Research on the construction and properties of waste cotton cellulose/carbon dots composite antibacterial hydrogel

The recycling and utilization of waste cotton fabrics is of great significance to implement green pollution reduction and carbon reduction. In order to realize the high value-added utilization of waste cotton cellulose (WCC) by introducing new carbon dots (CDs), WCC/CDs composite hydrogel with good antibacterial effect were prepared for the application in the field of antibacterial dressings. WCC/CDs composite hydrogel formed by doping different contents of carbon dots (CDs) with WCC hydrogel as raw material and epichlorohydrin (EPI) as crosslinking agent. The structure was characterized by X-ray diffraction (XRD) and infrared spectrum analysis, the antibacterial properties and swelling properties of WCC/CDs composite hydrogel were tested. The results show that the addition of CDs promoted the gelation of WCC hydrogel, enhanced its structure stability and endowed it with antibacterial properties. WCC/CDs composite hydrogel with 10wt% CDs was more stable, and its modulus reached 91 KPa. Compared with WCC hydrogel the swelling performance of WCC/CDs composite hydrogel was improved, and the swelling rate was 16.81 g·g-1. By destroying the cellular structure of bacteria and promoting the production of ROS, the inhibition rate of E. coil and S. aureus was more than 99%.


Introduction
With the development of science and technology and the rapid progress of consumer market, People's living standard has been continuously improved, and the consumption of textiles has gradually increased, thus causing a continuous increase in the number of waste textiles [1][2][3].By 2050, the amount of waste textiles is expected to reach 150 million tons.At present, the main recovery methods of waste cotton fiber are physical and chemical processing, compared with physical processing, chemical processing is more thorough, effective, and higher value-added products.However, due to the difference of cellulose varieties, it is difficult to realize the secondary utilization of cotton cellulose, no matter it is physical processing or chemical processing [4,5].In order to achieve the recycling and reuse of waste cotton fibers, converting the cellulose into hydrogel is one of the important ways of recycling and reusing waste cotton [6,7].Cellulose, as one of the most abundant natural polymers with good biocompatibility and degradability [8], is the best choice for hydrogel matrix, with two main advantages: (1) possessing a three-dimensional flexible network structure capable of encapsulating a high concentration of various guest molecules and nanoparticles [9,10]; (2) the abundance of hydroxyl groups on cellulose provides feasibility of synthesizing hydrogel with multiple structures [11].Therefore, cellulose bases hydrogel are valuable sustainable materials with important potential applications in biomedical fields such as cell therapy, wound healing, tissue transplantation, regenerative technologies and cellular drug delivery carriers [12][13][14].However, the poor mechanical strength and lack of active antimicrobial properties of conventional cellulose hydrogel limits its direct application in the biological field.The introduction of other polymers or inorganic materials by cross-linking can effectively enhance the physical properties of cellulose hydrogel, improve the usability and broaden its application areas while fully reflecting the microstructure and macroscopic properties of hydrogel [15].Among them, chemical cross-linking is a permanent connection formed by covalent bonds of polymers, providing stability close to that of natural hydrogel [16].The commonly chemical cross-linking agents are glutaraldehyde, formaldehyde, and epichlorohydrin (EPI).Besides, EPI is used to prepare hydrogel to increase the chemical stability, mechanical resistance, and adsorption/desorption capacity.Physical cross-linking is mainly a non-covalent bonding interaction to prepare cellulose hydrogel through intermolecular interaction forces or entanglement of molecular chains caused by hydrogen bonding, ionic coordination bonding, and hydrophobic interactions [17].Novel carbon nanomaterials have attracted much attention in the field of antimicrobials due to their excellent antibacterial activity and the fact that they are not easy to develop drug resistance [18,19].Among them, carbon dots (CDs) own good antibacterial effect, and its antibacterial mechanism is mainly caused by physical destruction, oxidative damage and photo-thermal effect on bacterial structure, and the abundant surface states of CDs enhance their interaction force with the substrate [20][21][22].Based on this, the aim of this paper is to convert waste cotton cellulose (WCC) into an antimicrobial hydrogel, and introduce CDs with good antimicrobial properties into the hydrogel structure by cross-linking to form the composite antimicrobial hydrogel of WCC/CDs with a view to achieving the dual purpose of enhancing the antimicrobial and mechanical properties of cellulosic hydrogel, thus broadening the application fields of cellulosic hydrogel.

