Hydrogel dressing based on near-infrared light controllable cascade reaction and preparation method thereof

Abstract

The invention relates to a hydrogel dressing based on near-infrared light controllable cascade reaction and a preparation method thereof, and belongs to the technical field of hydrogel dressings. The preparation method of the hydrogel dressing based on the near-infrared light controllable cascade reaction comprises the following steps: respectively grafting carboxymethyl-beta-cyclodextrin and L-arginine to carboxymethyl chitosan by adopting an EDC / NHS crosslinking method to prepare cyclodextrin grafted chitosan and L-Arg grafted chitosan; adding a photosensitizer into the cyclodextrin grafted chitosan solution I to obtain a C-C-I inclusion compound solution; adding hemin into the cyclodextrin grafted chitosan solution II to obtain a C-C-H inclusion compound solution; and mixing the oxidized hyaluronic acid solution, the L-Arg grafted chitosan solution, the C-C-I clathrate compound solution and the C-C-H clathrate compound solution. The CLIHO disclosed by the invention can be used for synergistically resisting bacteria through PTT / NO and synchronously adjusting a wound microenvironment, so that the healing speed and the healing quality of infectious chronic wounds are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of hydrogel dressings, and particularly relates to a hydrogel dressing based on near-infrared light controllable cascade reaction and a preparation method thereof. BACKGROUND

[0002] Skin, as the most critical natural barrier organ of the human body, its structural integrity is crucial to maintaining the health of the body. Under the action of various external factors, the structure of the skin may be damaged, thereby causing skin injury. Skin injury is divided into two categories: acute wounds and chronic wounds. Acute wounds usually can go through four orderly stages of hemostasis, inflammatory response, cell proliferation and tissue remodeling, thereby timely and effectively promoting wound healing. Chronic wounds, due to their complex microenvironment, including factors such as bacterial infection, hypoxic state and oxidative stress, cause the healing process to become slow, and even may not heal. Therefore, how to effectively treat chronic wounds with complex microenvironment is still a big challenge faced by the current medical field.

[0003] Bacterial infection is one of the main obstacles for chronic wound healing, which can form biofilm through extracellular polymeric substance (EPS). The presence of biofilm not only promotes the formation of drug-resistant bacteria, but also causes persistent inflammatory response, inhibits the formation of new tissue, and delays the wound healing rate. Photothermal antibacterial therapy (PTT) and photodynamic antibacterial therapy (PDT) kill bacteria by generating heat and reactive oxygen species (ROS) through laser-activated photosensitizers, with less side effects and strong penetration. However, PTT may damage the surrounding healthy tissue due to high temperature, and has limitations such as poor effect on heat-resistant bacteria and biofilm removal. The presence of oxidase in bacteria makes them resistant to ROS, which can weaken the bactericidal ability of ROS. In order to overcome these limitations, combining PTT with other antibacterial modes to exert synergistic antibacterial effect and effectively eliminate biofilm has become a promising research direction. As a new strategy, gas therapy has gradually emerged in the field of antibacterial therapy in recent years. Nitric oxide (NO) is a unique bioactive molecule, and low concentration of NO can activate immune cells, while high concentration of NO can covalently bind to the DNA, protein or lipid of bacteria, directly killing bacteria. NO can also activate phosphodiesterase and promote the degradation of cyclic di-GMP (c-di-GMP), thereby reducing bacterial adhesion and EPS production, and effectively destroying the biofilm structure. However, the physiological function of NO is restricted by its short half-life (1-5 s) and short diffusion distance (20-160 μm). Therefore, how to use NO donors to achieve controllable on-demand release of NO at the site of bacterial infection is crucial. In addition, due to the damage of the vascular system, the oxygen supply to the chronic wound is severely affected, resulting in a long-term hypoxic environment in the local wound. The formation of bacterial biofilm also exacerbates the hypoxia in the wound by consuming local oxygen. Hypoxic environment not only inhibits angiogenesis and re-epithelialization, two key healing processes, but also hinders the synthesis of extracellular matrix, thereby significantly delaying wound healing. Chronic wounds are often accompanied by high concentrations of hydrogen peroxide (H2O2). H2O2 is a strong oxidizing agent that can disrupt the balance of the wound microenvironment and further exacerbate inflammation. This inflammatory response not only exacerbates the wound, but also further delays the healing process. SUMMARY

[0004] The purpose of the present application is to provide a hydrogel dressing based on near-infrared light controllable cascade reaction and a preparation method thereof, which can remodel the wound microenvironment by reducing H2O2 levels, eliminating ROS and alleviating hypoxia, and release different concentrations of NO through the regulation of NIR "on / off", to exert the functions of PTT / NO synergistic antibacterial or promote angiogenesis, thereby improving the healing rate and quality of the wound.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0006] A preparation method of a hydrogel dressing based on near-infrared light controllable cascade reaction, the preparation method of the hydrogel dressing comprising the following steps:

[0007] S1: carboxymethyl-beta-cyclodextrin and L-arginine are grafted to carboxymethyl chitosan respectively by EDC / NHS crosslinking method to prepare cyclodextrin grafted chitosan and L-Arg grafted chitosan;

[0008] S2: a photosensitizer is added to cyclodextrin grafted chitosan solution one to obtain a C-C-I inclusion complex solution; hematin chloride is added to cyclodextrin grafted chitosan solution two to obtain a C-C-H inclusion complex solution;

[0009] S3: after mixing of oxidized hyaluronic acid solution, L-Arg grafted chitosan solution, C-C-I inclusion complex solution and C-C-H inclusion complex solution, the hydrogel dressing is obtained.

