Double-response antibacterial hydrogel as well as preparation and application thereof

Abstract

The invention belongs to the technical field of antibiosis and wound healing promotion, and relates to double-response (near-infrared light and hydrogen peroxide double-response photo-thermal / carbon monoxide therapy) antibacterial hydrogel as well as preparation and application thereof. The invention relates to a double-response antibacterial hydrogel. The antibacterial hydrogel is prepared from the following components: drug-loaded nano particles and a hydrogel matrix. The drug-loaded nano particles are prepared by loading dimanganese decacarbonyl (MnCO) into cavities of hollow mesoporous copper sulphide nano particles (HMCuS NPs) and coating the surfaces of the particles with a polydopamine layer (PDA). According to the invention, CO / PTT synergistic treatment of bacterial infected wounds is realized, and the antibacterial effect is significantly improved. An in-vitro antibacterial experiment result shows that the antibacterial hydrogel shows extremely strong inhibition capability on escherichia coli and staphylococcus aureus, and the problem of drug resistance caused by a bacterial biofilm is effectively solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial and wound healing promotion, and relates to a dual-response (near-infrared light and hydrogen peroxide dual-response photothermal / carbon monoxide treatment) antibacterial hydrogel and preparation and application thereof. BACKGROUND

[0002] When the skin tissue is damaged, a series of complex and dynamic wound healing processes occur, and in these processes, local infection caused by the invasion of pathogenic bacteria on the wound surface becomes one of the important factors hindering wound healing.

[0003] To overcome the multiple challenges of traditional antibiotic therapy in the context of wound infection, such as the increasing drug resistance of pathogenic bacteria and the drug penetration barrier, in recent years, photothermal therapy (PTT) and gas therapy (GT) have been widely used in the treatment of anti-infection due to their high efficiency, low toxicity and specificity. However, the limitations of single treatment method limit the clinical application of the above treatment methods. For example, the heat generated during the use of PTT may spread to the surrounding healthy tissues, causing side effects such as burning and inflammation. In addition, the heat may have difficulty penetrating the deep layer of the bacterial biofilm, resulting in the survival of deep layer bacteria and the recurrence of chronic infection. In GT, carbon monoxide (CO) is relatively stable in nature and has anti-inflammatory and antibacterial potential at a certain concentration. It is well known that continuous oxidative stress is the main cause of inflammation, and CO can prevent oxidative stress by inducing the activation of heme oxygenase (HO-1), thereby effectively inhibiting inflammation. In terms of antibacterial characteristics, CO can penetrate deep into the bacteria and inhibit bacterial respiration by interfering with the metabolic enzyme activity of the bacteria, thereby cutting off the synthesis of adenosine triphosphate (ATP) in the bacteria and causing bacterial death. Although CO has shown good antibacterial potential in laboratory studies, it faces a series of severe and inherent challenges in moving from theory to clinic. For example, the lack of targeting leads to potential systemic toxicity, and while CO acts on bacteria, it also affects normal human cells around the infection site, which may cause local tissue damage and delay the healing process. In addition, it is difficult to accurately control the release rate and total amount of CO, which may lead to "burst release" or insufficient release, thus there is a risk of poisoning or incomplete treatment. Therefore, in view of the limitations of single therapy, combined therapy (photothermal-photodynamic combination, photothermal-chemical dynamic combination, photothermal-gas therapy, and photodynamic-gas therapy combination, etc.) has gradually become an important trend in modern medicine to improve efficacy, overcome drug resistance, reduce side effects, and achieve more precise and individualized treatment. However, it is extremely difficult to integrate two functionally different molecules into one platform while meeting the optimal working conditions for the function of these two types of functions (such as excitation wavelength and reaction environment). In addition, the chemical synthesis of complex multifunctional molecules usually involves multiple steps, harsh reaction conditions, and difficult purification of intermediate products, resulting in a low overall yield of the final product. This limits its large-scale production and future clinical translation. Studies have shown that nanodelivery systems play a crucial role as carriers in the combined application of photothermal therapy (PTT) and carbon monoxide (CO) therapy, and they are not just simple "transport tools", but also key platforms for achieving synergistic effects, precise control and reducing toxicity. Through ingenious nanostructures, two functionally different and potentially conflicting therapies are organically integrated and synergistically controlled in "time-space", ultimately overcoming the limitations of single therapy and simple combination.

[0004] Studies have shown that traditional nanometer delivery systems can be injected into the wound site during the treatment of skin wounds, but they are easily washed away by body fluids, affecting the drug delivery effect. In addition, during the complex wound healing process, when the wound is exposed to the external environment, it is extremely vulnerable to the invasion of pathogens such as bacteria and fungi, and it is necessary to use a wound dressing as a physical barrier to reduce the risk of bacterial infection and promote wound healing. Compared with most traditional wound dressings, hydrogel dressings can keep the wound moist, cool the wound surface, and reduce the patient's pain, and are considered as ideal wound dressings. In addition, hydrogels have a three-dimensional network structure similar to the extracellular matrix (ECM) and good biocompatibility, and can be used as carriers for antibacterial agents to exert antibacterial effects. In order to make hydrogels better applied to the field of wound treatment, researchers have developed a kind of intelligent hydrogel, the biggest difference between the intelligent hydrogel and the traditional hydrogel is that the intelligent hydrogel will change its own properties in response to external environmental stimuli. According to the response mode to external environmental stimuli, intelligent hydrogels are mainly divided into temperature response, pH response, light response, and reactive oxygen species (ROS) response. Because it combines the good biocompatibility of traditional hydrogels and the advantages of responsive materials, it is widely used in chemical converters, drug release, tissue engineering and other fields, and in recent years it has gradually become a popular biomaterial in the field of drug delivery. However, most intelligent hydrogels respond slowly to external stimuli, making it difficult to meet the needs of high-precision and high-speed application scenarios. In addition, most current hydrogels can only respond to a single stimulus, while actual applications often require multiple stimuli to be synergistically controlled. SUMMARY

[0005] The purpose of the present application is to provide a dual-response (near-infrared light and hydrogen peroxide dual-response photothermal / carbon monoxide) antibacterial hydrogel and its preparation and application.

[0006] To achieve the above purpose, the technical solution adopted by the present application is:

[0007] A dual-response antibacterial hydrogel, the components of the antibacterial hydrogel are composed of drug-loaded nanoparticles and a hydrogel matrix.

[0008] The drug-loaded nanoparticles are MnCO loaded into the cavity of hollow mesoporous copper sulfide nanoparticles (HMCuSNPs), and a polydopamine layer (PDA) is coated on the surface of the particles.

[0009] The hydrogel matrix is prepared by reacting the phenylboronic acid bond of the H2O2-responsive crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA) with the diol group of polyvinyl alcohol (PVA) to form a dynamic crosslinking of phenylboronic ester bond.

[0010] Preparation method of dual-responsive antibacterial hydrogel

[0011] 1) Drug-loaded nanoparticles: Hollow mesoporous copper sulfide nanoparticles (HMCuS NPs) with photothermal properties are used as carriers to load carbon monoxide donor decacarbonyldimanganese (MnCO), and a polydopamine layer (PDA) is coated on the surface of the particles;

[0012] 2) Hydrogel matrix: A benzene boronic acid ester bond dynamic cross-linking is prepared by the reaction of the benzene boronic acid bond of the H2O2-responsive cross-linking agent (TSPBA) and the dihydric alcohol group of polyvinyl alcohol (PVA);

[0013] Among them, the mass percentage concentration ratio of TSPBA solution to PVA solution is 9:3.5-9:4.5.

