Preparation method and application of coixol and phellinus igniarius in-situ self-assembled hydrogel

Abstract

The invention discloses a preparation method and application of coixol and phellinus igniarius in-situ self-assembled hydrogel. The preparation method of the coixol and morin in-situ self-assembled hydrogel comprises the following steps: S1, carrying out reaction synthesis on coixol and morin by adopting an in-situ self-assembled crystallization method, so as to obtain Morin-Coixol Ce-MOF nanoparticles; s2, preparing a PNIPAM hydrogel by adopting a reversible addition-fragmentation chain transfer polymerization method; and S3, carrying out a polymerization reaction on the PNIPAM hydrogel and the Morin-Coixol (at) Ce-MOF nanoparticles, so as to obtain the coixol and phellinus igniarius in-situ self-assembled hydrogel. The coixol and phellinus linteus in-situ self-assembled hydrogel can be applied to the treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the field of drug preparation, and in particular to a preparation method and application of an in-situ self-assembled hydrogel of coixol and morin. Background Art

[0002] Osteoarthritis is currently the most common chronic joint disease, primarily caused by wear and tear of the joints, leading to degenerative changes in articular cartilage and secondary bone hyperplasia. The prevalence of osteoarthritis in people over 65 is reported to be approximately 80%, severely impacting the quality of life of middle-aged and elderly individuals. Clinical manifestations of osteoarthritis primarily include joint pain, stiffness, limited mobility, and deformities, which can be disabling in severe cases. The primary physiological pathology of osteoarthritis is articular cartilage damage, which can be caused by a variety of factors, including obesity, aging, strain, trauma, and congenital joint abnormalities. Cartilage is a highly differentiated elastic connective tissue that reduces friction between adjacent bones and cushions the shock of movement. Because articular cartilage is terminally differentiated and lacks vascular and lymphatic drainage, it is difficult to fully heal once damaged. In recent years, research on articular cartilage repair and restoration of damaged cartilage structure and function has become a hot topic in the field of osteoarthritis.

[0003] Traditional Chinese medicine hydrogels have shown significant potential in the treatment of osteoarthritis (OA). Combining traditional Chinese medicine active ingredients with modern biomaterial technologies, they exhibit anti-inflammatory, analgesic, and cartilage repair-promoting effects. These hydrogels not only prolong the drug's retention within the joint cavity but also exhibit excellent biocompatibility and mechanical properties, such as self-repair and lubrication, providing an innovative strategy for OA treatment. However, existing technologies simply physically mix the traditional Chinese medicine monomers with the hydrogels, resulting in reduced bioavailability of the traditional Chinese medicine and an inability to achieve synergistic therapeutic effects. Improvements are needed. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide a preparation method and application of an in situ self-assembled hydrogel of coixol and morin having excellent therapeutic effect on osteoarthritis.

[0005] In order to achieve the above object, the technical solution of the present invention is: A method for preparing an in-situ self-assembled hydrogel of coixol and morin comprises the following steps: S1. Coixol and Morin were synthesized by in situ self-assembly crystallization to obtain Morin-Coixol@Ce-MOF nanoparticles; S2, PNIPAM hydrogel was prepared by reversible addition-fragmentation chain transfer polymerization; S3. Polymerize PNIPAM hydrogel with Morin-Coixol@Ce-MOF nanoparticles to obtain in situ self-assembled hydrogel of coixol and morin.

[0006] Furthermore, the step S1 specifically includes the following steps: S11, directly adding coixol and morin into a three-necked flask filled with pure water, and adding ammonium cerium nitrate and glacial acetic acid to dissolve and react, and stirring at 300 rpm / min in a constant temperature magnetic water bath at 55-65°C for 30 minutes to obtain a Ce source solution, and cooling to room temperature in an ice-water bath; S12, adding terephthalic acid to a beaker containing N,N-dimethylformamide, and placing the beaker in a constant temperature water bath at 80° C. for ultrasonic-assisted dissolution to obtain a DMF solution; S13, slowly add the Ce source solution to the DMF solution, continue stirring for 30 min, then transfer the mixed solution to a polytetrafluoroethylene-lined hydrothermal reactor, seal the hydrothermal reactor, and place it in an oven at 100-150°C for crystallization reaction for 12-36 h; S14. After the hydrothermal reactor is naturally cooled to room temperature, the reaction product is taken out and centrifuged at a speed of 5000 rpm / min. The precipitate is washed with DMF, ethanol and deionized water in sequence. After each washing, the precipitate is centrifuged at a speed of 10000 rpm / min for 30 min. The washing is repeated three times and the product is vacuum dried at room temperature to obtain Morin-Coixol@Ce-MOF nanoparticles.

