A near-infrared light-exciteable composite nanosheet, its preparation method and application

Abstract

This invention discloses a near-infrared light-exciteable composite nanosheet, its preparation method, and its applications, belonging to the field of biomedical technology. The method includes the following steps: mixing epigallocatechin gallate with a Ti3C2 nanosheet dispersion, ultrasonically reacting, adding silver salt, and continuing stirring in the dark; after the reaction is complete, washing and separating to obtain MXene-EGCG@Ag composite nanosheets. The composite nanosheets provided by this invention possess near-infrared light-exciteable properties and can be used to prepare medically acceptable dosage forms such as hydrogels for the treatment of periodontitis. The preparation method provided by this invention is simple, the raw materials are readily available, and it exhibits significant antibacterial and anti-inflammatory effects, enabling further widespread application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a composite nanosheet that can be excited by near-infrared light, its preparation method and application. Background Technology

[0002] Periodontitis is a highly prevalent chronic inflammatory disease that not only leads to progressive destruction of periodontal supporting tissues and tooth loss, but is also closely related to various systemic diseases, making it a significant global public health challenge. Increasing research indicates that the occurrence and persistence of periodontitis are not solely driven by bacterial invasion, but rather result from the interaction of bacterial biofilms, host immune imbalance, and organelle dysfunction, exhibiting typical characteristics of immune metabolic abnormalities. Multi-species biofilms, with *Porphyromonas gingivalis* at their core, can deeply colonize periodontal pockets, constructing a highly structured pathogenic microecological barrier to evade immune clearance and antibiotic intervention. They continuously release virulence factors such as lipopolysaccharides and proteases, inducing a large accumulation of pro-inflammatory cytokines and reactive oxygen species, thereby promoting the irreversible process of gingival tissue destruction and alveolar bone resorption. Traditional treatments still require antibiotics for plaque control, easily leading to problems such as drug resistance.

[0003] Epigallocatechin gallate (EGCG) is a major polyphenolic compound in green tea, possessing triple activities of antioxidation, antibacterial activity, and anti-inflammation. Current in vitro and animal experiments have confirmed that it can inhibit the adhesion and biofilm formation of periodontal pathogens, downregulate the NF-κB pathway, reduce the release of TNF-α and IL-6, and block alveolar bone resorption. Simultaneously, it can protect periodontal ligament fibroblasts by scavenging ROS and promote osteogenic differentiation. Furthermore, it can synergistically enhance the efficacy of metronidazole and tetracycline, reduce drug resistance and side effects, and provide a safe new strategy for local adjuvant medication for periodontitis and modification of bone regeneration materials.

[0004] Currently, near-infrared photodynamic therapy (PDT) is one of the effective strategies for combating bacterial infections. MXene, as a photothermal converter, has been applied to PDT. However, the instability of MXene makes it difficult for it to play an effective role in treatment.

[0005] Therefore, how to provide a near-infrared light-exciteable MXene material for the treatment of periodontitis is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a near-infrared light-exciteable composite nanosheet with antibacterial and anti-inflammatory effects, along with its preparation method and applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing near-infrared light-exciteable composite nanosheets includes the following steps:

[0009] Epigallocatechin gallate (EGCG) was mixed with a Ti3C2 nanosheet dispersion and subjected to ultrasonic vibration. Silver salt was then added and the reaction was continued in the dark. After the reaction was completed, the nanosheets were washed and separated to obtain MXene-EGCG@Ag composite nanosheets.

[0010] Beneficial Effects: Traditional silver ion antibacterial materials mostly involve directly loading silver ions into hydrogels, which can easily lead to burst release of silver ions and biotoxicity. This invention innovatively reduces silver ions in situ to silver nanoparticles loaded onto MXene (Ti3C2 nanosheets) via EGCG. The phenol-quinone redox pair on EGCG reversibly oxidizes the silver nanoparticles back to silver ions, ensuring controlled release and improving the material's biocompatibility while maintaining antibacterial efficacy. Furthermore, the composite nanosheets provided by this invention combine photothermal antibacterial and silver ion antibacterial properties, achieving highly effective antibacterial action against bacterial biofilms that are difficult to remove in periodontitis sites. In addition, traditional methods of modifying MXene with silver are complex, while the method provided by this invention is simple to operate, effectively reducing costs and providing a convenient solution for treating periodontitis.

[0011] Preferably, the concentration of the Ti3C2 nanosheet dispersion is 12 mg / mL;

[0012] The mass ratio of epigallocatechin gallate to silver salt is 1:(1-3).

[0013] Preferably, the silver salt is silver nitrate.

[0014] Beneficial effects: The EGCG in this invention has reducing properties, which enables silver ions to be reduced in situ to silver nanoparticles on MXene, and the operation is simple.

[0015] Preferably, the ultrasonic oscillation response time is 10-20 minutes;

[0016] The continued stirring reaction is a light-protected stirring reaction, and the time is 2-4 hours.

[0017] Beneficial effects: The purpose of this process is to reduce silver ions to silver nanoparticles, and the sonication is to make MXene dispersed evenly.

[0018] A near-infrared light-exciteable composite nanosheet prepared by the above-described method.

[0019] The application of a near-infrared light-exciteable composite nanosheet in the preparation of a drug for treating periodontitis, wherein the drug for treating periodontitis is a medically acceptable dosage form.

[0020] More preferably, the medically acceptable dosage form includes one of injections, tablets, powders, and capsules.

[0021] Beneficial effects: In periodontal treatment, due to the narrow, deep, and irregularly shaped anatomical characteristics of periodontal pockets, traditional preparations are difficult to effectively penetrate deep into the lesion. Injectable hydrogels, with their shear-thinning ability and plasticity, can fully fill and conform to the irregular spaces of periodontal pockets, thus exhibiting unique advantages in local drug delivery and tissue repair.

[0022] A hydrogel comprising the aforementioned near-infrared light-exciteable composite nanosheets.

[0023] A method for preparing a hydrogel includes the following steps:

[0024] The composite nanosheets are mixed with carboxymethyl chitosan in water and reacted, and then mixed with oxidized fucoidan solution to obtain the hydrogel.

[0025] The composite nanosheets have a mass ratio of carboxymethyl chitosan to oxidized fucoidan of 1.2 mg: (0.4-0.6) g: (0.5-0.75) g.

[0026] More preferably, the reaction time of the composite nanosheets and carboxymethyl chitosan in water is 30 minutes.

[0027] Preferably, the oxidized fucoidan is obtained by oxidizing fucoidan with sodium periodate.

