- Synthesis of graphene oxide
To prepare GO, graphite powder was used as per the Hammers method ( 16). Firstly, 2 g of graphite (500 mesh) and 80 mL of concentrated sulfuric acid were poured and stirred. Gradually 2 g of sodium nitrate and 12 g of potassium permanganate were added to give a sludge-green mixture. Then 46 ml of deionized water was added to the reaction vessel and stirred for 30 minutes. Next, deionized water and 16 mL of 30% hydrogen peroxide was added to the reaction mixture. The obtained precipitate was placed in an ultrasonic bath for 30 minutes and the produced solution was washed and filtered with 10% volumetric HCL solution in water 3 to 4 times. The obtained brown solid precipitate was separated by centrifugation and dried for 24 hours in an oven at 50 ºC.
Transmission electron microscopy (TEM) was used to characterize the prepared GO and DLS test was used to determine the particle size and dispersion of the drug. Then all the treated cells were exposed to GO for 24 hours, then GO was removed and DOX was added for 24 hours (GO-DOX group).
The synthesis of GO particles was performed by Zeta Sizer (Maldon UK ZS series model) at room temperature and 90 degree angle.
In order to evaluate ad confirm the morphology and the size of GO, a drop of the particle suspension sample was placed on a carbon film grade and after drying at laboratory temperature using TEM KV 100 model (Leo 906 Zeiss, Germany), was photographed with an accelerator voltage of Kv120.
In this study, two breast cancer stem cells (MCF7 and BT474) were used. The cells were obtained from the Iranian Genetic Stores Center in frozen form and cultured into the DMEM (Dulbecco’s Modified Eagle Medium) medium with 10% fetal bovine serum (FBS) supplement. Cells acidic metabolites produced during their proliferation causes the culture medium to gradually change color from purple to yellow. For this reason, the cell culture medium was substituted every 24 to 48 hours. When cell density reached 80%, cell passage was performed by adding trypsin.
To freeze the cells, the flask supernatant materials was drained, and then washed with 1X phosphate buffered saline (PBS). Cell isolation was performed physically at 37 ºC. The contents of the flask were poured into a flask containing the culture medium until trypsin function cease. The Falcon containing the suspension was centrifuged at 1500 g for 6 minutes, then frozen in the microtube. Freezing medium contained 50% DMEM, 40% FBS, and 10% dimethyl sulfoxide (DMSO).
Table 1. Results of DLS analysis which represented particle diameter and GO dispersion
The 3- (4, 5-dimethylthiazol-2-yl) -2,5-diphynyltetrazolium bromide (MTT) test was based on subject of the cells to various concentrations of the drug (GO-DOX) and measuring the rate of cell death. For this purpose, the yellow active substance tetrazolium and the formation of insoluble purple crystals of formazan were used in the MTT method. In this method, 24 hours after culturing the cells with the desired concentration, the supernatant culture medium was substituted and the cells were cultured for 3 hours with culture medium containing tetrazolium dye. Then, the medium was combined using 100 μL of dimethyl sulfoxide and the amount and intensity of adsorption were read using an ELISA reader device at a wavelength of 570 nm.
Evaluation of apoptosis by flow cytometry
Flow cytometry was used to evaluate the extent of apoptosis in MCF7 and BT474 cancer cell lines as a result of incubation with GO and DOX (GO-DOX) compared to the control and DOX groups. After the relevant treatments, the cells were trypsinized, then centrifuged at 1200 rpm, followed by 5 mL of phosphate buffer. After re-centrifugation, 1 ml of kit buffer was added and after intense pipetting, 5 ml of Annexin V was added and incubated for 4 minutes in dark conditions. Finally, 4 µl of propidium iodide solution was added and analyzed by flow cytometry.
- invasion and cell migration rate
Firstly, 8 µL filters were covered with a matrix layer and 25,000 cells per well were transferred to the filter with serum-free culture medium. The cells inside the filter were then removed using a wet swab, and then the invading cells passed through the matrigel and filter were counted. To perform the test, the cell culture medium in which the cells had reached a obtained degree of growth were replaced with the new culture medium having 0.2% BSA and placed in the incubator to induce starvation in the cells for an overnight. The cells were then washed with PBS solution and separated from the flask surface by Tripsin / EDTA enzyme. the enzymatic activity was inhibited by adding the culture medium containing 5% FBS with twice amount of the enzyme volume and then cell sediment was prepared using centrifugation at 750 g for 5 minutes.
the cell precipitate was dissolved into 1 mL of culture medium and 50µL was taken to count using 0.04% trypan blue. We then adjusted the cell density to 125,000 viable cells per milliliter of culture medium and added 600 µL of new culture medium to each well. The coated filters were then incubated with 300 μL of serum-free culture medium at room temperature for one hour to perform the hydration. Then 200 μl of cell suspension in culture medium was added to each filter (about 25,000 cells per well), and incubated the cells in 37 ° C and 5% CO2 incubator for 10 hours. Finally, to calculate cell invasion and migration, the average of them was counted under a microscope.
- Colony formation ability test (sphericity test)
Firstly, 100,000 BT474 and MCF7 cells wre cultured into a T25 flask and then 5 ml of serum-free culture medium containing growth factors BFGF 20 ng / ml and EGF 20 ng / ml were added into th flask. In the next step, the culture dishes were transferred to an incubator with 37. 5°C and 5% CO2 without shaking for 48 hours. In order to change the environment of the spheres, the culture medium containing the spheres was pipetted and gently transferred to 15 ml tubes, and the spheres were slowly settled. the cells were centrifuged at 1000 rpm to precipitate, then the supernatant was removed and new medium was gently added from the side of the tube not to rupture the spheres. Then we transferred the spheres to our flask. Every three days, the culture medium was substituted and the new medium containing growth factors was added.
