Injectable hydrogel pharmaceutical composition for articular cavity as well as preparation method and application of injectable hydrogel pharmaceutical composition

Abstract

The invention discloses an injectable hydrogel pharmaceutical composition for an articular cavity as well as a preparation method and application of the injectable hydrogel pharmaceutical composition. The hydrogel pharmaceutical composition comprises gel particles and a medicine loaded in the gel particles, the gel particles are formed by cross-linking hyaluronic acid or salt thereof and silk fibroin through a cross-linking agent; the medicine comprises an anti-inflammatory drug and / or a growth factor. The preparation method is simple, good in safety and beneficial to industrial production. The uniform porous structure of the hydrogel is beneficial to adhesion and growth of bone cells, meanwhile, the double-load hydrogel loaded with the anti-inflammatory drug and the growth factor has good viscoelasticity and injectability and has the effects of synergistically resisting inflammation and promoting cartilage repair, and bone joint related diseases are treated in a bone joint intracavity injection mode; the drug residence time can be prolonged, and the bioavailability is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to an injectable hydrogel pharmaceutical composition for joint cavity, its preparation method and application. Background Technology

[0002] Osteoarthritis (OA) is a weakening joint disease that leads to damage to articular cartilage and underlying bone. Cartilage is composed of chondrocytes and the extracellular matrix (ECM). The ECM provides cells with a suitable environment and nutrients, and its reticular structure provides the cartilage with elasticity and shape-forming ability. Chondrocyte apoptosis and abnormal ECM degradation are hallmarks of OA pathological changes. A WHO survey indicates that 10% of men and 20% of women aged 60 and older have symptomatic OA, with 80% of these patients experiencing limitations in daily activities and 30% lacking the ability to live independently. OA is highly prevalent among middle-aged and elderly people, and the aging population in my country is exacerbating the increase in the OA patient population.

[0003] Commonly used oral medications for osteoarthritis (OA) treatment include nonsteroidal anti-inflammatory drugs (NSAIDs), selective cyclooxygenase (COX)-2 inhibitors, opioids, and glucocorticoids. However, these medications only relieve symptoms and do not improve cartilage defects.

[0004] Therefore, there is an urgent need for a safe and effective drug to treat osteoarthritis and other bone-related diseases. Summary of the Invention

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

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides an intra-articular injectable hydrogel drug composition comprising gel particles and a drug loaded in the gel particles; the gel particles comprising hyaluronic acid or its salt, silk fibroin and a cross-linking agent; the drug comprising an anti-inflammatory drug and / or a growth factor, preferably comprising an anti-inflammatory drug and a growth factor.

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

[0008] In a preferred embodiment, the growth factor is selected from one or more of insulin-like growth factor 1 (IGF-1), transforming growth factor (TGFs), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF).

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

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

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

[0012] In a preferred embodiment, the hyaluronic acid has a molecular weight of 400,000 to 2,500,000 Daltons, more preferably 800,000 to 2,500,000 Daltons, and even more preferably 1,500,000 to 2,500,000 Daltons.

[0013] In a preferred embodiment, the silk fibroin has a molecular weight of 0.8-500,000 Daltons, preferably 2.5-200,000 Daltons, more preferably 2.5-100,000 Daltons, and even more preferably 50,000-100,000 Daltons.

[0014] In a preferred embodiment, the elastic modulus of the gel particles is 1×10⁻⁶. 2 Pa-1×10 4 Pa; preferably 1×10 3 -1×10 4 Pa, more preferably 500-3000 Pa, and even 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, and most preferably 4.5:2.3:1.

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

[0017] In a preferred embodiment, the crosslinking agent is selected from 1,4-bisglycidoxybutane, 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), dicarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipamide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, or any combination thereof, 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 more of deionized water, physiological saline, phosphate buffer (PBS), Tris buffer, acetate buffer, and HEPES buffer, more preferably phosphate buffer, and even more preferably, the aqueous medium is an alkaline solution.

[0020] In a second aspect, the present invention provides a method for preparing the aforementioned intra-articular injectable hydrogel pharmaceutical composition, characterized by comprising the following steps:

[0021] (1) Preparation of gel particles: Dissolve HA and SF powder in an aqueous medium, stir evenly, defoam, add crosslinking agent for crosslinking, dialyze, granulate to obtain gel particles, wherein the aqueous medium is preferably an alkaline solution; further, the alkaline solution is a sodium hydroxide solution;

[0022] Furthermore, the defoaming treatment conditions are: treatment at 2-25℃ for more than 12 hours; even further, treatment at 4℃ for 24 hours.

[0023] Furthermore, the crosslinking conditions are incubation at 37-60°C; even further, incubation at 37°C for 4 hours.

