An injectable hydrogel for treating periodontitis with diabetes mellitus and a preparation method and application thereof

Abstract

The present application relates to periodontitis therapeutic drug preparation or biological material technical field, disclose a kind of for treating injectable hydrogel with diabetes periodontitis and its preparation method and application.The present application is prepared by combining raw material guanosine, 1, 4-phenyl boronic acid, adenosine and procyanidin B2, and the supramolecular hydrogel has excellent injectability, self-healing, biocompatibility and slow-release performance.The hydrogel itself has pharmacodynamic function, by adjusting immune microenvironment, blood vessel morphology, promoting bone formation, etc., significantly promote CPDM periodontal tissue regeneration.It can be seen that the injectable hydrogel provided by the present application has wide application prospect in the preparation of CPDM clinical treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of periodontitis treatment drugs or biomaterials, in particular to an injectable hydrogel for treating periodontitis with diabetes and a preparation method and application thereof. BACKGROUND

[0002] Periodontitis with diabetes (CPDM) is a complex chronic inflammatory disease formed by the bidirectional interaction of diabetes and periodontitis, and they are mutual risk factors. Compared with ordinary periodontitis, the treatment difficulty is significantly increased and the prognosis is poorer. Periodontitis is a periodontal tissue inflammatory destruction disease caused by dental plaque, and is the main cause of tooth loss in adults. The high blood glucose level of diabetic patients can exacerbate periodontal tissue inflammation, accelerate alveolar bone absorption and destruction, and cause deep periodontal pocket formation, tooth loosening and other symptoms to worsen, which seriously affects the oral health and masticatory function of patients. At the same time, the sustained inflammation of periodontitis with diabetes further aggravates insulin resistance, which is not conducive to blood glucose control, forming a vicious cycle of "diabetes aggravating periodontitis and periodontitis worsening diabetes", which not only reduces the quality of life of patients, but also increases the risk of complications of diabetes (such as cardiovascular disease and kidney disease), causing serious harm to the overall health of patients. Therefore, developing an efficient and safe treatment for periodontitis with diabetes has important clinical value and research significance for controlling inflammation, reconstructing periodontal tissue and improving the oral and overall health of patients.

[0003] In the prior art, the treatment of periodontitis with diabetes focuses on "controlling inflammation, eliminating pathogenic factors, and promoting periodontal tissue regeneration", forming multiple technical paths to try to solve the problems of high treatment difficulty and poor prognosis: first, basic periodontal treatment, which removes pathogenic factors such as dental plaque and calculus through scaling and curettage to relieve local inflammation, but has limited effect on the regeneration of damaged periodontal tissue; second, systemic drug treatment, which mainly uses antibiotics to control inflammation, but has limitations such as insufficient local drug concentration, obvious systemic side effects, and easy drug resistance with long-term use, and cannot meet the synergistic needs of blood glucose control and periodontal inflammation treatment; third, local drug delivery treatment, which uses antibiotic sustained-release agents for interventional drug delivery, trying to improve the local drug concentration and reduce systemic side effects, and is the current clinical local treatment method; fourth, local drug delivery system (LDDS) research, which uses hydrogel as the core carrier to encapsulate drugs (especially proteins and antibodies) by taking advantage of its hydrophilic three-dimensional network structure and porous characteristics, achieving sustained drug release, improving drug bioavailability, and reducing systemic side effects, and has become a research hotspot for local treatment of periodontitis with diabetes.

[0004] However, the existing technology for treating periodontitis with diabetes still has many significant defects, which is difficult to meet the actual needs of clinical treatment and cannot achieve the treatment goal of "inflammation control-tissue regeneration-blood glucose coordination": first, the limitation of basic periodontal therapy is obvious, which can only remove the pathogenic factors and cannot effectively control chronic inflammation, let alone promote the regeneration of damaged periodontal tissue, and cannot break the vicious cycle of diabetes and periodontitis; second, there are obvious shortcomings in systemic drug therapy and traditional local sustained-release agents. The local drug effect of systemic administration is insufficient, and the side effects are large. Traditional antibiotic sustained-release agents need to be administered during diagnosis, and the patient's compliance is poor. Moreover, there is a common problem of drug burst release, which leads to a large fluctuation in local drug concentration, seriously affecting the treatment effect. At the same time, long-term use of antibiotics can easily cause drug resistance, further increasing the difficulty of treatment; third, the existing hydrogel local drug delivery system still has some shortcomings. Most hydrogels lack specificity and are not designed in combination with the characteristics of periodontitis with diabetes, i.e. high blood sugar, heavy inflammation, and difficult tissue regeneration, so they cannot achieve precise and long-acting release of drugs; some hydrogels do not have injectability, which is difficult to adapt to the drug delivery requirements of complex anatomical sites such as periodontal pockets, and cannot ensure the precise enrichment of drugs in the lesion site; in addition, the existing hydrogels only focus on drug delivery and do not take into account the synergistic effect of anti-inflammatory, promotion of periodontal tissue regeneration and improvement of local microenvironment, which cannot fundamentally solve the core treatment pain points of periodontitis with diabetes and is difficult to achieve effective reconstruction of periodontal tissue and coordinated control of blood sugar. In summary, the existing treatment technology cannot break through the treatment bottleneck of periodontitis with diabetes, and a new injectable hydrogel and its preparation method are needed to achieve efficient, precise and long-acting treatment effect, and provide a new technical solution for the clinical treatment of periodontitis with diabetes. SUMMARY

[0005] The present application aims to provide an injectable hydrogel for treating periodontitis with diabetes and its preparation method and application, to solve the technical problem of poor drug efficacy in treating periodontitis with diabetes.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solution: an injectable hydrogel for treating periodontitis with diabetes, the raw materials of which include guanosine, 1,4-phenyl boronic acid, adenosine and procyanidin B2.

