Natural plant extract quercetin-based photo-thermal / nitric oxide synergistic antibacterial hydrogel wound dressing and preparation method thereof

Abstract

The invention relates to a photo-thermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on natural plant extract quercetin and a preparation method of the photo-thermal / nitric oxide synergistic antibacterial hydrogel wound dressing. The preparation method of the photo-thermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin comprises the following steps: embedding quercetin in cyclodextrin to prepare a cyclodextrin inclusion compound I, and embedding an NO donor in cyclodextrin to prepare a cyclodextrin inclusion compound II; dissolving the cyclodextrin inclusion compound I solution in a Fe < 3 + > solution to obtain a C-Q ferric chloride solution, and dissolving the cyclodextrin inclusion compound II solution in a carboxymethyl chitosan solution to obtain a C-B carboxymethyl chitosan solution; and uniformly mixing the carboxymethyl chitosan solution of C-B, the ferric chloride solution of C-Q and the oxidized hyaluronic acid solution, thereby obtaining the product. The preparation method of the CBCQ hydrogel dressing is simple, the CBCQ hydrogel dressing can be prepared through Schiff base reaction, host-guest interaction and coordination by simply mixing the components at room temperature, the gel forming time is short, and gel can be formed in about 25s.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on a natural plant extract quercetin and a preparation method thereof. Background Art

[0002] As the first line of defense of the human body, the skin plays an important role in sensing external stimuli, regulating body temperature, protecting various tissues and organs in the body from external damage, and resisting the invasion of pathogenic microorganisms. However, since the skin is in direct contact with the external environment, it is easily damaged by some external factors and causes skin damage. The repair process of damaged skin is a multi-factor complex process involving multiple cell types and bioactive factors in the body. Microbial invasion or bacterial infection is one of the biggest factors hindering wound healing. Open wounds are susceptible to bacterial infection, which leads to a prolonged inflammatory period and inhibits the formation of new granulation tissue. In addition, wound infection may cause systemic infection such as sepsis, which may even be life-threatening in severe cases. Wound dressings, as a physical barrier between skin wounds and the external environment, can prevent further damage and infection of the wound surface. Compared with traditional wound dressings (gauze, bandages, etc.), modern wound dressings such as hydrogels can provide a more moist environment for the wound, which is conducive to cell growth and wound repair. However, traditional hydrogel dressings lack sufficient antibacterial properties, and the use of metal nanoparticles as antibacterial components may have potential biological toxicity. Long-term use and abuse of antibiotics will produce toxic side effects and lead to dysbiosis and the emergence of drug-resistant bacteria. Therefore, it is urgent to develop new non-antibiotic antibacterial strategies to deal with complex bacterial infected wounds.

[0003] Photothermal therapy (PTT) has become a widely-watched non-antibiotic antibacterial method due to its physical antibacterial properties. In addition, near-infrared (NIR) can promote blood circulation in the wound site and reduce tissue inflammation. However, PTT antibacterial still has the following three limitations: First, the use of PTT alone requires a sufficiently high temperature to kill bacteria; second, the diffusion efficiency of commonly used nano-photothermal converters is worse than that of small molecule drugs, making it difficult to kill bacteria quickly and effectively, and the synthesis process of most nanomaterials is relatively complex, and it is impossible to simultaneously take into account good biocompatibility and high antibacterial activity; third, NIR can only act on the wound for a short time. After the light source is removed, the bacteria not killed by PTT will immediately multiply in the wound. Nitric oxide (NO) is a gas molecule with a broad-spectrum antibacterial effect. It causes bacterial lipid peroxidation, membrane protein nitration and DNA degradation by producing hydrophobic free radicals, and will not induce bacterial resistance. However, the antibacterial effect of NO is restricted by its short half-life (1-5s) and short diffusion distance (20-160μm), and the physiological function of NO is highly dependent on its concentration. High concentration of NO can kill bacteria and cancer cells, while low concentration of NO can promote angiogenesis and collagen production and accelerate wound healing. Therefore, it is crucial to use NO donors to achieve precise and controllable release of NO at the lesion site. Summary of the invention

[0004] The first objective of the present invention is to provide a photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin to solve the potential biological toxicity problem of nano-photothermal converters in the prior art and the technical problem that NO cannot be accurately and controllably released.

[0005] The second object of the present invention is to provide a method for preparing a photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A method for preparing a photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on natural plant extract quercetin comprises the following steps:

[0008] S1: Encapsulating quercetin in cyclodextrin to prepare cyclodextrin inclusion complex 1, and encapsulating NO donor in cyclodextrin to prepare cyclodextrin inclusion complex 2;

[0009] S2: Dissolve the cyclodextrin inclusion complex solution in Fe 3+ The ferric chloride solution of CQ is obtained in the solution, and the cyclodextrin inclusion compound II solution is dissolved in the carboxymethyl chitosan solution to obtain the carboxymethyl chitosan solution of CB;

[0010] S3: Evenly mix the carboxymethyl chitosan solution of CB, the ferric chloride solution of CQ and the oxidized hyaluronic acid solution to obtain.

[0011] Furthermore, the Fe-containing 3+ The solution is an anhydrous ferric chloride solution; the NO donor is N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine.

[0012] Furthermore, the mass concentration of the cyclodextrin inclusion compound solution 1 is 1.5-6%, the mass concentration of the cyclodextrin inclusion compound solution 2 is 1-5%; the mass concentration of the carboxymethyl chitosan solution is 5-20%, and the Fe 3+ The concentration of the solution is 10-50mM; the cyclodextrin inclusion complex solution and Fe 3+ The volume ratio of the solutions is 1-2:1, and the volume ratio of the second cyclodextrin inclusion compound solution to the carboxymethyl chitosan is 1:1-3.