[0007] Furthermore, in step S11, the mass ratio of coixol to morin is 1:0.5-1.5.

[0008] Furthermore, in step S11, the molar ratio of ammonium cerium phosphate to glacial acetic acid is 0.23:0.8-1.2.

[0009] Furthermore, the step S2 specifically includes the following steps: S21. Under nitrogen protection, the purified NIPAM monomer, CPDB, and AIBN initiator are added to anhydrous N,N-dimethylformamide to prepare a reaction solution; S22. The reaction solution was sealed in a Schlenk bottle and placed in a constant temperature oil bath at 70°C under nitrogen protection. The solution was magnetically stirred and polymerized for 6 h. After the polymerization reaction was completed, an inhibitor was added to terminate the polymerization. The gel solution was obtained after dialysis for 24 h.

[0010] Furthermore, the step S3 specifically includes the following steps: S31, adding the Morin-Coixol@Ce-MOF nanoparticles prepared in step S1 to DMF, and ultrasonically dispersing them for 30 minutes to form a nanoparticle dispersion, which is recorded as dispersion A; S32, adding the gel solution prepared in step S2 to DMF to prepare a RAFT polymerization reaction solution, which is recorded as solution B; S33. Under nitrogen protection and continuous magnetic stirring, slowly transfer dispersion A into solution B, and then continue to mix uniformly under nitrogen protection and magnetic stirring for 15-45 minutes to obtain a mixed dispersion; seal the mixed dispersion in a Schlenk flask, place it in a constant temperature oil bath at 65-75°C under nitrogen protection, and conduct a polymerization reaction with magnetic stirring for 4-8 hours; S34. After the polymerization reaction is completed, the polymerization reaction is quickly terminated by cooling in an ice bath, and the reactants are dialyzed and purified using ultrapure water for 12 to 36 hours to obtain an in situ self-assembled hydrogel of coixol and morin.

[0011] The invention discloses an application of an in-situ self-assembled hydrogel of coixol and morin. The in-situ self-assembled hydrogel of coixol and morin can be applied to the treatment of osteoarthritis.

[0012] Compared with the prior art, the present invention has the following advantages and positive effects: The present invention firstly crystallizes coixol and morpholin by in-situ self-assembly crystallization to obtain Morin-Coixol@Ce-MOF nanoparticles, and then polymerizes Morin-Coixol@Ce-MOF nanoparticles with PNIPAM hydrogel to finally obtain in-situ self-assembled hydrogel of coixol and morpholin. The hydrogel contains coixol and morpholin as main components. Morinolin is a yellow phytochemical extracted from the bark of moraceae plants such as yellow mulberry and orange tree and many Chinese herbal medicines. Ketone compounds have multiple effects such as antioxidant, antibacterial, anti-atherosclerotic, anti-inflammatory and immune, and anti-tumor. Coixin is a natural active ingredient extracted from coix seed (Job's tears), mainly containing polysaccharides, triterpenoid compounds, etc., with potential effects such as anti-inflammatory, antioxidant, immune regulation, and anti-tumor. By crystallizing coixin and mulberry yellow, and polymerizing them with PNIPAM hydrogel, the bioavailability of traditional Chinese medicine can be increased, achieving a synergistic treatment effect on diseases, thereby improving the therapeutic effect on osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0014] Figure 1 is the Zeta potential diagram of SA; Figure 2 is the transmission electron microscopy image of SA; Figure 3 Zeta potential diagram of Ce-MOF; Figure 4 Transmission electron microscopy image of Ce-MOF; Figure 5 Zeta potential diagram of SA@Ce-MOF; Figure 6 This is the scanning electron microscopy image of SA@Ce-MOF@hydrogel; Figure 7 Cytotoxicity diagram of SA@Ce-MOF@hydrogel; Figure 8 Schematic diagram of the OD value of each component's protective effect on OA chondrocytes; Figure 9 is the immunofluorescence image of COL2 in chondrocytes; Figure 10 This is a pathological staining image of the knee joint at 8 weeks. DETAILED DESCRIPTION

[0015] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts, any modifications, equivalent replacements, improvements, etc., shall be included in the scope of protection of the present invention.