[0007] Preferably, as an improvement, the molar ratio of the raw materials guanosine, 1,4-phenyl boronic acid, adenosine and procyanidin B2 is 4-8:4-8:2-4:1-2.

[0008] Preferably, as an improvement, the pore size of the hydrogel is 10-25 pm.

[0009] Preferably, as an improvement, the present application further provides a preparation method of an injectable hydrogel for treating periodontitis with diabetes, which comprises dispersing the above-mentioned raw materials in an aqueous solvent, heating and dissolving, and then cooling to obtain an injectable hydrogel.

[0010] Preferably, as an improvement, the aqueous solvent is PBS buffer.

[0011] Preferably, as an improvement, potassium hydroxide or sodium hydroxide is added to the PBS buffer so that the pH of the buffer is 7.4-8.

[0012] Preferably, as an improvement, the heating is to warm the solution to 75-90℃ until all reagents are dissolved, and then cool to room temperature.

[0013] Preferably, as an improvement, the present application also provides a use of an injectable hydrogel for treating periodontitis with diabetes in the preparation of a drug for treating periodontitis with diabetes.

[0014] Preferably, as an improvement, the injectable hydrogel is used by injection.

[0015] Preferably, as an improvement, the drug-loaded hydrogel includes any one of adenosine or procyanidin B2.

[0016] The principle and advantages of the present application are: 1. The present application combines the above raw materials to prepare a new type of supermolecular injectable hydrogel, wherein the vicinal diol (phenol) structure of the raw materials guanosine and procyanidin B2 (OPCB2) is condensed with 1,4-benzenediboronic acid (PBA) to form a dynamic benzene borate ester bond; under the action of intramolecular and intermolecular hydrogen bonds, G tetramer is formed, and then through π-π stacking, GAOPCB2 supermolecular hydrogel is finally formed by multi-level self-assembly, and the GAOPCB2 hydrogel has a loose pore structure, which lays a structural foundation for later drug loading and use in vivo.

[0017] 2. The hydrogel obtained by the present application has excellent injectability, self-healing, biocompatibility and sustained release performance. The hydrogel itself has a pharmacodynamic function, and can significantly promote the regeneration of CPDM periodontal tissue by adjusting the immune microenvironment, blood vessel morphology and promoting bone formation. Therefore, the injectable hydrogel provided by the present application has a broad application prospect in the preparation of CPDM clinical treatment drugs.

[0018] Specifically, the inventors found through experiments that the GAOPCB2 hydrogel obtained by the scheme has good injectability and potential for local application, ROS-responsive adenosine slow-release potential, no obvious cytotoxicity, in-vivo degradation within 4 days, no obvious hemolysis, no obvious acute toxicity to major organs, and good biocompatibility. The GAOPCB2 hydrogel has good antioxidant function, can inhibit macrophage M1 polarization, effectively regulate vascular endothelial cell migration, promote blood vessel formation, and effectively promote stem cell osteogenic differentiation, and has the potential for in-vivo bone regeneration application. The GAOPCB2 supramolecular hydrogel can effectively promote the epithelial sealing of CPDM and alveolar bone regeneration, and mainly plays a role in CPDM regeneration treatment by regulating the inflammatory immune microenvironment, regulating inflammation, blood vessel formation and osteogenic differentiation 3. The method for preparing the supramolecular hydrogel is simple, safe and non-toxic, and is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 ABTS for the embodiment of the present application + Fe 3+ ROS scavenging effect of OPCB2 in the reducing capacity determination experiment.

[0020] Figure 2 CCK8 experimental results of the OPCB2 hydrogel in the embodiment of the present application.

[0021] Figure 3 Formation principle of the phenylboronic ester bond in the embodiment of the present application.

[0022] Figure 4 Construction and stability results of the GAOPCB2 hydrogel in experimental example 1 of the present application.

[0023] Figure 5 Characterization of the GAOPCB2 hydrogel in experimental example 2 of the present application (a~b) FTIR spectrum of the GAOPCB2 hydrogel; c) 1H NMR spectrum of the GAOPCB2; d) 11B NMR spectrum of the GAOPCB2; e) Circular dichroism spectrum of the GAOPCB2; f) PXRD spectrum of the GAOPCB2 hydrogel; g) Variable temperature small-angle X-ray scattering spectrum of the GAOPCB2 hydrogel.

[0024] Figure 6(a) Viscosity scan; b) Strain scan; c) Self-healing scan; d-e) GAOPCB2 hydrogel injectability; f) Retention time of GAOPCB2 hydrogel under 37℃ oscillation; g) Inverted photograph of GAOPCB2 hydrogel co-incubated with H2O2 at specific time points; h) Active ingredient adenosine activity oxygen-responsive release curve chart.

[0025] Figure 7 (a) Experimental scheme; b) Live and dead cell staining of HUVEC cells treated with GAOPCB2 hydrogel; c) CCK8 cytotoxicity detection of HUVEC cells treated with GAOPCB2 hydrogel; d) Hemolysis rate of GAOPCB2 hydrogel at different concentrations; e) Visual diagram of hemolysis experiment of GAOPCB2 hydrogel at different concentrations; f) In vivo degradation of GAOPCB2 hydrogel; g) HE staining of skin and heart, liver, spleen, lung and kidney of mice at the injection site after injection of GAOPCB2 hydrogel.

