In-situ drug-loaded hydrogels based on nanobottle encapsulation technology, their preparation methods and applications

Abstract

This invention belongs to the field of biomedical materials technology, specifically relating to an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology, its preparation method, and its application. The in-situ drug-loaded hydrogel is formed by cross-linking copper chloride and reduced thiourea groups grafted onto hyaluronic acid within drug-loaded polydopamine nanobottles under near-infrared light irradiation. Diclofenac sodium is dispersed on the periphery and within the pores of the in-situ drug-loaded hydrogel; Kartogenin is encapsulated within the drug-loaded polydopamine nanobottles. Compared with existing technologies, this invention utilizes nanobottle encapsulation technology to achieve in-situ gelation and time-controlled drug release, solving the problems of non-in-situ hydrogels failing to adhere to irregular tissues and in-situ hydrogels struggling to simultaneously possess controllable time-controlled drug release capabilities. This solution achieves perfect adhesion of the hydrogel to the joint cavity through controllable in-situ gelation to protect the joint; simultaneously, it enables rapid release of diclofenac sodium and sustained release of Kartogenin, thereby achieving staged treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology, its preparation method, and its application. Background Technology

[0002] Osteoarthritis is a common degenerative joint disease characterized by progressive cartilage erosion, synovial inflammation, and subchondral bone remodeling. The staged pathological symptoms of osteoarthritis are: early stages are characterized by chondrocyte damage and extracellular matrix degradation caused by inflammation, while late stages are characterized by extensive cartilage loss and severely impaired repair capacity. Current clinical interventions—including nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections, and joint replacement—primarily only relieve symptoms and cannot stop disease progression; crucially, these treatments cannot sequentially address the different pathological stages of osteoarthritis to achieve staged repair.

[0003] Hyaluronic acid is considered an ideal material for treating osteoarthritis by acting on joints. Numerous studies have shown that hyaluronic acid is a natural joint lubricant, and hyaluronic acid-based hydrogels offer excellent joint lubrication and protection. Furthermore, controllable, in-situ hydrogel systems are another key component in the treatment of osteoarthritis. However, while most hyaluronic acid hydrogels offer joint protection, they cannot be directly injected into the joint cavity.

[0004] CN118873486A discloses an injectable hydrogel based on a gradient structure, its preparation method, and its application, comprising the following steps: dissolving polydopamine nanoparticles in a solvent, then adding genistein and stirring to dissolve, then adding copper chloride solution and stirring thoroughly to dissolve to obtain a drug-loaded polydopamine-copper chloride solution; dissolving a tetra-armed polyethylene glycol-mercapto group in a solvent to obtain a tetra-armed polyethylene glycol-mercapto solution; and mixing the drug-loaded polydopamine-copper chloride solution with the tetra-armed polyethylene glycol-mercapto solution to obtain an injectable hydrogel. This approach utilizes the π-π interaction between polydopamine and genistein to achieve efficient loading of hydrophobic drugs. A high-viscosity organic-inorganic composite network is initially formed between copper ions and polydopamine through Cu-N coordination. Further, a four-arm polyethylene glycol-thiol group is introduced into this system, and diffusion-controlled Cu-S coordination is used to prepare a polymeric hydrogel network with a unique gradient structure. However, this injectable hydrogel cannot precisely control the gelation time, often resulting in premature contact between the two solutions and gelation under suboptimal conditions. Furthermore, this injectable hydrogel only loads a single drug, failing to achieve dual drug loading and sequential release, thus failing to meet the needs of staged treatment for osteoarthritis.

[0005] CN113577376A discloses a dual-drug-loaded polysaccharide-based self-healing hydrogel and its preparation method. First, mesoporous polydopamine nanoparticles are prepared using a sacrificial template method and used to load antioxidant drugs. Then, the antioxidant-loaded polydopamine nanoparticles and anti-inflammatory drugs are added to a carboxymethyl chitosan solution, mixed thoroughly, and the carboxymethyl chitosan is chemically cross-linked with dialdehyde polysaccharide to obtain the dual-drug-loaded polysaccharide-based self-healing hydrogel. This method achieves the stepwise release of anti-inflammatory and antioxidant drugs by loading antioxidant active substances and active ingredients onto the mesoporous polydopamine nanoparticles and the polysaccharide-based self-healing hydrogel, respectively. However, while this stepwise release design achieves bilayer loading and sequential release of the drug, this sequential release is uncontrollable, merely a fast-slow release mechanism due to different release rates, and cannot meet the requirement of precise, on-demand, and timely release through artificial control.

[0006] It is evident that existing hydrogel-based osteoarthritis treatments have shown potential for local therapy and can effectively treat a specific pathological condition. However, they also fail to achieve a comprehensive strategy of early cartilage protection and late cartilage regeneration, and cannot implement precise site treatment. This leaves a huge unmet need in the field of osteoarthritis treatment.

[0007] In order to create a hydrogel that can treat the early and late-stage pathological symptoms of osteoarthritis in stages, this invention designs an in-situ drug-loaded hydrogel with the characteristics of in-situ adhesion to irregular tissues, long-term stability, and time-sequential drug release. Summary of the Invention

[0008] The purpose of this invention is to address at least one of the aforementioned problems by providing an in-situ drug-loaded hydrogel based on nano-bottle encapsulation technology, its preparation method, and its application. This addresses the issues of existing technologies where non-in-situ hydrogels cannot adhere to irregular tissues, and in-situ hydrogels struggle to simultaneously achieve controlled, sequential drug release. This solution designs an in-situ drug-loaded hydrogel based on nano-bottle encapsulation technology. Through controlled in-situ gelation, the hydrogel perfectly adheres to the joint cavity to protect the joint. Simultaneously, the introduction of diclofenac sodium, which has excellent anti-inflammatory properties, and Kartogenin (CAS No. 4727-31-5), which promotes cartilage differentiation, allows them to work in tandem with the entire drug-loaded hydrogel system under near-infrared light irradiation. This enables the controlled, sequential release of both drugs, achieving rapid release of diclofenac sodium and sustained release of Kartogenin, thereby achieving staged treatment of osteoarthritis.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] The first aspect of this invention discloses a method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology, comprising the following steps:

[0011] Step 1: Prepare drug-loaded polydopamine nanobottles using nanobottle encapsulation technology: Prepare polydopamine nanobottles using a template sacrificial method; Dissolve copper chloride, Kartogenin, and phase change material in a first solution, then add the polydopamine nanobottles and incubate to obtain drug-loaded polydopamine nanobottles;

[0012] Step 2, Preparation of reduced thiourea group-grafted hyaluronic acid: Sodium hyaluronate was dissolved in the first buffer solution, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole for activation, and then adipic acid dihydrazide was added to react and obtain an intermediate product; the intermediate product was dissolved in deionized water, mixed with a dimethyl sulfoxide solution containing methyl isothiocyanate and reacted to obtain reduced thiourea group-grafted hyaluronic acid;

[0013] Step 3, Preparation of in-situ drug-loaded hydrogel: Hyaluronic acid grafted with reduced thiourea groups and diclofenac sodium are dissolved in deionized water, and drug-loaded polydopamine nanobottles are added to form a hydrogel precursor solution; the hydrogel precursor solution is irradiated with near-infrared light, and copper chloride is released by photothermal response to gel in situ with hyaluronic acid grafted with reduced thiourea groups, crosslinking to form an in-situ drug-loaded hydrogel.

