Injectable adhesive hydrogel for treating chronic periodontitis

Abstract

The invention provides injectable adhesive hydrogel for treating chronic periodontitis, and belongs to the technical field of medical materials. The preparation method of the injectable adhesive hydrogel comprises the following steps: dropwise adding a Zn (NO3) 2.6 H2O aqueous solution into a grape seed extract aqueous solution, carrying out flocculent precipitation, and collecting the precipitate to obtain grape seed extract-zinc coordination nanoparticles; the preparation method comprises the following steps: dissolving methacrylic acid modified gelatin, oxidized hyaluronic acid and a photoinitiator in water to obtain a hydrogel precursor solution 1; adding grape seed extract-zinc coordination nanoparticles and melittin into the hydrogel precursor solution 1 to obtain a hydrogel precursor solution 2; and irradiating the hydrogel precursor solution 2 with blue light to obtain the injectable adhesive hydrogel. The hydrogel provided by the invention has good mechanical and rheological properties, can be effectively adhered to various fresh and moist tissues and materials, and has good cell compatibility. The excellent effects of resisting bacteria, diminishing inflammation and regulating and controlling the immune microenvironment are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of medical materials, and in particular to an injectable adhesive hydrogel for treating chronic periodontitis. Background Art

[0002] Periodontitis is a ubiquitous immune-inflammatory destructive disease. The presence of periodontitis not only causes gingival redness and swelling, but also leads to decreased attachment of periodontal soft tissues, alveolar bone resorption, impaired chewing function, and even tooth loss. Generally speaking, the main pathogenic factor of periodontitis is the formation of subgingival plaque biofilm, which can cause excessive proinflammatory immune response and oxidative stress, and ultimately lead to paracrine signals of bone resorption. At present, the periodontal treatment measures used in clinical practice include scaling and root planing, antibacterial photodynamic therapy, antibiotics and laser. Among them, drug therapy with antibiotics (such as minocycline, aristotelin, and amoxicillin) has been widely used in the treatment of periodontitis. The purpose of antibiotic use is to eliminate pathogens, such as pathogenic bacteria, and eliminate dental plaque biofilm to control infection and relieve inflammation. However, excessive use of antibiotics not only causes bacterial resistance and increases the risk of systemic toxicity, but also seriously disrupts the balance of oral microbiota. Microorganisms in the mouth play an important 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 to effectively avoid oral microbiota dysbiosis.

[0003] In recent years, various emerging tissue engineering composites, such as hydrogels, nanofibers, nanozymes, and microneedle patches, have shown great potential for the treatment of periodontitis due to their multiple biological functions. Among them, injectable hydrogels with good biocompatibility and bioavailability are considered to be the most ideal scaffolds because of their ability to be easily injected into the periodontal pocket and promote the controlled release of nanomedicine-assisted therapies. However, there is a lack of further research on the changes and regulation of the oral subgingival microbial community by hydrogels currently used for periodontitis treatment, which cannot guarantee that these innovative treatment strategies will not disrupt the richness and diversity of the normal oral microbial community. Summary of the invention

[0004] The existing hydrogel strategies for the treatment of periodontitis lack targeted treatment and repair of mitochondria in tissue cells at the site of inflammation, and cannot achieve efficient treatment of inflamed tissues. At the same time, the existing strategies for the treatment of periodontitis lack intervention and balance in the abundance and diversity of the periodontal microenvironment microbiota, which may damage the immune system of the oral microenvironment and have the potential risk of recurrence or other side effects. The present invention aims to provide an injectable adhesive hydrogel for the treatment of chronic periodontitis.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0007] The Zn(NO3)2·6H2O aqueous solution is added dropwise into the grape seed extract aqueous solution to flocculate and precipitate, and the precipitate is collected to obtain grape seed extract-zinc coordination nanoparticles (abbreviated as GZN);

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

[0009] Adding grape seed extract-zinc coordinated nanoparticles and bee venom peptide to the hydrogel precursor solution 1 to obtain the hydrogel precursor solution 2;