[0026] Figure 8 (a-b) Flow cytometry detection of ROS scavenging ability of GAOPCB2 hydrogel; c) ROS fluorescent probe experiment of GAOPCB2 hydrogel; d-g) Flow cytometry detection of macrophage polarization effect of GAOPCB2 hydrogel; h) Effect of GAOPCB2 hydrogel on RNA expression level of cell inflammatory factors.

[0027] Figure 9 (a-b) Scratch test of OPCB2 on HUVEC cell migration ability; c-e) Effect of GAOPCB2 on HUVEC cell tube formation ability; f-g) ALP and ARS detection of GAOPCB2 hydrogel promoting osteogenic differentiation function.

[0028] Figure 10 (a) Schematic diagram of animal model construction; b) Animal blood glucose level record; c) Animal periodontitis model establishment diagram; d) Animal experiment schematic diagram; e) 28-day Micro-CT alveolar bone imaging; f) 28-day buccal alveolar bone loss data statistical diagram; g) 28-day palatal alveolar bone loss data statistical diagram; h) 28-day distal adjacent surface alveolar bone loss data statistical diagram.

[0029] Figure 11The effect of the GAOPCB2 hydrogel in Example 6 of the present application on the closure of CPDM soft tissue was detected by HE staining (a~b); c) Masson staining of CPDM periodontal tissue in different treatment groups; d) Sirius red staining of CPDM periodontal tissue in different treatment groups. DETAILED DESCRIPTION

[0030] The present application will be further described in conjunction with the following examples, but the embodiments of the present application are not limited thereto. If not specifically indicated, the technical means used in the following examples and experimental examples are conventional means known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels.

[0031] EXAMPLE The present scheme provides a preparation method of an injectable hydrogel for treating periodontitis with diabetes, comprising the following steps: Guanosine (G), adenosine (A), OPCB2 and PBA were accurately weighed by a precision balance according to the molar ratio (the molar ratio of G, A, OPCB2 and PBA was 4:2:1:4, i.e. 5.6 mg:2.7 mg:2.9 mg:3.3 mg), and were added to a vial containing 360 μL of PBS and 40 μL of 0.5 M potassium hydroxide (KOH) solution (the pH of the obtained solution was 7.4~8), heated to clarify the solution (about 90°C), and cooled to room temperature (25°C) to construct a GAOPCB2 supramolecular hydrogel.

[0032] During the process, the vicinal diol (phenol) structure of G, A and OPCB2 condensed with PBA to form a dynamic phenylboronic acid ester bond; under the action of intramolecular and intermolecular hydrogen bonds, G tetramer was formed, and then through π-π stacking, GAOPCB2 supramolecular hydrogel was finally formed by multi-level self-assembly, and the GAOPCB2 hydrogel had a loose pore structure, which laid a structural foundation for later drug loading in vivo.

[0033] The key parameter screening experiment is as follows: (1) Selection of raw material types: Experimental design: Supramolecular hydrogel has good biocompatibility and good application potential in the treatment of periodontitis, but there are few reports on supramolecular hydrogel for the treatment of periodontitis.

[0034] The inflammatory environment of the periodontium is a significant factor hindering periodontal tissue regeneration, and regulating the periodontal immune microenvironment is a crucial foundation for achieving periodontal tissue regeneration. Proanthocyanidins B2 (OPCB2) is a plant polyphenol widely used in the medicinal and edible fields. It possesses significant antioxidant and ROS scavenging functions. Furthermore, OPCB2 has been shown to promote the recovery of vascular endothelial function under inflammatory conditions. Therefore, we hypothesize that OPCB2 has potential therapeutic effects on diabetic periodontitis (CPDM) with high oxidative stress. Firstly, we used DPPH free radical scavenging capacity assay and ABTS... + Scavenging capacity determination and Fe 3+ The reduction capacity assay was used to study the ROS scavenging ability of OPCB2; secondly, the tube formation assay was used to further verify the effect of OPCB2 on the tube-forming function of vascular endothelial cells, so as to evaluate the potential of OPCB2 in the treatment of CPDM.

[0035] Alveolar bone regeneration is a core requirement in the treatment of periodontitis. Adenosine, as an endogenous nucleoside in the human body, has good biocompatibility. Studies have shown that adenosine can effectively promote osteogenic differentiation of bone marrow mesenchymal stem cells and effectively promote bone formation, suggesting that adenosine has the potential for application in the treatment of periodontitis. Guanosine has hydrogen bond donors and acceptors and is a common supramolecular building block. Authoritative studies have shown that the introduction of borate ester bonds can effectively enhance the stability of guanosine supramolecular hydrogels. Therefore, we used OPCB2 and adenosine as the main therapeutic components, with guanosine as the gelling factor, and constructed a novel GAOPCB2 supramolecular hydrogel for the treatment of CPDM by introducing borate ester bonds.

[0036] Experimental Results: First, we used ABTS + Scavenging capacity determination and Fe 3+ The ROS scavenging ability of OPCB2 was studied by reducing its reductive capacity. The results showed that OPCB2 has a significant ROS scavenging function. Figure 1 (a~b). Secondly, we further verified the effect of OPCB2 on the tube-forming function of vascular endothelial cells, and the results showed that OPCB2 can effectively promote the tube-forming function of vascular endothelial cells (a~b). Figure 1 Therefore, OPCB2 has the potential to regulate the inflammatory microenvironment caused by high oxidative stress in CPDM.

