An injectable hydrogel for photothermal-assisted treatment of periodontitis and its preparation method

Abstract

This invention discloses an injectable hydrogel for photothermal-assisted treatment of periodontitis and its preparation method, belonging to the field of polymer material preparation technology. The preparation method includes the following steps: reacting MXene dispersion with epigallocatechin gallate and MgSO4; then sequentially performing ultrasonication, centrifugation, washing, and lyophilization to obtain multifunctional nanosheets MP-Mg; adding the multifunctional nanosheets MP-Mg to an aqueous solution of oxidized gellan gum for mixing to obtain solution A; mixing equal volumes of quaternary ammonium chitosan aqueous solution and carboxymethyl chitosan aqueous solution for reaction to obtain solution B; and mixing equal volumes of solution A and solution B for further reaction. The hydrogel prepared by this invention possesses injectability, photothermal responsiveness, biofilm penetration ability, synergistic antibacterial properties, and long-lasting anti-inflammatory effects, providing an innovative and practical photothermal-assisted treatment platform for the clinical treatment of periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of polymer material preparation technology, and particularly relates to an injectable hydrogel for photothermal-assisted treatment of periodontitis and its preparation method. 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] Recently, near-infrared photodynamic therapy (PDT) has become an effective strategy for combating bacterial infections. MXene, as a photosensitizer and photothermal agent, has been applied in PDT. However, the instability of MXene makes it difficult to achieve stable performance in treatment. Therefore, constructing a biomaterial that can stably exist in periodontal pockets and exert long-term antibacterial and anti-inflammatory effects for the treatment of periodontitis is of significant research value. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an injectable hydrogel for photothermal-assisted treatment of periodontitis and its preparation method. Specifically, this invention utilizes a layer-by-layer self-assembly process to react MXene with epigallocatechin gallate, thereby modifying MXene with a polyphenol layer to improve its stability. Combined with quaternary ammonium chitosan (QCS) carried within the hydrogel, this results in a stability of 1 W / cm². 2 Under irradiation with 808 nm near-infrared light, the precise and efficient combination of photothermal and cationic polymers is used to remove bacterial biofilm in periodontal pockets.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis includes the following steps:

[0007] (1) MXene dispersion was reacted with epigallocatechin gallate (EGCG) in an alkaline solution at pH 8.5 under dark conditions, and then MgSO4 was added to continue the reaction; then the reaction was carried out by sonication, centrifugation, washing and lyophilization to obtain multifunctional nanosheets MP-Mg.

[0008] (2) The multifunctional nanosheets MP-Mg were added to an aqueous solution of oxidized gellan gum and mixed to obtain solution A;

[0009] (3) Mix equal volumes of quaternary ammonium salt chitosan aqueous solution and carboxymethyl chitosan aqueous solution, and react to obtain solution B;

[0010] (4) Mix equal volumes of solution A and solution B and react to obtain multifunctional injectable hydrogel GQM.

[0011] Beneficial effects: This invention provides a method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis. Through the integration and synergistic effect of multiple components, it achieves precise local delivery while possessing both highly efficient photothermal sterilization and long-lasting anti-inflammatory effects, significantly improving the comprehensiveness and durability of periodontitis treatment.

[0012] In step (1), the MXene dispersion was reacted with epigallocatechin gallate (EGCG) under alkaline conditions at pH 8.5 in the dark, thereby loading EGCG onto the surface of MXene nanosheets. Subsequently, MgSO4 was added to chelate magnesium ions into the composite system. After ultrasonic dispersion, high-speed centrifugation, thorough washing, and lyophilization, multifunctional nanosheets MP-Mg were obtained. This step successfully constructed a composite system integrating photothermal conversion (MXene), antioxidant / anti-inflammatory (EGCG), and bone protection / immunomodulation (MgSO4). 2+ This multifunctional unit, which integrates photothermal destruction of biofilm and sustained-release anti-inflammatory effects, provides a core functional carrier for the subsequent realization of "photothermal destruction of biofilm + sustained-release anti-inflammatory".

[0013] In step (2), multifunctional nanosheets MP-Mg are uniformly dispersed in an aqueous solution of oxidized gellan gum to form solution A. Oxidized gellan gum is prepared by oxidizing gellan gum with sodium periodate. The aldehyde groups generated on its molecular chain not only enhance water solubility but also combine with subsequent chitosan substances to form imine bonds, thereby undergoing dynamic cross-linking and endowing the hydrogel with good injectability, self-healing ability, and sustained-release properties. More importantly, as a biocompatible natural polysaccharide matrix, oxidized gellan gum can effectively prolong the release time of EGCG and magnesium ions in MP-Mg within the periodontal pocket, ensuring a sustained anti-inflammatory effect for more than 7 days, thereby inhibiting inflammation-mediated alveolar bone resorption.