Materials
Waste cotton cloth; carbon dots (CDs) were homemade; epichlorohydrin (EPI), potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) were purchased from Tianjin Damao Chemical Reagent Factory; sodium hydroxide (NaOH), urea, and were purchased from Tianjin Beichen Founder's Reagent Factory, and all of the above experimental medicines were analytically pure.2,7-dichlorofluorescein diacetate, E. coli (ACTT25922) and S. aureus (ACTT25923) were obtained from Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences.

Preparation of WCC/CDs composite hydrogel
Preparation of WCC solution: Firstly, waste cotton cloth of a certain size was processed by cutting, cleaning and drying.At the same time, the alkali/urea system solution was prepared according to the mass ratio of sodium hydroxide: urea: distilled water as 7:12:81.The above solution was cooled in a refrigerator at -18°C for 15 min.Finally, 2 g of waste cotton cellulose was put into the mixed solution to prepare a WCC solution with a concentration of 2wt%.Preparation of WCC/CDs composite hydrogel: CDs were added to 10 mL of WCC solution in different ratios.After mixing, EPI was added to the above solution, which was subsequently heated at 40°C to obtain a WCC/CDs composite hydrogel.

Testing and characterization
(1) Microstructural characterization: The WCC hydrogel and WCC/CDs composite hydrogel were taken and freeze-dried on conductive gel respectively.After gold spraying treatment, the hydrogel was observed by a JSM-6700F field emission scanning electron microscope with an accelerating voltage of 10 kV to obtain SEM images of the hydrogel.
(2) Crystal structure characterization: The phase composition and crystallization of the dried WCC powder were characterized by DX-2700BH X-ray diffractometer.The X-ray scanning range was 10-80° and the scanning speed was 8°/min.
(3) Antimicrobial performance testing: The solid agar plate method was used to incubate different contents of hydrogel with bacterial cell suspensions in phosphate buffer solution (PBS) to test their antibacterial activities and explore the optimal ratios of antibacterial hydrogel.WCC/CDs composite hydrogel were added to the suspension of E. coli (or S. aureus) containing 1.5×10 5 CFU/mL.After incubation at 37°C for 24 h, 100 μL of the above-treated bacterial suspension was spread on the surface of solid agar plates.After 24 h of incubation at 37°C, the number of colonies was observed and the antibacterial results were obtained.(4) Detection of ROS in bacterial cells: The ROS content in the cells was detected by fluorescence staining using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe.E. coli or S. aureus (OD600~1.0)were incubated with WCC/CDs composite hydrogel in a constant temperature shaker at 37°C for 2.5 h.Then, DCFH-DA probe was added into the above mixture containing bacteria and hydrogel at a volume ratio of 1:1000 and the incubation was continued for 20 min at a constant temperature shaker at 37°C.Then, the bacterial suspensions were rotated at 6500 rpm centrifuged for 3 min, rinsed with water and resuspended in water to the original volume.Finally, the fluorescence intensity of each group was measured separately by flow cytometry.
(5) Morphological characterization of bacteria: WCC/CDs hydrogel was added to E. coli suspensions (OD600~1) and incubated in a constant temperature shaker (160 rpm) at 37°C for 30 min.The above mixture was centrifuged (7000 rpm, 5 min) to remove the supernatant, and 500 μL of 2.5% glutaraldehyde aqueous solution was added to fix it overnight.Subsequently, dehydration was performed sequentially with ethanol in volume fractions of 30, 50, 70, 80, 90, 95, and 100%.Changes in E. coli morphology was observed under a scanning electron microscope.E. coli was replaced with S. aureus by the same operation.( 6) Mechanical performance test: The hydrogel was taken and tested for compressive mechanical properties in UTM-1422 universal testing machine at a compression rate of 2 mm/min.(7) Swelling property test: After lyophilizing the hydrogel, the weight of the dry gel was m0, and then it was put into a beaker containing 50 mL of distilled water, and the swelling performance test was carried out at 37°C.The hydrogel was taken out and weighed at intervals mt, and it was noted that the water on the hydrogel surface had to be sucked out before each weighing.This cycle, until the weight of the hydrogel basically does not change, that is, to reach the swelling equilibrium.The swelling rate (SR) of the hydrogel was calculated according to equation (1).

