ROS (reactive oxygen species) response type oil-water two-phase hydrogel as well as preparation method and application thereof

Abstract

The invention discloses ROS response type oil-water two-phase hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials.The ROS response type oil-water two-phase hydrogel is characterized in that alendronate sodium and Ce < 3 + > form a hydrogel matrix, the hydrogel matrix degrades and reduces ROS of an inflammation part under the action of ROS, the alendronate sodium and Ce < 3 + > in soluble Ce < 3 + > salt form chemical cross-linked hydrogel, and the ROS response type oil-water two-phase hydrogel is prepared. The effect between Ce < 3 + > and alendronate sodium can be destroyed by ROS, because the oil-water two-phase hydrogel prepared by the invention has ROS responsiveness, the oil-water two-phase hydrogel is degraded in a high ROS environment, such as an inflammatory site, and can further release Ce < 3 + > with an ROS removal function, alendronate sodium with pharmacological activity, anti-inflammatory essential oil and a hydrophobic drug, so that oxidative stress is relieved, osteoclast is inhibited, and the effect of the alendronate sodium is inhibited. Subchondral bone repair is promoted, synovitis is inhibited, mesenchymal stem cells are induced to be differentiated into cartilage, and the synergistic effect of multiple mechanisms of oxidation resistance, inflammation resistance and repair is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a ROS-responsive oil-water two-phase hydrogel, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is one of the most common degenerative joint diseases worldwide, posing a serious threat to human health. It is estimated that there are approximately 500 million OA patients globally, and this number is increasing annually due to population aging. The disease primarily affects weight-bearing joints such as the knee and hip, severely impacting patients' motor function and quality of life, and imposing a significant medical burden.

[0003] The pathological mechanisms of osteoarthritis are complex, involving the coordinated degeneration of multiple tissues. Its core characteristics include extracellular matrix (ECM) degradation in articular cartilage cells, synovial inflammation, subchondral bone remodeling imbalance, and osteophyte formation. Specifically, chondrocytes exhibit decreased ability to synthesize type II collagen and proteoglycans, while the activity of degradative enzymes such as matrix metalloproteinases (MMPs) significantly increases, leading to progressive destruction of the cartilage ECM. Simultaneously, synovial macrophages secrete inflammatory factors such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), further exacerbating chondrocyte apoptosis and oxidative stress (excessive ROS production), forming a vicious cycle of inflammation-degradation. Furthermore, the increased activity of subchondral osteoclasts, leading to an imbalance between bone resorption and osteogenic formation, further promotes disease progression.

[0004] Injectable hydrogels, as an emerging minimally invasive treatment platform, have shown significant potential in the field of osteoarthritis. Their advantages include: long-term sustained release of drugs into the joint cavity through in-situ injection, reducing systemic side effects; a highly viscoelastic matrix that can mimic the lubricating function of synovial fluid, reducing cartilage friction; and the ability to achieve pH- and ROS-responsive drug release through chemical modification, precisely matching the pathological characteristics of OA. For example, patent CN119055584 A discloses a ROS-responsive chitosan gel that uses liposomes to load anti-inflammatory drugs, achieving controlled drug release while clearing ROS. Patent CN116421550A discloses a pH-responsive hyaluronic acid gel that simultaneously releases lubricating components and anti-inflammatory drugs through dual chemical bond cross-linking. However, these existing technologies still have significant bottlenecks: hydrogels are difficult to degrade in vivo, and the degradation products lack clear pharmacological effects; when using existing nanocarriers (liposomes or nanoparticles) to encapsulate hydrophobic drugs, the drug loading is low and the sustained-release efficiency is insufficient. Existing hydrogels have limited functions, such as only anti-inflammatory or only lubricating, and cannot simultaneously intervene in the multiple pathological mechanisms of OA, including oxidative stress, cartilage degeneration, and synovial inflammation. Summary of the Invention

[0005] This invention provides a ROS-responsive oil-water two-phase hydrogel, its preparation method, and its application. It effectively solves the technical problems of existing injectable hydrogels, such as the lack of activity of the hydrogel matrix, low hydrophobic drug loading efficiency, uncontrollable gelation time, and poor synergistic effect of multiple mechanisms. At the same time, it provides a ROS-responsive oil-water two-phase hydrogel that combines efficient delivery of hydrophobic drugs, controllable gelation time, and multi-effect synergy.