[0014] Preferably, in step 1, the polydopamine nanobottle is prepared by the following steps:

[0015] Polystyrene microspheres, used as templates, were dispersed in a dopamine / Tris-HCl buffer solution and reacted. The reaction product was collected by centrifugation and redispersed in a toluene / sodium dodecyl sulfonate aqueous emulsion for incubation. The incubated particles were collected and redispersed in tetrahydrofuran, followed by centrifugation to obtain polydopamine nanobottles.

[0016] Preferably, in step 1, the phase change material is a eutectic mixture of lauric acid and stearic acid; the first solution is methanol; and the mass ratio of Kartogenin, copper chloride, phase change material and polydopamine nanobottle is 1~10:1~50:25~100:1~5.

[0017] More preferably, in step 1, the phase change material is a eutectic mixture of lauric acid and stearic acid in a mass ratio of 4:1; the first solution is methanol; and the mass ratio of Kartogenin, copper chloride, phase change material and polydopamine nanobottle is 3:30:100:2.

[0018] The phase change material at this ratio has a corresponding phase change temperature of about 39°C, which can avoid overheating and damage to the body, and is also just slightly higher than the human body temperature (37°C), giving the entire drug-loaded hydrogel photothermally induced temperature response.

[0019] Preferably, in step 2, the first buffer solution is morpholine ethanesulfonic acid buffer; the mass ratio of sodium hyaluronate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and adipate dihydrazide is 1:2~3:1.5~2:3~4; the concentration of methyl isothiocyanate in the dimethyl sulfoxide solution containing methyl isothiocyanate is 1~3g:3~10mL, and the mass ratio of the intermediate product to the methyl isothiocyanate in the dimethyl sulfoxide solution containing methyl isothiocyanate is 1:1~3.

[0020] Preferably, in step 2, the grafting degree of the reduced thiourea group grafted onto hyaluronic acid is 25-40% (which may vary depending on the mass ratio of sodium hyaluronate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and adipic acid dihydrazide).

[0021] More preferably, in step 2, the first buffer solution is morpholine ethanesulfonic acid buffer; the mass ratio of sodium hyaluronate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, and adipate dihydrazide is 1:2.396:1.919:3.484. The concentration of methyl isothiocyanate in the dimethyl sulfoxide solution containing methyl isothiocyanate is 2 g:5 mL, and the mass ratio of the intermediate product to the methyl isothiocyanate in the dimethyl sulfoxide solution containing methyl isothiocyanate is 1:2.

[0022] More preferably, in step 2, the grafting degree of the reduced thiourea group grafted onto hyaluronic acid is 34% (corresponding to the mass ratio of sodium hyaluronate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and adipic acid dihydrazide being 1:2.396:1.919:3.484).

[0023] Preferably, in step 3, the concentration of the reduced thiourea group-grafted hyaluronic acid in the hydrogel precursor solution is 1-5 wt%, the concentration of diclofenac sodium in the hydrogel precursor solution is 10-100 μg / mL, and the concentration of the drug-loaded polydopamine nanobottle in the hydrogel precursor solution is 0.5-3 mg / mL; the wavelength of the near-infrared light is 808 nm, and the intensity is 0.5-1 W·cm. -2 The time is 3 to 8 minutes.

[0024] More preferably, in step 3, the concentration of the reduced thiourea group-grafted hyaluronic acid in the hydrogel precursor solution is 3 wt%, the concentration of diclofenac sodium in the hydrogel precursor solution is 50 μg / mL, and the concentration of the drug-loaded polydopamine nanobottle in the hydrogel precursor solution is 2 mg / mL; the wavelength of the near-infrared light is 808 nm, and the intensity is 0.75 W·cm. -2 The time is 4 minutes.

[0025] If the concentration of reduced thiourea group-grafted hyaluronic acid in the hydrogel precursor solution is too low, the grafting density in the drug-loaded hydrogel will be too low, resulting in decreased mechanical properties and weakened self-healing ability. Conversely, if the concentration of reduced thiourea group-grafted hyaluronic acid in the hydrogel precursor solution is too high, the prepared hydrogel precursor solution will be too viscous, which is not conducive to the dispersion of drug-loaded polydopamine nanobottles, leading to an overly hard final drug-loaded hydrogel and weakened adhesion. If the concentration of drug-loaded polydopamine nanobottles in the hydrogel precursor solution is too low, the content of the encapsulated material in the drug-loaded hydrogel will be low, affecting the effectiveness of in-situ gelation and drug release.

[0026] The second aspect of this invention discloses an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology, which is prepared by any of the preparation methods described above;

[0027] The drug-loaded polydopamine nanobottle is constructed by encapsulating copper chloride and Kartogenin with a phase change material.

[0028] The diclofenac sodium is dispersed in the periphery and pores of the in-situ drug-loaded hydrogel;

[0029] Under near-infrared light irradiation, copper ions are controllably released from drug-loaded polydopamine nanobottles and undergo ionic cross-linking with hyaluronic acid grafted with reduced thiourea groups to achieve in-situ gelation.

[0030] This in-situ drug-loaded hydrogel consists of polydopamine nanobottles that encapsulate copper ions (derived from copper chloride) and Kartogenin within their cavities using phase change materials. The inherent photothermal effect of polydopamine allows for the controlled release of the encapsulated material from the nanobottles under near-infrared light irradiation. The released copper ions then undergo ionic cross-linking with hyaluronic acid grafted with reduced thiourea groups to achieve in-situ gelation, ultimately forming an in-situ drug-loaded hydrogel.

[0031] The controlled release mechanism of the polydopamine nanobottle is as follows: the polydopamine shell of the nanobottle is locally heated under near-infrared light irradiation, causing the phase change material inside the cavity to change from a solid phase to a liquid phase, thereby releasing the encapsulated copper ions and Kartogenin in response.

[0032] Preferably, the concentration of the drug-loaded polydopamine nanobottle is 0.5~3 mg / mL; the concentration of the reduced thiourea group-grafted hyaluronic acid is 1~5 wt%; and the concentration of the diclofenac sodium is 10~100 μg / mL.

[0033] More preferably, in the drug-loaded polydopamine nanobottle, the mass ratio of Kartogenin, copper chloride, phase change material to polydopamine nanobottle is 1~10:1~50:25~100:1~5.

[0034] More preferably, the concentration of the drug-loaded polydopamine nanobottle is 2 mg / mL; the concentration of the reduced thiourea group-grafted hyaluronic acid is 3 wt%; and the concentration of diclofenac sodium is 50 μg / mL.

[0035] More preferably, in the drug-loaded polydopamine nanobottle, the mass ratio of Kartogenin, copper chloride, phase change material to polydopamine nanobottle is 3:30:100:2.

[0036] More preferably, the polydopamine nanobottle has an average diameter of 432.7 nm and an average opening size of 228.0 nm.