[0037] like Figure 1 As shown in f, adenosine alone cannot form a hydrogel, nor can adenosine and OPCB2 form a hydrogel. Figure 1 g), guanosine was introduced as a gelling agent, which participated in the formation of a stable GAOPCB2 supramolecular hydrogel in KOH / PBA solution. Figure 1 h).

[0038] (2) Selection of raw material dosage: Experimental design: Good biocompatibility is the most basic requirement for the in vivo application of biomaterials. We first used CCK8 experiment to study the half maximal inhibitory concentration (IC50) of each component, secondly, CCK8 and live and dead cell fluorescence staining were used to confirm the cytotoxicity of GAOPCB2, finally, GAOPCB2 hydrogel was injected into Balb / c mice, HE staining was used to confirm the effect of GAOPCB2 on the injected part of the skin and internal organs of the animals, and the final concentration and dosage of GAOPCB2 used in vivo were confirmed.

[0039] Experimental results: First, the CCK8 experiment results showed that the IC50 concentrations of OPCB2 were 5 mM ( Figure 2 ) respectively. On this basis, we used CCK8 experiment and live and dead cell staining experiment to study the cytotoxicity of GAOPCB2 hydrogel and its various components, and the results showed that GAOPCB2 hydrogel with a concentration of 0.35-0.7% w / v had no obvious cytotoxicity ( Figure 7 b~c). When injected into animals, organ HE staining showed no obvious toxicity ( Figure 7 g). Finally, we selected 0.7% w / v of GAOPCB2 for in vivo animal study.

[0040] (3) Selection of potassium hydroxide addition amount: According to the formation principle of phenylboronic ester bond ( Figure 3 ), the molar ratio of G:KOH:PBA is 1:1:1. Therefore, the addition amount of KOH is determined by the addition amount of guanosine, adenosine and OPCB2. According to the experimental results of raw material dosage in part (2), the addition amount of KOH is calculated; (4) Selection of reaction temperature and time: GAOPCB2 hydrogel is mainly self-assembled by hydrogen bond, phenylboronic ester bond and other interactions. After complete dissolution of each component, the temperature is 80-90℃ in the preparation of GAOPCB2 hydrogel. It is worth noting that OPCB2 is sensitive to temperature, and its antioxidant capacity will be significantly affected when the temperature exceeds 80℃, therefore the preparation temperature of GAOPCB2 is usually controlled below 80℃.

[0041] Experimental example 1: gel stability The state of raw materials adenosine (A), guanosine (G), OPCB2, G-A, G-OPCB2 and GAOPCB2 after heating, dissolving and cooling is shown in Figure 4 , all combinations did not form a gel; only GAOPCB2 formed a relatively stable gel, and its in vitro stability was more than 1 month, indicating that the GAOPCB2 hydrogel obtained by this scheme has good stability.

[0042] Experimental Example 2: Principle of GAOPCB2 Hydrogel Formation The experimental procedure is as follows: (1) Infrared spectroscopy (FTIR) GAOPCB2 hydrogel was prepared, freeze-dried for 24 h to form a powder, and pressed into a tablet with potassium bromide for detection on an infrared spectrometer.

[0043] (2) Nuclear magnetic resonance (NMR) GAOPCB2 hydrogel was prepared, freeze-dried for 24 h to form a powder. 10 mg of freeze-dried GAOPCB2 hydrogel powder, adenosine powder and PBA powder were weighed and heated to dissolve in 1 mL of DMSO-d6, respectively. 800 μL was taken into a nuclear magnetic resonance tube, and then the 1 H NMR spectrum was collected at 400 MHz. 20 mg of freeze-dried GAOPCB2 hydrogel powder and PBA powder were weighed and heated to dissolve in 1 mL of D2O, respectively. 800 μL was taken into a nuclear magnetic resonance tube, and then the 11 B NMR spectrum was collected at 600 MHz.

[0044] (3) Circular dichroism spectroscopy (CD) GAOPCB2 hydrogel dilute solution was prepared. The ellipticity change curve in the wavelength range of 200-400 nm at 25°C (parameter settings: scan rate 100 nm / min; scan bandwidth 2.0 nm; repeat three times).

[0045] (4) Powder X-ray diffraction experiment (PXRD) GAOPCB2 hydrogel freeze-dried powder was prepared. Analysis was performed at room temperature (parameter settings: voltage 40 kV; current 240 nm; working length 3.3482°).

[0046] (5) Variable temperature small angle scattering (VT-SAXS) GAOPCB2 hydrogel was prepared and placed in a Hilgenberg quartz capillary (outer diameter 2 mm, wall thickness 0.01 mm). A high-throughput and high-resolution Montel-P multi-layer optical pinhole collimation system and a low-background SCATEX double pinhole collimation system were used, and a VANETC-TM XP detector was used with an exposure time of 30 min. Software SAXS for Windows TM XP, DIFFRAC.SAXS and DIFFRAC.EVA were used for analysis, and the scattering intensity distribution graph was output after sample measurement.

