Injectable adhesive hydrogel for the treatment of chronic periodontitis

Abstract

The application provides an injectable adhesive hydrogel for treating chronic periodontitis, and belongs to the technical field of medical materials. A preparation method of the injectable adhesive hydrogel comprises the following steps: adding Zn(NO3)2.6H2O aqueous solution drop by drop into a grape seed extract aqueous solution, flocculating and precipitating, collecting the precipitate, and obtaining grape seed extract-zinc coordination nanoparticles; dissolving methacrylic acid modified gelatin, oxidized hyaluronic acid and a photoinitiator in water to obtain a hydrogel precursor solution 1; adding the grape seed extract-zinc coordination nanoparticles and melittin into the hydrogel precursor solution 1 to obtain a hydrogel precursor solution 2; irradiating the hydrogel precursor solution 2 with blue light to obtain the injectable adhesive hydrogel. The hydrogel provided by the application has good mechanical and rheological properties, can be effectively adhered to various fresh moist tissues and materials, has good cell compatibility, has excellent antibacterial, anti-inflammatory and immune microenvironment regulating effects.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to an injectable adhesive hydrogel for the treatment of chronic periodontitis. Background Technology

[0002] Periodontitis is a prevalent, immune-inflammatory, and destructive disease. It not only causes gingival redness and swelling but also leads to decreased attachment of periodontal soft tissues, alveolar bone resorption, impaired masticatory function, and even tooth loss. Typically, the main pathogenic factor of periodontitis is the formation of subgingival plaque biofilm, which can trigger excessive pro-inflammatory immune responses and oxidative stress, ultimately resulting in paracrine signals of bone resorption. Currently, clinical periodontal treatments include scaling and root planing, antimicrobial photodynamic therapy, antibiotics, and laser therapy. Among these, antibiotic therapy (such as minocycline, areolatin, and amoxicillin) is widely used in the treatment of periodontitis. The purpose of antibiotic use is to eliminate pathogens, such as pathogenic bacteria, and to remove dental plaque biofilm to control infection and alleviate inflammation. However, overuse of antibiotics not only leads to bacterial resistance and increases the risk of systemic toxicity but also severely disrupts the balance of the oral microbiota. Oral microorganisms play a crucial role in maintaining the oral barrier, and dysbiosis of the oral microbiota promotes the development of oral and systemic diseases. Therefore, it is crucial to develop an innovative treatment strategy that does not rely on antibiotics and effectively avoids oral microbiome dysbiosis.

[0003] In recent years, various emerging tissue-engineered composites, such as hydrogels, nanofibers, nanozymes, and microneedle patches, have shown great potential for treating periodontitis due to their diverse biological functions. Among them, injectable hydrogels, with their good biocompatibility and bioavailability, are considered the most ideal scaffolds because they can be easily injected into periodontal pockets and facilitate controlled release of nanomedicine-assisted therapies. However, current research on the alteration and regulation of the suboral gingival microbiota by hydrogels used in periodontitis treatment lacks further investigation, which cannot guarantee that these innovative treatment strategies will not disrupt the richness and diversity of the normal oral microbiota. Summary of the Invention

[0004] Existing hydrogel strategies for treating periodontitis lack targeted therapy and repair of mitochondria in inflamed tissue cells, failing to achieve efficient treatment of inflamed tissues. Furthermore, existing periodontitis treatment strategies lack intervention and balancing of the abundance and diversity of the periodontal microenvironment microbiota, which may disrupt the oral microenvironment's immune system, posing a potential risk of relapse or other side effects. The purpose of this invention is to provide an injectable adhesive hydrogel for treating chronic periodontitis.

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

[0006] One of the technical solutions of this invention is a method for preparing an injectable adhesive hydrogel, comprising the following steps:

[0007] A Zn(NO3)2·6H2O aqueous solution was added dropwise to an aqueous solution of grape seed extract, causing flocculation and precipitation. The precipitate was collected to obtain grape seed extract-zinc coordination nanoparticles (abbreviated as GZN).

[0008] Methacrylic acid modified gelatin (GelMA), oxidized hyaluronic acid (OHA) and photoinitiator were dissolved in water to obtain hydrogel precursor solution 1;

[0009] Grape seed extract-zinc coordination nanoparticles and bee venom peptide were added to hydrogel precursor solution 1 to obtain hydrogel precursor solution 2.

[0010] The hydrogel precursor solution 2 was irradiated with blue light to obtain the injectable adhesive hydrogel (abbreviated as GM / OHA-GZN&M).

[0011] The second technical solution of the present invention is an injectable adhesive hydrogel prepared by the above preparation method.

[0012] The third technical solution of the present invention is the application of the above-mentioned injectable adhesive hydrogel in the preparation of a drug for treating chronic periodontitis.