[0016] This embodiment discloses a method for preparing an in situ self-assembled hydrogel of coixol and morin, comprising the following steps: S1. Coixol and Morin were synthesized by in situ self-assembly crystallization to obtain Morin-Coixol@Ce-MOF nanoparticles; S11, directly adding coixol and morin into a three-necked flask filled with pure water, and adding ammonium cerium nitrate and glacial acetic acid to dissolve and react, and stirring at 300 rpm / min in a constant temperature magnetic water bath at 55-65°C for 30 minutes to obtain a Ce source solution, and cooling to room temperature in an ice-water bath; The mass ratio of coixol to morpholin is 1:0.5~1.5; the molar ratio of ammonium cerium acid to glacial acetic acid is 0.23:0.8~1.2.

[0017] S12, adding terephthalic acid to a beaker containing N,N-dimethylformamide, and placing the beaker in a constant temperature water bath at 80° C. for ultrasonic-assisted dissolution to obtain a DMF solution; S13, slowly add the Ce source solution to the DMF solution, continue stirring for 30 min, then transfer the mixed solution to a polytetrafluoroethylene-lined hydrothermal reactor, seal the hydrothermal reactor, and place it in an oven at 100-150°C for crystallization reaction for 12-36 h; S14. After the hydrothermal reactor is naturally cooled to room temperature, the reaction product is taken out and centrifuged at a speed of 5000 rpm / min. The precipitate is washed with DMF, ethanol and deionized water in sequence. After each washing, the precipitate is centrifuged at a speed of 10000 rpm / min for 30 min. The washing is repeated three times and the product is vacuum dried at room temperature to obtain Morin-Coixol@Ce-MOF nanoparticles.

[0018] S2, PNIPAM hydrogel was prepared by reversible addition-fragmentation chain transfer polymerization; S21. Under nitrogen protection, the purified NIPAM monomer, CPDB, and AIBN initiator are added to anhydrous N,N-dimethylformamide to prepare a reaction solution; S22. The reaction solution was sealed in a Schlenk bottle and placed in a constant temperature oil bath at 70°C under nitrogen protection. The solution was magnetically stirred and polymerized for 6 h. After the polymerization reaction was completed, an inhibitor was added to terminate the polymerization. The gel solution was obtained after dialysis for 24 h.

[0019] S3. Polymerize PNIPAM hydrogel with Morin-Coixol@Ce-MOF nanoparticles to obtain in situ self-assembled hydrogel of coixol and morin.

[0020] S31, adding the Morin-Coixol@Ce-MOF nanoparticles prepared in step S1 to DMF, and ultrasonically dispersing them for 30 minutes to form a nanoparticle dispersion, which is recorded as dispersion A; S32, adding the gel solution prepared in step S2 to DMF to prepare a RAFT polymerization reaction solution, which is recorded as solution B; S33. Under nitrogen protection and continuous magnetic stirring, slowly transfer dispersion A into solution B, and then continue to mix uniformly under nitrogen protection and magnetic stirring for 15-45 minutes to obtain a mixed dispersion; seal the mixed dispersion in a Schlenk flask, place it in a constant temperature oil bath at 65-75°C under nitrogen protection, and conduct a polymerization reaction with magnetic stirring for 4-8 hours; S34. After the polymerization reaction is completed, the polymerization reaction is quickly terminated by cooling in an ice bath, and the reactants are dialyzed and purified using ultrapure water for 12 to 36 hours to obtain an in situ self-assembled hydrogel of coixol and morin.

[0021] The prepared coixol and morin in situ self-assembled hydrogel can be used in the treatment of osteoarthritis.