[0014] In step (3), an aqueous solution of quaternary ammonium chitosan (QCS) and an aqueous solution of carboxymethyl chitosan are mixed in equal volumes to form solution B. QCS, carrying a permanent positive charge, can effectively disrupt bacterial cell membranes and exhibits broad-spectrum antibacterial activity; while carboxymethyl chitosan provides good water solubility, biocompatibility, and gelling ability. The two interact electrostatically to form a polyelectrolyte complex, which not only enhances the mechanical stability of the hydrogel network but also provides a controllable release platform for the antibacterial components.

[0015] In step (4), solutions A and B are mixed in equal volumes and reacted. Simultaneously, the multifunctional nanosheets MP-Mg are encapsulated in the formed three-dimensional network, which can rapidly form a uniform and flexible hydrogel GQM in situ. This hydrogel has excellent injectability and shape adaptability, and can penetrate and completely fill complex and irregular periodontal pockets, achieving precise adhesion to the lesion site and targeted drug enrichment.

[0016] Of particular note is the significant advantage of this invention in photothermal-assisted therapy: when the hydrogel is injected into the periodontal pocket, under near-infrared (NIR) light irradiation, the MXene component rapidly converts light energy into heat energy. The increased local temperature effectively breaks down the dense biofilm formed by periodontal pathogens—a key barrier that traditional antibiotics struggle to penetrate. The photothermal effect not only directly kills bacteria but also triggers the release of QCS, achieving synergistic "photothermal + chemical" sterilization and significantly improving antibacterial efficiency. After acute infection control, the hydrogel continuously releases EGCG and magnesium ions, providing long-lasting anti-inflammatory effects and inhibiting osteoclast activation, thereby significantly reducing alveolar bone loss. This progressive treatment strategy of "highly efficient photothermal sterilization in the early stage—slow-release long-lasting anti-inflammatory effect in the later stage" overcomes the limitations of existing therapies that are single, short-term, and prone to recurrence, achieving full-process intervention for periodontitis from infection clearance to tissue protection.

[0017] In summary, through ingenious material design and process integration, this invention is not only easy to operate and cost-controllable, but also achieves an organic unity of photothermal response, biofilm penetration, synergistic antibacterial and long-lasting anti-inflammatory effects, providing a novel, efficient, intelligent and practical photothermal-assisted treatment platform for the clinical treatment of periodontitis.

[0018] Optionally, the ratio of the MXene dispersion, epigallocatechin gallate, and MgSO4 is 1 mL: 30-60 mg: 30-60 mg.

[0019] Furthermore, the concentration of the MXene dispersion is 10 mg / mL MXene.

[0020] Furthermore, the MXene dispersion is obtained by dissolving MXene nanosheets in deionized water; the preparation process of the MXene nanosheets is as follows: LiF and Ti3AlC2 are reacted in hydrochloric acid solution, and then centrifuged, washed and lyophilized in sequence.

[0021] Optionally, the oxidized gellan gum in the aqueous solution is prepared by the sodium periodate oxidation method.

[0022] Furthermore, the preparation process of the oxidized gellan gum is as follows:

[0023] Gellan gum was dissolved in deionized water, sodium periodate was added, and the reaction was carried out in the dark. Then ethylene glycol was added to consume the unreacted sodium periodate, and the reaction was continued with stirring. Then, dialysis and freeze-drying were carried out in sequence.

[0024] Furthermore, the ratio of gellan gum, sodium periodate, and ethylene glycol is 1-3 g : 0.4-1.0 g : 1-3 mL.

[0025] Furthermore, the dialysis conditions are as follows: the product is added to M... W Dialyze in a dialysis bag with a capacity of 3500 for 72 hours.

[0026] Optionally, the concentration of the multifunctional nanosheets MP-Mg in solution A is 0.6-1.8 mg / mL.

[0027] Optionally, the concentration of the quaternary ammonium salt chitosan (QCS) aqueous solution is 0.5-3%;

[0028] The concentration of the carboxymethyl chitosan aqueous solution is 8-12%.

[0029] An injectable hydrogel for photothermal-assisted treatment of periodontitis is prepared by the above-described method.

[0030] The above-mentioned injectable hydrogel is used in the preparation of photothermal adjuvant therapy for periodontitis.

[0031] Compared with the prior art, the present invention has the following advantages and technical effects:

[0032] 1. The multifunctional injectable hydrogel prepared by this invention can be injected and penetrate deep into the environment of the periodontal pocket, and matches the complex anatomical structure and morphology of the periodontal pocket according to its topological morphology.

[0033] 2. The multifunctional injectable hydrogel prepared by this invention has good photothermal properties. It generates heat under near-infrared light (NIR) excitation, and breaks down the bacterial biofilm at the site of periodontitis through photothermal properties, releasing quaternary ammonium chitosan (QCS) to achieve effective antibacterial effect at the site of periodontitis in synergistic photothermal effect.