[0047] Fourier transform infrared spectroscopy (FTIR, Figure 5a) and proton nuclear magnetic resonance (1H NMR) 1 H NMR, Figure 5 c) The 2'-OH and 3'-OH of G, A were involved in the formation of GAOPCB2 hydrogel, and the ν(B-OC) absorption peak existing in GAOPCB2 hydrogel suggested that boron ester bond might exist in GAOPCB2 hydrogel; the nuclear magnetic resonance boron spectrum (11B NMR) experiment further confirmed that the ester bond existing in GAOPCB2 hydrogel was phenylboronic acid ester bond Figure 5 d). The CD experimental results proved that G-quadruplexes in head-to-head and head-to-tail forms existed in GAOPCB2 hydrogel Figure 5 e); PXRD results proved that π-π stacking existed in GAOPCB2 hydrogel Figure 5 f); Finally, we further detected the microstructure of GAOPCB2 hydrogel by VT-SAXS, and the results showed that at 65°C, nanofibers with a diameter of about 2 nm existed in GAOPCB2 hydrogel, which was consistent with the width of a G-quadruplex Figure 5 g). With the decrease of temperature, the fiber diameter in GAOPCB2 hydrogel increased continuously, and at 25°C, fibers with diameters of 2 nm, 8 nm, 10 nm, 16 nm and 24 nm existed in GAOPCB2 hydrogel. Therefore, we believe that with the decrease of temperature, quadruplexes form small-diameter fibers, these small-diameter fibers intertwine to form larger-diameter fibers, and finally form a three-dimensional network to encapsulate a large amount of water to form GAOPCB2 supramolecular hydrogel.

[0048] Experimental Example 3: Injectable and sustained-release properties of GAOPCB2 hydrogel Injectability is an important feature to ensure the biomedical application of hydrogel. Injectability facilitates clinical use and can achieve minimally invasive, reduce invasiveness, and at the same time reduce the adverse reactions related to systemic administration. To evaluate the injectability of GAOPCB2 hydrogel, rheological test was used to evaluate its self-healing ability.

[0049] The experimental procedure is as follows: (1) Rheological experiment: prepare GAOPCB2 hydrogel, heat to solution state, use a pipette to take 1.5 mL of gel solution and place it on a preheated rheometer parallel plate at 65°C, lower the cone PP50 to a gap distance of 0.5 mm, scrape off the excess sample, and add silicone oil around the parallel plate, seal the edge to prevent evaporation, and test at 25°C.

[0050] (2) Injection experiment: First, prepare the GAOPCB2 hydrogel, mix and suck into the syringe, and stand at room temperature until it is stable and gelled; inject the GAOPCB2 hydrogel in the syringe into the glass plate and vial, and observe whether the hydrogel can be injected and the properties and state after injection.

[0051] (3) Measurement of adhesion by flowing water method: To evaluate the adhesion of the GAOPCB2 gel, we first injected the gel into the isolated mouse skin by oscillation method, 10 μL per point, a total of 6 parallel points. Then the skin was immersed in neutral PBS and oscillated at 37°C at a speed of 100 rpm, and the retention rate of the gel was evaluated within a predetermined time.

[0052] (4) Reactive oxygen response degradation: Prepare 600 μL of GAOPCB2 hydrogel, and place 600 μL of H2O2 (10 μM, 25 μM, 50 μM, 100 μM) on the upper layer of the hydrogel sample to simulate the reactive oxygen response release properties of the gel in vitro at room temperature. At the predetermined time point, 20 μL of degradation products were collected from each hydrogel sample and diluted 40 times. At the same time, 20 μL of deionized water was added to the model. One of the active ingredients, adenosine (A), was quantitatively analyzed by high performance liquid chromatography (HPLC).

[0053] Figure 6 a-b indicates that when the strain reaches 100%, GAOPCB2 can change from a gel to a solution, and at the same time, its modulus can recover to its initial value in alternating strain changes. In addition, the hydrogel shows good shear thinning performance ( Figure 6 c). Figure 6 d shows that the GAOPCB2 hydrogel has a writable property after being sheared into a liquid by a needle. The above results confirm that the hydrogel has good injectability and rapid self-healing, and can quickly and completely recover its original modulus after being sheared. Good sustained-release potential is one of the important properties of the hydrogel for application in local treatment of CPDM to reduce adverse reactions related to systemic treatment. Therefore, HPLC experiments were used to evaluate the in vitro sustained-release potential of the GAOPCB2 hydrogel; Figure 6 e shows that the GAOPCB2 hydrogel has good in vitro sustained-release adenosine potential.

[0054] Experimental Example 4: Biocompatibility of GAOPCB2 Hydrogel To evaluate the biocompatibility of the GAOPCB2 hydrogel for application in CPDM treatment, live and dead cell staining, CCK8, in vivo degradation experiment and in vitro hemolysis experiment were used to detect the biocompatibility of the GAOPCB2 gel.

[0055] The experimental steps are as follows: (1) Cytotoxicity CCK8 experiment: Collect BMMSCs, MC-3T3-E1 and HUVEC cells in good condition and logarithmic growth phase, adjust the cell density, and inoculate in 96-well plates. After 12 h, the cells adhere, and different concentrations of G, A, OPCB2, PBA / KOH and GAOPCB2 are added to the aMEM medium. Incubate at 37°C, 5% CO2. Add 10 μL CCK8 reagent to the target well plate at a specific time point (24 h, 48 h, 72 h) after adding the drug, incubate at 37°C, 5% CO2 for 1.5 h, and detect the OD value at 450 nm.