[0010] Further, the final mass concentration of the C-C-I inclusion complex in the hydrogel dressing is 0.5-2.0%, the final mass concentration of the C-C-H inclusion complex is 0.1-1.0%, the final mass concentration of the L-Arg grafted chitosan is 1.0-3.0%, and the final mass concentration of the oxidized hyaluronic acid is 2.0-5.0%.

[0011] Further, the photosensitizer in S2 is indocyanine green.

[0012] Further, the preparation methods of the cyclodextrin grafted chitosan solution one and the cyclodextrin grafted chitosan solution two in S2 are both that 2-8 mg of cyclodextrin grafted chitosan is dissolved in 1 mL of PBS buffer solution to obtain; the mass ratio of the photosensitizer to the cyclodextrin grafted chitosan is 0.5-1.5:1.0; and the mass ratio of the hematin chloride to the cyclodextrin grafted chitosan is 0.1-0.5:1.0.

[0013] Further, the preparation method of the cyclodextrin grafted chitosan in S1 is that EDC and NHS are added to a carboxymethyl-beta-cyclodextrin solution for activation for 1-3 h, then a carboxymethyl chitosan solution one is added for reaction for 12-48 h, followed by dialysis purification, and then freeze-drying to obtain.

[0014] Further, the mass fraction of the carboxymethyl chitosan solution one is 1.0-5.0%; the mass fraction of the carboxymethyl-beta-cyclodextrin is 1.0-5.0%; the mass ratio of the carboxymethyl-beta-cyclodextrin to the carboxymethyl chitosan is 0.5-2:1.0, the mass ratio of the carboxymethyl-beta-cyclodextrin to the EDC is 1.0:1.5-3.0; and the mass ratio of the carboxymethyl-beta-cyclodextrin to the NHS is 1.0:1.0-2.0.

[0015] Further, the preparation method of the L-Arg grafted chitosan in S1 is as follows: EDC and NHS are added into the L-arginine solution to activate for 1-3 hours, then the carboxymethyl chitosan solution II is added to react for 12-48 hours, and then the product is obtained after dialysis purification and freeze-drying.

[0016] Further, the mass fraction of the carboxymethyl chitosan solution II is 0.5-3.0%, the L-arginine solution is obtained by dissolving 1.0-3.0 g of L-arginine in 30 mL of water, the mass ratio of L-arginine to carboxymethyl chitosan is 1.0-3.0:1.0, the mass ratio of L-arginine to EDC is 0.8-2.0:1.0, and the mass ratio of L-arginine to NHS is 1.0-5.0:1.0.

[0017] A hydrogel dressing based on near-infrared light controllable cascade reaction is prepared by the above-mentioned preparation method of the hydrogel dressing based on near-infrared light controllable cascade reaction.

[0018] The beneficial effects of the present application are as follows:

[0019] The hydrogel dressing (CLIHO) based on near-infrared light controllable cascade reaction of the present application can synergistically antibacterial and synchronously regulate the wound microenvironment through PTT / NO, so as to improve the healing speed and quality of the infected chronic wound. L-arginine (L-Arg) can generate NO under the catalysis of nitric oxide synthase (NOS) or in the presence of ROS, and can also be converted into ornithine by arginase, and then participate in the synthesis of collagen to promote wound healing. Hematin (Hemin) can effectively decompose high-level hydrogen peroxide at the wound and generate oxygen, so as to improve the oxidative stress and hypoxic microenvironment of the wound and provide oxygen for the generation of ROS and NO. Under near-infrared light (NIR) irradiation, ICG in the hydrogel can produce excellent photothermal effect, and ROS can be generated in the presence of oxygen, and ROS can further induce L-Arg to generate a large amount of NO, so that the hydrogel can efficiently antibacterial and eliminate biofilm through the synergistic effect of PTT and NO. When the NIR is turned off, the hydrogel dressing can remodel the wound microenvironment by reducing the local H2O2 level, removing the excess ROS generated in the NIR irradiation process, relieving hypoxia and promoting angiogenesis. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is the action principle diagram of the hydrogel dressing based on near-infrared light controllable cascade reaction;

[0021] Figure 2 It is the characterization diagram of the CMCS-CMCD graft and the CMCS-LA graft in Example 1, wherein A is the ultraviolet-visible spectrum diagram of the CMCS-CMCD graft, and B is the Fourier infrared spectrum diagram of the CMCS-LA graft;

[0022] Figure 3 The above are characterization diagrams of the hydrogel dressings in Examples 1 and Comparative Examples 1-3, where A is a visual diagram of the hydrogel dressing in Example 1, B is a SEM image of the hydrogel dressings in Examples 1 and Comparative Examples 1-3, and C is a pore size diagram of the hydrogel dressings in Examples 1 and Comparative Examples 1-3.

[0023] Figure 4 Fourier transform infrared spectra of the hydrogel dressings in Example 1 and Comparative Examples 1-3;

[0024] Figure 5 These are test diagrams for the self-healing, injectability, and plasticity of the hydrogel dressing in Example 1, where A is the self-healing test diagram, B is the injectability test diagram, and C is the plasticity test diagram.