[0034] 3. The MXene, epigallocatechin gallate, and magnesium ions used in the preparation process of this invention can exert anti-inflammatory functions, thereby reducing alveolar bone loss caused by inflammation and achieving effective treatment for periodontitis.

[0035] 4. The multifunctional injectable hydrogel prepared by this invention has the characteristic of sequential treatment. It can effectively kill bacteria in the early stage of bacterial infection and achieve long-lasting anti-inflammatory effect in the later stage of inflammation treatment, thereby reducing alveolar bone loss and treating periodontitis in a holistic and effective manner.

[0036] 5. The multifunctional injectable hydrogel prepared by this invention can remain stably in the periodontal pocket for 7 days, and continuously release anti-inflammatory components epigallocatechin gallate (EGCG) and magnesium ions to achieve long-lasting anti-inflammatory effects.

[0037] 6. The multifunctional injectable hydrogel prepared by this invention has an easy gelation process, a simple method, and a low cost. Attached Figure Description

[0038] 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:

[0039] Figure 1 This is a scanning electron microscope image of the multifunctional injectable hydrogel synthesized in Example 1;

[0040] Figure 2 Transmission electron microscopy and elemental distribution of the multifunctional nanosheets MP-Mg synthesized in Example 1;

[0041] Figure 3 Rheological diagram of the injectability and shear-thinning properties of the multifunctional injectable hydrogel synthesized in Example 1;

[0042] Figure 4 Photothermal cycling capability of the multifunctional injectable hydrogel synthesized in Example 1 under near-infrared (NIR) light excitation (a), and photothermal properties of the hydrogels synthesized in Example 1 and Comparative Example 3 under near-infrared (NIR) light excitation (b).

[0043] Figure 5 The image shows a comparison of the antibacterial properties of four groups of hydrogels—Comparative Example 1 (Gel), Comparative Example 2 (GQ), Example 1 (GQM), and Example 1 (GQM+NIR) under near-infrared light excitation—against two common pathogenic bacteria found in different periodontal sites. a represents Porphyromonas gingivalis; b represents Fusobacterium nucleatum.

[0044] Figure 6The graph shows a comparison of the antibacterial biofilm performance of four groups of hydrogels (Comparative Example 1 (Gel), Comparative Example 2 (GQ), Example 1 (GQM), and Example 1 (GQM+NIR) under near-infrared light excitation against two different bacterial biofilms. a is Porphyromonas gingivalis; b is Fusobacterium nucleatum.

[0045] Figure 7 The performance of the injectable hydrogels synthesized in Example 1 and Comparative Example 3 in removing ROS is shown in Figure 1. In Figure 3, a is the UV absorption spectrum of DPPH mixed with the injectable hydrogels synthesized in Example 1 and Comparative Example 3, and b is the ROS removal rate calculated based on the absorption peak intensity of a at 517 nm.

[0046] Figure 8 Images of alveolar bone after hydrogel treatment for periodontitis in four groups: Comparative Example 1 (Gel), Comparative Example 2 (GQ), Example 1 (GQM), and Example 1 (GQM+NIR) under near-infrared light excitation; where a is a statistical graph of the distance between the cementoenamel junction (CEJ) and alveolar ridge (ABC) of the second molar in each group; b is a Micro-CT image of each group;

[0047] Figure 9 Live / dead cell staining test of the hydrogels synthesized in Example 1 and Comparative Example 3. Detailed Implementation

[0048] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0050] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0051] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0052] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0053] This invention discloses a multifunctional injectable hydrogel, its preparation method, and its application. The hydrogel effectively kills bacteria by photothermal destruction of bacterial biofilms and has the advantage of long-lasting anti-inflammatory adjuvant treatment for periodontitis, reducing alveolar bone loss.

[0054] This invention discloses a method for preparing a multifunctional injectable hydrogel, the specific steps of which are as follows:

[0055] (1) Add 0.5-2 g LiF to a 9-12 mol / L hydrochloric acid solution to obtain a clear solution. Then add 0.5-2 g Ti3AlC2 black powder to the solution and stir for 20-30 h in a water bath at 35 ℃ to obtain a black solution. Centrifuge the solution at 3000-5000 rpm to collect the precipitate. Wash the solution repeatedly with deionized water and centrifuge until the pH of the supernatant is close to 6. Then, add deionized water to redissolve the precipitate and sonicate under an argon atmosphere for 1 h. Finally, centrifuge at low speed to collect the supernatant and freeze-dry it to obtain MXene nanosheets.