[0013] The present invention discloses the following technical effects:

[0014] This invention provides a novel dual-network crosslinked hydrogel of GM / OHA-GZN&M, designed for the treatment of periodontitis. Utilizing Schiff base dynamic crosslinking and a rich hydrogen bond network, the GM / OHA-GZN&M hydrogel exhibits excellent mechanical (injectability, adhesion, and mobility) and rheological properties, enabling it to effectively adhere to various fresh, moist tissues and materials. In subsequent antibacterial experiments, the hydrogel showed significant inhibitory effects against Staphylococcus aureus (gram-positive bacteria), MRSA, Escherichia coli (gram-negative bacteria), and Porphyromonas gingivalis, while simultaneously disrupting bacterial biofilm formation by interfering with their normal metabolism. Furthermore, the GZN-loaded GM / OHA hydrogel exhibits significant abilities to scavenge reactive oxygen species (mROS), restore mitochondrial function, and inhibit apoptosis. It can also significantly inhibit the expression of inflammatory mediators and promote macrophage polarization towards the M2 phenotype. In vitro cell experiments demonstrated that GM / OHA-GZN&M exhibited excellent cell compatibility, promoting cell proliferation, migration, and osteogenic activity. Finally, in a rat model of periodontitis, the combination of bee venom peptides and GZN hydrogel showed more significant effects in promoting alveolar bone regeneration and regulating the immune microenvironment, effectively inhibiting the overgrowth of harmful bacteria (Porphyromonas and Bacteroides) and alleviating dysbiosis in the periodontal pocket microenvironment. In conclusion, our research provides a feasible and promising therapeutic strategy for periodontitis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 SEM image A and TEM image B of the grape seed extract-zinc coordination nanoparticles in Example 1.

[0017] Figure 2 XPS image A, XRD image B, and FTIR spectrum C of grape seed extract-zinc coordination nanoparticles in Example 1.

[0018] Figure 3 For GelMA in Example 1 1 H nuclear magnetic resonance spectroscopy.

[0019] Figure 4 For OHA in Example 1 1H nuclear magnetic resonance spectroscopy.

[0020] Figure 5 Figure A shows the formation of GM / OHA-GZN&M hydrogel, Figure B shows its plasticity, Figure C shows its injectability, and Figure D shows its adhesiveness.

[0021] Figure 6 This is a SEM image of the GM / OHA-GZN&M hydrogel in Example 1.

[0022] Figure 7 The image shows the adhesion effect of the GM / OHA-GZN&M hydrogel in Example 1 on different organs (liver, spleen, heart, kidney, lung) and materials (metal, rubber, plastic, wood, glass).

[0023] Figure 8 The results are the pigskin overlap shear test A and adhesion strength B of the GM / OHA-GZN&M hydrogel in Example 1.

[0024] Figure 9 These are agar plate colonies of different hydrogels from Example 1.

[0025] Figure 10 In Figures A and B, the survival rates of Escherichia coli, Porphyromonas gingivalis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus were observed under different hydrogels.

[0026] Figure 11 The absorbance A and DPPH scavenging rate B of different gels after the DPPH scavenging test in Example 1, and the absorbance C and DPPH scavenging rate D of different gels after the PTIO scavenging test.

[0027] Figure 12 The figures show the expression of pro-inflammatory cytokines in the Raw264.7 cell inflammation model using different gels in Example 1. A represents TNF-α, B represents IL-6, C represents IL-1β, D represents MCP-1, E represents INF-γ, and F represents NO.

[0028] Figure 13 In Example 1, the M1 / M2 polarization rate of macrophages was detected by immunofluorescence staining (A) and flow cytometry (B) using different gels.

[0029] Figure 14 This is an in vitro mitochondrial targeting verification of the hydrogel in Example 1, where A is the fluorescence intensity of the mitochondrial suspension and B is the subcellular localization.

[0030] Figure 15 The fluorescence intensity (A) of MMP and ATP in RAW264.7 cells was evaluated using the JC-1 staining kit, the relative fluorescence intensity (B) of red / green fluorescence, and the relative ATP expression (C).

[0031] Figure 16 The compatibility of different hydrogels with MC3T3-E1 and RAW264.7 cells was evaluated. In this study, A represents MC3T3-E1 and B represents RAW264.7.

[0032] Figure 17 Micro-CT analysis of maxilla with teeth after different hydrogel treatments A, CEJ-ABC relative fold B.

[0033] Figure 18 H&E staining and Masson staining of maxilla with teeth after different hydrogel treatments (A), relative inflammatory infiltration area (B), and corresponding collagen deposition area (C).

[0034] Figure 19 The abundance and α-diversity index of oral microbiota in the periodontitis group and subgingival microbiota after treatment with GM / OHA-GZN&M hydrogel. Detailed Implementation

[0035] 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.