[0025] Figure 6 This is a graph showing the adhesion test of the hydrogel dressing in Example 1;

[0026] Figure 7 The graphs show the water content, swelling properties, and degradation properties of the hydrogel dressings in Example 1 and Comparative Examples 1-3, where A is the water content graph, B is the swelling rate graph, and C is the degradation property graph in PBS solution at 37°C.

[0027] Figure 8 The figures show the photothermal performance of the hydrogel dressings in Examples 1 and Comparative Examples 1-3, where A is a photothermal curve, B is a schematic photothermal photograph, C is a photothermal curve of the hydrogel dressing of Example 1 at different powers, D is a photothermal curve of the hydrogel dressing of Example 1 at different ICG concentrations, E is a photothermal cycling curve of the hydrogel dressing of Example 1, and F is a graph showing the effect of CMCD on the photothermal effect of the hydrogel dressing of Example 1.

[0028] Figure 9 Hydrogel dressings used in DPBF testing of Examples 1 and 1-3 1 O2 generation capacity diagram, where A represents the hydrogel dressings of Example 1 and Comparative Examples 1-3. 1 O2 generation capacity diagram, B represents the generation capacity of the hydrogel dressing in Example 1. 1 Graph showing the change of O2 over time;

[0029] Figure 10 The graphs show the NO generation detection of hydrogel dressings in Examples 1 and Comparative Examples 1-3, where A is the NO generation curve of the hydrogel dressing in deionized water, B is the NO generation curve of the hydrogel dressing in H2O2 solution (*P<0.05, n=3), and C is the NO generation curve of the hydrogel dressing in Example 1 under NIR on / off cycling.

[0030] Figure 11Intracellular O2 fluorescence staining patterns and fluorescence intensity statistics under different treatment conditions (no significant difference in ns, **P<0.01, n=3), scale bar: 100μm;

[0031] Figure 12 The graph shows the antioxidant properties of the hydrogel dressings in Example 1 and Comparative Examples 1-3 at the cellular level (no significant difference in ns, n=3), scale bar: 100μm;

[0032] Figure 13 Photographs of HUVEC cells with scratch marks and migration statistics (**P<0.01, n=3), scale bar: 100μm;

[0033] Figure 14 Diagrams showing the effect of hydrogel dressings on promoting HUVEC cell tubulation, and statistical graphs of tubule length, number of branches, and number of networks (*P<0.01, **P<0.01, ***P<0.001, n=3), scale bar: 100μm;

[0034] Figure 15 The graphs show the PTT / NO synergistic antibacterial properties of the hydrogel dressings in Examples 1 and Comparative Examples 1-3, where AB represents the plate coating and antibacterial rate of MRSA, CD represents the plate coating and antibacterial rate of S. aureus, EF represents the plate coating and antibacterial rate of S. aureus, and GH represents the plate coating and antibacterial rate of B. subtilis (**P<0.01, ***P<0.001, ****P<0.0001, n=3).

[0035] Figure 16 The graphs show the ability of hydrogel dressings in Examples 1 and 1-3 to remove bacterial biofilms. In Example 1, A is a crystal violet staining photograph of MRSA biofilm after treatment with hydrogel dressings; B is a crystal violet staining photograph of E. coli biofilm after treatment with hydrogel dressings; C is a quantitative statistical graph of crystal violet staining of MRSA biofilm; and D is a quantitative statistical graph of crystal violet staining of E. coli biofilm (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3).

[0036] Figure 17 Biocompatibility diagrams of the hydrogel dressings of Examples 1 and Comparative Examples 1-3 are shown, where A is a cell compatibility diagram, B is a blood compatibility diagram, and C is a tissue compatibility diagram. Scale bar: 100 μm.

[0037] Figure 18The images show the therapeutic effects of hydrogel dressings from Examples 1 and Comparative Examples 1-3 on full-thickness skin lesions of mice with MRSA infection. In the images, A is a diagram of the animal experiment protocol, B is a photograph of the temperature at the wound site in the animal experiment, and C is a temperature curve of the wound site in the animal experiment.

[0038] Figure 19 The following graphs illustrate the therapeutic effects of hydrogel dressings used in Examples 1 and 1-3 on full-thickness skin lesions infected with MRSA in mice. A shows photographs of wound healing, tracer images, and bacterial plate images of mice in each treatment group (scale bar: 0.3 cm); B shows a bacterial density statistical graph; C shows a wound healing curve; and D shows a body weight curve (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3).

[0039] Figure 20 The images show H&E and MASSON staining analysis of newly formed skin tissue at the defect site 14 days after treatment with hydrogel dressings in Examples 1 and 1-3, where A is the H&E staining analysis image and B is the MASSON staining analysis image.

[0040] Figure 21 This study assesses the quality of wound healing 14 days after treatment with hydrogel dressings in Examples 1 and 1-3. A represents the expression levels of IL-6, CD68, CD31, and α-SMA in newly formed tissue using immunofluorescence analysis (scale bar: 100 μm). BE represents the statistical analysis results of IL-6, CD68, CD31, and α-SMA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3). Detailed Implementation

[0041] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0042] Example 1

[0043] The preparation method of the hydrogel dressing based on near-infrared light-controlled cascade reaction in Example 1 includes the following steps:

[0044] S1: Dissolve 1g of carboxymethyl chitosan (CMCS) in 50mL of deionized water to obtain CMCS solution one; dissolve 1g of carboxymethyl-β-cyclodextrin (CMCD) in 50mL of deionized water to obtain CMCD solution; add 1.84g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.105g of N-hydroxysuccinimide (NHS) to the CMCD solution for 2h activation, then add CMCS solution one and stir for 24h reaction, dialyze at room temperature in a 3500D dialysis bag for 3d, and freeze-dry to obtain cyclodextrin-grafted chitosan (CMCS-CMCD graft).

