br Materials and Methods br Single layer graphene oxide
2. Materials and Methods
Single layer graphene oxide sheets and BSA were purchased from Biosharp Company. Trypsin-EDTA solution, Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM) solution, and Penicillin-streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were acquired from Sigma Aldrich (Shanghai, china). All the chemicals were used as re-ceived without any additional purification. Milli-Q Plus System was used to pretreat the experimentally used water.
Euphorbia milii leaf extract were formulated by boiling 20 mL water added with 0.01 g–0.3 g leaves at 80 °C. 0.45 μm filter was used to filter the mixture after 1 h and the obtained filtrate was preserved at 4 °C. In order to conduct reduction of GO in optimized state, 10 mg of GO was dispersed in 20 mL of distilled water for about 30 min by sonication. Further, 5 mL of Euphorbia milii leaf extracts were mixed with the as prepared GO solution and allowed for stirring at room temperature for 48 h. Then, the prepared materials were purified by washing thrice with distilled water, filtered with ethyl alcohol to get a clear solution. Finally, RGO were formed after drying by means of vacuum freeze dryer.
UV 1902, Phoenix, UV–Vis reflective spectrophotometer (Shanghai, China) was used to perform the optical density measurements over the range of 190–800 nm at room temperature. TEM (FEI Tecnai G20, USA) operating at 200 kV was used to study the morphology of the RGO. Nicolet, Magna-IR 750, Fourier transform infrared spectrometer (FTIR) was employed to collect the FTIR spectra. The KBr disc was arranged by mixing Euphorbia milii leaves extract (1 mL) and KBr powder (10 mg) and pressing it after being dried under a sodium lamp. At the same time, the specimens were mixed with KBr powder at a 1% (w/w) ratio and pressed into a sheer slice to prepare GO and RGO samples. An average of 9 scans per sample with a resolution of 2 cm was maintained while recording FTIR spectra.Multi-mode-8 atomic force microscope was used to acquire the AFM images. X'pert Pro X-ray diffractometer was used to record the X-ray diffraction (XRD) data with Cu Kα radiation. Renishaw in Via Reflex spectrometer was used to collect the Raman spectra.
RPMI-1640medium is employed in the culture ofA549 AMG-176 (Human lung cancer cell lines) and is complimented with 10% fetal bovine serum (FBS) in addition with 1% antibiotic mixture of Penicillin-Streptomycin. Further, the cells were allowed to incubate at 5% CO2 and 37 °C standard conditions. Later, the cells were cultured in the desired culture medium and incubated again.
2.5. Cytotoxicity Evaluation
MTT assay was performed for free RGO and RGO/PTX against A549 (Human lung cancer cell lines) . Enzyme-labeled Instrument (Mullikan FC, Thermo Scientific) was used for measuring the MTT cell toxicity at 490 nm. RGO and RGO/PTX with different concentrations Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 13–17
Fig. 1. UV–vis spectra of GO before and after reduction by Euphorbia milii plant extract for different reduction times 12 h, 24 h and 48 h.
were added to cells group (six wells) for 24 h respectively. Olympus CKX41 is employed to measure the fluorescence microscopy of viable cells.
3. Results and Discussion
Fig. 1(a) displayed the UV–vis absorption spectrum of GO, showing the plasmon peak at about 230 nm with narrow shoulder band at 300 nm which is seldom referred to n–π∗ transitions of the carbonyl groups . The plasmon peak eventually red-shifts to ∼270 nm with the time by Euphorbia milii leaves extract (Fig.1b–f), reflecting the consistently increased π -electron transition and structural ordering, with sp2 carbon restoration and feasible atom rearrangement . This might indicate the increase in the reduction of GO and the gradually restored aromatic structure with the increase in reaction time.
The structural elucidation of the graphene is mainly dependent on the distance between two layers. Fig.2 displayed the XRD patterns of graphite, GO, RGO prepared using Euphorbia milii leaves extract. Fig. 2, indicated that the d-spacing of the GO is around 0.78 nm (2θ ≈ 11.3), which is extremely larger than the d(002) value of graphite (d ≈ 0.34 nm, 2θ ≈ 26.2) as shown in figure which is because of the existence of oxygen-containing functionalities nailed on either side of the RGO along with the roughness of the atomic-scale .The (002) GO peak steadily disappears with the raise of reaction time, while the broad XRD peak at 24.0° (d ≈ 0.37 nm) gradually becomes significant (Fig. 2). This interlayer space shift can be ascribed to the GO deox-ygenation while the RGO pack becomes tighter than the GO due to reduction . On the other hand, the broad XRD peak of RGO is due to the presence of biomolecules of plant extracts that are adsorbed on the surface of formed graphene sheets.