Dual-drug-loading temperature-sensitive composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses dual-drug-loading temperature-sensitive composite hydrogel as well as a preparation method and application thereof. The composite hydrogel comprises a temperature-sensitive hydrogel matrix, an anti-inflammatory natural medicine extract and composite microspheres, wherein the anti-inflammatory natural medicine extract and the composite microspheres are dispersed in the temperature-sensitive hydrogel matrix; the composite microspheres comprise hydrogel microspheres loaded with bone-strengthening traditional Chinese medicine extracellular vesicles. The composite hydrogel disclosed by the invention has a'microsphere-gel 'composite system, local long-acting slow release and efficient anti-inflammation of an anti-inflammatory natural medicine extract are realized by utilizing a temperature-sensitive hydrogel matrix, and more importantly, bone-strengthening traditional Chinese medicine plant-derived extracellular vesicles are packaged in hydrogel microspheres as active ingredients; the nano-material can act on a bone joint interface in a targeting manner, and can promote osteogenesis and inhibit osteoclast enduringly, so that two major core pathological processes of RA inflammation and bone erosion are intervened at the same time.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a double drug-loaded temperature-sensitive composite hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis, cartilage destruction and bone erosion. Current clinical treatment drugs (such as DMARDs and biological agents) have problems such as short half-life, large systemic side effects, difficulty in sustained and effective release at the lesion site, and limited ability to promote bone repair. Specifically, these drugs have a short half-life, requiring frequent administration, and the drug accumulation concentration at the lesion joint site is limited after systemic administration, resulting in poor local treatment effect. Secondly, non-targeted distribution makes the drug easily distributed in non-diseased tissues, causing significant systemic side effects, such as increased risk of infection due to immune suppression, liver and kidney toxicity, etc. Furthermore, existing therapies mainly focus on inhibiting inflammation and immune response, and lack effective repair or reversal ability for cartilage destruction and bone erosion during the progression of RA, making it difficult to achieve true protection and reconstruction of joint structure. In addition, some potential natural active ingredients have not been able to fully exert their therapeutic potential due to their low solubility, fast in vivo metabolism, and poor bioavailability.

[0003] Hydrogels, especially temperature-sensitive hydrogels, have become ideal carriers for local drug delivery due to their good biocompatibility and controllable drug release characteristics. However, a single hydrogel system is difficult to achieve the synergistic delivery and differential release kinetics of different nature drugs (such as hydrophilic small molecule SIN and lipophilic biological macromolecule DR-EVs). SUMMARY

[0004] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the purpose of the present application is to provide a double drug-loaded temperature-sensitive composite hydrogel and a preparation method and application thereof.

[0005] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is: The first aspect of the present application provides a composite hydrogel, comprising a temperature-sensitive hydrogel matrix and an anti-inflammatory natural medicine extract and composite microspheres dispersed in the temperature-sensitive hydrogel matrix; the composite microspheres comprise hydrogel microspheres loaded with bone-strengthening traditional Chinese medicine extracellular vesicles.

[0006] The strong bone traditional Chinese medicine usually has problems of low solubility, poor stability, weak targeting, poor bioavailability, and non-specific in vivo distribution, which limits the full play of its curative effect. In the present application, the strong bone traditional Chinese medicine extracellular vesicles naturally load various active molecules (such as miRNA, protein, lipid) of the parent plant, have excellent biocompatibility, low immunogenicity and potential targeting, and can more efficiently and stably target deliver the therapeutic activity of the strong bone traditional Chinese medicine ingredients; the strong bone traditional Chinese medicine extracellular vesicles are loaded in the hydrogel microspheres, which not only can protect the biological activity of the strong bone traditional Chinese medicine extracellular vesicles, but also can provide a physical barrier for the slow release of the strong bone traditional Chinese medicine extracellular vesicles to achieve long-acting release; and the anti-inflammatory natural medicine extract and the composite microspheres are loaded in the temperature-sensitive hydrogel matrix, which can rapidly gel at a certain temperature when applied to lock the anti-inflammatory natural medicine extract and the composite microspheres in situ on the lesion, and can provide a carrier for the initial rapid release of the anti-inflammatory natural medicine extract, so as to realize the timely, appropriate and differential release of different therapeutic function drugs in different disease courses (acute attack period, chronic persistent period, tissue repair and reconstruction period), achieve the synchronous solution of efficient anti-inflammatory immune regulation and long-acting bone repair, and meet the whole treatment of the disease.

[0007] In some embodiments, the composite hydrogel is in a liquid state at 2-6℃ (such as 3-5℃); and the composite hydrogel can be converted into a solid state at 35-40℃. In the present application, the composite hydrogel in this state has flowability and injectability at low temperature, and gels at normal human body temperature to realize in situ gelation.

