (Wahab et al., 2016).

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Fig. 5

The microscopic images of cultured cells and their interaction with NCs at different concentrations, where Panel A at 24 h and Panel B at 48 h incubations respectively.

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3.5 MTT assay results with nanostructures

From the MTT assay cells proliferation rate was to measure under the manner of treated and untreated (control). The data received from the MTT assay shows that the cell death or inhibitory effects dependent on dose dependent manner against cancer cells. The cultured cells were incubated with NCs with different concentrations (0.5 μg/mL, 1 μg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL). With this, a series of different concentration also included control to compare the growth rate of cancer cells continuously. The cells were washed to remove the medium and % cell death was determined through MTT method (Wahab et al., 2016). A significant change was observed at low concentration of the material but as the doses were increased the cells viability was much influenced. The cells viability at different concentrations at 0.5 μg/mL, 1 μg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL of, NCs are 101%, 100%, 93%, 88%, 78%, 64%, 51% and 35% of the cells, respectively. The cells demise were varied at 48 h for the concentrations of 0.5 μg/mL, 1 μg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL of, NCs are 90%, 89%, 84%, 75%, 66%, 49% and 37%, 21%% of the cells, respectively (Fig. 6). The interaction of NCs with cancer cells and their % reduction express that NCs are much effective and can be possible to utilize as anticancer agents (Wahab et al., 2018).

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Fig. 6

MTT assay at different time (24 & 48 h) intervals with control solution. *p < 0.01, **p < 0.001 Vs Control. The data represent as of three identical experiments.

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3.6 qPCR study induced by NCs

RNA expression was also analyzed with qPCR with NCs in presence of caspase gens (3/7) with cancer cells at low and high concentration of the material and NCs (25 μg/mL) and high (50 μg/mL) at 24 and 48 h incubation time respectively. The obtained data reveals that in 24 and 48 h of incubation caspase 3 with NCs were express 2.52 and 3.75 fold changes respectively at low concentration of NCs (25 μg/mL). The data reveals that both the genes were up-regulated. At higher concentration of NCs (50 μg/mL), a sequential changed has been observed and it was examined from 3.64 to 5.85 fold change in 24 & 48 h incubation period respectively. The recovered data displayed that the employed NCs in causes the up-regulation expression and indicates the apoptosis in cells with caspase 3 genes (Fig. 7). The previous studies (Wahab et al., 2016; 2018) discloses that activities of caspases are responsible and narrates to affect the apoptotic cells, which are present in naturally growing cells. It also examined that the enhance fold change in caspases are liable in cancer cells, when treated with NCs and compared with the control gene (GAPDH). As per previous published literature (Wahab et al., 2016; 2018), that oxide based nanostructures play a deadly substance for the growth of cancer cells. The cells apoptosis are influenced directly or indirectly with various parameters such as higher doses of used nanostructured materials, culture medium concentrations, high rate of incubation and others, increases the apoptosis in cells and resulted an up-regulation in mRNA expression. The similar results were also found with caspase 7, once reacted with NCs at 24 & 48 h incubations. The cells were incubated at 24 h and at low concentration of NCs (25 μg/mL), the caspase 7 expressed 2.83 fold changes whereas at higher doses of NCs (50 μg/mL), the rate of apoptosis increased 4.48 fold change (Fig. 8) (Wahab et al., 2016; 2018). At low concentration of NCs (25 μg/mL), caspase 7 expressed to 3.59 whereas at higher concentration of NCs (50 μg/mL) its increased 7.28 fold change with 48 h incubation period.

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Fig. 7

Effect of NCs on mRNA expression level of C2C12 cells for caspase 3 at alternate incubation periods (24 & 48 h) in presence of control gene (GAPDH) for the normalization of data reacted with NCs at low (25 μg/mL) and high (50 μg/mL) concentration. The data represent as mean ± SD of three identical experiments with three replicates.

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Fig. 8

Effect of NCs on mRNA expression level mRNA expression level of C2C12 cells for caspase 7 at an alternate incubation periods (24 & 48 h) in presence of control gene (GAPDH) for the normalization of data reacted with NCs at low (25 μg/mL) and high (50 μg/mL) concentration. The data represent as mean ± SD of three identical experiments with three replicates.

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3.7 Discussions

On the basis of obtained results and their observations, a possible discussion related to cytotoxicity of cancer cells with NCs discussed. As according to the previously published research work (Wahab et al., 2014), the toxicity in cancer cells were much influenced with the inorganic based oxide nanostructures and other numerous physicochemical parameters for instance crystallite size, morphology of nano structures, utilized chemicals, different materials, concentration of nanostructures, incubation period of cells, cultured cells concentration, endocytic uptake of cells etc (Wahab et al., 2014). In the present case, the prepared NCs utilized and at initially the NCs were interacted on the upper layer of cancer cells. Due to very small size of the NCs and each cell, which have very small pores to provide easy passage for the entrance of NCs on the upper surface of cancer cells and due to very small size of each NC is ∼45 nm as compared to the individual cell size (∼18–20 µm), it can be enter easily in cells. In a culture medium the enhanced density of NCs sturdily favors formation of aggregated molecules (Wahab et al., 2014, 2012, 2020b) and postulates that these NCs destroy the internal cells organelles and are liable for cells death. The current approach is also justified with the available results such as microscopy, MTT and it reveals the cells death as with dose dependent manner (Wahab et al., 2021a,b). MTT assay provides the quantitative analysis of the control and treated cells and it shows that the initial concentration of applied NCs are not much affected on the growth of cancer cells were as once the concentrations of NCs roses, the cells growth were much influenced (Wahab et al., 2021a). The apoptosis in cells leads the cells death caused with the NCs and it analyzed via gene expression study, also justified and in consistent. Several other biochemical studies are required to disclose the phenomena why these structures (NCs) are responsible to control/regulate the growth or cell death of cancer cells (Wahab et al., 2021a). Why the NCs are responsible for the retardation of cancer cells growth and biochemical mechanism is under way to find out the exact research detail.