[0045] S2: Dissolve 1g of CMCS in 100mL of deionized water to obtain CMCS solution II, and dissolve 2.11g of L-Arg in 30mL of deionized water to obtain L-Arg solution; add 1.84g of EDC and 1.105g of NHS to the L-Arg solution to activate for 2h, add CMCS solution II and continue stirring for 24h, dialyze at room temperature in a 3500D dialysis bag for 3d, and freeze-dry to obtain L-Arg grafted chitosan (CMCS-LA graft).

[0046] S3: Dissolve 5 mg of CMCS-CMCD graft in 1 mL of PBS buffer to obtain CMCS-CMCD solution one; dissolve 5 mg of CMCS-CMCD graft in 1 mL of PBS buffer to obtain CMCS-CMCD solution two; add 5 mg of indocyanine green (ICG) to CMCS-CMCD solution one and stir in the dark for 6 h to obtain CMCS-CMCD-ICG (CCI) inclusion complex solution; add 1 mg of hemin chloride (Hemin) to CMCS-CMCD solution two and stir in the dark for 6 h to obtain CMCS-CMCD-Hemin (CCH) inclusion complex solution.

[0047] S4: Dissolve 10g of oxidized hyaluronic acid (OHA) in 100mL of water to obtain a 10% OHA solution. Dissolve 6g of CMCS-LA graft in 100mL of water to obtain a 6% CMCS-LA graft solution. Mix the CCI inclusion complex solution, CCH inclusion complex solution, CMCS-LA graft solution, and oxidized hyaluronic acid solution in S3 at a volume ratio of 2:1:4:3 and let stand to obtain a hydrogel dressing based on a near-infrared light-controlled cascade reaction. The hydrogel dressing of Example 1 is named CLIHO.

[0048] The mass fraction of the CCI inclusion complex solution is 5%, and the mass fraction of the CCH inclusion complex solution is 5%. The mass fractions of the CCI and CCH inclusion complex solutions are calculated based on the concentration of the CMCS-CMCD graft.

[0049] Comparative Example 1

[0050] The preparation method of the hydrogel dressing in Comparative Example 1 is roughly the same as that in Example 1. The difference between the preparation method of the hydrogel dressing in Comparative Example 1 and Example 1 is that the CMCS-LA grafting solution and OHA solution are mixed at a volume ratio of 2:3 to prepare the hydrogel dressing. The hydrogel dressing in Comparative Example 1 is named CLO.

[0051] Comparative Example 2

[0052] The preparation method of the hydrogel dressing in Comparative Example 2 is roughly the same as that in Example 1. The difference between the preparation method of the hydrogel dressing in Comparative Example 2 and Example 1 is that the hydrogel dressing is prepared by mixing the CCI inclusion complex solution and the OHA solution at a volume ratio of 1:4. The hydrogel dressing in Comparative Example 2 is named CIO.

[0053] Comparative Example 3

[0054] The preparation method of the hydrogel dressing in Comparative Example 3 is roughly the same as that in Example 1. The difference between the preparation method of the hydrogel dressing in Comparative Example 3 and that in Example 1 is that the hydrogel dressing is prepared by mixing the CCI inclusion complex solution, the CMCS-LA graft solution and the OHA solution in a volume ratio of 1:2:2. The hydrogel dressing in Comparative Example 3 is named CLIO.

[0055] In Examples 1 and Comparative Examples 1-3, the content of active components in the hydrogel dressings was consistent, with the final mass fraction concentrations of CCI inclusion complex, CCH inclusion complex, and CMCS-LA graft in each group of hydrogels being 1%, 0.5%, and 2.4%, respectively.

[0056] from Figure 2 It can be seen that CMCD exhibits a strong absorption peak at 260 nm, and the CMCS-CMCD graft also shows a similar absorption peak at the same wavelength, indicating that the CMCS-CMCD graft was successfully synthesized. The CMCS-LA graft shows an absorption peak at 1650 cm⁻¹. -1 A strong C=O stretching vibration absorption peak appeared at the specified position. The intensity and characteristic wavelength of the C=O stretching vibration absorption peak correspond to the C=O stretching vibration of the carboxyl group in the CMCD molecule. Furthermore, the absorption peak in this band is formed by the amidation reaction between the carboxyl group in the CMCD molecule and the amino group on L-Arg. This proves the successful grafting between CMCS and L-Arg and the formation of a new chemical bond with an amide structure. Figure 3B shows that the hydrogels of Examples 1 and Comparative Examples 1-3 all exhibit a distinct porous structure with a relatively uniform pore size distribution. Figure 3 As can be seen from C, CLIHO hydrogel exhibits a minimal pore size due to its high degree of cross-linking. This allows for better control of drug release rates during application and enhances its contact area with the wound through its fine pores, thereby improving absorption and permeability. Figure 4 It can be seen that the hydrogels in each group are within the range of 2900-3000 cm⁻¹ -1 Typical Schiff base CH deformation vibration peaks appeared in all ranges, confirming the successful formation of Schiff bases. This was especially true for the CLIHO hydrogel, where peaks were observed at 3200-3300 cm⁻¹. -1 The peak at 700-800 cm⁻¹ shows stretching vibrations associated with ICG molecules, while the peak at 700-800 cm⁻¹ shows... -1 The region also showed a CH bending vibration peak in the Hemin molecule. These characteristic absorption peaks further validated the successful synthesis of CLIHO hydrogel.