[0008] In some embodiments, the mass concentration of the composite microspheres in the composite hydrogel is 0.5-10% (w / v), such as 1-8wt% (w / v), 1-5wt% (w / v). In the present application, the composite microspheres with this loading amount can effectively disperse and regulate the effective concentration of the strong bone traditional Chinese medicine extracellular vesicles.

[0009] In some embodiments, the mass concentration of the anti-inflammatory natural medicine extract in the composite hydrogel is 0.5-5mg / mL, such as 1-4 mg / mL, etc.

[0010] In some embodiments, the loading amount of the strong bone traditional Chinese medicine extracellular vesicles in the composite microspheres is 50-200μg / mL.

[0011] In some embodiments, the average particle size of the hydrogel microspheres is 300-500μm, such as 350μm, 400μm, 450μm, etc. In the present application, the microspheres with this average particle size can be effectively dispersed in the temperature-sensitive hydrogel matrix and have good monodispersity, and can realize the controlled release of the strong bone traditional Chinese medicine extracellular vesicles.

[0012] In some embodiments, the hydrogel microspheres comprise three-dimensional porous structure hydrogel microspheres; the average pore size of the porous mesopores is 3-15 μm, such as 5-10 μm, etc. In the present application, the three-dimensional porous structure hydrogel microspheres not only can serve as the "nest" of the bone-strengthening traditional Chinese medicine extracellular vesicles, but also can regulate the release behavior to achieve long-acting release; and can provide a scaffold for cell migration and tissue ingrowth, which is conducive to tissue repair.

[0013] In some embodiments, the hydrogel microspheres comprise at least one of methacrylated gelatin (GelMA) hydrogel microspheres, methacrylated hyaluronic acid hydrogel microspheres, and methacrylated alginic acid hydrogel microspheres. In the present application, the methacrylated gelatin hydrogel microspheres have good biodegradability, cell adhesion (because they contain arginine-glycine-aspartic acid sequences) and adjustable physical properties, and can better play the role of carriers and scaffolds.

[0014] In some embodiments, the anti-inflammatory natural medicine extract comprises at least one of total paeonol, triptolide, curcumin, sinomenine (SIN), and naringenin. In the present application, the above anti-inflammatory natural medicine extract has both immunosuppressive and anti-inflammatory effects, and can play a therapeutic role in the early stage when applied, relieve pain, and prevent further tissue damage; among them, sinomenine can effectively inhibit the inflammatory pathways such as NF-κB and JAK-STAT by inhibiting M1 macrophage polarization and correcting Th17 / Treg immune cell imbalance, thereby relieving synovitis.

[0015] In some embodiments, the bone-strengthening traditional Chinese medicine extracellular vesicles comprise bone repair cell extracellular vesicles (DR-EVs) and / or epimedium extracellular vesicles. In the present application, the bone repair cell extracellular vesicles target the bone microenvironment and bidirectionally regulate bone metabolism by promoting osteoblast differentiation and inhibiting osteoclast generation; the epimedium extracellular vesicles have the ability to promote mesenchymal stem cell proliferation and osteogenic differentiation.

[0016] In some embodiments, the average particle size of the bone-strengthening traditional Chinese medicine extracellular vesicles is 50-150 nm, such as 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 90 nm, 100 nm, 120 nm, etc. In the present application, the particle size distribution is determined by Nanoparticle Tracking Analysis (NTA). In the present application, the bone-strengthening traditional Chinese medicine extracellular vesicles with the particle size can be effectively loaded into the three-dimensional porous structure hydrogel microspheres to provide a guarantee for subsequent long-acting release.

[0017] In some embodiments, the temperature-sensitive hydrogel matrix comprises a temperature-sensitive hydrophilic polymer, a glycosaminoglycan, and a polyol.

[0018] In some embodiments, the temperature-sensitive hydrogel base comprises 5-10% (e.g., 6%, 7%, 8%, 9%, etc.) temperature-sensitive hydrophilic polymer, 1-3% (e.g., 1.5%, 2.0%, 2.5%, etc.) glycosaminoglycan, and 10-25% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, etc.) polyol by mass concentration. In the present application, the hydrogel with such mass concentration composition can achieve good temperature-sensitive gelation behavior.

[0019] In some embodiments, the temperature-sensitive hydrophilic polymer comprises at least one of sodium carboxymethylcellulose, hydroxyethyl cellulose, and hydroxypropyl methylcellulose (HPMC), poly(hydroxyethyl methacrylate), poly(2-hydroxyethyl methacrylate), and ethyl cellulose.

[0020] In some embodiments, the glycosaminoglycan comprises hyaluronic acid and / or chondroitin sulfate. In the present application, hyaluronic acid (HA) is the main component of synovial fluid and cartilage, and has excellent lubricating, moisturizing, and anti-inflammatory properties.