[0036] 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. Any stated value or intermediate value within a stated range, as well as each smaller range between 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The main component and active substance of bee venom is melitin. As a natural insect antimicrobial cationic peptide, melitin has broad antibacterial activity. It can target peptidoglycans or lipopolysaccharides in bacterial cell walls through electrostatic attraction, disrupting the bacterial membrane and leading to bacterial death. In addition, it also has certain anti-inflammatory and analgesic effects. Recently, although melitin has been applied in biomedical engineering fields such as bacterial infection wound models and has shown good performance, its application in the treatment of periodontitis is rare. Grape seed extract (GSE) is a polyphenolic substance extracted and isolated from grape seeds, mainly composed of proanthocyanidins, catechins, epicatechins, gallic acid, gallic acid esters, and other polyphenolic substances. Previous reports indicate that polyphenols rich in phenolic hydroxyl groups can effectively bind to mitochondrial outer membrane proteins through various non-covalent interactions (including electrostatic attraction, hydrophobic interactions, hydrogen bonds, etc.). This binding helps to clear excess mROS, restore mitochondrial function, and inhibit inflammatory pathways including NLRP3 / caspase-1. Studies have shown that grape seed extract can significantly improve colonic damage and gut microbiota imbalance by regulating lipid metabolism, suggesting that it may have the potential to regulate oral microbiota.

[0041] Based on the above, this invention prepares a polysaccharide hydrogel with good mechanical properties by mixing modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) aqueous solutions in a specific ratio under specific conditions. This hydrogel exhibits good injectability and adhesion. Subsequently, polyphenol-based nanoparticles (grape seed extract@zinc) with mitochondrial targeting function and melittin, a bee venom peptide with broad-spectrum antibacterial activity, are loaded into the hydrogel, resulting in good antioxidant, antibacterial, and immunomodulatory effects. The polyphenols effectively mediate the targeted clearance of mitochondrial ROS (mROS), restoring mitochondrial function and disrupting the inflammatory free radical cycle. Furthermore, in an in vivo rat periodontitis model, the GM / OHA-GZN&M hydrogel effectively inhibits inflammation and promotes alveolar bone regeneration through immunomodulation. It also inhibits the overgrowth of pathogenic bacteria and alleviates oral flora imbalance in rats. In conclusion, this invention provides a promising treatment strategy for the clinical treatment of periodontitis.

[0042] The first aspect of this invention provides a method for preparing an injectable adhesive hydrogel, comprising the following steps:

[0043] A Zn(NO3)2·6H2O aqueous solution was added dropwise to a grape seed extract aqueous solution, and flocculation and precipitation were carried out. The precipitate was collected to obtain grape seed extract-zinc coordination nanoparticles.

[0044] Methacrylic acid-modified gelatin, oxidized hyaluronic acid, and a photoinitiator were dissolved in water to obtain hydrogel precursor solution 1;

[0045] Grape seed extract-zinc coordination nanoparticles and bee venom peptide were added to hydrogel precursor solution 1 to obtain hydrogel precursor solution 2.

[0046] The hydrogel precursor solution 2 was irradiated with blue light to obtain the injectable adhesive hydrogel.

[0047] In some embodiments of the present invention, the concentration of the Zn(NO3)2·6H2O aqueous solution is 1-5% w / v; the concentration of the grape seed extract aqueous solution is 1-3% w / v; the pH value of the grape seed extract aqueous solution is 9.0; and the volume ratio of the Zn(NO3)2·6H2O aqueous solution to the grape seed extract aqueous solution is 1:1. The present invention does not impose any special limitations on the source of the grape seed extract, which can be purchased or prepared using methods well known to those skilled in the art. Because grape seed extract has limited water solubility and cannot be completely dissolved at excessively high concentrations, the concentration of the grape seed extract aqueous solution in the present invention is 1-3% w / v.

[0048] In some embodiments of the present invention, after collecting the precipitate, the method further includes washing and freeze-drying the precipitate.

[0049] In some embodiments of the present invention, the method for preparing the methacrylic acid modified gelatin is as follows: methacrylic acid is added to a gelatin aqueous solution and reacted. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain the methacrylic acid modified gelatin. The concentration of the gelatin aqueous solution is 2-10% w / v. The volume ratio of the gelatin aqueous solution to methacrylic acid is 100:6.

[0050] In some embodiments of the present invention, the addition rate of the methacrylic acid is 1 mL / min; the reaction temperature is 50 °C and the reaction time is 1.5 h.

[0051] In some embodiments of the present invention, the method for preparing the oxidized hyaluronic acid is as follows: sodium periodate solution is added dropwise to an aqueous hyaluronic acid solution, and the reaction is carried out under dark conditions. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain the oxidized hyaluronic acid; the concentration of the aqueous hyaluronic acid solution is 2-10% w / v; the concentration of the sodium periodate solution is 0.5M; and the volume ratio of the aqueous hyaluronic acid solution to the sodium periodate solution is 100:5.

[0052] In some embodiments of the present invention, the reaction time under dark conditions is 2 hours.