[0028] More preferably, the method for preparing the oxidized fucoidan includes the following steps:

[0029] Fucoidan was dissolved in deionized water, sodium periodate was added, and the reaction was carried out at room temperature in the dark. Ethylene glycol was then added and the mixture was stirred to consume any unreacted sodium periodate. After the reaction was complete, the product was transferred to a dialysis bag (M...). W Dialysis was performed in 3500 g / L solution, followed by freeze-drying to obtain white, spongy oxidized fucoidan.

[0030] More preferably, the reaction time at room temperature in the dark is 4 hours, and the stirring time is 1 hour.

[0031] Beneficial effects: The above process can modify aldehyde groups on fucoidan, which can then react with the amino groups on carboxymethyl chitosan to form a hydrogel network. The polysaccharide and carboxymethyl chitosan have a large number of hydroxyl groups that can form hydrogen bonds with the hydroxyl groups on the nanosheets, thereby enabling uniform loading of nanosheets and slow release of nanosheets.

[0032] Application of a hydrogel in the preparation of a drug for treating periodontitis.

[0033] More preferably, in the process of application, the hydrogel is used for the preparation or use of an injectable.

[0034] Beneficial effects: This invention improves the stability of MXene by modifying it with EGCG and Ag nanoparticles, and combines this with hydrogel technology to achieve a stability of 1 W / cm². 2 Under irradiation with 808nm near-infrared light, it achieves antibacterial and anti-inflammatory functions through photothermal methods, which is of great value in the treatment of periodontitis.

[0035] Compared with the prior art, the present invention has the following advantages and technical effects:

[0036] The hydrogel provided by this invention can be injected deep into the periodontal pocket environment and matches the complex anatomical structure and morphology of the periodontal pocket according to its topological morphology. This hydrogel contains MXene-EGCG@Ag composite nanosheets with excellent photothermal properties, which can convert near-infrared light into heat. This photothermal property breaks down the bacterial biofilm at the periodontitis site, followed by silver ion diffusion and powerful bactericidal action, achieving effective antibacterial treatment at the periodontitis site. Furthermore, MXene and EGCG in this invention can exert anti-inflammatory functions, achieving effective treatment for periodontitis. The hydrogel provided by this invention can achieve long-lasting antibacterial and anti-inflammatory effects, comprehensively and effectively improving the periodontal microenvironment. This hydrogel can stably exist in the periodontal pocket site for 5 days, continuously releasing antibacterial and anti-inflammatory components for long-term improvement of periodontitis. In addition, the hydrogel formation process is easy, simple, and low-cost, thus facilitating its widespread application. Attached Figure Description

[0037] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0038] Figure 1 Here is a scanning electron microscope image of the hydrogel obtained in Example 1;

[0039] Figure 2 The elemental distribution diagram of the multifunctional nanosheets MXene-EGCG@Ag obtained in step (1) of Example 1 is shown below.

[0040] Figure 3 The potential results for MXene, MXene-EGCG, and MXene-EGCG@Ag in the Zeta mode of DLS are shown.

[0041] Figure 4 XRD characterization of MXene nanosheets and MXene-EGCG@Ag;

[0042] Figure 5The rheological diagram of viscosity-shear rate of the hydrogel obtained in Example 1 and the actual picture of the hydrogel injection are shown.

[0043] Among them, (a) is the viscosity-shear rate rheological diagram, and (b) is a physical image of the hydrogel injection.

[0044] Figure 6 The temperature-time curve of the hydrogel obtained in Example 1 and thermal images of different concentrations at different times are shown.

[0045] Among them, (a) is the temperature-time curve, and (b) is the thermal image of different concentrations at different times;

[0046] Figure 7 The in vitro antibacterial and antibiofilm effects of the hydrogel obtained in Example 1 against Porphyromonas gingivalis and multiple bacterial biofilms were evaluated.

[0047] In this diagram, AB represents representative CFU images of *Porphyromonas gingivalis* and multi-species biofilms after different treatments; CD represents the corresponding quantitative CFU counts; EF represents representative three-dimensional CLSM live / dead fluorescence images of *Porphyromonas gingivalis* and multi-species biofilms, where green fluorescence represents live bacteria and red fluorescence represents dead bacteria; G and I represent three-dimensional CLSM reconstructions of *Porphyromonas gingivalis* and multi-species biofilms, showing structural collapse and thickness changes (scale bar = 100 μm); H and J represent quantitative analysis of the thickness of *Porphyromonas gingivalis* and multi-species biofilms; KL represents representative SEM images of bacterial morphology and biofilm surface structure of *Porphyromonas gingivalis* and multi-species models (scale bar = 1 μm); MP represents representative crystal violet-stained images of *Porphyromonas gingivalis* and multi-species biofilms and corresponding quantitative analysis of biofilm biomass.

[0048] Figure 8 The results of GM+NIR group analysis of upregulated proteins in antimicrobial omics research;

[0049] Figure 9 Results of GM+NIR group analysis of downregulated proteins;

[0050] Figure 10 To investigate the cation transmembrane transport pathway and passive transmembrane transport pathway of Porphyromonas gingivalis between the control and GM+NIR groups through gene set enrichment analysis.

[0051] Among them, A is the cation transmembrane transport pathway, and B is the passive transmembrane transport pathway;

[0052] Figure 11 This is a schematic diagram of the antibacterial mechanism;

[0053] Figure 12 The ability of GM+NIR to regulate the in vitro immune microenvironment;

[0054] Among them, A) the relative mRNA levels of tumor necrosis factor α (TNFα), interleukin 1β (IL1β), interleukin 10 (IL10), and arginase (Arg1) in macrophages of each group; B) the proportion of M1 and M2 macrophages after different treatments; C) immunofluorescence staining images of the expression of inflammation-related proteins in macrophages after different treatments.

[0055] Figure 13 KEGG enrichment analysis for differentially expressed genes;

[0056] Figure 14 GO enrichment analysis for differentially expressed genes;

[0057] Figure 15 Gene enrichment analysis for the mitophagy pathway;

[0058] Figure 16 Differences in the expression of genes related to the mitophagy pathway among different groups;

[0059] Among them, L1, L2, and L3 are three parallel experimental groups of the control group; GM+NIR-1, GM+NIR-2, and GM+NIR-3 are three parallel experimental groups of the GM+NIR group.