[0014] 3) The drug-loaded nanoparticles obtained above are uniformly dispersed in the hydrogel matrix to obtain a dual-responsive antibacterial hydrogel; wherein the mass-volume concentration of the drug-loaded nanoparticles relative to the hydrogel matrix is 0.06% (w / v).

[0015] The preparation method of the drug-loaded nanoparticles is:

[0016] 1) Preparation method of HMCuS NPs: Polyvinylpyrrolidone is dispersed in deionized water, copper chloride solution is added and mixed, the system is adjusted to alkaline, then hydrazine hydrate and sodium sulfide are added, mixed, and heated in a water bath at 75-80°C for 2-3h;

[0017] 2) MnCO and the above obtained HMCuS NPs are dispersed in methanol, stirred overnight, and the mixed solution is placed in a vacuum drying machine for drying treatment as solution 1; wherein the mass ratio of HMCuS NPs to MnCO is 1:1-1:2;

[0018] 3) Dopamine hydrochloride is dissolved in Tris buffer as solution 2;

[0019] 4) Solution 1 is uniformly dispersed in solution 2, stirred at room temperature for 5-8h, washed, and drug-loaded nanoparticles (MnCO-CuS@PDA NPs, abbreviated as MCP NPs) are obtained.

[0020] The mass ratio of polyvinylpyrrolidone to copper chloride in step 1) is 20:1-30:1, the molar ratio of hydrazine hydrate to copper chloride is 1:1-2:1, and the molar ratio of sodium sulfide to copper chloride is 5:1-8:1.

[0021] The preparation method of the hydrogel matrix solution is:

[0022] 1) Preparation method of hydrogen peroxide responsive crosslinker TSPBA: N,N,N',N'-tetramethyl-1.3-propanediamine and 4-(bromomethyl)phenylboronic acid are dissolved in N,N-dimethylformamide together, stirred in a 60-80℃ oil bath for 12h, the reaction solution is cooled to room temperature, poured into tetrahydrofuran, white precipitate is precipitated, the product is collected and vacuum dried overnight to obtain the crosslinker TSPBA;

[0023] 2) Preparation of TSPBA precursor solution of antibacterial hydrogel: the prepared TSPBA is weighed and added to deionized water to obtain a solution;

[0024] 3) Preparation of PVA precursor solution of antibacterial hydrogel: polyvinyl alcohol is added to deionized water, heated to 80-90℃ and stirred to dissolve, and after complete dissolution, it is naturally cooled to room temperature, filtered to obtain a solution.

[0025] The molar ratio of N,N,N',N'-tetramethyl-1.3-propanediamine to 4-(bromomethyl)phenylboronic acid in step 1) is 1:2.5-1:3.

[0026] The application of the antibacterial hydrogel of the application, the application of the antibacterial hydrogel as an antibacterial infection disease preparation.

[0027] The bacterial infectious disease is a bacterial infectious disease caused by Escherichia coli and / or Staphylococcus aureus; the dual response is the combined response of near-infrared light and hydrogen peroxide.

[0028] As can be seen from the above, the application provides a near-infrared light and hydrogen peroxide dual-response photothermal / carbon monoxide antibacterial hydrogel (MCP@Gel), which can realize CO / PTT synergistic antibacterial under the conditions of NIR irradiation and H2O2 initiation.

[0029] Compared with the prior art, the application has the following beneficial effects:

[0030] The application is based on a near-infrared light and hydrogen peroxide dual-response photothermal / carbon monoxide antibacterial hydrogel, which loads nanoparticles with photothermal and CO release dual functions in a H2O2 responsive hydrogel, and under the combined action of NIR laser irradiation and H2O2, can generate local high temperature and release CO at the wound site, realize CO / PTT synergistic treatment of bacterial infected wounds, and significantly improve the antibacterial effect. The in vitro antibacterial experiment results show that the antibacterial hydrogel exhibits strong inhibitory ability to Escherichia coli and Staphylococcus aureus, and effectively solves the problem of drug resistance caused by bacterial biofilm.

[0031] The in-vivo experiment of the antibacterial hydrogel of the present application fully proves that it performs outstandingly in promoting wound healing. On the 10th day after treatment, the back wounds of the mice treated by the photothermal / carbon monoxide antibacterial hydrogel are almost completely healed compared with single therapy; through enzyme-linked immunoassay (ELISA) experiment and H&E and Masson staining analysis of skin tissue, the inflammation reaction of the mice in this group is extremely slight, the skin tissue repair is complete, the collagen deposition is significant and arranged in order, and the wound healing period is greatly shortened.

[0032] The antibacterial hydrogel of the present application has a unique H2O2 responsiveness, in which the phenylboronic ester bond can be broken in a high active oxygen environment, and the phenylboronic ester bond is broken at the same time, which consumes and removes ROS (such as H2O2) in the wound, automatically adjusts the local redox environment, relieves oxidative stress, promotes cell proliferation and angiogenesis, and at the same time promotes the degradation of the hydrogel. In addition, the hydrogel gradually degrades under the action of ROS and is automatically removed without additional intervention, avoiding the secondary trauma caused by the replacement of traditional dressings. In summary, the antibacterial hydrogel of the present application converts "structure destruction" into "treatment behavior", that is, in the phenylboronic ester hydrogel, the structure break itself is a kind of treatment behavior - not only releases drugs, but also removes ROS, antibacterial and anti-inflammatory.

[0033] It has been verified through experiments that the antibacterial hydrogel of the present application has excellent biological safety. During the treatment, the body weight of the mice remains stable and no obvious abnormalities are observed; the hemolysis test results show that the antibacterial hydrogel does not cause hemolysis and does not cause damage to the blood system, and can be safely applied to in-vivo treatment; in addition, the preparation does not depend on traditional antibiotics, providing a new non-antibiotic treatment approach to solve the problem of bacterial drug resistance, and having a broad application prospect in the field of antibacterial. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The preparation flowcharts of each step of the embodiments of the present application are shown in the figures; wherein, a. the preparation flowchart of hollow mesoporous copper sulfide nanoparticles; b. the preparation flowchart of composite nanoparticles; c. the preparation flowchart of near-infrared light and hydrogen peroxide dual-responsive photothermal / carbon monoxide antibacterial hydrogel.

[0035] Figure 2 The characterization figures of the products obtained in each step of the embodiments of the present application are shown in the figures; wherein, a. the transmission electron microscope figure of HMCuS NPs; b. the transmission electron microscope figure of MCP NPs; c. the Zeta potential of HMCuS NPs, MC NPs and MCP NPs; d. the ultraviolet-visible-near-infrared absorption spectrum figure of HMCuS NPs, MnCO, PDA and MCP NPs; e. the infrared absorption spectrum figure of HMCuS NPs, MnCO and MCP NPs.

[0036] Figure 3The characterization diagram of MCP@Gel is obtained for the embodiment of the application; wherein, a. The appearance diagram of MCP@Gel; b. The scanning electron microscope diagram of MCP@Gel; c. The swelling curve of Blank Gel and MCP@Gel; d. The degradation curve of Blank Gel in different concentrations of H2O2 aqueous solution.