[0053] In some embodiments of the present invention, the photoinitiator is LAP; in the hydrogel precursor solution 1, the concentrations of methacrylic acid modified gelatin, oxidized hyaluronic acid and photoinitiator are 5-10% w / v, 1-2% w / v and 0.1% w / v, respectively.

[0054] In some embodiments of the present invention, the concentrations of grape seed extract-zinc coordination nanoparticles and melitoxin in the hydrogel precursor solution 2 are 1 mg / mL and 50 mg / mL, respectively.

[0055] In some embodiments of the present invention, the parameters of the blue light irradiation are set as follows: wavelength 405nm, power 5W, and time 1min.

[0056] A second aspect of the present invention provides an injectable adhesive hydrogel prepared by the above-described preparation method.

[0057] A third aspect of the present invention provides the use of the above-mentioned injectable adhesive hydrogel in the preparation of a drug for treating chronic periodontitis.

[0058] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0059] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0060] Example 1

[0061] This embodiment provides an injectable adhesive hydrogel, and the preparation steps are as follows:

[0062] Step 1. Preparation of Grape Seed Extract-Zinc Coordination Nanoparticles (GZN): Grape seed extract was dissolved in pure water to a concentration of 2% w / v, preparing 50 mL of solution, and the pH was adjusted to 9.0. Then, 50 mL of 5% w / v Zn(NO3)2·6H2O aqueous solution was added dropwise to the above solution to induce flocculation and precipitation. After slow stirring for 15 minutes, the precipitate was collected by centrifugation, washed three times with pure water, and freeze-dried to obtain GZN.

[0063] Step 2. Synthesis of methacrylic acid modified gelatin (GelMA): Gelatin was dissolved in pure water at a concentration of 10% w / v to prepare 100 mL of solution. Then, 6 mL of methacrylic acid (MA) was gradually added at a rate of 1 mL / min. The reaction was carried out at 50 °C for 1.5 hours. The reaction was terminated after adding 200 mL of deionized water. After dialyzing, GelMA was obtained by freeze-drying.

[0064] Step 3. Synthesis of Oxidized Hyaluronic Acid (OHA): Hyaluronic acid (HA) was dissolved in pure water to a concentration of 2% w / v, and 100 mL of solution was prepared. Then, 5 mL of 0.5 M sodium periodate solution was added dropwise. After reacting in the dark for 2 hours, ethylene glycol was added to stop the reaction. OHA was obtained by dialysis and freeze-drying.

[0065] Step 4. Preparation of hydrogel:

[0066] GM / OHA hydrogel: First, GelMA, OHA, and photoinitiator (LAP) were dissolved in pure water to obtain hydrogel precursor solution 1, wherein the concentrations of GelMA, OHA, and photoinitiator (LAP) were 10% w / v, 2% w / v, and 0.1% w / v, respectively. Then, the GM / OHA hydrogel was prepared by irradiating hydrogel precursor solution 1 with blue light (wavelength 405 nm, power 5 W) for 1 minute.

[0067] GM / OHA-GZN hydrogel: GZN (1 mg / mL) was added to hydrogel precursor solution 1, and then the GM / OHA-GZN hydrogel was prepared by irradiating with blue light (wavelength 405 nm, power 5 W) for 1 minute.

[0068] GM / OHA-M hydrogel: Bee venom peptide (50 mg / mL) was added to hydrogel precursor solution 1, and then the GM / OHA-M hydrogel was prepared by irradiation with blue light (wavelength 405 nm, power 5 W) for 1 minute.

[0069] GM / OHA-GZN&M hydrogel: GZN (1 mg / mL) and melitoxin (50 mg / mL) were added to hydrogel precursor solution 1, and then the GM / OHA-GZN&M hydrogel was prepared by irradiating with blue light (wavelength 405 nm, power 5 W) for 1 minute.

[0070] 1. Characterization and Analysis:

[0071] 1.1 The successfully synthesized grape seed extract-zinc coordination nanoparticles (GZN) were characterized. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the GZN exhibited a spherical nanoparticle morphology (e.g., ...). Figure 1 (As shown in A and B). Figure 2 X-ray photoelectron spectroscopy (XPS) of sample A showed the presence of dual-mode peaks in Zn₂P (1020-1050 eV), confirming the successful synthesis of GZN. Figure 2In the B spectrum, a characteristic peak of GSE was observed, located at 2θ = 20°–30°, with a lower intensity than that of GSE in the X-ray diffraction (XRD) spectrum. This intensity reduction may be due to Zn 2+ The chelation with GSE reduces the exposure of phenolic hydroxyl groups in GSE. (See FTIR spectrum). Figure 2 As shown in C), the characteristic absorption peak of the GA benzene ring is as follows, such as at 1610 cm⁻¹. -1 1520cm -1 Other features, such as [missing information], were also observed in the GZN spectrum. Furthermore, the 755-870 cm⁻¹ range also appeared in the GZN spectrum. -1 The additional peaks within the range can be attributed to the CH vibration on the benzene ring. These findings support the association between GSE phenolic groups and Zn. 2+ Effective coordination between them.

