Hydrogel pharmaceutical composition for intra-articular injection, method for preparing same, and use thereof

Abstract

Provided are an injectable joint cavity hydrogel pharmaceutical composition and a method for preparing same. The hydrogel pharmaceutical composition comprises a gel particle and a drug loaded in the gel particle. The gel particle is formed by cross-linking hyaluronic acid or a salt thereof with fibroin via a cross-linking agent. The drug comprises an anti-inflammatory agent and / or a growth factor. The hydrogel pharmaceutical composition can treat a bone and joint-related disease by means of intra-articular injection.

Description

An injectable hydrogel drug composition for joint cavity and its preparation method and application TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to an injectable hydrogel drug composition for joint cavity and its preparation method and application. BACKGROUND

[0002] Osteoarthritis (OA) is a debilitating joint disease that causes damage to the articular cartilage and underlying bone. The cartilage is composed of chondrocytes and extracellular matrix (ECM), which provides a living environment and nutrient components for the cells, and the network structure in the ECM provides the cartilage with elastic shaping ability. Apoptosis of chondrocytes and abnormal degradation of ECM are one of the signs of pathological changes of OA. According to a survey result of WTO, 10% of men and 20% of women in the population of 60 years old suffer from symptomatic OA, of which 80% of the patients show that their daily activities are limited, and 30% of them are unable to take care of themselves. OA is highly prevalent in the middle-aged and elderly population, and the increase in the OA patient population is exacerbated by the aging of the population in China.

[0003] Commonly used oral drugs for OA treatment mainly include non-steroidal anti-inflammatory drugs (NSAIDs), selective cyclooxygenase (COX)-2 inhibitors, opioid drugs and glucocorticoids. However, these drugs can only relieve symptoms and cannot improve the cartilage defect.

[0004] Therefore, there is an urgent need for a safe and effective drug for treating osteoarthritis and other bone-related diseases. SUMMARY

[0005] The purpose of the present application is to provide an injectable hydrogel drug composition for joint cavity and its preparation method and application, which simultaneously encapsulates anti-inflammatory drugs and / or growth factors in HA / SF hydrogel, and the preparation method is simple, the hydrogel has good viscoelasticity, and the hydrogel can be used for intra-articular injection.

[0006] The technical scheme of the present application is as follows: in the first aspect, the present application provides an injectable hydrogel drug composition for joint cavity, which comprises gel particles and drugs loaded in the gel particles; the gel particles comprise hyaluronic acid or a salt thereof, silk fibroin and a crosslinking agent; the drugs comprise anti-inflammatory drugs and / or growth factors, preferably anti-inflammatory drugs and growth factors.

[0007] In a preferred embodiment, the anti-inflammatory agent is selected from the group consisting of non-steroidal anti-inflammatory drugs and / or glucocorticoids; preferably, the non-steroidal anti-inflammatory drug is selected from the group consisting of one or several of diclofenac, ibuprofen, celecoxib, acetaminophen, meloxicam, indomethacin, lornoxicam, etoricoxib, nimesulide; preferably, the glucocorticoid is selected from the group consisting of one or several of methylprednisolone, hydrocortisone, triamcinolone, prednisolone, betamethasone, dexamethasone.

[0008] In a preferred embodiment, the growth factor is selected from the group consisting of one or several of insulin-like growth factor 1 (IGF-1), transforming growth factors (TGFs), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs).

[0009] In a preferred embodiment, the anti-inflammatory agent is celecoxib and the growth factor is IGF-1.

[0010] In a preferred embodiment, the concentration of the anti-inflammatory agent is 0.01-10 wt%, preferably 0.01-1 wt%, more preferably 0.02-0.05 wt%, most preferably 0.03 wt%, based on the total mass of the gel particle.

[0011] In a preferred embodiment, the concentration of the growth factor is 10 -10 -10 -4 wt%, preferably 10 -9 -10 -5 wt%, more preferably 10 -8 -10 -6 wt%, most preferably 10- 7 wt%, based on the total mass of the gel particle.

[0012] In a preferred embodiment, the hyaluronic acid has a molecular weight of 4-25 million Dalton, preferably 8-25 million Dalton, more preferably 15-25 million Dalton.

[0013] In a preferred embodiment, the silk fibroin has a molecular weight of 0.8-5 million Dalton, preferably 2.5-20 million Dalton, more preferably 2.5-10 million Dalton, still more preferably 5-10 million Dalton.

[0014] In a preferred embodiment, the gel particle has an elastic modulus of 1 x 10 2 Pa - 1 x 10 4 Pa; preferably 1 x 10 3 -1 x 10 4 Pa, more preferably 500-3000 Pa, still more preferably 1000-3000 Pa.

[0015] In a preferred embodiment, the mass ratio of hyaluronic acid or its salt: silk fibroin: crosslinking agent in the gel particles is (2.5-7.5):(0.5-5):1, preferably (4-7.5):(1-5):1, more preferably (4-7.5):(2-4):1, most preferably 4.5:2.3:1.

[0016] In a preferred embodiment, the salt of hyaluronic acid is selected from one of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate, preferably sodium hyaluronate.

[0017] In a preferred embodiment, the crosslinking agent is selected from one or any combination of 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylene diamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, preferably BDDE.

[0018] In a preferred embodiment, the gel particles are prepared by chemical crosslinking of the hyaluronic acid or its salt, the silk fibroin and the crosslinking agent.

[0019] In a preferred embodiment, the intra-articular injectable hydrogel drug further comprises an aqueous medium, preferably the aqueous medium is selected from one or several of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, HEPES buffer, more preferably phosphate buffer, still more preferably the aqueous medium is an alkaline solution.

[0020] In a second aspect, the present application provides a preparation method of the aforementioned intra-articular injectable hydrogel drug composition, characterized in comprising the following steps:

[0021] (1) Preparation of gel particles: dissolve HA and SF powders in an aqueous medium, stir uniformly, defoam, add crosslinking agent for crosslinking, dialysis, granulation to obtain gel particles, wherein the aqueous medium is preferably an alkaline solution; further, the alkaline solution is a sodium hydroxide solution;

[0022] Further, the defoaming treatment condition is 2-25℃ for 12h or more; still further, 4℃ for 24h;

[0023] Further, the cross-linking condition is incubation at 37-60 °C; further, incubation at 37 °C for 4 h

[0024] (2) Preparation of drug-loaded hydrogel: adding a solution containing an anti-inflammatory drug and / or a growth factor into the gel particles.