[0060] Figure 17 Gene enrichment analysis for the FOXO pathway;

[0061] Figure 18 Differences in the expression of FOXO pathway-related genes among different groups;

[0062] Figure 19 mtROS levels in macrophages after different treatments;

[0063] Where A is the blank group, B is the control group, C is the gel group, D is the gel+NIR group, E is the GM group, and F is the GM+NIR group;

[0064] Figure 20 The state of mitochondria in macrophages after different treatments;

[0065] Figure 21 Mitochondrial membrane potential levels in macrophages from different groups;

[0066] Figure 22 The in vivo therapeutic effect of GM hydrogel on periodontitis in rats;

[0067] In this table, A represents representative three-dimensional micro-CT reconstructions of alveolar bone from different groups (scale bar = 1.0 mm); B represents quantitative analysis of the CEJ-ABC distance; C represents representative photographs of bacterial colonies collected from periodontal pockets; and D represents the corresponding quantitative CFU counts; E represents typical H&E staining images; and F represents the quantitative statistics of the number of immune cells in inflamed periodontal tissues; G represents typical Massen's trichrome staining images; and H represents the percentage of collagen degradation in inflamed periodontal tissues; I represents the pro-inflammatory fluorescence image of IL-6; and J represents the corresponding relative pro-inflammatory fluorescence intensity of IL-6; K represents the anti-inflammatory fluorescence image of Arg-1; and L represents the corresponding relative anti-inflammatory fluorescence intensity of Arg-1.

[0068] Note: Blank in the attached figures corresponds to the blank group; Control corresponds to the control group; Gel group is the group in which hydrogel of Comparative Example 1 is added during the experiment; Gel+NIR group is the group in which hydrogel of Comparative Example 1 is added during the experiment and then irradiated with near-infrared light (NIR, wavelength 808 nm) for 5 min; GM group is the group in which hydrogel of Example 1 is added during the experiment; GM+NIR group is the group in which hydrogel of Example 1 is added during the experiment and then irradiated with near-infrared light (NIR, wavelength 808 nm) for 5 min. Detailed Implementation

[0069] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0071] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels;

[0072] Among them, P. gingivalis (ATCC number 33277) and F. nucleatum (ATCC number 10953) were purchased from the American Standard Biological Collection Center.

[0073] Mouse L929 fibroblasts and RAW 264.7 cells were obtained from continuous culture in the laboratory using DMEM medium. The cells were cultured in a humidified incubator containing 5% (v / v) CO2 at 37 ℃.

[0074] The rats were purchased from the Deer Antler Research Institute of Changchun University of Science and Technology. They were healthy male SD rats aged 4 weeks, with batch number No. 210726240100149858.

[0075] The yeast powder was commercially available Sigma yeast powder.

[0076] Unless otherwise specified, room temperature or normal temperature in the embodiments of the present invention refers to 25±3℃.

[0077] Example 1

[0078] A method for preparing a hydrogel for treating periodontitis includes the following steps:

[0079] (1) 30 mg of epigallocatechin gallate (EGCG) was added to 2 mL of Ti3C2 nanosheet dispersion with a concentration of 12 mg / mL. After stirring evenly, 2 mL of Tris buffer was added to adjust the pH of the mixed solution to ≈ 8.0. Then, the mixture was ultrasonically vibrated for 20 min. 30 mg of silver nitrate was added to the reacted solution, and the mixture was stirred at room temperature in the dark for 4 h to generate silver nanoparticles. After the reaction, the resulting reactants were washed with deionized water and centrifuged at 8800 rpm to obtain MXene-EGCG@Ag. The MXene-EGCG@Ag was then lyophilized and stored at 4 °C under nitrogen for later use.

[0080] (2) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.4 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 1 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0081] (3) Weigh 1.2 mg of MXene-EGCG@Ag obtained in step (1) and 0.4 g of carboxymethyl chitosan and dissolve them in 5 mL of deionized water. Mix and react for 30 min to obtain solution A; dissolve the oxidized fucoidan obtained in step (2) in deionized water to obtain solution B with a mass fraction of 10 wt%. Mix solutions A and B in equal volume ratio thoroughly and react for 5 min to obtain hydrogel GM with antibacterial and anti-inflammatory effects.

[0082] Example 2

[0083] A method for preparing a hydrogel for treating periodontitis includes the following steps:

[0084] (1) 30 mg of epigallocatechin gallate (EGCG) was added to 2 mL of Ti3C2 nanosheet dispersion with a concentration of 12 mg / mL. After stirring evenly, 2 mL of Tris buffer was added to adjust the pH of the mixed solution to ≈ 8.0. Then, the mixture was ultrasonically vibrated for 20 min. 60 mg of silver nitrate was added to the reacted solution, and the mixture was stirred at room temperature in the dark for 4 h to generate silver nanoparticles. After the reaction, the resulting reactants were washed with deionized water and centrifuged at 8800 rpm to obtain MXene-EGCG@Ag. The MXene-EGCG@Ag was then lyophilized and stored at 4 °C under nitrogen for later use.

[0085] (2) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.6 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 1.5 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0086] (3) Weigh 1.2 mg of MXene-EGCG@Ag obtained in step (1) and 0.5 g of carboxymethyl chitosan and dissolve them in 5 mL of deionized water. Mix and react for 30 min to obtain solution A; dissolve the oxidized fucoidan obtained in step (2) in deionized water to obtain solution B with a mass fraction of 12 wt%. Mix solutions A and B in equal volume ratio thoroughly and react for 5 min to obtain a hydrogel with antibacterial and anti-inflammatory effects.

[0087] Example 3

[0088] A method for preparing a hydrogel for treating periodontitis includes the following steps:

[0089] (1) 30 mg of epigallocatechin gallate (EGCG) was added to 2 mL of Ti3C2 nanosheet dispersion with a concentration of 12 mg / mL. After stirring evenly, 2 mL of Tris buffer was added to adjust the pH of the mixed solution to 8.0. Then, the mixture was ultrasonically vibrated for 20 min. 90 mg of silver nitrate was added to the reacted solution, and the mixture was stirred at room temperature in the dark for 4 h to generate silver nanoparticles. After the reaction, the resulting reactants were washed with deionized water and centrifuged at 8800 rpm to obtain MXene-EGCG@Ag. The MXene-EGCG@Ag was then lyophilized and stored at 4 °C under nitrogen for later use.

[0090] (2) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.8 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 3 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0091] (3) Weigh 1.2 mg of MXene-EGCG@Ag obtained in step (1) and 0.6 g of carboxymethyl chitosan and dissolve them in 5 mL of deionized water. Mix and react for 30 min to obtain solution A; dissolve the oxidized fucoidan obtained in step (2) in deionized water to obtain solution B with a mass fraction of 15 wt%. Mix solutions A and B in equal volume ratio thoroughly and react for 5 min to obtain a hydrogel with antibacterial and anti-inflammatory effects.