[0056] 10 mg of MXene nanosheets were dissolved in 1 mL of deionized water to obtain a 10 mg / mL MXene dispersion. 30-60 mg of epigallocatechin gallate (EGCG) powder was dissolved in an alkaline aqueous solution at pH 8.5, and 1 mL of the 10 mg / mL MXene dispersion was added. The mixture was reacted in the dark for 2-4 h to obtain a mixed solution. Then, 30-60 mg of MgSO4 powder was added to the above mixed solution and reacted for 2-4 h. After ultrasonic vibration of the mixed solution, the precipitate was collected by centrifugation at 8000-10000 rpm, washed with deionized water, and then lyophilized to obtain multifunctional nanosheets MP-Mg.

[0057] (2) Oxidized gellan gum was prepared by sodium periodate oxidation. The preparation steps were as follows: 1-3 g of gellan gum was dissolved in 100 mL of deionized water, and 0.4-1.0 g of sodium periodate was added. The mixture was reacted at room temperature in the dark for 4-8 h. Then, 1-3 mL of ethylene glycol was added to consume the unreacted sodium periodate, and the mixture was stirred for another 1 h. The product was then placed into a dialysis bag (M... W Dialysis in 3500 g / L for 72 hours, followed by freeze-drying, yielded a white spongy oxidized gellan gum.

[0058] (3) Add the multifunctional nanosheets MP-Mg to a 3-6wt% aqueous solution of oxidized gellan gum, mix and react for 30 min to obtain solution A with a concentration of 0.6-1.8 mg / mL of multifunctional nanosheets MP-Mg;

[0059] Solution B is obtained by mixing equal volumes of 0.5-3 wt% quaternary ammonium salt chitosan (QCS) aqueous solution and 8-12 wt% carboxymethyl chitosan aqueous solution and reacting for 2 h.

[0060] Equal volumes of solution A and solution B were thoroughly mixed and reacted for 5 minutes to obtain the multifunctional injectable hydrogel GQM.

[0061] This invention first prepares an MXene dispersion, then epigallocatechin gallate (EGCG) and magnesium sulfate are sequentially added to the MXene dispersion for reaction, yielding multifunctional nanosheets MP-Mg with stable photothermal and anti-inflammatory properties. The multifunctional nanosheets MP-Mg are reacted with oxidized gellan gum (OG) to obtain solution A; quaternary ammonium chitosan (QCS) solution and carboxymethyl chitosan solution are reacted to obtain solution B. Solutions A and B are mixed and reacted to obtain a multifunctional injectable hydrogel GQM. The hydrogel GQM prepared by this invention can be injected into periodontal pockets, matching the complex anatomical structure and morphology of the periodontal pockets. Furthermore, it can utilize photothermal properties to break down bacterial biofilms at the periodontitis site, releasing quaternary ammonium chitosan (QCS) to synergistically achieve effective antibacterial activity at the periodontitis site. Moreover, the MXene, epigallocatechin gallate, and magnesium ions used in the preparation process can exert a sustained anti-inflammatory function in subsequent treatment, thereby reducing alveolar bone loss caused by inflammation and achieving excellent therapeutic effects for periodontitis. This invention constructs a biomaterial that can stably exist in periodontal pockets and exert long-term antibacterial and anti-inflammatory effects.

[0062] Unless otherwise specified, "room temperature" in this invention refers to 20-30℃.

[0063] All raw materials used in this invention were purchased from the market.

[0064] The technical solution of the present invention will be further illustrated by the following embodiments.

[0065] Example 1

[0066] A method for preparing a multifunctional injectable hydrogel includes the following steps:

[0067] (1) 1 g of LiF was added to a 9 mol / L hydrochloric acid solution to obtain a clear solution. 1 g of Ti3AlC2 black powder was added, and the mixture was heated to 35 ℃ and stirred for 26 h in a water bath to obtain a black solution. The precipitate was collected by centrifugation at 3500 rpm, and the solution was repeatedly washed with deionized water and centrifuged until the pH of the supernatant was close to 6. Then, an appropriate amount of deionized water was added to redissolve the precipitate, and the mixture was ultrasonically vibrated for 1 h under an argon atmosphere. Finally, the supernatant was collected by low-speed centrifugation and lyophilized to obtain MXene nanosheets. 10 mg of MXene nanosheets were dissolved in 1 mL of deionized water to obtain a 10 mg / mL MXene dispersion.

[0068] 30 mg of epigallocatechin gallate (EGCG) powder was dissolved in an alkaline aqueous solution at pH 8.5, and 1 mL of 10 mg / mL MXene dispersion was added. The mixture was reacted in the dark for 2 h to obtain a mixed solution. Then, 30 mg of MgSO4 powder was added to the above mixed solution and reacted for 4 h. After ultrasonic vibration of the mixed solution, the precipitate was collected by centrifugation at 8000 rpm, washed with deionized water, and then lyophilized to obtain multifunctional nanosheets MP-Mg.