[0056] (2) Live and dead cell fluorescence staining experiment: Collect BMMSCs, MC-3T3-E1 and HUVEC cells in good condition and logarithmic growth phase, adjust the cell density, and inoculate in 6-well plates. After 12 h, the cells adhere, and different concentrations of G, A, OPCB2, PBA / KOH and GAOPCB2 are added to the aMEM medium. Incubate at 37°C, 5% CO2 for 24 h. Collect the cells in the well plate and place them in a 15 mL centrifuge tube. Wash with PBS, resuspend with 500 μL live and dead cell fluorescence staining working solution, incubate at room temperature for 40 min, stop staining with PBS, centrifuge and discard the supernatant, and resuspend with PBS. Take 20 μL of cell suspension onto a clean glass slide, cover with a cover glass, seal with nail polish, and observe and photograph under a fluorescence microscope.

[0057] (3) In vivo degradation experiment of GAOPCB2 hydrogel: First, prepare GAOPCB2 hydrogel, and inject 100 μL of 27 GAOPCB2 hydrogel subcutaneously on the back of 6-8 week old BABL / c female mice with an insulin needle. PBS is used as a control. Observe the retention of the gel on the back of the mice at a specific time point and take photos to evaluate its degradation.

[0058] (4) HE staining to evaluate the biocompatibility of GAOPCB2 hydrogel: First, prepare GAOPCB2 hydrogel, and inject 100 μL of GAOPCB2 hydrogel subcutaneously on the back of 6-8 week old BABL / c female mice with an insulin needle. PBS is used as a control. At a specific time point (euthanize the experimental animals, take the skin and important organs (heart, liver, spleen, lung, kidney) at the injection site of the mice, fix, embed, cut 4 um sections, and perform HE staining for histopathological analysis.

[0059] (5) Hemolysis experiment: BABL / c mouse orbital blood was taken and added to an anticoagulant vacuum blood collection tube containing sodium heparin, shaken up and down, and mixed thoroughly; after adding pre-cooled PBS, repeated centrifugation was performed to prepare 5% packed red blood cells; then co-incubated with an equal volume of different concentrations of GAOPCB2 hydrogel (PBS and 0.1% Triton-100 were used as negative and positive controls, respectively), the supernatant was aspirated, and the absorbance (OD) value at 562 nm was detected by a microplate reader, and the hemolysis rate was calculated.

[0060] As shown in Figure 7 a, live and dead cell staining and CCK8 experiment showed that GAOPCB2 hydrogel had no obvious cytotoxicity ( Figure 7 b~c); Figure 7 d~e) in vitro hemolysis experiment proved that GAOPCB2 hydrogel did not cause obvious hemolysis phenomenon; Figure 7 f), and had no obvious toxicity to important organs Figure 7 g). The above shows that GAOPCB2 hydrogel has good biocompatibility for CPDM treatment.

[0061] Experimental Example 5: Anti-inflammatory, pro-osteogenic and pro-angiogenic effects of GAOPCB2 hydrogel To further study the immune regulation and tissue regeneration effects of GAOPCB2 in CPDM treatment, we treated BMMSCs and HUVEC cells with LPS-p.g. and D(+)-glucose to simulate the inflammatory and high glucose environment in vivo.

[0062] The experimental steps are as follows: (1) GAOPCB2 hydrogel ROS fluorescence staining experiment and flow cytometry analysis experiment: LPS-P.g. and D(+)-glucose were used to treat cells to simulate the inflammatory and high glucose environment in vivo. DCFH-DA was used to label intracellular reactive oxygen species, and flow cytometry and fluorescence microscopy were used to quantify and visualize the level of intracellular ROS changes.

[0063] (2) GAOPCB2 hydrogel regulates macrophage polarization flow cytometry experiment: LPS-P.g. and D(+)-glucose were used to treat cells to simulate the inflammatory and high glucose environment in vivo. GAOPCB2 hydrogel was used to treat mouse macrophage Raw264.7 cells, and after 24 h, flow cytometry was used to detect the expression of M1 macrophage surface molecule CD86 and M2 macrophage surface marker molecule CD206.

[0064] (3) qRT-PCR experiment of GAOPCB2 hydrogel regulating macrophage polarization: LPS-P.g. and D(+)-glucose were used to treat cells to simulate the inflammatory and high glucose environment in vivo. After GAOPCB2 hydrogel treated mouse macrophage Raw264.7 cells for 24 h, the expression levels of related cytokines were detected by qRT-PCR.

[0065] (4) HUVEC cell scratch test: Collect HUVEC cells in good condition and logarithmic growth phase, adjust the cell density, and inoculate in a 6-well plate. After 12 h, the cells adhere to the bottom, use a 1000 μL sterile gun head to make a scratch on the bottom of the well, give the corresponding treatment to the corresponding well plate, incubate at 37°C with 5% CO2. Observe the cell growth in the scratch area under a microscope at different time intervals and take pictures. Use ImageJ software to quantitatively analyze the area of the scratch area.

[0066] (5) Cell tube formation experiment: Collect HUVEC cells in good condition and logarithmic growth phase, adjust the cell density, and inoculate in a 6-well plate. After 12 h, the cells adhere to the bottom, give the corresponding treatment to the corresponding well plate, incubate at 37°C with 5% CO2 for 24 h. Then collect the HUVEC cells in each treatment group, adjust the cell density, and inoculate in a BioCoat Matrigel pre-coated 96-well plate. Observe the cell tube formation under a microscope at 0, 2, 4, and 6 h, take pictures, and use ImageJ software to quantitatively analyze the relevant indicators of the experiment.