[0021] In some embodiments, the polyol comprises at least one of glycerol, propylene glycol, polyethylene glycol, sorbitol, and xylitol. In the present application, glycerol acts as a humectant and plasticizer, reduces the phase transition temperature of the gel, increases the toughness and ductility of the material, thereby adjusting the mechanical properties and degradation rate of the gel.

[0022] In a second aspect of the present application, a preparation method of the composite hydrogel is provided, comprising the following steps: At low temperature, the anti-inflammatory natural medicine extract and the composite microspheres are added to the temperature-sensitive hydrogel base, and the composite hydrogel is prepared after dispersion.

[0023] In some embodiments, the low temperature is at a temperature of 1-6°C, such as 2-5°C, 4°C, etc.

[0024] In some embodiments, the preparation method of the composite microspheres comprises the following steps: adding a photoinitiator and bone-strengthening traditional Chinese medicine extracellular vesicles to a hydrogel microsphere precursor solution, performing microfluidic emulsification and photocrosslinking, and obtaining the composite microspheres.

[0025] In some embodiments, the specific operation of the microfluidic emulsification comprises: using the hydrogel microsphere precursor solution containing the photoinitiator and the bone-strengthening traditional Chinese medicine extracellular vesicles as the water phase, using liquid paraffin containing 1-5% (v / v) Span 80 as the oil phase, and injecting the water phase at a flow rate of 400-600 μL / h and the oil phase at a flow rate of 1300-1700 μL / h into the microfluidic chip for shearing to obtain droplets.

[0026] In some embodiments, the specific operation of the photo-crosslinking comprises crosslinking reaction of the microfluidic emulsification product under irradiation of 350-410 nm ultraviolet light; the intensity of the ultraviolet light is 8-15 mW / cm 2 , such as 10 mW / cm 2 ; the irradiation time is 20-30 s.

[0027] In some embodiments, the preparation method of the bone-strengthening traditional Chinese medicine extracellular vesicle comprises the following steps: homogenizing the bone-strengthening traditional Chinese medicine in a buffer solution, then performing differential centrifugation, and then performing ultracentrifugation on the supernatant to obtain the bone-strengthening traditional Chinese medicine extracellular vesicle.

[0028] In some embodiments, the specific operation of the differential centrifugation comprises: 300×g-700×g (such as 400×g, 500×g, 600×g) centrifugation for 10-30 minutes (such as 15 minutes, 20 minutes, 25 minutes), 1000×g-3000×g (such as 1500×g, 2000×g, 2500×g) centrifugation for 20-40 minutes (such as 25 minutes, 30 minutes, 35 minutes), and 8000×g-15000×g (such as 10000×g, 12000×g, 14000×g) centrifugation for 40-70 minutes (such as 50 minutes, 60 minutes).

[0029] In some embodiments, the specific operation of the ultracentrifugation comprises: 80,000×g-200,000×g (such as 100,000×g, 150,000×g) centrifugation for 60-80 minutes.

[0030] In some embodiments, the differential centrifugation and / or the ultracentrifugation are performed at low temperature; the temperature of the low temperature is 1-6℃, such as 2-5℃, 4℃, etc.

[0031] In some embodiments, the preparation method of the hydrogel matrix comprises the following steps: adding a temperature-sensitive hydrophilic polymer into a glycosaminoglycan solution, and then adding a polyol after hydration to obtain the hydrogel matrix.

[0032] In a third aspect of the present application, the composite hydrogel is provided for use in the preparation of a bone repair drug.

[0033] In some embodiments, the drug comprises at least one of a drug for promoting bone repair, treating bone erosion and / or cartilage damage, preventing and / or treating arthritis (such as rheumatoid arthritis).

[0034] In some embodiments, the drug inhibits osteoclast activity and / or promotes osteoblast activity.

[0035] The present application has the following beneficial effects: The composite hydrogel of the application has a multi-level and three-dimensional system of ''plant vesicles + chemical drugs + composite gel carrier (microspheres / temperature-sensitive matrix)'', successfully realizes the synergy and integration of ''immune regulation'' and ''bone repair'' functions, and realizes the synergistic effect and functional complementation of 1+1>2.

[0036] The composite hydrogel of the application has a ''microsphere-gel'' composite system, which not only realizes the local long-acting release and high-efficiency anti-inflammation of anti-inflammatory natural medicine extracts by using a temperature-sensitive hydrogel matrix, but also, more importantly, encapsulates the bone-strengthening plant-derived extracellular vesicles as active ingredients in the hydrogel microspheres, so that the extracellular vesicles can target the bone-joint interface, durably promote osteogenesis and inhibit osteoclasts, thereby simultaneously intervening in the two core pathological processes of inflammation and bone erosion of RA. This bidirectional regulation overcomes the singleness of the existing treatment strategies and provides a new radical treatment approach that can not only quickly relieve symptoms but also long-term improve the joint structure.