[0037] The third aspect of this invention discloses the application of an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology, as described above, in the preparation of a medicament for treating osteoarthritis.

[0038] Preferably, the in-situ drug-loaded hydrogel is used in conjunction with thermotherapy, wherein the thermotherapy consists of applying the in-situ drug-loaded hydrogel to a temperature of 808 nm and 0.5~1 W·cm⁻¹ daily. -2 Irradiate with near-infrared light of high intensity once, for 3-8 minutes each time.

[0039] More preferably, the in-situ drug-loaded hydrogel is used in conjunction with thermotherapy, wherein the thermotherapy consists of daily application of the in-situ drug-loaded hydrogel at an 808 nm wavelength and a temperature of 0.75 W·cm⁻¹. -2 Irradiate with near-infrared light of high intensity once, for 5 minutes each time.

[0040] The working principle of this invention is as follows:

[0041] Polydopamine nanobottles utilize phase change materials to stably encapsulate copper chloride and Kartogenin, enabling both substances to be released only upon near-infrared light irradiation. Specifically, under near-infrared light irradiation, the polydopamine nanobottles effectively convert light energy into heat energy, causing the phase change material to transform from a solid phase to a liquid phase, thereby releasing the encapsulated substances and achieving controllable in-situ gelation and drug release.

[0042] The resulting in-situ drug-loaded hydrogel contains diclofenac sodium in its pores and periphery, enabling rapid anti-inflammatory effects through its explosive release. Meanwhile, the hydrogel retains numerous polydopamine nanobottles within, encapsulating Kartogenin which is slowly released only after multiple rounds of near-infrared light irradiation, thus achieving a long-lasting chondrogenic effect and ultimately realizing the sequential release of both drugs.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] Firstly, the polydopamine nanobottle and the hyaluronic acid grafted with reduced thiourea groups in this invention can effectively remove reactive oxygen species in the oxidative stress environment of osteoarthritis.

[0045] Secondly, the reduced thiourea group grafted with hyaluronic acid in this invention can provide joint lubrication and prevent joint movement injuries.

[0046] Thirdly, in this invention, diclofenac sodium can significantly alleviate the inflammatory response during the onset of osteoarthritis.

[0047] Fourth, the Kartogenin in this invention can significantly promote cartilage regeneration.

[0048] Fifth, the introduction of nano-bottle encapsulation technology in this invention can stably encapsulate the cross-linking agent copper chloride and the cartilage regeneration drug Kartogenin, releasing them only through near-infrared light response, thereby effectively achieving controllable in-situ gelation and slow, continuous release of Kartogenin, and achieving a sustained cartilage regeneration effect.

[0049] Sixth, the in-situ drug-loaded hydrogel of the present invention can overcome the shortcomings of non-in-situ hydrogels in being unable to adhere to irregular tissues, enhance joint lubrication and reduce joint movement damage, and can effectively target different pathological stages of osteoarthritis through time-sequential drug release, thereby achieving staged treatment of osteoarthritis. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the preparation process of in-situ drug-loaded hydrogels constructed based on nanobottle encapsulation technology.

[0051] Figure 2 Transmission electron microscopy image of a polydopamine nanobottle.

[0052] Figure 3 EDS image (A) and UV-Vis image (B) of the drug-loaded polydopamine nanobottle.

[0053] Figure 4 To reduce thiourea groups grafted with hyaluronic acid 1 H NMR image.

[0054] Figure 5 Cryo-scanning electron microscopy images of the in-situ drug-loaded hydrogels (HDPCK, HPC, and HDPC) prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0055] Figure 6A The results show the self-healing ability verification of the in-situ drug-loaded hydrogel (HDPCK) prepared in Example 1.

[0056] Figure 6B The relaxation curves of the in-situ drug-loaded hydrogels (HDPCK, HPC, and HDPC) prepared in Example 1, Comparative Example 1, and Comparative Example 2 are shown.

[0057] Figure 6C The adhesion properties of the in-situ drug-loaded hydrogel (HDPCK) prepared in Example 1 are shown.

[0058] Figure 6D The tensile peel test results are for the in-situ drug-loaded hydrogels (HDPCK, HPC, and HDPC) prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0059] Figure 6E The Young's modulus is given by the in-situ drug-loaded hydrogels (HDPCK, HPC, and HDPC) prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0060] Figure 6F The swelling rate of the in-situ drug-loaded hydrogels (HDPCK, HPC and HDPC) prepared for Example 1, Comparative Example 1 and Comparative Example 2 was determined.

[0061] Figure 6G The degradation test results are for the in-situ drug-loaded hydrogels (HDPCK, HPC, and HDPC) prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0062] Figure 6H The results are the free radical scavenging effects of the in-situ drug-loaded hydrogels (HDPCK, HPC, HDPC and Control) prepared in Example 1, Comparative Example 1 and Comparative Example 2 and Control Example.

[0063] Figure 6I The results of the photothermal performance test of the in-situ drug-loaded hydrogel (HDPCK) prepared in Example 1 are shown.

[0064] Figure 6J The results of the photothermal stability test of the in-situ drug-loaded hydrogel (HDPCK) prepared in Example 1 are shown.

[0065] Figure 6K The temperature change results of the in-situ drug-loaded hydrogel (HDPCK) prepared in Example 1 during the photothermal performance test.

[0066] Figure 6L The drug (diclofenac sodium DS) release curve of the in-situ drug-loaded hydrogel (HDPCK) prepared in Example 1 during the photothermal performance test.

[0067] Figure 6M The drug (KGN) release curve of the in-situ drug-loaded hydrogel (HDPCK) prepared in Example 1 during the photothermal performance test.

[0068] Figure 7 The results of the biosafety test of co-culture of in-situ drug-loaded hydrogels (HDPCK, HPC, and HDPC) prepared in Examples 1, Comparative Examples 1, and Comparative Examples 2, and chondrocytes (A) and bone marrow mesenchymal stem cells (B) subjected to hyperthermic conditions (HDPCK+, HPC+, and HDPC+).

[0069] Figure 8 The results of reactive oxygen species scavenging tests on in-situ drug-loaded hydrogels (HDPCK, HPC, HDPC and Control, PBS) prepared for Example 1, Comparative Example 1, Comparative Example 2, Control Example, and Blank Example 1, as well as under hyperthermic conditions (HDPCK+, HPC+ and HDPC+).

[0070] Figure 9 Flow cytometry images of in-situ drug-loaded hydrogels (HDPCK, HPC, HDPC and Control, PBS) prepared for Examples 1, Comparative Examples 1, Comparative Examples 2, Control Examples, and Blank Examples 1, and apoptosis assays under hyperthermic conditions (HDPCK+, HPC+ and HDPC+).

[0071] Figure 10 The results of staining experiments show the effects of in situ drug-loaded hydrogels (HDPCK, HPC, HDPC, and Control) prepared in Examples 1, Comparative Examples 1, Comparative Examples 2, and Control Examples on the chondrogenic differentiation of bone marrow mesenchymal stem cells under hyperthermic conditions (HDPCK+, HPC+, and HDPC+).