[0067] (6) Alizarin red mineralized nodule staining experiment: Collect BMMSCs and MC-3T3-E1 cells in good condition and logarithmic growth phase, adjust the cell density, and inoculate in a 6-well plate. When the cell density reaches 70-80%, give the corresponding treatment to the corresponding well plate, incubate at 37°C with 5% CO2. Change the liquid every 3 days, and after 4 weeks, use the alizarin red mineralized nodule staining kit to stain and observe the cell osteogenic differentiation, and take pictures.

[0068] (7) Alkaline phosphatase staining experiment: Collect BMMSCs and MC-3T3-E1 cells in good condition and logarithmic growth phase, adjust the cell density, and inoculate in a 6-well plate. When the cell density reaches 70-80%, give the corresponding treatment to the corresponding well plate, incubate at 37°C with 5% CO2. Change the liquid every 3 days, and after 7 days, use the alkaline phosphatase staining kit to stain and take pictures.

[0069] As Figure 8As shown in a~b, flow cytometry results showed that both LPS-pg and HG could increase the intracellular ROS level in BMMSCs and HUVECs. After treatment with GAOPCB2 hydrogel, the intracellular ROS level decreased. ROS fluorescent probe experiments further demonstrated that GAOPCB2 hydrogel has the function of scavenging ROS. Figure 8 c). ROS plays an important role in macrophage polarization homeostasis. To further evaluate the effect of GAOPCB2 on macrophage polarization, such as Figure 8 As shown in d~e, macrophages polarize towards the M1 pro-inflammatory phenotype under inflammatory and high-glucose environments, while GAOPCB2 hydrogel can inhibit macrophage M1 polarization and promote macrophage M2 polarization to some extent. Figure 8 f~g). We further used qRT-PCR to detect the RNA expression levels of cytokines in each treatment group, and the results are as follows. Figure 8 As shown in h, an inflammatory environment can significantly increase the expression levels of intracellular pro-inflammatory factors TNF-α and IL-1β, while decreasing the expression level of anti-inflammatory factor IL-10. After treatment with GAOPCB2 hydrogel, the expression levels of TNF-α and IL-1β decreased, while the level of IL-10 increased.

[0070] Building upon the established immunomodulatory effects of GAOPCB2 hydrogel, we further investigated its role in angiogenesis and osteogenic differentiation at the cellular level. First, we used a scratch assay to study the effect of GAOPCB2 hydrogel on the migration ability of HUVEC cells. For example... Figure 9 As shown in a~b, the migration ability of HUVEC cells decreased after treatment with LPS-pg and HG, while GAOPCB2 hydrogel effectively restored the migration ability of HUVEC cells. The effect of GAOPCB2 hydrogel on the tubule formation ability of HUVEC cells was also studied using a tubule formation assay. Figure 9 As shown in Figures c-e, treatment with LPS-pg and HG reduced the angiogenesis capacity of HUVEC cells, while GAOPCB2 hydrogel effectively restored their angiogenesis capacity. The effects of GAOPCB2 hydrogel on osteogenic differentiation capacity were investigated using ARS and ALP staining assays. Figure 9 As shown in f~g, the osteogenic differentiation ability of cells decreased after treatment with LPS-pg and HG, while GAOPCB2 hydrogel could effectively restore the osteogenic differentiation ability of cells.

[0071] Experimental Example 6: GAOPCB2 hydrogel promotes CPDM soft tissue closure and hard tissue formation To further determine the biological application of GAOPCB2 hydrogel in vivo, in this experiment, we constructed the SD rat model of periodontitis with diabetes mellitus, and used Micro CT, HE, Masson and Sirius red staining to evaluate the therapeutic effect of GAOPCB2 hydrogel in the hard and soft tissue regeneration of CPDM.

[0072] As shown in Figure 10 a, 2 mm orthodontic ligature wire and 4-0 nylon surgical silk were used to ligate the first maxillary molar of SD rats, and streptozotocin (STZ, 60 mg / kg) was injected intraperitoneally to construct the SD rat model of periodontitis with diabetes mellitus, and another 6 SD rats were not treated as blank controls. After 4 weeks, the blood glucose levels of the rats were randomly measured in the morning, and the average blood glucose level was 24.93 mmol / L ( Figure 10 b), which was significantly higher than that of the blank control group, in addition, the first maxillary molar of the rats was red and swollen, and the furcation was exposed, and Micro-CT showed that the alveolar bone was absorbed ( Figure 10 c), thus the SD rat model of periodontitis with diabetes mellitus was successfully constructed. The experimental animals were randomly divided into 4 groups, namely the physiological saline group (Control, Ctr group), OPCB2 group, Minocycline Hydrochloride Ointment (MiNo) group and GAOPCB2 hydrogel group, each group of 6, and the same time, the SD rats without any treatment were used as blank control group (Blank). The above groups were treated every 5 days, each 50 uL (25 uL on each side), and the body weight and blood glucose of the rats were measured and recorded. At 14 days and 28 days (end of the experiment), 3 rats were randomly sacrificed ( Figure 10 d), we used Micro-CT to evaluate the effect of GAOPCB2 hydrogel on the regeneration of alveolar bone in CPDM. As shown in Figure 10 e, compared with the blank control group, the model group showed obvious absorption of alveolar bone, and after 4 weeks of treatment, the alveolar bone of the MiNo group, OPCB2 group and GAOPCB2 group recovered to different degrees, and the alveolar bone of the GAOPCB2 group recovered most significantly. As shown in Figure 10 f, after 28 days of treatment, the buccal alveolar bone height of the MiNo group, OPCB2 group and GAOPCB2 group recovered to different degrees, and compared with the Ctr group, the GAOPCB2 hydrogel group recovered most significantly, with a statistically significant difference (p=0.045). In terms of palatal alveolar bone, after 28 days of treatment, the buccal alveolar bone loss distance of the blank control group, physiological saline group, MiNo group, OPCB2 group and GAOPCB2 group also recovered, and compared with the Ctr group, the palatal alveolar bone height of the GAOPCB2 hydrogel group recovered most significantly, but the difference was not statistically significant (p=0.348,Figure 10 g). Finally, we evaluated the alveolar bone loss of the distal surface of the first maxillary molar of SD rats, as shown in Figure 10 h). As shown in Figure 9h, the distal alveolar bone of SD rats was recovered to different degrees after MiNo, OPCB2 and GAOPCB2 hydrogel treatment. Compared with the Ctr group, the recovery of the GAOPCB2 hydrogel group was the most obvious, and the difference was statistically significant (p = 0.026). The above results prove that the GAOPCB2 hydrogel can significantly promote the recovery of the alveolar bone of diabetic SD rats with periodontitis, and has certain application value for the treatment of periodontitis with diabetes.