[0092] Comparative Example 1

[0093] A method for preparing a hydrogel, differing from Example 1 only in that it excludes step (1), and specifically includes the following steps:

[0094] (1) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.4 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 1.5 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0095] (2) Weigh 0.4g of carboxymethyl chitosan and dissolve it in 5mL of deionized water. Mix and react for 30 min to obtain solution A. Dissolve the oxidized fucoidan obtained in step (1) in deionized water to obtain solution B with a mass fraction of 10wt%. Mix solutions A and B in equal volume ratio thoroughly and react for 5 min to obtain hydrogel.

[0096] Comparative Example 2

[0097] A method for preparing a hydrogel, differing from Example 1 only in that the amount of MXene-EGCG@Ag added in step (3) is changed, specifically including the following steps:

[0098] (1) 30 mg of epigallocatechin gallate (EGCG) was added to 2 mL of Ti3C2 nanosheet dispersion with a concentration of 12 mg / mL. After stirring evenly, 2 mL of Tris buffer was added to adjust the pH of the mixed solution to ≈ 8.0. Then, the mixture was ultrasonically vibrated for 20 min. 30 mg of silver nitrate was added to the reacted solution, and the mixture was stirred at room temperature in the dark for 4 h to generate silver nanoparticles. After the reaction, the resulting reactants were washed with deionized water and centrifuged at 8800 rpm to obtain MXene-EGCG@Ag. The MXene-EGCG@Ag was then lyophilized and stored at 4 °C under nitrogen for later use.

[0099] (2) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.4 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 1 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0100] (3) Weigh 0.4 mg of MXene-EGCG@Ag obtained in step (1) and 0.4 g of carboxymethyl chitosan and dissolve them in 5 mL of deionized water. Mix and react for 30 min to obtain solution A; dissolve the oxidized fucoidan obtained in step (2) in deionized water to obtain solution B with a mass fraction of 10 wt%. Mix solutions A and B in equal volume ratio thoroughly and react for 5 min to obtain hydrogel.

[0101] Comparative Example 3

[0102] A method for preparing a hydrogel, differing from Example 1 only in that the amount of MXene-EGCG@Ag added in step (3) is changed, specifically including the following steps:

[0103] (1) 30 mg of epigallocatechin gallate (EGCG) was added to 2 mL of Ti3C2 nanosheet dispersion with a concentration of 12 mg / mL. After stirring evenly, 2 mL of Tris buffer was added to adjust the pH of the mixed solution to ≈ 8.0. Then, the mixture was ultrasonically vibrated for 20 min. 30 mg of silver nitrate was added to the reacted solution, and the mixture was stirred at room temperature in the dark for 4 h to generate silver nanoparticles. After the reaction, the resulting reactants were washed with deionized water and centrifuged at 8800 rpm to obtain MXene-EGCG@Ag. The MXene-EGCG@Ag was then lyophilized and stored at 4 °C under nitrogen for later use.

[0104] (2) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.4 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 1 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0105] (3) Weigh 0.8 mg of MXene-EGCG@Ag obtained in step (1) and 0.4 g of carboxymethyl chitosan and dissolve them in 5 mL of deionized water. Mix and react for 30 min to obtain solution A; dissolve the oxidized fucoidan obtained in step (2) in deionized water to obtain solution B with a mass fraction of 10 wt%. Mix solutions A and B in equal volume ratio thoroughly and react for 5 min to obtain hydrogel.

[0106] Comparative Example 4

[0107] A method for preparing a hydrogel, differing from Example 1 only in that Ag is not introduced in step (1), specifically including the following steps:

[0108] (1) Add 30 mg of epigallocatechin gallate (EGCG) to 2 mL of Ti3C2 nanosheet dispersion with a concentration of 12 mg / mL, stir well, add 2 mL of Tris buffer to adjust the pH of the mixture to ≈ 8.0, and then react with sonication for 20 min. After the reaction is complete, wash the resulting reactant with deionized water and centrifuge at 8800 rpm to prepare MXene-EGCG, which is then dried for later use.

[0109] (2) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.4 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 1 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0110] (3) Weigh 1.2 mg of MXene-EGCG obtained in step (1) and 0.4 g of carboxymethyl chitosan and dissolve them in 5 mL of deionized water. Mix and react for 30 min to obtain solution A; dissolve the oxidized fucoidan obtained in step (2) in deionized water to obtain solution B with a mass fraction of 10 wt%. Mix solutions A and B in equal volume ratio thoroughly and react for 5 min to obtain a hydrogel with antibacterial and anti-inflammatory effects.

[0111] Comparative Example 5

[0112] A method for preparing a hydrogel, differing from Example 1 only in that EGCG and Ag are not introduced in step (1), specifically including the following steps:

[0113] (1) Preparation method of MXene: 1g of MAX (the precursor of MXene) and 1g of LiF were added to 9 M hydrochloric acid solution and reacted for 26 h. After repeated centrifugation and washing, a precipitate was obtained. The precipitate was dispersed in the solution again and sonicated for 1 h to make the precipitate evenly dispersed in the solution. After centrifugation again, a uniform black solution was obtained. After freeze-drying, black MXene powder was obtained.

[0114] (2) Dissolve 1 g of fucoidan in 100 mL of deionized water, add 0.4 g of sodium periodate, and react at room temperature in the dark for 4 h. Then add 1 mL of ethylene glycol and continue stirring for 1 h to consume unreacted sodium periodate. After the reaction is complete, transfer the product into a dialysis bag (M). W Dialysis in 3500 g / L for 72 h, followed by freeze-drying, yielded white spongy oxidized fucoidan.

[0115] (3) Weigh 1.2 mg of MXene obtained in step (1) and 0.4 g of carboxymethyl chitosan and dissolve them in 5 mL of deionized water. Mix and react for 30 min to obtain solution A. Dissolve the oxidized fucoidan obtained in step (2) in deionized water to obtain solution B with a mass fraction of 10 wt%. Mix solutions A and B in equal volume ratio and react for 5 min to obtain a hydrogel with antibacterial and anti-inflammatory effects.

[0116] Technical effects:

[0117] 1. Performance Characterization

[0118] Figure 1 The image shown is a scanning electron microscope image of the hydrogel obtained in Example 1. It can be seen that the hydrogel has a porous structure and can support multifunctional nanosheets MXene-EGCG@Ag.

[0119] Figure 2 The elemental distribution diagram of the multifunctional nanosheet MXene-EGCG@Ag obtained in step (1) of Example 1 shows that epigallocatechin gallate (EGCG) and silver ions have been successfully modified onto the nanosheet.