[0057] Experimental Example 1

[0058] Tests on the self-healing, injectability, plasticity and adhesion of hydrogel dressings

[0059] Figure 5 and Figure 6 It can be seen that CLIHO hydrogel possesses good self-healing, injectability, plasticity, and adhesion. Even after undergoing mechanical damage, CLIHO hydrogel can restore its physical properties through its dynamic Schiff base bond repair structure, thus exhibiting excellent self-healing ability. The good adhesion properties of CLIHO hydrogel can be attributed to the imine bonds formed during its synthesis via Schiff base reactions, which not only enhance the chemical stability of CLIHO hydrogel but also improve its surface hydrophilicity and biocompatibility.

[0060] Experimental Example 2

[0061] Testing of water content, swelling properties and degradation properties of hydrogel dressings

[0062] Figure 7 As can be seen, CLIHO hydrogel has a high water content of over 90%, providing a continuously moist environment for the wound area, effectively preventing wound dryness and scab formation, thereby helping to promote cell growth and accelerate the wound healing process. CLIHO hydrogel reaches swelling equilibrium within 12 hours, exhibiting excellent moisture absorption capacity, enabling it to rapidly absorb tissue exudate from the wound area and effectively regulate the humidity of the wound site. CLIHO hydrogel remains stable for 14 days in PBS solution at 37°C, indicating its good stability and ability to meet the long-term stability requirements of wound dressings during chronic wound healing.

[0063] Experimental Example 3

[0064] Photothermal performance testing of hydrogel dressings

[0065] from Figure 8 It can be seen that the temperature rise of the hydrogel dressing with added ICG was significantly enhanced during light irradiation, indicating that the photothermal effect of ICG was effectively utilized in the hydrogel. The temperature of the hydrogel dressing was positively correlated with the NIR power and the ICG concentration. Figure 8 As shown in Figure E, the temperature of the CLIHO hydrogel rapidly rises to approximately 45°C within 3 minutes, and even after 5 NIR on / off cycles, its temperature change shows no significant difference. This indicates that the CLIHO hydrogel not only possesses good photothermal effects but also exhibits strong photothermal stability, maintaining a stable thermal effect after multiple light exposures. Figure 8 As shown in Figure F, in the hydrogel dressing without CMCD, the temperature curve of ICG gradually decreased after 4 minutes, possibly due to the low solubility of ICG, resulting in poor persistence of its photothermal effect. However, after adding CMCD, the temperature curve of the hydrogel dressing remained stable at around 45℃ within 15 minutes, indicating that the addition of CMCD effectively enhanced the solubility and stability of ICG, thereby improving the utilization rate of its photothermal effect.

[0066] Test Example 4

[0067] ROS, NO and O2 generation capacity of hydrogel dressings

[0068] use 1 The O2-specific fluorescent probe (DPBF) was used to detect the ROS generated by CLIHO hydrogel in solution. Figure 9 A indicates that only hydrogels containing ICG can be formed under NIR irradiation. 1 O2; CLIHO hydrogel formation 1 The amount of O2 gradually increases with the extension of light exposure time. Figure 9 B).

[0069] The NO release from the hydrogel in solution was quantitatively detected using the Griess reagent method. Figure 10It can be seen that almost no NO generation was detected in hydrogels without ICG or L-Arg, while NO release was significantly increased in hydrogels containing both ICG and L-Arg. This indicates that under NIR irradiation, ROS generated by ICG reacts with L-Arg to successfully generate NO. In 1 mM H2O2 solution, all hydrogels containing L-Arg released NO, indicating that H2O2, as a type of ROS, can also react with L-Arg to promote NO generation. CLIHO hydrogel showed the highest NO generation in 1 mM H2O2, significantly different from CLIO hydrogel (P < 0.05). This is because Hemin, with catalase-like activity, can release O2 by consuming H2O2, thereby accelerating ROS generation from ICG under NIR, increasing the efficiency of the cascade reaction, and thus significantly increasing NO generation. Through this pathway, L-Arg not only functions as a precursor to NO but also participates in the regulation of oxidative stress and wound healing through its reaction with ROS.

[0070] The release of intracellular O2 from the hydrogel dressing was detected using the hypoxia probe [Ru(dpp3)]Cl2. Figure 11 As shown, significant red fluorescence was observed in both the CIO and CLIO groups, indicating that ICG consumed intracellular O2 during ROS generation, exacerbating cellular hypoxia. In contrast, the intensity of red fluorescence decreased in the CLIHO group, and there was no significant difference compared to the control group (serum-free medium), suggesting that Hemin effectively alleviates cellular hypoxia by consuming H2O2 and releasing O2 during the cascade reaction within the CLIHO hydrogel.