[0037] Figure 4 The antibacterial mechanism schematic diagram of the near-infrared light and hydrogen peroxide double-response photothermal / carbon monoxide antibacterial hydrogel is provided for the embodiment of the application.

[0038] Figure 5 The effect diagram of the hydrogel prepared by different PVA and TSPBA ratios is provided for the comparative example of the application.

[0039] Figure 6 The effect diagram of the hydrogel prepared by PVA and TSPBA ratio of 9:3 is provided for the comparative example of the application; wherein, a. The effect diagram of gelation; b. The rheological behavior curve.

[0040] Figure 7 The effect diagram of MCP@Gel under 808nm near-infrared laser irradiation under different changes is provided for the application example of the application; wherein, a. The temperature change curve of MCP@Gel doped with different mass-volume concentrations of MCP NPs under near-infrared laser (808nm, 0.5W / cm 2 ) irradiation; b. The temperature change curve of MCP@Gel doped with 0.06%(w / v) MCP NPs under different power 808nm near-infrared laser irradiation; c. The photothermal cycle curve (irradiated with 808nm, 0.5W / cm 2 near-infrared laser, 10min each time, the laser is turned off for 10min, 4 cycles of light-shielding cycle, and the temperature is recorded every 30s during the cycle).

[0041] Figure 8 The CO release behavior effect diagram of MCP@Gel and blank hydrogel is provided for the application example of the application; wherein, a. The CO release behavior curve of MCP@Gel under different time responses of 100μM H2O2; b. The CO cumulative release amount curve; c. The CO release behavior curve of Blank Gel; d. The CO cumulative release amount curve of MCP@Gel under different concentrations of H2O2 response.

[0042] Figure 9Antibacterial effect diagram of different formulations provided for the application example; wherein, a. antibacterial activity diagram of different formulations on escherichia coli; b. antibacterial activity diagram of different formulations on staphylococcus aureus (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001); c. activity diagram of different formulations against escherichia coli biofilm; d. activity diagram of different formulations against staphylococcus aureus biofilm (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0043] Figure 10 Live / dead bacterial staining diagram of escherichia coli and staphylococcus aureus after treatment of different formulations provided for the application example.

[0044] Figure 11 Scanning electron microscope diagram of escherichia coli and staphylococcus aureus after treatment of different formulations provided for the application example.

[0045] Figure 12 In vivo thermal imaging diagram of mice under near-infrared light irradiation after mice wounds were given Blank Gel and MCP@Gel provided for the application example.

[0046] Figure 13 Effect diagram of near-infrared light and hydrogen peroxide double-responsive photothermal / carbon monoxide treatment hydrogel and different experimental groups; wherein, a. animal experiment scheme diagram; b. healing condition of mouse wounds after treatment of different treatment groups; c. quantification result diagram of healing condition (n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0047] Figure 14 Effect diagram of expression of different inflammatory factors at mouse wound of each experimental group after treatment provided for the application example; wherein, a. content diagram of tumor necrosis factor-α (TNF-α); b. content diagram of inflammatory factor interleukin-1β (IL-1β); c. content diagram of interleukin-6 (IL-6) (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

[0048] Figure 15 Effect diagram of histological analysis of mouse wound tissue of each experimental group after treatment provided for the application example; wherein, a. representative H&E staining; b. masson staining picture.

[0049] Figure 16 Safety evaluation effect diagram of near-infrared light and hydrogen peroxide double-responsive photothermal / carbon monoxide treatment hydrogel provided for the application example; wherein, a. body weight change curve diagram; b. hemolysis rate of MCP@Gel doped with different volume fractions of MCP NPs. DETAILED DESCRIPTION

[0050] The application will be further described below with reference to the drawings, and the following examples are only used to more clearly illustrate the technical solutions of the application, and cannot be used to limit the protection scope of the application.

[0051] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0052] Based on the limitations of antibiotic therapy in antibacterial treatment, the present application proposes a near-infrared light and hydrogen peroxide dual-responsive photothermal / carbon monoxide antibacterial hydrogel, which realizes synergistic antibacterial effect through the synergistic effect of PTT and CO treatment, and promotes wound healing. The present application uses hollow mesoporous copper sulfide nanoparticles with photothermal properties as a carrier, loads carbon monoxide donors, and then wraps them with biocompatible materials to construct nanoparticles with dual functions of photothermal and carbon monoxide therapy. The multifunctional nanoparticles are doped in H2O2-responsive hydrogel to construct a near-infrared light and hydrogen peroxide dual-responsive photothermal / carbon monoxide antibacterial hydrogel (MCP@Gel), which realizes efficient antibacterial and wound healing effect, reduces damage to normal tissues, overcomes the shortcomings of traditional antibiotic therapy, and provides a new strategy for non-antibiotic bacterial infection treatment.

[0053] Example 1

[0054] The preparation of the NIR and H2O2-responsive combined antibacterial material is as shown in Figure 1

[0055] S1, Preparation of HMCuS NPs

[0056] Specific method: 240 mg of PVP-K30 was added to 25 mL of deionized water and dissolved, then 150 μL of 0.5 mol / L CuCl2 solution was added, and stirred at room temperature for 5 min. 25 mL of NaOH (pH=9.0) solution was added and stirred for 2 min, then 6.4 μL of N2H4·H2O was added, and after stirring for 5 min, a yellow Cu2O suspension was formed. 200 μL of 160 mg / mL Na2S aqueous solution was added to the suspension and stirred for 5 min, and the reaction was refluxed at 75°C for 2 h. After the reaction was completed, ice bath cooling was performed, centrifugation was performed at 10000 rpm for 15 min, and water washing was performed twice, and storage was performed at 4°C (see Figure 1 a).

[0057] S2, Preparation of MCP NPs

[0058] ​To obtain MCP NPs, first, 1.5 mg of the above HMCuS NPs and 2.25 mg of MnCO3 were dissolved in 1 mL of methanol and stirred overnight, and then the mixed solution was placed in a vacuum drying machine to evaporate the volume of the mixed solution to 0.2 mL, and MnCO3 was encapsulated in the HMCuS NPs by the solvent evaporation method to obtain the product MnCO3@CuS NPs (MC NPs). Subsequently, the MC NPs were re-dispersed in 1 mL of Tris buffer solution (0.1 M, pH = 8.5) containing 0.75 mg of dopamine hydrochloride (DA·HCl), and after ultrasonic dispersion, 2 mL of Tris buffer solution was added, stirred at room temperature for 5 h, and washed twice with deionized water to obtain MnCO3@CuS-PDA NPs (MCP NPs) (see Figure 1 b).

[0059] The characterization of the product obtained in each step is described in Figure 2 , wherein, from Figure 2 (a) it can be seen that the prepared HMCuS NPs are spherical, the surface is rough, have obvious hollow structure, the size of the nanoparticles is relatively uniform, and the average particle size is 70-90 nm. The MCP NPs are spherical structure, and the diameter is 100-140 nm Figure 2 (b). From Figure 2 (c) it can be seen that the potential of HMCuS NPs is about -22.18 mV, and when MnCO3 is loaded, since it is electrically neutral, the potential of MC NPs remains almost unchanged, about -22.36 mV. After the outer layer of the nanoparticles is coated with PDA, due to the presence of a large number of phenolic hydroxyl groups on the surface of PDA, the potential of HMCuS NPs decreases slightly, and the potential of MCP NPs is about -26.56 mV. By inductively coupled plasma-mass spectrometry, it is calculated that the drug loading of MnCO3 is 26.7%. As shown in Figure 2 (d), the characteristic absorption peak of MnCO3 appears in MCP NPs with slight red shift, indicating the successful loading of MnCO3. As shown in Figure 2 (e), in the infrared spectrum of MCP NPs, the stretching vibration characteristic peak of the terminal carbonyl group in MnCO3 appears at 2003 cm -1 , and the sulfur-carbon stretching vibration peak of CuS appears at 1107 cm -1 , indicating the successful preparation of MCP NPs.