[0072] 1.2 in 1 H nuclear magnetic resonance spectroscopy ( Figure 3 and Figure 4 As can be seen in the diagram, gelatin was modified by grafting methacrylate groups onto the C=C bonds, while the sugar ring of HA was opened by treatment with sodium peroxide, forming a linear chain ending with a dialdehyde. These results demonstrate the successful synthesis of GelMA and OHA.

[0073] 1.3 From Figure 5 As observed in Figure A, when 10% GelMA and 2% OHA were mixed in a pot, the hydrogel precursor solution became viscous, indicating the formation of Schiff base bonds (the first layer of cross-linking network) between the aldehyde groups on OHA and the amino groups on GelMA. Subsequently, after irradiation with blue light (405 nm) for 1 minute, a second layer of cross-linking network was established via free radical polymerization. Then, grape seed extract-zinc coordination nanoparticles (GZN) (1 mg / mL) and bee venom (50 mg / mL) were added to the above system to form a GM / OHA-GZN&M hydrogel. Figure 5 As shown in Figure B, the GM / OHA-GZN&M hydrogel exhibits good migration properties and can form various shapes. Furthermore, it displays shear-thinning behavior, facilitating injection via a syringe needle. Figure 5 (C). Furthermore, due to its flexible dynamic cross-linking and abundant hydrogen bond structure... Figure 5 (D) The GM / OHA-GZN&M hydrogel adhered firmly to the finger. To further investigate the morphology and structural mechanism of the GM / OHA-GZN&M hydrogel, we performed SEM, SEM-eds, and XPS analyses. SEM images show ( Figure 6 As the content of OHA, GZN and melittin increases, the porous structure becomes more compact and regular.

[0074] 2. Mechanical property testing of hydrogels

[0075] Cut the pigskin into rectangular strips 30 mm long and 10 mm wide. Apply 100 μL of hydrogel to both ends of two strips of skin, then overlap them to form a 10 × 10 mm strip. 2 The lap joints were sheared and allowed to stabilize for 1 hour. Adhesion strength was measured using an electronic tensile testing machine (Zwick Z250), calculated by dividing the maximum load by the overlap area. Stress-strain curves and compressive strength at 50% strain were also obtained at a crosshead speed of 2 mm / min. At least three specimens were tested for each sample, and the average value was taken.

[0076] Results analysis: such as Figure 7 As shown, the hydrogel of the GM / OHA-GZN&M sample exhibited good adhesion properties, demonstrating excellent adhesion to various organs (liver, spleen, heart, kidney, lung) and materials (metal, rubber, plastic, wood, glass). Furthermore, in the pigskin overlap shear test, with increasing content of OHA, GZN&M, and bee venom, the hydrogel showed higher adhesion than commercially available fibrin glue. Figure 8 The adhesion strength of A and B was 4.99 ± 0.37 kPa. This good adhesion is mainly attributed to the abundant carboxyl, amino, and hydroxyl functional groups in GM, OHA, GZN, and Melittin. These functional groups endow the hydrogel with the ability to adhere to different substrates through hydrogen bonding, π-π stacking interactions, and electrostatic interactions.

[0077] 3. In vitro antibacterial activity test of hydrogel

[0078] The in vitro antibacterial activity of CFE hydrogel was evaluated using *Escherichia coli* (ATCC 8739) and *Porphyromonas gingivalis* (ATCC 33277) as representative Gram-negative bacteria, and *Staphylococcus aureus* (ATCC 6538) and methicillin-resistant *Staphylococcus aureus* (MRSA) (ATCC 43300) as representative Gram-positive bacteria. The test samples were divided into five groups (PBS as the negative control group, and GM / OHA, GM / OHA-GZN, GM / OHA-M, and GM / OHA-GZN&M as the experimental groups). Before the experiment, equal masses of hydrogel (1 g) in 24-well plates were sterilized under UV irradiation for 4 h and washed with PBS. The diluted bacterial suspension (10 μL, 10...) was then... 6 CFU mL -1The bacterial suspension was evenly added to the surface of the hydrogel. After incubation at 37°C for 4 hours, 1 mL of PBS was added to the bacterial suspension to dilute it 100 times. 10 μL of the bacterial suspension was spread onto an LB agar plate. Colony growth was observed after 12 hours.

[0079] Results analysis: Figure 9 The results showed that, compared with the PBS group, the GM / OHA hydrogel group loaded with melitoxin had a significantly reduced number of colonies on agar plates. Figure 10 Figures A and B show that the GM / OHA-GZN&M group had the lowest bacterial survival rates, with Staphylococcus aureus survival rate of 0.96±0.25%, methicillin-resistant Staphylococcus aureus survival rate of 1.26±0.29%, Escherichia coli survival rate of 0.71±0.20%, and Porphyromonas gingivalis survival rate of 0.92±0.19%. This indicates that the hydrogel loaded with melitoxin and grape seed extract-zinc coordination nanoparticles has good antibacterial activity.