[0010] The hydrogel precursor solution 2 is 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] A third technical solution of the present invention is a use 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] The present invention provides a novel double-network cross-linked hydrogel of GM / OHA-GZN&M, which is designed for the treatment of periodontitis. With Schiff base dynamic cross-linking and rich hydrogen bond network, GM / OHA-GZN&M hydrogel has good mechanical (injectability, adhesion and mobility) and rheological properties, and can effectively adhere to various fresh and moist tissues and materials. In subsequent antibacterial experiments, the hydrogel showed significant inhibitory effects on bacteria such as Staphylococcus aureus (gram-positive bacteria), MRSA, Escherichia coli (gram-negative bacteria) and Porphyromonas gingivalis (P.gingivalis), while disrupting the formation of bacterial biofilms by interfering with the normal metabolism of these bacteria. In addition, GM / OHA hydrogel loaded with GZN has significant ability to remove reactive oxygen molecules (mROS), restore mitochondrial function and inhibit cell apoptosis. It can also significantly inhibit the expression of inflammatory mediators and promote the polarization of macrophages to the M2 phenotype. In vitro cell experiments showed that GM / OHA-GZN&M had good cell compatibility and could promote cell proliferation, migration and osteogenesis. Finally, in the rat periodontitis model, the combination of bee venom peptide and GZN hydrogel showed a more significant effect in promoting alveolar bone regeneration and regulating the immune microenvironment by effectively inhibiting the overgrowth of harmful bacteria (Porphyromonas and Bacteroides) and alleviating the dysbiosis of the periodontal pocket microenvironment. In conclusion, our study provides a feasible and promising therapeutic strategy for the treatment of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

[0018] Figure 3 is the GelMA in Example 1 1 H NMR spectroscopy.

[0019] Figure 4 is the OHA in Example 1 1H NMR 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 the SEM image of GM / OHA-GZN&M hydrogel in Example 1.

[0022] Figure 7 The diagram shows the adhesion effect of 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 These are the pig skin lap shear test A and adhesion strength B of the GM / OHA-GZN&M hydrogel in Example 1.

[0024] Fig. 9 These are the agar plate colonies of different hydrogels in Example 1.

[0025] Fig.10 A and B are the survival rates of Escherichia coli, Porphyromonas gingivalis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus under different hydrogels.

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

[0027] Fig.12 The expression of proinflammatory cytokines of different gels in the Raw264.7 cell inflammation model in Example 1, wherein A is TNF-α, B is IL-6, C is IL-1β, D is MCP-1, E is INF-γ, and F is NO.

[0028] Fig.13 The M1 / M2 polarization rate of macrophages was detected by immunofluorescence staining (A) and flow cytometry (B) for different gels in Example 1.

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

[0030] Fig.15 A is the fluorescence intensity of MMP and ATP in RAW264.7 cells evaluated by JC-1 staining kit, B is the relative fluorescence intensity of red / green, and C is the relative ATP expression.

[0031] Fig.16 Evaluation of the cell compatibility of different hydrogels for MC3T3-E1 and RAW264.7 cells, where A is MC3T3-E1 and B is RAW264.7.

[0032] Fig.17 Micro-CT analysis of maxillary bones with teeth after different hydrogel treatments (A), CEJ-ABC relative magnification (B).

[0033] Fig.18 H&E staining and Masson staining of maxillary bones with teeth after treatment with different hydrogels (A), relative inflammatory infiltration area (B), and corresponding collagen deposition area (C).

[0034] Fig.19 The abundance and α-diversity index of the oral flora in the periodontitis group and the subgingival microbiota after GM / OHA-GZN&M hydrogel treatment. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The main component and active substance of bee venom is melidin. As a natural insect antimicrobial cationic peptide, melidin has a wide range of antibacterial activities and can target the peptidoglycan or lipopolysaccharide of bacterial cell walls through electrostatic attraction, destroying the bacterial membrane and causing bacterial death. In addition, it also has certain anti-inflammatory and analgesic effects. Recently, although melidin 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 separated from grape seeds, mainly composed of polyphenolic substances such as proanthocyanidins, catechins, epicatechins, gallic acid, gallic acid esters, etc. It has been previously reported that polyphenols rich in phenolic hydroxyl groups can effectively bind to mitochondrial outer membrane proteins through a variety of non-covalent interactions (including electrostatic attraction, hydrophobic interaction, hydrogen bonding, etc.). This binding helps to clear excessive mROS, restore mitochondrial function, and inhibit inflammatory pathways including NLRP3 / caspase-1. Studies have shown that grape seed extract can significantly improve colon damage and intestinal flora imbalance by regulating lipid metabolism, suggesting that it may have the potential to regulate oral microorganisms.

