Preparation method and application of tung oil smoke nanocomposite hydrogel

Abstract

The invention discloses a preparation method and application of a tung oil smoke nanocomposite hydrogel, comprising: adding AgNO3 solution to tung oil smoke, ultrasonic treatment, then adding N,N′-bis(acryloyl)cystamine, ultrasonic dissolving, and obtaining a CWOS@Ag+@BACA nanocomposite crosslinker; adding acrylamide to the CWOS@Ag+@BACA nanocomposite crosslinker, ultrasonic dissolving, adding a thermal initiator, ultrasonicating in an ice bath, and obtaining a CAB hydrogel precursor solution; heating the CAB hydrogel precursor solution in an oven to obtain a CAB hydrogel. The nanocomposite hydrogel prepared by the invention has excellent mechanical properties, photothermal conversion and antibacterial properties, and has higher biocompatibility. Without introducing drugs such as antibiotics, the purpose of promoting wound healing is achieved by relying on the advantages of the hydrogel material itself, providing a new idea for acute wound dressings, and having broad clinical application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to a preparation method and application of tung oil smoke nano composite hydrogel. Background Art

[0002] Wound healing is a long and complex process, which also faces the long-term threat of bacterial infection. Its healing mainly includes hemostasis, inflammation, proliferation, and remodeling. In the process of healing, it may be infected by bacteria. Once the number of bacteria in the wound exceeds a critical threshold of 10 5 When the wound is infected, it may turn into a systemic infection and may even lead to death. At present, gauze is the most widely used dressing in clinical practice, but gauze alone cannot achieve the antibacterial and antibacterial effects, which is insufficient for preventing wound infection. For this reason, non-antibiotic substances with antibacterial properties are added to the dressing, which can inhibit the growth and infection of bacteria to a certain extent, such as chitosan, silver and zinc oxide. However, problems related to their solubility and cytotoxicity limit the widespread use of these materials. In addition, high cost and low biocompatibility also limit their development and application in clinical practice. Therefore, it is necessary to study a wound dressing with good biocompatibility, low cost, microbial barrier and effective promotion of wound healing.

[0003] Hydrogels are widely considered to be the best choice among various wound dressing materials due to their excellent flexibility, elasticity, biocompatibility, high water content, and responsiveness to physiological conditions, as well as the ability to provide a microenvironment similar to the extracellular matrix (ECM). However, it is reported that hydrogels used for wound dressings often exhibit unsatisfactory mechanical properties and are susceptible to external damage. In order to overcome these problems, researchers have proposed different strategies to construct hydrogels with better mechanical properties. For example, nanocomposite hydrogels, which are usually composed of organic and inorganic components and have complex nanoscale structures, can effectively combine the unique properties and structures of inorganic or organic components. Thanks to the introduction of nanoparticles, in addition to enhancing the mechanical properties of hydrogels, hydrogels are also given more properties. However, the introduced nanoparticles have disadvantages such as biocompatibility, cumbersome preparation process and high cost, which limit their wide application. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing tung oil fume nanocomposite hydrogel. The nanocomposite hydrogel prepared by this method has excellent mechanical properties, photothermal conversion and antibacterial properties, providing a new direction for clinical wound dressings.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing tung oil smoke nanocomposite hydrogel comprises the following steps:

[0007] (1) Adding AgNO3 solution to tung oil smoke and ultrasonic treatment to obtain CWOS@Ag+ nanoparticles;

[0008] (2) adding N,N′-bis(acryloyl)cystamine to CWOS@Ag+ nanoparticles and dissolving them by ultrasonication to obtain CWOS@Ag+@BACA nanocomposite crosslinker;

[0009] (3) adding acrylamide to the CWOS@Ag+@BACA nanocomposite crosslinker, dissolving it by ultrasonication, adding a thermal initiator, and ultrasonicating it in an ice bath to obtain a CAB hydrogel precursor solution;

[0010] (4) The CAB hydrogel precursor solution is heated in an oven to obtain a CAB hydrogel.