[0013] Furthermore, the volume ratio of the oxidized hyaluronic acid solution, the CB carboxymethyl chitosan solution, and the CQ ferric chloride solution is 1-3:1:1.

[0014] Furthermore, the cyclodextrin is mono-(6-hexanediamino-6-deoxy)-β-cyclodextrin.

[0015] Furthermore, the mass concentration of the oxidized hyaluronic acid solution is 5-20%.

[0016] The photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin is prepared by the above-mentioned preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin.

[0017] Beneficial effects of the present invention:

[0018] The present invention selects natural plant extract quercetin (Que) and Fe 3+ The formed complex was used as a photothermal conversion agent, N,N′-di-sec-butyl-N,N′-dinitroso-1,4-phenylenediamine (BNN6) was used as a NO release precursor, and mono-(6-hexanediamino-6-deoxy)-β-cyclodextrin (Et-βCD) was used as a carrier of Que and BNN6 to improve the solubility and loading rate of Que and BNN6 in the hydrogel. Bio-derived oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCs) were used as hydrogel matrix materials. The chemical cross-linking network of the hydrogel was formed by the Schiff base reaction between -CHO on OHA and CMCs and -HN2 on Et-βCD, the host-guest interaction between Et-βCD and Que and BNN6, and the interaction between Que and Fe 3+The metal coordination bonds between them form a physical cross-linked network of the hydrogel, thereby preparing a physical / chemical double cross-linked photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing (CBCQ hydrogel dressing) based on the natural plant extract quercetin.

[0019] The CBCQ hydrogel dressing of the present invention is formed by combining Que and Fe 3+ The PTT effect generated by coordination acts as a photothermal switch for BNN6 to release NO, exerting a synergistic antibacterial effect of PTT / NO and exerting a sustained antibacterial function through Que; at the same time, by regulating the release of NO through NIR, NO can exercise different physiological functions such as antibacterial, anti-inflammatory, promotion of collagen formation and angiogenesis, thereby more effectively promoting wound healing.

[0020] The present invention uses a natural plant extract, quercetin, which is a flavonol natural plant extract with multiple biological activities such as tumor inhibition, anti-oxidation, antibacterial, and anti-inflammatory. Near infrared (NIR) cannot act on wounds for a long time. After the NIR is removed, the antibacterial properties of quercetin itself can also protect the wound.

[0021] The preparation method of the CBCQ hydrogel dressing of the present invention is simple. The components can be simply mixed at room temperature to obtain the dressing through Schiff base reaction, host-guest interaction and coordination. The gelling time is short, and the dressing can be gelled in about 25 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the construction process and experimental principle of the CBCQ hydrogel dressing in Examples 1-4;

[0023] Figure 2 Characterization of CQ and CB in Example 4, wherein A is a comparison diagram of Que before and after inclusion with Et-βCD, B is a comparison diagram of CB before and after ultraviolet irradiation, C is a Fourier transform infrared spectrum diagram of CQ, and D is a Fourier transform infrared spectrum diagram of CB;

[0024] Figure 3 The figures are gelation diagrams, gelation time diagrams and microstructure diagrams of CBCQ hydrogel dressings, wherein A is the gelation diagram of the CBCQ hydrogel dressing in Example 4, B is the gelation time diagram of the CBCQ hydrogel dressings in Examples 1-4 and Comparative Example 1, and C is the SEM diagram of the interior of the CBCQ hydrogel dressings in Examples 1-4 and Comparative Example 1;

[0025] Figure 4The figures are the result diagrams of injectability, plasticity, self-healing and adhesion of CBCQ hydrogel dressings, wherein A is a visual diagram of the injectability writing performance of the CBCQ hydrogel dressing in Example 4, B is a visual diagram of the injectability of the CBCQ hydrogel dressing in water in Example 4, C is a visual diagram of the plasticity of the CBCQ hydrogel dressing in Example 4, and D is a qualitative characterization of the self-healing performance of the CBCQ hydrogel dressing in Example 4; E is a continuous step strain scanning diagram of the CBCQ hydrogel dressing in Example 4, and F is a qualitative characterization of the adhesion performance of the CBCQ hydrogel dressing in Example 4;

[0026] Figure 5 The antioxidant performance diagram of CBCQ hydrogel dressings in Examples 1-4 and Comparative Example 1, wherein A is a statistical diagram of the intracellular reactive oxygen content of L929 cells under different treatment conditions, and B is a fluorescence staining diagram of the intracellular reactive oxygen of L929 cells under different treatment conditions;

[0027] Figure 6 The photothermal performance and NO release performance of CBCQ hydrogel dressings, where A is the photothermal performance and NO release performance of CBCQ hydrogel dressings in Examples 1-4 and Comparative Example 1 under NIR (1.0 W / cm 2 ) after 5 min of irradiation, B is the temperature change curve of CBCQ1 hydrogel NIR (1.0 W / cm 2 ) is the temperature change curve under on / off cycle irradiation, C is the change curve of NO release in the solution of CBCQ hydrogel dressing in Examples 1-4 and Comparative Example 1 with NIR on / off and NIR irradiation time, D is the effect of Et-βCD on the PTT effect of CBCQ hydrogel dressing, and E is the effect of Et-βCD on NO release of CBCQ hydrogel dressing;