[0041] Based on the above, the present invention mixes modified gelatin (GelMA) and oxidized hyaluronic acid (OHA) aqueous solutions in proportion and prepares a polysaccharide hydrogel with good mechanical properties under specific conditions. The hydrogel has good injectability and adhesion ability. Afterwards, polyphenol-based nanoparticles (grape seed extract @ zinc) with mitochondrial targeting function and bee venom polypeptide (Melittin) with broad-spectrum antibacterial activity are loaded therein, so that the hydrogel has good antioxidant, antibacterial and immunomodulatory effects. Polyphenols effectively mediate the targeted clearance of mitochondrial ROS (mROS), restore mitochondrial function, and break the inflammatory free radical cycle. In addition, the in vivo rat periodontitis model shows that GM / OHA-GZN&M hydrogel effectively inhibits inflammation and promotes alveolar bone regeneration through immunomodulation. Inhibit the overgrowth of pathogenic bacteria and alleviate the dysbiosis of the oral flora in rats. In summary, the present invention provides a promising therapeutic strategy for the clinical treatment of periodontitis.

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

[0043] The Zn(NO3)2·6H2O aqueous solution is added dropwise into the grape seed extract aqueous solution to flocculate and precipitate, and the precipitate is collected to obtain grape seed extract-zinc coordinated nanoparticles;

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

[0045] Adding grape seed extract-zinc coordinated nanoparticles and bee venom peptide to the hydrogel precursor solution 1 to obtain the hydrogel precursor solution 2;

[0046] The hydrogel precursor solution 2 is 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; 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 specifically limit the source of the grape seed extract, which can be purchased or prepared by a preparation method well known to those skilled in the art. Since the water solubility of grape seed extract is limited and the concentration is too high to be completely dissolved, 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 comprises the steps of washing the precipitate and freeze-drying the precipitate.

[0049] In some embodiments of the present invention, the preparation method of the methacrylic acid modified gelatin is: methacrylic acid is added to a gelatin aqueous solution and reacted, and 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 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 preparation method of the oxidized hyaluronic acid is: adding sodium periodate solution dropwise to a hyaluronic acid aqueous solution, reacting in dark conditions, and after the reaction is completed, dialyzing and freeze-drying the reaction solution to obtain the oxidized hyaluronic acid; the concentration of the hyaluronic acid aqueous solution is 2-10% w / v; the concentration of the sodium periodate solution is 0.5M; the volume ratio of the hyaluronic acid aqueous solution to the sodium periodate solution is 100:5.

[0052] In some embodiments of the present invention, the reaction time in 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, in the hydrogel precursor solution 2, the concentrations of grape seed extract-zinc coordinated nanoparticles and melittin 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: wavelength 405nm, power 5W, time 1min.

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

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

[0058] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0059] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, 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): Dissolve grape seed extract in pure water at a mass concentration of 2% w / v, prepare a total of 50 mL, and adjust its pH value to 9.0. Then, add 50 mL of 5% w / v Zn(NO3)2·6H2O aqueous solution dropwise to the above solution to cause flocculation and precipitation. After slowly stirring for 15 minutes, collect the precipitate by centrifugation, wash it with pure water 3 times, and freeze-dry it 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, and 100 mL was prepared. 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. After adding 200 mL of deionized water, the reaction was terminated. After dialysis, GelMA was obtained by freeze drying.

[0064] Step 3. Synthesis of oxidized hyaluronic acid (OHA): Dissolve hyaluronic acid (HA) in pure water at a mass concentration of 2% w / v, prepare 100 mL, and then add 5 mL of 0.5M sodium periodate solution dropwise. After reacting for 2 hours in the dark, add ethylene glycol to stop the reaction. Obtain OHA 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, 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 (added amount was 1 mg / mL) was added to the hydrogel precursor solution 1, and then 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: Melittin (50 mg / mL) was added to the hydrogel precursor solution 1, and then GM / OHA-M hydrogel was prepared by irradiating with blue light (wavelength 405 nm, power 5 W) for 1 minute.

