Triamcinolone acetonide-loaded hydrogel as well as preparation method and application thereof

Abstract

The invention provides a triamcinolone acetonide-loaded hydrogel as well as a preparation method and application thereof. The anti-inflammatory and cartilage repair effects of the triamcinolone acetonide-loaded hydrogel in arthritis are evaluated through an in-vitro cell experiment and an in-vivo animal model, and the triamcinolone acetonide-loaded hydrogel has excellent clinical potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a triamcinolone acetonide-loaded hydrogel, its preparation method and application. Background Art

[0002] Arthritis generally refers to inflammatory diseases that occur in human joints and their surrounding tissues, caused by inflammation, infection, degeneration, trauma or other factors. Arthritis-related diseases or symptoms include rheumatoid arthritis, osteoarthritis, chronic non-rheumatoid arthritis. Among them, osteoarthritis (OA) is a chronic joint disease characterized by degenerative destruction of articular cartilage, synovial inflammation, and bone remodeling around the joint. According to statistical data, the number of OA patients worldwide has exceeded 300 million, and its prevalence continues to rise due to population aging and the increasing obesity rate. The pathogenesis of OA is complex and involves intricate interactions between multiple biological processes, among which the inflammatory response plays a core role. In early OA, mechanical stress or biochemical stimuli can induce chondrocytes and synoviocytes to release pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). These inflammatory mediators not only accelerate the degradation of the cartilage matrix, but also inhibit the anabolic activity of chondrocytes, impairing their repair ability and forming a vicious cycle. Therefore, inhibiting the inflammatory response is considered an important strategy for alleviating OA symptoms and delaying disease progression. Currently, the treatment methods for OA mainly include non-drug intervention, drug treatment and surgical treatment.

[0003] Triamcinolone acetonide (TA) is a potent glucocorticoid and is widely used clinically due to its anti-inflammatory and immunosuppressive properties, especially in the treatment of OA. Traditional TA injections have the problem of short-term retention and require frequent injections. Frequent intra-articular injections not only increase the pain and economic burden of patients, but also increase the risk of infection and joint injury. Therefore, there is an urgent need for a method to improve the therapeutic effect of TA and reduce side effects. Summary of the Invention

[0004] In view of this, in order to make up for the deficiencies of the prior art, the present invention is specifically proposed.

[0005] In the first aspect of the present invention, a hydrogel system is provided, and the hydrogel system includes triamcinolone acetonide and a hyaluronic acid derivative.

[0006] In the present invention, the hyaluronic acid derivative refers to a class of substances obtained by chemically or physically modifying natural hyaluronic acid. The hyaluronic acid derivatives include, but are not limited to, one or a combination of several of vinylated hyaluronic acid derivatives, mercaptoylated hyaluronic acid derivatives, esterified hyaluronic acid derivatives, sulfonated hyaluronic acid derivatives, acylated hyaluronic acid derivatives, phosphorylated hyaluronic acid derivatives, oxidized hyaluronic acid derivatives, and quaternized hyaluronic acid derivatives.

[0007] Furthermore, the hyaluronic acid derivative is selected from one or a combination of several of vinylated hyaluronic acid derivatives and mercaptoylated hyaluronic acid derivatives.

[0008] Furthermore, the hyaluronic acid derivative is a hyaluronic acid crosslink of vinylated hyaluronic acid derivative and mercaptoylated hyaluronic acid derivative.

[0009] In the present invention, the triamcinolone acetonide may be itself or its derivative, and the derivatives include, but are not limited to, triamcinolone acetonide acetate.

[0010] Furthermore, the triamcinolone acetonide is selected from triamcinolone acetonide acetate.

[0011] Furthermore, the mass ratio of the triamcinolone acetonide to the hyaluronic acid derivative is (0.5 - 2):(1 - 10).

[0012] Furthermore, the mass ratio of the triamcinolone acetonide to the hyaluronic acid derivative is 1:4.

[0013] Furthermore, the mass concentration of the triamcinolone acetonide is 1 - 10 mg / mL.

[0014] Furthermore, the mass concentration of the triamcinolone acetonide is 5 mg / mL.

[0015] Furthermore, the mass concentration of the hyaluronic acid derivative is 0.5 - 5% (w / v).

[0016] Furthermore, the mass concentration of the hyaluronic acid derivative is 1 - 2% (w / v).

[0017] Furthermore, the mass concentration of the hyaluronic acid derivative is 1% (w / v).

[0018] Furthermore, the mass ratio of the vinylated hyaluronic acid derivative to the mercaptoylated hyaluronic acid derivative in the hyaluronic acid crosslink is (1 - 10):(0.5 - 2).

[0019] Furthermore, the mass ratio of the vinylated hyaluronic acid derivative to the mercaptoylated hyaluronic acid derivative in the hyaluronic acid crosslink is (3 - 5):1.

[0020] Further, the mass ratio of the vinylated hyaluronic acid derivative to the mercapto - modified hyaluronic acid derivative in the hyaluronic acid cross - linker is 4:1.

[0021] Further, the mass concentration of the vinylated hyaluronic acid derivative in the hydrogel system is 0.2 - 2% (w / v).

[0022] Further, the mass concentration of the vinylated hyaluronic acid derivative in the hydrogel system is 0.8% (w / v).

[0023] Further, the mass concentration of the mercapto - modified hyaluronic acid derivative in the hydrogel system is 0.05 - 0.5% (w / v).

[0024] Further, the mass concentration of the mercapto - modified hyaluronic acid derivative in the hydrogel system is 0.2% (w / v).

[0025] In the second aspect of the present invention, a pharmaceutical composition is provided, and the pharmaceutical composition includes the hydrogel system described in the first aspect of the present invention.

[0026] Further, the pharmaceutical composition includes pharmaceutically acceptable carriers and / or excipients.

[0027] In the present invention, pharmaceutically acceptable carriers and / or excipients may include pharmaceutically acceptable substances such as excipients, stabilizers, solubilizing agents, emulsifiers, suspending agents, buffers, isotonic agents, antioxidants or preservatives. In addition, high - molecular materials such as polyethylene glycol (PEG) or compounds such as cyclodextrin can also be used. Specific examples are listed below, but the present invention is not limited thereto, and well - known substances can be used. As excipients, excipients that do not have pharmacological effects themselves, such as starch or lactose, are preferred. As stabilizers, albumin, gelatin, sorbitol, mannitol, lactose, sucrose, trehalose, maltose, glucose, etc. can be cited. Among the above substances, sucrose or trehalose is preferred. As solubilizing agents, ethanol, glycerol, propylene glycol, polyethylene glycol, etc. can be cited. As emulsifiers, lecithin, aluminum stearate or sorbitan sesquioleate, etc. can be cited. As suspending agents, polyethylene glycol, polyvinylpyrrolidone (PVP) or carboxymethyl cellulose (CMC), etc. can be cited. As isotonic agents, sodium chloride, glucose, etc. can be cited. As buffers, citrates, acetates, boric acid or phosphates, etc. can be cited. As antioxidants, ascorbic acid, sodium bisulfite, sodium metabisulfite, etc. can be cited. As preservatives, phenol, thimerosal, benzalkonium chloride, etc. can be cited.

[0028] In the present invention, the pharmaceutical composition further comprises other medicaments for treating arthritis. Examples of the other medicaments for treating arthritis include well-known medicaments used in joint diseases, such as joint disease therapeutics, anti-inflammatory agents, analgesics, bone regenerating agents, bone resorption inhibitors, antibiotics or growth agents, etc. Additionally, when administering the hydrogel system described in the first aspect of the present invention by injection or the like, since it can cause pain due to injection, a pain reliever can also be included. The above-mentioned medicaments can be combined in one kind or two or more kinds.