[0071] Experimental Example 5

[0072] Antioxidant performance test of hydrogel dressing

[0073] An intracellular oxidative stress model was established using 1 mM H₂O₂ in L929 cells, and the scavenging effect of CLIHO hydrogel on intracellular ROS was investigated using the DCFH-DA probe. Figure 12 It can be seen that the cells treated with H2O2 exhibited the strongest fluorescence intensity and showed obvious green fluorescence, indicating that the cells treated with H2O2 had a high level of ROS. In contrast, the cells treated with the hydrogel dressing showed weaker green fluorescence, and the fluorescence intensity of each hydrogel group was not significantly different from that of the normal group, indicating that CLIHO hydrogel can reduce excessive ROS in cells to normal levels. This demonstrates that the hydrogel dressing also has a significant antioxidant effect in cells, effectively reducing the accumulation of excessive ROS and thus protecting cells and tissues from oxidative damage.

[0074] Experimental Example 6

[0075] In vitro angiogenesis-promoting capacity test of hydrogel dressings

[0076] The angiogenesis capabilities of CIO and CLIHO hydrogels were systematically evaluated using HUVEC cell scratch assays and tube formation assays. Figure 13 and 14 As shown, the cell migration rate in the CLIHO hydrogel-treated group was significantly higher than that in the CIO hydrogel group and the control group (P<0.01). The CLIHO hydrogel-treated group also exhibited the longest tubular structure, the most branches, and the most complex network structure in the tube formation experiment (P<0.01). These results indicate that L-Arg in the CLIHO hydrogel significantly enhances angiogenesis through its NOS-catalyzed NO production.

[0077] Experimental Example 7

[0078] Test of PTT / NO synergistic antibacterial function of hydrogel dressing under NIR irradiation

[0079] like Figure 15 As shown, when NIR was off, there was no significant difference in the inhibition rate of each group of hydrogels after 10 min or 15 min of interaction with the four bacteria MRSA, E. coli, S. aureus, and B. subtilis. After 10 min of NIR irradiation, the CLO group, lacking ICG, could not trigger the cascade reaction through NIR, therefore its inhibition rate against the four bacteria was not significantly different from that without NIR. However, the other three groups of ICG-containing hydrogels all showed high antibacterial rates. The CIO group had antibacterial rates of 64.16±3.06%, 77.51±2.04%, and 70.97±2.11% against MRSA, E. coli, and S. aureus, respectively; the CLIO group had rates of 75.84±1.18%, 83.09±0.82%, and 78.96±1.63%, respectively; while the CLIHO group showed the highest antibacterial rate, at 82.43±0.60%, 87.76±0.64%, and 84.72±1.40%, respectively, which were significantly higher than those of the CIO and CLIO groups (P<0.01).

[0080] For the thermostable bacterium *B. subtilis*, the inhibition rates of CLIO and CLIHO hydrogels were 66.71±0.90% and 74.18±0.75%, respectively, while the inhibition rate of CIO hydrogel was only 40.90±1.30%. This difference was more pronounced after 15 min of NIR treatment, indicating that the synergistic effect of PTT / NO can significantly enhance the antibacterial effect and effectively eliminate thermostable bacteria. Furthermore, under short-term NIR treatment, the CLIHO group exhibited the highest inhibition rate among the four bacteria, indicating that CLIHO hydrogel, under NIR irradiation, can consume the bacteria's endogenous H2O2 through Hemin, promoting a more efficient cascade reaction, thereby rapidly generating large amounts of ROS and NO in a short time, significantly enhancing its antibacterial effect.

[0081] The effectiveness of each hydrogel group in eliminating MRSA and E. coli biofilms was evaluated using crystal violet staining. Results are as follows: Figure 16 As shown, without NIR irradiation, the biofilms of MRSA and E. coli treated with PBS and CIO exhibited a deep purple color and relatively intact structure, indicating poor biofilm removal efficiency. In contrast, the biofilms of the two bacteria in the CLO, CLIO, and CLIHO groups containing L-Arg were lighter in color, suggesting that these hydrogels promoted biofilm removal to some extent. This may be due to the role of intracellular NOS catalyzing the conversion of L-Arg to NO. Under NIR irradiation conditions, the CIO group, exhibiting a PTT effect, showed some biofilm removal ability; while the CLIO and CLIHO groups, with their PTT / NO synergistic antibacterial effect, showed significantly better biofilm removal efficiency against MRSA and E. coli than the CIO group (P<0.0001), and the CLIHO treatment group exhibited the lightest color and highest OD. 590 The lowest value indicates that CLIHO hydrogel has the strongest biofilm removal ability. This may be because, compared to PTT alone, NO can not only kill bacteria in the biofilm by inducing oxidative and nitrification stress, but also promote the depolymerization of bacterial biofilm by regulating c-di-GMP levels.