[0120] Figure 3The potentials for MXene, MXene-EGCG, and MXene-EGCG@Ag in the Zeta mode of DLS (Dynamic Light Scattering) are -48.51 mV, -50.90 mV, and -23.71 mV, respectively. When MXene is used to modify EGCG alone, the surface potential of MXene increases; when Ag is added, the potential decreases.

[0121] Figure 4 XRD characterization of MXene nanosheets and MXene-EGCG@Ag shows that, compared with MXene nanosheets, MXene-EGCG@Ag exhibits obvious peaks at 38.08°, 44.32° and 64.36°, which correspond to the characteristic peaks of Ag (111), (200) and (220) respectively, proving that EGCG in situ reduces Ag to MXene nanosheets.

[0122] The shear-thinning properties of the prepared hydrogel were analyzed using a rheometer (TA Discovery DHR-2). An 8 mm diameter upper parallel plate fixture was used, with a fixed measurement gap of 1 mm. The injectability and shear-thinning properties of the hydrogel were tested in viscosity mode. The results are as follows: Figure 5 As shown, Figure 5 The rheological diagram (a) of viscosity-shear rate of the hydrogel obtained in Example 1 and the actual picture (b) of the hydrogel injection show that the hydrogel has good injectability and can be injected into and match the complex morphology of periodontal pockets.

[0123] At 1 W / cm 2 The hydrogel was irradiated with a laser, and its temperature was recorded every 30 seconds. Infrared imaging was used to capture images to test the photothermal cycling ability and photothermal properties of the hydrogel under near-infrared (NIR) excitation. The results are as follows: Figure 6 As shown, Figure 6 The temperature-time curve (a) and thermal images (b) of the hydrogel obtained in Example 1 at different times and concentrations (0 mg / mL, 0.2 mg / mL, 0.4 mg / mL and 0.6 mg / mL) demonstrate the photothermal cycling ability and photothermal performance of the hydrogel under near-infrared (NIR) excitation. This indicates that the antibacterial and anti-inflammatory hydrogel has good and stable photothermal performance. It can heat up to about 60°C after 3 min of NIR light excitation. Such photothermal function can effectively destroy the bacterial biofilm in the periodontal pocket and achieve effective sterilization.

[0124] 2. Cell biocompatibility assessment:

[0125] The effects of the hydrogels obtained in Example 1 and Comparative Example 3 on L929 cells were evaluated using a live / dead cell staining assay, specifically including the following steps:

[0126] The hydrogels obtained in Example 1 and Comparative Example 3 were co-cultured with L929 cells. After 24 and 72 hours, the cells were incubated for 30 min with calcein and PI from the live / dead cell staining kit. The stained cells were observed under a fluorescence microscope; live cells emitted green fluorescence (from calcein), and dead cells emitted red fluorescence (from PI).

[0127] 3. Antibacterial biofilm experiment:

[0128] 3.1 Preparation of biofilms:

[0129] *Porphyromonas gingivalis* (ATCC number 33277) and *Fusobacterium nucleatum* (ATCC number 10953) were both purchased from the U.S. National Collection of Standard Biological Products. *P. gingivalis* was cultured using TSB medium (TSB powder 30.12 g / L). -1 5 g L of yeast powder -1 L-cysteine ​​hydrochloride 0.5 g L -1 Vitamin K1 mg L -1 Heme chloride 5 mg L -1 F. nucleatum was cultured using BHI medium (BHI powder 36 g / L). -1 5 g L of yeast powder -1 L-cysteine ​​hydrochloride 0.5 g L -1 Vitamin K1 mg L -1 Heme chloride 5mg / L -1 The two types of bacteria were cultured separately under anaerobic conditions at 37 °C (85% N2, 10% H2, 5% CO2). Once the bacteria reached the logarithmic growth phase, the OD600 value was measured, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFUmL -1 It is used for subsequent antibacterial biofilm experiments.

[0130] For the formation of single-species biofilms of *P. gingivalis* and *F. nucleatum*, the bacterial concentration in the logarithmic phase was adjusted to 1 × 10⁻⁶. 8 CFU mL -1Circular cell smears were placed on the bottom of 24-well plates and cultured anaerobically for 96 h to form biofilms. The two types of biofilms were divided into four groups: a control group (bacteria only, no hydrogel), a gel group, a GM group, and a GM+NIR group. The amount of hydrogel obtained in Example 1 or Comparative Example 1 added was 300 μL. The biofilms were then co-cultured for 24 h, and subsequent experiments were conducted to verify the antibacterial effects of each group.

[0131] 3.2 Colony forming unit count

[0132] The biofilm was washed with CPW to remove non-adhesive bacteria. Cell smears with attached bacterial biofilm were transferred to a glass vial containing 1 mL of CPW. Bacteria on the biofilm were collected by pipetting and vortexing. 10 μL of the bacterial suspension was serially diluted and inoculated onto blood agar plates. The plates were incubated anaerobically at 37 °C for an appropriate time. CFU (Cellular Units) were calculated based on the number of bacterial colonies on the blood agar plates and the corresponding dilution factor.

[0133] 3.3 Fluorescent staining for live / dead bacteria:

[0134] Biofilms were washed with CPW to remove non-adhesive bacteria. A mixture of SYTO 9 (2.5 μM) and propidium iodide (2.5 μM) was prepared and dropped onto the cell smear surface, then incubated in the dark for 15 min. 3D images of the biofilm were acquired using a CLSM. Five regions were randomly collected from each sample group, and images of 254 μm × 254 μm size and all z-sections were acquired at 1.1 μm intervals. All samples were observed using the same parameters and analyzed using NIS Elements Viewer and ImageJ software.

[0135] Figure 7This study evaluates the in vitro antibacterial and antibiofilm effects of hydrogels on *Porphyromonas gingivalis* and multi-species biofilms. Images AB show representative CFU images of *Porphyromonas gingivalis* and multi-species biofilms after different treatments. Images CD show the corresponding quantitative CFU counts. Images EF show representative three-dimensional CLSM live / dead fluorescence images of *Porphyromonas gingivalis* and multi-species biofilms, where green fluorescence represents live bacteria and red fluorescence represents dead bacteria. Images G and I show three-dimensional CLSM reconstructions of *Porphyromonas gingivalis* and multi-species biofilms, showing structural collapse and thickness changes (scale bar = 100 μm). Images H and J show quantitative analysis of the thickness of *Porphyromonas gingivalis* and multi-species biofilms. Images KL show representative SEM images of bacterial morphology and biofilm surface structure of *Porphyromonas gingivalis* and multi-species models (scale bar = 2 μm). MP represents representative crystal violet-stained images of Porphyromonas gingivalis biofilms and multiple bacterial species, along with corresponding quantitative analysis of biofilm biomass. Data are expressed as mean ± SD (n = 3). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001; n, not significant.