[0080] 4. In vitro antioxidant activity test of hydrogel:

[0081] The in vitro antioxidant activity of the hydrogels was evaluated using DPPH· and PTIO· scavenging assays. For the DPPH· scavenging assay, freeze-dried hydrogels of different components were ground into powder. 30 mg of each component of the freeze-dried hydrogel powder was treated with DPPH·ethanol solution (3 mL, 100 μM) and incubated in the dark for 30 min. After centrifugation at 3000 rpm for 10 min, the absorbance of the supernatant at 517 nm was measured using a microplate reader. Similarly, in the PTIO· scavenging assay, 30 mg of freeze-dried hydrogel was added to PTIO· solution (3 mL, 100 μM) and incubated in the dark for 60 min. The absorbance of the supernatant at 557 nm was recorded. PBS (300 μL) served as the negative control for both experiments.

[0082] Results analysis: such as Figure 11 As shown in the results from AD, the DPPH (PTIO) scavenging rate was significantly improved in both the GM / OHA-GZN and GM / OHA-GZN&M groups. Specifically, the DPPH scavenging rate in the GM / OHA-GZN&M group reached 84.39±0.77%, and the PTIO scavenging rate was 85.72±3.14%. This indicates that the GZN-loaded hydrogels possess good antioxidant activity.

[0083] 5. In vitro anti-inflammatory activity test of hydrogel:

[0084] RAW 264.7 cells (1×10⁻⁶) 6Macrophages were co-cultured in hydrogel extract (30 μL) and LPS (100 ng / mL) for 24 hours. The levels of inflammatory factors (IL-6, IL-1β, MCP-1, TNF-α, INF-γ) in the culture supernatant were measured using an ELISA kit. Furthermore, the expression and quantity of CD86 and CD206 were detected by immunofluorescence staining and flow cytometry. For flow cytometry, macrophages polarized to M1 / M2 were quantified using APC anti-mouse CD86 (CD206) antibody. For immunofluorescence staining, macrophages were fixed in 4% paraformaldehyde, permeated with 0.5% Tween, blocked with 10% BSA, incubated with antibody, and observed by confocal microscopy. Quantitative analysis was performed using ImageJ. All experiments were performed three times, and the average value was taken.

[0085] Results Analysis: First, the expression of pro-inflammatory cytokines, including interferon-γ (INF-γ), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and NO release, was investigated in a Raw264.7 cell inflammation model. Figure 12 As shown in the AF, compared with the GM / OHA group, the pro-inflammatory cytokines in the GZN-loaded GM / OHA group were significantly reduced, with a decrease ranging from 40% to 60%. Specifically, the relative expression levels of TNF-α, IL-6, IL-1β, MCP-1, INF-γ, and NO in the GM / OHA-GZN&M group were 34.40±1.38%, 38.74±2.08%, 35.19±4.38%, 34.95±3.50%, 39.43±1.78%, and 25.82±1.54%, respectively. Furthermore, the M1 / M2 polarization rate of macrophages was detected by immunofluorescence staining and flow cytometry. Figure 13 As shown in Figures A and B, the positive levels of M1 macrophages (CD86-labeled) in the GM / OHA-GZN and GM / OHA-GZN&M groups were significantly lower than those in other groups, while the positive levels of M2 macrophages (CD206-labeled) were significantly higher than those in other groups. These experiments demonstrate that the GZN-loaded GM / OHA hydrogel not only effectively scavenges reactive oxygen species (ROS) and restores mitochondrial function, but also possesses significant immunomodulatory properties, inhibiting the expression of pro-inflammatory cytokines and promoting the increase of M2 phenotype macrophages.

[0086] 6. In vitro mitochondrial targeting validation of hydrogels

[0087] Cultured cells were collected, mechanically homogenized, and centrifuged twice to separate mitochondria and other cellular components. The separated mitochondria were then incubated with Cy5.5-labeled GZN nanoparticles at 37°C for 1 hour, followed by washing three times with PBS to remove unbound particles. The fluorescence intensity of the mitochondrial suspension was then measured using flow cytometry. To analyze nanoparticle internalization, RAW264.7 cells were incubated with Cy5.5-labeled GZN, and images were taken using confocal microscopy at different time points. For subcellular localization analysis, cells were seeded in 12-well plates and co-cultured with GZN for 4 hours. Cells were then stained with ER-tracker, Mito-tracker, Golgi-tracker, Lyso-tracker, and Hoechst 33342 according to the manufacturer's instructions. Fluorescence was captured using laser confocal microscopy, and colocalization values ​​were determined from the images.

[0088] Results analysis: such as Figure 14 As shown in Figure A, with the increase of GZN incubation amount, the signal of Cy5.5 in the extracted mitochondria became more obvious, indicating a strong binding affinity between GZN and mitochondria. Subcellular localization experiments were then performed on RAW264.7 cells (see Figure A). Figure 14 The results showed that the fluorescence signal of cy5.5-labeled GZN significantly overlapped with that of mitochondria, and the Pearson correlation coefficient (R) was 0.77, exceeding that of other organelles.