[0025] In a preferred embodiment, the concentration of the anti-inflammatory drug in step (2) is 100-1000 pg / mL, preferably 200-500 pg / mL, more preferably 250 pg / mL.

[0026] In a preferred embodiment, the concentration of the growth factor in step (2) is 50-500 ng / mL, preferably 75-200 ng / mL, more preferably 100 ng / mL.

[0027] In a third aspect, the present application provides a kit comprising:

[0028] (1) an anti-inflammatory drug and / or a growth factor; and

[0029] (2) gel particles comprising hyaluronic acid or a salt thereof, fibroin, and a cross-linking agent,

[0030] Preferably, the kit comprises both an anti-inflammatory drug and a growth factor.

[0031] In a preferred embodiment, the anti-inflammatory drug is selected from non-steroidal anti-inflammatory drugs and / or glucocorticoids; preferably, the non-steroidal anti-inflammatory drug is selected from one or more of diclofenac, ibuprofen, celecoxib, acetaminophen, meloxicam, indomethacin, clonixin, etoricoxib, nimesulide; preferably, the glucocorticoid is selected from one or more of methylprednisolone, hydrocortisone, triamcinolone acetonide, prednisolone, betamethasone, dexamethasone.

[0032] In a preferred embodiment, the growth factor is selected from one or more of insulin-like growth factor 1 (IGF-1), transforming growth factors (TGFs), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs).

[0033] In a preferred embodiment, the anti-inflammatory drug is celecoxib and the growth factor is IGF-1.

[0034] In a preferred embodiment, the components in the kit are mixed before use, such that the concentration of the anti-inflammatory drug is 0.01-10 wt%, preferably 0.01-1 wt%, more preferably 0.02-0.05 wt%, most preferably 0.03 wt%, based on the total mass of the gel particles.

[0035] In a preferred embodiment, the components in the kit are mixed prior to use such that the concentration of the growth factor is 10 -10 -10 -4 wt%, preferably 10 - 9 -10 -5 wt%, more preferably 10 -8 -10 -6 wt%, most preferably 10 -7 wt%.

[0036] In a preferred embodiment, the hyaluronic acid has a molecular weight of 4-25 million Dalton, preferably 8-25 million Dalton, more preferably 15-25 million Dalton.

[0037] In a preferred embodiment, the silk fibroin has a molecular weight of 0.8-5 million Dalton, preferably 2.5-20 million Dalton, more preferably 2.5-10 million Dalton.

[0038] In a preferred embodiment, the gel particles have an elastic modulus of 1 x 10 2 Pa - 1 x 10 4 Pa; preferably 1 x 10 3 - 1 x 10 4 Pa, more preferably 500-3000 Pa, still more preferably 1000-3000 Pa.

[0039] In a preferred embodiment, the mass ratio of hyaluronic acid or a salt thereof : silk fibroin : crosslinking agent in the gel particles is (2.5-7.5) : (0.5-5) : 1, preferably (4-7.5) : (1-5) : 1, more preferably (4-7.5) : (2-4) : 1, most preferably 4.5 : 2.3 : 1.

[0040] In a preferred embodiment, the salt of hyaluronic acid is selected from one of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate, preferably sodium hyaluronate.

[0041] In a preferred embodiment, the crosslinking agent is selected from one or any combination of 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylene diamine (HMDA), 1-(2,3- epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, preferably BDDE.

[0042] In a preferred embodiment, the gel particles are prepared by chemical crosslinking of the hyaluronic acid or salt thereof, the fibroin and the crosslinking agent.

[0043] In a preferred embodiment, the kit further comprises an aqueous medium, preferably, the aqueous medium is selected from one or more of deionized water, normal saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, HEPES buffer, more preferably, the aqueous medium is phosphate buffer, still more preferably, the aqueous medium is an alkaline solution.

[0044] In a fourth aspect, the present application provides use of the aforementioned injectable hydrogel composition for articular cavity or the composition prepared according to the aforementioned method or the aforementioned kit in the preparation of a medicament for treating bone joint related diseases.

[0045] In a preferred embodiment, the bone joint related disease is selected from one or more of postoperative fracture, osteoarthritis, rheumatoid arthritis, degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, periarthritis of shoulder, osteoporosis, osteonecrosis of the femoral head and other various joint dysfunction diseases. Advantages:

[0046] The present application firstly simultaneously loads anti-inflammatory drugs (celecoxib) and growth factors (IGF-1) in HA / SF hydrogel, and in vitro experiments show that, compared with the single drug-loaded hydrogel group, the dual (double-loaded hydrogel) group of the present application significantly inhibits the expression of pro-inflammatory factors, increases the expression of anti-inflammatory factors, up-regulates the mRNA expression level of cartilage formation related genes (COL II, Sox9 and Aggrecan), and down-regulates the mRNA expression level of related enzymes (MMP1 and ADAMTS5) genes that promote the degradation of cartilage extracellular matrix, has a synergistic anti-inflammatory and cartilage repair promoting effect, and is expected to provide a potential drug delivery system for the treatment of OA.

[0047] The uniform porous structure of the hydrogel is conducive to the adhesion and growth of bone cells, and the hydrogel still has good viscoelasticity after loading anti-inflammatory drugs and growth factors, has good injectability, can treat bone joint related diseases through intra-articular injection, can prolong the drug retention time, and improve the bioavailability. The preparation method of the intra-articular injectable hydrogel drug composition is simple, safe, and conducive to industrialized production.

[0048] BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 shows the elastic modulus of HA / SF hydrogel prepared using different contents of gel particles.

[0050] Figure 2 shows the elastic modulus of HA / SF hydrogel with different HA molecular weights, wherein HA-SF-S represents HA / SF hydrogel prepared using S-HA, HA-SF-M represents HA / SF hydrogel prepared using M-HA, and HA-SF-L represents HA / SF hydrogel prepared using L-HA.