[0069] GM / OHA-GZN&M hydrogel: GZN (added amount was 1 mg / mL) and bee venom peptide (added amount was 50 mg / mL) were added to the hydrogel precursor solution 1, and then 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 GZN was in the form of spherical nanoparticles (e.g. Figure 1 A and B in the figure). Figure 2 The X-ray photoelectron spectroscopy (XPS) spectrum in Figure A shows the presence of a dual-mode peak of Zn2P (1020-1050 eV), confirming the successful synthesis of GZN. Figure 2In Figure B, the characteristic peak of GSE is observed, which is located at 2θ = 20°-30°, and its intensity is lower than that of GSE in the X-ray diffraction (XRD) spectrum. This intensity reduction may be due to the Zn 2+ Chelation with GSE results in reduced exposure of phenolic hydroxyl groups in GSE. As shown in the FTIR spectrum ( Figure 2 As shown in C), the characteristic absorption peaks of GA benzene ring, such as 1610cm -1 、1520cm -1 In addition, the GZN spectrum also showed the presence of 755-870cm -1 The additional peaks in the range of 2.5 and 1.5 can be attributed to the CH vibration on the benzene ring. These findings support the relationship between the GSE phenolic group and Zn 2+ The effective coordination between .

[0072] 1.2 In 1 H NMR spectroscopy ( Figure 3 and Figure 4 ), gelatin was modified by grafting methacrylate groups on the C=C bonds, while the sugar rings of HA were opened by treatment with sodium peroxide to form linear chains terminated with dialdehydes. The above results indicate the successful synthesis of GelMA and OHA.

[0073] 1.3 From Figure 5 In Figure A, it can be observed that when 10% GelMA and 2% OHA were mixed in a pot, the hydrogel precursor solution became viscous, indicating that a Schiff base bond (the first layer of cross-linked network) was formed between the aldehyde group on OHA and the amino group on GelMA. Subsequently, after irradiation with blue light (405nm) for 1 minute, a second layer of cross-linked network was established by free radical polymerization. Then, grape seed extract-zinc coordinated nanoparticles (GZN) (1mg / mL) and bee venom (50mg / mL) were added to the above system to form GM / OHA-GZN&M hydrogel. Figure 5 As shown in Figure B, the GM / OHA-GZN&M hydrogel has good mobility and can be formed into various shapes. In addition, it also exhibits shear thinning behavior, which facilitates injection through a syringe needle ( Figure 5 In addition, due to its flexible dynamic cross-linking and rich hydrogen bond structure ( Figure 5 D), GM / OHA-GZN&M hydrogel can be firmly attached to the finger. To further explore the morphology and structural mechanism of GM / OHA-GZN&M hydrogel, we performed SEM, SEM-eds and XPS analysis. SEM images show ( Figure 6 ), with the increase of OHA, GZN and melittin component contents, the porous structure becomes more compact and regular.

[0074] 2. Mechanical properties test of hydrogel

[0075] The pig skin was cut into rectangular strips with a length of 30 mm and a width of 10 mm. 100 μl of hydrogel was applied to both ends of the two strips and overlapped to form a 10×10 mm 2 The lap shear joints were then placed on the lap shear joints and allowed to stabilize for 1 hour. Adhesion strength was measured using an electronic tensile machine (Zwick Z250) and the strength was calculated by dividing the maximum load by the overlap area. At a crosshead speed of 2 mm / min, stress-strain curves and compressive strength at 50% strain were also obtained. At least 3 specimens were tested for each sample and the average was taken.