[0072] Figure 11 Images of articular cartilage histology in SD rats with an anterior cruciate ligament transection-induced osteoarthritis model after local treatment with in situ drug-loaded hydrogels (HDPCK, HPC, HDPC, Control, PBS) prepared in Examples 1, Comparative Examples 1, Comparative Examples 2, Control Examples, and Blank Examples 1, and images of articular cartilage histology after local treatment with anterior cruciate ligament transection-induced osteoarthritis model under hyperthermic conditions (HDPCK+, HPC+, and HDPC+). (Scale bar: 400 μm)

[0073] Figure 12Images of cartilage histology in SD rats with a full-thickness cartilage defect model after local treatment of the cartilage defect site with in situ drug-loaded hydrogels (HDPCK, HPC, HDPC, Control, and Defect) prepared in Examples 1, Comparative Examples 1, Comparative Examples 2, Control Examples, and Blank Examples 2, and images of cartilage histology after local treatment of the cartilage defect site with hyperthermic conditions (HDPCK+, HPC+, and HDPC+). Scale bar: 400 μm. Detailed Implementation

[0074] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0075] To accomplish the purpose of this invention, the experimental reagents, materials, or instruments used in the embodiments were all purchased from commercially available products unless otherwise specified.

[0076] To address the problems existing in the prior art, this invention provides an in-situ drug-loaded hydrogel constructed based on nano-bottle encapsulation technology, its preparation method, and its application. The in-situ drug-loaded hydrogel provided by this invention can be used to treat osteoarthritis. The in-situ drug-loaded hydrogel can improve the problems of non-in-situ hydrogels being unable to adhere to irregular tissues and in-situ hydrogels being unable to simultaneously possess the ability to release drugs in a controlled sequence. It enhances joint lubrication and tissue joint movement damage, and can effectively target different pathological stages of osteoarthritis through time-sequential drug release, thereby achieving staged treatment of osteoarthritis.

[0077] The specific technical solution of the present invention is as follows:

[0078] An in-situ drug-loaded hydrogel constructed based on nanobottle encapsulation technology, wherein the in-situ drug-loaded hydrogel is formed by controlling the release of copper ions encapsulated in polydopamine nanobottles through near-infrared light response, and crosslinking with reduced thiourea groups in the precursor solution grafted with hyaluronic acid.

[0079] in,

[0080] The precursor solution uses deionized water as a solvent, the concentration of reduced thiourea group-grafted hyaluronic acid is 3wt%, the concentration of drug-loaded polydopamine nanobottles encapsulating copper chloride (providing copper ions) and Kartogenin is 2mg / mL, and the concentration of diclofenac sodium is 50μg / mL.

[0081] The drug-loaded polydopamine nanobottle was obtained at 40°C by mixing a eutectic mixture (phase change material) of lauric acid and stearic acid in a mass ratio of 4:1, polydopamine nanobottle, copper chloride, and Kartogenin in methanol; the polydopamine nanobottle was obtained by the polystyrene microsphere template sacrificial method.

[0082] The reduced thiourea group grafted onto hyaluronic acid is obtained by a two-step reaction of hyaluronic acid, adipic dihydrazide, and methyl isothiocyanate.

[0083] A method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology, comprising the following steps:

[0084] Step 1, Preparation of drug-loaded polydopamine nanobottles:

[0085] Preparation of polydopamine nanobottles: Using polystyrene microspheres as templates, they were dispersed in dopamine / Tris-HCl buffer and reacted overnight. The particles were then collected by centrifugation and redispersed in deionized water. The collected particles were then dispersed in a toluene / sodium dodecyl sulfate aqueous emulsion and incubated for six hours. The process was terminated with ethanol, and the particles were collected by centrifugation. The collected particles were then dispersed in tetrahydrofuran, and centrifuged after four hours to obtain polydopamine nanobottles.

[0086] Preparation of drug-loaded polydopamine nanobottles: At 40°C, a eutectic mixture of copper chloride, Kartogenin, and lauric acid and stearic acid in a mass ratio of 4:1 was dissolved in methanol. Then, the polydopamine nanobottles were dispersed in the mixed solution and incubated for half an hour. After centrifugation, the particles were collected, washed, and dispersed in pre-cooled deionized water to obtain drug-loaded polydopamine nanobottles.

[0087] Step 2, Preparation of hyaluronic acid grafted with reduced thiourea groups:

[0088] Sodium hyaluronate was dissolved in morpholine ethanesulfonic acid buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole were added. After activation at 37°C for two hours, adipate dihydrazide was added, and the reaction was carried out at room temperature for twenty-two hours. The resulting product was dialyzed against deionized water and lyophilized, then dissolved in deionized water and mixed with a dimethyl sulfoxide solution containing methyl isothiocyanate. The mixture was reacted for three days under nitrogen protection. The product was dialyzed against sodium chloride solution and deionized water for three days, and then lyophilized to obtain reduced thiourea-grafted hyaluronic acid.

[0089] Step 3, Preparation of in-situ drug-loaded hydrogel:

[0090] Preparation of in-situ hydrogel precursor solution: Hyaluronic acid grafted with reduced thiourea groups and diclofenac sodium were dissolved in deionized water to form a solution, and then drug-loaded polydopamine nanobottles were dispersed in the solution to form a hydrogel precursor solution.

[0091] Preparation of in-situ drug-loaded hydrogels: The hydrogel precursor solution was irradiated with near-infrared light to form an in-situ drug-loaded hydrogel.

[0092] In step 1: 0.5 mL of 400 nm, 2.5 w / v polystyrene microspheres were dispersed in 10 mL of 2 mg / mL dopamine / Tris-HCl buffer (10 mM, pH 8.5). The nanoparticles were collected by centrifugation at 8000 rpm for 5 min. The nanoparticles were incubated with 10 mL of 1 v / v toluene / sodium dodecyl sulfonate aqueous emulsion, where the concentration of sodium dodecyl sulfonate in the emulsion was 1 w / v%. At 40 °C, a eutectic mixture of 150 mg copper chloride, 15 mg Kartogenin, and 500 mg lauric acid and stearic acid (mass ratio 4:1) was dissolved in 0.5 mL of methanol. Then, 10 mg of polydopamine nanobottles were dispersed in the mixed solution and incubated for half an hour. The particles were then collected by centrifugation at 8000 rpm for 5 min, washed twice with dimethyl sulfoxide, and dispersed in pre-cooled deionized water to obtain drug-loaded polydopamine nanobottles.

[0093] In step 2: 2g of sodium hyaluronate was dissolved in 200mL of buffer solution (pH 6.5) containing 1g of morpholine ethanesulfonic acid. 4.792g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 3.838g of 1-hydroxybenzotriazole were added. After activation at 37°C for two hours, 6.968g of adipate dihydrazide was added, and the reaction was carried out at room temperature for twenty-two hours. The resulting product was purified by dialyzing with deionized water (MWCO: 14 kDa) for three days. After lyophilization, 2g of the purified product was dissolved in 200mL of deionized water and mixed with 10mL of dimethyl sulfoxide solution containing 4g of methyl isothiocyanate. The mixture was reacted under nitrogen protection for three days. The product was dialyzed against sodium chloride solution and deionized water (MWCO: 14 kDa) for three days, and then lyophilized to obtain reduced thiourea-grafted hyaluronic acid. The grafting degree of the reduced thiourea-grafted hyaluronic acid was 34%.