[0037] The composite hydrogel of the application is an injectable sol, which has good fluidity at room temperature (25 DEG C) and is convenient for injection operation; after entering the body and reaching the joint cavity, the composite hydrogel can be rapidly converted into a non-flowing gel in response to body temperature (37 DEG C), so as to encapsulate the drugs in situ at the lesion site, realize long-acting release, immune regulation and bone repair, and achieve multiple goals.

[0038] The composite hydrogel of the application not only effectively inhibits the inflammatory response and abnormal immune activation of arthritis, but also simultaneously promotes osteogenic differentiation and inhibits osteoclast activity, so as to repair bone and cartilage damage while regulating the immune microenvironment, and finally provides a safe, efficient and local treatment new strategy for rheumatoid arthritis, which has the functions of immune regulation and bone repair.

[0039] The preparation method of the composite hydrogel of the application can prepare a system with uniform composite microsphere particle size and good monodispersity. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a schematic diagram of the preparation process and functional effect mechanism of the composite hydrogel of the application.

[0041] Figure 2 It is a form diagram (A) and a particle size distribution diagram (B) of DR-EVs in Example 1 of the application.

[0042] Figure 3 From left to right, it is a SEM diagram of GelMA@DR-EVs microspheres in Example 1 of the application with a scale of 200 μm, 100 μm and 20 μm.

[0043] Figure 4 It is a curve (A) of the storage modulus and loss modulus of HPA-SIN changing with temperature and an FTIR spectrum (B) in Example 1 of the application.

[0044] Figure 5 Degradation graph of each group of HPA-SIN, GelMA@DR-EVs and HPA@SIN-GM@DR in Example 1 of the present application.

[0045] Figure 6 Cumulative release curve graph of SIN in HPA-SIN in Example 1 of the present application.

[0046] Figure 7 Cumulative release curve graph of DR-EVs in GelMA@DR-EVs and HPA@SIN-GM@DR in Example 1 of the present application.

[0047] Figure 8 Effect of each group on M1 polarization of macrophages in Example 3 of the present application.

[0048] Figure 9 Effect of each group on Ctsk (A), Mmp9 (B), Bmp2 (C), RUNX2 (D) secretion of osteoblasts in Example 4 of the present application.

[0049] Figure 10 Effect of each group on differentiation inhibition of osteoclasts in Example 4 of the present application.

[0050] Figure 11 Effect of each group on expression of TNF-α (A), IL-6 (B), IL-1β (C), IL-10 (D) in joint tissues of CIA rats in Example 5 of the present application.

[0051] Figure 12 Effect of each group on ankle bone erosion of CIA rats in Example 5 of the present application (A) and its bone volume fraction graph (B). DETAILED DESCRIPTION

[0052] The content of the present application will be further described in detail through specific examples. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels, or can be obtained by existing technical methods, unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0053] Example 1 In this example, a composite hydrogel was prepared, and the specific process was as follows: Figure 1 The preparation process of the composite hydrogel and its treatment mechanism schematic diagram are shown in Figure 1 The preparation process schematic diagram of the composite hydrogel.

[0054] Extraction of extracellular vesicles derived from Drynaria roosii (DR-EVs): Fresh Drynaria roosii material was added to phosphate-buffered saline (PBS, pH=7.4) at a ratio of 1:10 (g / mL). Homogenization was performed using a high-speed homogenizer at 12,000 rpm in a 20-second / 30-second cycle for a total of 10 minutes. After solvent extraction, the homogenate was subjected to differential centrifugation: 500×g for 20 minutes, 2000×g for 30 minutes, and 10000×g for 50 minutes (4℃) to remove cell debris and large particles. The supernatant was further purified by ultracentrifugation (100,000×g, 70 minutes, 4℃) to precipitate the extracellular vesicles. The precipitate was resuspended in PBS and purified again by ultracentrifugation. Finally, the DR-EVs were filtered through a 0.22 μm filter, aliquoted, and stored at -80℃.

[0055] The morphology of DR-EVs was observed using a transmission electron microscope (TEM), revealing a typical cup-shaped structure with an intact membrane structure. Figure 2 A). Nanoparticle tracking analysis (NTA) shows that the DR-EVs particle size is mainly distributed around 67.8 nm, and the distribution is uniform. Figure 2 (B in the text) The extract was tested using Western blotting (protein immunoblotting) to detect its marker proteins (such as CD63, TSG101, and HSP70 were positive), and negative staining markers (such as Golgi protein GM130 were negative) were excluded to confirm that the extract was EVs rather than cell debris.

