br Corresponding author at The Third Department of Tumor Sur
Corresponding author at: The Third Department of Tumor Surgery, Tangshan Gongren Hospital, No. 34, longze south Road, Lubei District, Tangshan City 063000, People's Republic of China. E-mail address: [email protected] (X. Feng).
Available online 20 November 2018
similar methods have been developed for the graphene synthesis by making use of peptides, proteins, plants, bacteria, fungi, and others . In particular, the application of plant extracts as reducing agents is well renowned as they are effective bio-reductants due to the ex-istence of phyto-compounds like flavonoids and polyphenols. These Oxidopamine hydrochloride are an abundant source of natural antioxidants present in our diet from plant sources with high reducing ability . Their reducing nature is chiefly attributed to the number of free hydroxyl groups in their molecular structure . Several green reducing agents such as plant extracts, plant biomolecules have been already used for the production of graphene sheets and metal nanoparticles [21–26]. Sorafenib (SRF) is a well-known tyrosine kinase inhibiting antic-ancer drug which is effectual against multiple carcinomas especially gastric cancers by targeting the Ras/Raf/Mek/Erk cascade pathway and blocking the tumor cell proliferation . In the present work, we have reduced GO in presence of sorafenib using ascorbic as green reducing agent. The resulting RGO will have sorafenib on its surface as ad-sorbant. This sorafenib coated RGO (SRGO) have been used to study their cytotoxicity against gastric cancer cells.
2. Experimental section
Sorafenib, Graphite flakes, Phosphate buffer solution (PBS), and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] were obtained from Sigma–Aldrich (St. Louis, MO, USA). Dubelcco's modified Eagle's medium (DMEM), penicillin-streptomycin mixtures, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco®BRL (Carlsbad, CA, USA).
2.2. Preparation of graphene oxide
In this experiment, graphene oxide was synthesized by making use of modified Hummers method. 1 g of graphite was added to 23 ml of concentrated H2SO4 in a conical flask and was placed on an ice bath. The reaction mixture was slowly added with about 3 g of KMnO4 at a temperature of 0–5 °C under constant stirring and stirred for about 30 min. A hot plate with a pre-set temperature of 35 °C was used to transfer the flask on to it and allowed to stand for 1 h. About 46 ml of deionized water was then added to the reaction mixture after comple-tion of heating time, followed by dynamic stirring. Thus obtained re-action mixture was then transferred to a boiling water bath for about 30 min. In order to stop the reaction, the reaction mixture was added
with 10 ml of 30% H2O2 followed by the addition of 140 ml of deio-nized water to achieve yellow coloured graphene oxide. The product was then filtered and repeatedly washed by using 10% HCl followed by and deionized water.
2.3. Preparation of reduced graphene oxide
About 50 ml of Ascorbic was added to 30 mg GO,along with 50 mg of sorafenib and vigorously stirred to obtain uniformly dispersed medium. The above reaction mixture was added with 180 μl aqueous NH3 solution until a change in colour from red to dark green is ob-served. The reaction mixture was then heated on an oil bath with a water condenser at 95 °C for variable time intervals of 1, 3, and 6 h. Thus formed SRGO samples were filtered after completion of heating, washed, and dried at 70 °C.by means of hot air oven
2.4. In vitro cytotoxicity studies
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to investigate the cytotoxicity of blank RGO (prepared without adding drug), SRF, and SRGO formulations on SGC7901 cancer cell lines. A96-well plate was seeded with 1 × 104 cells per well and were incubated for one day. The following day, old media was replaced with fresh media containing blank NP, free SRF, and SRGONPs and incubated at 37 °C for one more day. The media was then removed on the next day and was washed with PBS twice. MTT solution (5 mg/ml) was then suspended into individual wells and in-cubated for the next 4 h. Each well was lateradded with 100 μl of di-methylsulfoxide (DMSO) in order to dissolve the MTT formazan crys-tals, after removing the unreduced MTT and medium. A microplate reader (Bio-Rad, CA, USA) was used to record the absorbance of for-mazan crystal at 570 nm.
2.5. Hoechst staining
Hoechst 33382 staining was performed to investigate the apoptosis of blank NP, SRF, and SRGO on SGC7901 gastric cancer cell lines.A6-well plate was seeded with treated with the blank NP, SRF, and SRGO nanoparticles and was incubated for 24 h. Phosphate buffer saline (PBS) was used to wash twice and then followed by cold methonal: acetone (1:1) fixation for about 5 min. These cells were stained with Hoechst 33382 for at least 10 min at room temperature after washing thrice in PBS. Then, the cells were again washed twice by using PBS and studied under fluorescence microscope.