[0051] Figure 3 shows the in vitro anti-inflammatory effect of HA / SF hydrogel with different SF molecular weights, wherein inner, middle and outer respectively represent HA / SF hydrogel prepared using 8kDa-25kDa silk fibroin, 25kDa-100kDa silk fibroin and >100kDa silk fibroin. Control group: C28 / I2 cells are induced into inflammatory cells, and normal cell culture solution is given. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0052] Figure 4 shows the elastic modulus of gel particles with different HA and SF concentrations at a shear frequency of 10Hz.

[0053] Figure 5 shows the appearance of HA / SF hydrogel without drug loading, HA / SF hydrogel loaded with IGF-1, HA / SF hydrogel loaded with CLX, and double-drug-loaded HA / SF hydrogel.

[0054] Figure 6 shows the SEM images of HA / SF hydrogel without drug loading, HA / SF hydrogel loaded with IGF-1, HA / SF hydrogel loaded with CLX, and double-drug-loaded HA / SF hydrogel, with the left column scale being 300.0μm and the right column scale being 2.0μm.

[0055] Figure 7 shows the swelling properties of HA / SF hydrogel without drug loading, HA / SF hydrogel loaded with IGF-1, HA / SF hydrogel loaded with CLX, and double-drug-loaded HA / SF hydrogel.

[0056] Figure 8 shows the rheological properties of the unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel and dual loaded HA / SF hydrogel.

[0057] Figure 9 shows the elastic modulus of the unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel and dual loaded HA / SF hydrogel at a shear frequency of 10 Hz.

[0058] Figure 10 shows the drug release profile of the Dual dual loaded hydrogel.

[0059] Figure 11 shows the cell proliferation activity of the leachate of the unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel and dual loaded HA / SF hydrogel.

[0060] Figure 12 shows the haemocompatibility of the unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel and dual loaded HA / SF hydrogel.

[0061] Figure 13 shows the in vitro anti-inflammatory effect of the unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel and dual loaded HA / SF hydrogel. PC: control group, C28 / I2 cells induced to be inflammatory cells, normal cell culture solution was given.

[0062] Figure 14 shows the effect of the unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel and dual loaded HA / SF hydrogel on the gene expression level of the C28 / I2 cells induced to be inflammatory cells. PC: control group, C28 / I2 cells induced to be inflammatory cells, normal cell culture solution was given; NC: C28 / I2 cells not induced to be inflammatory, normal cell culture solution was given.

[0063] Figure 15 is the SDS gel electrophoresis result of the three molecular weight range of silk fibroin lyophilized blocks.

[0064] In the figures, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; #P<0.05, ##P<0.01, ####P<0.0001; @@P<0.01.

[0065] DETAILED DESCRIPTION

[0066] DEFINITIONS

[0067] As used herein, the term "silk fibroin" includes Bombyx mori silk fibroin, insect or spider silk proteins, or recombinant silk fibroin. In one embodiment, the silk fibroin is obtained from Bombyx mori.

[0068] As used herein, the term "hyaluronic acid" is a biodegradable polymer component consisting of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. This water-soluble polymer is naturally present in almost all tissues, especially in the extracellular matrix, the vitreous of the eye, and the synovial fluid of joints. HA is commercially available in pure form. Small gel particle HA fillers can be used to stimulate the production of natural collagen, which is believed to be induced by mechanical stretching of the dermis and activation of dermal fibroblasts.

[0069] As used herein, the term "crosslinking" refers to intermolecular bonds that link individual polymer molecules, macromolecules, and / or monomer chains into more stable structures such as gels. The term "crosslinking agent" refers to a substance that links individual polymer molecules, macromolecules, and / or monomer chains into intermolecular bonds. Representative crosslinking agents include 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic hydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylene diamine (HMDA), 1-(2,3- epoxypropyl)-2,3-epoxycyclohexane, carbodiimides, and the like.

[0070] As used herein, the term "gel" refers to a thick liquid or semisolid preparation of solution, suspension, or emulsion type. Gel bases are single-phase dispersion systems, which are divided into aqueous and oily. Aqueous gel bases are generally composed of water, glycerin or propylene glycol, and cellulose derivatives, carbomers, and alginates, tragacanth, gelatin, starch, and the like; oily gel bases are composed of liquid paraffin and polyethylene or fatty oils and colloidal silica or aluminum soap, zinc soap, and the like.

[0071] As used herein, the term "anti-inflammatory agent" refers to a drug that treats inflammation that occurs after tissue is damaged. Anti-inflammatory agents include steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents, wherein steroidal anti-inflammatory agents exert anti-inflammatory effects by inhibiting the synthesis of prostaglandins, inhibiting the aggregation of leukocytes, reducing the formation of bradykinin, inhibiting the aggregation of platelets, etc. Non-steroidal anti-inflammatory agents (NSAIDs) are a class of anti-inflammatory agents that do not contain a steroidal structure, and this class of drugs includes aspirin, acetaminophen, indomethacin, naproxen, nambumetone, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib, etc., which have anti-inflammatory, anti-rheumatic, analgesic, antipyretic, and anti-coagulant effects, and are widely used in the clinic for the relief of osteoarthritis, rheumatoid arthritis, various fevers, and various pain symptoms.

[0072] As used herein, the term "growth factor" refers to a class of polypeptide substances that have multiple effects, such as regulating cell growth and other cell functions, by binding to specific, high-affinity cell membrane receptors. They are cytokines secreted by various cells, which act on specific target cells to regulate cell division, matrix synthesis, and tissue differentiation. There are various growth factors, such as platelet-derived growth factors (platelet-derived growth factor, PDGF; osteosarcoma-derived growth factor ODGF), epidermal growth factor (epidermal growth factor EGF, transforming growth factor TGFα and TGFβ), fibroblast growth factors (αFGF, βFGF), insulin-like growth factors (IGF-I, IGF-II), nerve growth factors (NGF), interleukin growth factors (IL-1, IL-1, IL-3, etc.), erythropoietin (EPO), colony-stimulating factors (CSF), etc.