[0136] The results showed that in *P. gingivalis* and multi-species biofilms, dense colonies grew in both the control group and the Gel group at all levels of serial dilution, while the colony count in the GM group was significantly reduced, and the GM+NIR group only had sporadic colonies remaining at low dilutions. Figure 7 (Parts A and C). This stepwise enhanced bactericidal trend stems from the Ag introduced into the GM hydrogel network. + Synergistic effect of MXene and EGCG: Ag + MXene itself possesses broad-spectrum bactericidal activity. Under NIR irradiation, it generates a mild photothermal effect, accelerating the growth of Ag. + Release and diffusion, while EGCG weakens biofilm aggregation, making bacteria more susceptible to Ag. + They are more sensitive to attacks. Multi-species biofilms also showed the same trend. Figure 7 (Parts B and D). As can be seen from Parts E and F, Example 1 hydrogel biofilm thickness was the lowest and dead bacterial density (red part) was the highest under near-infrared (NIR) excitation, demonstrating its best antibacterial biofilm function. Comparative Example 1 had no antibacterial properties, and Comparative Example 2 had a weak ability to break down bacterial biofilms. The three-dimensional reconstruction and quantitative analysis of biofilm thickness were highly consistent with the fluorescence results. Whether it was a single-strain or multi-strain system, the control group and the Gel group formed a thick and continuous three-dimensional biofilm, while the thickness of the Gel+NIR group only decreased slightly ( Figure 7 (GJ part).

[0137] SEM observation provided direct morphological evidence for the above results. In the control and Gel groups, *P. gingivalis* and various biofilm bacteria were densely packed with smooth cell surfaces and intact rod- or coccobacillus morphology. In the Gel+NIR group, only a few bacteria showed slightly rough surfaces. In the GM group, a large number of bacteria exhibited membrane wrinkling, volume shrinkage, and partial lysis, while the GM+NIR group showed the most severe morphological damage: blurred cell outlines, membrane structure fracture and collapse, and localized areas almost entirely lacking intact cells. Figure 7 (Parts K and L).

[0138] Crystal violet staining further validated the anti-biofilm effect of GM+NIR at the level of overall adhesion biomass. Figure 7 (MP). In the *P. gingivalis* and multi-species models, the control group and Gel group showed uniform and deep purple staining on the well walls, indicating biofilm enrichment. The staining in the Gel+NIR group was slightly weakened, while the staining in the GM group was significantly lighter and showed large areas of vacancy. The GM+NIR group had the weakest purple signal, and a large area of ​​unstained area was visible at the bottom of the well, corresponding to the lowest biomass in the quantitative bar chart.

[0139] Figure 8-11 These are the results of an antimicrobial omics study. Among them, Figure 8 The results of GM+NIR group analysis of upregulated proteins in antimicrobial omics research; Figure 9 Results of GM+NIR group analysis of downregulated proteins; Figure 10 To investigate the cation transmembrane transport pathway and passive transmembrane transport pathway of *Porphyromonas gingivalis* between the control and GM+NIR groups through gene set enrichment analysis; where A represents the cation transmembrane transport pathway and B represents the passive transmembrane transport pathway. Figure 11 This is a schematic diagram of the antibacterial mechanism.

[0140] from Figure 8-11 According to the results of antimicrobial omics research, GM+NIR achieves a synergistic antimicrobial effect of energy depletion, membrane structure damage and biofilm disintegration by simultaneously promoting metabolic overload and blocking repair pathways, causing bacteria to gradually move from short-term compensation to systemic collapse, and finally forming a strong and stable antimicrobial effect.

[0141] 4. Anti-inflammatory properties:

[0142] 4.1 The anti-inflammatory properties of the product were tested using the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) method:

[0143] RAW 264.7 cells were divided into 6 different groups (blank group, control group, gel group, gel+NIR group, GM group, and GM+NIR group), and were divided into 6-well plates at a density of 5 × 10⁶ cells per well. 6 Cells were cultured at high density for 24 hours (LPS was added to the medium at a concentration of 1 μg / mL, and after 3 hours of culture, the medium was replaced with the corresponding conditioned medium for each group. For the control group, LPS was treated for 3 hours, and then the medium was replaced with ordinary medium. The blank group was not treated with LPS and used ordinary medium directly). After removing the cell culture medium, RNA was extracted from the cells using TRIzol according to the manufacturer's instructions and reverse transcribed using a reverse transcriptase kit (TAKARA, Osaka, Japan). PCR was performed using a Prime Script™ RT-PCR kit (TAKARA, Tokyo, Japan) and an Applied Biosystems 7300 (ThermoScientific, Waltham, MA) according to the manufacturer's instructions. Gene primers are shown in Table 1. Data normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression were analyzed using the 2-ΔΔCt method. Each experiment was repeated three times.

[0144] Table 1

[0145]

[0146] 4.2 Flow Cytometry: RAW264.7 cells were divided into 6 different groups (blank group, control group, gel group, gel+NIR group, GM group, and GM+NIR group), and were cultured in 6-well plates at a rate of 5 × 10⁶ cells per well. 6 Cells were cultured at high density for 24 hours (LPS was added to the culture medium at a concentration of 1 μg / mL, and after 3 hours of culture, the medium was replaced with the corresponding conditioned medium for each group. For the control group, LPS treatment was performed for 3 hours, followed by replacement with ordinary medium. The blank group received no LPS treatment and used ordinary medium directly). Cells were co-cultured with CD86 antibody (1:100 dilution) and CD206 antibody (1:400 dilution). Results were analyzed using FlowJo software (version 7.6; TreeStar, USA).