[0082] Experimental Example 8

[0083] Biocompatibility testing of hydrogel dressings

[0084] The cytotoxicity of hydrogels with different components to L929 cells was evaluated using the CCK-8 assay. Figure 17 A indicates that after co-culturing L929 cells with the hydrogel extract for 24 h and 48 h, the cell viability of the CLO, CIO, CLIO, and CLIHO hydrogel treatment groups was greater than 85%, meeting the international standard requirements for cell compatibility, namely, a cell proliferation rate greater than 70%, indicating that all hydrogel components have good cell compatibility. The blood compatibility of the hydrogels was assessed using a hemolysis test.Figure 17 B indicates that the supernatant color of each hydrogel treatment group was similar to that of the physiological saline group, appearing colorless and transparent; while the supernatant of the deionized water group was bright red, indicating that blood cells had ruptured. Calculations showed that the hemolysis rate of all hydrogel groups was less than 5%, meeting international safety standards, indicating that CLIHO hydrogel has good blood compatibility. Tissue compatibility was assessed by subcutaneous injection of different hydrogel components into mice. Figure 17 C indicates that during the seven-day observation period, no mice in any of the experimental groups died and no significant adverse reactions occurred. Compared with normal mice, no immune responses or pathological changes were observed in the major organs of mice in each hydrogel-treated group, further demonstrating the good tissue compatibility of the hydrogel. These results indicate that CLIHO hydrogel not only has good blood and cell compatibility but also showed no significant toxic reactions in vivo, making it safe for the treatment of infected wounds.

[0085] Experimental Example 9

[0086] The therapeutic effect of hydrogel dressings on full-thickness skin lesions caused by MRSA infection in mice.

[0087] Male Kunming mice weighing approximately 30g were anesthetized and their back hair was shaved. A circular skin defect with a diameter of 8mm was created on their backs using surgical scissors. Then, 50μL of MRSA bacterial suspension (1×10⁻⁶) was applied. 8 Mice were infected with CFU / mL wounds for 24 hours to establish a full-thickness skin injury model of MRSA infection. Mice were randomly divided into six groups (n=10) and received different treatments: Control, CLO+NIR, CIO+NIR, CLIO+NIR, CLIHO, and CLIHO+NIR. Figure 18 A). Figure 18 BC shows the temperature change at the wound site during treatment, which is consistent with the temperature of the in vitro photothermal effect of the hydrogel dressing, with the wound temperature maintained at around 45°C during treatment. Figure 19 A shows the wound healing status of different treatment groups at different time points, intuitively reflecting the role of hydrogel dressings in promoting wound repair. On days 3 and 7 post-treatment, the wounds of mice in the Control group, which had not received treatment, showed a light yellow biofilm of MRSA infection and wound ulceration, while the wounds of all hydrogel-treated groups had shown significant shrinkage. By day 14, the wounds treated with CLIO+NIR and CLIHO+NIR had essentially healed, with the CLIHO+NIR treatment group showing the most significant wound shrinkage. Bacterial density at the wound site was assessed on day 3, and the plate colony count results are as follows: Figure 19As shown in Figure B, the in vivo antibacterial effect and the in vitro antibacterial effect of each component hydrogel showed similar trends. Among them, the bacterial density of the CLIHO+NIR treatment group was the lowest (P<0.001), indicating that the efficient cascade reaction in CLIHO hydrogel gives it a significant advantage in inhibiting wound infection.

[0088] The remaining wound area of ​​mice in each treatment group is as follows: Figure 19 As shown in Figure C, the wound healing speed of the CLIO+NIR group and the CLIHO+NIR group was significantly faster than that of the CLIO+NIR group, indicating that synergistic PTT / NO treatment accelerates wound healing more effectively than PTT treatment alone. This may be because synergistic PTT / NO treatment has a higher antibacterial effect, and in the absence of NIR, the CLIO and CLIHO hydrogels utilize the trace amounts of NO generated from endogenous H2O2 and NOS at the wound site, which can effectively promote wound healing. The wound healing effect of the CLIHO+NIR group was significantly better than that of the CLIO+NIR group (P<0.05), with the remaining wound area being only 0.79±0.40% of the initial wound area. This is because the CLIHO hydrogel generates O2 by consuming H2O2 at the wound site, which, on the one hand, improves the efficiency of the cascade reaction and exhibits more efficient antibacterial properties; on the other hand, it reduces the H2O2 level at the wound site, simultaneously alleviating oxidative stress and local hypoxia, creating a more favorable microenvironment for wound healing, thus achieving optimal wound healing results.

[0089] H&E staining was performed on the wound tissue on day 14. The results are as follows: Figure 20 As shown in Figure A, all hydrogel treatment groups exhibited the formation of new epidermis; compared with the control group, the inflammatory response in the hydrogel treatment groups was significantly reduced. In particular, the CLIHO+NIR group showed more orderly arrangement of new epidermis and the thinnest epidermal thickness, indicating that CLIHO hydrogel combined with NIR has optimal wound healing ability, thereby effectively promoting the wound repair process. MASSON staining results are as follows... Figure 20 As shown in Figure B, the effect of hydrogel on collagen deposition was further confirmed. The CLIHO+NIR group showed a significant increase in collagen fiber deposition on day 14, and the collagen fibers were dark blue, indicating that the treatment group played a significant role in promoting collagen synthesis and deposition.

[0090] Experimental Example 10

[0091] In vivo anti-inflammatory and angiogenesis-promoting capacity test of hydrogel

[0092] from Figure 21AC analysis showed that, compared with the Control group, all hydrogel treatment groups exhibited lower IL-6 and CD68 expression levels, with the CLIHO+NIR group showing the lowest IL-6 and CD68 expression levels (P<0.05), demonstrating its significant advantage in reducing inflammation. The advantage of CLIHO hydrogel in modulating the inflammatory response mainly stems from its ability to effectively inhibit bacterial growth and eliminate biofilms under the synergistic antibacterial effect of PTT / NO, reducing infection and thus lowering the inflammatory response. On the other hand, the trace amounts of NO produced by L-Arg under the catalysis of endogenous NOS during NIR switching have anti-inflammatory and immunomodulatory effects. Furthermore, CLIHO's antioxidant properties can effectively remove excess ROS from wounds, eliminating the adverse effects of excessive oxidative stress on the inflammatory response.