[0024] (2) Preparation of drug-loaded hydrogel: Add a solution containing anti-inflammatory drugs and / or growth factors to the gel particles.

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

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

[0028] (1) Anti-inflammatory drugs and / or growth factors; and

[0029] (2) Gel particles, said gel particles comprising hyaluronic acid or its salt, silk fibroin and a cross-linking agent.

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

[0031] In a preferred embodiment, the anti-inflammatory drug is selected from nonsteroidal anti-inflammatory drugs and / or glucocorticoids; preferably, the nonsteroidal anti-inflammatory drug is selected from one or more of diclofenac, ibuprofen, celecoxib, acetaminophen, meloxicam, indomethacin, cloxicam, etoricoxib, and nimesulide; preferably, the glucocorticoid is selected from one or more of methylprednisolone, hydrocortisone, triamcinolone, prednisolone, betamethasone, and 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 factor (TGFs), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF).

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

[0034] In a preferred embodiment, the ingredients 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%, and most preferably 0.03 wt%, based on the total mass of the gel particles.

[0035] In a preferred embodiment, the components of the kit are mixed prior to use such that the concentration of the growth factor is 10 based on the total mass of the gel particles. -10 -10 -4 wt%, preferably 10 -9 -10 -5 wt%, more preferably 10 -8 -10 -6 wt%, optimal choice 10 -7 wt%.

[0036] In a preferred embodiment, the hyaluronic acid has a molecular weight of 400,000 to 2,500,000 Daltons, more preferably 800,000 to 2,500,000 Daltons, and even more preferably 1,500,000 to 2,500,000 Daltons.

[0037] In a preferred embodiment, the silk fibroin has a molecular weight of 0.8-500,000 Daltons, preferably 2.5-200,000 Daltons, and more preferably 2.5-100,000 Daltons.

[0038] In a preferred embodiment, the elastic modulus of the gel particles is 1×10⁻⁶. 2 Pa-1×10 4 Pa; preferably 1×10 3 -1×10 4 Pa, more preferably 500-3000 Pa, and even more preferably 1000-3000 Pa.

[0039] 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, and most preferably 4.5:2.3:1.

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

[0041] In a preferred embodiment, the crosslinking agent is selected from 1,4-bisglycidoxybutane, 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), dicarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipamide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, or any combination thereof, preferably BDDE.

[0042] 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.

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

[0044] In a fourth aspect, the present invention provides the use of the aforementioned intra-articular injectable hydrogel pharmaceutical composition or the composition prepared according to the aforementioned method or the aforementioned kit in the preparation of a medicament for treating osteoarthritis-related diseases.

[0045] In a preferred embodiment, the bone and joint related diseases are selected from one or more of the following: post-fracture surgery, osteoarthritis, rheumatoid arthritis, degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, frozen shoulder, osteoporosis, femoral head necrosis, and other joint dysfunction diseases.

[0046] Beneficial effects:

[0047] This invention is the first to simultaneously load an anti-inflammatory drug (celecoxib) and a growth factor (IGF-1) into an HA / SF hydrogel. In vitro experiments showed that, compared with the hydrogel group loaded with a single drug, the Dual (dual-loaded hydrogel) group of this invention significantly inhibited the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors. At the same time, it upregulated the mRNA expression levels of cartilage formation-related genes (COLII, Sox9, and Aggrecan) and downregulated the mRNA expression levels of genes related to enzymes that promote the degradation of extracellular matrix of chondrocytes (MMP1 and ADAMTS5). It has a synergistic anti-inflammatory effect and promotes cartilage repair, and is expected to provide a potential drug delivery system for the treatment of OA.

[0048] The uniform porous structure of the hydrogel of this invention promotes osteocyte adhesion and growth. Even after loading with anti-inflammatory drugs and growth factors, it retains good viscoelasticity and excellent injectability. When administered via intra-articular injection, it can prolong drug retention time and improve bioavailability for treating osteoarthritis-related diseases. The intra-articular injectable hydrogel drug composition of this invention has a simple preparation method, good safety profile, and is conducive to industrial production. Brief description of the attached diagram

[0049] Figure 1 This indicates the elastic modulus of HA / SF hydrogels made using different amounts of gel particles.

[0050] Figure 2 The values ​​represent the elastic modulus of HA / SF hydrogels with different HA molecular weights, where HA-SF-S represents HA / SF hydrogels prepared using S-HA, HA-SF-M represents HA / SF hydrogels prepared using M-HA, and HA-SF-L represents HA / SF hydrogels prepared using L-HA.

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

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

[0053] Figure 5 The images show the appearance of unloaded HA / SF hydrogels, IGF-1 loaded HA / SF hydrogels, CLX loaded HA / SF hydrogels, and dual-drug loaded HA / SF hydrogels.