[0056] Preparation of GelMA@DR-EVs microspheres: Gelatin was dissolved in PBS (50℃) to form a 10% (w / v) solution. Methacrylic anhydride (MA) was slowly added, and the pH was controlled at 8.0. The reaction was carried out for 3 hours. After the reaction, the microspheres were purified by dialysis and lyophilized to obtain the GelMA precursor. The GelMA precursor was dissolved in PBS containing 0.5% (w / v) LAP photoinitiator to form a 10% (w / v) solution. DR-EVs were added to a final concentration of 100 μg / mL, and the mixture was gently mixed on ice. Microspheres were prepared using a microfluidic emulsion method: The above mixture and a liquid paraffin oil phase containing 3% (v / v) Span 80 were injected into a microfluidic chip at specific flow rates (e.g., aqueous phase: 500 μL / h, oil phase: 1500 μL / h) using a syringe pump. In the microfluidic chip, the aqueous phase formed highly uniform droplets under the shear of the oil phase. The droplets were then exposed to 365nm ultraviolet light (10 mW / cm²). 2Crosslinking. The microspheres were crosslinked by irradiation for 20-30 s. The crosslinked microspheres were washed with petroleum ether and PBS alternately to obtain GelMA@DR-EVs microspheres, which were stored at 4 °C for later use.

[0057] The microspheres were observed under an optical microscope to be uniform spherical shape with an average diameter of about 400 μm. Scanning electron microscopy (SEM) showed that the microspheres had a porous structure with a pore size of about 5-10 μm, which was conducive to drug loading and release Figure 3 ).

[0058] Preparation of thermo-sensitive hydrogel matrix (HPA-SIN): Hyaluronic acid (HA, 2% w / v) was dissolved in deionized water, and hydroxypropyl methyl cellulose (HPMC, 7% w / v) was gradually added and stirred at 80 °C for 30 min. The mixture was left to stand overnight at 4 °C to hydrate and degas. Glycerol (20% v / v) was added to adjust the rheological properties, and sinomenine (SIN, 2 mg / mL) was added and uniformly dispersed by ultrasonication to obtain the HPA-SIN thermo-sensitive hydrogel precursor.

[0059] The storage modulus (G') and loss modulus (G") were determined by a rheometer, and the results showed that a sol-gel transition occurred near 35 °C, and G' increased significantly to 100 kPa at 37 °C, indicating good temperature sensitivity Figure 4 Fourier transform infrared spectroscopy (FTIR) showed that no new peaks were observed during gelation, but the characteristic peaks shifted (e.g., O-H shifted from 3342 cm -1 to 3296 cm -1 ), indicating that gelation was a physical crosslinking process with enhanced hydrogen bonding Figure 4 .

[0060] Preparation of composite hydrogel (HPA@SIN-GM@DR): The prepared GelMA@DR-EVs microspheres were slowly added to the HPA@SIN pre-gel solution under low temperature (4 °C) conditions, and uniformly dispersed by gentle vortex or repeated inversion. The addition amount of microspheres was usually 5% (w / v) of the total mass of the final composite system, and the HPA@SIN-GM@DR composite hydrogel was prepared.

[0061] 37 °C, each group of hydrogel samples was immersed in PBS solution (pH = 7.4), and extracted, freeze-dried and weighed at the preset time points to obtain the degradation rate as Figure 5The degradation rate of HPA-SIN alone was rapid, with a residual mass of about 60-70% on day 7, about 20-25% on day 14, and almost completely disappeared on day 21. The degradation rate of GelMA@DR-EVs microspheres was slower (about 85-90% on day 7, about 45-50% on day 14, and almost completely degraded on day 21). In contrast, the degradation rate of the composite hydrogel HPA@SIN-GM@DR was the slowest in the early and middle stages (about 80-85% on day 7, about 55-60% on day 14), and its residual mass also decreased to near zero level on day 21.

[0062] Through in vitro release experiments, the cumulative release rate of SIN and DR-EVs (which can be tracked by protein content or specific fluorescent markers) was detected by periodic sampling in PBS solution (pH = 7.4) at 37°C oscillation, and the release curve was drawn.

[0063] The cumulative release rate of SIN in HPA is shown in Figure 6 The cumulative release rate of DR-EVs in GelMA@DR-EVs microspheres and composite hydrogel HPA@SIN-GM@DR is shown in Figure 7

[0064] It can be seen that the release of DR-EVs in HPA@SIN-GM@DR peaked at 14 days, with a release rate of about 40%, allowing the strong bone active ingredients of the composite hydrogel to continue to act in the chronic period, meeting the treatment needs of the disease in the later stage of bone destruction. This release behavior is closely related to the rapid gelation of the temperature-sensitive matrix and the regulation of the porous structure of the microspheres, verifying the advantages of the present application in synergistic delivery and controlled release.