[0022] The following specific experiments are used to verify the effect of the in situ self-assembly hydrogel of coixol and morin in the present invention:

[0023] Experimental Example 1 Preparation of Morin-Coixol self-assembled nanoparticles: The pH gradient-induced non-covalently driven self-assembly method was adopted. The specific method was as follows: 8 mg of coixol and 8 mg of morin (coixol: morin = 1:0.5~1.5 (mass ratio)) were accurately weighed and dissolved in 1 ml of anhydrous ethanol respectively. The mixture was ultrasonicated for 30 min (15-30 min) until completely dissolved to prepare coixol ethanol solution and morin ethanol solution. After the prepared coixol and morin ethanol solutions were mixed evenly, 20 ml (15-30 ml) of phosphate buffered saline (PBS) adjusted to pH 6.0 (5.8-6.4) was precisely measured and placed in a beaker. Under magnetic stirring, the drug-mixed ethanol solution was slowly injected into the PBS buffer at a rate of 1 ml / min (a microinjection pump precisely controlled the dripping speed). Stirring was maintained for 1 h (0.5-1.5 h). The mixture was transferred to a dialysis bag and dialyzed against PBS buffer at pH 7.4 for 12 h (8-24 h). The dialysate was replaced every 4 h to remove ethanol and unassembled drug molecules. After dialysis, the dialysate was filtered using a 0.22 μm filter membrane to remove large unassembled particles and impurities. The filtrate was collected to obtain a dispersion of Morin-Coixol self-assembled nanoparticles. Centrifuge at 13000 rpm (8000~15000 rpm) for 3 h (2-4 h) to obtain the bottom precipitate. After vacuum drying, Morin-Coixol self-assembled nanoparticles can be obtained; this is the SA in the following comparative experiment, and its performance and structure are as follows Figure 1 、 Figure 2 shown.

[0024] Experimental Example 2 Ce-MOF was prepared using a solvothermal method. The following steps were taken: 1.17 g of ammonium cerium nitrate and 122 μl of glacial acetic acid (ammonium cerium nitrate: glacial acetic acid = 0.23:0.8-1.2 (molar ratio)) were accurately weighed and added to a three-necked flask containing 4 ml of pure water. The mixture was placed in a magnetic water bath at 60°C (55-65°C) and stirred at 300 rpm for 30 min to prepare a Ce source solution. The solution was then cooled to room temperature in an ice-water bath. 0.355 g of terephthalic acid (BDC) was accurately weighed and added to a beaker containing 18.7 ml of N,N-dimethylformamide (DMF). The solution was then ultrasonically dissolved in a water bath at 80°C to prepare a DMF solution. The Ce source solution was slowly added to the DMF solution and stirred continuously for 30 min. The mixed solution was then transferred to a hydrothermal reactor, sealed, and placed in a 120°C oven for crystallization for 24 h. After the reactor was cooled to room temperature naturally, the reaction product was taken out and centrifuged at 5000 rpm. The precipitate was washed with DMF, ethanol and deionized water in sequence. After each washing, the precipitate was centrifuged at 10000 rpm for 30 min. The washing was repeated 3 times to obtain Ce-MOF material; this is the Ce-MOF in the following comparative experiment. Its performance and structure are shown in FIG. Figure 3 、 Figure 4 shown.

[0025] Experimental Example 3 Preparation of Morin-Coixol@Ce-MOF: The in situ self-assembly crystallization method was adopted. The specific method was as follows: during the preparation stage of the Ce source solution for the synthesis of Ce-MOF materials, 8 mg of high-purity coixol and 8 mg of high-purity morin (coixol: morin = 1:0.5~1.5 (mass ratio)) were directly added to a three-necked flask containing 4 ml of pure water, and dissolved and reacted simultaneously with 1.17 g of ammonium cerium nitrate and 122 μl of glacial acetic acid (ammonium cerium nitrate: glacial acetic acid = 0.23:0.8~1.2 (molar ratio)). The mixture was stirred at 300 rpm in a constant temperature magnetic water bath at 60°C (55~65°C) for 30 min. According to the Ce-MOF material synthesis steps in Experimental Example 2, the above Ce source solution was slowly added to the pre-prepared DMF solution containing BDC (0.355 g BDC dissolved in 18.7 ml DMF), mixed evenly and transferred to a polytetrafluoroethylene-lined hydrothermal reactor. The sealed reactor was then placed in a 120 ° C (100~150 ° C) oven for crystallization reaction for 24 h (12~36 h). After the reactor was naturally cooled to room temperature, the reaction product was taken out and centrifuged at 5000 rpm. The precipitate was washed with DMF, ethanol and deionized water in sequence. After each washing, it was centrifuged at 10000 rpm for 30 min. The washing was repeated 3 times and vacuum dried at room temperature to obtain a Ce-MOF composite material with self-assembled co-loaded Morin + Coixol; this is the SA@Ce-MOF in the following comparative experiment; its performance is as follows Figure 5 shown.