[0060] S3, Preparation of MCP@Gel and Blank Gel

[0061] To obtain MCP@Gel, first, the crosslinker TSPBA needs to be synthesized. The specific method is as follows: 0.1 g of N,N,N',N'-tetramethyl-1.3-propanediamine and 0.5 g of 4-(bromomethyl)phenylboronic acid are dissolved in 10 mL of N,N-dimethylformamide, and stirred in a 60°C oil bath for 12 h. After the reaction is completed, the solution is cooled to room temperature, and the mixed solution is poured into tetrahydrofuran, and white precipitate is precipitated. The product is collected and vacuum dried overnight to obtain the crosslinker TSPBA, which is stored at -20°C for standby use.

[0062] Then the precursor solution of the hydrogel is prepared. 9 g of PVA is weighed into 91 g of deionized water, heated to 90°C and stirred to dissolve, and after complete dissolution, it is naturally cooled to room temperature. The insoluble material is filtered out with a 0.22 μm cellulose acetate filter membrane to obtain a 9 wt% PVA solution, which is stored at room temperature for standby use. 0.04 g of the above prepared TSPBA is weighed into 0.96 g of deionized water to obtain a 4 wt% TSPBA solution, which is prepared for immediate use. Finally, 0.3 mg of MCP NPs prepared in S2 is uniformly mixed into 250 μL of 4 wt% TSPBA solution, and 250 μL of 9 wt% PVA solution is vortexed to prepare a mass volume concentration of 0.06% (w / v) MCP@Gel (see Figure 1 c) To compare the performance, 250 μL of 4 wt% TSPBA solution is vortexed with 250 μL of 9 wt% PVA solution to prepare a blank hydrogel (Blank Gel) without MCP NPs.

[0063] The above obtained products are characterized, from Figure 3 As can be seen from (a), after gelation, MCP@Gel is a uniform black gel. The internal pore structure of MCP@Gel is observed by SEM, as shown in Figure 3 As shown in (b), MCP@Gel has an ordered porous structure, and the pore structure is relatively dense. As shown in Figure 3 As shown in (c), the hydrogel has good swelling performance and reaches swelling equilibrium after 12 h, indicating that MCP@Gel as a three-dimensional porous network structure material has a certain water absorption capacity. In the treatment of wound infection, it can absorb the exudate of the wound, avoid wound infection, and thus clean the wound and maintain the humidity of the environment, which is conducive to the healing of the wound.

[0064] The degradability of the hydrogel was then determined. Aqueous solutions containing 50, 100, and 150 μM H2O2 were prepared respectively. 1.0 g of Blank Gel was weighed and placed in 10 mL of the above aqueous solutions containing different concentrations of H2O2, ensuring that the hydrogel was completely immersed in the water. The gel was removed at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h, respectively. The solution on the surface of the gel was removed with dry and clean filter paper and the gel was weighed again. After weighing, the gel was put back into the H2O2 aqueous solution and the solution was added to 10 mL.

[0065] Depend on Figure 3 As shown in (d), the degradation rate of the hydrogel increases with increasing H2O2 concentration. However, in a pure aqueous solution without H2O2, the hydrogel showed almost no degradation after 24 hours, remaining stable. This responsive degradability will also prevent secondary damage to the wound during gel removal, thus promoting wound healing.

[0066] Furthermore, such as Figure 4 As shown, the local thermal effect generated by HMCuS NPs in MCP@Gel under NIR irradiation can not only directly destroy bacterial cell membranes, proteins, and other biomolecules, but also alter the structure of the PDA coating layer, increasing its permeability and promoting the release of CO from MnCO in response to H2O2 for gas therapy, achieving CO / PTT synergistic antibacterial action. CO, as a gaseous signaling molecule, exerts its antibacterial effect by inhibiting the bacterial respiratory chain and disrupting cell membrane integrity; it also exerts its anti-inflammatory effect through multiple pathways, including inhibiting inflammatory factor expression, regulating immune cell function, and antioxidation. Furthermore, in CO / PTT synergistic antibacterial action, CO can alleviate the inflammatory response induced by PTT and promote tissue repair and regeneration. In addition, the H2O2-responsive TSPBA-PVA hydrogel, as an ideal wound dressing, can act as a carrier to improve local drug delivery performance. Its high water content keeps the wound moist, accelerating wound healing, and it can also respond to the degradation of endogenous and exogenous ROS, avoiding secondary damage to the wound when removing the gel.

[0067] Comparative Example 1

[0068] The effects of different mass percentage concentrations of PVA and TSPBA solutions on gel formation were investigated, following the procedure for preparing Blank Gel in Example 1 above, with the difference being the adjustment of the concentrations of the PVA and TSPBA solutions. Specifically, the TSPBA concentration was fixed at 3 wt%, and the PVA concentration was 3 wt%, 6 wt%, or 9 wt% (see [link to example]). Figure 5 ).

[0069] like Figure 5As shown, when the concentration of TSPBA is fixed at 3 wt%, and the concentrations of PVA are 3 wt% and 6 wt%, the two solutions cannot form a gel when mixed. However, TSPBA at a concentration of 3 wt% can crosslink with 9 wt% PVA to form a flexible gel. Furthermore, the rheological behavior of the formed flexible gel was investigated. Specifically, a rheometer was used to perform frequency scanning on the gel at 25°C, with the frequency scanning range set to 0.1–100 rad / s and the strain fixed at 1%. The G′ and G″ of the hydrogel are as follows: Figure 6 As shown in (b), within the angular frequency test range of 0.1–100 rad / s, by Figure 6 As can be seen, 3 wt% TSPBA can crosslink with 9 wt% PVA to form a flexible gel, but the prepared gel is relatively soft, and the transferability of the gel after molding is poor (see [reference]). Figure 6 ).

[0070] Comparative Example 2

[0071] The effects of different mass percentage concentrations of PVA and TSPBA solutions on gel formation were investigated, following the procedure for preparing Blank Gel in Example 1 above, with the difference being the adjustment of the concentrations of the PVA and TSPBA solutions. Specifically, when the TSPBA concentration was higher (i.e., 6 wt% or 9 wt%), the PVA concentration was 3 wt%, 6 wt%, or 9 wt% (see [link to example]). Figure 5 ).

[0072] like Figure 5 As shown, when the concentration of TSPBA is high (above 6 wt%), PVA concentrations of 3%, 6 wt%, and 9 wt% will form a solid gel. This gel crumbles easily when rubbed and cannot be used as a dressing.

[0073] Application Example 1

[0074] like Figure 7 and Figure 8 As shown, the in vitro photothermal properties and CO release properties of the gel material described in Example 1 were investigated.