Microstructure of WCC/CDs composite hydrogel
The surface morphology of WCC hydrogel and WCC/CDs composite hydrogel is shown in Figure 1.
From Figure 1(a), it can be observed that the WCC hydrogel appears an obvious network structure, showing a lot of pores with high porosity and an uneven pore distribution, which is caused by the solvent sublimation of the hydrogel during the freeze-drying process.Relative to WCC hydrogel, WCC/CDs composite hydrogel had a rougher and denser surface structure (Figure 1(b)), and the porous structure turned out to be smaller pore sizes and the overall structure did not collapse, which was mainly due to the fact that a large number of hydrogen bonds formed between the abundant -NH2, -OH groups of CDs and -OH group of the cellulose.This in turn increased its cross-linking degree of WCC hydrogel.

Crystal structure analysis of WCC/CDs composite hydrogel
The X-ray diffractograms of CDs, WCC hydrogel, and WCC/CDs composite hydrogel are shown in Figure 2. A broad diffraction peak appears at around 2θ = 20.29°for CDs, which is the C (002) crystal plane, indicating that CDs is with the amorphous carbon structure [23].For the WCC hydrogel, the diffraction peak appearing in at 2θ = 22.3° corresponds to the cellulose (002) crystal plane, and there is a distinct diffraction peak of cellulose (002) at 2θ=34.90° [24].For WCC/CDs hydrogel, there is also a distinct cellulose (002) diffraction peak at 2θ = 34.90°,but the C (002) diffraction peak at 2θ = 20.29°has slightly enhanced compared to WCC hydrogel.In addition, the WCC and the WCC/CDs composite hydrogel had a significant diffraction peak at 47.10°, corresponding to the diffraction peak of NaOH .H2O, which is presumed to be the NaOH dissolve system, and it coincides with the standard card of NaOH .H2O (JCPDS 02-0706).The results showed that the WCC/CDs composite hydrogel was successfully prepared by loading CDs into WCC hydrogel.

Antimicrobial properties of WCC/CDs composite hydrogel
In order to investigate the effect of CDs content on the antimicrobial performance of WCC/CDs composite hydrogel, the antibacterial effect of WCC/CDs composite hydrogel on E. coil and S. aureus were presented in Figure 3. From the figure, it can be seen that the antimicrobial effect of WCC/CDs composite hydrogel strengthened with the increase of the content of CDs, and the antimicrobial inhibition rate can reach 99.7% for E. coil and 99.9% for S. aureus, when the mass fraction of CDs is 8%.The antimicrobial activity of the WCC/CDs composite hydrogel against both E. coil and S. aureus almost 100%.This was mainly due to the fact that CDs destroy the bacteria structure of E. coil and S. aureus as well as caused irreversible damage to the cells through oxidative stress.