[0054] Figure 6 SEM images of unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel, and dual-drug loaded HA / SF hydrogel are shown. The scale bar in the left column is 300.0 μm, and the scale bar in the right column is 2.0 μm.

[0055] Figure 7 The swelling properties of unloaded HA / SF hydrogels, IGF-1 loaded HA / SF hydrogels, CLX loaded HA / SF hydrogels, and dual-drug loaded HA / SF hydrogels are indicated.

[0056] Figure 8 The rheological properties of unloaded HA / SF hydrogels, IGF-1 loaded HA / SF hydrogels, CLX loaded HA / SF hydrogels, and dual-drug loaded HA / SF hydrogels are represented.

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

[0058] Figure 10 The image shows the drug release curve of the Dual dual-load hydrogel.

[0059] Figure 11 The cell proliferation activity of extracts from unloaded HA / SF hydrogels, IGF-1 loaded HA / SF hydrogels, CLX loaded HA / SF hydrogels, and dual-drug loaded HA / SF hydrogels is indicated.

[0060] Figure 12 The blood compatibility of unloaded HA / SF hydrogels, IGF-1 loaded HA / SF hydrogels, CLX loaded HA / SF hydrogels, and dual-drug loaded HA / SF hydrogels is indicated.

[0061] Figure 13 The in vitro anti-inflammatory effects of unloaded HA / SF hydrogel, IGF-1 loaded HA / SF hydrogel, CLX loaded HA / SF hydrogel, and dual-drug loaded HA / SF hydrogel are shown. PC: Control group, C28 / I2 cells induced into inflammatory cells and given normal cell culture medium.

[0062] Figure 14 This study illustrates the effects of unloaded HA / SF hydrogels, IGF-1-loaded HA / SF hydrogels, CLX-loaded HA / SF hydrogels, and dual-drug-loaded HA / SF hydrogels on gene expression levels in chondrocyte C28 / I2 cells induced to become inflammatory cells. PC: Control group, C28 / I2 cells induced to become inflammatory cells, given normal cell culture medium; NC: C28 / I2 cells not induced to become inflammatory, given normal cell culture medium.

[0063] Figure 15 SDS gel electrophoresis results for lyophilized blocks of silk fibroin in three molecular weight ranges.

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

[0066] definition

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

[0068] As used herein, the term "hyaluronic acid" is a biodegradable polymeric component composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. This water-soluble polymer is naturally found in almost all tissues, particularly in the extracellular matrix, synovial fluid of the eye, and 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 the mechanical stretching of the dermis and the activation of dermal fibroblasts.

[0069] As used herein, the term "crosslinking" refers to the intermolecular bonds that link individual polymer molecules, macromolecules, and / or monomer chains into a more stable structure such as a gel. 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-bisglycidoxybutane, 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), dicarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic hydrazide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, etc.

[0070] As used herein, the term "gel" refers to a thick liquid or semi-solid formulation in the form of a solution, suspension, or emulsion. Gel matrices are single-phase dispersion systems and can be aqueous or oil-based. Aqueous gel matrices are generally composed of water, glycerol or propylene glycol with cellulose derivatives, carbomer and alginate, tragacanth gum, gelatin, starch, etc.; oil-based gel matrices are composed of liquid paraffin with polyethylene or fatty oils with colloidal silica or aluminum soaps, zinc soaps, etc.

[0071] As used in this article, the term "anti-inflammatory drug" refers to medications that treat inflammation resulting from tissue damage. Anti-inflammatory drugs include steroidal anti-inflammatory drugs (NSAIDs) and nonsteroidal anti-inflammatory drugs (NSAIDs). Steroidal NSAIDs exert their anti-inflammatory effects by inhibiting prostaglandin synthesis, suppressing leukocyte aggregation, reducing bradykinin formation, and inhibiting platelet aggregation. Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of anti-inflammatory drugs that do not contain a steroidal structure. These drugs include aspirin, acetaminophen, indomethacin, naproxen, naproxenone, diclofenac, ibuprofen, nimesulide, rofecoxib, and celecoxib. These drugs have anti-inflammatory, antirheumatic, analgesic, antipyretic, and anticoagulant effects and are widely used clinically to relieve symptoms of osteoarthritis, rheumatoid arthritis, various types of fever, and various types of pain.

[0072] As used in this article, the term "growth factor" refers to a class of polypeptides that regulate cell growth and other cellular functions through binding to specific, high-affinity cell membrane receptors. These are cytokines secreted by various cells, acting on specific target cells to regulate cell division, matrix synthesis, and tissue differentiation. There are many types of growth factors, such as platelet-derived growth factors (PDGF; osteosarcoma-derived growth factor ODGF), epidermal growth factors (EGF, TGFα, and TGFβ), fibroblast growth factors (αFGF, βFGF), insulin-like growth factors (IGF-Ⅰ, IGF-Ⅱ), nerve growth factor (NGF), interleukin-like growth factors (IL-1, IL-3, etc.), erythropoietin (EPO), and colony-stimulating factor (CSF).