[0147] 4.3 Immunofluorescence Staining: Immunofluorescence was used to detect the expression levels of TNF-α, IL-1, IL-10, and Arg proteins to evaluate macrophage polarization. RAW264.7 cells were divided into six different groups (blank group, control group, gel group, gel+NIR group, GM group, and GM+NIR group), and were seeded in 24-well plates at a concentration of 1 × 10⁶ cells per well. 5The cells were cultured at high density for 24 hours (LPS was added to the culture medium at a concentration of 1 μg / mL, and after culturing for 3 hours, the medium was replaced with the corresponding conditioned medium for each group. For the control group, LPS was treated for 3 hours, and then the medium was replaced with ordinary medium. The blank group was not treated with LPS and used ordinary medium directly). After removing the cell culture medium, RAW 264.7 cells were fixed with 4% PFM (paraformaldehyde) for 30 minutes, washed 3 times with PBS, and incubated in immunostaining blocking buffer (Beyotime, Shanghai, China) for 2 hours. Subsequently, they were incubated overnight at 4°C with anti-IL1 primary antibody (1:500, sc-57315, Santa), TNFα primary antibody (1:500, sc-28318, Santa), IL10 primary antibody (1:500, sc-32815, Santa), and Arg primary antibody (1:500, sc-271430, Santa). After overnight incubation, the cells were stained with IgG secondary antibodies conjugated to Alexa488 (1:1000, A0428, Beyotime) and Alexa647 (1:1000, A0473, Beyotime) for 1 hour at room temperature. DAPI staining was performed for 6 minutes. Finally, fluorescence images were acquired using a confocal microscope. The results are as follows: Figure 12 As shown:

[0148] Figure 12 This study aimed to assess the in vitro immune microenvironment regulation capabilities of GM+NIR. A represents the relative mRNA levels of tumor necrosis factor α (TNFα), interleukin 1β (IL1β), interleukin 10 (IL10), and arginase (Arg) in macrophages from each group; B represents the proportion of M1 and M2 macrophages after different treatment methods; and C represents immunofluorescence staining images of inflammation-related protein expression in macrophages after different treatment methods.

[0149] It can be seen that GM+NIR has the ability to reprogram macrophages. To further determine the polarization state of macrophages, immunofluorescence staining was used to detect the expression of M1 biomarkers (TNF-α and IL-1β) and M2 biomarkers (Arg-1 and IL-10) in the LPS-induced inflammatory environment. As shown in Part B, the Example 1 (GM+NIR) group under near-infrared light excitation had the lowest M1 biomarker content and the highest M2 biomarker content, exhibiting the best anti-inflammatory performance.

[0150] 4.4. RNA Sequencing: Following the same cell grouping and culture methods as described in Part 4.1, after cell culture, total RNA was extracted from macrophages using RNAiso reagent for RNA isolation and library preparation. RNA purity and quantification were assessed using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using an Agilent 2100 bioanalyzer. Transcriptome libraries were constructed using the VAHTS Universal V5 RNA-seq Library Prep Kit according to the manufacturer's instructions. RNA sequencing and differentially expressed gene analysis were performed. The libraries were sequenced using an Illumina Novaseq 6000 sequencing platform, yielding 150 bp paired-end reads. Approximately 50 raw reads were obtained per sample. The raw reads in FASTQ format were processed using FastP software, and clean reads were obtained after removing low-quality reads for subsequent data analysis. The clean reads were mapped to a reference genome using HISAT2. The number of bases per million exons (FPKM) of each gene was calculated, where FPKM < 1 was defined as low expression, 1 ≤ FPKM < 10 as moderate expression, and FPKM ≥ 10 as high expression. Readings for each gene were obtained using HTSeq-count. Principal component analysis (PCA) was performed using R (v 3.2.0) to assess the biological reproducibility of the samples. Differentially expressed genes were analyzed using DESeq2 software; genes meeting the p-value < 0.05 and foldchange > 2 or foldchange < 0.5 thresholds were defined as significantly differentially expressed genes (DEGs). Systematic cluster analysis of DEGs was performed using R (v 3.2.0) to demonstrate gene expression patterns across different sample combinations. Based on the hypergeometric distribution algorithm, we performed Gene ontology (GO) and Kyotoencyclopedia of genes and genomes (KEGG) pathway enrichment analyses on DEGs, and used R (v3.2.0) to screen for significant enriched terms.

[0151] Mitochondrial staining: RAW 264.7 cells were divided into 6 different groups (blank group, control group, gel group, gel+NIR group, GM group, and GM+NIR group), and 1×10⁶ cells were injected into each well of a 24-well plate. 5Cells were cultured at high density for 24 hours (LPS was added to the culture medium at a concentration of 1 μg / mL, and after 3 hours of culture, the medium was replaced with the corresponding conditioned medium for each group. For the control group, LPS treatment was performed for 3 hours, followed by replacement with ordinary culture medium. The blank group received no LPS treatment and used ordinary culture medium directly). After culture, the cell culture medium was removed, and Mito-Tracker staining solution pre-incubated at 37°C was added, followed by incubation at 37°C for 30 minutes. The Mito-Tracker staining solution was then removed, and fresh cell culture solution pre-incubated at 37°C was added. The cells were then observed using a fluorescence microscope.

[0152] Mitochondrial membrane potential assay: RAW 264.7 cells were divided into 6 different groups (blank group, control group, gel group, gel+NIR group, GM group, and GM+NIR group). Cells were then divided into 24-well plates at a concentration of 1 × 10⁻⁶ cells per well. 5 Cells were cultured at high density for 24 hours (LPS was added to the culture medium at a concentration of 1 μg / mL, and after 3 hours of culture, the medium was replaced with the corresponding conditioned medium for each group. For the control group, LPS treatment was performed for 3 hours, followed by replacement with ordinary culture medium. The blank group was not treated with LPS and used ordinary culture medium directly). After culture, the culture medium was removed, and 1 mL of cell culture medium and 1 mL of JC-1 staining working solution were added and mixed well. The cells were incubated at 37°C for 20 minutes in a cell culture incubator. After incubation, the supernatant was removed and the cells were washed twice with JC-1 staining buffer. 2 mL of cell culture medium was added, and the cells were observed under a fluorescence microscope.

[0153] Mitochondrial reactive oxygen species (ROS) content detection: RAW 264.7 cells were divided into 6 different groups (blank group, control group, gel group, gel+NIR group, GM group, and GM+NIR group). 1 × 10⁻⁶ cells were injected into each well of a 24-well plate. 5 The cells were cultured at high density for 24 hours (LPS was added to the culture medium at a concentration of 1 μg / mL, and after 3 hours of culture, the medium was replaced with the corresponding conditioned medium for each group. For the control group, LPS was treated for 3 hours, and then the medium was replaced with ordinary medium. The blank group was not treated with LPS and used ordinary medium directly). After incubation, 500 μl of MitoSOX working solution was added to each group, and the cells were incubated at 37°C for 30 minutes. After incubation, DAPI staining was performed for 5 minutes. The cells were then observed under a fluorescence microscope and detected by flow cytometry.