[0094] In step 3, the concentration of the reduced thiourea group-grafted hyaluronic acid in the hydrogel precursor solution is 3 wt%, the concentration of diclofenac sodium in the hydrogel precursor solution is 50 μg / mL, and the concentration of the drug-loaded polydopamine nanobottle in the hydrogel precursor solution is 2 mg / mL. The near-infrared light irradiation conditions are 808 nm wavelength and 0.75 W·cm⁻¹. -2 strength.

[0095] Therefore, step 3 is:

[0096] Preparation of the in-situ hydrogel precursor solution: 3 wt% of reduced thiourea-grafted hyaluronic acid was mixed with 50 μg / mL diclofenac sodium to obtain a premix. Then, drug-loaded polydopamine nanobottles were dispersed in the premix to a concentration of 2 mg / mL, forming the in-situ hydrogel precursor solution. It should be noted that all concentrations mentioned here refer to the final concentrations in the precursor solution.

[0097] Preparation of in-situ drug-loaded hydrogels: The in-situ hydrogel precursor solution was heated at 808 nm wavelength and 0.75 W·cm⁻¹. -2 Cross-linking is achieved by irradiating with near-infrared light of high intensity for four minutes.

[0098] Application of an in-situ drug-loaded hydrogel constructed based on nanobottle encapsulation technology in the preparation of a drug for treating osteoarthritis.

[0099] This invention provides an in-situ drug-loaded hydrogel constructed based on nanobottle encapsulation technology and its preparation method. The in-situ drug-loaded hydrogel provided by this invention can be used to treat osteoarthritis. The in-situ drug-loaded hydrogel can improve the problems of non-in-situ hydrogels being unable to adhere to irregular tissues and in-situ hydrogels being unable to simultaneously possess the ability to release drugs in a controlled sequence. It enhances joint lubrication and reduces joint movement damage. Furthermore, diclofenac sodium and Kartogenin have excellent anti-inflammatory and cartilage differentiation-promoting abilities, respectively. By combining with nanobottle encapsulation technology and hydrogel systems, they can effectively achieve time-sequential release, achieving the effects of rapid release of diclofenac sodium and sustained release of Kartogenin. Thus, the time-sequential release of drugs can effectively target different pathological stages of osteoarthritis, thereby achieving the goal of first rapidly reducing inflammation to protect early cartilage degeneration in osteoarthritis and long-term promoting cartilage differentiation to improve late-stage cartilage defects in osteoarthritis, ultimately realizing staged treatment of osteoarthritis.

[0100] This invention provides a novel material option for staged treatment of persistent pathological symptoms of osteoarthritis in clinical practice.

[0101] It should be noted that the above scheme only provides the optimal data combination. In other embodiments, the parameter combinations involved can be adjusted. For example, the mass ratio of Kartogenin, copper chloride, phase change material and polydopamine nanobottle can be selected in the range of 1~10:1~50:25~100:1~5; the mass ratio of sodium hyaluronate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and adipic acid dihydrazide can be selected in the range of 1:2~3:1.5~2:3~4; and the corresponding grafting degree of reduced thiourea groups grafted onto hyaluronic acid is 25~40%. (Isothiocyanate is also mentioned.) The concentration of methyl isothiocyanate in the dimethyl sulfoxide solution of methyl ester was selected within the range of 1-3 g: 3-10 mL; the mass ratio of the intermediate product to methyl isothiocyanate in the dimethyl sulfoxide solution containing methyl isothiocyanate was controlled within the range of 1:1-3; the concentration of hyaluronic acid grafted with reduced thiourea groups in the hydrogel precursor solution was selected within the range of 1-5 wt%; the concentration of diclofenac sodium in the hydrogel precursor solution was selected within the range of 10-100 μg / mL; the concentration of drug-loaded polydopamine nanobottles in the hydrogel precursor solution was selected within the range of 0.5-3 mg / mL; the intensity of near-infrared light was selected within the range of 0.5-1 W·cm. -2 The time can be selected within the range of 3 to 8 minutes. Moreover, any combination of selections within the above range will result in in-situ drug-loaded hydrogels with basically the same performance (see Example 1 below); among them, the only option with relatively obvious performance differences is the option that changes the drug loading amount.

[0102] Example 1

[0103] Preparation of in-situ drug-loaded hydrogels.

[0104] Step 1, Preparation of drug-loaded polydopamine nanobottles:

[0105] Preparation of polydopamine nanobottles: 0.5 mL of 400 nm, 2.5 w / v polystyrene microspheres were dispersed in 10 mL of 2 mg / mL dopamine / Tris-HCl buffer (10 mM, pH 8.5) and reacted overnight. The mixture was then collected by centrifugation at 6000 rpm for 5 min and re-dispersed in deionized water. The collected particles were then dispersed in 10 mL of 1% (v / v) toluene / sodium dodecyl sulfate aqueous emulsion and incubated for six hours. The process was terminated with ethanol, and the particles were collected by centrifugation. The collected particles were then dispersed in tetrahydrofuran and centrifuged after four hours to obtain polydopamine nanobottles.

[0106] Preparation of drug-loaded polydopamine nanobottles: At 40°C, a eutectic mixture of 150 mg copper chloride, 15 mg Kartogenin, and 400 mg lauric acid and stearic acid in a mass ratio of 4:1 was dissolved in 0.5 mL methanol. Then, 10 mg of polydopamine nanobottles were dispersed in the mixed solution and incubated for half an hour. After centrifugation at 6000 rpm for 5 min, the particles were collected. After washing with dimethyl sulfoxide, the particles were dispersed in pre-cooled deionized water to obtain drug-loaded polydopamine nanobottles.

[0107] Step 2, Preparation of hyaluronic acid grafted with reduced thiourea groups:

[0108] 2 g of sodium hyaluronate was dissolved in 200 mL of buffer solution (pH 6.5) containing 1 g of morpholine ethanesulfonic acid. 4.792 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 3.838 g of 1-hydroxybenzotriazole were added. After activation at 37 °C for two hours, 6.968 g of adipate dihydrazide was added, and the reaction was carried out at room temperature for twenty-two hours. The resulting product was purified by dialyzing with deionized water (MWCO: 14 kDa) for three days. After lyophilization, 2 g of the purified product was dissolved in 200 mL of deionized water and mixed with 10 mL of dimethyl sulfoxide solution containing 4 g of methyl isothiocyanate. The mixture was reacted under nitrogen protection for three days. The product was dialyzed against sodium chloride solution and deionized water (MWCO: 14 kDa) for three days, and then lyophilized to obtain reduced thiourea-grafted hyaluronic acid.

[0109] Step 3, Preparation of in-situ drug-loaded hydrogel:

[0110] Preparation of in-situ hydrogel precursor solution: 3 wt% of reduced thiourea group-grafted hyaluronic acid and 50 μg / mL diclofenac sodium were dissolved in deionized water to form a solution. Then, drug-loaded polydopamine nanobottles were dispersed in the solution to a final concentration of 2 mg / mL, thus forming the hydrogel precursor solution.