[0065] Example 2 In this embodiment, a composite hydrogel was prepared, and the specific process was as follows: A similar preparation method as in Example 1 was used, but the loading amount of active ingredients was adjusted to verify the practicability of the present application under different parameters. Specifically: (1) The extraction method of Drynariae Rhizoma-derived extracellular vesicles (DR-EVs) was the same as in Example 1, but when preparing GelMA@DR-EVs microspheres, the final concentration of DR-EVs was adjusted to 50 μg / mL.

[0066] (2) In the preparation of the thermosensitive hydrogel matrix (HPA), curcumin was selected as an anti-inflammatory natural drug extract, and its mass concentration was adjusted to 1 mg / mL.

[0067] (3) The mass concentration of the composite microspheres (GelMA@DR-EVs) in the final composite hydrogel was adjusted to 1% (w / v).

[0068] ​The remaining steps, such as microfluidic emulsification, photocrosslinking, matrix mixing, etc., were all performed according to the method described in Example 1. The final composite hydrogel was liquid at 4°C and rapidly turned into solid at 37°C, showing good temperature sensitivity and injectability. It can be seen that even at a lower loading amount, a drug-loaded system with a "microsphere-gel" composite structure can be successfully prepared.

[0069] Example 3 This example uses the composite hydrogel prepared in Example 1 to study the inhibition of macrophage M1 polarization. The specific process is as follows: The effect on LPS-induced macrophage M1 polarization was detected by flow cytometry. Method: Primary macrophages were extracted from the abdominal cavity of SD rats and seeded in a 6-well plate at a density of 1× 10 6 mg / mL ratio at 37°C for 24 hours, and then the supernatant was prepared by centrifugation. After the cells adhered, they were divided into the following groups: Control group (Ctrl group, complete culture medium), LPS group (Vehicle group, 100 ng / mL LPS), LPS+HPA@SIN-GM@DR group (HPA@SIN-GM@DR group, 100 ng / mL LPS + 200 μg / mL composite hydrogel extract). After 24 hours of treatment, the cells were collected and stained with anti-rat CD86-FITC antibody (CUSABIO, CSB-PA004965EC01HU) and anti-rat CD206-PE antibody (CUSABIO, CSB-YP014783HU). After staining in the dark for 30 minutes, the cells were detected by flow cytometry (BD FACSCanto II), and the proportion of CD86 positive cells was analyzed using FlowJo software.

[0070] Results: Compared with the Control group, LPS stimulation significantly promoted the polarization of macrophages to the M1 phenotype, and the proportion of CD86 positive cells increased significantly. After intervention with HPA@SIN-GM@DR composite hydrogel, the proportion of CD86 positive cells was significantly lower than that of the LPS group, indicating that the composite hydrogel of the application can effectively inhibit LPS-induced macrophage M1 polarization Figure 8 ).

[0071] Example 4 This example uses the composite hydrogel prepared in Example 1 to study the effect of the composite hydrogel on osteoblast proliferation and osteoclast differentiation. The specific process is as follows: (1) The effect on the secretion of Bmp2, RUNX2 by osteoblasts and Ctsk, Mmp9 by osteoclasts was detected by qPCR: SD rat bone marrow-derived mesenchymal stem cells (BMSCs) were used as an osteogenic model and seeded at a density of 1 x 10 5 cells / well in 12-well plates and cultured in osteogenic induction medium (containing 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate sodium, and 100 nM dexamethasone) for 7 days. At the same time, primary macrophages extracted from the abdominal cavity of SD rats were used as an osteoclastic model and seeded at a density of 4 x 10 5 cells / well in 12-well plates and induced for osteoclastic differentiation using medium containing 50 ng / mL RANKL and 30 ng / mL M-CSF. The experiment was divided into the following groups: Control group (complete medium), induction group (osteogenic or osteoclastic induction conditions), induction + HPA@SIN-GM@DR group (induction conditions + 200 μg / mL complex hydrogel extract). Fresh medium containing the corresponding stimulants and extracts was replaced every 2 days. After 7 days of culture, the cells were collected, total RNA was extracted using the TRIzol method, and cDNA was synthesized after reverse transcription. Using β-actin as an internal reference, qPCR analysis was performed using TB Green Premix Ex Taq II on the QuantStudio 5 system. The reaction conditions were pre-denaturation at 95°C for 30 seconds, cycle stage (95°C for 5 seconds, 60°C for 30 seconds) repeated 40 times, and finally melt curve analysis. The relative expression of genes (Bmp2, RUNX2, Ctsk, Mmp9) was calculated using the 2^–ΔΔCt method Figure 9 ).