[0075] S1. Investigation of in vitro photothermal properties

[0076] To investigate the effect of MCP NP concentration on photothermal performance, pure water was used as a control group. High-precision digital thermometers were used to measure the temperature of four MCP@Gel samples doped with different mass-volume concentrations of MCP NPs (0.015%, 0.03%, 0.06%, and 0.09%) after irradiation with an 808nm laser (0.5W / cm²). 2 The temperature rise was recorded every 30 seconds for 10 minutes, and the temperature rise curve was plotted (see [reference]). Figure 7 a).

[0077] The MCP@Gel doped with different volume fractions of MCP NPs (0.015%, 0.03%, 0.06%, 0.09%) was prepared according to the preparation process of S3 in Example 1 above.

[0078] To investigate the effect of NIR laser power density on the photothermal performance, a high-precision digital thermometer and an 808 nm laser were used to record the temperature change of MCP@Gel containing 0.06% (w / v) MCP NPs under irradiation of different power densities (0.5 W / cm 2 , 0.75 W / cm 2 , 1.0 W / cm 2 ). The temperature was recorded every 30 s, and the temperature rise curve was plotted (see Figure 7 b).

[0079] To investigate the photothermal stability of MCP@Gel, 1 mL of MCP@Gel containing 0.06% (w / v) MCP NPs was taken in a plastic cuvette and irradiated with an 808 nm, 0.5 W / cm 2 laser for 10 min each time, and the laser was turned off for 10 min. Four cycles of light-avoidance were performed, and the temperature was recorded every 30 s during the period. The temperature change curve was plotted (see Figure 7 c).

[0080] Figure 7 (a) shows that the heat generated by MCP@Gel under near-infrared light irradiation is positively correlated with the concentration of MCP NPs, showing a certain dose dependence, and also shows that the prepared hydrogel has good photothermal effect, which can help to produce PTT efficacy and kill bacteria. Figure 7 (b) shows that with the increase of laser power density, the temperature of MCP@Gel gradually rises, indicating that the photothermal performance of the gel depends to a certain extent on the power density of the laser, and has good photothermal controllability. From Figure 7 (c) it can be seen that after four cycles of light-avoidance, the temperature of the gel cannot be reduced to the initial temperature during the 10 min cooling process, so the highest temperature of each cycle rises compared with the last one as the laser on / off irradiation cycle proceeds, but the temperature rise and fall curves are basically symmetrical, indicating that MCP@Gel has good photothermal stability and photothermal reproducibility.

[0081] Evaluation of CO release performance

[0082] Hb solution has a characteristic absorption peak at 430 nm, while CO has a strong affinity for Hb and can generate carboxyhemoglobin (HbCO), at this time, the characteristic absorption peak of Hb at 430 nm disappears, and a characteristic absorption peak of HbCO appears at 410 nm, so the release of CO can be indirectly determined according to this characteristic of Hb. The specific detection method is as follows: 1.08 mg of bovine hemoglobin particles is completely dissolved in 4 mL of PBS (10 mM, pH = 7.4) to prepare a 4.2 μM Hb solution, then 2.0 mg of sodium dithionite (SDT) is added under the condition of nitrogen flow, and stirring is performed for reduction. Add H2O2 solution to make the concentration of H2O2 in the Hb solution 100 μM. Put MCP@Gel in a quartz cuvette, then add the above prepared 4.2 μM Hb solution, and immediately seal the system to prevent air from entering, and use a UV spectrophotometer to measure the UV absorption curve of 350-600 nm at a certain time (see Figure 8 ), and the concentration of released CO (c CO ) is calculated by formula 1:

[0083]

[0084] In formula 1, c Hb is the initial concentration of the Hb solution (4.2 μM), I 410 nm and I 430 nm are the UV absorption values of the solution at 410 nm and 430 nm at each specific time, respectively.

[0085] Blank Gel is used as a negative control, and the above experimental steps are repeated to explore whether the CO release behavior of the hydrogel is derived from MCP NPs. In addition, Hb solutions with different concentrations of H2O2 (50, 100, 150 μM) are prepared, and the above experimental steps are repeated to explore whether the CO release behavior of MCP@Gel depends on the concentration of H2O2.

[0086] From Figure 8 (a), it can be seen that the characteristic absorption peak of the solution at 0 h is at 430 nm, and at 0.25-0.75 h, the characteristic absorption peak at 430 nm decreases and blue shifts gradually to 410 nm, with a peak value of 1.12, indicating that CO is released at this time. According to the UV results and formula 1, the cumulative release of CO from MCP@Gel reaches 3.7 μM at 4 h Figure 8 (b)), the released CO can play an antibacterial and anti-inflammatory effect, and the small amount of CO released will not harm the body. Figure 8(c) shows that the characteristic absorption peak of Hb solution in the negative control group always remains at 430 nm, without blue shift, and the peak value is constantly decreasing. It shows that the Hb solution is constantly degrading over time, but there is no CO release, and the Blank Gel has no CO release behavior because it does not contain MCP NPs. From Figure 8 (d) shows that the amount of CO released depends on the concentration of H2O2, and the higher the concentration of H2O2, the faster the rate of CO release from MCP@Gel within 30 min, and the CO release has H2O2 responsiveness.

[0087] Application Example 2

[0088] As shown in Figure 9 , Figure 10 and Figure 11 , the in vitro antibacterial activity and anti-biofilm activity of the gel material described in Example 1 were investigated.

[0089] S1, Evaluation of Antibacterial Activity

[0090] Two conventional standard strains of E. coli and S. aureus were selected as representative bacteria, and the in vitro antibacterial performance of different preparations was evaluated by bacterial plate counting method, specifically:

[0091] Different gel experimental groups are as follows: (1) PBS; (2) PBS+NIR; (3) Blank Gel; (4) Blank Gel+NIR; (5) CuS@Gel; (6) CuS@Gel+NIR; (7) MCP@Gel; (8) MCP@Gel+NIR; (9) MCP@Gel+H2O2; (10) MCP@Gel+H2O2+NIR. The specific method is: 500 μL of prepared hydrogel is placed in a 48-well plate, 100 μL of PBS is added to the hole, and the hole plate is sterilized and disinfected by ultraviolet irradiation for 40 min. Add 100 μL of 1×10 8 CFU / mL bacterial solution to each well; wherein the NIR group is irradiated with 0.5 W / cm 2 808 nm laser for 10 min; the H2O2 group is added with H2O2 solution to make the H2O2 concentration in each well 100 μM, incubated for 4 h, and the non-irradiation group is directly placed in the room temperature environment for incubation for 4 h.

[0092] Three kinds of gels were prepared:

[0093] (1) The gel composed of 250 μL of 4 wt% TSPBA solution and 250 μL of 9 wt% PVA is Blank Gel;

[0094] (2) CuS@Gel consisting of 250 μL of 4 wt% TSPBA solution containing 0.0447 mg of HMCuS NPs and 250 μL of 9 wt% PVA;

[0095] (3) MCP@Gel consisting of 250 μL of 4 wt% TSPBA solution containing 0.3 mg of MCP NPs and 250 μL of 9 wt% PVA.

[0096] After corresponding treatment of each group, all bacterial liquid in each well was taken out and coated in LB agar plates. The coated plates were incubated in a 37 °C incubator for 20 min, and then incubated for 12-16 h in an inverted position. The colony morphology was observed, the number of bacteria was recorded, a column chart was drawn, and the bacteriostatic effect was judged by comparison with the negative control group (see Figure 9 a and Figure 9 b).