[0011] The method of the present invention takes natural nanoparticles of tung oil smoke with a negative charge on the surface as the core, and through electrostatic interaction with silver ions, the silver ions are wrapped on the surface of the tung oil smoke to obtain CWOS@Ag+ nanoparticles; then N,N′-bis(acryloyl)cystamine is introduced, and the CWOS@Ag+@BACA nanocomposite crosslinker is formed through coordination with the silver ions; finally, a thermal initiator and an acrylamide monomer are introduced, and the thermal initiator generates a large number of free radicals at high temperature, which are polymerized with the acrylamide monomer, and a nanocomposite hydrogel is prepared under the connection of the nanocomposite crosslinker CWOS@Ag+@BACA. The nanocomposite hydrogel has a stable and uniform three-dimensional network structure, and exhibits excellent mechanical properties, photothermal conversion and antibacterial properties.

[0012] Preferably, in step (1), the ultrasonic treatment time is 20-40 min.

[0013] Preferably, in step (2), the mass ratio of tung oil smoke to AgNO3 is 1-5:2-3, preferably 1:2.78.

[0014] Preferably, in step (3), the mass ratio of the thermal initiator to acrylamide is 3-15:1000, preferably 15:1000.

[0015] Preferably, in step (3), the thermal initiator is one or more of potassium persulfate, ammonium persulfate, azobisisobutyronitrile or cumene hydroperoxide.

[0016] Preferably, in step (3), the temperature of the ice bath is ≤15°C, and the ultrasonic time is 2-5 min.

[0017] Preferably, in step (4), the temperature of the oven is 60-70° C., and the heating time is 5-6 h.

[0018] The present invention also provides a tung oil fume nanocomposite hydrogel, which is prepared by the above-mentioned preparation method. The tung oil fume nanocomposite hydrogel prepared by the present invention has a three-dimensional pore structure, and the pores of its microstructure are uniform in size and evenly distributed.

[0019] The present invention also provides the use of the above-mentioned tung oil fume nanocomposite hydrogel in the preparation of a drug for treating acute wound healing. Through in vitro biocompatibility and antibacterial experiments, the tung oil fume nanocomposite hydrogel prepared by the present invention exhibits excellent biocompatibility and outstanding antibacterial properties, and exhibits a significant effect of promoting wound healing in a rat acute wound model.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The method of the present invention integrates natural nanoparticles of tung oil smoke with negative charges on the surface as a cross-linking agent into biocompatible nanomaterials, and forms a nanocomposite hydrogel through in situ polymerization. The introduction of tung oil smoke gives the hydrogel the ability of photothermal conversion. Under near-infrared irradiation, it absorbs light energy and converts heat energy and synergizes with silver ions in the hydrogel, showing excellent antibacterial properties.

[0022] The nanocomposite hydrogel prepared by the present invention effectively improves its mechanical properties and has higher biocompatibility. Without introducing drugs such as antibiotics, the purpose of promoting wound healing is achieved by relying on the advantages of the hydrogel material itself, providing a new idea for acute wound dressings and having broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the SEM image of CWOS;

[0024] Figure 2 TEM image of CWOS;

[0025] Figure 3 TEM image of CWOS@Ag+;

[0026] Figure 4 This is the SEM image of the internal structure of CAB hydrogel;

[0027] Figure 5 EDS images of S and Ag elements in CAB hydrogel;

[0028] Figure 6 The effects of different formulations on the adhesion of hydrogels;

[0029] Figure 7 The temperature rise curve of CAB hydrogel under different powers;

[0030] Figure 8The total blood loss of the hydrogel attached to the injured liver;

[0031] Fig. 9 The diameter of the inhibition zone of the hydrogel for E.coli, S.aureus, and C.albicans;

[0032] Fig.10 Results of CCK-8 test on HaCaT cells with different concentrations of CWOS;

[0033] Fig.11 This is a trend chart of rat wound healing rate;

[0034] Fig.12 HE staining images of skin tissues in different experimental groups. DETAILED DESCRIPTION