[0028] Figure 7 Figures 1 and 2 are test diagrams of the antibacterial performance of CBCQ hydrogel dressings, wherein A is a statistical diagram of the inherent antibacterial results of the CBCQ hydrogel dressings of Examples 1-4 and Comparative Example 1, B, C, and D are statistical diagrams of the antibacterial results of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and methicillin-resistant Staphylococcus aureus (MRSA) under different treatment conditions, E, F, and G are images of the antibacterial effects of E. coli, S. aureus, and MRSA under different treatment conditions, and H, I, and J are statistical diagrams of the antibacterial results of the CBCQ hydrogel dressings of Examples 1-4 and Comparative Example 1 against E. coli, S. aureus, and MRSA;

[0029] Figure 8The biocompatibility results of the CBCQ hydrogel dressings in Examples 1-4 and Comparative Example 1 are shown in Figure 1, wherein A is a visual diagram of the hemolysis experiment, B is a statistical diagram of the cell survival rate of L929 cells, and C is a HE staining result diagram of the skin and muscle at the subcutaneous injection site and the heart, liver, spleen, lung, and kidney of mice (Scalebar = 100 μm);

[0030] Fig. 9 The figure shows the promoting healing effect of the CBCQ hydrogel dressing on the full-thickness skin infection wounds of mice in Example 4, wherein A is a schematic diagram of the treatment grouping and experimental flow of the full-thickness infection model of mice, B is a visual diagram of the wound healing of the full-thickness skin infection wounds of mice on days 0, 3, 7, and 14 under different treatment conditions and a schematic diagram of the wound area, C is the proportion of the wound area of ​​mice on days 0, 3, 7, and 14 to the initial wound area under different treatment conditions, D is the HE staining result of the newborn skin tissue of mice on days 7 and 14, and E is the Masson staining result of the newborn skin tissue of mice on days 7 and 14 (Scale bar = 100 μm);

[0031] Fig.10 The expression diagrams of angiogenesis-related and inflammatory factor-related proteins in the new tissue, where A is the positive expression fluorescence image of α-SMA, IL-10 and TNF-β on the 14th day under different treatment conditions (Scalebar = 50 μm), B, C, and D are the statistical results of the positive expression of α-SMA, IL-10 and TNF-β on the 14th day under different treatment conditions. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0033] Tegaderm TM Film dressings come from 3M Company in the United States. The Film group uses Tegaderm TM Film dressings for wound treatment.

[0034] Example 1

[0035] The preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin of this embodiment comprises the following steps:

[0036] S1: Add quercetin (Que) solid and Et-βCD solid into 100 mL of deionized water, stir in the dark at room temperature for 24 h, and after the reaction reaches equilibrium, filter with a 0.22 μm polytetrafluoroethylene filter (PTFE filter) to remove the precipitate. After filtration, freeze in a -80°C refrigerator and freeze-dry in a freeze dryer to obtain cyclodextrin inclusion complex 1, named CQ, and place in a 4°C refrigerator for use; N,N′-di-sec-butyl-N,N′-dinitroso-1,4 -phenylenediamine (BNN6) solid and Et-βCD solid were added to 100 mL of deionized water, stirred at room temperature in the dark for 24 h, and after the reaction reached equilibrium, filtered with a 0.22 μm polytetrafluoroethylene filter (PTFE filter) to remove the precipitate, placed in a -80°C refrigerator after filtration, and freeze-dried with a freeze dryer to obtain cyclodextrin inclusion complex II, named CB, and placed in a 4°C refrigerator for use; the final concentrations of quercetin, Et-βCD, and BNN6 were all 2.5 mM;

[0037] S2: Mix the CB solution with the carboxymethyl chitosan (CMCs) solution to obtain the CB carboxymethyl chitosan solution, and mix the CQ solution with the anhydrous ferric chloride solution to obtain the CQ ferric chloride solution. The mass concentration of the CB solution is 2%, the mass concentration of the CMCs solution is 10%, and the volume ratio of the CB solution to the CMCs solution is 1:3; the mass concentration of the CQ solution is 1.5%, the concentration of the anhydrous ferric chloride solution is 27mM, and the volume ratio of the CQ solution to the anhydrous ferric chloride solution is 2:1;

[0038] S3: Mix the oxidized hyaluronic acid solution, the carboxymethyl chitosan solution of CB, and the ferric chloride solution of CQ in a volume ratio of 2:1:1 and blow evenly to obtain a CBCQ hydrogel dressing; the CBCQ hydrogel dressing in Example 1 is named CBCQ 0.25 .

[0039] The oxidized hyaluronic acid was prepared by a sodium periodate oxidation method, and the oxidized hyaluronic acid solution was prepared by dissolving 1.2 g of oxidized hyaluronic acid in 10 mL of PBS solution.

[0040] Example 2

[0041] The preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin in Example 2 is substantially the same as that in Example 1. The difference between the preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin in Example 2 and Example 1 is that the mass concentration of the CQ solution in Example 2 is 3%, and the CBCQ hydrogel dressing in Example 2 is named CBCQ 0.5 .

[0042] Example 3

[0043] The preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin in Example 3 is substantially the same as that in Example 1. The difference between the preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin in Example 3 and Example 1 is that the mass concentration of the CQ solution in Example 3 is 4.5%, and the CBCQ hydrogel dressing in Example 3 is named CBCQ 0.75 .

[0044] Example 4

[0045] The preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin in Example 4 is roughly the same as that in Example 1. The difference between the preparation method of the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin in Example 4 and Example 1 is that the mass concentration of the CQ solution in Example 4 is 6%, and the CBCQ hydrogel dressing in Example 4 is named CBCQ1.

[0046] Comparative Example 1

[0047] The preparation method of the antibacterial hydrogel wound dressing of Comparative Example 1 is substantially the same as that of Example 1. The difference between the preparation method of the antibacterial hydrogel wound dressing of Comparative Example 1 and Example 1 is that in Comparative Example 1, the OHA solution, the carboxymethyl chitosan solution of CB and the solution 1 are mixed in a volume ratio of 2:1:1 and blown evenly to obtain the antibacterial hydrogel wound dressing of Comparative Example 1, which is named CBCQ0 or CB. The preparation method of the solution 1 is to dissolve 0.0146 g of anhydrous ferric chloride in 10 mL of PBS solution.