[0073] At the end of the experiment, we used HE, Masson staining and Sirius red hot staining to detect the periodontal soft tissue of the experimental animals in each group. As shown in Figure 11 a-b, compared with the blank control group, the epithelial attachment of the model group was damaged to varying degrees and migrated apically. After 4 weeks of treatment, the epithelial attachment of the MiNo, OPCB2 and GAOPCB2 groups was recovered to varying degrees. Compared with the Ctr group, the recovery of the GAOPCB2 hydrogel group was the most obvious, and the difference was statistically significant (p = 0.045). We further used Masson trichrome staining (c) and Sirius red staining (d) to evaluate the fibrogenesis and maturity of the soft tissue in each treatment group. The fibers in the soft tissue of the Ctr group were sparse and poorly matured, while the fibers in the soft tissue of the MiNo, OPCB2 and GAOPCB2 groups were arranged and developed to varying degrees. Among them, the fibers in the GAOPCB2 group were the most dense and mature. Figure 11 Figure 11

[0074] ​​In summary, these results, combined with the results of injectability, sustained-release potential and biocompatibility evaluation, show the application prospect of GAOPCB2 hydrogel in immunotherapy. Specifically, in the hydrogel obtained by the scheme, the synergistic effect of G, A and OPCB2 effectively improves the therapeutic effect of the hydrogel in CPDM. Specifically, adenosine (A) can effectively promote osteogenic differentiation, and OPCB2 has significant antioxidant, anti-inflammatory and pro-osteogenic effects. However, considering that the bioavailability of adenosine (A) and OPCB2 is very low, the bioavailability needs to be increased in actual use, thereby increasing its therapeutic effect. Local drug delivery systems can achieve constant drug concentration and minimize systemic side effects, and are commonly used treatment methods, and the injectable hydrogel obtained by the scheme can be uniformly injected into the gingival pocket, which is convenient for improving drug utilization and data effect. Compared with the widely studied polymer hydrogel, the supramolecular hydrogel obtained by the scheme has better biocompatibility and degradability; in addition, other hydrogels currently used for CPDM are carriers and have no therapeutic function, while the scheme introduces guanosine as a gel factor, and adenosine and OPCB2 are introduced into the gel system as a gel skeleton, and the obtained GAOPCB2 supramolecular hydrogel has both carrier and therapeutic functions, and has a significant effect when used for treating CPDM.

[0075] The above is only an embodiment of the present application, and the specific technical solutions and / or common knowledge of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. An injectable hydrogel for treating periodontitis with diabetes, characterized in that: The raw materials include guanosine, 1,4-phenylboronic acid, adenosine, and proanthocyanidin B2.

2. The injectable hydrogel for treating diabetic periodontitis according to claim 1, characterized in that: The molar ratio of the raw materials guanosine, 1,4-phenylboronic acid, adenosine and proanthocyanidin B2 is 4~8:4~8:2~4:1~2.

3. The injectable hydrogel for treating diabetic periodontitis according to claim 2, characterized in that: The pore size of the hydrogel is 10~25 μm.

4. A method for preparing an injectable hydrogel for treating diabetic periodontitis according to any one of claims 1 to 3, characterized in that: This includes dispersing raw materials in an aqueous solvent, heating to dissolve them, and then cooling to obtain an injectable hydrogel.

5. A method for preparing an injectable hydrogel for treating diabetic periodontitis according to claim 4, characterized in that: The aqueous solvent is PBS buffer.

6. A method for preparing an injectable hydrogel for treating diabetic periodontitis according to claim 5, characterized in that: Potassium hydroxide or sodium hydroxide is added to the PBS buffer to make the pH of the buffer 7.4-8.

7. A method for preparing an injectable hydrogel for treating diabetic periodontitis according to claim 6, characterized in that: The heating process involves raising the solution to 75-90°C until all reagents dissolve, and then cooling it to room temperature.

8. The use of the injectable hydrogel according to any one of claims 1 to 3 or the injectable hydrogel prepared by the method according to any one of claims 5 to 7 in the preparation of a medicament for treating periodontitis with diabetes.

9. The application according to claim 8, characterized in that: The hydrogel is used by injection.

10. The application according to claim 9, characterized in that: The hydrogel drug loading includes either adenosine or proanthocyanidin B2.

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