[0029] As a joint disease therapeutic, for example, an antagonist of extracellular matrix degradation of articular chondrocytes such as an adrenocorticotropic hormone agent or a chondroprotective agent such as sodium chondroitin sulfate, or a p21-activated kinase (PAK) antagonist as an antagonist of a signal cascade amplification system, etc. can be cited.

[0030] As an anti-inflammatory agent, a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAIDs) etc. can be cited. Examples of the steroidal anti-inflammatory agent include dexamethasone, cortisone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinonide, beclomethasone, ethamide, etc. As the non-steroidal anti-inflammatory agent, for example, aspirin, ibuprofen, naproxen, diclofenac, indomethacin, nabumetone, phenylbutazone, rofecoxib, celecoxib, oxicam, piroxicam, pyrazolone, azapropazone, etc. can be cited.

[0031] As an analgesic, in addition to NSAIDs which are also anti-inflammatory analgesics, opioid analgesics etc. can be cited. Examples of the opioid analgesics include endorphin, dynorphin, enkephalin, codeine, dihydrocodeine, dextropropoxyphene, etc.

[0032] As a bone resorption inhibitor, any one or a mixture of two or more kinds selected from estrogen agents, calcitonin and bisphosphonates can be cited.

[0033] As antibiotics, examples include penicillin antibiotics, cephalosporin antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, peptide antibiotics and other antibiotics. As penicillin antibiotics, examples include benzylpenicillin, phenoxymethylpenicillin, methicillin, flucloxacillin, amoxicillin, ampicillin, piperacillin, azlocillin, ticarcillin and the like. As cephalosporin antibiotics, examples include cefazolin, cefuroxime, cefamandole, cefotaxime, cefoperazone, cefpiramide, cefalexin, cefaclor, cefixime, cefditoren and the like. As aminoglycoside antibiotics, examples include gentamicin, netilmicin, tobramycin, streptomycin, neomycin, kanamycin, amikacin and the like. As macrolide antibiotics, examples include erythromycin, clarithromycin, roxithromycin, rokitamycin, clindamycin, azithromycin and the like. As tetracycline antibiotics, examples include tetracycline, minocycline, doxycycline and the like. In addition, as β-lactam antibiotics, examples include latamoxef, flomoxef, aztreonam, imipenem, panipenem. In addition, examples also include vancomycin, rifampicin, chloramphenicol and the like.

[0034] As growth agents, examples include osteogenic factor (BMP), bone growth factor (BGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), insulin, insulin-like growth factor (IGF), hormones, cytokines or transforming growth factor (TGF) and the like. The above growth agents may include one or two or more kinds, and may further be combined with known drugs having other pharmaceutical effects.

[0035] When the action of the pharmaceutical composition itself causes pain, a pain reliever such as a local anesthetic can be used. As local anesthetics, examples include benzyl alcohol, chlorobutanol, procaine hydrochloride, lidocaine hydrochloride, dibucaine hydrochloride, mepivacaine hydrochloride and the like, as long as known drugs are used. In addition, when injecting, if the pain is caused by the pH and osmotic pressure of the liquid agent being significantly different from those of body fluids, it is preferably formulated into a liquid agent containing a buffer or an isotonic agent or the like.

[0036] The pharmaceutical composition of the present invention is used as an oral or parenteral preparation, and is preferably used as a parenteral preparation such as an injection or a drip. The administration method of the parenteral preparation may be any known method and is not particularly limited. For example, intravenous injection, arterial injection, subcutaneous injection, intramuscular injection, drip and the like can be cited. In addition, the pharmaceutical composition of the present invention can be directly injected into the affected part (such as a joint), or can also be administered by opening the affected part through a surgical operation. Those skilled in the art can appropriately select a suitable administration method for the patient.

[0037] In the present invention, oral or parenteral preparations can be made into sustained-release preparations, which can reduce the number of daily administrations. To prepare the above-mentioned sustained-release preparations, known methods can be used. By separate administration or making sustained-release preparations, it is easy to maintain a constant drug concentration in the body, so it is easy to obtain a sustained drug effect, and further, side effects can be reduced, thus reducing the burden on patients.

[0038] The third aspect of the present invention provides a method for preparing the hydrogel system described in the first aspect of the present invention, the method comprising mixing a triamcinolone acetonide solution with a hyaluronic acid derivative solution.

[0039] Further, the method comprises mixing a triamcinolone acetonide solution with a vinylated hyaluronic acid derivative to obtain solution A, mixing a triamcinolone acetonide solution with a mercaptoylated hyaluronic acid derivative to obtain solution SH, and mixing solution A with solution SH.

[0040] Further, the volume ratio of the triamcinolone acetonide solution to the vinylated hyaluronic acid derivative solution is (0.5 - 2):(0.5 - 2).

[0041] Further, the volume ratio of the triamcinolone acetonide solution to the vinylated hyaluronic acid derivative solution is 1:1.

[0042] Further, the volume ratio of the triamcinolone acetonide solution to the mercaptoylated hyaluronic acid derivative solution is (0.5 - 2):(0.5 - 2).

[0043] Further, the volume ratio of the triamcinolone acetonide solution to the mercaptoylated hyaluronic acid derivative solution is 1:1.

[0044] Further, when mixing solution A and solution SH, the mass ratio of the vinylated hyaluronic acid derivative in solution A to the mercaptoylated hyaluronic acid derivative in solution SH is 3:1 - 5:1.

[0045] Further, when mixing solution A and solution SH, the mass ratio of the vinylated hyaluronic acid derivative in solution A to the mercaptoylated hyaluronic acid derivative in solution SH is 4:1.

[0046] Further, the mass concentration of the triamcinolone acetonide solution is 2 - 20 mg / mL.

[0047] Further, the mass concentration of the triamcinolone acetonide solution is 10 mg / mL.

[0048] Further, the method includes mixing 1 mL of triamcinolone acetonide injection (10 mg / mL) with 1 mL of 2% w / v HA-A and 1 mL of 2% w / v HA-SH in equal volumes respectively to obtain a mixture containing 5 mg / mL TA and 1% w / v HA-A and a mixture containing 5 mg / mL TA and 1% w / v HA-SH. The mixture solution containing 5 mg / mL TA and 1% w / v HA-A and the mixture solution containing 5 mg / mL TA and 1% w / v HA-SH are mixed at a mass ratio of HA-A to HA-SH of 5:1, 4:1 and 3:1 to form a hydrogel system.

[0049] The fourth aspect of the present invention provides the use of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a medicament for treating arthritis-related diseases or symptoms.

[0050] In the present invention, "treating" arthritis means improving the signs and / or symptoms of arthritis and / or preventing the development of arthritis and / or preventing the development of arthritis in a subject at risk of developing arthritis.

[0051] In the present invention, the arthritis-related diseases or symptoms include but are not limited to rheumatoid arthritis, osteoarthritis, chronic non-rheumatoid arthritis. All types of arthritis are characterized by joint inflammation, causing pain, swelling, redness, stiffness, fever, degeneration of joints (such as in the wrist, fingers / toes (metacarpophalangeal or metatarsophalangeal joints), elbows, shoulders, hips, knees, ankles, feet, neck, or back) or extra-articular tissues (such as iritis, uveitis, oral ulcers, gastrointestinal inflammation, genitourinary inflammation, or skin damage). The subject has reduced mobility due to pain and stiffness, and arthritis can indirectly lead to obesity, high cholesterol, and / or heart disease. Arthritis can also cause extra-articular diseases, such as iritis, uveitis, oral ulcers, gastrointestinal inflammation, genitourinary inflammation, and skin damage.