In order to calculate the percentage of cell line spherogenesis, the medium containing spheres was transferred from the culture dish to a sterile tube and pipetting was conducted gently so that the cell aggregates be disintegrated and a uniform solution achieved remaining the spheres. Then, using a suitable micropipette, 100 μl of the suspension containing the spheres was removed and placed in a non-stick container. We then counted the spheres using a stereo microscope with 40x magnification.
- Graphene oxide properties
GO was prepared from graphite powder following Hammers method. The results revealed that the synthesized solution contained proper conditions in terms of physicochemical properties. According to the TEM image, GO multilayer with a size of 2 µm was formed (Figure 1).
DLS results outlined that the average particle diameter was 1302 nm, and polydispersity of 0.07, represented the efficient particle dispersion (due to <0.1), indicating the single dispersion (table 1). Accordingly, GO particles were dispersed properly in the solution without any accumulation.
The effect of GO on MCF7 and BT474 cancer cell lines revealed that IC50 for GO was 40 μg / mL which was considered for further tests. MTT assay was implemented to determine cell survival and IC50 concentration of GO-DOX on MCF7 and BT474 cells in 24 hours applying various concentrations of drug against cells.
Figure 2A depicts the MTT test at concentrations of 0, 1, 2.5, 5, and 10 μg / mL GO-DOX after 24 hours on MCF7 cells. Additionally, as represented in figure 2B, by increasing the concentration of GO-DOX, the survival rate of BT474 and MCF7 cancer cell lines was significantly decreased compared to the control group. Figure 2C demonstrates a diagram of both cells where MCF7 cancer line cells are more affected by GO-DOX in a concentration-dependent behavior compared to BT474 cells. Interestingly, there was a difference between the survival rates for both, which was significant for concentrations of 1 (p = 0.003), 2.5 (p = 0.003), 5 (p= 0.00009), and 10µg/mL (p = 0.0001), respectively.
- Determination of LC50 concentration
The concentration at which 50% of cell death occurs is known as 50% lethal concentration (LC50). The stock concentration of doxorubicin was 10 mg / mL, which according to the results of the MTT test within 24 hours for MCF7 was ~1 μL of stock and 10 mg / mL GO-DOX caused 50% survival which was equivalent to 0.1 mg / mL. However, for the BT474 LC50 cell line, it was not reported for GO-DOX because of lower than 50% death rate at high concentrations. It should be noted that a concentration of 0.1 mg / mL was used in subsequent experiments.
- Aptosis rate using flow cytometry
Concentration of GO-DOX IC50 was used for flow cytometry test and determination of the amount of apoptosis. Figure 4A shows the results obtained from flow cytometry and the results of Annexin and PI (Propidium iodide) test showed a significant increase in the apoptosis in MCF7 cancer cell line compared to the control group. The same is true for BT474 cells (Figure 4B). This suggests that GO-DOX increased apoptosis in the BT474 cancer cell line compared to the control group. In both cell lines, the percentage of apoptotic cells was significantly increased compared to the control (P <0.001).
According to the results of the Figure 5 for the both cell lines, there was a significant increase in apoptosis and necrosis following incubation with graphene and doxorubicin, which resulted in a mortality rate in MCF-7 cells. However, in BT474 cells the mortality was mainly related to the apoptosis. The paired t-test analysis between GO-DOX group and the other two groups, namely DOX and control for both cell lines, showed a significant higher rate of apoptosis in the GO-DOX group compared to the other two groups. In MCF-7 cells, the rate of necrosis was also significantly higher compared to the other two groups.
- The ability of colony formation
Colony formation potential in MCF7 cell group treated with DOX, GO, GO-DOX was evaluated and compared with control. Morphologically, the size of colonies from MCF7 cell group treated with control was completely different. According to figure 6, the colony forms in the treated groups were different from those of the control. For instance, the number of single cells in the treated groups was substantially increased, and this disclosed that the cells formed colonies during the 7 days. The treatment prevented the formation of colonies, while in control cells they were able to form colonies with the well-known forms of holocolon, paracolon, and morocolon. Additionally, with the presence of GO-DOX, the inhibitory effect on the cells was significantly higher in comparison with the control cells (p <0.05).
In fact, in the spheroid test, the ability of spheres to form in a non-sticky environment was examined. In this test, the number of spheres produced after formation and treatment with GO and GO-DOX was evaluated. Figure 7 depicts two-cell spheroids under the influence of GO and GO-DOX compared to the control group. The t-test analysis on the number of spheroids formed in the control of two cell lines also outlined a significant difference between spheroids with an increase in the number of spheroids in BT-474 cells compared to the inhibited MCF-7.
- Cell invasion and migration
As shown in the figures 8 and 9, the transvel plate containing the cell with or without matrigel have been represented. In the figure 8, the rate of cell migration and invasion was determined by counting the remaining cells after staining on the transvel plate without the presence and presence of matrigel, respectively. MCF7 cells had a higher percentage of invasion than BT474 cells due to the lack of their presence in the cell count on transvel without matrigel, while they had a high percentage of presence in transval containing matrigel. In case of BT474 cells in both conditions, the percentage of cells was similar to each other, so they showed a low percentage of invasion. The number of MCF7 cells count was significantly different in DOX, GO, GO-DOX groups with and without matrigel, while in BT474 cells only in DOX group there was a significant difference between the both conditions.