[0048] Comparative Example 2

[0049] The preparation method of the antibacterial hydrogel wound dressing of Comparative Example 2 is substantially the same as that of Example 4. The difference between the preparation method of the antibacterial hydrogel wound dressing of Comparative Example 2 and Example 4 is that in Comparative Example 2, the OHA solution, the ferric chloride solution of CQ and the solution 2 are mixed in a volume ratio of 2:1:1 and blown evenly to obtain the antibacterial hydrogel wound dressing of Comparative Example 2, which is named CQ1. The preparation method of the solution 2 is to dissolve 0.75 g of CMCs in 10 mL of PBS solution.

[0050] Comparative Example 3

[0051] The preparation method of the antibacterial hydrogel wound dressing of Comparative Example 3 is substantially the same as that of Example 1. The difference between the preparation method of the antibacterial hydrogel wound dressing of Comparative Example 3 and Example 1 is that in Comparative Example 3, the OHA solution, the carboxymethyl chitosan solution of CB and the solution 3 are mixed in a volume ratio of 2:1:1 and blown evenly to obtain the antibacterial hydrogel wound dressing of Comparative Example 3, which is named CBQ1. The preparation method of solution 3 is to mix the Que saturated solution and 27 mM Fe 3+ Mix in a volume ratio of 2:1.

[0052] Comparative Example 4

[0053] The preparation method of the antibacterial hydrogel wound dressing of comparative example 4 is substantially the same as that of example 4. The difference between the preparation method of the antibacterial hydrogel wound dressing of comparative example 4 and example 4 is that in comparative example 4, the OHA solution, the ferric chloride solution of CQ, and solution 4 are mixed at a volume ratio of 2:1:1 and blown evenly to obtain the antibacterial hydrogel wound dressing of comparative example 4, which is named BCQ1. The preparation method of solution 4 is to mix a saturated solution of BNN6 with 10% CMCs at a volume ratio of 1:3.

[0054] Comparative Example 5

[0055] The preparation method of the antibacterial hydrogel wound dressing of Comparative Example 5 comprises the following steps:

[0056] Et-βCD and 10% carboxymethyl chitosan were mixed in a volume ratio of 1:3 to obtain solution five, and Et-βCD and 27 mM anhydrous ferric chloride solution were mixed in a volume ratio of 1:2 to obtain solution six; OHA solution, solution five, and solution six were mixed in a volume ratio of 2:1:1 and blown evenly to obtain the antibacterial hydrogel wound dressing of comparative example 5, named Gel.

[0057] The construction process and principle of CBCQ hydrogel Figure 1 As shown, CBCQ hydrogels are 3+ The PTT effect generated by coordination acts as a photothermal switch for BNN6 to release NO, exerting a synergistic antibacterial effect of PTT / NO and exerting a sustained antibacterial function through Que; at the same time, by regulating the release of NO through NIR, NO can exercise different physiological functions such as antibacterial, anti-inflammatory, promotion of collagen formation and angiogenesis, thereby more effectively promoting wound healing.

[0058] The low solubility of Que and BNN6 in water limits their application. First, the CD derivative Et-βCD was used to encapsulate Que and BNN6 in order to improve the solubility of Que and BNN6. After Que was encapsulated by Et-βCD, the color of its solution changed from a yellow suspension to a clear orange solution, which intuitively showed that the solubility of Que could be improved after being encapsulated by Et-βCD ( Figure 2 A). After ultraviolet irradiation, the CB inclusion complex changes from colorless to yellow-pink. This is because CB generates red BPA (N,N'-di-sec-butyl-1,4-phenylenediamine) and NO under ultraviolet irradiation ( Figure 2 B), indirectly indicating that BNN6 is encapsulated into the hydrophobic cavity of Et-βCD. The above results show that Et-βCD is of great significance for increasing the effective concentration of hydrophobic Que and BNN6 in aqueous solution or hydrogel. Figure 2 C and Figure 2 D shows that the FTIR spectrum of Que has peaks at 1521 and 1561 cm -1 The orange arrow shows the stretching vibration absorption peak of the A and B rings in the benzene ring, 3406 cm -1 The strong absorption peak at 3405 cm is caused by the stretching vibration of the intramolecular hydrogen bond formed by the hydroxyl group and the carbonyl group. -1 A characteristic peak appeared at 1645cm -1 The FTIR spectrum of CQ has the characteristic peaks of Que and Et-βCD; similarly, the FTIR spectrum of BNN6 has the peak at 1451 cm -1 There is a characteristic peak caused by the stretching vibration of the N=O bond, and the FTIR spectrum of CB also has the characteristic peaks of BNN6 and Et-βCD. The above results indicate the successful preparation of CQ and CB.

[0059] The host-guest interactions between BNN6 and Que and Et-βCD, as well as the host-guest interactions between Que and Fe 3+ The CBCQ hydrogel is prepared by the metal coordination between Figure 3 As shown in A, the OHA solution, CB carboxymethyl chitosan solution and CQ ferric chloride solution were mixed evenly in a sample bottle and solid CBCQ hydrogel was formed after standing for a few seconds. Figure 3 B shows that the gelation time of CBCQ hydrogels containing different CQ concentrations is about 25s, which is faster than that of CBCQ0 hydrogels without CQ loading. This may be due to the fact that the cross-linking strength of CBCQ hydrogels is increased after the addition of CQ inclusion complexes. The microscopic morphology of CBCQ hydrogels was observed by SEM. Figure 3 As shown in Figure C, the interior of the CBCQ hydrogel is a loose and porous three-dimensional network structure with an internal pore size of about 130-150μm. This loose and porous structure helps to improve the air permeability of wound dressings and the ability to absorb wound exudate; on the other hand, it is also beneficial to the transmission and diffusion of nutritional metabolites, providing a good environment for cell growth and tissue regeneration, which in turn helps to promote wound healing.