[0052] Further, the arthritis-related diseases or symptoms are selected from osteoarthritis.

[0053] Further, the osteoarthritis includes but is not limited to knee osteoarthritis, hip osteoarthritis, hand osteoarthritis, spinal osteoarthritis, foot osteoarthritis, shoulder osteoarthritis.

[0054] Further, the osteoarthritis is selected from knee osteoarthritis.

[0055] In the present invention, the subjects of the pharmaceutical composition can include humans, dogs, cats, horses, cows, sheep, goats, and pigs. Human subjects can be male, female, adult, child, elderly (65 years old or older).

[0056] The fifth aspect of the present invention provides any one of the following applications:

[0057] (1) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in regulating iNOS, IL-6, TNF-α, CD68, CD206 or IL-10;

[0058] The hydrogel system or the pharmaceutical composition reduces the expression of iNOS, IL-6, TNF-α or CD68 and promotes the expression of CD206 or IL-10.

[0059] (2) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug for improving cartilage repair;

[0060] (3) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug for reducing cartilage surface fibrosis;

[0061] (4) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug for reducing synovial hyperplasia;

[0062] (5) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in maintaining cartilage matrix homeostasis;

[0063] (6) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in promoting cartilage regeneration;

[0064] (7) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in reducing cartilage matrix degradation;

[0065] (8) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of an anti-inflammatory drug;

[0066] (9) The application of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in regulating Col2 expression;

[0067] The hydrogel system or the pharmaceutical composition promotes Col2 expression.

[0068] (10) The application of the hyaluronic acid derivative in increasing the efficacy of triamcinolone acetonide;

[0069] The hyaluronic acid derivative and triamcinolone acetonide play a synergistic role.

[0070] (11) The application of the hyaluronic acid derivative in the controlled release or sustained release of triamcinolone acetonide;

[0071] Further, the hyaluronic acid derivative is selected from one or a combination of several of vinylated hyaluronic acid derivatives and thiolated hyaluronic acid derivatives.

[0072] Further, the hyaluronic acid derivative is a hyaluronic acid crosslink of vinylated hyaluronic acid derivative and thiolated hyaluronic acid derivative.

[0073] (12) Use of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in regulating macrophage polarization;

[0074] (13) Use of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in downregulating Beclin-1 and PINK1;

[0075] (14) Use of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in upregulating TOM20.

[0076] The sixth aspect of the present invention provides any one of the following methods:

[0077] (1) A method for regulating the expression of iNOS, IL-6, TNF-α, CD68, CD206 or IL-10, the method comprising administering the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention;

[0078] (2) A method for maintaining the homeostasis of cartilage matrix, the method comprising administering the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention;

[0079] (3) A method for promoting cartilage regeneration, the method comprising administering the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention;

[0080] (4) A method for reducing the degradation of cartilage matrix, the method comprising administering the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention;

[0081] (5) A method for regulating the expression level of Col2, the method comprising administering the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention;

[0082] (6) A method for increasing the efficacy of triamcinolone acetonide, the method comprising administering a hyaluronic acid derivative;

[0083] (7) A method for controlled release or sustained release of triamcinolone acetonide, the method comprising administering a hyaluronic acid derivative;

[0084] (8) A method for downregulating Beclin-1 and PINK1, the method comprising administering the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention;

[0085] (9) A method for upregulating TOM20, the method comprising administering the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.

[0086] Advantages and beneficial effects of the present invention:

[0087] The present invention provides a hydrogel loaded with triamcinolone acetonide, its preparation method and application. In the present invention, the anti-inflammatory and cartilage repair effects of the hydrogel loaded with triamcinolone acetonide in arthritis were evaluated through in vitro cell experiments and in vivo animal models. In vitro experiments showed that the hydrogel loaded with triamcinolone acetonide did not affect the mortality rate of chondrocytes, could significantly inhibit the expression of pro-inflammatory genes and promote the expression of anti-inflammatory genes, and the hyaluronic acid derivative and triamcinolone acetonide had a synergistic effect, demonstrating its strong anti-inflammatory properties. In vivo animal model studies further revealed that the hydrogel loaded with triamcinolone acetonide effectively alleviated joint synovial inflammation and reduced cartilage matrix degradation through continuous drug release, while promoting cartilage tissue regeneration and repair. Histological analysis and gait behavior evaluation further confirmed its therapeutic effect. The hydrogel loaded with triamcinolone acetonide not only effectively alleviated the inflammatory response, but also enhanced cartilage repair, highlighting its significant clinical potential. Description of the Drawings

[0088] Figure 1 It is a graph of the mechanical properties results of HA-A and HA-SH hydrogels with different mass ratios. (A) Schematic diagram of the formation of a hyaluronic acid hydrogel based on thiol chemical crosslinking; (B) Appearance diagrams of HA-A before and after crosslinking with HA-SH; (C) Graph of the storage modulus (G′) and loss modulus (G″) of the HA hydrogel measured at 1% strain with frequency scanning (0.1~10 Hz) after gelation at room temperature for 24 h; (D) Graph of the storage modulus (G′) and loss modulus (G″) of the HA hydrogel with strain scanning (0.1~100%) at a constant frequency of 1 Hz after gelation at room temperature for 24 h.

[0089] Figure 2Swelling, degradation and microstructure diagrams of HA-A / HA-SH hydrogels. (A) Swelling ratio diagrams of hydrogels with different mass ratios (HA-SH / HA-A = 1:5, 1:4, 1:3 w / w); (B, C) Degradation rate diagrams of HA hydrogels in PBS with different hyaluronidase concentrations: (B) within 24 h, (C) over 14 days; (D, E) Microstructure and safety diagrams of HA hydrogels without triamcinolone, with the red arrow indicating the loaded triamcinolone, scale bars = 300 μm and 50 μm; (F, G) Release curve diagrams of triamcinolone at different time points in vitro.

[0090] Figure 3 In vitro biocompatibility results diagrams of HA-A / HA-SH@TA hydrogels. (A) Dynamic light scattering (DLS) analysis diagram of triamcinolone microparticles, with an average particle size of 3279 nm; (B) Schematic diagram of the Transwell co-culture system; (C - D) THP-1 macrophages (C) and chondrocytes (D) co-cultured with HA or HA-A / HA-SH@TA hydrogels for 24, 48, and 72 h, and cell viability was detected by the CCK-8 method; (E - F) Live / dead cell staining of THP-1 macrophages (E) and chondrocytes (F).

[0091] Figure 4 In vitro anti-inflammatory effect diagrams of HA-A / HA-SH@TA hydrogels. (A - E) qRT-PCR analysis result diagrams of pro-inflammatory factors (iNOS, IL-6, TNF-α) and anti-inflammatory factors (CD206, IL-10) in THP-1 macrophages; (F, N) Western blot detection of the effect of HA-A / HA-SH@TA hydrogels on LPS-induced macrophage polarization; (G - J) Flow cytometry analysis of THP-1 macrophage M1 (CD86) and M2 (CD206) markers; (K) ELISA quantification of pro-inflammatory and anti-inflammatory cytokines in the supernatant of THP-1 macrophages; (L) Immunofluorescence images showing the ROS level in THP-1 macrophages; (M, O) Western blot analysis of THP-1 macrophage mitophagy-related proteins (Beclin-1, PINK1) and mitochondrial membrane protein TOM20.