[0073] As used herein, the term "salt" includes, for example, salts of inorganic acids and salts of organic acids. Examples of salts may include hydrochlorides, phosphates, pyrophosphates, hydrobroms, sulfates, sulfinates, nitrates, malates, maleates, fumarates, tartrates, succinates, citrates, acetates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, and alkylates (e.g., acetates, HOOC-(CH2)). n -COOH, where n is 0-4). Furthermore, if the compound herein is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acid salt. Conversely, if the compound herein is a free base, the 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 acid, according to conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will understand the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts.

[0074] As used herein, the term “treatment” and its related expressions mean therapeutic treatment. When referring to a specific condition, treatment means: (1) improving the condition or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that cause or contribute to the condition, or (b) one or more biological manifestations of the condition, (3) alleviating one or more symptoms, effects or side effects associated with or related to the condition or its treatment, or (4) slowing the progression of the condition, or slowing one or more biological manifestations of the condition.

[0075] As used herein, “prevention” means prophylactic administration of medication to substantially reduce the likelihood or severity of a disease or its biological manifestations, or to delay the onset of such a disease or its biological manifestations. Those skilled in the art will understand that “prevention” is not an absolute term. For example, prophylactic treatment is appropriate when a subject is considered 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 carcinogen.

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

[0077] As used herein, the term "kit" may include two or more single-dose or multi-dose pharmaceutical agents, each individually packaged or formulated; or two or more single-dose or multi-dose pharmaceutical agents packaged or formulated in combination. Thus, one or more pharmaceutical agents may be present in a first container, and the kit may optionally include one or more pharmaceutical agents in a second container. One or more containers are housed within a package, and the package may optionally include instructions for administration or dosage. The kit may include additional components, such as syringes or other components for administering the pharmaceutical agents and diluents, or other components for formulation. Detailed Implementation Plan

[0078] Insulin-like growth factor (IGF) is the main anabolic growth factor in cartilage. It protects the NF-κB pathway by inhibiting the PI3K / Akt and MAPKs-specific pathways. Macroscopic and pathological studies have shown that it has a chondroprotective effect by promoting the formation of hyaline cartilage.

[0079] Celecoxib is a selective cyclooxygenase inhibitor. Inflammatory stimuli can induce the production of cyclooxygenase-2 (COX-2), leading to the synthesis and accumulation of inflammatory prostaglandins, especially prostaglandin E2, causing inflammation, edema, and pain. Celecoxib can inhibit COX-2 to prevent the production of inflammatory prostaglandins, thus achieving anti-inflammatory, analgesic, and antipyretic effects. Clinically, it can be used to treat, prevent, and alleviate osteoarthritis, rheumatoid arthritis, various types of fever, and various pain symptoms.

[0080] Intra-articular injectable hydrogels are commonly made from either natural or synthetic materials. Common natural materials include hyaluronic acid, chondroitin sulfate, chitosan, and gelatin; synthetic materials mainly include PEG and PVA. Hyaluronic acid (HA), a natural polysaccharide, is a major component of the extracellular matrix of articular cartilage cells. Studies have shown that the content and viscoelasticity of hyaluronic acid in the synovial fluid of osteoarthritis (OA) patients are significantly reduced. Therefore, intra-articular injection of hyaluronic acid solution or hydrogel can replenish the missing components in the synovial fluid, making it an excellent viscoelastic supplement.

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

[0082] In one embodiment, this application provides a dual-drug-loaded hydrogel containing HA and SF hydrogels co-encapsulating anti-inflammatory drugs and growth factors, which is used for intra-articular injection to treat osteoarthritis-related diseases.

[0083] Example

[0084] The technical solution of the present invention will be described in detail below with reference to the embodiments. However, those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0085] Materials and their sources used in the following embodiments:

[0086] Hyaluronic acid from Shanghai Maclean Biochemical Technology Co., Ltd. (molecular weight 400,000-800,000 Daltons, batch number C14499337; molecular weight 800,000-1,500,000 Daltons, batch number C14504635; molecular weight 1,500,000-2,500,000 Daltons, batch number C14554777).

[0087] Silk fibroin, produced by Shenzhen Huasi Biotechnology Co., Ltd. (batch number SLPSXK1N005);

[0088] Celecoxib Beijing Bailingwei Technology Co., Ltd.;

[0089] Insulin-like growth factor (IGF-1) Abbkine;

[0090] BDDE Shanghai Maclean Biotechnology Co., Ltd.