[0035] The present invention is further described below by means of specific implementation methods. The following examples are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0036] The sources of the raw materials used in the embodiments of the present invention are shown in Table 1 below:

[0037] Table 1

[0038]

[0039] Example 1: Preparation of a tung oil smoke nanocomposite hydrogel (CAB)

[0040] (1) 1.0 mg of CWOS was weighed and placed in an EP tube, and then 2.0 mL of AgNO3 (1.39 mg / mL) and 3.0 mL of deionized water were added dropwise to the EP tube, and the tube was placed in an ultrasonic cleaner (power 300 W, frequency 40 kHz) for 30 min to obtain CWOS@Ag+ nanoparticles coated with a large amount of Ag+;

[0041] (2) After the ultrasonic treatment, 2.0 mg of BACA was weighed and placed in an EP tube, and ultrasonic treatment was performed again for 6 min to completely dissolve it, thereby obtaining a CWOS@Ag+@BACA nanocomposite crosslinker;

[0042] (3) Weigh 1.0 g of AM and place it in an EP tube and sonicate for 3 min to completely dissolve it; weigh 15 mg of KPS and place it in an EP tube, add a certain amount of ice cubes and place it in an ultrasonic machine, and sonicate for 3 min in an ice bath (≤15°C) to obtain a CAB hydrogel precursor solution;

[0043] (4) The obtained CAB hydrogel precursor solution was divided into a vial and placed in an oven (60° C.) for heating for 6 h to obtain a CAB hydrogel.

[0044] The shape, surface morphology, and agglomeration degree of CWOS and CWOS@Ag+ nanoparticles were observed by SEM and TEM images. Figure 1-2 As shown in the figure, the tung oil smoke CWOS particles are uniform spherical particles with a particle size of about 50nm, and there is a certain aggregation phenomenon. When Ag+ is introduced, Ag+ is wrapped on the surface of CWOS under the action of electrostatics, which makes CWOS well dispersed to a certain extent, such as Figure 3 shown.

[0045] The prepared CAB hydrogel was placed in a -20°C refrigerator for 24 hours, and the freeze-dried sample was cut into a cross section with a knife, then subjected to gold spraying, and its internal structure morphology was observed under a scanning electron microscope (SEM). Figure 4 As shown, CAB hydrogel has a three-dimensional pore structure, and its microstructure pore size is uniform and evenly distributed.

[0046] The distribution of Ag+ and S elements in CAB hydrogel was observed by elemental analysis (EDS). Figure 5 , they are uniformly dispersed in the hydrogel due to the coordination effect of S and Ag.

[0047] Comparative Example 1: Preparation of MBA hydrogel

[0048] (1) Weigh 1.0 g of AM and place it in an EP tube. Then, add 5.0 mL of deionized water into the EP tube and place it in an ultrasonic cleaner (power 300 W, frequency 40 kHz) for 3 min.

[0049] (2) After the ultrasound was completed, 2.0 mg of MBA was weighed and placed in an EP tube, and ultrasound was performed again for 6 min to completely dissolve it;

[0050] (3) After the ultrasound is completed, 15 mg of KPS is weighed and placed in an EP tube, and a certain amount of ice is poured into the tube and placed in an ultrasound machine. The tube is ultrasounded in an ice bath (≤15°C) for 3 min to obtain an MBA hydrogel precursor solution.

[0051] (4) The obtained MBA hydrogel precursor solution was divided into a vial and placed in an oven (60° C.) for heating for 6 h to obtain MBA hydrogel.

[0052] Comparative Example 2: Preparation of CB hydrogel

[0053] (1) Weigh 1.0 mg of CWOS and place it in an EP tube. Then, add 5.0 mL of deionized water into the EP tube and place it in an ultrasonic cleaner (power 300 W, frequency 40 kHz) for 30 min.