[0111] Preparation of in-situ drug-loaded hydrogels: Pre-in-situ hydrogel precursor solutions were prepared at 808 nm wavelength and 0.75 W·cm⁻¹. -2 Irradiate with near-infrared light of high intensity for four minutes to achieve cross-linking and form an in-situ drug-loaded hydrogel.

[0112] The in-situ drug-loaded hydrogel prepared in Example 1 is designated as HDPCK.

[0113] The process for preparing the in-situ drug-loaded hydrogel in this embodiment is as follows: Figure 1 As shown, the successful preparation of in-situ drug-loaded hydrogels was achieved through... Figures 2-5 The successful fabrication of polydopamine nanobottles was verified using transmission electron microscopy, with images showing that they are hollow, single-opening nanoparticles. Figure 2ImageJ analysis showed that the average diameter of the polydopamine nanobottle was 432.7 nm, and the average opening size was 228.0 nm. EDS analysis verified the effective encapsulation of copper chloride in the drug-loaded polydopamine nanobottle, which contains Cu and Cl elements. Figure 3 A). The effective encapsulation of Kartogenin in the drug-loaded polydopamine nanobottle was verified by UV-Vis spectroscopy. The drug-loaded polydopamine nanobottle exhibited the characteristic absorption peak of Kartogenin at 280 nm. Figure 3 B). Through 1 H NMR analysis confirmed the successful preparation of hyaluronic acid grafted with reduced thiourea groups. Figure 4 The successful preparation of the in-situ drug-loaded hydrogel was verified by cryo-scanning electron microscopy. The images showed that the in-situ drug-loaded hydrogel had a uniform porous structure and contained a large number of nanoparticles. Figure 5 ).

[0114] Comparative Examples 1-2

[0115] Referring to the description in Example 1, the main differences in preparing different in-situ drug-loaded hydrogels are whether diclofenac sodium is added during the preparation of the hydrogel precursor solution and whether Kartogenin is present in the drug-loaded polydopamine nanobottle.

[0116] in:

[0117] Without adding diclofenac sodium and without encapsulating Kartogenin in the drug-loaded polydopamine nanobottles, the 2 mg / mL polydopamine nanobottles encapsulated only with copper chloride, prepared in step 1 of Example 1, were directly mixed with 3 wt% of reduced thiourea group-grafted hyaluronic acid to obtain a hydrogel precursor solution. The solution was analyzed at 808 nm wavelength and 0.75 W·cm⁻¹. -2 The in-situ drug-loaded hydrogel obtained by irradiation with near-infrared light of high intensity for four minutes is denoted as HPC.

[0118] Diclofenac sodium was added, but the drug-loaded polydopamine nanobottles did not encapsulate Kartogenin. The 2 mg / mL polydopamine nanobottles encapsulated only with copper chloride, prepared in step 1 of Example 1, were mixed with 3 wt% reduced thiourea-grafted hyaluronic acid and 50 μg / mL diclofenac sodium to obtain a hydrogel precursor solution. The solution was analyzed at 808 nm wavelength and 0.75 W·cm⁻¹. -2 The in-situ drug-loaded hydrogel obtained by irradiation with near-infrared light of high intensity for four minutes is denoted as HDPC.

[0119] Comparison Example

[0120] In the experimental model for studying the free radical scavenging ability of hydrogels, no in-situ drug-loaded hydrogels were added, and only a deionized aqueous solution containing DPPH was used, which was designated as the Control group.

[0121] In the IL-1β-induced inflammatory model, the normal group in the non-inflammatory model serves as the negative control group, denoted as the Control group.

[0122] In the ACLT surgery-induced osteoarthritis model, the healthy normal group that did not participate in the model served as the negative control group and was designated as the Control group.

[0123] For the full-thickness cartilage defect model, the healthy normal group that did not participate in the model was the negative control group, denoted as the Control group.

[0124] Blank example 1

[0125] In an IL-1β-induced inflammation model / ACLT surgery-induced osteoarthritis model, the positive control group that received only PBS without in-situ drug-loaded hydrogel treatment was designated as the PBS group.

[0126] Blank example 2

[0127] In a full-thickness cartilage defect model, the positive control group that did not receive in-situ drug-loaded hydrogel treatment but only received PBS was designated as the Defect group.

[0128] Figures 6A-6M Characterization of different in-situ drug-loaded hydrogels prepared in Example 1, Comparative Examples 1 and 2, and Control Examples.

[0129] Freshly prepared hydrogel samples were cut in half and gently reconnected under light contact to assess the self-healing ability of the hydrogels and observe the restoration of their integrity. Figure 6A Meanwhile, its relaxation curve was further verified by rheological determination. Figure 6B The hydrogel was applied to freshly cut pigskin, and its adhesion was observed by twisting and other actions. Figure 6C Then, a tensile peel test was performed using a universal testing machine to evaluate the adhesive properties. Figure 6D As can be seen, all three in-situ drug-loaded hydrogels exhibit good self-healing and adhesion capabilities, and these properties do not change with the introduction of drugs.

[0130] Subsequently, nanoindentation was used to study the mechanical strength of the three in-situ drug-loaded hydrogels. The results showed that changes in the drug did not significantly affect the Young's modulus of the three hydrogels. Figure 6E ).

[0131] In addition, the swelling properties of three in-situ drug-loaded hydrogels were determined by gravimetric analysis. The initial dry weight (M0) and the weight after swelling in PBS at room temperature (M1) of the hydrogels were recorded, ensuring that excess surface water was removed with absorbent paper before weighing. The swelling ratio was determined using the following formula: Swelling ratio = (M1 / M0) × 100%. In the swelling experiment, all samples exhibited similar behavior, with swelling ratios of approximately 180–190%. Figure 6F ).

[0132] The degradation capacity of the hydrogels was determined by gravimetric method: To assess the degradation, the initial mass of the hydrogel for degradation was (M10). The hydrogel was then immersed in PBS at 37°C and placed on a shaker. At different time intervals, the remaining hydrogel was freeze-dried and weighed (M11). The gravimetric degradation capacity of the hydrogel was calculated using the formula: Gravimetric degradation rate = [(M10-M11) / M10] × 100%. All hydrogels exhibited similar degradation behavior, with approximately 60% degradation occurring after about 14 days. Figure 6G ).

[0133] The hydrogels were incubated in 0.1 mM DPPH solution in the dark for 10 hours, and then the absorbance at 517 nm was measured using a UV-Vis spectrophotometer to determine the antioxidant activity of the in-situ hydrogels. This indicated that the three in-situ drug-loaded hydrogels had good free radical scavenging effects. Figure 6H ).

[0134] In-situ hydrogel samples were subjected to different power densities (0.5~1.0 W·cm). -2 Irradiate the light with 808 nm near-infrared light for 5 minutes to evaluate its photothermal properties. Figure 6I ); The photothermal stability of the in-situ hydrogel was tested by multiple rounds of irradiation. Figure 6J The temperature change of the in-situ hydrogel was monitored using an infrared camera during the testing process. Figure 6K As can be seen from the test results, under all test conditions, 0.75 W·cm -2 The photothermal heating effect is optimal (approximately 40°C, which matches the photothermal response temperature of the phase change material, and does not damage the body or cause discomfort).