[0076] Result analysis: Figure 7 As shown in the figure, the hydrogel of GM / OHA-GZN&M sample showed good adhesion properties and had excellent adhesion effects on different organs (liver, spleen, heart, kidney, lung) and materials (metal, rubber, plastic, wood, glass). In addition, in the pig skin lap shear test, as the content of OHA, GZN&M and bee venom increased, the hydrogel showed a higher adhesion than the commercial fibrin glue ( Figure 8 The adhesion strength of A and B in Figure 1 was 4.99±0.37 kPa. This good adhesion is mainly attributed to the abundant functional groups such as carboxyl, amino and hydroxyl groups in GM, OHA, GZN and Melittin. These functional groups give the hydrogel the ability to adhere to different substrates through hydrogen bonding forces, 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 5 groups (PBS as the negative control group, GM / OHA group, GM / OHA-GZN group, GM / OHA-M group and GM / OHA-GZN&M group as experimental groups). Before the experiment, the same mass of hydrogel (1 g) in a 24-well plate was sterilized under ultraviolet irradiation for 4 h and washed with PBS. The diluted bacterial suspension (10 μL, 10 6 CFU mL -1) were evenly added to the hydrogel surface. After culturing at 37°C for 4 hours, 1 mL of PBS was added to the bacterial suspension and diluted 100 times. 10 μL of the bacterial suspension was spread on an LB agar plate. The colony growth was observed after 12 hours.

[0079] Result analysis: Fig. 9 It was shown that the number of colonies on the agar plate in the GM / OHA hydrogel group loaded with bee venom peptide was significantly reduced compared with that in the PBS group. Fig.10 A and B show that the bacterial survival rate of the GM / OHA-GZN&M group was the lowest, with the survival rate of Staphylococcus aureus being 0.96±0.25%, the survival rate of methicillin-resistant Staphylococcus aureus being 1.26±0.29%, the survival rate of Escherichia coli being 0.71±0.20%, and the survival rate of Bacteroides gingivalis being 0.92±0.19%. This indicates that the hydrogel loaded with melittin 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 by DPPH· and PTIO· scavenging assays. For the DPPH· scavenging assay, freeze-dried hydrogels of different components were ground into powders. 30 mg of freeze-dried hydrogel powders of different components were 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 value of the supernatant at 517 nm was detected by a microplate reader. Similarly, in the PTIO· scavenging assay, freeze-dried hydrogels (30 mg) were added to PTIO· solution (3 mL, 100 μM), incubated in the dark for 60 min, and the absorbance value of the supernatant at 557 nm was recorded. The negative control for the above two experiments was PBS (300 μL).

[0082] Result analysis: Fig.11 As shown in Figures AD, the scavenging rates of DPPH (PTIO) were significantly increased in the GM / OHA-GZN and GM / OHA-GZN&M groups. The DPPH scavenging rate of 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 hydrogel has good antioxidant activity.

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

[0084] RAW 264.7 cells (1×10 6), hydrogel extract (30 μL) and LPS (100 ng / mL) were co-cultured 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 ELISA kits. In addition, the expression and quantity of CD86 and CD206 were detected by immunofluorescence staining and flow cytometry. For flow cytometry, APC anti-mouse CD86 (CD206) antibody was used to quantify macrophages polarized to M1 / M2. For immunofluorescence staining, macrophages were fixed in 4% paraformaldehyde, permeabilized with 0.5% Tween, blocked with 10% BSA, then incubated with antibodies and observed by confocal microscopy. Image J was used for quantitative analysis. All experiments were performed three times and the average value was taken.

[0085] Results Analysis: First, the expression of proinflammatory 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, were studied in the Raw264.7 cell inflammation model. Fig.12 As shown in Figure AF, compared with the GM / OHA group, the proinflammatory cytokines in the GM / OHA group loaded with GZN decreased significantly, with a decrease of 40%-60%. Among them, 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. In addition, immunofluorescence staining and flow cytometry were used to detect the M1 / M2 polarization rate of macrophages. As Fig.13 As shown in A and B, the positive level of M1 macrophages (CD86 marker) in the GM / OHA-GZN and GM / OHA-GZN&M groups was significantly lower than that in other groups, while the positive level of M2 macrophages (CD206 marker) was significantly higher than that in other groups. These experiments show that the GM / OHA hydrogel loaded with GZN not only has a good effect of scavenging reactive oxygen species (ROS) and restoring mitochondrial function, but also has significant immunomodulatory properties, which can inhibit the expression of proinflammatory cytokines and promote the increase of M2 phenotype macrophages.