[0054] (2) After the ultrasound was completed, 2.0 mg of BACA was weighed and placed in an EP tube, and the tube was ultrasounded again for 6 min to completely dissolve it;

[0055] (3) After the ultrasonic treatment, 1.0 g AM was weighed and placed in an EP tube for 3 min to completely dissolve it; 15 mg KPS was weighed and placed in an EP tube, and a certain amount of ice cubes were poured into the tube and placed in an ultrasonic machine. The tube was ultrasonicated in an ice bath (≤15°C) for 3 min to obtain a CB hydrogel precursor solution;

[0056] (4) The obtained CB hydrogel precursor solution was divided into a vial and placed in an oven (60° C.) for heating for 6 h to obtain a CB hydrogel.

[0057] Comparative Example 3: Preparation of AB hydrogel

[0058] (1) Weigh 1.0 g of AM and place it in an EP tube. Then, add 2.0 mL of AgNO3 (1.39 mg / mL) and 3.0 mL of deionized water into the EP tube and place it in an ultrasonic cleaner (power 300 W, frequency 40 kHz) for 3 min.

[0059] (2) After the ultrasound was completed, 2.0 mg of BACA was weighed and placed in an EP tube, and the tube was ultrasounded again for 6 min to completely dissolve it;

[0060] (3) After the ultrasound is completed, 15 mg of KPS is weighed and placed in an EP tube, and a certain amount of ice is poured into the tube and placed in an ultrasound machine. The tube is ultrasounded in an ice bath (≤15°C) for 3 min to obtain an AB hydrogel precursor solution;

[0061] (4) The obtained AB hydrogel precursor solution was divided into a vial and placed in an oven (60° C.) for heating for 6 h to obtain AB hydrogel.

[0062] Example 2: CAB hydrogel formulation screening test

[0063] CAB hydrogel was prepared according to the preparation method of Example 1, and the effect of the amount of each component on the tensile and adhesive properties of the hydrogel was investigated and the prescription was screened. The prescription ratio is shown in Table 2.

[0064] Table 2

[0065]

[0066] (1) Cut the prepared hydrogels 1, 2, 3, 4, 5, and 6 into rectangular strips with a length: width: height of 15:5:3 mm for later use;

[0067] (2) The two ends of the cut hydrogel were clamped on a 50N force sensor on a computerized tensile and compression testing machine, and the prepared hydrogel was subjected to a tensile test at room temperature at a tensile speed of 100 mm / min. Each group of samples was tested three times in parallel to investigate the effect of different CWOS and KPS on the tensile properties of the hydrogel, as well as the tensile properties of the prepared hydrogel.

[0068] (3) Similarly, the prepared hydrogels 1, 2, 3, 4, 5, and 6 were cut into hydrogel sheets with a length: width: thickness = 10:10:3 mm respectively.

[0069] (4) The hydrogel was bonded between two pieces of clean pig skin to ensure tight adhesion between the prepared hydrogel and the pig skin, which was previously bonded and fixed on the sensor. The samples were stretched at a speed of 50 mm / min at room temperature. Each group of samples was stretched 3 times in parallel to investigate the effects of different concentrations of CWOS and KPS on the adhesion properties of the hydrogel, as well as the adhesion properties of the prepared hydrogel.

[0070] The results are shown in Table 3. The stress-strain of hydrogel 3 reached the best (1.88MPa, 1986.21%). In the case of no addition of Ag+, but only adding CWOS and BACA to mix the CB hydrogel (hydrogel 6), its stress-strain was (0.51MPa, 708.60%), which was far lower than the mechanical properties of the prepared CAB nanocomposite hydrogel.

[0071] At the same time, for the investigation of adhesion performance, such as Figure 6 As shown, hydrogel 3 exhibits the strongest bonding strength among all proportions, reaching 34.72±2.34KPa; without the addition of Ag+, the bonding strength of the CB hydrogel (hydrogel 6) prepared at the same proportion only reached 22.40±2.40KPa. The results show that when silver ions are not introduced, CWOS is only physically mixed and does not form a nanocomposite crosslinker with BACA. The mechanical properties of the hydrogel constructed by the macromolecular CWOS@Ag+@BACA nanocomposite crosslinker are significantly better than those of the hydrogel constructed by the small molecule BACA crosslinker. Therefore, hydrogel 3 is preferred as the preferred formulation of the CAB hydrogel of the present invention.