[0092] Figure 5It is the in vivo treatment effect diagram of HA-A / HA-SH@TA hydrogel on OA mice. (A) Schematic diagram of the mouse osteoarthritis model; (B) Experimental flow chart of osteoarthritis mice; (C) Footprint diagram, color coding: green, right front (RF); yellow, right hind limb (RH); blue, left front limb (LF); purple, left hind limb (LH); (D, E) Total speed (D) and stride length (E) of the left hind limb of OA mice; (F) H&E, Masson and Saf-O / FG staining images of the OA-damaged knee joints treated under different conditions.

[0093] Figure 6 It is the diagram showing the anti-inflammatory and cartilage regeneration effects of HA-A / HA-SH@TA hydrogel in osteoarthritis mice. (A-C) Representative immunohistochemical (IHC) staining sections and quantitative analysis, CD68 (A), TNF-α (B) and Col2 (C); (D) Immunofluorescence staining diagram of macrophage polarization markers iNOS (M1 phenotype, green) and CD206 (M2 phenotype, red). Specific implementation manners

[0094] The following further describes the present invention in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make equivalent embodiments with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0095] Embodiment

[0096] I. Materials and methods

[0097] 1. Materials

[0098] The vinylated derivative of hyaluronic acid HA-A (200 kDa) and the mercaptoylated derivative of hyaluronic acid HA-SH (220 kDa) were purchased from Blafar Company. Hyaluronidase was purchased from Bloomage Biotechnology Co., Ltd. Other reagents used were all of analytical grade.

[0099] 2. Preparation of HA-A / HA-SH@TA hydrogel

[0100] HA-A and HA-SH were separately dissolved in 0.1 M Na2HPO4 (pH = 9.0) at a concentration of 1% w / v to prepare injectable hyaluronic acid (HA) hydrogels. Next, HA-A and HA-SH were crosslinked at mass ratios of 5:1, 4:1, and 3:1 (HA-A / HA-SH, w / w) to form HA-A / HA-SH blends. The mixtures were vortexed at low speed for 20 s to ensure uniform distribution of the components. The synthesis of the hydrogels and subsequent experiments were conducted at room temperature.

[0101] To prepare drug-loaded hydrogels, 1 mL of triamcinolone acetonide injection (10 mg / mL) was mixed with 1 mL of 2% w / v HA-A and 1 mL of 2% w / v HA-SH in equal volumes to obtain a mixture containing 5 mg / mL TA and 1% w / v HA-A, and a mixture containing 5 mg / mL TA and 1% w / v HA-SH. The mixture solution containing 5 mg / mL TA and 1% w / v HA-A was mixed with the mixture solution containing 5 mg / mL TA and 1% w / v HA-SH at mass ratios of HA-A to HA-SH of 5:1, 4:1, and 3:1 to form HA-A / HA-SH@TA, and then incubated at room temperature for a certain time before testing.

[0102] 3. Gel time assessment

[0103] The gelation time was determined using the vial inversion method. HA-A and HA-SH were mixed at a concentration of 1% and a mass ratio of 4:1 to form a homogeneous solution, and then transferred to a glass bottle. The gelation time was the time when the hydrogel no longer flowed after being inverted for 1 minute. The experiment was repeated 3 times.

[0104] 4. Rheological testing

[0105] The rheological properties of the HA-A / HA-SH hydrogels were characterized using an HR-2 rheometer (TA Instruments) equipped with 8 mm parallel steel plates. Before testing, different volumes of the mixtures were incubated in a flat cylindrical mold for 24 h. The dynamic behavior of the materials was evaluated at a fixed frequency (1 Hz), while the flow properties of the materials were evaluated at a fixed strain (1%). The storage modulus (G′) and loss modulus (G″) were monitored simultaneously. The critical strain value was defined as the intersection point of G′ and G″. Each experiment was repeated 3 times.

[0106] 5. Swelling ratio

[0107] Weigh hydrogels with different mass ratios having the same initial weight (Wi), and place them in pre-weighed glass bottles, with each bottle containing 2 mL of PBS. Then incubate the vials on a shaker at 37 °C at 50 rpm. Remove the hydrogels at specific time points, remove the PBS, reweigh, and record the swollen weight (Ws). Swelling ratio = (Ws / Wi) × 100%. All experiments were repeated three times and the average value was taken.

[0108] 6. Degradation by hyaluronidase

[0109] Soak dry hydrogels with different mass ratios in 2 mL of PBS solution containing 0, 0.1, 1, 10, 50, or 100 U / mL of hyaluronidase, and incubate on a shaker at 37 °C at 50 rpm. Remove the hydrogels at specific time points, rinse with deionized water to remove degradation products and enzyme residues, and dry to a constant weight. Record the dry weight (Ws) after degradation. Degradation rate = (Ws / Wi) × 100%, where Wi represents the initial weight of the hydrogel.

[0110] 7. Scanning electron microscopy (SEM)

[0111] The crosslinked hydrogels were quickly frozen in liquid nitrogen for freeze-drying. Cut the dried samples along the cross-section, bond the conductive resin with double-sided tape, and sputter gold. Then observe the samples using SEM (FEI Company, USA).

[0112] 8. In vitro release of triamcinolone acetonide

[0113] Collect the supernatant of the drug-loaded hydrogels with different mass ratios at specific time points, and replace with fresh medium. Analyze the release of TA by measuring the absorbance of the supernatant.

[0114] 9. In vitro biocompatibility and bioactivity

[0115] 9.1 Cell culture

[0116] THP-1 macrophages and mouse chondrocytes were obtained from the cell bank. THP-1 macrophages and chondrocytes were cultured in DMEM medium (KeyGen Biotech, Jiangsu, China) containing 10% fetal bovine serum (Clark Biosciences, Houston, USA), and placed in an incubator at 37 °C and 5% CO2.

[0117] 9.2 CCK-8 assay

[0118] Seed THP-1 macrophages or chondrocytes in 96-well plates at a density of 1 × 10^5 cells / well, and culture at 37 °C and 5% CO2 for 24 h to allow the cells to adhere. Then co-culture the cells with the hydrogel or DMSO for 24, 48, and 72 h to evaluate cell proliferation.

[0119] According to the instructions of the CCK-8 kit (VC5001, VicMed), add 100 µL of CCK-8 solution to each well and incubate for 1 - 4 h. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the percentage of viable cells. The experiment was repeated 3 times.

[0120] 9.3 Live / Dead cell staining

[0121] THP-1 macrophages or chondrocytes were cultured in a medium containing hydrogel or DMSO for 12 h. The cells were digested, washed, and centrifuged. The precipitate was resuspended in 1× Assay Buffer to form a cell suspension with a density of 1×10^5 cells / mL. According to the instructions of the calcein-AM / PI kit (CA1630, Solarbio), add 2 µL of calcein-AM and 5 µL of PI to each 1 mL of cell suspension for staining. Observe the stained cells under a fluorescence inverted microscope (Olympus, Center Valley, USA).

[0122] 10. Inflammatory response of THP-1 macrophages

[0123] 10.1 Establishment of an in vitro inflammation model

[0124] To evaluate the in vitro anti-inflammatory ability of the hydrogel, an inflammation model was first established. THP-1 macrophages were seeded in a 6-well plate and cultured in DMEM medium containing 10% fetal bovine serum. When the cell confluence reached approximately 70 - 80%, LPS was added to the cell culture plate at a final concentration of 10 ng / mL.