[0060] from Figure 4 A- Figure 4 As can be seen from Figure C, CBCQ hydrogel has good injectability and plasticity. The CBCQ hydrogel dressing of the present invention can be accurately injected into the damaged part by injection, which is convenient for it to adapt to irregular wounds. It can not only provide more effective protection, but also broaden its application range. Figure 4 D shows that the CBCQ hydrogel is divided into two parts, and after standing for a period of time, they healed into a whole again, indicating that the CBCQ hydrogel has good healing ability. The CBCQ hydrogel was tested for structural recovery using a rotational rheometer. The parameters of the rotational rheometer were 40 mm, 37 ° C, 1 rad / s, and the strain was scanned from 1-1000%. The results are shown in Figure 4 As shown in Figure E, when the elastic modulus (G′) is less than the loss modulus (G″), the CBCQ hydrogel network collapses. After a period of self-healing under low strain, G′ can almost recover to the state before the CBCQ hydrogel broke. Even after 3 alternating cycles, G′ and G″ can almost recover to their original states. The above results show that CBCQ hydrogel has good self-healing properties. This is because CBCQ hydrogel is mainly formed by Schiff base reaction, host-guest interaction and metal coordination, which are all reversible reactions. After being destroyed by external force, cross-linking can occur again, so that the broken CBCQ hydrogel can heal into a whole again, forming a wound dressing with self-healing properties. When damaged, the CBCQ hydrogel can automatically repair the damaged area and maintain the integrity of the material, thereby improving the durability of the wound dressing, helping to reduce the frequency of replacement and reducing costs. Figure 4 As can be seen in Figure F, CBCQ hydrogel has good adhesion to some biological tissue materials such as mouse heart, liver, spleen, lung, and kidney, as well as some non-biological tissue materials such as plastic, glass, and rubber. When CBCQ hydrogel dressings are applied to skin wounds, they can act as a sealant to reduce blood loss and form a closed environment to prevent wounds from being contaminated or infected, which is beneficial to tissue regeneration.

[0061] Reactive oxygen species (ROS) and reactive nitrogen free radicals are beneficial for controlling infection and promoting tissue regeneration, but excessive free radicals in the wound or untimely removal can lead to oxidative stress, causing lipid peroxidation, DNA breakage, enzyme inactivation, and ultimately cell damage and tissue necrosis. It can also aggravate the inflammatory response and ultimately hinder the normal healing process of the wound. Therefore, hydrogels with antioxidant properties have great application prospects in the field of inflammation clearance and wound healing. CFH-DA staining was used to evaluate the antioxidant effect of CBCQ hydrogel at the cellular level. The results are as follows: Figure 5 A and Figure 5 As shown in B, compared with the group without H2O2 treatment, the cells treated with H2O2 produced stronger green fluorescence, indicating that H2O2 induced L929 cells to produce a large amount of reactive oxygen species (ROS). Adding CBCQ hydrogel to the cells treated with H2O2 can weaken the production of green fluorescence, indicating that CBCQ hydrogel has a certain scavenging effect on ROS induced by H2O2. According to statistical analysis, compared with the group treated with 1mM H2O2, the fluorescence reduction of the group treated with H2O2 combined with CBCQ hydrogel was statistically significant (P<0.05), and CBCQ hydrogel was able to reduce the ROS induced by 1mM H2O2 to the level of normal cells. The above results show that CBCQ hydrogel has good antioxidant properties. CBCQ hydrogel can effectively scavenge intracellular ROS free radicals, reduce oxidative stress reactions, and help protect cells and tissues from oxidative damage.

[0062] Que is a flavonoid compound with excellent properties such as antibacterial, anti-inflammatory and antioxidant properties; its molecular structure has a catechol bond and Fe 3+ Theoretically, the complex has good photothermal conversion efficiency. The photothermal performance of CBCQ hydrogel dressing is mainly due to the interaction between Que and Fe 3+ Therefore, the effect of CQ concentration on the photothermal properties of CBCQ hydrogel was investigated experimentally. The laser power was 1.0 W / cm 2 The CBCQ hydrogel dressings of Examples 1-4 and Comparative Example 1 were irradiated with NIR at 808 nm and 808 nm for 5 min respectively. The results are as follows Figure 6 As shown in A, after 5 min of NIR irradiation, CBCQ 0.25 , CBCQ 0.5 , CBCQ 0.75 The temperature of CBCQ0 hydrogel increased from about 22℃ at room temperature to 37.2℃, 42.6℃, 46.8℃ and 52.1℃ respectively, while the temperature of CBCQ1 hydrogel increased from 22.8℃ to 24.6℃ under NIR light irradiation, with almost no change, which proved that Que and Fe 3+ After complexation, CBCQ hydrogel was endowed with good photothermal conversion performance. 2 The photothermal cycling experiment was conducted on the CBCQ1 hydrogel dressing, which can reach a relatively mild temperature (about 50°C) after 5 minutes of NIR irradiation. That is, the photothermal stability of the CBCQ hydrogel dressing was evaluated by measuring its temperature change under 808nm NIR on / off cycle irradiation. Figure 6 B It can be seen that the temperature of CBCQ1 increased to about 55 °C within 5 min, and there was no obvious difference in the temperature change under 5 on / off cycle operations, indicating that CBCQ1 hydrogel has good photothermal stability under NIR irradiation.