[0072] Table 3

[0073]

[0074] Example 3: Performance investigation of CAB hydrogel

[0075] 1 Main reagents and instruments (see Table 4)

[0076] Table 4 Reagents and consumables used in the experiment

[0077]

[0078] 2 Test methods

[0079] 2.1 Investigation of photothermal conversion performance

[0080] The prepared CAB hydrogel was irradiated with near-infrared light of 808 nm, and the temperature rise of the hydrogel was recorded using an infrared thermal imager.

[0081] First, the prepared hydrogel was cut into gel slices with a length: width: height = 10:10:3 mm. Then, the cut gel slices were placed on a glass plate, and a near-infrared lamp was placed about 15 cm above the gel slice for irradiation. At the same time, the distance between the thermal imager and the gel slice was adjusted to be appropriate so that a clear infrared thermal image can be presented and its temperature change can be recorded. 2 , 1.0W / cm 2 , 1.5W / cm 2 All the above experimental data were processed by Smart View.

[0082] The results are as follows Figure 7 As shown, at 0.5, 1.0 and 1.5 W / cm 2 When irradiated with the power of 0.5W / cm 2 When the temperature rises after 6 minutes of irradiation, it is not very obvious, but at 1.0 and 1.5 W / cm 2 When NIR was applied, the temperature began to rise rapidly, indicating that the hydrogel had excellent photothermal conversion ability. When the local temperature exceeded 50°C, it would physically damage the bacteria and kill the microorganisms through thermal damage. At about 100s, the temperature of the CAB hydrogel tended to stabilize at 1.0W / cm 2 Under irradiation, the hydrogel can heat up to about 60°C.

[0083] 2.2 Hemostatic performance investigation

[0084] The in vivo hemostatic performance experiment was conducted using a liver bleeding model of adult male SD rats (200-220g). First, the experimental rats were weighed in turn, and then injected with 20% urethane (0.6-0.7mL / 100g) to anesthetize the rats. After the rats fell into a coma, they were fixed on a 30° tilted operating board. The abdominal skin was disinfected with 75% ethanol, and then the skin was carefully cut open with surgical scissors to expose the liver. After wiping the body fluid on the surface of the liver, the pre-weighed filter paper was placed under the liver to ensure that it was not soaked by other liquids. Then, a 10mL syringe with a No. 7 needle was used to carefully create an artificial wound with a diameter of about 2.0mm on the rat liver, and then different types of hydrogels MBA, CB, and CAB prepared in advance were immediately applied to the wound, and optical photos were taken at 0s, 30s, 60s, 90s, and 120s to record the hemostasis. After the end, the filter paper was weighed and the hemostasis of each gel was counted.

[0085] The results are as follows Figure 8 As shown, compared with MBA, CB and blank groups, CAB hydrogel can effectively inhibit continuous bleeding of the liver, so that the total blood loss of the liver is only 35.97±2.33 mg, showing excellent hemostatic performance.

[0086] 2.3 Antibacterial performance investigation

[0087] Use a pipette to draw 100 μL (CFU = 10 7 ) was added dropwise to the culture dish of the solid culture medium, and the bacterial solution was evenly coated on the surface of the solid culture medium with a sterilized applicator. The culture dish was rotated during the coating step to make the coating more uniform. Subsequently, the CB (without Ag+), AB (without CWOS) and CAB hydrogels prepared in the embodiment and the comparative example were divided into 4 groups, namely, the CB, AB, CAB and CAB-NIR groups were carefully taken out after the coating was completed, and placed in the center of the culture dish after coating. At the same time, the CAB-NIR group was irradiated with near-infrared light for 8 minutes. After it was stably adhered to the culture medium, the dish was covered with a lid, sealed with a sealing film and disinfected with alcohol, placed in an incubator for 24 hours, and then taken out for observation. The size of the inhibition zone was calculated by Image J to evaluate its antibacterial effect. SPSS18.0 analysis software was used for data statistical analysis, and Origin 2018 was used to create the result data chart. Measurement data were expressed as mean ± standard deviation (x ± s), and one-way analysis of variance (ANOVA) was used for inter-group comparison. Dunnett's Test was used for inter-group comparison when the data were in accordance with normal distribution, and rank sum test was used when the data were not in accordance with normal distribution. P < 0.05 was considered statistically significant.