[0097] S2, Anti-biofilm activity evaluation

[0098] Two conventional standard strains of E. coli and S. aureus were selected as representative bacteria. 100 μL of 1 x 10 8 CFU / mL bacterial liquid and 100 μL of liquid medium were added to a 96-well plate, and incubated in a constant temperature incubator for 48 h to form a biofilm. After carefully aspirating the waste liquid, it was washed twice with PBS. Then 100 μL of each group of preparations was added to each well: (1) PBS; (2) CuS@Gel+NIR; (3) MCP@Gel+NIR; (4) MCP@Gel+H2O2; (5) MCP@Gel+H2O2+NIR, and 100 μL of PBS was additionally added to the gel group to keep the biofilm moist. The H2O2 group needed to add 100 μL of PBS containing 100 μM H2O2, and the NIR group was irradiated with a 0.5 W / cm 2 808 nm laser for 10 min. After the light irradiation, it was incubated for 4 h. Then each group of preparations was taken out, washed once with PBS, dried at room temperature for 1 h, 200 μL of methanol was added to each well for 15 min, the methanol was aspirated and discarded, washed once with PBS, 200 μL of 0.1% crystal violet staining solution was added for 30 min in the dark, the crystal violet staining solution was washed off, and after drying at room temperature, 200 μL of 30% acetic acid solution was added. The plate was incubated in a shaker at 100 rpm for 30 min, and the OD value was measured at 570 nm with a microplate reader. The value was recorded, and the anti-biofilm effect was judged by comparison with the PBS group (see Figure 9 c and Figure 9 d).

[0099] S3, Live / dead bacterial staining

[0100] Live / dead bacterial staining experiment was performed using fluorescent dyes SYTO-9 and PI to evaluate the bacterial viability after treatment with different formulations. First, SYTO-9 and PI fluorescent dyes were prepared according to the kit instructions. The experimental groups were as follows: (1) PBS; (2) PBS + NIR; (3) Blank Gel; (4) Blank Gel + NIR; (5) CuS@Gel; (6) CuS@Gel + NIR; (7) MCP@Gel; (8) MCP@Gel + NIR; (9) MCP@Gel + H2O2; (10) MCP@Gel + H2O2 + NIR (the experimental group part was set the same as the preparation method described in S1 above). The specific method was as follows: 500 μL of E. coli bacterial solution (2 x 10 7 CFU / mL) was incubated with each group of formulations for 4 h, and 100 μL of PBS containing 100 μM H2O2 was added to the H2O2 group. The NIR group was irradiated with a 0.5 W / cm 2 808 nm laser for 10 min. After co-incubation, the treated bacterial solution was mixed with an equal volume of PI / SYTO-9 solution and incubated at room temperature in the dark for 30 min. The fluorescence imaging of each group of bacterial samples was observed using a laser confocal microscope. SYTO-9 stained live bacteria green, while dead bacteria were stained red by PI due to cell membrane rupture (see Figure 10 ).

[0101] In addition, the same S. aureus bacterial solution (2 x 10 8 CFU / mL) was used to perform the above experimental steps to investigate the viability of S. aureus after treatment with different formulations (see Figure 10 ).

[0102] S4, Characterization of bacterial morphology

[0103] The morphological changes of E. coli and S. aureus treated with different formulations were observed by SEM. The bacterial solution (1 x 10 8 CFU / mL) was incubated with different experimental groups (1) PBS; (2) PBS + NIR; (3) Blank Gel; (4) Blank Gel + NIR; (5) CuS@Gel; (6) CuS@Gel + NIR; (7) MCP@Gel; (8) MCP@Gel + NIR; (9) MCP@Gel + H2O2; (10) MCP@Gel + H2O2 + NIR (the experimental group part was set the same as the preparation method described in S1 above), and 100 μL of PBS containing 100 μM H2O2 was added to the H2O2 group. The NIR group was irradiated with a 0.5 W / cm 2808 nm laser irradiation for 10 min. After the completion of the co-incubation, the bacterial suspension was collected and centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. The bacteria were fixed by adding 2.5% glutaraldehyde solution quickly, and stored at 4°C overnight. The samples were washed with PBS three times for 15 min each time, and the bacteria in each group were dehydrated with a gradient concentration (30%, 50%, 70%, 80%, 90%, and 95%) of ethanol solution. Finally, the bacteria were resuspended in anhydrous ethanol and adjusted to an appropriate concentration, dropped on a silicon wafer, dried at room temperature, gold-sprayed, and observed by SEM (see Figure 11 ).

[0104] As shown in Figure 9 (a, b), according to the statistical results of plate counting, the CO released by MCP@Gel under the initiation of H2O2 could cause a certain degree of damage to E. coli and S. aureus, and the survival rates of the bacteria were 36.2% and 41.6%, respectively. After NIR irradiation, the bacteria were almost all killed due to the combination of PTT, and the survival rates were only 0.47% and 0.75%. It is shown that MCP@Gel has good synergistic broad-spectrum antibacterial properties under the co-activation of NIR irradiation and H2O2, and the effect is significantly better than that of the single mode antibacterial therapy.

[0105] The statistical results of the OD value measurement of E. coli crystal violet are shown in Figure 9 (c), compared with the PBS control group, 45.27% and 45.58% of the biofilm in the MCP@Gel+NIR group and the MCP@Gel+H2O2 group were removed, respectively, and 72.51% of the biofilm in the MCP@Gel+H2O2+NIR treatment group was removed, showing strong anti-biofilm ability. Figure 9 (d) shows the OD value of S. aureus crystal violet. Compared with the control group, the MCP@Gel+H2O2+NIR treatment group removed 77.56% of the biofilm. MCP@Gel has good anti-biofilm ability under the co-activation of NIR irradiation and H2O2. On the one hand, the generated CO can regulate the metabolic process of bacteria, affect the electron transfer of the respiratory chain by combining with cytochrome oxidase, thereby inhibiting the growth and reproduction of bacteria, making it difficult for bacteria to form a biofilm, or making the bacteria in the biofilm difficult to maintain the structure and function of the biofilm due to energy shortage. On the other hand, the local high temperature generated by PTT can also destroy the structure of the biofilm, further improving the inhibitory effect of CO on bacteria, so the synergistic treatment of the two methods produces the greatest damage to the biofilm.

[0106] As shown in Figure 10As shown, the CuS@Gel+NIR and MCP@Gel+NIR groups treated with NIR light, compared with the non-illuminated MCP@Gel+H2O2 group, showed more red fluorescence in E. coli and S. aureus, indicating that a single treatment mode can kill some bacteria. Furthermore, E. coli and S. aureus treated with MCP@Gel under both NIR light and H2O2 were almost entirely stained red, demonstrating that MCP@Gel has a high broad-spectrum bactericidal efficiency under the combined excitation of NIR and H2O2, and its antibacterial effect is significantly better than that of single antibacterial therapy.

[0107] like Figure 11 As shown, compared with the PBS group and the Blank Gel group treated with E. coli and S. aureus, the bacteria in the CuS@Gel+NIR group and the MCP@Gel+NIR group treated with NIR light and the non-light-treated MCP@Gel+H2O2 group showed a certain degree of shrinkage. The bacteria in the MCP@Gel group treated with NIR light and H2O2 were rougher on the surface and more curled in shape (E. coli) or collapsed in structure (S. aureus). This indicates that under the triggering of H2O2, the CO released by MCP@Gel and PTT work synergistically to destroy the bacterial cell membrane and cause intracellular leakage, resulting in the most severe damage to the bacterial morphology. This leads to S. aureus and E. coli being unable to maintain normal physiological activities, thereby inhibiting bacterial growth and reproduction and achieving the antibacterial purpose.

