br Preparation of carboxymethyl cellulose CMC COOH br In
2.2. Preparation of carboxymethyl cellulose (CMC-COOH)
In order to obtain the carboxymethyl cellulose, 1 g carboxymethyl cellulose sodium was suspended in 100 mL of distilled water, and then 0.5 mL of concentrated hydrochloric Z-VAD-FMK was injected. The suspension was stirred for 48 h at 25 °C. The functionalized CMC (CMC-COOH) were purified by dialysis against water for 1 week with solvent changes every 24 h.
2.3. Preparation of Eu(III) complex-CMC
50 mL of DMF, 100 mg CMC-COOH, 25.7 mg AMBA, and 24.7 mg EEDQ was added into a 100 mL Schlenk flask. The mixture was stirred at 25 °C for 72 h. The obtained AMBA-CMC was purified by DMF and ethanol and then dissolved in DMF. Subsequently, 5 mg EuCl3 was added above mixed solution and stirred for 48 h. The resulting Eu complex-CMC was precipitated by adding ethanol. The precipitate was dispersed in water and dialyzed for one week in water.
100 mg CMC-COOH was dissolved in 50 mL DMF, and then 12.2 mg DPY and 24.7 mg EEDQ were added into the above solution. The mix-ture was stirred at 25 °C for 72 h. And then, the mixture was centrifuged and the precipitate was dispersed in water. 31.6 mg KMnO4 was added and continued to stirrer for 48 h at 25 °C. The obtained K-DPY-CMC was purified by dialyzed against water for one week.
2.5. Rheology of CDEAC hydrogels
The mechanical properties of the CDEAC hydrogels were char-acterized using a rheometer with a cone and plate geometry, with a cone angle and diameter of 0.9675° and 40 mm, respectively (AR-1000 TA Instruments). For all experiments, an integrated Peltier plate
Fig. 1. (A) FTIR spectra of CMC-COOH (black line), K-DPY-CMC (red line), AMBA-CMC (blue line) and Eu(III) complex-CMC. (B-D) XPS spectra of survey scan (B) and high-resolution of N1s (C) and O1s (D) XPS spectra of AMBA-CMC and Eu(III) complex-CMC. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
was used to control the temperature and a solvent trap was utilized to minimize solvent evaporation. Strain and frequency sweep experiments were conducted to determine the linear viscoelastic response of the hydrogel. Strain sweep experiments were performed with the amplitude varying from 0.1 to 100% strain and a frequency of 1 Hz. Similarly, frequency sweep experiments were performed with an amplitude of 1–100 Hz and a strain of 1%. It was determined that at a strain of 1% and a frequency of 1 Hz the hydrogels were within their linear viscoe-lastic response. These conditions were subsequently used in all further rheological experiments. Time sweep experiments were conducted to determine the storage modulus, G′, and the loss modulus, G″, of the hydrogels with varying CMC concentrations. Prior to the measure-ments, the hydrogel was allowed to equilibrate in the rheometer for 15 min.
2.6. Characterization of the hydrogel structure
FT-IR spectra were achieved on an infrared spectrometer (Nicolet FT-170SX) using a KBr pellet. Morphology of nanocellulose hydrogel was performed by gold sputtered using an SC7640 High-Resolution Sputter Coater (Quorum Technologies) for 30 s at 2.0 kV and 20 mA. Subsequently, the gold-coated samples were imaged by Scanning Electron Microscopy (SEM, FEI, Sirion 200). X-ray photoelectron spectroscopy (XPS) measurements were investigated on a PHI-5702 multifunctional spectrometer using Al Kα radiation. Emission spectra were recorded on a Shimadzu RF-5301 spectrophotometer. 1H NMR spectrums were collected on a JEOL ESC 400 M instrument. The me-chanical properties of the nanocellulose hydrogels were characterized
by a rheometer with a cone and plate geometry (AR-1000 TA Instruments). Fluorescence microscopic images were observed under a fluorescent microscope (Leica DR) equipped with a digital camera (ORCA-ER, Hamamatsu). Photos were processed using Photoshop software (Adobe, CA).
2.7. Cell culture experiments
MCF-7 cells (Human breast cancer cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS, Invitrogen) in an incubator supplied with 5% CO2 at 37 °C. Trypsin-EDTA solution (GIBCO) was used to detach the cells from the flask. After cell detachment, fresh nutrition medium was added, and the cell suspension was centrifuged at 1000 rpm for 3 min. The supernatant was then removed, and the cells were re-dispersed in fresh medium.
For cell culture in the CDEAC hydrogel, 10 wt% Eu(III) complex-CMC and K-DPY-CMC suspension in water were sterilized by UV light for 20 min. The Eu(III) complex-CMC was mixed with sterilized deio-nized water and concentrated PBS (10 × ). Subsequently, the cell sus-pension was added. The final CMCs concentration in the mixed sus-pension was 1.0, 2.0, 3.0, or 4.0 wt%. The resulting mixed suspension at a cell density of 1 × 105 cells/mL was transferred into a 12-well plate (300 μL/well) and incubated at 37 °C for 30 min. Then, 100 μL of DMEM medium and K-DPY-CMC with the same concentration as Eu(III) complex-CMC in the mixed suspension were added to the wells. The plates were incubated with 5% CO2 at 37 °C. The cell culture medium was changed every 3 days.