[0088] The results are as follows Fig. 9 As shown in the figure, it can be seen from the CB, AB and CAB groups that CWOS itself has no antibacterial effect, so the size of the inhibition zone of the CB group is 0, and its antibacterial ability mainly comes from Ag+. The antibacterial experimental results for E. coli, S. aureus and C. albicans show that the AB, CAB and CAB-NIR groups all show excellent antibacterial properties and have broad-spectrum antibacterial properties. By comparing the AB, CAB and CAB-NIR groups, it can be seen that under the irradiation of near-infrared light, due to the excellent photothermal conversion ability of CAB hydrogel, the CAB hydrogel that converts light energy into heat energy shows a synergistic antibacterial effect with Ag+. Under the synergistic effect of photothermal and Ag+, its antibacterial effect is significantly stronger than that of AB containing only Ag+ and CAB hydrogel without near-infrared light irradiation.

[0089] In summary, the CAB nanocomposite hydrogel designed based on the CWOS@Ag+@BACA nanocomposite crosslinker exhibits excellent photothermal conversion performance, hemostasis and antibacterial properties, and is expected to meet application requirements as a medical dressing.

[0090] Application Example 1:

[0091] 1 Experimental animals

[0092] Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Anhui University of Chinese Medicine and approved by the Animal Ethics Procedures and Guidelines of the People's Republic of China (Animal Ethics Number: AHUCM-rats-2019001). Male Sprague-Dawley (SD) rats, weighing 250 ± 50 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with an experimental animal production license number of SCXK (Liao) 2020-0001. Rats were grouped under standardized conditions in animal facilities, with 3 rats in each group (25 ° C, 12 h light-dark cycle). They were fed standard laboratory food and water ad libitum. This was used for the establishment of a full-thickness wound model in rats and the application of CAB hydrogels in the later stage.

[0093] 2 Test methods

[0094] 2.1 CAB hydrogel biocompatibility test

[0095] CAB hydrogels with different CWOS concentrations were prepared according to the method of Example 1. First, CAB hydrogels with different CWOS concentrations (0.04, 0.2, 1.0 mg / mL) were soaked in cell culture medium and co-cultured for 24 hours. At the same time, the HaCaT cell suspension was inoculated in a 96-well plate, with 100 μL (0.5×104~1.0×104) in each well, and placed in a cell culture incubator for 24 hours. After the culture, 100 μL of the gel soaking solution was aspirated into a 96-well plate, 6 replicates were set for each group, and continued to be placed in an incubator for 24 hours. After the culture was completed, the excess liquid in the 96-well plate was aspirated, and 10 μL of CCK-8 reagent was added thereto. It was placed in an incubator for incubation for 1-2 hours and then taken out. The absorbance of each group at a wavelength of 450 nm was detected by an ELISA instrument to evaluate the cytotoxicity of the CAB hydrogel.

[0096] The cell viability calculation formula is as follows:

[0097]

[0098] Where As is the absorbance of the experimental well (culture medium containing cells, CCK-8, and the drug to be tested); Ac is the absorbance of the control well (culture medium containing cells, CCK-8, and no drug to be tested); Ab is the absorbance of the blank well (culture medium without cells and the drug to be tested, CCK-8).

[0099] The results of CCK-8 test were as follows Fig.10 As shown in the figure, the cell survival rate of hydrogels prepared with different concentrations of CWOS was higher than 100%. However, when the CWOS concentration was gradually increased to 1.0 mg / mL, there was no significant difference in cell survival rate compared with the blank group. According to the cytotoxicity grading standard (ISO 10993.5-2009), when the RGR value is greater than 75%, it is considered to be non-cytotoxic.