[0063] As a photoresponsive NO donor, BNN6 can produce a large amount of NO under the photothermal effect induced by NIR and release trace amounts of NO under visible light. The NO release performance of CBCQ hydrogel in solution was identified using the Griess kit. Figure 6 As shown in C, when there is no NIR irradiation, CBCQ 0.25 , CBCQ 0.5 , CBCQ 0.75 There was no significant difference in the NO release of CBCQ1, CBCQ0 hydrogels, which was about 1 μM. When irradiated by NIR, the NO release of CBCQ hydrogel increased with the increase of CQ inclusion complex concentration and the extension of NIR irradiation time. This may be because the increase of CQ concentration and the extension of irradiation time can lead to the enhancement of PTT effect, thereby promoting the release of NO. Among them, the NO release of CBCQ1 hydrogel can reach 3.05±0.17 μM after 60 minutes of NIR irradiation.

[0064] In the present invention, Et-βCD forms an inclusion complex with Que and BNN6 and then is loaded into the hydrogel system. In order to further verify that Et-βCD can improve the efficacy of Que and BNN6 in the hydrogel system, whether the introduction of Et-βCD affects the hydrogel PTT effect and NO release level is investigated. The results are as follows: Figure 6 D and 6E, NIR (808nm, 1.0W / cm 2 After irradiation for 5 min, the temperature of CBQ1 hydrogel remained almost unchanged (from 17.8 to 19.5 °C), while the temperature of CBCQ1 hydrogel increased to 51.7 °C. The PTT effects of the two hydrogels were significantly different (P < 0.05), which may be related to the fact that Et-βCD increased the solubility of Que. This once again verifies that the photothermal performance of CBCQ hydrogel is the best of Que and Fe. 3+ Similarly, compared with the BCQ1 hydrogel directly encapsulating BNN6, the NO release of CBCQ1 hydrogel with CB added increased from 2.37±0.17μM to 2.88±0.34μΜ, which also confirmed that Et-βCD can increase the encapsulation amount of BNN6 in the hydrogel.

[0065] Wound dressings with superior antibacterial properties are essential for promoting wound healing. The inherent antibacterial effect of CBCQ hydrogel dressings was first evaluated. Figure 7As shown in A. After the bacteria were in contact with non-NIR irradiated CBCQ hydrogel at 37°C for 4 hours, the antibacterial effect of CBCQ hydrogel on E.coli, S.aureus and MRSA gradually increased with the increase of CQ inclusion complex concentration. Among them, the antibacterial rates of CBCQ1 hydrogel on E.coli, S.aureus and MRSA were 72.37±0.41%, 76.65±0.67% and 74.33±0.12%, respectively, which showed that CBCQ hydrogel had a certain antibacterial effect on E.coli, S.aureus and MRSA, and the inherent antibacterial property of CBCQ hydrogel mainly originated from the antibacterial property of Que and CMCs themselves. Among them, the positively charged amino groups in CMCs will destroy the bacterial cell wall, thereby inhibiting the growth and reproduction of bacteria; in addition, Que can also destroy the bacterial cell membrane and cell wall, thereby killing bacteria. Photothermal antibacterial is to kill bacteria by converting light energy into heat energy, raising the local temperature; while NO produces hydrophobic free radicals that enter the cell membrane and damage DNA, thereby inhibiting or killing microorganisms. CBCQ hydrogel dressing has excellent photothermal effect and NO release activity. Therefore, CBCQ1 hydrogel dressing was selected to investigate whether the CBCQ hydrogel dressing of the present invention has PTT / NO synergistic antibacterial ability. The results are as follows Figure 7 As shown in BG, when CBCQ1 hydrogel was irradiated with NIR for 6 minutes, the three groups of Con (PBS instead of hydrogel in equal volume), Gel and CB did not have photothermal activity because they did not contain CQ inclusion complexes, and their antibacterial effects on E. coli, S. aureus and MRSA were no different from those without NIR irradiation; without NIR irradiation, there was no significant difference in the antibacterial rate of CQ1 hydrogel without CB inclusion complex and CBCQ1 hydrogel containing both CQ and CB inclusion complexes against each bacteria, and the antibacterial effects of both depended on the antibacterial properties of Que and CMCs themselves. 3+The coordination of CQ and CB inclusion complexes produced PTT antibacterial activity, and the inhibition rates against E. coli, S. aureus and MRSA increased to 74.62±0.37%, 77.43±1.95% and 81.44±0.56%, respectively; CBCQ1 hydrogel containing CQ and CB inclusion complexes could release a large amount of NO through PTT-mediated CB inclusion complexes, producing PTT / NO synergistic antibacterial properties, and the inhibition rates against E. coli, S. aureus and MRSA increased to 84.82±0.21%, 86.82±0.26% and 89.65±0.42%, respectively. The inhibition rate of CBCQ1 hydrogel was higher than that of CQ1 hydrogel without CB inclusion complexes through the single PTT effect. The above results show that the CBCQ hydrogel dressing prepared by the present invention has the synergistic antibacterial function of PTT / NO, and its antibacterial effect is better than the antibacterial effect of PTT alone. This synergistic effect can not only enhance the antibacterial effect of CBCQ hydrogel dressing, but also reduce the drug resistance problem caused by a single treatment method. Considering that the synergistic antibacterial effect of PTT / NO in CBCQ hydrogel dressing is triggered by CQ inclusion complex, the effect of CQ inclusion complex concentration on the antibacterial performance of CBCQ hydrogel was investigated. The results are as follows: Figure 7 As shown in HJ, with the increase of CQ inclusion complex concentration in CBCQ hydrogel, the antibacterial effect of CBCQ hydrogel on E. coli, S. aureus and MRSA gradually increased.