[0135] Further high-performance liquid chromatography was used to study the drug release profiles of diclofenac sodium and Kartogenin in the in-situ drug-loaded hydrogel, and to determine their time-series drug release effects. Figure 6L , 6MAs can be seen, diclofenac sodium, being freely dispersed around the hydrogel and within its pores, experiences a rapid release in aqueous solution within a short period, unaffected by the presence of the hydrogel. In contrast, Kartogenin is stably encapsulated in nanobottles, protected by solidified phase change material. It is only released under near-infrared light irradiation, where the phase change material melts due to the heating of the nanobottles. As the irradiation ends and the ambient temperature cools, the phase change material solidifies and is stably encapsulated again.

[0136] Therefore, it can be seen that the in-situ drug-loaded hydrogel of this scheme achieves the sequential release of two drugs through this drug loading design.

[0137] Test Example 1

[0138] To investigate the effects of in-situ drug-loaded hydrogels on the behavior of rat chondrocytes and bone marrow mesenchymal stem cells, rat chondrocytes and bone marrow mesenchymal stem cells were both loaded at 5 × 10⁻⁶. 3 Cells were cultured at a density of 1 cell / mL in well plates, and in situ drug-loaded hydrogels were added for co-culture. After three days, the cells were stained in the dark for 30 minutes using a live-dead reagent to observe cell growth.

[0139] Rat chondrocytes and bone marrow mesenchymal stem cells were divided into groups of 5 × 10⁻⁶. 3 The method for co-culturing in-situ drug-loaded hydrogels at a density of cells / mL in well plates is as follows:

[0140] Using the Transwell plate culture system, rat chondrocytes and bone marrow mesenchymal stem cells were cultured at 5 × 10⁻⁶ wells. 3 Cells were added to the lower wells at a density of cells / mL, and then HPC, HDPC, and HDPCK in situ drug-loaded hydrogels were placed in the upper Transwell chamber to form a cell-hydrogel co-culture system.

[0141] Simultaneously, three in-situ drug-loaded hydrogels were co-cultured with cells under conditions of with and without hyperthermia. The groups receiving hyperthermia were further designated as HPC+, HDPC+, and HDPCK+. For the hyperthermia groups, the hydrogels were exposed to 808 nm wavelength at 0.75 W·cm⁻¹ daily. -2 Irradiate with near-infrared light of high intensity once, for 5 minutes each time.

[0142] The different biomimetic matrix hydrogels obtained above were used to culture chondrocytes in vitro using DMEM / F12 medium and bone marrow mesenchymal stem cells using MEM medium.

[0143] Experimental results are as follows Figure 7 As shown, it can be seen that the three in-situ drug-loaded hydrogels interact with chondrocytes under conditions of given / no thermotherapy ( Figure 7A) and bone marrow mesenchymal stem cells ( Figure 7 B) After co-culture, the cells were able to grow normally and their proliferation and death were not inhibited by the introduction of the in-situ drug-loaded hydrogel, which proves the high biosafety of the in-situ drug-loaded hydrogel and the thermotherapy method.

[0144] Test Example 2

[0145] To investigate the protective effect of co-culturing chondrocytes with in situ drug-loaded hydrogels under inflammatory conditions, chondrocytes were co-cultured with the in situ drug-loaded hydrogels in Transwell chambers. The upper chamber contained the in situ drug-loaded hydrogel, and the lower chamber contained 5 × 10⁶ cells. 4 Chondrocytes at a density of [number] cells / mL were cultured. An inflammatory environment was created by stimulating the in situ drug-loaded hydrogel with / without hyperthermia. After two days of co-culture, reactive oxygen species in the environment were measured using a DCFH-DA fluorescent probe. Fluorescence signals were observed after 30 minutes of incubation with the probe. Experimental results are as follows: Figure 8 As shown in the figure, the experimental results indicate that the in-situ drug-loaded hydrogel can significantly remove reactive oxygen species in the inflammatory environment.

[0146] Simultaneously, the apoptosis status of chondrocytes co-cultured for two days was assessed using the Annexin V-APC / 7-AAD Apoptosis Kit. The experimental results are as follows: Figure 9 As shown in the experimental results, the in-situ drug-loaded hydrogel can effectively alleviate cell apoptosis. Its efficacy is mainly attributed to the use of diclofenac sodium, which can effectively alleviate the inflammatory environment.

[0147] Test Example 3

[0148] To investigate the effect of in-situ drug-loaded hydrogels on the chondrogenic ability of bone marrow mesenchymal stem cells (BMSCs), BMSCs were co-cultured with in-situ drug-loaded hydrogels in Transwell chambers. The upper chamber contained the in-situ drug-loaded hydrogel, and the lower chamber contained 5 × 10⁶ cells / mL of hydrogel. 4 Bone marrow mesenchymal stem cells were cultured at a density of [number] cells / mL. Using chondrogenic induction medium, in situ drug-loaded hydrogels were treated with / without hyperthermia. After seven days of co-culture, cells were fixed with 4% paraformaldehyde and stained with alexandrite blue for 30 minutes to obtain stained images. Experimental results are as follows: Figure 10As shown, the experimental results indicate that the in-situ drug-loaded hydrogel can significantly accelerate the chondrogenic differentiation capacity of bone marrow mesenchymal stem cells. Furthermore, the HDPCK+ group receiving thermotherapy showed better results than the HDPCK group without thermotherapy. This therapeutic effect induced by thermotherapy is attributed to two aspects: firstly, thermotherapy based on regular near-infrared light irradiation effectively promotes cell metabolism and growth, thereby enhancing the directed differentiation capacity of cells; secondly, regular near-infrared light irradiation effectively stimulates the continuous release of Kartogenin from the hydrogel, enhancing the therapeutic effect in response to the release.

[0149] Test Example 4

[0150] (1) Construction of an anterior cruciate ligament transection model in SD rats: Four-week-old male SD rats were anesthetized, the joint capsule was opened, and the anterior cruciate ligament was transected to induce joint instability. After surgery, the rats were fed for four weeks to induce osteoarthritis.

[0151] (2) Application of in-situ drug-loaded hydrogel for the treatment of osteoarthritis: After four weeks of rearing, the in-situ drug-loaded hydrogel precursor solution was injected into the joint cavity, and the solution was applied using an 808 nm wavelength and a 0.75 W·cm⁻¹ pressure. -2 Rats were irradiated with near-infrared light of high intensity for four minutes to form an in-situ drug-loaded hydrogel within the joint cavity. Afterward, the rats were fed normally, and injected every two weeks. A group receiving hyperthermia received daily treatment of the joints with 808 nm wavelength light at 0.75 W·cm⁻¹. -2 They were irradiated with near-infrared light for five minutes, and then sacrificed six weeks later to analyze the repair effect.

