Anticancer Activity of Graphene Oxide / 5-FU on CT 26 dsRED Adenocarcinoma Cell Line

Cancer is one of the greatest health challenges in the world. Every year, many people die because of cancer. Chemotherapy is one of the treatment options in cancer disease. Fluorouracil) (5-FU) is one of the chemotherapy drugs, but it has relatively low toxic effect on tumor cells when it is used on free form, which also results in its poor efficacy. GO (graphene oxide) has a singleatomic layer and has several functional groups such as epoxide, carbonyl, carboxyl and hydroxyl which makes it a suitable carrier for drug loading. In the present study, we loaded 5-FU on GO nano-carrier to produce GO/5-FU, and characterized it by FT-IR. CT26 dsRED adenocarcinoma cell line was treated with GO/5-FU, free 5-FU, GO, and PBS (Phosphate buffer saline). The results showed significant inhibition of the CT26 dsRED cells using GO/5-FU compared to free 5-FU (P<0.05). Therefore, loaded 5-FU on GO (GO/5-FU) could be a new approach for optimization of 5-FU tumor cytotoxicity.


INTRODUCTION
Cancer is one of the health-threatening diseases.In 2015, about 90.5 million people were affected by cancer 1 .About 14.1 million new cases occur a year and it causes about 8.8 million deaths (15.7% of deaths) 2 .Fluorouracil (5-FU), is one of the choice drugs for chemotherapy of several cancers [3][4][5] .5-FU acts in several ways, but principally as a thymidylate synthase (TS) inhibitor, synthesis of the thymidine, and the conversion of (dUMP) to (dTMP) which inhibits.Administration of 5-FU stops cancer cell dividing and causes cancer cell death [6][7][8] .Loading of cancer drugs on nano-carriers is one of the ways to increase their therapeutic effect and biocompatibility.Graphene oxide is hydrophilic and monomolecular layer of graphite with various of functional groups such as epoxide, carbonyl, carboxyl and hydroxyl [9][10][11][12][13] .The unique structural properties of GO has caused its wide application by many researchers as a nano-carrier to loading of several drugs.In prior studies 5-FU loaded on several nano-carriers, co-poly (d,l-lactic/glycolic acid) (PLGA) by Ywu-Jang Fu et al., 14 , poly (HEMA) nanoparticles by Chouhan et al., 15 , Pectin nanocarrier by Kumar Dutta et al., 16 , polyamidoamine (PAMAM) by Ly, Uyen et al., 17 , and poly(ε-caprolactone) nanoparticles (PCL NPs) by Ortiz et al., 18 .Although previous studies had relative success to improve the antitumor activity of 5-FU by loading it on several nano-carriers, but more studies are need to improve the efficacy of 5-FU through loading it on new nano-carriers.The purpose of present study was the loading of 5-FU on GO to improve its antitumor activity.We loaded 5-FU on GO nano-carrier.New compound (GO/5-FU), free 5-FU, GO and PBS treated with CT26 Ds-Red adenocarcinoma cells and the results of GO/5-FU group compared with the results of 5-FU, GO and PBS treated groups.

Materials
5-FU was provided from Ebewe Pharma Co.The CT26 dsRED colon adenocarcinoma cell line purchased from Pasture Institute of Iran, then they were cultured in RPMI 1640 medium supplemented with L-glutamine (Biowest), 10% fetal bovine serum and penicillin-streptomycin (100 U/ml and 100 mg/ml, respectively) at 37°C in a 5% CO 2 humidified atmosphere.Graphite powder was purchased from Merck & Co.Other materials provided from local suppliers.

Synthesis of graphene oxide
Graphene oxide (GO) was synthesized by Hammer modified method 19 .Briefly, graphite powder (3g) was oxidized in 12 ml of H 2 SO 4 at the presence of 2.5 g of K 2 S 2 O 8 and 2.5g of P 2 O 5 at 80°C for 4.5 hours.Then the graphite oxide was neutralized using 120 ml of precooled sulfuric acid.KMnO 4 (15g) progressively added into the solution under vigorous stirring under 20°C.Temperature was raised to 40°C and continued stirring for 2 h, two times deionized water 250 ml, 700 ml and 20 ml of H 2 O 2 was added under strong stirring, respectively.For elimination of excess materials, the mixture was washed and neutralized with 100 ml of HCl (37%), HCl aqueous solution 100 ml (1: 10 v/v), and deionized water (1000 ml), respectively.Neutralized graphene oxide was dried at the ambient temperature, dispersed (0.5% wt) in deionized water, and dialyzed with dialysis bag (7000 Da) for one week.