[0100] 2.2 In vivo wound healing test of CAB hydrogel

[0101] Each rat was weighed and injected with 20% urethane intraperitoneally at 0.6-0.7 mL / 100 g. After the rat was fully anesthetized, its limbs were fixed on the mouse board, the hair on the back of the rat was removed, and then an appropriate amount of depilatory cream was evenly applied. The depilatory cream on the back was removed after 10 minutes of depilation. After the hair was removed, the back was wiped clean with 70% alcohol, and a full-thickness acute wound with a diameter of about 10 mm was artificially made on the clean area of ​​the rat's back with a circular punch. The animals were randomly divided into a control group, a CAB group, and a CAB-NIR group. Among them, the wounds of the rats in the control group did not receive any wound auxiliary treatment during the entire animal experiment; the wounds of the rats in the CAB group were applied with CAB hydrogel, which was removed and replaced with new CAB hydrogel every 24 hours, and no other wound auxiliary treatment was performed; the wounds of the rats in the CAB-NIR group were applied with CAB hydrogel and the surface of the gel was exposed to a near-infrared lamp of 1.0 W / cm 2 The wound was irradiated for 8 min, and the CAB hydrogel was removed and replaced every 24 h and irradiated for 8 min using near-infrared light. No other wound treatment was performed. During the experiment, the wound healing of the rats was observed daily and photographed on days 0, 4, 8, and 12. The wound area was analyzed and processed using Image J software, and the wound healing rate was calculated using the following formula:

[0102]

[0103] S0 is the wound area of ​​rats on day 0, S t It is the size of the wound area that has not healed on the corresponding days.

[0104] SPSS 18.0 analysis software was used for statistical analysis, and Origin 2018 was used to create the result data chart. The measurement data were expressed as mean ± standard deviation (x ± s), and one-way analysis of variance (ANOVA) was used for inter-group comparison. When the data were in accordance with the normal distribution, Dunnett's Test was used for inter-group comparison; when the data were not in accordance with the normal distribution, the rank sum test was used. P < 0.05 was considered statistically significant.

[0105] The results are as follows Fig.11As shown in the figure, the wound healing rates of the CAN-NIR group and the CAB group were significantly higher than those of the control group. On the 4th day, the wound healing rates of the CAN-NIR group and the CAB group reached 60.94% and 44.42%, respectively, while the wound healing rate of the control group was 16.28%. On the 12th day, the experimental group almost recovered completely, with wound healing rates of 98.11% and 92.14%, respectively, while blood scabs were still visible in the wounds of the control group, and the healing rate was only 85.61%. The CAB-NIR group was 12.5% ​​ahead of the control group, and the difference was statistically significant (P<0.01).

[0106] 2.3 Wound tissue staining

[0107] On the 12th day, the skin tissue of the rat wound site was carefully removed, cleaned with saline, and the excess saline was absorbed with filter paper. It was then placed in the pre-prepared fixative and stored at room temperature overnight for use in frozen sectioning.

[0108] Carefully remove the skin tissue from the fixative and rinse it several times with PBS solution until it is clean. Then soak it in 75% ethanol for 12 hours, dehydrate it with 85% ethanol for 1 hour, then dehydrate it with 95% ethanol for 30 minutes, dehydrate it twice, and then dehydrate it with anhydrous ethanol for 25 minutes, dehydrate it twice. After the end, put it in xylene to make it transparent for 30 minutes, and do it twice. After the dehydration and transparency are completed, the tissue is embedded. First, put the tissue into melted soft wax (melted at 58°C), let it stand for about 90 minutes, then put it into hard wax (65°C), let it stand and cool and solidify for about 2 hours to form a block, and finally use a tissue embedding machine to embed the tissue to form a wax block, take it out and store it in a 4°C refrigerator, and take it out when slicing. When slicing, fix the embedded tissue wax block on the paraffin slicer, adjust the slice thickness to about 5μm and cut it into thin slices. Place the slices carefully and gently on the slide and bake them in a 37℃ oven for 12h to allow the horizontal volatilization to be complete. After completion, take them out and place them in a slice box for storage at room temperature until they are ready for staining.

