Cytotoxic assessment of liver cancer cells (HepG2) with raw, functionalized multiwalled carbon nanotubes and their comparison with nanohydroxyapatite

2.1 Experimental

2.2 Cell culture (HepG2 cells) and treatment with CNTs and HAs

The HepG2 cells were obtained from ATCC (American Type Culture collection, Manassas, U.S.A) and thawed it for 2–3 min at 37 °C. The cells were transferred to the culture flasks with cultured medium (DMEM) including 12% fetal bovine serum (FBS), 0.2% sodium bicarbonate (NaHCO₃), and antibiotic–antimycotic solution (100X, 1 mL/100 mL of medium) with 5% CO₂ environment at 37 °C. The culture medium was refilled every other day and cells were subcultured once it reached to their maximum confluence (90%). Initially, the CNTs and HAs were used at high concentration and then after, diluted it at desired concentrations for the exposure of cells. The cells were plated in 6 and 96 well plates as per the condition and requirement of experiment.

2.3 Reagents and consumables for the biological study

The chemical such as MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bro mide], was procured from Sigma Chem. Co. USA and used without any further modification except dilution. On the other hand, the cells culture medium Dulbecco's Modified Eagle Medium (DMEM), antibiotics-antimycotic and fetal bovine serum (FBS) were acquired from Invitrogen, Waltham, Massachusetts, USA. The plastic wares such as well plates (6 and 96), pipets, bottles and other consumables products for the cells culture were used from Nunc, Roskilde, Denmark.

2.4 MTT assay

The cells viability of control and treated samples were accessed via MTT assay as per the followed protocol previously (Siddiqui et al., 2008; Mosmann, 1983). Briefly, the cells were cultured in a specialized 96 well plates (1 × 10⁴/well) with permissible to follow for 24 h at 37 °C with humidified environment. The cells were exposed with RMWCNTs, FMWCNTs and HAs from 1 to 100 μg/mL for 24 h. Once the cells were completely mixed in well plates, stock solution of MTT (5 mg/mL in PBS) was incorporated with the rate of 10–20 μL/well in 100 μL of cell suspension and further incubated for 4 h. In this well plates, MTT salt was reduces and to form a purple formazan salt in mitochondria of living cells. When the incubation period was completed, the well plate’s solutions was with draw from the pipette and in these wells ∼200 µL/well of DMSO was added for to aspirate the formazan product and mixed gently, a purple color visible at this stage and deepen over pipeting. The optical density of the solution was examined at 550 nm through multiwall micro-plate reader (Multiskan Ex, Thermo Scientific, Finland). The untreated or control cells were also employed as reference and to run with the same conditions. The value of maximum absorbance depends upon the employed solvent in sample solution and the percentage (%) viability of cells was calculated as per the mentioned below equation: $$\%\text{viability}=\left[\left(\text{total}\text{cells}-\text{viable}\text{cells}\right)/\text{total}\text{cells}\right]\times 100$$

2.5 RNA isolation

The RNA was mined from RNeasy mini Kit (Qiagen) as per the manufacturer’s protocol. Initially, the RNA was extracted from the cancer cells ( HepG2) in a 6-well plates with control and treated samples (RMWCNTs, FMWCNTs and HA) at a concentration of 50 µg/ml for 24 h. The cDNA was formed from treated and untreated cells taking 1 µg of RNA by Reverse Transcriptase kit using MLV reverse transcriptase (GE Health Care, UK) as per the manufactures’ protocol. The qPCR was performed on Roche®Light Cycler®480 (96-well block) (USA) following the cycling program recommended. 2 μL (40 ng) of cDNA template added to the volume of 20 µL of reaction mixture. Relative ratios were calculated based on the 2^–△△CT method. qPCR was monitored using the CFX96TM Real-Time qPCR Detection Systems (Bio-Rad) (Humes et al., 2017).

2.6 Statistical analysis

The recovered data is displayed as a mean ± SD. Statistical analysis was performed by student T-tests. Results were considered significant when P < 0.05.

3.1 FTIR spectroscopy

The FTIR spectroscopy was used to analyse the chemical functional groups of RMWCNTs, FMWCNTs and HAs for chemical characteristics and illustrated as Fig. 1. Initially RMWCNTs were analyzed, where few peaks were observed at 3200–3600 cm⁻¹ and 1631 cm⁻¹, which indicates water molecule (H-O-H) and C⚌C peak in CNTs consequently (Fig. 1 (a)). The acid digested or functional CNTs shows the different signals at 3459 cm⁻¹, depicts the –OH stretching vibration mode whereas signal at 1639 cm⁻¹ denotes the C⚌C group of CNTs. Another signal was noticed at 1215 cm⁻¹, which assign to C–H stretching band (Fig. 1 (b). The signal at 1049 cm⁻¹ denotes to the carboxylic acid (CO OH–) functional group whereas the signal at 875 cm⁻¹ signifies C–C band (Wahab et al., 2009) (Fig. 1(b)). The recovered bands and their positions designates that the CNTs are functionalized with carboxylic (–COOH) group. Fig. 1(c) shows the bands at 3400 cm⁻¹, 1805 cm⁻¹, 1072 cm⁻¹, 856 cm⁻¹ and 536 cm⁻¹ are related to the hydroxyl (–OH), CO₃²⁻ions, Ca(OH)₂, HPO₄²⁻ and PO₄³⁻ groups respectively, in the prepared HA powder.