[0089] 7. Detection of mitochondrial membrane potential (MMP) and ATP content

[0090] MMPs of RAW264.7 cells were evaluated using the JC-1 staining kit. Cells were pretreated overnight with a specific drug (hydrogel extract (30 μL)) and then incubated with 1000 μM H2O2 for 2 h. They were then stained with JC-1 dye for 20 min, washed twice with JC-1 buffer, and fluorescence was observed under an inverted fluorescence microscope. RAW264.7 cells were cultured in 6-well plates and subjected to different treatments (pretreatment with hydrogel extract (30 μL) overnight followed by incubation with 1000 μM H2O2 for 2 h). Cells were then lysed according to the reagent manufacturer's instructions. Lysis buffer was added to each well, and the precipitate was collected by centrifugation. 100 μM ATP was added to the working solution, and the cells were incubated at 37 °C for 5 min. Fluorescence intensity was measured using a chemiluminescence analyzer.

[0091] Results analysis: Excessive accumulation of ROS disrupts the mitochondrial membrane potential, leading to mitochondrial depolarization and subsequent functional impairment. For example... Figure 15As shown in Figure A, the red-green fluorescence ratio of the H2O2 group was significantly reduced, indicating mitochondrial membrane depolarization. However, both the GM / OHA-GZN and GM / OHA-GZN&M groups showed better improvement, with significantly reduced red-green fluorescence ratios in both groups. Quantification of red-green fluorescence ratios for GM / OHA-GZN and GM / OHA-GZN&M (…) Figure 15 The concentrations in groups B and B were 10.28 times and 13.06 times that of H2O2, respectively. Considering that ATP production depends on mitochondrial membrane depolarization generated by the proton pump, the ATP production capacity of each hydrogel group was further examined. Figure 15 In the C, GM / OHA-GZN and GM / OHA-GZN&M groups, the levels recovered to 56.33% and 65.01% of the control group, respectively, indicating that GZN has a protective effect and can maintain mitochondrial ATP production function.

[0092] 8. Cell compatibility evaluation

[0093] 1 mL of hydrogel was immersed in 5 mL of DMEM medium and incubated for 24 hours. The extract was then collected to obtain the hydrogel extract DMEM solution. MC3T3-E1 and RAW264.7 cells were cultured at 4 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and co-cultured in DMEM for 12 hours. Different hydrogel extract DMEM solutions were added to the wells, and cell viability was calculated using the CCK-8 assay according to the manufacturer's instructions. Control cells were cultured separately in DMEM.

[0094] Results Analysis: Figure 16 It was found that all MC3T3-E1 and RAW264.7 cells exhibited excellent viability, with cell viability exceeding 80% after 5 days of co-culturing with the hydrogel extract alone. Notably, the GM / OHA-GZN and GM / OHA-GZN&M groups showed higher cell viability compared to the control, GM / OHA, and GM / OHA-M groups. In MC3T3-E1 cells, the GM / OHA-GZN group (308.52±5.21%) and the GM / OHA-GZN&M group (310.45±7.32%) showed higher cell viability than the control group (223.02±6.54%), the GM / OHA group (266.56±8.56%), and the GM / OHA-M group (274.52±6.25%).

[0095] 9. Animal experiments

[0096] Eighteen SD rats (6-8 weeks old) were randomly assigned to six groups (n=3 per group): healthy rats (healthy group), periodontitis + PBS (PBS group), periodontitis + GM / OHA (GM / OHA group), periodontitis + GM / OHA-M (GM / OHA-M group), periodontitis + GM / OHA-GZN (GM / OHA-GZN group), and periodontitis + GM / OHA-GZN&M (GM / OHA-GZN&M group). *Porphyromonas gingivalis* strain (10⁹ CFU / mL, 500 μL) was administered every other day after ligating the maxillary second molar (M2) with silk sutures. After a total of 14 days, 200 μL of PBS, GM / OHA, GM / OHA-M, GM / OHA-GZN, or GM / OHA-GZN&M solution was injected into the periodontal pocket every three days. Three weeks later, maxillary bone with teeth was collected for analysis. The samples underwent micro-computed tomography (Micro-CT) analysis, H&E staining, Masson staining, immunofluorescence staining, and immunohistochemical staining.

[0097] Results analysis: Compared to the periodontitis group, the hydrogel treatment group showed less alveolar bone and a shorter periodontal ligament-cementum distance. Furthermore, it was observed that the efficacy of hydrogel treatment groups containing only melitoxin or GZN was relatively inferior to those containing both ingredients. The CEJ-ABC distance in the GM / OHA-GZN&M group was only 1.18 times that of the healthy group, lower than that in the periodontitis group (2.43 times), the GM / OHA group (2.20 times), the GM / OHA-M group (1.92 times), and the GM / OHA-GZN group (1.65 times).