[0072] (2) The effect on osteoclast differentiation was evaluated by TRAP staining. Methods: Primary macrophages extracted from the abdominal cavity of SD rats were seeded at a density of 4 x 10 5 cells / well in 12-well plates and divided into the following groups: Control group (complete medium), RANKL group (50 ng / mL RANKL + 30 ng / mL M-CSF), RANKL + HPA@SIN-GM@DR group (induction conditions + 200 μg / mL complex hydrogel extract). Fresh medium containing the corresponding stimulants and extracts was replaced every 2 days. After 7 days of culture, the cells were stained using a TRAP staining kit (Sigma-Aldrich) according to the instructions, observed under a microscope, and the number of TRAP-positive cells containing 3 or more nuclei was counted.

[0073] Results: (1) Compared with the Control group, the mRNA expression levels of osteogenesis-related genes Bmp2 and RUNX2 in the osteogenesis induction group were significantly up-regulated (p<0.01). After HPA@SIN-GM@DR intervention, the expression levels of Bmp2 and RUNX2 were further significantly increased (p<0.05) compared with the induction group. In the osteoclast model, RANKL induction significantly increased the expression of osteoclast-related genes Ctsk and Mmp9 (p<0.01), while HPA@SIN-GM@DR treatment effectively inhibited the expression of these two genes (p<0.05). (2) RANKL stimulation can successfully induce RAW 264.7 cells to differentiate into a large number of TRAP-positive multinucleated osteoclasts. Compared with the RANKL group, the number of TRAP-positive multinucleated osteoclasts in the HPA@SIN-GM@DR treatment group was significantly reduced, proving that the composite hydrogel of the application can effectively inhibit the differentiation of osteoclasts Figure 10 ).

[0074] Example 5 This example uses the composite hydrogel prepared in Example 1 to study the therapeutic effect on arthritis in CIA rats, and the specific process is as follows: A collagen-induced arthritis (CIA) rat model was established and evaluated from two aspects of inflammatory factors and bone erosion. Methods: Male SD rats were randomly divided into Sham group (normal control group), CIA model group, and CIA+HPA@SIN-GM@DR treatment group. The rats were immunized intradermally at the base of the tail with bovine type II collagen emulsion to establish the CIA model. The immunization scheme was as follows: 0.2 mL bovine type II collagen (Biolead, BLD-BCL2) + incomplete Freund's adjuvant (Sigma, F5881) containing 100 μg of bovine type II collagen per rat was injected intradermally at the base of the tail as the primary immunization on day 0; 0.2 mL bovine type II collagen + incomplete Freund's adjuvant (Sigma, F5506) containing 100 μg of bovine type II collagen per rat was injected again at the same site on day 7 for booster immunization. The success criteria for modeling were as follows: after booster immunization, the rats developed multiple arthritis, the arthritis index (scoring criteria: 0 points for normal, 1 point for mild redness or swelling, 2 points for obvious swelling, and 3 points for joint ankylosis. The joint score was the sum of the scores for the four paws (maximum 12 points)), and the ankle joint diameter increased significantly (>20% compared with before modeling) were considered as successful modeling. After successful modeling, the treatment group was injected with 100 μL of HPA@SIN-GM@DR composite hydrogel (equivalent to SIN 0.2 mg and DR-EVs 10 μg) into the ankle joint cavity, once every 7 days for a total of 3 times. After the last administration, the samples were taken for detection.

[0075] (1) Inflammatory factor detection (qPCR): RNA extraction: About 30 mg of ankle synovial membrane tissue was taken and added with 1 mL of TRIzol reagent, and homogenized thoroughly. Chloroform was added at a ratio of chloroform: TRIzol = 0.2:1, and after thorough shaking and mixing, centrifugation was performed at 4°C and 12000 g for 15 minutes. The upper aqueous phase was aspirated, and an equal volume of isopropanol was added to precipitate the RNA, and centrifugation was performed at 4°C and 12000 g for 10 minutes. After washing the precipitate with 75% ethanol, it was dissolved in RNase-free water. Reverse transcription was performed using PrimeScript TM RT Master Mix (TaKaRa, RR036A). 1 μg of total RNA was taken, 5x PrimeScript RT Master Mix 4 μL was added, and RNase-free water was added to make up to 20 μL of the system. The reaction conditions were: 37°C for 15 minutes, 85°C for 5 seconds. The obtained cDNA was stored at -20°C for standby use. qPCR: TB Green TM Premix Ex Taq TM II (TaKaRa, RR820A) was used to detect in QuantStudio 5 real-time fluorescent quantitative PCR instrument (Applied Biosystems). The reaction system (20 μL) was composed as follows: TB Green Premix Ex Taq II (2X) 10 μL, upstream and downstream primers (10 μM) 0.8 μL each, ROX Reference Dye II (50X) 0.4 μL, cDNA template 2 μL, and RNase-free water to 20 μL. Reaction program: pre-denaturation: 95°C for 30 seconds; PCR cycle (40 cycles): 95°C for 5 seconds, 60°C for 30 seconds (annealing / extension); melting curve analysis: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds. β-actin was used as an internal reference gene, and the relative expression of genes (TNF-α, IL-6, IL-1β, IL-10) was calculated by the method of 2^-ΔΔCt Figure 11 ).