[0026] Experimental Example 4 PNIPAM hydrogels were prepared using the reversible addition-fragmentation chain transfer (RAFT) method. 4-Cyanovaleranodithiobenzoic acid (CPDB), N-isopropylacrylamide (NIPAM) monomers (purified by recrystallization before use), and azobisisobutyronitrile (AIBN) initiator (purified by recrystallization) were prepared in advance. All reagents were deoxygenated before use. Under nitrogen, 10.00 g of purified NIPAM monomer, 0.27 g of CPDB, and 0.033 g of AIBN initiator were added to 10 ml of deoxygenated anhydrous N,N-dimethylformamide (DMF) to prepare a reaction solution. The reaction solution was then sealed in a Schlenk flask and placed in a 70°C oil bath under nitrogen for 6 hours with magnetic stirring. After the polymerization was completed, the polymerization inhibitor hydroquinone was added to terminate the polymerization, and the mixture was dialyzed for 24 hours to remove unreacted monomer, RAFT reagent, initiator, and byproducts.

[0027] Experimental Example 5 Preparation of Morin-Coixol@Ce-MOF@PNIPAM: An in situ encapsulation method was used. Specifically, 50 mg (25-75 mg) of the Morin-Coixol@Ce-MOF nanoparticles prepared in Experimental Example 3 were dispersed in 10 ml of DMF and ultrasonically dispersed for 30 minutes to form a uniform and stable nanoparticle dispersion, designated as Dispersion A. The reaction solution from the previous PNIPAM hydrogel preparation method was added to 10 ml of DMF to prepare a RAFT polymerization reaction solution, designated as Solution B. Under nitrogen protection and continuous magnetic stirring, Dispersion A was slowly transferred to Solution B, and then mixed for 30 minutes (15-45 minutes) under nitrogen protection and magnetic stirring to ensure uniform dispersion of Morin-Coixol@Ce-MOF in the RAFT polymerization reaction system. The mixed dispersion was sealed in a Schlenk flask and placed in a constant temperature oil bath at 70°C (65-75°C) under nitrogen protection. The mixture was stirred magnetically for 6 hours (4-8 hours) to allow the PNIPAM hydrogel to in situ encapsulate Morin-Coixol@Ce-MOF during the RAFT polymerization process. After the polymerization reaction was completed, the mixture was quickly terminated by cooling in an ice bath and then dialyzed with ultrapure water for 24 hours (12-36 hours) to obtain a PNIPAM composite hydrogel loaded with Morin-Coixol@Ce-MOF nanoparticles (Morin-Coixol@Ce-MOF@PNIPAM), which is the SA@Ce-MOF@hydrogel in the comparative experiment below. Its structure is shown below. Figure 6 shown.

[0028] The following comparative experiments are conducted on the finished products prepared according to the above experimental examples to demonstrate their technical effects. 1. In vitro experiments 1. Cell culture: 1.1 Primary chondrocytes were isolated from the knee articular cartilage of newborn mice.

[0029] 1.2 Cut the cartilage tissue into 1 mm 3 Small pieces were digested with 0.25% trypsin for 0.5 h and then with 0.2% type II collagenase for 4 h in a shaking incubator at 90 rpm.

[0030] 1.3 Collect the released chondrocytes and culture them in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin solution in a 37°C, 5% CO2 incubator.

[0031] 1.4 Use chondrocytes within the second generation for experiments.

[0032] 2. Cytotoxicity Assay (CCK8) 2.1 Chondrocytes were seeded into 96-well plates at a density of 0.5 × 10^4 / ml, with 6 replicate wells per group, and cultured for 12 hours.

