Materials
Pectin (from citrus peel) was supplied by Sigma-Aldrich. Silver nitrate (AgNO3, AR, ≥ 99.8%), calcium chloride (CaCl3, AR, ≥ 96.0%) and glycerin(C3H8O3, AR, ≥ 99.0%) were purchased from Xilong Scientific Co., Ltd. (Shantou, China). Nutrient broth for antibacterial activity test was purchased from Qingdao Hope bio-Technology Co., Ltd. (Qingdao, China). E. coli (strain number: 133264) and S. aureus (strain number: 186335) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China).
Preparation Of Pe-s And Pe-s/ag
Using deionized water, prepare 1 g/L NaOH solution, 0.5 mol/L AgNO3 solution, 2% CaCl2 solution and 3% glycerol solution for reserve, respectively. Hereafter referred to as NaOH solution, AgNO3 solution, CaCl2 solution and glycerol solution, respectively.
Preparation Of Pe-s
1, 1.5, 2 and 2.5 g of pectin were dissolved in 100 mL of deionised water and stirred magnetically for 30 min to make a homogeneous aqueous pectin solution. Afterwards, 50 mL of CaCl2 solution and 50 mL of glycerol solution were added and the cross-linking reaction was carried out with magnetic stirring for 30 min. After forming a homogeneous solution, the pectin sponge was poured into a 12-well plate and dried using a freeze dryer. The pectin sponges (PE-S) were named as PE-S1, PE-S2, PE-S3 and PE-S4, respectively.
Preparation Of Pe-s/ag
PE-S/Ag sponges were prepared according to the ratios in Table 1. After the pectin was added to the NaOH solution and stirred using magnetic force for 30 min to make a homogeneous pectin base solution, AgNO3 solution was added and magnetic stirring was maintained to allow the reaction solution to fully react. After the reaction was completed, anhydrous ethanol was added at a ratio of 1:2 between the reaction solution and anhydrous ethanol to allow sufficient precipitation of the pectin nanosilver complex. The precipitate was centrifuged at 10,000 r/min for 5 min. The precipitate was washed twice using anhydrous ethanol to remove unreacted AgNO3. 100 mL of deionised water was added to the precipitate and the solution was homogenised by magnetic stirring for 30 min,then 50 mL of CaCl2 solution and 50 mL of glycerol solution were added. After forming a homogeneous solution, it was poured into a 12-well plate and dried using a freeze-dryer to obtain a nano-silver pectin composite sponge. The PE-S/Ag sponges were named PE-S/Ag1, PE-S/Ag2, PE-S/Ag3 and PE-S/Ag4, respectively.
Table 1
Preparation ratios of pectin nano-silver complex materials.
Sample number | Pectin (g) | NaOH (mL) | AgNO3 (mL) |
PE-S/Ag1 | 1 | 200 | 4 |
PE-S/Ag2 | 1.5 | 300 | 6 |
PE-S/Ag3 | 2 | 400 | 8 |
PE-S/Ag4 | 2.5 | 500 | 10 |
Characterisation Of Pe-s And Pe-s/ag Sponges
Moisture absorption capacity evaluation
According to a previous method [12], equal volumes of sponge samples (volume approx. 1.5 cm in diameter and 1 cm in height) were pre-weighed at room temperature and weight marked as M1. The samples were immersed in PBS (pH 7.4) buffer. A gauze of the same quality was used as a control and removed after approximately 60 seconds and the residual buffer was blotted off the surface of the sample using filter paper. Immediately afterwards, the samples were weighed and marked as M2. The water absorption (L) was calculated by means of Eq. (1):
$$\text{L}\left(\text{\%}\right)=\frac{{M}_{2}{-M}_{1 }}{{M}_{1}}\times 100 \left(1\right)$$
Porosity
The porosity of the sponges was measured using previous methods [18–19]. An equal volume of sponge sample (volume approx. 1.5 cm in diameter and 1 cm in height) was taken at room temperature and the sample was pre-weighed and marked as M0. The sample was immersed in anhydrous ethanol and removed after about 60 seconds and the residual anhydrous ethanol was blotted off the surface of the sample with filter paper and weighed immediately afterwards and marked as M1. The porosity (P) was calculated by Eq. (2):
$$\text{P}\left(\text{\%}\right)=\frac{{M}_{1}{-M}_{0 }}{\rho V}\times 100 \left(2\right)$$
Where V (cm3) is the volume of the sponge and ρ (g/cm3) is the density of ethanol (0.7893 g/cm3).
In Vitro Biodegradation Experiments
According to previous methods [20], quantitative sponge samples (10 mg) were accurately measured and this weight was labelled M0, the samples were immersed in 10 mL of PBS (pH 7.4) containing a concentration of 2×104 U/mL of lysozyme and kept in incubation at 37 ℃. Nine sets of samples were prepared and removed at 1, 3, 5, 7, 9, 12, 18, 24 and 30 days of incubation, washed with de-anhydrous ethanol and dried and weighed, with the weight labelled Mt. The degradation rate (D) of the sponge samples was calculated by Eq. (3):
$$\text{D}\left(\text{\%}\right)=\frac{{M}_{0}{-M}_{t }}{{M}_{0}}\times 100 \left(3\right)$$
Solution Stability Testing
According to previous methods, equal volumes of PE-S and PE-S/Ag sponge samples were added to PBS (pH 7.4) buffer and photographed after 0 min, 1 min, 1 h and 48 h respectively to record the state of the sponge samples.
