Materials
Chitosan (Mw 190–310 kDa), pectin from citrus peels (galacturonic acid ≥ 74.0%), graphite powder, succinic anhydride, sodium periodate, ethylene glycol, hydroxylamine hydrochloride, methanol and sodium hydroxide were obtained from Sigma–Aldrich. Ethanol, acetone, potassium permanganate, sodium nitrate, sulfuric acid 98%, and hydrogen peroxide, were obtained from commercially available suppliers (Merck KGaA). All chemicals were of analytical grade purity and they were used as received without any further purification. The aqueous solutions were prepared in deionized water.
Synthesis of N-succinyl chitosan (NSC)
As previously described, NSC was synthesized with slight modifications (Bashir et al., 2017). In short, the purified chitosan (0.5 g) was suspended in 75 mL aqueous solution of acetic acid (5.0% v/v). This mixture was placed magnetic stirring at 50 °C for 1 h. After that, dilution of the solution was done by adding 75 mL methanol. Subsequently, succinic anhydride (2.5 g) was dissolved in 60 mL acetone and mixed with chitosan solution. The stirring was continued at 50 °C for 24 hours. After 24 h, the pH of the mixture was adjusted to 12 with NaOH (1.0 mol/L) solution, and the formation of a clear solution was observed. The stirring of this clear solution was continued for further 5 hours. Afterward, 150mL ethanol (96%) was added to form precipitates followed by filtration to separate the precipitates. These precipitates were dispersed in ethanol for 24 hours. Then, dispersed precipitated product was washed with several times with ethanol and acetone to remove excess reagents and dried using freeze-dryer for 4 hours. The final product was stored at room temperature. The degree of the substitution (DS) of NSC was determined to be 0.52 according to the described previously (Bashir et al., 2017).
Synthesis of oxidized pectin (OP)
Synthesis of OP was according to the previously reported procedure with slight modifications (Ahadi et al., 2019). Initially, 0.5 g of pectin was dispersed in 10 mL ethanol (96%). Then 10 mL of sodium periodate solution (0.5 M) was added and the temperature was controlled at 40 °C. After the reaction was carried out for 8 h in a dark environment. The appropriate amount of ethylene glycol was added to continue the reaction for 2 h to remove the unreacted oxidant. After the reaction, the product was dialyzed in deionized water (dialysis bag, molecular weight cutoff: 8–12 kDa) for 3 days to remove the excess sodium periodate, then frozen at − 20 ℃ and lyophilized at − 80 ℃ for 24 h. The degree of oxidation (DO) was determined by potentiometric titration, and DO value was 42.284 ± 0.897 %.
Synthesis of graphene oxide (GO)
GO was prepared using graphite powder via the modified Hummers method (Shahriary and Athawale, 2014). In brief, the graphite powder (1 g) and sodium nitrate (0.5 g) were added to sulfuric acid 98% (23 mL) under stirring. While using an ice bath and keeping the temperature at 0 °C (to prevent overheating and explosion), potassium permanganate (3g) was added gradually to the mixture under the same conditions. Subsequently, the mixture was stirred at 35 °C for 24 h. Then, the solution was diluted with distilled water, and the reaction was finally completed by adding 30% hydrogen peroxide (5mL). The resulting mixture was washed with distilled water until the filtrate showed neutral pH. The obtained solid was freeze-dried for 24 h.
Preparation of the OP/NCS/GO hydrogels
NSC and OP were dissolved in the phosphate-buffered saline (PBS, pH 7.4 and the room temperature for 5 h) separately to form a 3 wt. % solution. The various contents of GO (0mg/ml, 2mg/ml, 4mg/ml, and 6mg/ml) was dispersed into OP solutions (3 wt. %) with the aid of ultrasound. Then, the solutions were stored at 4℃ for further use. The preparation of OP/NSC/GO hydrogels was carried out using a system of two interconnected syringes. Equal volumes of the NSC and OP/GO solution were loaded into two separate Luer-Lock syringes, respectively, and then both syringes were connected via a connector. The solutions were thoroughly mixed by pressing alternately on each of the plungers (more than three times). Upon thorough mixing, the entire syringe contents were pushed into one of the syringes, the connector in conjunction with the empty syringe was disengaged, and the prepared hydrogel was ejected and liberally deposited in suitable mold (10 mm diameter) for further investigation. According to the difference in the contents of GO, the hydrogels were coded as OP/NSC/GO-0, OP/NSC/GO-2, OP/NSC/GO-4, and OP/NSC/GO-6 hydrogels. The procedures of synthesizing the hydrogels have been shown in Figs. 1 and 2.