[0069] (2) Oxidized gellan gum was prepared by sodium periodate oxidation. The preparation steps were as follows: 1 g of gellan gum was dissolved in 100 mL of deionized water, 0.5 g of sodium periodate was added, and the mixture was reacted at room temperature in the dark for 5 h. Then, 1 mL of ethylene glycol was added to consume the unreacted sodium periodate, and the mixture was stirred for another 1 h. The product was then placed into a dialysis bag (M). W Dialysis was performed in 3500 g / L solution for 72 h, followed by freeze-drying to obtain a white, spongy oxidized gellan gum.

[0070] (3) Add the multifunctional nanosheets MP-Mg to a 3% aqueous solution of oxidized gellan gum and mix for 30 min to obtain solution A with a concentration of 1.8 mg / mL of multifunctional nanosheets MP-Mg;

[0071] Solution B was obtained by mixing 3% quaternary ammonium salt chitosan (QCS) aqueous solution and 12% carboxymethyl chitosan aqueous solution in equal volume ratio and reacting for 2 h.

[0072] Equal volumes of solution A and solution B were thoroughly mixed and reacted for 5 min to obtain the multifunctional injectable hydrogel GQM (the MP-Mg concentration in the multifunctional injectable hydrogel was 0.9 mg / mL).

[0073] Example 2

[0074] A method for preparing a multifunctional injectable hydrogel includes the following steps:

[0075] (1) 2 g of LiF was added to a 12 mol / L hydrochloric acid solution to obtain a clear solution. 2 g of Ti3AlC2 black powder was added, and the mixture was heated to 35 ℃ and stirred for 30 h to obtain a black solution. The precipitate was collected by centrifugation at 4500 rpm, and the solution was repeatedly washed with deionized water and centrifuged until the pH of the supernatant was close to 6. Then, an appropriate amount of deionized water was added to redissolve the precipitate, and the mixture was ultrasonically vibrated for 1 h under an argon atmosphere. Finally, the supernatant was collected by low-speed centrifugation and lyophilized to obtain MXene nanosheets. 10 mg of MXene nanosheets were dissolved in 1 mL of deionized water to obtain a 10 mg / mL MXene dispersion.

[0076] 60 mg of epigallocatechin gallate (EGCG) powder was dissolved in an alkaline aqueous solution at pH 8.5, and 1 mL of 10 mg / mL MXene dispersion was added. The mixture was reacted in the dark for 4 h to obtain a mixed solution. Then, 60 mg of MgSO4 powder was added to the above mixed solution and reacted for 2 h. After ultrasonic vibration of the mixed solution, the precipitate was collected by centrifugation at 10,000 rpm, washed with deionized water, and then lyophilized to obtain multifunctional nanosheets MP-Mg.

[0077] (2) Oxidized gellan gum was prepared by sodium periodate oxidation. The preparation steps were as follows: 3 g of gellan gum was dissolved in 100 mL of deionized water, 1.0 g of sodium periodate was added, and the mixture was reacted at room temperature in the dark for 8 h. Then, 3 mL of ethylene glycol was added to consume the unreacted sodium periodate, and the mixture was stirred for another 1 h. The product was then placed into a dialysis bag (M). W Dialysis was performed in 3500 g / L solution for 72 h, followed by freeze-drying to obtain a white, spongy oxidized gellan gum.

[0078] (3) Add the multifunctional nanosheets MP-Mg to a 6% aqueous solution of oxidized gellan gum and mix for 30 min to obtain solution A with a concentration of 1.8 mg / mL of multifunctional nanosheets MP-Mg;

[0079] Solution B was obtained by mixing equal volumes of 1.5% quaternary ammonium salt chitosan (QCS) aqueous solution and 10% carboxymethyl chitosan aqueous solution and reacting for 2 h.

[0080] Equal volumes of solution A and solution B were thoroughly mixed and reacted for 5 minutes to obtain the multifunctional injectable hydrogel GQM.

[0081] Example 3

[0082] A method for preparing a multifunctional injectable hydrogel includes the following steps:

[0083] (1) 1.5 g LiF was added to a 10 mol / L hydrochloric acid solution to obtain a clear solution. 1.5 g Ti3AlC2 black powder was added, and the mixture was heated to 35 ℃ and stirred for 24 h in a water bath to obtain a black solution. The precipitate was collected by centrifugation at 4000 rpm, and the solution was repeatedly washed with deionized water and centrifuged until the pH of the supernatant was close to 6. Then, an appropriate amount of deionized water was added to redissolve the precipitate, and the mixture was ultrasonically vibrated for 1 h under an argon atmosphere. Finally, the supernatant was collected by low-speed centrifugation and lyophilized to obtain MXene nanosheets. 10 mg of MXene nanosheets was dissolved in 1 mL of deionized water to obtain a 10 mg / mL MXene dispersion.