Material Characterisation
According to previous methods [18, 20], AgNPs in pectin colloidal solutions were tested using a UV-vis spectroscopy TU-1950 spectrophotometer (Beijing, China) and Fourier transform infrared spectroscopy (FT-IR, Thermo Fisher Scientific, USA) was performed in the scan range of 4000 to 500 cm− 1. The samples were heated from 10 to 800 oC with a thermogravimetric analyser (TGA, SDTQ600) in a nitrogen atmosphere (20.0 mL/min) at a heating rate of 10 oC/min. X-ray diffraction (XRD, PANalytical B.V., X'Pert3 Powder) 2θ scans ranging from 30°-80°. The microstructures of PE-S and PE-S/Ag sponges were analysed using scanning electron microscopy (SEM, S-5000, Hitachi Co., Ltd., Matsuda, Japan) and energy dispersive X-ray spectrometry (EDS). The elements in the composite sponges were examined using X-ray photoelectron spectroscopy (Thermo Fisher Scientific K-Alpha, USA). The specific surface area of the sponge samples was determined using a specific surface area pore size analyser (BET, ASAP 2460, Micromeritics, USA).
Antibacterial Test
Preparation of bacterial suspensions
According to previous methods [15–16], LB (Lennox broth) broth liquid medium was sterilized at 121 oC for 20 min. E. coli and S. aureus were inoculated, and the activated bacteria solution was shaked in an oscillating incubator at 37 oC for 18–24 h.
Bacterial suspension dilution: Take 0.1 mL of activated bacterial suspension and add it to 29.9 mL of sterile water, which is diluted 300 times to obtain a concentration of 107 CFU/mL of bacterial suspension.
Minimum Inhibitory Concentration (Mic)
The PE-S/Ag sponge samples were dispersed in 5 mL LB broth at a concentration of 8 mg/mL. The concentration gradients prepared by using LB were: 0, 0.5, 1, 2, 4, and 8 mg/mL. 0.2 mL LB broth, 0.05 mL sample broth and 0.05 mL bacterial suspension were then mixed completely and added to a 96-well plate. The concentrations of the PE-S/Ag sponge samples in the system were 0, 21, 42, 83, 167, 333 and 667 µg/mL. The incubation times were controlled at 0, 3, 6, 9, 12, 15 and 24 h, respectively.
Cytotoxicity Assay
Based on previous methods[21], LO2 cells (concentration: 1×105) were grown in 96-well plates and 100 µL of samples (PE-S and PE-S/Ag) were taken to replace DMEM medium with samples at concentrations of 5, 10, 20, 30 and 40% for 24h, 48h and 72h. CCK-8 (5 µL/well) was next added to the wells and absorbance at 450nm was measured after 1 hour Promotion of LO2 cells by sponge samples.
Whole Blood Clotting
The blood clotting experiment was referred to the previous reference[22]. A 10mg sponge sample was placed in a test tube and heated in a 37°C water bath for 5min. 0.2 mL of blood sample (0.2 mL rabbit whole blood, 0.1 mL 0.1mol/L CaCl2) was placed in the test tube. After a 37°C water bath for 1, 2, 4, 6 and 10 min, 10 mL of deionised water was slowly added and soaked for 30 s. 5 mL of the liquid was removed and centrifuged at 1000 r/min for 1 min and the supernatant was placed in a test tube and stored at 37°C for 60 min. The absorbance of the solution was measured at 542 nm and labelled as A1. The absorbance value of the same concentration of whole blood in deionised water was used as control A0. The blood clotting index (BCI) of the sponge was calculated by Eq. (4):
$$\text{B}\text{C}\text{I}\left(\text{\%}\right)=\frac{{\text{A}}_{1}}{{\text{A}}_{0}}\times 100 \left(4\right)$$
Pe-s/ag Sponge Ag Release Curve
Based on previous methods [23], the Ag+ release pattern of PE-S/Ag sponges was investigated. 10 mg of PE-S/Ag samples were accurately weighed in 50 mL of PBS buffer and incubated at 37 oC in an oscillating incubator. After incubation for 3, 6, 9, 12, 24, 48, 72 and 96 hours, 5 mL of the sample solution was taken and filtered using a 0.22 µm membrane to obtain the sample Ag+ clear solution. Finally, 5 mL of PBS buffer was re-added to the original reaction solution and the incubation continued. The concentration of Ag+ in the solution was determined by inductively coupled plasma emission spectrometry (ICP-OES).
Statistical analysis
Each experiment was repeated at least 3 times and the results were shown as mean ± stand deviation (SD) and analyzed by Student t-test and ANOVA test. A value of P < 0.05 was considered statistically.