[0125] After 24 h of LPS induction, the medium was aspirated. Fresh medium was added to the blank group and the control group, and HA hydrogel, TA, or HA-A / HA-SH@TA was added to the experimental groups respectively. After further incubation for 3 days and 7 days, qRT-PCR was used to evaluate the mRNA expression levels of inflammation-related genes (iNOS, IL-6, TNF-α, CD206, IL-10).

[0126] 10.2 qRT-PCR

[0127] Total RNA was extracted from THP-1 macrophages using TRIzol reagent (VR603, VicMed). RNA was reverse transcribed into cDNA using a reverse transcription kit (HY-K0501, MCE). qPCR reactions were performed on a LightCycler 480 instrument (Roche) using SYBR Green Master Mix (VFP101, VicMed). The mRNA expression levels of iNOS, IL-6, TNF-α, CD206, and IL-10 were detected using qRT-PCR. Each qRT-PCR reaction was independently repeated at least 3 times. Data were analyzed using the 2 -ΔΔCT method. RT-qPCR primers were designed using an online tool (Primer3web), and the primer sequences are shown in Table 1.

[0128] Table 1 Primer sequences

[0129] Gene Name Sequence SEQ ID NO. ACTB 5´-GATGCGTTGTTACAGGAAGTCC-3´ (Forward) SEQ ID NO.1 ACTB 5´-GGCACGAAGGCTCATCATTCA-3´ (Reverse) SEQ ID NO.2 IL-6 5´-TGAGGAGACTTGCCTGGTGAA-3´ (Forward) SEQ ID NO.3 IL-6 5´-CAGCTCTGGCTTGTTCCTCAC-3´ (Reverse) SEQ ID NO.4 TNF-α 5´-TATCCTGGGGGACCCAATGT-3´ (Forward) SEQ ID NO.5 TNF-α 5´-AAAAGAAGGCACAGAGGCCA-3´ (Reverse) SEQ ID NO.6 iNOS 5´-TTCAGTATCACAACCTCAGCAAG-3´(Forward) SEQ ID NO.7 iNOS 5´-TGGACCTGCAAGTTAAAATCCC-3´(Reverse) SEQ ID NO.8 CD206 5´-TCCGGGTGCTGTTCTCCTA-3´(Forward) SEQ ID NO.9 CD206 5´-CCAGTCTGTTTTTGATGGCACT-3´(Reverse) SEQ ID NO.10 IL-10 5´-GACTTTAAGGGTTACCTGGGTTG-3´(Forward) SEQ ID NO.11 IL-10 5´-TCACATGCGCCTTGATGTCTG-3´(Reverse) SEQ ID NO.12

[0130] 10.3 Macrophage polarization

[0131] THP-1 macrophages were induced to mature with phorbol 12-myristate 13-acetate (PMA) at a concentration of 100 ng / ml. After 48 hours of culture, the mature adherent macrophages were named M0 macrophages. Then, the mature macrophages were digested with Accutase and polarized into M1-like and M2-like phenotypes. To induce M1 polarization, cells were incubated with IFN-γ (20 ng / ml) and LPS (100 ng / ml) for more than 24 h. For M2 polarization, IL-4 (20 ng / ml) and IL-13 (20 ng / ml) were used for more than 24 h. Once polarization was completed, the cells were used for subsequent experiments in serum-free RPMI without additional stimulation.

[0132] 10.4 ELISA assay

[0133] According to the instructions provided by the ELISA kit (Abcam), ELISA was used to determine the production of iNOS, TNF-α, IL-10, IL-6, and CD206. The optical density values of each well were recorded using an AMR-100 automatic microplate reader (Hangzhou Ausheng Instruments). The same indicators were measured in cells as in animal models. After 24 hours of cell treatment, the supernatant was collected and the levels of related markers were determined.

[0134] 10.5 Western blot analysis

[0135] Cell extracts were separated on a 4 - 12% precast gel SDS - PAGE, and then the proteins were transferred to a nitrocellulose membrane. The membrane was incubated with the primary antibody (diluted 1:2000) overnight at 4°C, and then incubated with the secondary antibody (diluted 1:4000) for 1.5 hours at room temperature. Immunoreactive bands were identified using a Tanon Scanning device (Tanon Science & Technology).

[0136] 11. In Vivo Animal Experiments and Histological Evaluation

[0137] 11.1 Animal Model Construction and In Vivo Implantation

[0138] Six - week - old C57 / B6J mice were obtained from GemPharmatech (Nanjing, China) and housed under pathogen - free conditions. All animal experiments were approved by the Animal Experiment Ethics Committee of Xuzhou Medical University (Project Number 202504T009). To establish a mouse OA model, the mice were anesthetized by inhaling isoflurane. After complete anesthesia, the hair around the left hindlimb knee joint was shaved, and the surgical area was disinfected with 75% ethanol. A longitudinal incision about 5 mm long was made on the medial side of the knee joint to expose the joint cavity. After separating the soft tissues, the anterior cruciate ligament was transected with surgical scissors, and part of the medial meniscus was resected.

[0139] Thirty - two OA mice and eight normal mice were divided into 5 groups (n = 8 per group): Sham group (no severe trauma to the knee joint), OA group (induced to have OA, without any other treatment), HA group (induced to have OA, injected with HA hydrogel), TA group (induced to have OA, injected with free TA), HA - A / HA - SH@TA group (induced to have OA, injected with HA - A / HA - SH@TA hydrogel). The above groups were treated continuously for 4 weeks. The mice were euthanized 8 weeks after surgery, and their knee joints were collected for histological analysis.

[0140] 11.2 Gait Assessment

[0141] Gait analysis was performed on the mice after 4 weeks of intervention. The mice were placed at the starting point of the gait analysis system and allowed to freely walk across the glass plate to the end point. Images of the paw contact with the glass plate were taken by a high - speed camera, and gait parameters such as the total speed and stride length of the left and right hindlimbs were recorded.

[0142] 11.3 Histological Analysis

[0143] The collected joint samples were fixed in 4% paraformaldehyde for 48 h, and then slowly decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution at 4 °C for one week. The decalcified tissues were dehydrated through an ethanol gradient, embedded in paraffin, and sectioned into 5-7 μm thick slices. The tissue sections were stained with H&E, Masson, and Saf-O / FG, observed, and photographed under a microscope. The paraffin sections were dewaxed with xylene and dehydrated through an ethanol gradient. After antigen retrieval using an EDTA solution, non-specific binding was blocked with 5% bovine serum albumin (BSA) at room temperature for 30 minutes. Then the sections were incubated with the corresponding primary antibody and incubated overnight at 4 °C, and then incubated with the secondary antibody at room temperature for 1 hour. After washing 3 times with PBS (5 min each time), DAB was developed for 5 minutes and hematoxylin was counterstained for 2 minutes. Finally, the sections were dehydrated, mounted, and observed under an optical microscope.

[0144] 12. Statistical analysis

[0145] Statistical analysis was performed using OriginPro 2024 and GraphPad Prism9. The data were expressed as mean ± standard deviation (SD). Statistical differences were analyzed using Student's t-test or one-way analysis of variance (ANOVA). A P value < 0.05 was considered statistically significant.

[0146] II. Experimental results

[0147] 1. Preparation and mechanical properties of HA-A / HA-SH hydrogel

[0148] An injectable HA-A / HA-SH hydrogel was successfully prepared through a thiol-Michael addition reaction. As Figure 1 shown in A, this in-situ crosslinking reaction can proceed spontaneously at room temperature without a catalyst, and the gelation time is about 10 minutes. The injectability of the hydrogel was verified through a 27G needle, indicating that it can pass through the needle smoothly and rapidly form a stable gel after extrusion ( Figure 1 B).