[0033] 2.2 The experiment was divided into the following 6 groups: 1) Normal group: culture medium only 2) Culture medium + SA@Ce-MOF@hydrogel (500ng / mL) 3) Culture medium + SA@Ce-MOF@hydrogel (1000ng / mL) 4) Culture medium + SA@Ce-MOF@hydrogel (2000ng / mL) 5) Culture medium + SA@Ce-MOF@hydrogel (4000ng / mL) 6) Culture medium + SA@Ce-MOF@hydrogel (8,000 ng / mL) 2.3 Cultivation time: 1, 3, 5, 7 days.

[0034] 2.4 Cell viability assay: CCK-8 kit was used to detect the viability of cells in each group and the cell survival rate was calculated. The results are as follows: Figure 7 shown.

[0035] 2.5 Method: Remove the culture medium from the 96-well plate, rinse once with PBS, prepare complete culture medium containing 10% CCK8, add 100 μl of the prepared culture medium to each well, protect from light, and incubate in an incubator for 2 hours.

[0036] 2.6 Experimental results: SA@Ce-MOF@hydrogel has a proliferative effect on chondrocytes at concentrations of 500ng / ml to 2000ng / ml, and has an inhibitory effect on chondrocytes at concentrations of 4000ng / ml and 8000ng / ml.

[0037] 3. Chondrocyte Proliferation Assay (CCK8) 3.1 Chondrocytes were seeded into 96-well plates at a density of 0.5 × 10 / ml, with 6 replicate wells per group.

[0038] 3.2: Divide cells into the following 6 groups (CCK8): 1) Normal group: culture medium only 2) OA group: culture medium + TNF-α (10 ng / mL) 3) SA group: TNF-α +SA (500ng / ml) 4) Ce-MOF group: TNF-α + Ce-MOF (500ng / ml) 5) SA@Ce-MOF group: TNF-α +SA@Ce-MOF (500ng / ml) 6) SA@Ce-MOF@hydrogel group: TNF-α +SA@Ce-MOF@hydrogel (500ng / ml) 3.3 Cultivation time: 1, 3, 5, 7 days.

[0039] 3.4 Cell viability assay: CCK-8 kit was used to detect the viability of cells in each group and the cell survival rate was calculated. The results are as follows: Figure 8 shown.

[0040] 3.5 Method: Remove the culture medium from the 96-well plate, rinse once with PBS, prepare complete culture medium containing 10% CCK8, add 100 μl of the prepared culture medium to each well, protect from light, and incubate in an incubator for 2 hours. (Assay duration: 1, 3, 5, or 7 days.) 3.6 Results: SA and Ce-MOF had a certain protective effect on TNF-α-treated chondrocytes, but no significant proliferative effect. SA@Ce-MOF and SA@Ce-MOF@hydrogel not only had a protective effect on TNF-α-treated chondrocytes, but also had a proliferative effect.

[0041] 4. Immunofluorescence Staining of Chondrocyte COL2 4.1 Methods: Macrophages were seeded into 96-well plates at a density of 0.5× 10^4 / ml, with three replicates per experimental group, and cultured for 12 hours.

[0042] 4.2 M0 macrophages are divided into the following 6 groups: 1) Normal group: culture medium only 2) LPS group (M1 inducer): LPS (100 ng mL) 3) SA group: LPS + SA (500 ng / ml) 4) Ce-MOF group: LPS+Ce-MOF (500ng / ml) 5) SA@Ce-MOF group: LPS+SA@Ce-MOF (500ng / ml) 6) SA@Ce-MOF@hydrogel group: LPS+SA@Ce-MOF@hydrogel (500ng / ml) 4.3 After 24 hours of co-culture, macrophages and chondrocytes cultured using the above method were re-seeded in a 96-well plate at a 1:1 ratio and co-cultured for 3 days. The cells were then immunostained for COL2.

[0043] 4.4 Staining method: 1) Remove the culture medium and add PBS to wash for 3 minutes.

[0044] 2) Add 4% paraformaldehyde to fix for 15 minutes, remove the fixative, and add PBS to rinse for 3 x 3 minutes.