[0076] (2) Bone microstructure detection: The rat ankle joint was fixed in 4% paraformaldehyde. A high-resolution Micro-CT system (Skyscan 1176) was used to scan the sample. After scanning, three-dimensional reconstruction was performed using the matching software (CTAn), and parameters such as bone volume fraction (Bone Volume / Total Volume, BV / TV) were analyzed to evaluate the degree of bone destruction.

[0077] Results: (1) qPCR results showed that the mRNA expression levels of TNF-α, IL-6 and IL-1β in the ankle joint tissues of the CIA model group were significantly up-regulated (p<0.01) compared with the Sham group, while the expression of IL-10 was down-regulated. After treatment with HPA@SIN-GM@DR, the expression of pro-inflammatory factors was significantly inhibited, while the expression of IL-10 was significantly increased, indicating that it had good anti-inflammatory effect ( Figure 11 ).(2) Micro-CT three-dimensional reconstruction showed that the CIA model group rats had severe bone erosion in the ankle joint, and the bone trabecula was sparse and fractured, and the BV / TV value was significantly reduced. The joint bone structure integrity of the HPA@SIN-GM@DR treatment group was obviously improved, the bone erosion was reduced, and the BV / TV value was significantly higher than that of the model group (p<0.01), proving that the composite hydrogel of the application can effectively relieve the joint bone destruction of CIA rats ( Figure 12 ).

[0078] From Figure 1 It can also be seen that after the composite hydrogel of the application is injected into the joint cavity, the temperature-sensitive matrix rapidly gels at body temperature, locking the drug in situ; SIN is rapidly released from the matrix, inhibits M1 macrophage polarization and corrects Th17 / Treg immune cell imbalance, efficiently inhibits inflammatory pathways such as NF-κB and JAK-STAT, thereby relieving synovitis; at the same time, DR-EVs are slowly and continuously released from the GelMA microspheres, target the bone microenvironment, promote osteoblast differentiation and inhibit osteoclast generation, bidirectionally regulate bone metabolism, and ultimately achieve the dual effects of synergistic anti-inflammatory and bone repair.

[0079] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A composite hydrogel, characterized in that: It includes a thermosensitive hydrogel matrix and an anti-inflammatory natural drug extract and composite microspheres dispersed in the thermosensitive hydrogel matrix; the composite microspheres include hydrogel microspheres loaded with extracellular vesicles of bone-strengthening traditional Chinese medicine.

2. The composite hydrogel according to claim 1, characterized in that: The composite hydrogel satisfies at least one of the following conditions: (I) In the composite hydrogel, the mass concentration of the composite microspheres is 0.5-10% (w / v). (II) The mass concentration of the anti-inflammatory natural drug extract in the composite hydrogel is 0.5-5 mg / mL; (III) The loading amount of extracellular vesicles of bone-strengthening Chinese medicine in the composite microspheres is 50-200 μg / mL.

3. The composite hydrogel according to claim 1, characterized in that: The average particle size of the hydrogel microspheres is 300-500 μm.

4. The composite hydrogel according to claim 1, characterized in that: The hydrogel microspheres include hydrogel microspheres with a three-dimensional porous structure; preferably, the average pore size of the porous microspheres is 3-15 μm.

5. The composite hydrogel according to claim 1, characterized in that: The hydrogel microspheres include at least one of methacrylamide gelatin hydrogel microspheres, methacrylamide hyaluronic acid hydrogel microspheres, and methacrylamide alginate hydrogel microspheres.

6. The composite hydrogel according to claim 1, characterized in that: The anti-inflammatory natural drug extract includes at least one of total paeoniflorin, triptolide, curcumin, sinomenine, and naringenin.

7. The composite hydrogel according to claim 1, characterized in that: The bone-strengthening Chinese herbal extracellular vesicles include bone-strengthening extracellular vesicles (DR-EVs) and / or epimedium extracellular vesicles; preferably, the average particle size of the bone-strengthening Chinese herbal extracellular vesicles is 50-150 nm.

8. The composite hydrogel according to claim 1, characterized in that: The thermosensitive hydrogel matrix comprises a thermosensitive hydrophilic polymer, glycosaminoglycans, and polyols.

9. A method for preparing the composite hydrogel according to any one of claims 1-8, characterized in that: Includes the following steps: At low temperature, anti-inflammatory natural drug extracts and composite microspheres are added to a temperature-sensitive hydrogel matrix and dispersed to obtain the composite hydrogel.

10. The use of the composite hydrogel according to any one of claims 1-8 in the preparation of bone repair drugs.

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