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

[0087] Cultured cells were harvested, mechanically homogenized, and subjected to two centrifugation steps to separate mitochondria and other cellular components. The isolated mitochondria were then incubated with Cy5.5-labeled GZN nanoparticles for 1 hour at 37°C and washed three times with PBS to remove unbound particles. The fluorescence intensity of the mitochondrial suspension was then measured using flow cytometry. To analyze the internalization of nanoparticles, RAW264.7 was incubated with Cy5.5-labeled GZN and imaged 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. They were then stained with ER-tracker, Mito-tracker, Golgi-tracker, Lyso-tracker, and Hoechst 33342 according to the instructions. Fluorescence was captured using a laser confocal microscope, and colocalization values ​​were determined by Image J.

[0088] Result analysis: Fig.14 As shown in Figure A, as the amount of GZN incubation increased, the Cy5.5 signal in the extracted mitochondria became more obvious, indicating that there was a strong binding affinity between GZN and mitochondria. Fig.14 Middle B), the results showed that the fluorescence signal of GZN labeled with cy5.5 had a significant overlap with mitochondria, and the Pearson correlation coefficient (R) was 0.77, exceeding that of other organelles.

[0089] 7. Mitochondrial membrane potential (MMP) and ATP content detection

[0090] The JC-1 staining kit was used to evaluate the MMP of RAW264.7 cells. The cells were pretreated with specific drugs (hydrogel extract (30 μl)) overnight and then incubated with 1000 μM H2O2 for 2 hours. Subsequently, they were stained with JC-1 dye for 20 minutes, washed twice with JC-1 buffer, and the fluorescence was observed under an inverted fluorescence microscope. RAW264.7 cells were cultured in 6-well plates and received different treatments (cells were pretreated with hydrogel extract (30 μl) overnight and then incubated with 1000 μM H2O2 for 2 hours). Subsequently, the cells were lysed according to the instructions of the reagent manufacturer. The lysate was added to each well, and the precipitate was collected by centrifugation. 100 μM ATP was added to the working solution, and then incubated at 37°C for 5 minutes, and the fluorescence intensity was measured by a chemiluminometer.

[0091] Results: Excessive accumulation of ROS can disrupt the mitochondrial membrane potential, leading to mitochondrial depolarization and subsequent dysfunction. Fig.15As shown in 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 good improvement, and the red-green fluorescence ratio of both groups was significantly reduced. Quantification of the red-green fluorescence ratio corresponding to GM / OHA-GZN and GM / OHA-GZN&M ( Fig.15 In B), the ATP production capacity of each group of hydrogels was further tested, considering that ATP production depends on the mitochondrial membrane depolarization produced by the proton pump. Fig.15 In C, the GM / OHA-GZN and GM / OHA-GZN&M groups recovered to 56.33% and 65.01% of the control group level, respectively, indicating that GZN has a protective effect and is able to maintain mitochondrial ATP production function.

[0092] 8. Cytocompatibility Assessment

[0093] 1 mL of hydrogel was immersed in 5 mL of DMEM medium and incubated for 24 hours. Then, the extract was collected to obtain a hydrogel extract DMEM solution. MC3T3-E1 and RAW264.7 cells were cultured at 4 × 10 4 Cells were seeded at a density of 100 μL / mL (100 μL per well) 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 method according to the manufacturer's instructions. Control cells were cultured alone in DMEM.

[0094] Result analysis: Fig.16 It can be seen that all groups of MC3T3-E1 and RAW264.7 cells showed excellent activity, and the cell survival rate could reach more than 80% after being co-cultured with the hydrogel extract alone for 5 days. It should be pointed out that compared with the control group, GM / OHA group and GM / OHA-M group, the cell survival rate of the GM / OHA-GZN group and GM / OHA-GZN&M group was higher. In MC3T3-E1 cells, the cell survival rates of the GM / OHA-GZN group (308.52±5.21%) and the GM / OHA-GZN&M group (310.45±7.32%) were higher than those of 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 Experimentation

[0096] Eighteen SD rats (6-8 weeks old) were randomly divided into 6 groups (3 rats in each group), including 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 once every other day after silk thread ligation of the maxillary second molar (M2) of the rats. 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, the maxillary bones with teeth were collected for analysis. The samples were subjected to micro-computed tomography (Micro-CT) analysis, H&E staining, Masson staining, immunofluorescence staining, and immunohistochemical staining.