[0045] 3) Add 0.5% Triton 100 to permeabilize for 20 minutes, and then add PBS to rinse for 3 x 3 minutes.

[0046] 4) Block with 10% goat serum for 30 minutes, remove the goat serum, and add 50 μl of 1:100 COL2 primary antibody for overnight incubation at 4°C.

[0047] 5) Recover the primary antibody and rinse with PBS for 3 x 3 minutes. Add 50 μl of red fluorescent-labeled goat anti-rabbit secondary antibody and incubate at 37°C in the dark for 1 hour. Wash with PBS for 3 x 3 minutes.

[0048] 6) Add DAPI to stain the nucleus for 10 minutes, then add PBS to wash for 3 x 3 minutes. Take pictures with a fluorescence microscope. The results are as follows Figure 9 shown.

[0049] 4.5 Results: SA@Ce-MOF@hydrogel and SA@Ce-MOF can promote the synthesis of chondrocyte matrix by promoting the polarization of macrophages to M2 type.

[0050] 2. In vivo experiments 1. Establishment of rat osteoarthritis model.

[0051] 1.1 Animal model establishment: Adult male SD rats were used to establish an OA model by transection of the anterior cruciate ligament and medial meniscus.

[0052] 1.2 All animals were randomly divided into 6 groups 1) Normal group: normal rats 2). OA group: PBS solution only 3) SA@Ce-MOF group: SA@Ce-MOF 4)SA@Ce-MOF@Hydrogel group: SA@Ce-MOF@Hydrogel 2. Collection, embedding and preparation of knee joint specimens.

[0053] 2.1 The knee joints of the rats were removed at 8 weeks.

[0054] 2.2 Fix in 10% formalin for 24 hours, rinse with running water for 2 minutes, add 10% EDTA decalcification solution, and decalcify for 4-6 weeks. Wait until the bone tissue can be pierced by a needle without resistance. Dehydrate with graded alcohol, clear with xylene, and dip in wax for 3 hours. Embed and cut into 4 μm thick sections. Dewax and hydrate the sections.

[0055] 2.3 Masson and Safranin-Fast Green Staining Steps: 1) Dewaxing and hydrating sections 2) Stain according to the kit instructions.

[0056] 3) Dehydrate and transparent the slices.

[0057] 4) Seal the slides with neutral gum and take photos. Figure 10 shown.

[0058] 2.4 Results: SA@Ce-MOF@hydrogel and SA@Ce-MOF demonstrated significant therapeutic effects on rat osteoarthritis induced by anterior cruciate ligament and medial meniscus transection. The joint surfaces of rats treated with SA@Ce-MOF@hydrogel were smoother and the cartilage layer thicker than those treated with SA@Ce-MOF. This suggests that SA@Ce-MOF@hydrogel exhibits superior therapeutic efficacy compared to SA@Ce-MOF.

[0059] The present invention firstly crystallizes coixol and morpholin by in-situ self-assembly crystallization to obtain Morin-Coixol@Ce-MOF nanoparticles, and then polymerizes Morin-Coixol@Ce-MOF nanoparticles with PNIPAM hydrogel to finally obtain in-situ self-assembled hydrogel of coixol and morpholin. The hydrogel contains coixol and morpholin as main components. Morinolin is a yellow phytochemical extracted from the bark of moraceae plants such as yellow mulberry and orange tree and many Chinese herbal medicines. Ketone compounds have multiple effects such as antioxidant, antibacterial, anti-atherosclerotic, anti-inflammatory and immune, and anti-tumor. Coixin is a natural active ingredient extracted from coix seed (Job's tears), mainly containing polysaccharides, triterpenoid compounds, etc., with potential effects such as anti-inflammatory, antioxidant, immune regulation, and anti-tumor. By crystallizing coixin and mulberry yellow, and polymerizing them with PNIPAM hydrogel, the bioavailability of traditional Chinese medicine can be increased, achieving a synergistic treatment effect on diseases, thereby improving the therapeutic effect on osteoarthritis.