Mechanical properties of WCC/CDs composite hydrogel
The stress-strain curves of the WCC hydrogel and WCC/CDs composite hydrogel are shown in Figure 6.The stresses of the hydrogel samples all increased with the increase of strain.At the same strain, the stresses rose with the introduction of CDs, and the inclination of the curves was significantly larger than that of the WCC hydrogel.When the strain was in the range of 60%-80%, the stress-strain curves varied nearly linearly, so the modulus of the hydrogel was calculated using the stress-strain curves in this stress range.Compared with WCC hydrogel, the modulus of WCC/CDs composite hydrogel increased from 18.45 KPa to 91 KPa when the content of CDs was 10%.And according to the theory of rubber elasticity, the modulus of the hydrogel is directly proportional to the number density of polymer chains in its network.As the number density of polymer chains depends on the number of crosslinking points and the volume fraction of the polymer network, the modulus of hydrogel is also proportional to the product of crosslinking points and the volume fraction of the polymer network [26].Therefore, the introduction of CDs is beneficial to the WCC hydrogel formation and improves the mechanical strength of traditional cellulose hydrogel, which is "soft and weak".

Swelling properties of WCC/CDs composite hydrogel
Hydrogel with excellent swelling performance is beneficial for absorbing wound exudate and avoiding bacterial growth on the wound.The swelling performance of WCC/CDs composite antimicrobial hydrogel affects the biological application of hydrogel.Therefore, the hydrogel was tested for swelling performance, and the swelling rate curves of WCC hydrogel and WCC/CDs composite hydrogel are shown in Figure 7.The WCC hydrogel can be rapidly swollen within 1 min, and the swelling rate reaches 9.21 g /g, and the swelling equilibrium is reached when the weight of the hydrogel no longer changes significantly around 90 min.The maximal swelling rate reaches 12.63 g /g.Compared to WCC hydrogel, the WCC/CDs composite antimicrobial hydrogel is more effective in absorbing wound exudate.The maximum swelling rate of WCC/CDs composite hydrogel was 16.81 g /g.The introduction of CDs increases the hydrophilicity of the hydrogel, which leads to an increase in the swelling rate of the hydrogel.This is due to the large number of hydrophilic functional groups of CDs such as carboxylic acid, hydroxyl, amino, and amide groups [27].In addition, the swelling ability of the hydrogel is directly proportional to the degree of cross-linking [28].The doping of CDs increased the degree of cross-linking of WCC hydrogel.2. When 10% CDs are added, the WCC/CDs composite hydrogel has excellent antibacterial performance, with an antibacterial rate of 100% against E. coli and S. aureus, which kills the cells mainly by destroying the bacterial cellular structure and causing oxidative damage through the production of ROS.
3. Compared with WCC hydrogel, the mechanical properties and swelling properties of WCC/CDs hydrogel also significantly enhanced.The WCC/CDs antimicrobial hydrogel shows potential application prospect in the fields of recycling waste textiles and medical antimicrobial dressings.

Figure 3 .
Figure 3. Antibacterial effect of composite hydrogel doped with different levels of CDs on E. coil and S. aureus.

Figure 4 .
Figure 4. Bacterial morphology of E. coli (a) and S. aureus (b) after antimicrobial hydrogel treatment.

3. 5 .
Effect of WCC/CDs composite hydrogel on the intracellular ROS of bacterialTreated with WCC/CDs antimicrobial hydrogel, reactive oxygen species (ROS) in E. coli and S. aureus were quantitatively characterized by flow cytometry.And the ROS in the cells were stained using a 2',7'dichlorodihydrofluorescein diacetate (DCFH-DA) probe, in which the strength of the mean fluorescence intensity (MFI) responded to the content of ROS in the cells[25], and the test results are shown in Figure5.Compared with the control group, the MFI increased from 127 to 686 in E. coli cells and from 146 to 523 in S. aureus cells after treatment with antimicrobial hydrogel, which means that the ROS produced in bacterial cells increased significantly.The above results indicated that the WCC/CDs can be hydrogel to promote the production of ROS in bacterial cells, and this promotional effect is mainly attributed to the introduction of CDs.The level of ROS was elevated, which can cause some irreversible damages to the bacterial cells such as leading to lipid peroxidation and inactivation of proteins to inhibit bacterial growth, thus killing the bacterial cells.

Figure 5 .
Figure 5. ROS content produced in E. coli and S. aureus cells.

Figure 7 .
Figure 7. Dissolution curves of WCC and WCC/CDs composite hydrogel in distilled water.
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