[0149] The mechanical properties of the hydrogel were further characterized through rheological analysis. Under the conditions of a fixed frequency (1 Hz) and a fixed stress (1%) ( Figure 1(C-D), the storage modulus (G') and loss modulus (G") of the hydrogel were monitored over time. The results showed that under the same conditions, the storage modulus of the hydrogel increased significantly with the increase in the mass ratio of HA-SH / HA-A. In addition, hydrogels with different ratios of HA-A / HA-SH exhibited stable mechanical properties in the frequency range of 0.1 - 100 rad / s and the deformation range of 0.1 - 10%. Based on these results, the HA hydrogel with a HA-SH:HA-A ratio of 1:4 w / w had the best viscoelastic balance: the moderate elasticity provided continuous lubrication and shock absorption during joint movement, effectively reducing the friction between bone and bone, while the appropriate viscosity ensured that the hydrogel could be evenly distributed within the joint to form a stable lubricating film. This viscoelastic profile met the requirements of joint treatment, enabling effective pressure buffering and friction reduction while maintaining structural integrity under dynamic loads. Therefore, the HA hydrogel with a HA-SH:HA-A ratio of 1:4 w / w was determined as the optimal choice.

[0150] 2. Swelling, Degradation and Microstructure of HA-A / HA-SH Hydrogels

[0151] The carboxyl and amino functional groups of HA endow it with extraordinary water absorption capacity, and thus it has inherent hydrophilicity. However, excessive swelling of the HA hydrogel may increase the fluid pressure in the joint cavity, which may cause discomfort, pain or edema. Therefore, regulating the cross-linking density of the hydrogel system is crucial for controlling the swelling and degradation rates.

[0152] To evaluate the swelling behavior of the hydrogel, the hydrogel was incubated in PBS at 37 °C. The results showed that HA hydrogels with different mass ratios all reached swelling equilibrium within 4 - 6 h ( Figure 2 A), and the swelling rate decreased significantly with the increase in the mass ratio of HA-SH / HA-A.

[0153] To further investigate the degradation characteristics of the hydrogel, we conducted in vitro degradation experiments. The 1:4 w / w HA hydrogel was immersed in PBS containing different concentrations of hyaluronidase. The short-term observation results at 24 h showed that the degradation rate accelerated with the increase in enzyme concentration ( Figure 2 B). The long-term observation results showed that in the absence of hyaluronidase, the hydrogel maintained mass stability within two weeks ( Figure 2 C). However, with the increase in the concentration of hyaluronidase, the time for complete degradation of the HA hydrogel gradually shortened. These results confirmed that the degradation rate of the HA hydrogel could be precisely regulated in vitro by adjusting the concentration of hyaluronidase.

[0154] To further investigate the microscopic structural characteristics of the hydrogel, we characterized the 1:4 w / w HA-SH / HA-A@TA hydrogel by scanning electron microscopy (SEM). The results showed that the hydrogel presented a highly ordered three-dimensional porous network structure ( Figure 2 D), with an average pore size of approximately 50 μm. This porous structure allows for the effective loading and uniform distribution of TA, supporting a controllable drug release behavior ( Figure 2 E). These structural characteristics are typical of many hydrogels, but the in-situ crosslinking of HA-A / HA-SH used in this study enables a more targeted release to achieve a sustained therapeutic effect.

[0155] The controllable swelling, stable degradation, and structured porous network properties of the HA-A / HA-SH hydrogel provide a solid foundation for evaluating its potential as a sustained drug delivery platform. To evaluate the TA release performance of the HA-A / HA-SH@TA hydrogel, an in vitro drug release experiment was conducted. The TA-loaded hydrogel was immersed in PBS buffer (pH 7.4, 37 °C), and the absorbance of the supernatant was measured at different time intervals to quantify the released TA ( Figure 2 F-G). The results demonstrated the ability of the drug to be released slowly. Within the first 24 hours, the release amount of TA was relatively low, and the cumulative release amount was less than 20%. As time passed, the release amount gradually increased, and the cumulative release amount did not exceed 80% by the 10th day. The HA-A / HA-SH@TA hydrogel showed no obvious burst release when incubated in PBS, indicating its ability to achieve a steady and sustained drug release and making it a promising high-efficiency drug delivery system.

[0156] 3. In vitro biocompatibility of the HA-A / HA-SH@TA hydrogel

[0157] The in vitro biocompatibility of the HA-A / HA-SH@TA hydrogel was systematically evaluated using a Transwell co-culture system. Dynamic light scattering (DLS) characterization showed that the average diameter of the TA particles was 3279 nm ( Figure 3 A), enabling effective diffusion through an 8-μm pore size Transwell membrane. In the established evaluation platform, the HA-A / HA-SH hydrogel, free TA, or HA-A / HA-SH@TA hydrogel was placed in the upper chamber, and THP-1 macrophages and chondrocytes were cultured in the lower chamber to simulate physiological drug release conditions ( Figure 3 B).

[0158] Cell cytotoxicity was evaluated by the CCK-8 method and live / dead staining. After 72 h of co-culture, the CCK-8 results showed that the HA-A / HA-SH@TA hydrogel maintained cell viability above 90% (the viability of THP-1 macrophages was 93.1% and that of chondrocytes was 97.0%), showing no significant difference compared with the control group ( Figure 3 C–D). Live / dead staining further confirmed the above findings ( Figure 3 E–F), and significantly higher survival rates were observed in the HA and HA-A / HA-SH@TA hydrogel treatment groups compared with the DMSO control group (where a large number of cells died due to solvent toxicity). These data demonstrated the excellent biocompatibility of the HA-A / HA-SH@TA hydrogel and confirmed that the loading of TA did not introduce additional cytotoxic effects.

[0159] 4. In vitro anti-inflammatory effect of HA-A / HA-SH@TA hydrogel

[0160] To investigate the in vitro anti-inflammatory properties of TA, an inflammatory model was established by stimulating THP-1 macrophages with lipopolysaccharide (LPS), and the HA-A / HA-SH@TA hydrogel was used for treatment. The gene expression levels of inflammatory cytokines (iNOS, IL-6, TNF-α) were quantified by qRT-PCR on the 3rd and 7th days after treatment. The results showed that LPS stimulation significantly upregulated the expression of pro-inflammatory cytokines ( Figure 4 A–C), confirming the successful establishment of the inflammatory model. On the 7th day after treatment, the levels of inflammatory factors in both the HA hydrogel group and the TA group were significantly reduced, and the expression of inflammatory markers in the HA-A / HA-SH@TA hydrogel group was the lowest. Meanwhile, the expression of anti-inflammatory factors CD206 and IL-10 also increased significantly ( Figure 4 D–E). The q values were calculated by the King formula, and the calculation results are shown in Table 1. All q values were greater than 1.15, indicating a synergistic effect. These research results showed that the HA-A / HA-SH@TA hydrogel plays a crucial role in regulating the immune response, effectively alleviating inflammation, and promoting the anti-inflammatory response. The HA-A / HA-SH@TA hydrogel achieved the best anti-inflammatory effect through the synergistic effect of TA sustained release and HA microenvironment regulation.