[0109] Take out the slices prepared in the early stage and place them in a 66℃ oven for 20-30min, then take them out and soak them in xylene for 5min, soak them 3 times, then pass through (100%, 95%, 85%) ethanol in turn, pass through for 3min each time, finally put the slices in a beaker, rinse slowly with running water, wash away the ethanol, until the slices are clean and transparent. After the end, take them out and dye them in hematoxylin (H) dye solution for 2-5min, then differentiate them with 1% hydrochloric acid alcohol for a few seconds, rinse them with running water, then blue them with saturated lithium carbonate solution for a few seconds, rinse them with running water; then dehydrate the slices in 95% ethanol for 2min, shake them up and down several times. After the end, dye them in eosin (Eosin, E) dye solution for a few seconds, then pass through 95% ethanol for 5-10s each, and dehydrate them in 100% ethanol for 1min each. After the end, pass through phenol-xylene, xylene I, and II to make them transparent, each for 2min. After the xylene evaporates, drop neutral gum on the slide and cover with a coverslip to complete HE staining.

[0110] On day 12, H&E staining was used to determine the morphological status of the regenerated tissue. Fig.12 As shown. On the 12th day, the epidermis of the control group had not grown completely and there were still gaps, while the epidermis of the CAN-NIR and CAB groups had grown completely, and the stratum corneum and the basal layer at the bottom of the epidermis could be observed. Basal cells, epidermal stem cells, can proliferate and differentiate, while new cells continue to move toward the surface and differentiate into cells of all other layers. The formation of the granular layer located above allows effective tissue substances to pass through the epidermis. In the dermis, new capillaries and granulation tissue were observed in all three groups, and the papillary layer closely connected to the basal layer was observed in the CAN-NIR and CAB groups. The formation of the papillary layer expands the junction between the epidermis and the dermis, making it easier for the epidermis to obtain nutrients from the vascular system of the dermis. In the CAB-NIR group, dense connective tissue and reticular layer of hair follicles were observed.

Claims

1. A method for preparing tung oil smoke nanocomposite hydrogel, characterized in that: The steps include: (1) Adding AgNO3 solution to tung oil smoke and ultrasonic treatment to obtain CWOS@Ag+ nanoparticles; (2) Add N, N′-bis(acryloyl)cystamine to CWOS@Ag+ nanoparticles and dissolve them by ultrasonic to obtain CWOS@Ag+@BACA nanocomposite crosslinker; (3) Add acrylamide to the CWOS@Ag+@BACA nanocomposite crosslinker, dissolve it by ultrasonication, add a thermal initiator, and ultrasonicate it in an ice bath to obtain a CAB hydrogel precursor solution; (4) The CAB hydrogel precursor solution is heated in an oven to obtain a CAB hydrogel.

2. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 1, characterized in that: In step (1), the ultrasonic treatment time is 20-40 min.

3. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 1, characterized in that: In step (1), the mass ratio of tung oil smoke to AgNO3 is 1-5:2-3.

4. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 3, characterized in that: In step (1), the mass ratio of tung oil smoke to AgNO3 is 1:2.

78.

5. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 1, characterized in that: In step (3), the mass ratio of the thermal initiator to acrylamide is 3-15:1000.

6. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 5, characterized in that: In step (3), the mass ratio of the thermal initiator to acrylamide is 15:1000.

7. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 5, characterized in that: In step (3), the thermal initiator is one or more of potassium persulfate, ammonium persulfate, azobisisobutyronitrile or cumene hydroperoxide.

8. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 1, characterized in that: In step (3), the temperature of the ice bath is ≤15°C, and the ultrasonic time is 2-5 min.

9. The method for preparing the tung oil smoke nanocomposite hydrogel according to claim 1, characterized in that: In step (4), the temperature of the oven is 60-70° C. and the heating time is 5-6 hours.

10. A tung oil smoke nanocomposite hydrogel prepared by the preparation method according to any one of claims 1 to 9.

11. Use of the tung oil smoke nanocomposite hydrogel according to claim 10 in preparing a medicine for treating acute wound healing.

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