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Fig. 1

Fourier transform infrared spectroscopy (FTIR) shows functional detail of RMWCNTs (a), FMWCNTs (b) and HA (c).

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3.2 Morphology of CNTs and HA

The structural scrutiny was performed via SEM and TEM for RMWCNTs, FMWCNTs and HA powder and presented as Fig. 2. The acquired image display several carbon particle structures with (Fig. 2(a)) an aggregation, whereas other image (Fig. 2(b)) shows FMWCNTs, which are separated and seems to be several ropes likes structures. From the observation, the estimated size of an individual coiled rope like structure is ∼2–4 μm length and ∼10–15 nm diameters respectively. The HA powder seems lumpy, aggregated and arranged as a network like structures (Fig. 2(c)). Further for more clarification, the RMWCNTs, FMWCNTs and HA were again analyzed with TEM and presented as Fig. 2 (d-f). The recovered information from the SEM are in consistent and shows that the RMWCNTs (Fig. 2d) exhibit smooth surfaces, whereas the acid digested CNTs surfaces were rough and granulated on the surfaces of NTs (Fig. 2e) due to acid treatment (Fig. 2e). From TEM image, it’s very clear that an individual CNT exhibit 2–4 μm long whereas the diameter reaches to ∼10–15 nm (Fig. 2e). The HA powder molecules are interlinked with other molecule and form a leaf/network shaped structure and analogous SEM observation (Fig. 2f).

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Fig. 2

The scanning electron microscopic (SEM) images of RMWCNTs (a), FMWCNTs (b) and HA (c) powder images consequently. Also transmission electron microscopic (TEM) image display the RMWCNTs (d) and FMWCNTs (e) including HA powder (f) respectively.

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3.3 Morphology of HepG2 cells and their dose dependent treatment effect

The cells were cultured in a specified 75 mm² flask and grow at maximum confluence, it was further harvested with trypsin and treated with RMWCNTs, FMWCNTs and HAs at different concentration from 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/ mL, 25 μg/ mL, 50 μg/ mL and 100 μg/mL with control to check the effect of CNTs, HA and incubated further at 24 h incubation time (Fig. 3). As per the images, it seems that cells were initially mono nucleated and once the inculcation time was reached at 24 h, start to further proliferate. Further inculcation completely damages the cells. In this experiment, the small amount of CNTs covered the surface of selected positions, whereas once the CNTs doses were increases from 50 μg/mL and 100 μg/mL and it roofed the entire surface of the cells (Fig. 3). This is hypothesized that the thin ropes of CNTs (RMWCNTs and FMWCNTs) structures exhibit the capability to enter easily to towards cell membranes, cytoplasm, cells organelles and creates the possibility of damage of cells. A similar trend was also found in case of HA, which shows the dose dependent toxicity with cancer cells. From the microscopic images, it’s realized that the as the conc of CNTs and HA increases the viability of cells were damaged with interference of NTs (Fig. 3) (Aoki and Saito, 2020).

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Fig. 3

Microscopic images of HepG2 cells with RMWCNTs, FMWCNTs and HAs shown at different concentrations (1 to 100 μg/mL) at 24 h incubation period.

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3.4 MTT assay or cytotoxicity assessment

The recovered data from MTT assay reveals that the viability of cells is in concentration/dose dependent (Fig. 4). The viability of cells with the RMWCNTs doses at 1 μg/ mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL and 100 µg/mL recorded as 103%, 103%, 103%, 81%, 75%, 70% and 65% respectively by MTT assay at 24 h (Fig. 4). In case of FMWCNTs viability of cells are more effective as compared to the RMWCNTs at different doses are 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/ mL, 25 μg/mL, 50 μg/mL and 100 µg/mL recorded as 99%, 93%, 91%, 84%, 79%, 71% and 63% respectively by MTT assay at 24 h (Fig. 4). The viability of cells with polymeric HAs shown a similar inclination for the cancer cells at different doses are 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/ mL, 25 μg/mL, 50 μg/mL and 100 µg/mL recorded as 95%, 93%, 93%, 90%, 83%, 71% and 62% respectively by MTT assay at 24 h (Fig. 4). The current work also corroborated with previously published literature (Aoki and Saito, 2020) and indicates that polymeric material express the efficacy to control the proliferation of cancer cells at dose dependent manner.