[0073] As used herein, the term "salt" includes, for example, salts of inorganic acids and salts of organic acids. Examples of salts can include hydrochloride, phosphate, pyrophosphate, hydrobromide, sulfate, sulfinate, nitrate, malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoate (e.g., acetate, HOOC-(CH2) n -COOH, where n is 0-4). Furthermore, if the compounds herein are obtained in the form of an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the compounds herein are a free base, an addition salt (particularly a pharmaceutically acceptable addition salt) can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for making acid addition salts from base compounds. Those skilled in the art will appreciate that a variety of synthetic methods can be used to prepare the nontoxic pharmaceutically acceptable addition salts.

[0074] As used herein, the term "treatment" and related terms, refer to therapeutic treatment. When used in reference to a specific condition, treatment indicates: (1) improvement of the condition or one or more biological manifestations of the condition, (2) interference with (a) one or more points in a biological cascade that leads to or causes the condition, or (b) one or more biological manifestations of the condition, (3) reduction of one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing of the progression of the condition, or slowing of one or more biological manifestations of the condition.

[0075] As used herein, "prevention" refers to prophylactic administration to substantially reduce the likelihood or severity of a condition or a biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. Those skilled in the art will appreciate that "prevention" is not an absolute term. For example, prophylactic treatment is appropriate when a subject is deemed to be at high risk of developing cancer, such as when the subject has a strong family history of cancer or when the subject has been exposed to a carcinogenic substance.

[0076] As used herein, the term "subject" refers to an animal, such as a mammal (including a human), who has been or will be the object of treatment, observation or experiment. The methods described herein can be useful in human therapy and / or veterinary applications. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

[0077] As used herein, the term "kit" can include single or multiple doses of two or more agents, each individually packaged or formulated; or single or multiple doses of two or more agents packaged or formulated in combination. Thus, one or more agents can be present in a first container, and the kit can optionally include one or more agents in a second container. The container(s) is placed in a package, and the package can optionally include instructions for administration or dosing. The kit can include additional components, such as a syringe or other means for administering the agents and diluents or other means for formulation. Specific embodiments

[0078] Insulin-like growth factor (IGF) is a major anabolic growth factor in cartilage, which protects the NF-κΒ pathway by inhibiting PI3K / Akt and MAPKs specific pathways, macroscopic and pathological studies have shown that it has a cartilage protective effect by promoting the formation of hyaline cartilage.

[0079] Celecoxib is a selective cyclooxygenase inhibitor. Inflammation stimulates the production of cyclooxygenase-2 (COX-2), which leads to the synthesis and accumulation of inflammatory prostaglandins, especially prostaglandin E2, causing inflammation, edema and pain. Celecoxib can prevent the production of inflammatory prostaglandins by inhibiting COX-2, achieving anti-inflammatory, analgesic and antipyretic effects, and can be used in the treatment, prevention and management of osteoarthritis, rheumatoid arthritis, various fevers and various pain symptoms in clinical practice.

[0080] Commonly used materials for intra-articular injection of hydrogel can be divided into natural materials and synthetic materials. Common natural materials include hyaluronic acid, chondroitin sulfate, chitosan, gelatin, etc. Synthetic materials mainly include PEG and PVA, etc. Among them, hyaluronic acid (Hyaluronic acid, HA) is also known as hyaluronic acid, which is a natural polysaccharide and the main component of the extracellular matrix of articular cartilage. Studies have shown that in the synovial fluid of OA patients, the content and viscoelasticity of hyaluronic acid are significantly decreased, therefore, intra-articular injection of hyaluronic acid solution or hydrogel can supplement the missing components in the synovial fluid, and is an excellent viscoelastic supplement.

[0081] Silk fibroin (SF) is a natural polymer material, which has good biocompatibility, adjustable mechanical properties and drug loading capacity, and has been widely used in bone tissue engineering.

[0082] In one embodiment, the present application provides a double-drug-loaded hydrogel containing HA and SF hydrogel combined with anti-inflammatory drugs and growth factors for intra-articular injection of bone joints to treat bone joint-related diseases.

[0083] Examples

[0084] The technical solutions of the present application will be described in detail below in conjunction with the examples, but those skilled in the art should understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application.

[0085] The materials used in the following examples and their sources are as follows:

[0086] Example 1: Preparation of HA / SF hydrogel

[0087] 1. Preparation of HA / SF hydrogel

[0088] (1) Precisely weigh 0.2 g of sodium hydroxide, add Wahaha water 20 mL, stir to dissolve, and prepare 1% sodium hydroxide solution for use;

[0089] (2) Precisely take 0.2 g of HA (molecular weight 1.5-2.5 million Dalton) and 0.1 g of SF (molecular weight 25-100 thousand Dalton) into a 10 mL beaker, add 2 mL of 1% sodium hydroxide solution, stir uniformly, and defoam at 4°C for 24 h;

[0090] (3) After defoaming, add 0.044 g of BDDE to form a mixed solution, and incubate in a 37°C water bath for 4 h;

[0091] (4) Take out the gel and put it into a 8000 molecular weight dialysis bag for 24 h dialysis;

[0092] (5) After dialysis, granulate through a 60 mesh sieve to obtain HA / SF gel particles;

[0093] (6) Take 0.8 g of HA / SF gel particles, add 1 mL of PBS solution, stir uniformly, and obtain HA / SF hydrogel.

[0094] 2. Rheological property of hydrogel of different concentrations

[0095] Take 0.6 g, 0.8 g, and 1.0 g of HA / SF gel particles, respectively, add 1 mL of PBS solution to resuspend, and use a kinex rheometer to detect the elastic modulus G' at a shear frequency of 0.1-10 Hz, and the elastic modulus G' of HA / SF hydrogel and HA hydrogel at a shear frequency of 10 Hz (see Figure 1).

[0096] The results show that the hydrogel prepared by resuspending 0.8 g of HA / SF gel particles in 1 mL of PBS solution has good elastic modulus and good injectability.

[0097] Example 2: Investigation of HA of different molecular weights

[0098] In order to further explore which molecular weight of HA is more suitable for joint cavity injection, three molecular weights of HA were investigated, namely 40-80 million Dalton, 80-150 million Dalton, and 150-250 million Dalton, denoted as S-HA, M-HA, and L-HA. Different molecular weights of HA were crosslinked with SF (molecular weight 2.5-10 million Dalton) according to the method of Example 1, and the elastic modulus (G') of HA / SF gel particles was detected by a rheometer, and the results are shown in Figure 2.