[0091] Example 1: Preparation of HA / SF hydrogel

[0092] 1. Preparation of HA / SF hydrogel

[0093] (1) Accurately weigh 0.2g of sodium hydroxide, add 20mL of Wahaha water, stir to dissolve, and prepare a 1% sodium hydroxide solution for later use;

[0094] (2) Accurately weigh 0.2g of HA (molecular weight 1.5-2.5 million Daltons) and 0.1g of SF (molecular weight 2.5-10 million Daltons) into a 10mL beaker, add 2mL of 1% sodium hydroxide solution, stir evenly, and then defoam at 4℃ for 24h.

[0095] (3) After defoaming, add 0.044g BDDE to form a mixed solution and incubate in a water bath at 37℃ for 4h;

[0096] (4) Remove the gel and place it in an 8000 molecular weight dialysis bag for dialysis for 24 hours;

[0097] (5) After dialysis, the HA / SF gel particles were granulated by passing them through a 60-mesh sieve.

[0098] (6) Weigh 0.8g of HA / SF gel particles, add 1mL of PBS solution, and stir until homogeneous to obtain HA / SF hydrogel.

[0099] 2. Investigation of the rheological properties of hydrogels at different concentrations

[0100] Weigh out 0.6 g, 0.8 g, and 1.0 g of HA / SF gel particles, respectively, and resuspend them in 1 mL of PBS solution. Use a Kinex rheometer to determine the elastic modulus G' at shear frequencies 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 [link to Kinex rheometer]). Figure 1 ).

[0101] The results showed that the hydrogel prepared by resuspending 0.8g HA / SF gel particles in 1mL PBS solution had good elastic modulus and good injectability.

[0102] Example 2: Investigation of HA with different molecular weights

[0103] To further explore which molecular weight of HA is more suitable for intra-articular injection, three molecular weights of HA were investigated: 400,000-800,000 Daltons, 800,000-1,500,000 Daltons, and 1,500,000-2,500,000 Daltons, denoted as S-HA, M-HA, and L-HA. Following the method in Example 1, HA of different molecular weights were crosslinked with SF (molecular weight 25,000-100,000 Daltons). The elastic modulus (G') of the HA / SF gel particles was measured using a rheometer. The results are shown in [Figure 1]. Figure 2 .

[0104] The results showed that the gel particles prepared by L-HA had a high elastic modulus, so HA with a strength of 1.5-2.5 million Daltons was selected for the following experiments.

[0105] Example 3: Investigation of SF with different molecular weights

[0106] A fractional dialysis bag with an outer, middle, and inner layer structure was prepared using dialysis bags with three different molecular weight cutoffs of 100 kDa, 25 kDa, and 8 kDa. Silk fibroin solution was added to the inner dialysis bag, and dialysis was initiated. Ultimately, the outer, middle, and inner dialysis bags yielded silk fibroin solutions with different molecular weight ranges. The silk fibroin solutions from the outer, middle, and inner dialysis bags were then removed and freeze-dried to obtain lyophilized blocks of silk fibroin with different molecular weight ranges: 8 kDa-25 kDa silk fibroin, 25 kDa-100 kDa silk fibroin, and >100 kDa silk fibroin. Figure 15 The results of SDS gel electrophoresis of lyophilized blocks of silk fibroin in three molecular weight ranges are shown.

[0107] The anti-inflammatory effects of hydrogels prepared with three different molecular weights (0.8-2.5 million Daltons, 2.5-10 million Daltons, and over 100,000 Daltons) of SF were compared. HA / SF hydrogels were prepared according to the method in Example 1, and designated as inner (containing 0.8-2.5 million Daltons of SF), middle (containing 2.5-10 million Daltons of SF), and outer (containing over 100,000 Daltons of SF), respectively. After freeze-drying, appropriate complete culture medium was added to prepare an 8 mg / ml gel extract. An appropriate amount of IL-1β was added to the extract to prepare an IL-1β extract with a final concentration of 50 ng / ml. The extract 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 measured using an ELISA kit. The results are shown below. Figure 3 .

[0108] The results showed that, compared with the control group (C28 / I2 cells induced into inflammatory cells without drug administration, given normal cell culture medium), HA / SF hydrogel prepared with medium molecular weight (25,000-100,000 Daltons) SF significantly increased 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 internal and external groups, the medium group had the effect of inhibiting the expression of pro-inflammatory factor IL-6.