Cells culture and MTT assay
CT26 Ds-Red colon adenocarcinoma cell line was cultured in RPMI1640 medium supplemented with L-glutamine (Biowest), fetal bovine serum 10% and penicillin-streptomycin (100 U/ml and 100 mg/ml, respectively) at 37°C, 5% CO 2 and humidified atmosphere.MTT assay applied to measure the cytotoxicity of (GO/5-FU), free 5-FU, GO and PBS 21,22 .CT26 dsRED cells seeded and incubated overnight at 37°C, 5% CO 2 humidified atmosphere, then the cells were treated by GO/5-FU, free 5-FU, GO and PBS.For each groups we considered four well (quadruplicate).After 24 h incubation at 37°C, 5% CO 2 and humidified atmosphere, 20 µl MTT solution was added and incubated for 4 h at 37°C, then medium and MTT were removed.DMSO (200 µl) was added to all wells.Incubation was done for 15 min.in ambient temperature and darkness.Absorbance was measured by a microplate reader (Anthos 2020, 540 nm).To calculate the cell viability (%) the following formula was used 23 .

Statistical analyses
Data were analyzed with Graphpad Prism 6. Student's t-test was used for analysis of the results between two groups.Data was presented as mean ± standard deviation (SD).Results were considered statistically significant at P < 0.05.

Synthesis and characterization of GO
Graphite used for synthesis of GO according of modified Hummers' method 10 .Obtained GO was characterized by atomic force microscopy (AFM), transition electron microscopy (TEM), X-ray powder diffraction (XRD) and FTIR spectroscopy methods to detect the shape, size, and chemical structure of obtained GO 24 (Figure 1).

Drug loading and characterization
5-FU loaded on GO nano-carrier according as described in materials and methods.Loaded 5-FU calculated by UV-Vis spectrophotometry (CECIL CE 2041, 265 nm).5-FU standard curve provided according to calculated absorbance for serial dilutions of 5-FU (Table 1, Fig. 2).Loaded 5-FU concentration was calculated according the following formula: Loaded 5-FU = (initial 5-FU concentration -5-FU concentration in supernatant) The concentration of 5-FU on supernatant was (µg/ml) and initial concentration of 5-FU was 1746 (µg/ml).Dilution was performed for high concentration samples.

Cytotoxic activity of GO/5-FU
MTT assay was used to detect the cytotoxicity effect of GO/5-FU.We assayed GO/5-FU, free 5-FU, GO and PBS (100mM) on CT26 dsRED adenocarcinoma cell line, the results are shown in (Fig. 3, Table 2).The inhibition of CT26 dsRED cell line by GO/5-FU was significantly higher compared to free 5-FU (P<0.017)(Figure 3).

GO/5-FU IC 50
IC 50 is a drug concentration which inhibit the growth of 50% cells 25 .Calculated IC 50 in present study for GO/5-FU and free 5-FU was 5.2 µg ml -1 and 8.1 µg ml -1 , respectively.These results showed the IC 50 of GO/5-FU was 35.8% lower compared to free 5-FU.In the other words, inhibition potency of GO/5-FU was higher (35.8%) in comparison with free 5-FU (Table 2).Loading of anticancer drugs on several nano-carriers is a new concept on chemotherapy and in the recent years, extensive research has been conducted on this subject.Fluorouracil (5-FU) is a common anticancer drug which is used in several cancers but its efficacy is low.The purpose of present study was improving the efficacy of 5-FU by loading it on GO nano-carrier.Our results showed that loaded 5-FU on GO (GO/5-FU) significantly inhibited CT26 dsRED adenocarcinoma cell line compared to its free form (5-FU).This improvement may be due to slow and steady release of 5-FU when it loaded on GO nano-carrier.The results are in agreement with those of TU et al., 17 , NQ et al., 26 , El-Ghannam et al., 27 , Ganguly et al., 28 and Liu et al., 29 .However, our findings do not support those of Wang et al., study 30 , in that, they found the amount of loaded 5-FU on KGM/SA/GO nano-carrier was 32% while in our study it was 58%.One possible explanation for this discrepancy is that our incubation condition (24 h, 37°C, shaking 50 RPM vs 24 h, room temperature and without any shaking) or it may be related to the nano-carriers differences (GO vs KGM/SA/GO).Our results regarding cell viability do not support those of Rana et al., study 31 (26% vs 80%, respectively).This difference may be due to the application of different cancer cell lines (CT26 Ds-Red vs CEM, respectively).The results of present study showed the potential of GO/5-FU as novel compound for the enhancement of anti-cancer activity of Florouracil.

Fig. 3 .
Fig. 3. Cytotoxic effect of GO/5-FU, free 5-FU, GO and PBS.MTT assay was used to assay of cytotoxic effect (see material and method).Data presented as mean ± SD.Inhibition of CT26 dsRED cell by GO/5-FU was significant, (P<0.017).