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Fig. 4

The MTT assay of RMWCNTs, FMWCNTs and HAs at different concentration (1–100 μg/mL) at 24 h incubation period with control solution. Experiments were performed in triplicate manner. *p < 0.05, **p < 0.01 Vs Control.

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3.5 mRNA gene expressions and activity of caspase with RMWCNTs, FMWCNTs and HAs

The qPCR was used to know the mRNA expression levels of apoptotic marker genes (e.g. p53, Bax, caspase-3 and Bcl2) in HepG2 cells exposed with RMWCNTs, FMWCNTs and HAs at 50 µg/mL for 24 h. From the recovered data’s, it can be easily seen that a noteworthy change was examined in mRNA level of apoptotic markers (p53, Bax, casp-3 and Bcl2) genes in HepG2 cells, when exposes with RMWCNTs, FMWCNTs and HAs (Fig. 5). The suppression gene of mRNA tumor supports the apoptosis induction by foreign material and observed the enzymatic activities of caspase-3 at the concentrations of 50 µg/ml. In case of RMWCNTs, the selected gene were express the up regulations whereas Bcl2 express down-regulation and fold changes for p53(2.0), Bax (4.0), Casp3 (1.7) and Bcl2 (0.62) correspondingly (Fig. 5), Similar treand of up and down (Bcl2) regulations were also found in case of FMWCNTs sample and the fold changes for p53 (2.1), Bax (4.0), Casp3 (1.8) and Bcl2 (0.67) congruently (Fig. 5). The HAs express the up-regulation in mRNA expression and the fold changes for p53 (2.2), Bax (4.2), Casp3 (1.9) exept the Bcl2 (0.70) respectively (Fig. 5). The obtained results displayed RMWCNTs, FMWCNTs and HAs induces the apoptosis in cells in presence of marker genes (Fig. 5) (Srivastava et al., 2011).

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Fig. 5

mRNA expression of apoptosis marker genes by quantitative polymerase chain reaction (qPCR) analysis in HepG2 cells with RMWCNTs (a), FMWCNTs (b) and HA (c) at 50 mg/mL concentration for 24 h. qPCR was achieved with Roche LightCycler ®480 soft-ware (version 1.5). The GAPDH gene was used as a control to normalize data. The data is accessible as the mean ± SD of three identical experiments with three replicates manner. Significantly different compared with the control group (p < 0.05 for each).

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3.6 Discussion

The CNTs and HAs both are unique material with polymeric and physicochemical characteristics including high surface, electrical conductivity and including functional properties. Although, with unique physicochemical characteristic limited information is available towards the direction to control the proliferation of cancer cells. Based on the recovered results such as FTIR, SEM, TEM, microscopy, MTT and qPCR study, the cells toxicity influences with used RMWCNTs, FMWCNTs, and HAs and are depends upon their unique size, shape, chemical composition, surface functionalization’s, endocytic uptake of cells, DNA interactions and others various factors (Bergamaschi, 2008). Based on these observations, a possible mechanism and related cytotoxicity of cancer cells with RMWCNTs, FMWCNTs, and HAs are described. As it’s illustrious that the size of RMWCNTs and FMWCNTs are having very small (D = 10–15 nm, L = 2–4 µm), whereas the HAs are also exhibit a similar characteristics and comes in the nanorange with network like structure. The rope like structures, which have very small diameter and longer length are capable to penetrate the cell membrane of cancer cells. Once it enters in to the cancer cells, attack to the cells internal organelles surface of cells. Regarding this, several hypothesis related to the internalization of foreign material towards cancer cells were documented (Wahab et al., 2014). As per the previous study, cell membranes exhibit small pore like passages which facilitates to enter CNTs in to cells and damaged the upper layer of cell walls (Coccini et al., 2010). In other reports, the small nanostructures can be easily entered in to the cells cytoplasm and these small nanostructured adducts react with cell’s organelles and diminished the growth of cancer cells. In this case, CNTs were used from low to high concentration and shows the damage of cancer cells as dose dependent. Similar observations were also found in HAs, where very small particles are combined together and to form adduct and these adducts react to cells also their high density of materials in liquid medium strongly favors the interaction with cells and to enhanced the damage of cancer cells (Wahab et al., 2009; Bergamaschi, 2008; Coccini et al., 2010). The qPCR results also confirm that the apoptosis in cells caused with foreign material (RMWCNTs, FMWCNTs and HAs), which further confirms the cytotoxicity of cancer cells.