[0109] Example 4: Investigation of HA and SF concentrations

[0110] Accurately weigh 0.1g of HA and 0.1g of SF; 0.16g of HA and 0.1g of SF; 0.2g of HA and 0.1g of SF; 0.2g of HA and 0.02g of SF; 0.2g of HA and 0.04g of SF; 0.2g of HA and 0.16g of SF; 0.2g of HA and 0.2g of SF; and 0.3g of HA and 0.1g of SF into 10mL beakers respectively. Add 2mL of 1% sodium hydroxide solution and stir well to obtain HA and SF solutions of different concentrations, which are recorded as samples 1-8. Prepare HA / SF gel particles from the above solutions according to the method in Example 1. Measure their elastic modulus (G') using a Kinex rheometer. The results are shown in Table 1 and 2. Figure 4 .

[0111] The results showed that the G' of gel particles with different HA and SF concentrations varied greatly. Gel particles with 10%-15% HA and 2%-10% SF had higher G', but the hydrogel prepared with 15% HA and 5% SF had poor injectability and could not be easily injected within 30 seconds. Considering that it is used for intra-articular injection, the requirements for injectability and elastic modulus are higher. Gel particles with 10% HA and 5%-8% SF have better performance.

[0112] Table 1 Elastic modulus (G') of gel particles with different HA and SF concentrations

[0113] sample HA (mass percentage) SF (Percentage by weight) At 10 Hz G'(Pa) 1 5％ 5％ 73.9 2 8％ 5％ 463.9 3 10％ 5％ 2895.3 4 10％ 1％ 589.9 5 10％ 2％ 641.4 6 10％ 8％ 1551.0 7 10％ 10％ 773.5 8 15％ 5％ 3026.7

[0114] Example 5: Preparation of drug-loaded hydrogel

[0115] 1. Preparation of HA / SF hydrogel loaded with celecoxib (CLX) liposomes

[0116] (1) Preparation of CLX liposomes: Accurately weigh 4 mg of CLX, 80 mg of phospholipids and 20 mg of cholesterol into a 50 mL round bottom flask, add 20 mL of chloroform and dissolve it completely; remove the organic solvent chloroform by rotary evaporation under reduced pressure at 60 °C, and then vacuum dry for 24 h to further remove the organic solvent; then add 16 mL of PBS buffer, and sonicate with the probe for 20 min (power 100 W, total working time 20 min, sonication on 0.2 s, sonication off 0.1 s), and the CLX liposome solution is ready;

[0117] (2) Preparation of HA / SF hydrogel loaded with CLX: Weigh 0.8 g of HA / SF gel particles from Example 1, add 1 mL of CLX liposome solution (CLX concentration 0.25 mg / mL), and stir until homogeneous. The mass percentage of CLX in the gel particles is 0.03 wt%.

[0118] 2. Preparation of HA / SF hydrogels loaded with IGF-1

[0119] Take 1 mL of PBS solution, add an appropriate amount of IGF-1, and prepare an IGF-1 solution with a concentration of 100 ng / mL for later use;

[0120] Weigh 0.8 g of the HA / SF gel particles from Example 1, add 1 mL of 100 ng / mL IGF-1 solution, and stir until homogeneous. The mass percentage of IGF-1 in the gel particles is 10%. -7 wt%.

[0121] 3. Preparation of HA / SF hydrogels loaded with CLX and IGF-1:

[0122] Take 1 mL of CLX liposome solution and add an appropriate amount of IGF-1 to obtain IGF-1 / CLX solution (CLX concentration 0.25 mg / mL, IGF-1 concentration 100 ng / mL);

[0123] Weigh 0.8 g of the HA / SF gel particles from Example 1, add 1 mL of IGF-1 / CLX solution, and stir until homogeneous to obtain the CLX / IGF-1 dual-carrier hydrogel. The mass percentage of CLX in the gel particles is 0.03 wt%, and the mass percentage of IGF-1 is 10 wt%. -7 wt%.

[0124] Example 6: Appearance Transparency Test

[0125] On a blank A4 sheet of paper, write HA / SF, CLX, IGF-1, and Dual with a black marker. Take an appropriate amount of HA / SF hydrogel (HA / SF) from Example 1, CLX hydrogel (CLX) from Example 5, IGF-1 hydrogel (IGF-1), and CLX / IGF-1 dual hydrogel (Dual) and cover them over the corresponding text. Observe the appearance and transparency of the hydrogels.

[0126] The results are as follows Figure 5 As shown, all four types of hydrogels prepared exhibited good transparency.

[0127] Example 7: Injectability Test

[0128] The HA / SF hydrogel of Example 1 and the CLX / IGF-1 dual hydrogel of Example 5 were labeled with tartrazine and rhodamine B, respectively, and the injectability of the two hydrogels in a 1 mL syringe was observed.

[0129] The results confirmed that both hydrogels could be easily injected with a 1mL syringe within 30 seconds, demonstrating good injectability.