[0099] The results show that the gel particles prepared from L-HA have a higher elastic modulus, so 150-250 million Dalton of HA is selected for the following experiments.

[0100] Example 3: Investigation of SF of different molecular weights

[0101] Three different gradient dialysis bags with molecular weight cut-off of 100 kDa, 25 kDa and 8 kDa were used to prepare a hierarchical dialysis bag with an outer layer, a middle layer and an inner layer. The dialysis was started after the silk fibroin solution was added to the inner layer dialysis bag. The final outer layer, middle layer and inner layer dialysis bags would obtain silk fibroin solutions with different molecular weight ranges, respectively. The silk fibroin solutions in the outer layer, middle layer and inner layer dialysis bags were taken out and freeze-dried, respectively, to obtain silk fibroin lyophilized blocks with different molecular weight ranges, i.e. 8 kDa-25 kDa silk fibroin, 25 kDa-100 kDa silk fibroin and >100 kDa silk fibroin. Figure 15 shows the SDS gel electrophoresis results of the silk fibroin lyophilized blocks with three molecular weight ranges.

[0102] The anti-inflammatory effects of hydrogels prepared from three molecular weights (0.8-2.5 kDa, 2.5-10 kDa and above 10 kDa) of SF were compared. The HA / SF hydrogels were prepared according to the method of Example 1 and were recorded as inner (containing 0.8-2.5 kDa SF), middle (containing 2.5-10 kDa SF) and outer (containing above 10 kDa SF), respectively. After freeze-drying, the appropriate complete medium was added to prepare a gel extract solution of 8 mg / ml, and the appropriate IL-1β was added to prepare an IL-1β extract solution with a final concentration of 50 ng / ml, which was then co-cultured with C28 / I2 cells for 48 hours. The cell supernatant was collected, and the levels of anti-inflammatory factors IL-4 and IL-10 and pro-inflammatory factor IL-6 were determined using an Elisa kit. The results are shown in Figure 3.

[0103] The results showed that compared with the control group (C28 / I2 cells induced to inflammatory cells without drug administration, and normal cell culture solution was given), the HA / SF hydrogel prepared from the middle molecular weight (2.5-10 kDa) SF could significantly increase the expression levels of anti-inflammatory factors IL-4 and IL-10 in the cell supernatant of the in vitro induced arthritis model; compared with the inner group and the outer group, the middle group had the effect of inhibiting the expression of pro-inflammatory factor IL-6.

[0104] Example 4: Investigation of HA and SF concentrations

[0105] Precisely weigh HA 0.1 g, SF 0.1 g; HA 0.16 g, SF 0.1 g; HA 0.2 g, SF 0.1 g; HA 0.2 g, SF 0.02 g; HA 0.2 g, SF 0.04 g; HA 0.2 g, SF 0.16 g; HA 0.2 g, SF 0.2 g; HA 0.3 g, SF 0.1 g in a 10 mL beaker, respectively, add 2 mL of 1% sodium hydroxide solution, stir evenly, and then different concentrations of HA and SF solutions are obtained, which are recorded as samples 1-8, respectively. The above solutions are prepared into HA / SF gel particles according to the method of Example 1, and the elastic modulus (G') is detected by kinex rheometer. The results are shown in Table 1 and Figure 4.

[0106] The results show that the G' of gel particles with different HA and SF concentrations is quite different. The G' of gel particles with 10%-15% HA and 2%-10% SF is higher, but the hydrogel prepared with 15% HA and 5% SF has poor injectability and cannot be easily pushed out within 30 seconds. Considering the higher requirements for injectability and elastic modulus for joint cavity injection, the gel particles with 10% HA and 5%-8% SF have better effects.

[0107] Table 1 Elastic modulus (G') of gel particles with different HA and SF concentrations

[0108] Example 5: Preparation of drug-loaded hydrogel

[0109] 1. Preparation of HA / SF hydrogel loaded with celecoxib (CLX) liposomes

[0110] (1) Preparation of CLX liposomes: precisely weigh CLX 4 mg, phospholipid 80 mg, and cholesterol 20 mg in a 50 mL round-bottom flask, add 20 mL of chloroform to dissolve completely; remove the organic solvent chloroform by rotary evaporation under reduced pressure at 60°C, and then dry in vacuum for 24 h to further remove the organic solvent; then add 16 mL of PBS buffer, and sonicate for 20 min (power 100 W, total working time 20 min, sonication on 0.2 s, sonication off 0.1 s) to prepare the CLX liposome solution;

[0111] (2) Preparation of HA / SF hydrogel loaded with CLX: weigh 0.8 g of HA / SF gel particles in Example 1, add 1 mL of CLX liposome solution (CLX concentration 0.25 mg / ml), and stir evenly to obtain the product. The mass percentage of CLX in the gel particles is 0.03 wt%.

[0112] 2. Preparation of HA / SF hydrogel loaded with IGF-1

[0113] Take 1 mL PBS solution, add appropriate amount of IGF-1, prepare IGF-1 solution with concentration of 100 ng / mL for standby;

[0114] Take 0.8 g HA / SF gel particles in Example 1, add 1 mL IGF-1 / CLX solution, stir evenly, and obtain CLX / IGF-1 dual-loaded hydrogel. The mass percentage of CLX in the gel particles is 0.03 wt%, and the mass percentage of IGF-1 is 10 -7 wt%.

[0115] 3. Preparation of HA / SF hydrogel loaded with CLX and IGF-1:

[0116] Take 1 mL CLX liposome solution, add appropriate amount of IGF-1, and obtain IGF-1 / CLX solution (CLX concentration 0.25 mg / mL, IGF-1 concentration 100 ng / mL);

[0117] Take 0.8 g HA / SF gel particles in Example 1, add 1 mL IGF-1 / CLX solution, stir evenly, and obtain CLX / IGF-1 dual-loaded hydrogel. The mass percentage of CLX in the gel particles is 0.03 wt%, and the mass percentage of IGF-1 is 10 -7 wt%.