[0108] Application Example 3

[0109] The in vivo antibacterial activity and safety of the near-infrared light and hydrogen peroxide dual-responsive photothermal / carbon monoxide antibacterial hydrogel described in Example 1 were investigated. In this invention, a mouse dorsal infection wound model was established using *S. aureus* as a model bacterium to continuously observe wound healing and evaluate the hydrogel's wound repair effect.

[0110] To investigate the PTT effect of the formulation in vivo, two model mice were selected and designated as: (1) Blank Gel group; (2) MCP@Gel group. 500 μL of Blank Gel and MCP@Gel were applied to the wounds of the mice, respectively, and administered 0.5 W / cm² of medication. 2 The wound was irradiated with an 808nm laser, and temperature changes at the wound site were monitored and photographed using an infrared thermal imager. The results are as follows: Figure 12As shown, the temperature of the wound in mice in the MCP@Gel+NIR group could rise to 48.3℃ over time. This temperature can kill most bacteria and will not cause burns to the skin tissue. In contrast, the wound temperature in mice in the Blank Gel+NIR group only changed slightly, with the highest temperature rising to 38℃. This indicates that MCP@Gel has good photothermal properties in vivo.

[0111] Figure 13 (a) is a schematic diagram of the animal experimental protocol for the near-infrared light and hydrogen peroxide-responsive photothermal / carbon monoxide therapy hydrogel material and other experimental groups. Specific protocol: Mice with wound infection were randomly divided into 8 groups, with 6 mice in each group. The groups are as follows: (1) PBS; (2) PBS+NIR; (3) Blank Gel+H2O2; (4) Blank Gel+H2O2+NIR; (5) MCP@Gel; (6) MCP@Gel+NIR; (7) MCP@Gel+H2O2; (8) MCP@Gel+H2O2+NIR. 50 μL of PBS was administered subfascially to the PBS group. Different hydrogel groups were applied to the wound infection sites of mice in each group for treatment. 200 μL of 100 μM H2O2 was added to the gel in the H2O2 group. 0.5 W / cm² H2O2 was used in the NIR group. 2 The mice were irradiated with an 808nm laser for 5 minutes, and the temperature was monitored using a thermal imager during the treatment, being kept below 50℃ to prevent burns. All treatment sites were bandaged with 3M transparent sterile dressings to prevent the mice from biting and dislodging the hydrogel; any residual hydrogel was removed after 24 hours. Mouse weights and wound site photographs were recorded on days 1, 3, 5, 7, and 10 after treatment. Figure 13 (b) It can be seen that on day 10, the wound area of ​​mice in all treatment groups gradually decreased and healed slowly. Among them, the PBS group and PBS+NIR group, which do not contain antibacterial components, showed poor wound healing with scab formation. The Blank Gel+H2O2 group and Blank Gel+NIR group showed poor wound healing. The Gel+H2O2+NIR group and the MCP@Gel group showed faster wound healing compared to the PBS and PBS+NIR groups, possibly because the hydrogel's ability to absorb exudate and maintain wound moisture improved the wound healing environment. The MCP@Gel+NIR and MCP@Gel+H2O2 groups, due to their antibacterial properties, exhibited better wound healing than all other groups by day ten, with the wounds essentially healed. The MCP@Gel+H2O2+NIR group, benefiting from the synergistic antibacterial effect of CO / PTT and the multiple advantages of hydrogel (including anti-infection, improved microenvironment, and wound healing promotion), showed the best healing, with almost complete wound healing and no obvious scarring. This indicates that MCP@Gel, under the combined action of H2O2 and NIR, can exert better anti-infection and wound healing promotion effects.Figure 13 (c) shows that the relative area of each group of mice wounds on the 10th day was quantitatively analyzed, and the relative wound area of the MCP@Gel+H2O2+NIR group on the 10th day was only 1.47% of that on the 0th day, and compared with other groups, the wound closure effect of this group was the best, indicating that MCP@Gel has a better effect on promoting wound healing under the combined action of NIR and H2O2.

[0112] After the treatment was completed, the mice were sacrificed, and the wound tissues of the mice in each group were separated and collected. After being washed with PBS, tissue homogenate was prepared, and the contents of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) at the wound site after treatment were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 14 As shown in Fig. 8, the levels of inflammatory factors in the wounds of mice in the PBS group, the PBS+NIR group, the Blank Gel+H2O2 group, the Blank Gel+H2O2+NIR group and the MCP@Gel group were higher than those in the remaining treatment groups on the 10th day after infection, indicating that infection triggered a strong inflammatory response. Compared with the MCP@Gel+NIR group, the levels of inflammatory factors in the wounds of mice in the MCP@Gel+H2O2 group were slightly lower, which may be because MCP@Gel can release CO in response to H2O2, and CO can inhibit the expression and release of inflammatory factors through multiple signaling pathways, resulting in a decrease in the levels of inflammatory factors. The levels of inflammatory factors in the wounds of mice treated by MCP@Gel+NIR+H2O2 were the lowest, which may be due to the synergistic antibacterial and anti-inflammatory effects of CO / PTT. The high-temperature environment can directly destroy the cell membranes, proteins and other biological macromolecules of bacteria, thereby achieving the purpose of bactericidal and anti-inflammatory. CO plays an anti-inflammatory role through multiple pathways such as inhibiting the expression of inflammatory factors, regulating the function of immune cells and antioxidant effects. And in CO combined with PTT, CO can reduce the inflammatory response caused by PTT and promote tissue repair and regeneration.

[0113] After the treatment was completed, the mice were sacrificed, and the wound tissues of the mice in each group were separated and collected. After being washed with PBS, tissue homogenate was prepared, and the contents of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) at the wound site after treatment were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 15(a), the skin around the wound of the PBS group, PBS+NIR group, Blank Gel group, Blank Gel+NIR group and MCP@Gel group lacks the adnexal organs of the skin, such as hair follicles and sebaceous glands, and inflammatory cells and granulation tissue disorder can be observed. The wound healing of the MCP@Gel+NIR group and the MCP@Gel+H2O2 group under PTT or CO treatment is relatively good, a small amount of regenerated tissue appears, inflammatory cells are relatively reduced, and the thickness of the epidermal layer also increases to a certain extent, but the repair is not complete. The wound of the MCP@Gel+H2O2+NIR group of mice already has a relatively complete epidermal layer, and the repair of the hair follicles of the skin tissue at the wound is relatively complete, and the inflammatory cells of the skin tissue are relatively few, which is the good antibacterial and anti-inflammatory effect of the synergistic treatment of PTT and CO. Masson staining results Figure 15 (b), after 10 days of treatment, the collagen deposition (blue) of the PBS group and the PBS+NIR group is less and loose, the collagen deposition of the Blank Gel group, the Blank Gel+NIR group and the MCP@Gel group increases, but it is not as good as that of the MCP@Gel+NIR group and the MCP@Gel+H2O2 group. The main reason is that these three groups do not have effective antibacterial effect, although the presence of hydrogel can provide a good environment for wound healing, but bacterial infection will interfere with the generation of collagen and affect the normal healing of the wound. The collagen structure of the skin around the wound of the MCP@Gel+H2O2+NIR group of mice is denser compared with other treatment groups, and the collagen is arranged in order, and the number of collagen fibers is the most, indicating that the skin tissue healing state of this treatment group is the best.