[0098] like Figure 17 As shown in Figures A and B, alveolar bone recovery after treatment with GM / OHA-GZN&M hydrogel (indicated by the yellow dashed line) showed a more significant improvement, with a recovery level comparable to that of healthy alveolar bone. Furthermore, both the GM / OHA-GZN and GM / OHA-GZN&M groups showed good restoration of junctional epithelial connections, a significant reduction in inflammatory cell infiltration (indicated by the black arrows), and the periodontal ligament (PDL) tending towards normal alignment. Corresponding quantitative values ​​( Figure 18 The trends in staining images (A and B) were consistent with those in the control group. Compared to the control group, the GM / OHA-GZN&M group (29.67 ± 3.30%) showed a significant decrease in inflammatory cell infiltration. Masson staining ( Figure 18Figures A and C show that the relative collagen density of the GM / OHA-GZN&M group (87.67±3.68%) was significantly higher than that of the periodontitis group (27.33±2.05%), the GM / OHA group (46.67±4.11%), the GM / OHA-m group (58.33±1.25%), and the GM / OHA-gzn group (70.1±5.26%). These results demonstrate that the combined use of bee venom and GZN has a synergistic effect in promoting alveolar bone regeneration and regulating the immune microenvironment in the treatment of periodontitis in rats.

[0099] 10. Microbial Analysis

[0100] Oral swab samples were collected from periodontal pockets, carefully collected, and frozen with dry ice before 16S rRNA sequencing. Following DNA extraction, PCR amplification, and product purification, a 16S rRNA library was constructed using specific microbiome-specific primers and processed using the Illumina NovaSeq sequencing platform. Bioinformatics analysis was performed using OECloud tools (https: / / cloud.oebiotech.com). Specifically, the abundance and alpha diversity index of observed species were calculated and analyzed based on species richness and evenness.

[0101] Results analysis: such as Figure 19 As shown, the oral microbiota diversity in the periodontitis group was significantly reduced, indicating an imbalance in the microbial ecosystem. However, after GM / OHA-GZN&M hydrogel treatment, the abundance and α-diversity of the subgingival microbiota were substantially improved.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of an injectable adhesive hydrogel in the preparation of a drug for treating chronic periodontitis, characterized in that, The method for preparing the injectable adhesive hydrogel includes the following steps: A Zn(NO3)2·6H2O aqueous solution was added dropwise to a grape seed extract aqueous solution, causing flocculation and precipitation. The precipitate was collected to obtain grape seed extract-zinc coordination nanoparticles. Methacrylic acid-modified gelatin, oxidized hyaluronic acid, and a photoinitiator were dissolved in water to obtain hydrogel precursor solution 1; Grape seed extract-zinc coordination nanoparticles and bee venom peptide were added to hydrogel precursor solution 1 to obtain hydrogel precursor solution 2. The hydrogel precursor solution 2 was irradiated with blue light to obtain the injectable adhesive hydrogel; The concentration of the Zn(NO3)2·6H2O aqueous solution is 1-5% w / v; the concentration of the grape seed extract aqueous solution is 1-3% w / v; the pH value of the grape seed extract aqueous solution is 9.0; the volume ratio of the Zn(NO3)2·6H2O aqueous solution to the grape seed extract aqueous solution is 1:

1. In the hydrogel precursor solution 1, the concentrations of methacrylic acid modified gelatin, oxidized hyaluronic acid, and photoinitiator are 5-10% w / v, 1-2% w / v, and 0.1% w / v, respectively.

2. The application according to claim 1, characterized in that, The method for preparing the methacrylic acid modified gelatin is as follows: methacrylic acid is added to a gelatin aqueous solution and reacted. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain the methacrylic acid modified gelatin. The concentration of the gelatin aqueous solution is 2-10% w / v. The volume ratio of the gelatin aqueous solution to methacrylic acid is 100:

6.

3. The application according to claim 1, characterized in that, The method for preparing the oxidized hyaluronic acid is as follows: sodium periodate solution is added dropwise to an aqueous hyaluronic acid solution, and the reaction is carried out under dark conditions. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain the oxidized hyaluronic acid. The concentration of the aqueous hyaluronic acid solution is 2-10% w / v; the concentration of the sodium periodate solution is 0.5M; and the volume ratio of the aqueous hyaluronic acid solution to the sodium periodate solution is 100:

5.

4. The application according to claim 1, characterized in that, The photoinitiator is LAP.

5. The application according to claim 1, characterized in that, In the hydrogel precursor solution 2, the concentrations of grape seed extract-zinc coordination nanoparticles and melitin peptides are 1 mg / mL and 50 mg / mL, respectively.

6. The application according to claim 1, characterized in that, The parameters for the blue light irradiation were set as follows: wavelength 405nm, power 5W, and time 1min.

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