[0152] This embodiment mainly investigates the repair effect of in-situ drug-loaded hydrogels on early osteoarthritis. Figure 11 Histological analysis results of cartilage tissue in the joints after treatment in each group (scale bar: 400 μm). Among them, the in-situ drug-loaded hydrogel containing diclofenac sodium and drug-loaded polydopamine nanobottles encapsulating copper chloride and Kartogenin, which was given with heat therapy, showed the strongest repair effect. This corresponds to the hydrogel's own time-sequential drug release capability. In the early stage, a large amount of diclofenac sodium is released to effectively alleviate the inflammatory environment. After the environment stabilizes, Kartogenin is slowly released to promote cartilage recovery, which proves the cartilage-protective effect of in-situ drug-loaded hydrogel in the early treatment of osteoarthritis.

[0153] Test Example 5

[0154] (1) Construction of a full-thickness cartilage defect model in SD rats: Six-week-old male SD rats were taken, and after general anesthesia, the joint capsule was opened to expose the patellar groove of the femur. A cylindrical defect (2 mm in diameter and 1.5 mm in depth) was created in the patellar groove of the left femur using a sterile skin punch under saline irrigation.

[0155] (2) Application of in-situ drug-loaded hydrogel for repairing full-thickness cartilage defects: The in-situ drug-loaded hydrogel precursor solution was injected into the defect site, and immediately treated with 808 nm wavelength and 0.75 W·cm -2 Near-infrared light of high intensity was applied for four minutes to form an in-situ drug-loaded hydrogel. Rats were then fed normally post-surgery. The group receiving hyperthermia received daily treatment of the joints with 808 nm wavelength light at 0.75 W·cm⁻¹. -2 They were irradiated with near-infrared light for five minutes, and then sacrificed six weeks later to analyze the repair effect.

[0156] This embodiment mainly investigates the cartilage regeneration effect of in-situ drug-loaded hydrogels on cartilage defects caused by late-stage osteoarthritis. Figure 12 Histological analysis results of cartilage tissue in the joints after treatment in each group (scale bar: 400μm) show that the in-situ drug-loaded hydrogel containing diclofenac sodium and drug-loaded polydopamine nanobottles encapsulating copper chloride and Kartogenin, which received thermotherapy, showed the strongest repair effect. This proves that the dual treatment approach of thermotherapy combined with Kartogenin can effectively promote cartilage regeneration, and the constructed in-situ drug-loaded hydrogel can effectively promote cartilage regeneration to treat advanced osteoarthritis.

[0157] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology, characterized in that, Includes the following steps: Step 1: Preparation of drug-loaded polydopamine nanobottles using nanobottle encapsulation technology: Polydopamine nanobottles are prepared using a template sacrificial method; copper chloride, Kartogenin, and a phase change material are dissolved in a first solution, then the polydopamine nanobottle is added and incubated to obtain drug-loaded polydopamine nanobottles; wherein, the phase change material is a eutectic mixture of lauric acid and stearic acid in a mass ratio of 4:1, and its corresponding phase change temperature is higher than human body temperature; Step 2, Preparation of reduced thiourea group-grafted hyaluronic acid: Sodium hyaluronate was dissolved in the first buffer solution, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole for activation, and then adipic acid dihydrazide was added to react and obtain an intermediate product; the intermediate product was dissolved in deionized water, mixed with a dimethyl sulfoxide solution containing methyl isothiocyanate and reacted to obtain reduced thiourea group-grafted hyaluronic acid; Step 3, Preparation of in-situ drug-loaded hydrogel: Hyaluronic acid grafted with reduced thiourea groups and diclofenac sodium are dissolved in deionized water, and drug-loaded polydopamine nanobottles are added to form a hydrogel precursor solution. The concentration of the reduced thiourea group-grafted hyaluronic acid in the hydrogel precursor solution is 1-5 wt%, the concentration of diclofenac sodium in the hydrogel precursor solution is 10-100 μg / mL, and the concentration of the drug-loaded polydopamine nanobottles in the hydrogel precursor solution is 0.5-3 mg / mL. The hydrogel precursor solution is irradiated with near-infrared light to raise the temperature above the phase transition temperature. Copper chloride is released using the photothermal response of polydopamine to gel in situ with the reduced thiourea group-grafted hyaluronic acid, crosslinking to form an in-situ drug-loaded hydrogel.

2. The method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology according to claim 1, characterized in that, In step 1, the polydopamine nanobottle is prepared through the following steps: Polystyrene microspheres, used as templates, were dispersed in a dopamine / Tris-HCl buffer solution and reacted. The reaction product was collected by centrifugation and redispersed in a toluene / sodium dodecyl sulfonate aqueous emulsion for incubation. The incubated particles were collected and redispersed in tetrahydrofuran, followed by centrifugation to obtain polydopamine nanobottles.

3. The method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology according to claim 1, characterized in that, In step 1, the first solution is methanol; the mass ratio of Kartogenin, copper chloride, phase change material and polydopamine nanobottle is 1~10:1~50:25~100:1~5.

4. The method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology according to claim 1, characterized in that, In step 2, the first buffer solution is morpholine ethanesulfonic acid buffer; the mass ratio of sodium hyaluronate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and adipate dihydrazide is 1:2~3:1.5~2:3~4; the concentration of methyl isothiocyanate in the dimethyl sulfoxide solution containing methyl isothiocyanate is 1~3g:3~10mL, and the mass ratio of the intermediate product to the methyl isothiocyanate in the dimethyl sulfoxide solution containing methyl isothiocyanate is 1:1~3.

5. The method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology according to claim 1, characterized in that, In step 2, the grafting degree of the reduced thiourea group grafted onto hyaluronic acid is 25-40%.

6. The method for preparing an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology according to claim 1, characterized in that, In step 3, the near-infrared light has a wavelength of 808 nm and an intensity of 0.5~1 W·cm. -2 The time is 3 to 8 minutes.

7. An in-situ drug-loaded hydrogel based on nano-bottle encapsulation technology, characterized in that, It was prepared by the preparation method described in any one of claims 1 to 6; The drug-loaded polydopamine nanobottle is constructed by encapsulating copper chloride and Kartogenin with a phase change material. The diclofenac sodium is dispersed in the periphery and pores of the in-situ drug-loaded hydrogel; Under near-infrared light irradiation, copper ions are controllably released from drug-loaded polydopamine nanobottles and undergo ionic cross-linking with hyaluronic acid grafted with reduced thiourea groups to achieve in-situ gelation.

8. The in-situ drug-loaded hydrogel based on nano-bottle encapsulation technology according to claim 7, characterized in that, The concentration of the drug-loaded polydopamine nanobottle is 0.5~3 mg / mL; the concentration of the reduced thiourea group grafted hyaluronic acid is 1~5 wt%; and the concentration of the diclofenac sodium is 10~100 μg / mL.

9. The application of an in-situ drug-loaded hydrogel based on nanobottle encapsulation technology as described in claim 7 or 8 in the preparation of a medicament for treating osteoarthritis.

10. The application according to claim 9, characterized in that, The method of using the in-situ drug-loaded hydrogel is as follows: The in-situ drug-loaded hydrogel is subjected to 808 nm wavelength and 0.5~1 W·cm⁻¹ temperature daily. -2 Irradiate with near-infrared light of high intensity once, for 3-8 minutes each time.

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