[0114] As shown in Figure 16 (a), during the treatment, the body weight of each group of mice did not appear obvious fluctuation under the irradiation with or without NIR and the treatment of H2O2, indicating that MCP@Gel did not produce obvious physical toxicity to the mice, and the irradiation of NIR and H2O2 had no effect on the growth of the mice, and both the preparation and the treatment method showed good biological safety.

[0115] To investigate the biocompatibility of MCP@Gel, hemolysis test was used as an index to evaluate the blood toxicity of MCP@Gel. Fresh blood of mice was taken in centrifuge tubes, and after standing at 4°C for 2h, the blood was centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was discarded to obtain red blood cells (RBC). Then the RBC was washed three times with PBS, and each time the RBC was centrifuged under the same conditions. The obtained RBC was resuspended in PBS to prepare a 4% RBC suspension. 2 mL of the RBC suspension was taken in a centrifuge tube, and 0.5 mL of MCP@Gel doped with different mass-volume concentrations of MCPNPs (0.03%, 0.045%, 0.06%, 0.075%, and 0.09%) was added. PBS was used as a negative control, and distilled water was used as a positive control. The mixture was incubated at 37°C for 4h, and then centrifuged at 3000 rpm for 10 min at 4°C. The hemolysis phenomenon was photographed and recorded. 200 μL of the supernatant of each group was taken in a 96-well plate, and the OD value was measured at 540 nm. The hemolysis rate was calculated according to the numerical value. If the hemolysis rate was greater than 5%, it was determined that hemolysis had occurred. The hemolysis rate was calculated by formula 2:

[0116]

[0117] In formula 2, OD 样品 is the OD value of different sample groups, OD H2O is the OD value of the positive control group, and OD PBS is the OD value of the negative control group.

[0118] The pictures of centrifugation of fresh blood incubated with different concentrations of MCP@Gel and the hemolysis rate are shown in Figure 16 (b). No obvious hemolysis phenomenon occurred in each MCP@Gel group, and the hemolysis rate was less than 5%, indicating that the preparation had good blood compatibility. This provides a safety guarantee for the application of MCP@Gel in vivo treatment and further clinical application.

Claims

1. A dual-responsive antibacterial hydrogel, characterized in that: The antibacterial hydrogel is composed of drug-loaded nanoparticles and a hydrogel matrix.

2. The dual-responsive antibacterial hydrogel according to claim 1, characterized in that: The drug-loaded nanoparticles consist of manganese decacarbonyl dimanganese (MnCO) loaded into the cavity of hollow mesoporous copper sulfide nanoparticles (HMCuS NPs) and coated with a polydopamine (PDA) layer on the particle surface.

3. The dual-responsive antibacterial hydrogel according to claim 1, characterized in that: The hydrogel matrix is ​​prepared by dynamic crosslinking of phenylboronic acid bonds formed by the reaction of the H2O2-responsive crosslinking agent N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA) with the diol groups of polyvinyl alcohol (PVA).

4. The method for preparing the dual-responsive antibacterial hydrogel according to claim 1, characterized in that: 1) Drug-loaded nanoparticles: Hollow mesoporous copper sulfide nanoparticles (HMCuS NPs) with photothermal properties are used as carriers to load carbon monoxide donor decacarbonyl dimanganese (MnCO) and the surface of the particles is coated with a polydopamine layer (PDA). 2) Hydrogel matrix: prepared by dynamic crosslinking of phenylboronic acid bonds formed by the reaction of H2O2-responsive crosslinking agent (TSPBA) with the diol groups of polyvinyl alcohol (PVA); 3) The drug-loaded nanoparticles obtained above are uniformly dispersed in the hydrogel matrix to obtain a dual-response antibacterial hydrogel; wherein the mass-volume concentration of the drug-loaded nanoparticles relative to the hydrogel matrix is ​​0.06% (w / v).

5. The method for preparing the dual-responsive antibacterial hydrogel according to claim 4, characterized in that: The method for preparing the drug-loaded nanoparticles is as follows: 1) Preparation method of HMCuS NPs: Disperse polyvinylpyrrolidone in deionized water, add copper chloride solution and mix. After mixing, adjust the system to alkaline, then add hydrazine hydrate and sodium sulfide to the system, mix, and heat in a water bath at 75-80℃ for reflux reaction for 2-3 hours. 2) Disperse MnCO and the obtained HMCuS NPs in methanol, stir overnight, and dry the mixed solution in a vacuum dryer to obtain solution 1; wherein the mass ratio of HMCuS NPs to MnCO is 1:1 to 1:

2. 3) Dissolve dopamine hydrochloride in Tris buffer to prepare solution 2; 4) Disperse solution 1 uniformly in solution 2, stir at room temperature for 5-8 hours, wash, and obtain drug-loaded nanoparticles (MnCO-CuS@PDANPs, abbreviated as MCP NPs).

6. The method for preparing the dual-responsive antibacterial hydrogel according to claim 5, characterized in that: In step 1), the mass ratio of polyvinylpyrrolidone to copper chloride is 20:1 to 30:1, the molar ratio of hydrazine hydrate to copper chloride is 1:1 to 2:1, and the molar ratio of sodium sulfide to copper chloride is 5:1 to 8:

1.

7. The method for preparing the dual-responsive antibacterial hydrogel according to claim 4, characterized in that: The preparation method of the hydrogel matrix solution is as follows: 1) Preparation method of hydrogen peroxide responsive crosslinking agent TSPBA: N,N,N',N'-tetramethyl-1,3-propanediamine and 4-(bromomethyl)phenylboronic acid are dissolved together in N,N-dimethylformamide and stirred under reflux in an oil bath at 60-80℃ for 12h. After the reaction solution is cooled to room temperature, it is poured into tetrahydrofuran, and a white precipitate is precipitated. The product is collected and dried under vacuum overnight to obtain the crosslinking agent TSPBA. 2) Preparation of TSPBA precursor solution for antibacterial hydrogel: Weigh the TSPBA prepared above and add it to deionized water to obtain a solution; 3) Preparation of PVA precursor solution for antibacterial hydrogel: Add polyvinyl alcohol to deionized water, heat to 80-90℃ and stir to dissolve. After complete dissolution, allow it to cool naturally to room temperature, filter, and obtain the solution.

8. The method for preparing the dual-responsive antibacterial hydrogel according to claim 7, characterized in that: In step 1), the molar ratio of N,N,N',N'-tetramethyl-1,3-propanediamine to 4-(bromomethyl)phenylboronic acid is 1:2.5 to 1:

3.

9. The application of the dual-responsive antibacterial hydrogel according to claim 1, characterized in that: Application of the dual-responsive antibacterial hydrogel in formulations for treating bacterial infectious diseases.

10. The application of the dual-responsive antibacterial hydrogel according to claim 9, characterized in that: The bacterial infectious disease is a bacterial infectious disease caused by Escherichia coli and / or Staphylococcus aureus; the dual response is a combined response to near-infrared light and hydrogen peroxide.

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