[0084] 45 mg of epigallocatechin gallate (EGCG) powder was dissolved in an alkaline aqueous solution at pH 8.5, and 1 mL of 10 mg / mL MXene dispersion was added. The mixture was reacted in the dark for 3 h to obtain a mixed solution. Then, 45 mg of MgSO4 powder was added to the above mixed solution and reacted for 3 h. After ultrasonic vibration of the mixed solution, the precipitate was collected by centrifugation at 8800 rpm, washed with deionized water, and then lyophilized to obtain multifunctional nanosheets MP-Mg.

[0085] (2) Oxidized gellan gum was prepared by sodium periodate oxidation. The preparation steps were as follows: 2 g of gellan gum was dissolved in 100 mL of deionized water, 0.6 g of sodium periodate was added, and the mixture was reacted at room temperature in the dark for 6 h. Then, 1-3 mL of ethylene glycol was added to consume the unreacted sodium periodate, and the mixture was stirred for another 1 h. The product was then placed into a dialysis bag (M). W Dialysis was performed in 3500 g / L solution for 72 h, followed by freeze-drying to obtain a white, spongy oxidized gellan gum.

[0086] (3) Add the multifunctional nanosheets MP-Mg to a 5% aqueous solution of oxidized gellan gum and mix for 30 min to obtain solution A with a concentration of 1.8 mg / mL of multifunctional nanosheets MP-Mg;

[0087] Solution B was obtained by mixing 1% quaternary ammonium salt chitosan (QCS) aqueous solution and 8% carboxymethyl chitosan aqueous solution in equal volumes and reacting for 2 h.

[0088] Equal volumes of solution A and solution B were thoroughly mixed and reacted for 5 minutes to obtain the multifunctional injectable hydrogel GQM.

[0089] Comparative Example 1

[0090] A method for preparing a hydrogel includes the following steps:

[0091] (1) Oxidized gellan gum was prepared by sodium periodate oxidation. The preparation steps were as follows: 3 g of gellan gum was dissolved in 100 mL of deionized water, 1.0 g of sodium periodate was added, and the mixture was reacted at room temperature in the dark for 8 h. Then, 3 mL of ethylene glycol was added to consume the unreacted sodium periodate, and the mixture was stirred for another 1 h. The product was then placed into a dialysis bag (M). W Dialysis was performed in 3500 g / L solution for 72 h, followed by freeze-drying to obtain a white, spongy oxidized gellan gum.

[0092] (2) A 6% aqueous solution of oxidized gellan gum is solution A;

[0093] Solution B is a 10% carboxymethyl chitosan aqueous solution;

[0094] Equal volumes of solution A and solution B were thoroughly mixed and reacted for 5 min to obtain a hydrogel without multifunctional nanosheets MP-Mg and quaternary ammonium salt chitosan QCS.

[0095] Comparative Example 2

[0096] A method for preparing a hydrogel includes the following steps:

[0097] (1) Oxidized gellan gum was prepared by sodium periodate oxidation. The preparation steps were as follows: 3 g of gellan gum was dissolved in 100 mL of deionized water, 1.0 g of sodium periodate was added, and the mixture was reacted at room temperature in the dark for 8 h. Then, 3 mL of ethylene glycol was added to consume the unreacted sodium periodate, and the mixture was stirred for another 1 h. The product was then placed into a dialysis bag (M). W Dialysis was performed in 3500 g / L solution for 72 h, followed by freeze-drying to obtain a white, spongy oxidized gellan gum.

[0098] (2) A 6% aqueous solution of oxidized gellan gum is solution A;

[0099] Equal volumes of 3% quaternary ammonium salt chitosan (QCS) aqueous solution and 10% carboxymethyl chitosan aqueous solution were mixed and reacted for 2 hours to obtain solution B;

[0100] Equal volumes of solution A and solution B were thoroughly mixed and reacted for 5 min to obtain hydrogel GQ without multifunctional nanosheets MP-Mg.

[0101] Comparative Example 3

[0102] The difference from Example 1 is that the concentration of the multifunctional nanosheets MP-Mg in the injectable hydrogel GQM was changed to 0 mg / mL, 0.3 mg / mL, and 0.6 mg / mL. The other preparation procedures are the same as in Example 1.

[0103] Effect verification:

[0104] 1. Cell biocompatibility assessment:

[0105] The effects of the hydrogels prepared in Examples 1, 2, and 3 on L929 cells were evaluated using a live / dead cell staining assay. The hydrogels prepared in Examples 1, 2, and 3 were co-cultured with L929 cells. After 24 and 72 hours, the cells were incubated for 30 minutes 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).