[0118] Example 6: Transparency test

[0119] On a blank A4 paper, write HA / SF, CLX, IGF-1, Dual with a black marker pen, take appropriate amount of HA / SF hydrogel (HA / SF) in Example 1, CLX hydrogel (CLX) in Example 5, IGF-1 hydrogel (IGF-1) and CLX / IGF-1 dual-loaded hydrogel (Dual) and cover them on the corresponding fonts, and observe the transparency of the hydrogel.

[0120] The results are shown in Figure 5, and the transparency of the four prepared hydrogels is good.

[0121] Example 7: Injectability test

[0122] Label the HA / SF hydrogel of Example 1 and the CLX / IGF-1 dual-loaded hydrogel (Dual) of Example 5 with citrine and rhodamine B respectively, and observe the injectability of the two hydrogels in a 1 mL syringe.

[0123] The results show that both hydrogels can be easily pushed out of a 1 mL syringe within 30 seconds, and the injectability is good.

[0124] Example 8: Micro-morphology characterization

[0125] Take the appropriate amount of HA / SF hydrogel (HA / SF) in Example 1, CLX hydrogel (CLX), IGF-1 hydrogel (IGF-1) and CLX / IGF-1 dual-loaded hydrogel (Dual) in Example 5 into the freeze dryer, and observe the surface morphology of the two hydrogels by scanning electron microscope (SEM), and the results are shown in Figure 6.

[0126] The results show that the HA / SF hydrogel prepared in the application has high porosity, and the pore structure is uniform and dense. The addition of IGF-1 does not affect the appearance and pore distribution of the HA / SF hydrogel. The uniform distribution of drug-loaded liposomes on the pore wall of the HA / SF hydrogel and the Dual dual-loaded hydrogel (indicated by arrows in the figure) shows that the drug-loaded liposomes are successfully loaded into the HA / SF hydrogel.

[0127] Example 9: Swelling experiment

[0128] Take the appropriate amount of HA / SF hydrogel (HA / SF) in Example 1, CLX hydrogel (CLX), IGF-1 hydrogel (IGF-1) and CLX / IGF-1 dual-loaded hydrogel (Dual) in Example 5, record the initial weight, then put it into Wahaha water completely submerged, and take it out and weigh at 15, 30, 45, 60, 75, 90, 105, 120 minutes, respectively, record the weight of the hydrogel at different time points, and observe its swelling property, and the results are shown in Figure 7.

[0129] The results show that the hydrogels prepared in the application have good water absorption and swelling property. The addition of drugs, especially the addition of celecoxib liposomes, reduces the swelling property of the hydrogel to some extent, which may be related to the hydrophobicity of the liposomes and the blockage of the liposomes to part of the pores of the hydrogel.

[0130] Example 10: Rheological property test

[0131] Take the appropriate amount of HA / SF hydrogel (HA / SF) in Example 1, CLX hydrogel (CLX), IGF-1 hydrogel (IGF-1) and CLX / IGF-1 dual-loaded hydrogel (Dual) in Example 5, and perform rheological test, record the elastic modulus G' and viscous modulus G" of each group at shear frequency of 0.1-10Hz (see Figure 8).

[0132] Figure 9 is the elastic modulus G' at a shear frequency of 10 Hz, which shows that the elastic modulus of the HA / SF hydrogel is about 900 Pa, meeting the mechanical property requirements of the hydrogel for intra-articular injection. For the drug-loaded hydrogel, especially the loading of celecoxib liposomes, the elastic modulus of the HA / SF hydrogel can be significantly increased to about 1100 Pa.

[0133] Example 11: Drug release test of Dual

[0134] An appropriate amount of Dual double-loaded hydrogel in Example 5 was placed in a dialysis bag (MWCO = 8000-14000) for dialysis, and the release liquid was PBS (pH = 7.0-7.2). At the specified time points (1st, 3rd, 5th, 7th, 10th, 14th, 21st, 28th, 35th day), an appropriate amount of dialysate was taken to determine the drug release, and an equal volume of fresh release liquid was supplemented. The results are shown in Figure 10.

[0135] The results show that the Dual double-loaded hydrogel can partially function as a drug depot in the joint cavity, achieving the effect of controlled release of growth factor IGF-1 and anti-inflammatory drug CLX.

[0136] Example 12: Cell compatibility test

[0137] Human chondrocyte cells (C28 / I2 cells) were used for the test. First, an appropriate amount of HA / SF hydrogel in Example 1 was freeze-dried and added to complete culture medium, and then placed at 37°C for 1, 2, and 3 days to obtain HA / SF hydrogel leachate on the 1st, 2nd, and 3rd days, respectively. An appropriate amount of CLX liposome solution, IGF-1 solution, and CLX / IGF-1 solution was added to the HA / SF hydrogel leachate on the 1st, 2nd, and 3rd days, respectively, to obtain leachate of three kinds of drug-loaded hydrogels, which were co-cultured with C28 / I2 cells, respectively. Then, the CCK-8 (Cell Counting Kit-8) kit was used to detect the effect of the four kinds of hydrogels on the proliferation activity of the cells, and the results are shown in Figure 11.

[0138] The results show that the HA / SF hydrogel, CLX liposome hydrogel, IGF-1 hydrogel, and Dual double-loaded hydrogel all have no obvious cytotoxicity, and with the extension of time, the CLX liposome hydrogel and the double-loaded hydrogel show better ability to promote the proliferation of chondrocytes.

[0139] Example 13: Blood compatibility test

[0140] Fresh blood was taken from the abdominal aorta of rats, mixed well and transferred to a clean centrifuge tube. The red blood cell precipitate was obtained by centrifugation at 4°C, 3000 rpm for 15 min. The supernatant was discarded and washed with PBS solution for 3 times. Then the red blood cell precipitate was resuspended with PBS solution (2%, V / V) for use. 250 μL of 2% red blood cell suspension was mixed with 500 μL of HA / SF hydrogel, IGF-1 hydrogel, CLX hydrogel, Dual hydrogel of Example 1 and 5, PBS and 1% Triton (n=4) respectively, and incubated in a 37°C water bath for 1 h. After incubation, the samples were centrifuged at 3000 rpm for 15 min to observe whether hemolysis occurred. The supernatant was taken for OD value detection. The hemolysis rate was calculated according to the OD value, and the formula was as follows:

[0141] Hemolysis rate (%) = [(A-A0) / (A1-A0)]x100.