[0097] Results: Compared with the periodontitis group, the hydrogel-treated group showed less alveolar bone volume and shorter periodontal ligament-cementum distance. In addition, it can be observed that the efficacy of the hydrogel-treated group containing only melittin or GZN is relatively inferior to that of the hydrogel-treated group containing both components. The CEJ-ABC distance of the GM / OHA-GZN&M group was only 1.18 times that of the healthy group, which was lower than that of the periodontitis group (2.43 times), GM / OHA group (2.20 times), GM / OHA-M group (1.92 times) and GM / OHA-GZN group (1.65 times).

[0098] like Fig.17 As shown in A and B, the alveolar bone recovery after GM / OHA-GZN&M hydrogel treatment (indicated by the yellow dotted line) showed a more significant improvement, and its recovery level was comparable to that of healthy alveolar bone. In addition, the GM / OHA-GZN and GM / OHA-GZN&M groups showed good restoration of junctional epithelial connections, significantly reduced inflammatory cell infiltration (indicated by the black arrows), and the periodontal ligament (PDL) tended to be normally arranged. The corresponding quantitative values ​​( Fig.18 (A and B) are consistent with the staining image trend. Compared with the control group, the inflammatory cell infiltration in the GM / OHA-GZN&M group (29.67±3.30%) was significantly reduced. Masson staining ( Fig.18A 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%), GM / OHA group (46.67±4.11%), GM / OHA-m group (58.33±1.25%), and GM / OHA-gzn group (70.1±5.26%). The above results prove that the combination of melittin and GZN has the effect of synergistically promoting alveolar bone regeneration and regulating the immune microenvironment in the treatment of rat periodontitis.

[0099] 10. Microbiological analysis

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

[0101] Result analysis: Fig.19 As shown in Figure 3, the diversity of oral flora in the periodontitis group decreased significantly, indicating an imbalance in microbial ecology. However, after treatment with GM / OHA-GZN&M hydrogel, both the abundance and α-diversity of the subgingival microbiota were substantially improved.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an injectable adhesive hydrogel, characterized in that: The following steps are involved: The Zn(NO3)2·6H2O aqueous solution is added dropwise into the grape seed extract aqueous solution to flocculate and precipitate, and the precipitate is collected to obtain grape seed extract-zinc coordinated nanoparticles; Dissolving methacrylic acid-modified gelatin, oxidized hyaluronic acid and a photoinitiator in water to obtain a hydrogel precursor solution 1; Adding grape seed extract-zinc coordinated nanoparticles and bee venom peptide to the hydrogel precursor solution 1 to obtain the hydrogel precursor solution 2; The hydrogel precursor solution 2 is irradiated with blue light to obtain the injectable adhesive hydrogel.

2. The method for preparing the injectable adhesive hydrogel according to claim 1, characterized in that: 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.

3. The method for preparing the injectable adhesive hydrogel according to claim 1, characterized in that: The preparation method of the methacrylic acid modified gelatin is as follows: methacrylic acid is added to a gelatin aqueous solution and reacted, and 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.

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

5.

5. The method for preparing the injectable adhesive hydrogel according to claim 1, characterized in that: 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.

6. The method for preparing the injectable adhesive hydrogel according to claim 1, characterized in that: In the hydrogel precursor solution 2, the concentrations of grape seed extract-zinc coordinated nanoparticles and bee venom peptide are 1 mg / mL and 50 mg / mL, respectively.

7. The method for preparing the injectable adhesive hydrogel according to claim 1, characterized in that: The parameters of the blue light irradiation are set as: wavelength 405nm, power 5W, time 1min.

8. An injectable adhesive hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the injectable adhesive hydrogel according to claim 8 in the preparation of a drug for treating chronic periodontitis.

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