[0130] Example 8: Microscopic Morphology Characterization

[0131] Appropriate amounts of the HA / SF hydrogel (HA / SF) from Example 1, and the CLX hydrogel (CLX), IGF-1 hydrogel (IGF-1), and CLX / IGF-1 dual hydrogel (Dual) from Example 5 were freeze-dried. The surface morphology of the two hydrogels was observed using a scanning electron microscope (SEM). The results are as follows: Figure 6 As shown.

[0132] The results showed that the HA / SF hydrogel prepared in this invention has high porosity and a uniform and dense pore structure. The addition of IGF-1 did not affect the appearance morphology or pore distribution of the HA / SF hydrogel. Uniform distribution of drug-loaded liposomes was observed on the pore walls of both the HA / SF hydrogel loaded with CLX liposomes and the Dual dual-loaded hydrogel (as shown by arrows in the figure), indicating that the drug-loaded liposomes were successfully loaded into the HA / SF hydrogel.

[0133] Example 9: Swelling Test

[0134] Appropriate amounts of the HA / SF hydrogel (HA / SF) from Example 1, and the CLX hydrogel (CLX), IGF-1 hydrogel (IGF-1), and CLX / IGF-1 dual-loaded hydrogel (Dual) from Example 5 were taken and their initial weights were recorded. They were then completely immersed in Wahaha water and weighed after 15, 30, 45, 60, 75, 90, 105, and 120 minutes, respectively. The weights of the hydrogels at different time points were recorded, and their swelling properties were observed. The results are as follows: Figure 7 As shown.

[0135] The results showed that the hydrogels prepared in this invention all exhibited good water absorption and swelling properties. The addition of drugs, especially celecoxib liposomes, reduced the swelling properties of the hydrogels to some extent, possibly due to the hydrophobicity of the liposomes and their blocking of some pores in the hydrogel.

[0136] Example 10: Rheological property test

[0137] Appropriate amounts of the HA / SF hydrogel (HA / SF) from Example 1, the CLX hydrogel (CLX), the IGF-1 hydrogel (IGF-1), and the CLX / IGF-1 dual-loaded hydrogel (Dual) from Example 5 were taken and subjected to rheological tests. The elastic modulus G' and viscous modulus G of each group at shear frequencies of 0.1-10Hz were recorded (see [link to article]). Figure 8 ).

[0138] Figure 9 The elastic modulus G' is the elastic modulus at a shear frequency of 10 Hz. The results show that the elastic modulus of HA / SF hydrogel is about 900 Pa, which meets the mechanical property requirements of intra-articular injection hydrogel. For drug-loaded hydrogels, especially the loading of celecoxib liposomes, the elastic modulus of HA / SF hydrogel can be significantly improved, increasing it to about 1100 Pa.

[0139] Example 11: Drug release test in Dual

[0140] An appropriate amount of the Dual dual-carrier hydrogel from Example 5 was placed in a dialysis bag (MWCO = 8000-14000) and dialyzed. The release solution was PBS (pH = 7.0-7.2). At specified time points (days 1, 3, 5, 7, 10, 14, 21, 28, and 35), appropriate amounts of dialysate were collected to measure drug release. Simultaneously, an equal volume of fresh release solution was added. The results are as follows: Figure 10 As shown.

[0141] The results showed that the Dual dual-load hydrogel could act as a drug reservoir in the joint cavity, achieving a sustained-release effect on growth factor IGF-1 and anti-inflammatory drug CLX.

[0142] Example 12: Cell compatibility test

[0143] Human chondrocytes (C28 / I2 cells) were used in the experiment. First, an appropriate amount of HA / SF hydrogel from Example 1 was taken, freeze-dried, and added to complete culture medium. The mixture was incubated at 37°C for 1, 2, and 3 days to obtain HA / SF hydrogel extracts on days 1, 2, and 3, respectively. Appropriate amounts of CLX liposome solution, IGF-1 solution, and CLX / IGF-1 solution were added to the HA / SF hydrogel extracts on days 1, 2, and 3, respectively, to obtain extracts of three drug-loaded hydrogels. These extracts were co-cultured with C28 / I2 cells, and the effects of the four hydrogels on cell proliferation activity were then detected using the CCK-8 (Cell Counting Kit-8) assay. The results are as follows: Figure 11 As shown.

[0144] The results showed that HA / SF hydrogel, CLX liposome hydrogel, IGF-1 hydrogel and Dual dual-load hydrogel had no significant cytotoxicity, and CLX liposome hydrogel and dual-load hydrogel showed better ability to promote chondrocyte proliferation over time.