[0106] 2. Antibacterial biofilm experiment:

[0107] P. gingivalis ATCC 33277 (Porphyromonas gingivalis) and F. nucleatum ATCC 10953 (Fusobacterium nucleatum) were both purchased from the American Center for Standard Biological Collections. P. gingivalis was cultured using TSB medium (TSB powder 30.12 g / L). -1 5 g / L yeast powder -1 L-cysteine ​​hydrochloride 0.5 g L -1 Vitamin K1 mg L -1 Heme chloride 5mg / 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 bacteria were cultured anaerobically 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.

[0108] 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-1 Circular cell smears were placed on the bottom of 24-well plates and cultured anaerobically for 96 h to form a biofilm. The cells were divided into five groups: no treatment (Control group), addition of Comparative Example 1 hydrogel (Gel group), addition of Comparative Example 2 hydrogel (GQ group), addition of Example 1 hydrogel (GQM group), and addition of Example 1 hydrogel followed by near-infrared (NIR) irradiation for 5 min (GQM+NIR group). The cells were then co-cultured with the bacterial biofilm for 24 h, and subsequent experiments were conducted to verify the antibacterial effects of each group.

[0109] 2.1 Colony forming unit count:

[0110] 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, and bacteria were collected from the biofilm 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, and the CFU (Cellular Units) were calculated based on the number of bacterial colonies on the blood agar plates and the corresponding dilution factor.

[0111] 2.2 Fluorescent staining for live / dead bacteria:

[0112] 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 according to the Invitrogen SYTO9 green fluorescent nucleic acid staining kit instructions, dropped onto the cell smear surface, and incubated in the dark for 15 min. 3D images of the biofilm were acquired using a CLSM, with five regions randomly collected from each sample group. 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.

[0113] 3. In vivo experiments in rats:

[0114] Six-week-old male SD rats, weighing 180-200 grams, were acclimatized for 7 days before the experiment and randomly divided into 7 groups: Control group (healthy), Periodontitis group (periodontitis), Gel group, GQ group, GQM group, and GQM+NIR group. After general anesthesia via intraperitoneal injection of sodium pentobarbital, the left maxillary second molar was ligated using 0.25 mm suture. A combined bacterial suspension of *Porphyromonas gingivalis* and *Fusobacterium 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 groups received an injectable hydrogel (200 μg / mL) injected into the periodontal pocket. -1Processed under NIR conditions: 1.0 MHz, 1 W / cm² 2 Treatment lasted 5 minutes. Treatment was administered every 2 days for 28 days (14 applications in total). The control group received an equal volume of physiological saline. All rats were euthanized after completing the relevant experiments. Soft and hard tissues of the left maxilla were removed, fixed with 4% paraformaldehyde for 24 hours, and the left maxilla was scanned using a mini-CT scanner at 90 kV and 0.04 mA. 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.

[0115] Figure 1 The image shows a scanning electron microscope (SEM) image of the multifunctional injectable hydrogel synthesized in Example 1. As can be seen from the image, the hydrogel has a porous structure and is capable of loading and releasing multifunctional nanosheets MP-Mg.

[0116] Figure 2 The image shows the transmission electron microscopy and elemental distribution of the multifunctional nanosheet MP-Mg synthesized in Example 1. As can be seen from the image, epigallocatechin gallate and magnesium ions have been successfully modified onto the nanosheet.

[0117] Figure 3 The rheological diagram shows the injectability and shear-thinning properties of the multifunctional injectable hydrogel synthesized in Example 1. As can be seen from the figure, the multifunctional injectable hydrogel synthesized in Example 1 has good injectability and can be injected to match the complex morphology of periodontal pockets.

[0118] Figure 4 The figures show the photothermal cycling ability of the multifunctional injectable hydrogel synthesized in Example 1 under near-infrared light (NIR) excitation (a), and the photothermal performance of the hydrogels synthesized in Example 1 and Comparative Example 3 under near-infrared light (NIR) excitation (b). As can be seen from the figures, the multifunctional injectable hydrogel prepared by the present invention has good and stable photothermal performance, and can be heated to above 45°C, effectively destroying the bacterial biofilm in the periodontal pocket and achieving effective sterilization.

[0119] Figure 5 This is a comparative graph showing the antibacterial properties of four hydrogels—Comparative Example 1 (Gel), Comparative Example 2 (GQ), Example 1 (GQM), and Example 1 (GQM+NIR) under near-infrared light excitation—against two common pathogenic bacteria found in different periodontal disease sites. a) Porphyromonas gingivalis; b) Fusobacterium nucleatum. As can be seen from the graph, the hydrogel synthesized in Example 1 under near-infrared light (NIR) excitation exhibits a more than three-order-of-magnitude improvement in antibacterial performance, achieving excellent bactericidal effects. Comparative Example 1 shows no antibacterial properties, while Comparative Example 2 exhibits weak antibacterial activity.