[0161] Table 1 Calculation of synergistic effect

[0162] D3 iNOS IL-6 TNF-α CD206 IL-10 Effect of HA -0.027995334 0.034029547 0.038392051 0.045088567 -0.004793289 Effect of TA 0.113314448 0.304228222 0.198735321 3.476650564 4.55122828 Effect of HA@TA 0.264622563 0.647987774 0.316922614 4.892914654 7.188136609 q 2.990376408 1.976144718 1.380941375 1.330139879 1.588672018 D7 iNOS IL-6 TNF-α CD206 IL-10 Effect of HA -0.007703704 -0.014333368 0.018336112 0.098837209 0.014588859 Effect of TA 0.334074074 0.534608569 0.27852705 10.24563953 13.71352785 Effect of HA@TA 0.494222222 0.748879391 0.435065919 14.01162791 21.25066313 q 1.502451041 1.418498902 1.49119755 1.233729948 1.525731354

[0163] Based on these findings, we further elucidated the anti-inflammatory mechanism of the HA-A / HA-SH@TA hydrogel. In this study, its regulatory effects on macrophage polarization, oxidative stress level, and mitophagy were systematically evaluated. Using the THP-1 macrophage inflammatory model, Western blot analysis (Figure 4 F, N) It was demonstrated that the HA-A / HA-SH@TA hydrogel not only inhibited LPS-induced M1 macrophage polarization but also promoted M2 macrophage polarization. Flow cytometry ( Figure 4 G-J) and ELISA ( Figure 4 K) quantification showed that the expressions of M1 markers (such as CD86, iNOS, IL-6, TNF-α, etc.) were significantly decreased, while the expressions of M2 markers (such as CD206, IL-10, etc.) were significantly increased, confirming that the HA-A / HA-SH@TA hydrogel effectively regulated macrophage polarization. Immunofluorescence analysis ( Figure 4 L) indicated that the HA-A / HA-SH@TA hydrogel effectively reduced the LPS-induced reactive oxygen species (ROS) level, significantly alleviated oxidative stress, and thus mitigated the local inflammatory response. Further analysis revealed that the HA-A / HA-SH@TA hydrogel significantly downregulated the expressions of mitophagy-related proteins (Beclin-1 and PINK1), ( Figure 4 M, O), and simultaneously restored the level of mitochondrial membrane protein TOM20, suggesting that the hydrogel effectively blocked excessive inflammatory responses by regulating the ROS-mediated mitophagy pathway.

[0164] These results provided cellular and molecular evidence that the HA-A / HA-SH@TA hydrogel exerted a synergistic anti-inflammatory effect by reprogramming macrophage polarization, alleviating oxidative stress, and maintaining mitochondrial homeostasis, providing new theoretical support for the treatment of OA.

[0165] 5. In Vivo Treatment of OA Mice with HA-A / HA-SH@TA Hydrogel

[0166] As Figure 5 shown in A, an osteoarthritis mouse model was established by unilateral anterior cruciate ligament transection (ACLT) combined with medial meniscus destabilization (DMM) of the knee joint to evaluate the therapeutic effect of the HA-A / HA-SH@TA hydrogel ( Figure 5 B). The experiment was divided into 5 groups: sham operation group (Sham), OA model group (OA), HA hydrogel group (HA), free triamcinolone acetonide group (TA), and HA-A / HA-SH@TA hydrogel group. Four weeks after surgery, a gait analysis system was used to analyze the gait behavior of mice in each group. The results showed that compared with the Sham group, the plantar area of the left hind limb of rats in the OA group was significantly reduced ( Figure 5 C). In addition, the movement speed and stride length of the left hind limb were significantly decreased ( Figure 5 D-E). After treatment with HA, TA, and HA-A / HA-SH@TA hydrogels, the gait parameters were significantly improved. The movement speed of the left hind limb in the HA-A / HA-SH@TA group recovered to 84.5% of the Sham group, and the stride length was significantly increased, indicating that the movement dysfunction caused by OA was alleviated.

[0167] H&E staining was used to evaluate the degree of cartilage regeneration and inflammatory cell infiltration. As Figure 5 shown in Fig. F, the mice in the OA group showed severe cartilage surface fibrosis, synovial hyperplasia and inflammatory cell infiltration. However, the treatments with HA, TA and HA-A / HA-SH@TA hydrogels significantly alleviated these pathological changes. Notably, among all the treatment groups, the HA hydrogel loaded with TA showed the most obvious effect on cartilage repair and inflammation reduction. Masson staining further indicated that compared with the OA group, the HA-A / HA-SH@TA group showed good tissue collagen fibers and reduced fibrosis area, which could significantly promote tissue repair. In addition, safranin O staining showed that compared with the OA group, the content of glycosaminoglycans (GAGs) in the HA-A / HA-SH@TA group increased significantly, and the cartilage structure integrity was the best, confirming the effective maintenance of cartilage matrix homeostasis. The potential mechanism for this phenomenon may be closely related to the sustained-release effect of the HA-A / HA-SH@TA hydrogel. First, the loaded TA can inhibit inflammation and reduce cartilage degradation through its controlled release, providing a better environment for the survival and repair of chondrocytes. Second, the HA matrix, as a natural cartilage matrix material, has good biocompatibility, which can promote cell adhesion and proliferation, and further stimulate the synthesis of GAGs. The increase in GAGs indicates enhanced repair and remodeling of the cartilage matrix, which helps to retain the elasticity and structure of cartilage, ultimately leading to the best cartilage repair effect. Therefore, the HA-A / HA-SH@TA hydrogel not only alleviates OA progression through its anti-inflammatory effect, but also promotes cartilage regeneration by providing a supportive biological environment, significantly improving the structural integrity of cartilage.

[0168] To clarify the anti-inflammatory and cartilage regeneration mechanisms of the HA-A / HA-SH@TA hydrogel, immunohistochemistry was used to detect the expressions of TNF-α, CD68 (a macrophage marker) and type II collagen (Col2) in the joint tissues. The results showed that compared with the OA group, the number of TNF-α positive cells in the HA-A / HA-SH@TA group decreased by 86.3%, and the CD68 expression decreased by 84.3%, which was also higher than that in the group treated with TA alone, indicating a significant inhibition of inflammation ( Figure 6 A-C). In addition, the expression intensity of Col2 in the HA-A / HA-SH@TA group was 2.5 times that of the OA group, confirming its role in promoting cartilage regeneration by enhancing Col2 synthesis.

[0169] In addition, immunofluorescence analysis further confirmed the regulatory effect of the HA-A / HA-SH@TA hydrogel on the expressions of iNOS and CD206. The results showed that while promoting the expression of CD206, the HA-A / HA-SH@TA group significantly inhibited the expression of iNOS ( Figure 6D). As a marker of M2 macrophages, the upregulation of CD206 indicates that the HA-A / HA-SH@TA hydrogel promotes the polarization of macrophages towards the anti-inflammatory M2 phenotype, thereby further inhibiting the inflammatory response.

[0170] In this study, a dynamically covalently crosslinked injectable HA-A / HA-SH@TA hydrogel delivery system was developed for the local sustained release of TA to address clinical challenges such as short TA retention time, multiple injection times, and potential cartilage degeneration, and to regulate the joint microenvironment to improve the safety and effectiveness of OA treatment. Through various characterization methods, this application demonstrated that the hydrogel has controllable swelling properties, stable degradation characteristics, and a structured porous network, laying a solid foundation for its use as a drug delivery platform. In vitro experiments showed that the HA-A / HA-SH@TA hydrogel significantly inhibited the release of inflammatory factors without significantly affecting chondrocyte viability, indicating its significant anti-inflammatory effect. In vivo experiments further confirmed that the HA-A / HA-SH@TA hydrogel could effectively reduce synovial inflammation in an OA mouse model, decrease cartilage matrix degradation, and promote the regeneration and repair of cartilage tissue. Histological analysis and gait behavior assessment also confirmed its therapeutic effect. In summary, the drug-loaded hydrogel not only effectively alleviated inflammation in OA but also promoted cartilage repair, showing significant potential for clinical application. This hydrogel system can achieve local sustained release of TA, extend the therapeutic activity, and precisely regulate the drug release kinetics while minimizing systemic side effects. In addition, the minimally invasive injection property of the hydrogel provides further advantages for its clinical application. This study provides theoretical and practical guidance for the development of more advanced and safer OA treatment regimens.