Gelation time test
The gelation time of hydrogels was measured by the tube inversion method. A mixture of 1 ml of OP/NSC/GO solution was poured into a glass bottle with a diameter of 20 mm, and it was placed at room temperature. The gelatin time is determined by the time it takes for the mixture to stop flowing when the glass bottle is inverted.
Swelling measurements
To evaluation the swelling ratio, the same shape and size of freeze-dried hydrogel samples were weighed (Wd) and soaked in PBS (pH = 7.4) at 37°C. After 48 h, samples were removed and the water remaining on the surface was absorbed by filter paper. Then, the swollen hydrogel was immediately weighed (Ws) and the swelling ratio was calculated from the formula:
In vitro degradation analysis
The degradation of the OP/NSC/GO hydrogels was examined for the weight loss under the aqueous condition (PBS at 37oC) for 4 weeks. Firstly, the initial weight of lyophilized OP/NSC/GO hydrogels was accurately weighed (W0). After predetermined time intervals, hydrogels were removed from the medium and, they were freeze-dried. Then, the sample was accurately weighed and recorded as (W1). The PBS was replaced by fresh PBS every 3 days. All measurements were performed in duplicate. The following formula calculated the degradation ratios of the OP/NSC/GO hydrogels:
Characterizations
Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR–ATR) spectroscopy was carried out with a JASCO-4700 FTIR spectrometer, and the wavelength range was set at 500–4000 cm− 1 at room temperature. Scanning electron microscopy (SEM) and transmission electron spectroscopy (TEM) images were taken with a JEOL JSM-IT300S microscope (Tokyo, Japan), which works at a 5kV accelerating voltage and a JEM-100CX electron microscope, respectively. X-ray diffraction (XRD) analysis was performed using Philips PW1730 X-ray diffractometer with Cu Kα radiation (λ = 0.15405 nm) which operates at 40 kV and 40 mA. Data were collected from 5 to 50° 2θ at room temperature. The hydrogel (600µL) was formed in a cubic container, and an avometer was used to measure the conductivity using the electric circuit.
Rheological Analysis
Rheological properties of hydrogels were carried out on an Anton Paar MCR-302 rheometer at room temperature using a 25 mm diameter parallel plate. The frequency sweep test was analyzed over the range of 1–100 rad/s at a fixed strain rate of 1%, and in the strain sweep test, the strain ranged from 0 to 1000%.
Considering the strain sweep results, the self-healing properties were quantitatively evaluated by the damage-healing cycles which were continuous step switches from 1% (120 s for each interval) to 300% (60 s for each interval), with a constant frequency of 1 Hz at room temperature.
The viscosity of hydrogels was monitored at the different shear rates to characterize the injectability quantitatively. The viscosity-shear rate curves were obtained at room temperature, and the viscosity was recorded when the corresponding shear rate was in the range of 1-100 s− 1.
Hemolysis rate
Hemolysis testing of the prepared hydrogels was performed according to the previously reported procedure with slight modifications (Iqbal et al., 2017). Fresh human blood from a healthy donor was collected in 5ml EDTA Vacutainer. The freeze-dried hydrogel samples of OP/NSC/GO0, OP/NSC/GO2, OP/NSC/GO4, and OP/NSC/GO6 were ground into powder. 25 mg of the powdered sample was weighed and poured into the test tube, then 10 mL of normal saline was slowly added. All tubes were incubated at 37 °C) for 30 min. Subsequently, 0.1 mL EDTA blood was added to the test tube with a micropipette. After shocking and mixing, well, it was kept at a constant temperature at 37°C for 60 min. Then, the tubes were removed, and they were centrifuged at 1500 rpm for 10 min. The absorbance values of the supernatants at 545 nm were measured with an ultraviolet spectrophotometer using Lambda 25 UV/Vis spectrophotometer. The following formula calculated the hemolysis rate of hydrogel samples:
Where A1, A2, A3 are the absorbance of the hydrogel sample group, the positive control group (10 mL distilled water, 0.1 mL human EDTA blood, without hydrogel sample material), and the negative control group (10 mL normal saline, 0.1 mL human EDTA blood, without hydrogel sample material), respectively.
Cytotoxicity assay
Evaluation of cytotoxicity of the hydrogels was conducted using MTT (3-[4, [5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and in terms of ISO 10993-5:2009 (Biological evaluation of medical devices: Tests for in vitro cytotoxicity) using mouse fibroblast L-929 cells. Firstly, the prepared hydrogels were sterilized by UV irradiation for 2 h, then they were added to a 24-well culture plate. Also, L-929 cells with a density of 1.0 × 106 cells were seeded on each hydrogel to be assessed using the standard MTT test. After culturing for 24 h, MTT assays were conducted to test the cell growth. The wells without hydrogels were set as the controls.