[0066] The intuitive results of the hemolytic performance test of CBCQ hydrogels containing different concentrations of CQ inclusion complexes are as follows Figure 8 As shown in A, the positive control group (with H2O added) is uniformly red as a whole, at which time the blood cell membrane is broken, hemoglobin escapes from it, and hemolysis occurs; while the blood cells of the NaCl group and the CBCQ hydrogel dressing group of Examples 1-4 and Comparative Example 1 are concentrated at the bottom of the centrifuge tube as a whole, and the supernatant is transparent, which intuitively shows that the CBCQ hydrogel dressing does not cause obvious hemolysis. The OD value of the supernatant at 540nm was further measured, and statistical analysis showed that the hemolysis rate of the CBCQ hydrogel was less than 1%, which met the international standard (<5%), indicating that the CBCQ hydrogel did not cause hemolysis.

[0067] Cytocompatibility is one of the important indicators for evaluating the quality of wound dressings. Good cytocompatibility can promote cell growth and proliferation, and is also beneficial to wound healing. The CCK-8 method was used to evaluate the cytotoxicity of CBCQ hydrogels containing different concentrations of CQ inclusion complexes on L929 cells. The results are as follows: Figure 8 As shown in B, L929 cells and CBCQ0, CBCQ 0.25 , CBCQ 0.5 , CBCQ 0.75After 24 hours of co-culture with CBCQ1 hydrogel extract, the cell viability was 102.36±2.01%, 103.02±0.89%, 105.31±2.03%, 98.75±1.81% and 103.66±0.71% respectively; after 48 hours, the cell viability was 97.91±1.59%, 96.03±1.34%, 92.56±1.48% and 91.59±0.56% respectively. There was no statistical difference between the Con group (using an equal volume of fresh culture medium instead of hydrogel extract) and the CBCQ hydrogel group, indicating that CBCQ hydrogel has good cell compatibility. The tissue compatibility of the prepared CBCQ hydrogel was evaluated by subcutaneous injection of mice. On the 14th day, the skin and muscles of the mice in contact with the hydrogel, as well as the main organs such as the heart, liver, spleen, lung and kidney were stained with HE, such as Figure 8 As shown in Figure C, after the injection of each group of hydrogels, the connective tissue of the skin tissue was intact, and the structures such as hair follicles and blood vessels were clearly visible; the muscle tissue had clear horizontal stripes, and no obvious inflammatory infiltration was found in the skin tissue and muscle tissue, indicating that the CBCQ hydrogel dressing would not cause rejection in mice. At the same time, there were no obvious abnormalities in the main organs of the mice compared with the Con group (normal mice), no obvious inflammatory infiltration, and no obvious damage or toxicity, indicating that the degradation products of the CBCQ hydrogel had no significant effect on the organs of the mice, nor did they cause lesions in the organs. The above results show that the CBCQ hydrogel has good tissue compatibility and can be safely used in the treatment of infected wounds while reducing adverse reactions and risks.

[0068] The application value of the prepared CBCQ1 hydrogel in the healing of chronic infectious wounds was evaluated by using a mouse full-thickness skin infection model. The specific grouping and experimental procedures are as follows: Fig. 9 As shown in A, the wound healing images of mice at 0, 3, 7, and 14 days after the start of treatment are shown in Fig. 9 As shown in B, on day 0, the wounds of each group were relatively uniform; on day 3 of treatment, scabs could be observed; on day 7 of treatment, the wound area of ​​each group decreased, and the wounds of the CBCQ hydrogel group were smaller than those of the Con group (mice that did not receive any treatment after infection) and the Film group; on day 14 of treatment, the scabs of the Con group had not completely fallen off, while the scabs of the other treatment groups had all fallen off, and the wound area of ​​the mice in the CBCQ1+NIR group was the smallest, and was basically completely healed. The statistical results of the wound area of ​​each group of mice on days 0, 3, 7, and 14 are shown in the figure. Fig. 9C. Compared with the CBCQ1 group, the CBCQ1+NIR group had better wound healing effect. The mouse skin wound was almost completely healed, and new skin tissue appeared at the wound site, indicating that the antibacterial effect of the CBCQ1+NIR group (PTT / NO synergistic antibacterial and inherent antibacterial) was better than that of the CBCQ1 group (intrinsic antibacterial), and it could more effectively promote the healing of infected wounds. The above results show that the CBCQ1+NIR group is superior to the commercial dressing TedadermTMFilm in the treatment of skin infection injuries. The HE staining results of different treatment groups on the 7th and 14th days after full-thickness skin infection in mice are shown in the figure. Fig. 9 As shown in D, on the 7th day, there was still inflammatory cell infiltration in each treatment group, and the inflammatory reaction in the CBCQ1+NIR group was milder; less granulation tissue and epidermis were formed in each group, and there was still a wound gap. The gap in the CBCQ1+NIR group was the smallest (blue arrow in the figure) and the healing speed was the fastest. On the 14th day, new epidermal regeneration was found in each group, and dermal gaps were visible. The dermal gap in the Con group was significantly the longest, while the dermal gap in the CBCQ1+NIR group was the shortest (yellow arrow in the figure). In addition, compared with the Con group, each group showed the formation of skin appendages such as hair follicles and subcutaneous glands, and the CBCQ1+NIR hydrogel group had the most skin appendages.