[0093] Angiogenesis is crucial for the efficient transport of nutrients and O2 to the wound site. CD31, an endothelial marker, is used to identify vascular endothelial cells; α-SMA, an angiogenesis-related protein, is used to assess the distribution of vascular smooth muscle. Therefore, combining the expression levels of CD31 and α-SMA can be used to assess angiogenesis in wound tissue. Figure 21 A and Figure 21 As shown in the DE, CLIHO+NIR exhibited the highest levels of CD31 and α-SMA expression compared to other treatment groups (P<0.01). This phenomenon can be attributed to the excellent antibacterial and biofilm-eliminating effects of this treatment group, and the presence of Hemin provides favorable healing conditions for angiogenesis by alleviating the hypoxic environment.

Claims

1. A method for preparing a hydrogel dressing based on a near-infrared light-controlled cascade reaction, characterized in that, The preparation method of the hydrogel dressing includes the following steps: S1: Using the EDC / NHS crosslinking method, carboxymethyl-β-cyclodextrin and L-arginine were grafted onto carboxymethyl chitosan to prepare cyclodextrin-grafted chitosan and L-Arg-grafted chitosan, respectively. S2: Add photosensitizer to cyclodextrin-grafted chitosan solution one to obtain CCI inclusion complex solution; add heme chloride to cyclodextrin-grafted chitosan solution two to obtain CCH inclusion complex solution; S3: This is obtained by mixing oxidized hyaluronic acid solution, L-Arg grafted chitosan solution, CCI inclusion complex solution and CCH inclusion complex solution.

2. The method for preparing hydrogel dressings based on near-infrared light-controlled cascade reactions according to claim 1, characterized in that, The final mass concentration of CCI inclusion complex in the hydrogel dressing is 0.5–2.0%, the final mass concentration of CCH inclusion complex is 0.1–1.0%, the final mass concentration of L-Arg grafted chitosan is 1.0–3.0%, and the final mass concentration of oxidized hyaluronic acid is 2.0–5.0%.

3. The method for preparing hydrogel dressings based on near-infrared light-controlled cascade reactions according to claim 1, characterized in that, The photosensitizer mentioned in S2 is indocyanine green.

4. The method for preparing a hydrogel dressing based on a near-infrared light-controlled cascade reaction according to claim 1, characterized in that, The preparation methods for both cyclodextrin-grafted chitosan solution one and cyclodextrin-grafted chitosan solution two in S2 are as follows: 2-8 mg of cyclodextrin-grafted chitosan is dissolved in 1 mL of PBS buffer; the mass ratio of the photosensitizer to the cyclodextrin-grafted chitosan is 0.5-1.5:1.0; and the mass ratio of the heme chloride to the cyclodextrin-grafted chitosan is 0.1-0.5:1.

0.

5. The method for preparing a hydrogel dressing based on a near-infrared light-controlled cascade reaction according to claim 1, characterized in that, The preparation method of cyclodextrin-grafted chitosan described in S1 is as follows: EDC and NHS are added to a carboxymethyl-β-cyclodextrin solution for activation for 1-3 hours, followed by the addition of a carboxymethyl chitosan solution and reaction for 12-48 hours. After dialysis purification, the chitosan is obtained by freeze-drying.

6. The method for preparing a hydrogel dressing based on a near-infrared light-controlled cascade reaction according to claim 5, characterized in that, The mass fraction of the carboxymethyl chitosan solution is 1.0–5.0%; the mass fraction of the carboxymethyl-β-cyclodextrin is 1.0–5.0%; the mass ratio of the carboxymethyl-β-cyclodextrin to the carboxymethyl chitosan is 0.5–2:1.0; the mass ratio of the carboxymethyl-β-cyclodextrin to the EDC is 1.0:1.5–3.0; and the mass ratio of the carboxymethyl-β-cyclodextrin to NHS is 1.0:1.0–2.

0.

7. The method for preparing a hydrogel dressing based on a near-infrared light-controlled cascade reaction according to claim 1, characterized in that, The preparation method of L-Arg grafted chitosan described in S1 is as follows: EDC and NHS are added to L-arginine solution for activation for 1-3 hours, followed by the addition of carboxymethyl chitosan solution II and reaction for 12-48 hours. After dialysis purification, the chitosan is obtained by freeze drying.

8. The method for preparing a hydrogel dressing based on a near-infrared light-controlled cascade reaction according to claim 7, characterized in that, The mass fraction of the carboxymethyl chitosan solution is 0.5-3.0%, and the L-arginine solution is obtained by dissolving 1.0-3.0 g of L-arginine in 30 mL of water; the mass ratio of L-arginine to carboxymethyl chitosan is 1.0-3.0:1.0; the mass ratio of L-arginine to EDC is 0.8-2.0:1.0; and the mass ratio of L-arginine to NHS is 1.0-5.0:1.

0.

9. A hydrogel dressing based on a near-infrared light-controlled cascade reaction, characterized in that, The hydrogel dressing was prepared using the method for preparing near-infrared light-controlled cascade reactions as described in any one of claims 1-8.

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