[0154] Figure 13-21 The effects and mechanisms of GM+NIR on macrophage mitophagy. Figure 13 KEGG enrichment analysis for differentially expressed genes; Figure 14GO enrichment analysis for differentially expressed genes; Figure 15 Gene enrichment analysis for the mitophagy pathway; Figure 16 Differences in the expression of genes related to the mitophagy pathway among different groups; Figure 17 Gene enrichment analysis for the FOXO pathway; Figure 18 Differences in the expression of FOXO pathway-related genes among different groups; Figure 19 The mtROS levels of macrophages after different treatments are shown; where A is the blank group, B is the control group, C is the gel group, D is the gel+NIR group, E is the GM group, and F is the GM+NIR group. Figure 20 The state of mitochondria in macrophages after different treatments (red: normal mitochondria, green: dysfunctional mitochondria). Figure 21 Mitochondrial membrane potential levels in macrophages from different groups;

[0155] It can be seen that GM+NIR can restore the mitochondrial autophagy function of macrophages under inflammatory conditions, clear damaged mitochondria, drive macrophage phenotypic transformation through metabolic reprogramming, and achieve regulation of the immune microenvironment in periodontitis.

[0156] 5. In vivo experiments in rats:

[0157] Six-week-old male SD rats, weighing 180-200g, were acclimatized for 7 days before the experiment and randomly divided into 6 groups: Blank group, Control group, Periodontitis group (no hydrogel injection), Gel group, GM group, and GM+NIR group. After general anesthesia via intraperitoneal injection of sodium pentobarbital, the left maxillary second molar was ligated with a 0.25 mm suture. A suspension of *Porphyromonas gingivalis* and *Clostridium nucleatum* was injected into the periodontal tissue every other day for a total of 7 injections. Two weeks later, redness and swelling of the periodontal tissue and bleeding on probing indicated successful model establishment. The experimental group (GM+NIR group) received an injectable hydrogel (20 μL) injected into the periodontal pocket and was treated under NIR conditions: 1.0 MHz, 0.5 W / cm². 2 The treatment was administered every 2 days for 28 days (14 treatments in total), with each treatment lasting 5 minutes. The control group received an equal volume of physiological saline. All rats were euthanized after completing the experiment. The soft and hard tissues of the left maxilla were removed and fixed with 4% paraformaldehyde for 24 hours. The left maxilla of the rats was scanned using a miniature CT scanner at 90 kV and 0.04 mA. The scan data were reconstructed and analyzed using N-Recon software. The distance between the cementoenamel junction (CEJ) and alveolar ridge (ABC) of the second molar was measured to assess alveolar bone resorption.

[0158] Figure 22The results of GM hydrogel's in vivo treatment of periodontitis are shown below. A represents representative three-dimensional micro-CT reconstructions of alveolar bone from different groups (scale bar = 1.0 mm). B shows quantitative analysis of the CEJ-ABC distance. C shows representative photographs of bacterial colonies collected from periodontal pockets, and D shows the corresponding quantitative CFU counts. E shows typical H&E staining images (scale bar = 500 μm, black box indicates bone and soft tissue areas; scale bar = 50 μm, yellow arrows indicate inflammatory cells), and F shows the quantitative statistics of immune cell counts in inflamed periodontal tissue. G shows typical Massen's trichrome staining images (scale bar = 500 μm; black box: bone and soft tissue areas; scale bar = 50 μm), and H shows the percentage of collagen degradation in inflamed periodontal tissue. I represents the pro-inflammatory fluorescence image of IL-6 (green fluorescence represents IL-6-positive cells, blue fluorescence represents cell nuclei), and J represents the corresponding relative pro-inflammatory fluorescence intensity of IL-6; K represents the anti-inflammatory fluorescence image of Arg-1 (red fluorescence represents Arg-1-positive cells, blue fluorescence represents cell nuclei), and L represents the corresponding relative anti-inflammatory fluorescence intensity of Arg-1 (data are expressed as mean ± SD (n = 6). Statistical significance: *P < 0.05, **P < 0.01, **P < 0.001; n, not significant).

[0159] Micro-CT reconstruction results showed that the Control group exhibited significant vertical alveolar bone resorption and ridge collapse, with a significantly increased CEJ-ABC distance (p < 0.001), indicating successful model construction. Both the GM and GM+NIR groups significantly inhibited bone resorption (p < 0.01 vs. Control), with the GM+NIR group showing the most outstanding performance. Its CEJ-ABC distance essentially recovered to near-healthy levels, indicating that it not only reduced bone loss but also achieved orderly reconstruction of bone structure.

[0160] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing near-infrared light-exciteable composite nanosheets, characterized in that, Includes the following steps: Epigallocatechin gallate ester was mixed with Ti3C2 nanosheet dispersion and subjected to ultrasonic vibration reaction. Silver salt was then added and the reaction was continued in the dark. After the reaction was completed, the nanosheets were washed and separated to obtain the near-infrared light-excited composite nanosheets. The concentration of the Ti3C2 nanosheet dispersion was 12 mg / mL; The mass ratio of epigallocatechin gallate to silver salt is 1:(1-3).

2. The method for preparing a near-infrared light-exciteable composite nanosheet according to claim 1, characterized in that, The silver salt is silver nitrate.

3. The method for preparing near-infrared light-exciteable composite nanosheets according to claim 1, characterized in that, The duration of the ultrasonic oscillation response is 10-20 minutes; The continued stirring reaction is a light-protected stirring reaction, and the time is 2-4 hours.

4. The near-infrared light-exciteable composite nanosheets prepared by the preparation method according to any one of claims 1-3.

5. The application of the near-infrared light-exciteable composite nanosheets as described in claim 4 in the preparation of a drug for treating periodontitis, characterized in that, The medication for treating periodontitis is in a medically acceptable dosage form.

6. A hydrogel, characterized in that, Including the near-infrared light-exciteable composite nanosheets as described in claim 4; The preparation method of the hydrogel includes the following steps: The composite nanosheets are mixed with carboxymethyl chitosan in water and reacted, and then mixed with oxidized fucoidan solution to obtain the hydrogel. The mass ratio of the composite nanosheets, carboxymethyl chitosan, and oxidized fucoidan is 1.2:(400-600):(500-750).

7. A method for preparing the hydrogel as described in claim 6, characterized in that, Includes the following steps: The composite nanosheets are mixed with carboxymethyl chitosan in water and reacted, and then mixed with oxidized fucoidan solution to obtain the hydrogel. The mass ratio of the composite nanosheets, carboxymethyl chitosan, and oxidized fucoidan is 1.2:(400-600):(500-750).

8. The method for preparing a hydrogel according to claim 7, characterized in that, The oxidized fucoidan is obtained by oxidizing fucoidan with sodium periodate.

9. The use of the hydrogel as described in claim 6 in the preparation of a medicament for treating periodontitis.

Images