[0145] Example 13: Blood compatibility test

[0146] Fresh blood was collected from the abdominal aorta of rats, thoroughly mixed, and transferred to a clean centrifuge tube. The tube was centrifuged at 3000 rpm for 15 min at 4°C to obtain erythrocyte pellet. The supernatant was discarded, and the pellet was washed three times with PBS solution. The pellet was then resuspended in PBS solution (2%, V / V) for later use. 250 μL of the 2% erythrocyte suspension was mixed thoroughly with 500 μL of HA / SF hydrogel, IGF-1 hydrogel, CLX hydrogel, and Dual hydrogel from Examples 1 and 5, respectively, along with PBS and 1% Triton (n=4), and incubated in a 37°C water bath for 1 h. After incubation, the pellet was centrifuged at 3000 rpm for 15 min to observe for hemolysis. The supernatant was collected for OD value analysis. The hemolysis rate was calculated based on the OD value using the following formula:

[0147] Hemolysis rate (%) = [(A-A0) / (A1-A0)] × 100.

[0148] A0 represents the OD value of the negative control group (PBS group), A1 represents the OD value of the positive control group (Wahaha Water), and A represents the OD value of the experimental group. A hemolysis rate of less than 5% can be considered as no hemolysis has occurred. Results are as follows... Figure 12 As shown, the hemolysis rates of the four hydrogels prepared—HA / SF hydrogel, IGF-1 hydrogel, CLX hydrogel, and Dual hydrogel—were all less than 5%, indicating that they did not undergo hemolysis in vivo.

[0149] Example 14: In vitro pharmacodynamic test

[0150] The HA / SF hydrogel, IGF-1 hydrogel, CLX hydrogel, and Dual hydrogel from Examples 1 and 5 were freeze-dried and then added to an appropriate complete culture medium to prepare a gel extract at a concentration of 5 mg / ml. An appropriate amount of IL-1β was added to each of the four extracts to prepare an IL-1β extract with a final concentration of 50 ng / ml. These extracts were then co-cultured with C28 / I2 cells at 37°C in a 5% CO2 cell culture incubator for 48 hours.

[0151] 1. Collect cell supernatant and use an ELISA kit to measure the levels of anti-inflammatory factors IL-4 and IL-10, and pro-inflammatory factors TNF-α and IL-6. The results are as follows: Figure 13 As shown.

[0152] The results showed that, compared with the positive control group (PC group, induced chondrocytes were an inflammatory OA cell model, no drug was administered), HA / SF blank hydrogel, IGF-1 hydrogel, CLX hydrogel, and Dual dual-load hydrogel all significantly inhibited the expression levels of pro-inflammatory factors TNF-α and IL-6, and increased the expression levels of anti-inflammatory factors IL-4 and IL-10. Compared with IGF-1 hydrogel, Dual dual-load hydrogel showed a significant difference in the inhibitory effect on TNF-α and IL-6; compared with CLX hydrogel, Dual dual-load hydrogel showed a significant difference in the inhibitory effect on IL-6 and the promoting effect on IL-10.

[0153] 2. Cells were collected, and RNA was extracted from the cells using the Trizol method and reverse transcribed into cDNA. RT-PCR was then used to further investigate the gene expression of Sox9, COLII, MMP13, ADAMTS5, and Aggrecan. The results are as follows: Figure 14 As shown, NC is the negative control group, which uses normal cells that have not been induced to cause inflammation, without drug administration, and is given normal cell culture medium.

[0154] The results showed that, compared with PC, HA / SF blank hydrogel, IGF-1 hydrogel, CLX hydrogel and Dual dual-load hydrogel could significantly increase the gene expression level of cartilage formation-related gene COLII (collagen II), and downregulate the gene expression levels of enzymes that promote the degradation of extracellular matrix of chondrocytes, MMP13 (matrix metalloproteinase 13) and ADAMTS5 (platelet-reactive protein integrin metallopeptidase 5); and the Dual group significantly increased the gene expression levels of cartilage formation-related genes Sox9 and Aggrecan.

[0155] Compared with the IGF-1 group, the Dual group significantly downregulated the expression of MMP13 and upregulated the gene expression of COLII, Aggrecan and Sox9.

[0156] Compared with the CLX group, the Dual group significantly upregulated the gene expression of COLII, Aggrecan, and Sox9.

[0157] In summary, the Dual dual-load hydrogel exerts a synergistic effect between the anti-inflammatory drug celecoxib and the growth factor IGF-1, significantly promoting cartilage formation and inhibiting the degradation of the extracellular matrix of chondrocytes, which is beneficial for the treatment of osteoarthritis-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 method of manufacturing according to claim 15 or 16, wherein, 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, loxoprofen, 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, periarthritis of shoulder, osteoporosis, osteonecrosis of the femoral head and other various joint dysfunction diseases.

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