[0120] Figure 6 This is a comparative graph showing the antibacterial biofilm performance of four hydrogels—Comparative Example 1 (Gel), Comparative Example 2 (GQ), Example 1 (GQM), and Example 1 (GQM+NIR) under near-infrared light excitation—against two different bacterial biofilms. a) represents *Porphyromonas gingivalis*; b) represents *Fusobacterium nucleatum*. The graph shows that the hydrogel of Example 1 under near-infrared (NIR) excitation exhibits the lowest biofilm thickness and the highest density of dead bacteria (red area), demonstrating its superior antibacterial biofilm function. Comparative Example 1 shows no antibacterial performance, while Comparative Example 2 has weak antibacterial ability and no ability to break down bacterial biofilms.

[0121] Figure 7 The ROS scavenging performance of the injectable hydrogels synthesized in Example 1 and Comparative Example 3 is shown in Figure 1. Here, a represents the UV absorption spectrum of the mixture of DPPH and the injectable hydrogels synthesized in Example 1 and Comparative Example 3, and b represents the ROS scavenging rate calculated based on the absorption peak intensity of a at 517 nm. The calculation formula is: ROS scavenging rate = 100% * (absorbance A) 空白 -Absorbance A 样品 ) / Absorbance A 空白 As can be seen from the figure, the hydrogel of Example 1 can remove more than 60% of ROS and has the best anti-inflammatory properties.

[0122] Figure 8 Images of alveolar bone after treatment with hydrogels in four groups—Comparative Example 1 (Gel), Comparative Example 2 (GQ), Example 1 (GQM), and Example 1 (GQM+NIR) under near-infrared light excitation—are shown. Figure a shows a statistical graph of the distance between the cementoenamel junction (CEJ) and alveolar ridge (ABC) of the second molar in each group; Figure b shows Micro-CT images of each group. The images show that Example 1 (GQM+NIR) under near-infrared light excitation retained the most alveolar bone, effectively reducing alveolar bone loss and demonstrating effective treatment for periodontitis. Comparative Example 1 showed no antibacterial properties and the greatest alveolar bone loss; Comparative Example 2 had weak antibacterial ability but no anti-inflammatory ability, and also experienced significant alveolar bone loss.

[0123] Figure 9 Live / dead cell staining tests were performed on the hydrogels synthesized in Example 1 and Comparative Example 3. As can be seen from the figures, the hydrogels in both Example 1 and Comparative Example 3 exhibit good biocompatibility.

[0124] 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.

Claims

1. A method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis, characterized in that, Includes the following steps: (1) MXene dispersion was reacted with epigallocatechin gallate under dark conditions, followed by the addition of MgSO4 to continue the reaction; then, the mixture was subjected to sonication, centrifugation, washing and lyophilization to obtain multifunctional nanosheets MP-Mg. The ratio of MXene dispersion, epigallocatechin gallate, and MgSO4 is 1 mL: 30-60 mg: 30-60 mg. (2) The multifunctional nanosheets MP-Mg were added to an aqueous solution of oxidized gellan gum and mixed to obtain solution A; The concentration of the multifunctional nanosheets MP-Mg in solution A is 0.6-1.8 mg / mL; (3) Mix equal volumes of quaternary ammonium salt chitosan aqueous solution and carboxymethyl chitosan aqueous solution, and react to obtain solution B; (4) Mix equal volumes of solution A and solution B and react to obtain the injectable hydrogel for photothermal-assisted treatment of periodontitis; The concentration of the oxidized gellan gum aqueous solution is 3-6 wt%; The concentration of the quaternary ammonium salt chitosan aqueous solution is 0.5-3 wt%; The concentration of the carboxymethyl chitosan aqueous solution is 8-12 wt%.

2. The method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis according to claim 1, characterized in that, The MXene dispersion was obtained by dissolving MXene nanosheets in deionized water; the preparation process of the MXene nanosheets is as follows: The product was prepared by reacting LiF with Ti3AlC2 in hydrochloric acid solution, followed by centrifugation, washing, and freeze-drying.

3. The method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis according to claim 1, characterized in that, The oxidized gellan gum in the aqueous solution was prepared by the sodium periodate oxidation method.

4. The method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis according to claim 3, characterized in that, The preparation process of the oxidized gellan gum is as follows: Gellan gum was dissolved in water, sodium periodate was added, and the reaction was carried out in the dark. Then ethylene glycol was added and the reaction was continued with stirring. The mixture was then subjected to dialysis and freeze-drying in sequence.

5. The method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis according to claim 4, characterized in that, The ratio of gellan gum, sodium periodate, and ethylene glycol is 1-3 g: 0.4-1.0 g: 1-3 mL.

6. A method for preparing an injectable hydrogel for photothermal-assisted treatment of periodontitis according to claim 4, characterized in that, The dialysis conditions are as follows: the product is added to M... W Dialyze in a dialysis bag with a capacity of 3500 for 72 hours.

7. An injectable hydrogel for photothermal-assisted treatment of periodontitis, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.

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