[0142] A0 is the OD value of the negative control group (PBS group), A1 is the OD value of the positive control group (Ora-ha-ha water), and A is the OD value of the experimental group. Hemolysis rate less than 5% can be considered as no hemolysis phenomenon. The results are shown in Figure 12. The hemolysis rates of the four prepared hydrogels, HA / SF hydrogel, IGF-1 hydrogel, CLX hydrogel and Dual hydrogel, are all less than 5%, indicating that they do not occur hemolysis in vivo.

[0143] Example 14: In vitro pharmacodynamic test

[0144] The HA / SF hydrogel, IGF-1 hydrogel, CLX hydrogel and Dual hydrogel of Example 1 and 5 were freeze-dried and added with appropriate complete medium to prepare a gel extract solution of 5 mg / ml. Appropriate IL-1β was added to the four extract solutions to prepare an IL-1β extract solution with a final concentration of 50 ng / ml. Then the C28 / I2 cells were co-cultured with the IL-1β extract solution at 37°C in a 5% carbon dioxide cell incubator for 48 hours.

[0145] 1. The cell supernatant was collected, and the levels of anti-inflammatory factors IL-4 and IL-10 and pro-inflammatory factors TNF-α and IL-6 were determined by Elisa kit. The results are shown in Figure 13.

[0146] Results showed that compared with the positive control group (PC group, induced chondrocytes as inflammatory OA cell model, no drug), HA / SF blank hydrogel, IGF-1 hydrogel, CLX hydrogel and Dual double-loaded hydrogel can significantly inhibit the expression level of pro-inflammatory factors TNF-α and IL-6, and improve the expression level of anti-inflammatory factors IL-4 and IL-10. Compared with IGF-1 hydrogel, Dual double-loaded hydrogel has significant difference in inhibiting TNF-α and IL-6; compared with CLX hydrogel, Dual double-loaded hydrogel has significant difference in inhibiting IL-6 and promoting IL-10.

[0147] 2, collect cells, extract RNA in cells by Trizol method, and reverse transcribe into cDNA, and further explore the gene expression of Sox9, COLII, MMP13, ADAMTS5 and Aggrecan by RT-PCR experiment, the results are shown in Figure 14, wherein NC is the negative control group, which uses normal cells without inducing inflammation, no drug, and normal cell culture solution is given.

[0148] Results showed that compared with PC, HA / SF blank hydrogel, IGF-1 hydrogel, CLX hydrogel and Dual double-loaded hydrogel can significantly improve the gene expression level of cartilage formation related gene COLII (collagen II), and down-regulate the gene expression level of related enzymes MMP13 (matrix metalloproteinase 13) and ADAMTS5 (platelet reaction protein disintegrin metallopeptidase 5) which promote the degradation of cartilage extracellular matrix; and Dual group significantly increases the gene expression level of cartilage formation related genes Sox9 and Aggrecan.

[0149] Compared with IGF-1 group, Dual group can significantly down-regulate the expression of MMP13, and up-regulate the gene expression of COLII, Aggrecan and Sox9.

[0150] Compared with CLX group, Dual group can significantly up-regulate the gene expression of COLII, Aggrecan and Sox9.

[0151] In summary, Dual double-loaded hydrogel plays a synergistic effect of anti-inflammatory drug celecoxib and growth factor IGF-1, significantly promotes the formation of cartilage and inhibits the degradation of cartilage extracellular matrix, which is beneficial to the treatment of bone and joint related diseases.

Claims

1. An intra-articular injectable hydrogel pharmaceutical composition comprising gel particles and a drug loaded in the gel particles; the gel particles comprising hyaluronic acid or a salt thereof, fibroin and a crosslinking agent; the drug comprising an anti-inflammatory drug and / or a growth factor, preferably comprising an anti-inflammatory drug and a growth factor.

2. The synovial joint injectable hydrogel pharmaceutical composition of claim 1, wherein, The anti-inflammatory drug is selected from non-steroidal anti-inflammatory drugs and / or glucocorticoids; preferably, the non-steroidal anti-inflammatory drug is selected from one or more of diclofenac, ibuprofen, celecoxib, acetaminophen, meloxicam, indomethacin, lornoxicam, etoricoxib, nimesulide; preferably, the glucocorticoid is selected from one or more of methylprednisolone, hydrocortisone, triamcinolone acetonide, prednisolone, betamethasone, dexamethasone.

3. The synovial joint injectable hydrogel pharmaceutical composition according to claim 1 or 2, wherein, The growth factor is selected from one or more of insulin-like growth factor 1 (IGF-1), transforming growth factors (TGFs), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs).

4. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-3, wherein, The anti-inflammatory drug is celecoxib and the growth factor is IGF-1.

5. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-4, wherein, The concentration of the anti-inflammatory drug is 0.01-10 wt%, preferably 0.01-1 wt%, more preferably 0.02-0.05 wt%, most preferably 0.03 wt%, based on the total mass of the gel particles.

6. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-5, wherein, The concentration of the growth factor is 10 -10 -10 -4 wt%, preferably 10 -9 -10 -5 wt%, more preferably 10 -8 -10 -6 wt%, most preferably 10 -7 wt%.

7. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-6, wherein, The molecular weight of the hyaluronic acid is 40-2.5 million Dalton, preferably 80-2.5 million Dalton, more preferably 150-2.5 million Dalton.

8. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-7, wherein, The molecular weight of the fibroin is 0.8-0.5 million Dalton, preferably 2.5-0.2 million Dalton, more preferably 2.5-0.1 million Dalton, still more preferably 5-0.1 million Dalton.

9. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-8, wherein, The elastic modulus of the gel particles is 1 x 10 2 Pa-1 x 10 4 Pa; preferably 1 x 10 3 -1 x 10 4 Pa, more preferably 500-3000 Pa, still more preferably 1000-3000 Pa.

10. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-9, wherein, The mass ratio of hyaluronic acid or a salt thereof: fibroin: crosslinking agent in the gel particles is (2.5-7.5):(0.5-5):1, preferably (4-7.5):(1-5):1, more preferably (4-7.5):(2-4):1, most preferably 4.5:2.3:

1.

11. The synovial joint injectable hydrogel pharmaceutical composition according to any one of claims 1-10, wherein, The salt of the hyaluronic acid is selected from one of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate, preferably sodium hyaluronate.

12. The synovial joint injectable hydrogel pharmaceutical composition of any one of claims 1-11, wherein, The crosslinking agent is selected from one or any combination of 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis-carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, preferably BDDE.

13. The intra-articular injectable hydrogel pharmaceutical composition according to any one of claims 1-12, wherein the gel particles are prepared by chemical crosslinking of the hyaluronic acid or a salt thereof, the fibroin and the crosslinking agent.

14. The synovial injectable hydrogel pharmaceutical composition according to any one of claims 1-13, wherein the synovial injectable hydrogel pharmaceutical further comprises an aqueous medium, preferably, the aqueous medium is one or several selected from the group consisting of deionized water, normal saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, HEPES buffer, more preferably, the aqueous medium is a phosphate buffer, still more preferably, the aqueous medium is a basic solution.

15. A method of preparing the injectable hydrogel pharmaceutical composition for joint cavity use according to any one of claims 1 to 14, characterized by, comprising the following steps: (1) Preparation of gel particles: dissolving HA and SF powders in an aqueous medium, preferably a basic solution, stirring uniformly, defoaming, adding a crosslinking agent for crosslinking, dialyzing, and granulating to obtain gel particles; (2) Preparation of drug-loaded hydrogel: adding a solution containing an anti-inflammatory drug and / or a growth factor to the gel particles.

16. The method of claim 15, wherein, The concentration of the anti-inflammatory drug in step (2) is 100-1000 pg / mL, preferably 200-500 pg / mL, more preferably 250 pg / mL.

17. The production method according to claim 15 or 16, characterized by, The concentration of the growth factor in step (2) is 50-500 ng / mL, preferably 75-200 ng / mL, more preferably 100 ng / mL.

18. A kit comprising: (1) an anti-inflammatory drug and / or a growth factor; and (2) gel particles comprising hyaluronic acid or a salt thereof, silk fibroin, and a crosslinking agent, Preferably, the kit comprises both an anti-inflammatory drug and a growth factor.

19. The kit of claim 18, wherein The anti-inflammatory drug is selected from the group consisting of non-steroidal anti-inflammatory drugs and / or glucocorticoids; preferably, the non-steroidal anti-inflammatory drug is selected from the group consisting of one or several of diclofenac, ibuprofen, celecoxib, acetaminophen, meloxicam, indomethacin, clodanac, etoricoxib, nimesulide; preferably, the glucocorticoid is selected from the group consisting of one or several of methylprednisolone, hydrocortisone, triamcinolone acetonide, prednisolone, betamethasone, dexamethasone.

20. The kit of claim 18 or 19, wherein, The growth factor is selected from the group consisting of one or several of insulin-like growth factor 1 (IGF-1), transforming growth factors (TGFs), bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs).

21. The kit of any one of claims 18-20, wherein, The anti-inflammatory drug is celecoxib, and the growth factor is IGF-1.

22. The kit of any one of claims 18-21, wherein, The components in the kit are mixed before use, so that the concentration of the anti-inflammatory drug is 0.01-10 wt%, preferably 0.01-1 wt%, more preferably 0.02-0.05 wt%, most preferably 0.03 wt%, based on the total mass of the gel particles.

23. The kit of any one of claims 18-22, wherein, The components in the kit are mixed prior to use such that the concentration of the growth factor is 10 -10 -10 -4 wt%, preferably 10 -9 -10 -5 wt%, more preferably 10 -8 -10 -6 wt%, most preferably 10 -7 wt%.

24. The kit of any one of claims 18-23, wherein, The hyaluronic acid has a molecular weight of 4-25 million Daltons, preferably 8-25 million Daltons, more preferably 15-25 million Daltons.

25. The kit of any one of claims 18-24, wherein, The silk fibroin has a molecular weight of 0.8-5 million Daltons, preferably 2.5-20 million Daltons, more preferably 2.5-10 million Daltons.

26. The kit of any one of claims 18-25, wherein, The elastic modulus of the gel particles is 1 x 10 2 Pa - 1 x 10 4 Pa; preferably 1 x 10 3 - 1 x 10 4 Pa, more preferably 500 - 3000 Pa, still more preferably 1000 - 3000 Pa.

27. The kit of any one of claims 18-26, wherein, The mass ratio of hyaluronic acid or its salt: silk fibroin: crosslinking agent in the gel particles is (2.5-7.5):(0.5-5):1, preferably (4-7.5):(1-5):1, more preferably (4-7.5):(2-4):1, most preferably 4.5:2.3:

1.

28. The kit of any one of claims 18-27, wherein, The salt of hyaluronic acid is selected from one of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate, preferably sodium hyaluronate.

29. The kit of any one of claims 18-28, wherein, The crosslinking agent is selected from one or any combination of 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylene diamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, preferably BDDE.

30. The kit of any one of claims 18-29, wherein the gel particles are prepared by chemical crosslinking of the hyaluronic acid or its salt, the silk fibroin and the crosslinking agent.

31. The kit of any one of claims 18-30, wherein the kit further comprises an aqueous medium, preferably the aqueous medium is selected from one or more of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, HEPES buffer, more preferably phosphate buffer, still more preferably the aqueous medium is an alkaline solution.

32. Use of the intra-articular injectable hydrogel pharmaceutical composition of any one of claims 1-14 or the composition prepared by the method of any one of claims 15-17 or the kit of any one of claims 18-31 in the manufacture of a medicament for the treatment of bone joint related diseases.

33. Use according to claim 32, characterized in that, The bone joint related diseases are selected from one or more of postoperative fracture, osteoarthritis, rheumatoid arthritis, degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, frozen shoulder, osteoporosis, osteonecrosis of the femoral head and other various joint dysfunction diseases.

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