[0069] Masson staining is used to evaluate the degree of collagen fibrosis in skin tissue, where collagen fibers appear blue, muscle fibers appear red, and cell nuclei are stained blue-black. The results of Masson staining on the 7th and 14th days after full-thickness skin infection in mice under different treatment conditions are shown in the figure. Fig. 9 As shown in E, on the 7th day, the Con group, Film group, CQ1 group, and CBCQ1 group had less collagen deposition and more muscle fiber generation. Compared with these treatment groups, the CBCQ1+NIR group had more collagen deposition and a small amount of muscle fiber generation. On the 14th day, the collagen deposition of each treatment group increased, among which the CBCQ1+NIR group had more regular collagen deposition, the structural morphology was closer to normal tissue, and more skin appendages were formed. These results indicate that CBCQ1+NIR can better promote the proliferation and remodeling of infected skin wounds and better promote skin regeneration.

[0070] Angiogenesis has an important influence on the wound healing process. New blood vessels can provide necessary nutrients and oxygen, remove metabolic waste, and participate in the repair of damaged tissues. Immunofluorescence staining of angiogenesis-related protein α-SMA was used to evaluate the regeneration of blood vessels in skin wound healing tissues. Fig.10As shown in AD, compared with the Con group (mice without any treatment after infection), the positive expression of α-SMA in the Film group, CQ1 group, CBCQ1 group and CBCQ1+NIR group gradually increased, and there was a statistical difference (P<0.05). Among them, the positive expression of α-SMA in the CBCQ1+NIR group was the highest, indicating that the wound area of ​​the mice treated with the CBCQ1+NIR group showed more neovascularization. This is mainly due to the natural photothermal conversion material Que contained in the CBCQ hydrogel. Que and Fe 3+ The complex has a high photothermal conversion efficiency and can generate heat under NIR irradiation to stimulate angiogenesis. In addition, when NIR is off, the continuous release of trace NO also plays a key role. Trace NO can promote angiogenesis, relax vascular smooth muscle, help improve the local microenvironment of the wound, and accelerate wound healing by promoting the proliferation of fibroblasts and endothelial cells and promoting their migration to the wound site.

[0071] Inflammatory response is an important stage in the wound healing process, but excessive inflammatory response often leads to tissue damage and delayed healing. IL-10 can inhibit the differentiation of proinflammatory lymphocytes, thereby playing an anti-inflammatory role. TNF-β is a cytokine secreted by lymphocytes that can mediate a variety of inflammatory, immune stimulation and antiviral responses. Therefore, IL-10 and TNF-β were selected as indicators of inflammatory response, and the inflammatory response in the new tissue of skin wounds was investigated by immunofluorescence experiments. The positive expression of IL-10 in the Film group, CQ1 group, CBCQ1 group and CBCQ1+NIR group gradually increased, which was statistically different from the Con group (mice without any treatment after infection) (P<0.05); however, the corresponding TNF-β expression in the Film group, CQ1 group, CBCQ1 group and CBCQ1+NIR group gradually decreased, which was statistically different from the Con group (P<0.05). Among them, the CBCQ1+NIR group had the highest IL-10 expression level and the lowest TNF-β expression level, and the wound area of ​​the treated mice showed a lower inflammatory response, proving that it has significant advantages in reducing inflammation. This is because, on the one hand, the PTT / NO synergistic antibacterial effect of CBCQ hydrogel can effectively inhibit bacterial growth, reduce infection, and thus reduce inflammatory response. On the other hand, the trace NO continuously released when NIR is turned off has anti-inflammatory and immunomodulatory effects, which can inhibit the production of inflammatory factors and reduce inflammatory response.

Claims

1. A method for preparing a photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on natural plant extract quercetin, characterized in that: The following steps are involved: S1: Encapsulating quercetin in cyclodextrin to prepare cyclodextrin inclusion complex 1, and encapsulating NO donor in cyclodextrin to prepare cyclodextrin inclusion complex 2; S2: Dissolve the cyclodextrin inclusion complex solution in Fe 3+ The ferric chloride solution of CQ is obtained in the solution, and the cyclodextrin inclusion compound II solution is dissolved in the carboxymethyl chitosan solution to obtain the carboxymethyl chitosan solution of CB; S3: Evenly mix the carboxymethyl chitosan solution of CB, the ferric chloride solution of CQ and the oxidized hyaluronic acid solution to obtain.

2. The method for preparing the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin according to claim 1, characterized in that: The Fe-containing 3+ The solution is an anhydrous ferric chloride solution; the NO donor is N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine.

3. The method for preparing the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin according to claim 1, characterized in that: The mass concentration of the cyclodextrin inclusion compound solution 1 is 1.5-6%, the mass concentration of the cyclodextrin inclusion compound solution 2 is 1-5%; the mass concentration of the carboxymethyl chitosan solution is 5-20%, and the Fe 3+ The concentration of the solution is 10-50mM; the cyclodextrin inclusion complex solution and Fe 3+ The volume ratio of the solutions is 1-2:1, and the volume ratio of the second cyclodextrin inclusion compound solution to the carboxymethyl chitosan is 1:1-3.

4. The method for preparing the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin according to claim 1, characterized in that: The volume ratio of the oxidized hyaluronic acid solution, the CB carboxymethyl chitosan solution, and the CQ ferric chloride solution is 1-3:1:

1.

5. The method for preparing the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin according to claim 1, characterized in that: The cyclodextrin is mono-(6-hexanediamino-6-deoxy)-β-cyclodextrin.

6. The method for preparing the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin according to claim 1, characterized in that: The mass concentration of the oxidized hyaluronic acid solution is 5-20%.

7. A photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on natural plant extract quercetin, characterized in that: The wound dressing is prepared by the method for preparing the photothermal / nitric oxide synergistic antibacterial hydrogel wound dressing based on the natural plant extract quercetin as described in any one of claims 1 to 6.

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