Claims

1. A method for preparing an in situ self-assembled hydrogel of coixol and morin, characterized in that: The following steps are involved: S1. Coixol and Morin were synthesized by in situ self-assembly crystallization to obtain Morin-Coixol@Ce-MOF nanoparticles; S2, PNIPAM hydrogel was prepared by reversible addition-fragmentation chain transfer polymerization; S3. Polymerize PNIPAM hydrogel with Morin-Coixol@Ce-MOF nanoparticles to obtain in situ self-assembled hydrogel of coixol and morin.

2. The method for preparing the in situ self-assembled hydrogel of coixol and morin according to claim 1, wherein: The step S1 specifically includes the following steps: S11, directly adding coixol and morin into a three-necked flask filled with pure water, and adding ammonium cerium nitrate and glacial acetic acid to dissolve and react, and stirring at 300 rpm / min in a constant temperature magnetic water bath at 55-65°C for 30 minutes to obtain a Ce source solution, and cooling to room temperature in an ice-water bath; S12, adding terephthalic acid to a beaker containing N,N-dimethylformamide, and placing the beaker in a constant temperature water bath at 80° C. for ultrasonic-assisted dissolution to obtain a DMF solution; S13, slowly add the Ce source solution to the DMF solution, continue stirring for 30 min, then transfer the mixed solution to a polytetrafluoroethylene-lined hydrothermal reactor, seal the hydrothermal reactor, and place it in an oven at 100-150°C for crystallization reaction for 12-36 h; S14. After the hydrothermal reactor is naturally cooled to room temperature, the reaction product is taken out and centrifuged at a speed of 5000 rpm / min. The precipitate is washed with DMF, ethanol and deionized water in sequence. After each washing, the precipitate is centrifuged at a speed of 10000 rpm / min for 30 min. The washing is repeated three times and the product is vacuum dried at room temperature to obtain Morin-Coixol@Ce-MOF nanoparticles.

3. The method for preparing the in situ self-assembled hydrogel of coixol and morin according to claim 2, wherein: In step S11, the mass ratio of coixol to morin is 1:0.5-1.

5.

4. The method for preparing the in situ self-assembled hydrogel of coixol and morin according to claim 3, wherein: In step S11, the molar ratio of ammonium cerium phosphate to glacial acetic acid is 0.23:0.8-1.

2.

5. The method for preparing the in situ self-assembled hydrogel of coixol and morin according to claim 4, wherein: The step S2 specifically includes the following steps: S21. Under nitrogen protection, the purified NIPAM monomer, CPDB, and AIBN initiator are added to anhydrous N,N-dimethylformamide to prepare a reaction solution; S22. The reaction solution was sealed in a Schlenk bottle and placed in a constant temperature oil bath at 70°C under nitrogen protection. The solution was magnetically stirred and polymerized for 6 h. After the polymerization reaction was completed, an inhibitor was added to terminate the polymerization. The gel solution was obtained after dialysis for 24 h.

6. The method for preparing the in situ self-assembled hydrogel of coixol and morin according to claim 5, wherein: The step S3 specifically includes the following steps: S31, adding the Morin-Coixol@Ce-MOF nanoparticles prepared in step S1 to DMF, and ultrasonically dispersing them for 30 minutes to form a nanoparticle dispersion, which is recorded as dispersion A; S32, adding the gel solution prepared in step S2 to DMF to prepare a RAFT polymerization reaction solution, which is recorded as solution B; S33. Under nitrogen protection and continuous magnetic stirring, slowly transfer dispersion A into solution B, and then continue to mix uniformly under nitrogen protection and magnetic stirring for 15-45 minutes to obtain a mixed dispersion; seal the mixed dispersion in a Schlenk flask, place it in a constant temperature oil bath at 65-75°C under nitrogen protection, and conduct a polymerization reaction with magnetic stirring for 4-8 hours; S34. After the polymerization reaction is completed, the polymerization reaction is quickly terminated by cooling in an ice bath, and the reactants are dialyzed and purified using ultrapure water for 12 to 36 hours to obtain an in situ self-assembled hydrogel of coixol and morin.

7. An application of an in situ self-assembled hydrogel of coixol and morin, wherein the in situ self-assembled hydrogel of coixol and morin is prepared by the preparation method of the in situ self-assembled hydrogel of coixol and morin according to claim 6; the in situ self-assembled hydrogel of coixol and morin can be used in the treatment of osteoarthritis.

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