[0171] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A hydrogel system, characterized in that: The hydrogel system comprises triamcinolone acetonide and a hyaluronic acid derivative.

2. The hydrogel system according to claim 1, characterized in that The hyaluronic acid derivatives include one or a combination of hyaluronic acid vinyl derivatives, hyaluronic acid thiol derivatives, hyaluronic acid acid ester derivatives, hyaluronic acid sulfonated derivatives, hyaluronic acid acylated derivatives, hyaluronic acid phosphorylated derivatives, hyaluronic acid oxidized derivatives, and hyaluronic acid quaternary ammonium derivatives; Preferably, the hyaluronic acid derivative is selected from one or a combination of hyaluronic acid vinyl derivatives and hyaluronic acid thiolated derivatives; Preferably, the hyaluronic acid derivative is a hyaluronic acid cross-linked product of a vinyl derivative of hyaluronic acid and a thiolated derivative of hyaluronic acid; Preferably, the triamcinolone acetonide comprises triamcinolone acetonide acetate.

3. The hydrogel system according to claim 1, characterized in that The mass ratio of triamcinolone acetonide to the hyaluronic acid derivative is (0.5-2):(1-10); Preferably, the mass ratio of triamcinolone acetonide to the hyaluronic acid derivative is 1:4; Preferably, the mass concentration of triamcinolone acetonide is 1-10 mg / mL; Preferably, the mass concentration of triamcinolone acetonide is 5 mg / mL; Preferably, the mass concentration of the hyaluronic acid derivative is 0.5-5% (w / v); Preferably, the mass concentration of the hyaluronic acid derivative is 1-2% (w / v); Preferably, the mass concentration of the hyaluronic acid derivative is 1% (w / v).

4. The hydrogel system according to claim 2, characterized in that The mass ratio of the vinyl derivative of hyaluronic acid to the thiolated derivative of hyaluronic acid in the cross-linked hyaluronic acid is (1-10):(0.5-2); Preferably, the mass ratio of the vinyl derivative of hyaluronic acid to the thiolated derivative of hyaluronic acid in the hyaluronic acid cross-linked product is (3-5):1; Preferably, the mass ratio of the vinyl derivative of hyaluronic acid to the thiolated derivative of hyaluronic acid in the cross-linked hyaluronic acid is 4:1; Preferably, the mass concentration of the vinyl derivative of hyaluronic acid in the hydrogel system is 0.2-2% (w / v); Preferably, the mass concentration of the vinyl derivative of hyaluronic acid in the hydrogel system is 0.8% (w / v); Preferably, the mass concentration of the thiolated hyaluronic acid derivative in the hydrogel system is 0.05-0.5% (w / v); Preferably, the mass concentration of the thiolated hyaluronic acid derivative in the hydrogel system is 0.2% (w / v).

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the hydrogel system according to any one of claims 1 to 4; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

6. A method for preparing the hydrogel system according to any one of claims 1 to 4, characterized in that: The method comprises mixing a solution of triamcinolone acetonide with a solution of a hyaluronic acid derivative.

7. The method according to claim 6, characterized in that The method comprises mixing a triamcinolone acetonide solution with a hyaluronic acid vinyl derivative to obtain a solution A, mixing the triamcinolone acetonide solution with a hyaluronic acid thiolated derivative to obtain a solution SH, and mixing the solution A with the solution SH; Preferably, the volume ratio of the triamcinolone acetonide solution to the hyaluronic acid vinyl derivative solution is (0.5-2):(0.5-2); Preferably, the volume ratio of the triamcinolone acetonide solution to the hyaluronic acid vinyl derivative solution is 1:1; Preferably, the volume ratio of the triamcinolone acetonide solution to the thiolated hyaluronic acid derivative solution is (0.5-2):(0.5-2); Preferably, the volume ratio of the triamcinolone acetonide solution to the thiolated hyaluronic acid derivative solution is 1:1; Preferably, when the solution A and the solution SH are mixed, the mass ratio of the hyaluronic acid vinyl derivative in the solution A to the hyaluronic acid thiolated derivative in the solution SH is 3:1-5:1; Preferably, when the solution A and the solution SH are mixed, the mass ratio of the vinyl derivative of hyaluronic acid in the solution A to the thiolated derivative of hyaluronic acid in the solution SH is 4:

1.

8. Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug for treating arthritis-related diseases or symptoms; Preferably, the arthritis-related diseases or symptoms include rheumatoid arthritis, osteoarthritis, chronic non-rheumatoid arthritis; Preferably, the arthritis-related disease or symptom is selected from osteoarthritis; Preferably, the osteoarthritis includes knee osteoarthritis, hip osteoarthritis, hand osteoarthritis, spine osteoarthritis, foot osteoarthritis, and shoulder osteoarthritis; Preferably, the osteoarthritis is selected from knee osteoarthritis.

9. Any of the following applications: (1) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in regulating the expression of iNOS, IL-6, TNF-α, CD68, CD206 or IL-10; (2) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug for improving cartilage repair; (3) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug for reducing cartilage surface fibrosis; (4) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug for reducing synovial hyperplasia; (5) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in maintaining cartilage matrix homeostasis; (6) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in promoting cartilage regeneration; (7) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in reducing cartilage matrix degradation; (8) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of anti-inflammatory drugs; (9) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in regulating Col2 expression; (10) Application of hyaluronic acid derivatives in increasing the efficacy of triamcinolone acetonide; (11) Application of hyaluronic acid derivatives in controlled or sustained release of triamcinolone acetonide; (12) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in regulating macrophage polarization; (13) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in downregulating Beclin-1 and PINK1; (14) Use of the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in up-regulating TOM20. Preferably, the hyaluronic acid derivative is selected from one or a combination of hyaluronic acid vinyl derivatives and hyaluronic acid thiolated derivatives; Preferably, the hyaluronic acid derivative is a hyaluronic acid cross-linked product of a vinylated hyaluronic acid derivative and a thiolated hyaluronic acid derivative.

10. Any of the following methods: (1) A method for regulating the expression of iNOS, IL-6, TNF-α, CD68, CD206 or IL-10, characterized in that: The method comprises administering the hydrogel system of any one of claims 1 to 4 or the pharmaceutical composition of claim 5; (2) A method for maintaining cartilage matrix homeostasis, characterized in that the method comprises administering the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5; (3) A method for promoting cartilage regeneration, characterized in that the method comprises administering the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5; (4) A method for reducing cartilage matrix degradation, characterized in that the method comprises administering the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5; (5) A method for regulating Col2 expression, characterized in that the method comprises administering the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5; (6) A method for increasing the efficacy of triamcinolone acetonide, characterized in that the method comprises administering a hyaluronic acid derivative; (7) A method for controlled or sustained release of triamcinolone acetonide, characterized in that the method comprises administering a hyaluronic acid derivative; (8) A method for downregulating Beclin-1 and PINK1, characterized in that the method comprises administering the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5; (9) A method for upregulating TOM20, characterized in that the method comprises administering the hydrogel system according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5.

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