[0006] The first objective of this invention is to provide a method for preparing a ROS-responsive oil-water two-phase hydrogel, characterized by comprising the following steps: The hydrophobic drug was dissolved in the anti-inflammatory essential oil to obtain an oil phase loaded with the hydrophobic drug.

[0007] According to alendronate sodium and Ce 3+ A molar ratio of 1:0.5 to 3 was used to mix an aqueous solution of alendronate sodium with soluble Ce. 3+ The salt solution is mixed to obtain the aqueous phase.

[0008] The oil phase was added to the aqueous phase and ultrasonically emulsified to obtain a precursor solution, which was then gelled. Sodium alendronate and Ce were then added. 3+ A gel matrix is ​​formed by chemical cross-linking, and the oil phase loaded with hydrophobic drugs is dispersed in the gel matrix in the form of droplets to obtain a ROS-responsive oil-water two-phase hydrogel.

[0009] In a preferred embodiment, the hydrophobic drug comprises 1% to 10% of the oil phase. The hydrophobic drug dissolves well in the oil phase and is released slowly, thereby promoting the differentiation of mesenchymal stem cells into chondrocytes and promoting cartilage tissue regeneration. However, if the hydrophobic drug comprises more than 10% of the oil phase, drug precipitation occurs, making it difficult to achieve proper emulsification and gel formation in the later stages.

[0010] In a preferred embodiment, the hydrophobic drug is a poorly soluble nonsteroidal anti-inflammatory drug or 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid.

[0011] In a preferred embodiment, the anti-inflammatory essential oil is cinnamon oil, clove oil, or turmeric oil.

[0012] In a preferred embodiment, the oil phase accounts for 1% to 10% of the mass of the precursor solution.

[0013] In a preferred embodiment, the gelation time is 5 min to 30 min, depending on the ratio of sodium alendronate to Ce. 3+ Different proportions, Ce 3+ The higher the mass percentage, the faster the gelation time.

[0014] As a preferred embodiment, the soluble Ce3+ The salt is cerium nitrate or cerium chloride.

[0015] In a preferred embodiment, the ultrasonic emulsification is performed at 25°C to 35°C with an ultrasonic power of 70W to 80W for 1 to 3 minutes.

[0016] The second objective of this invention is to provide a ROS-responsive oil-water two-phase hydrogel prepared by the above-described preparation method.

[0017] A third objective of this invention is to provide the application of the above-mentioned ROS-responsive oil-water two-phase hydrogel in the preparation of a drug for treating osteoarthritis.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for preparing a ROS-responsive oil-water two-phase hydrogel. First, a hydrophobic drug is dissolved in an anti-inflammatory essential oil to obtain an oil phase loaded with the hydrophobic drug. Following the reaction of alendronate sodium and Ce... 3+ A molar ratio of 1:0.5 to 3 was used to mix an aqueous solution of alendronate sodium with soluble Ce. 3+ The salt solution was mixed to obtain an aqueous phase. The oil phase was added to the aqueous phase and ultrasonically emulsified to obtain a precursor solution, which was then gelled. Sodium alendronate and Ce were added. 3+ A hydrogel matrix is ​​formed through chemical cross-linking, and an oil phase loaded with a hydrophobic drug is dispersed in the hydrogel matrix as droplets to obtain a ROS-responsive oil-water two-phase hydrogel. This invention uses sodium alendronate and Ce... 3+ It forms a hydrogel matrix that degrades under ROS, reducing ROS at inflammatory sites, alendronate sodium, and soluble Ce. 3+ Ce in salt 3+ Chemically cross-linked hydrogels are formed between Ce through complexation. 3+ The interaction between alendronate sodium and ROS is disrupted. Since the oil-water two-phase hydrogel prepared in this invention is ROS-responsive, it degrades under high ROS conditions, such as at inflammatory sites, and can further release Ce, which has ROS-scavenging capabilities. 3+ It also contains pharmacologically active alendronate sodium, anti-inflammatory essential oils, and hydrophobic drugs, thereby alleviating oxidative stress, inhibiting osteoclasts, promoting subchondral bone repair, inhibiting synovial inflammation, and inducing mesenchymal stem cells to differentiate into cartilage, achieving a synergistic effect of multiple mechanisms of antioxidation, anti-inflammation, and repair.

[0019] This invention achieves efficient loading and delivery of hydrophobic drugs. Oil-phase droplets are stably dispersed via an emulsion method, increasing the encapsulation efficiency of the hydrophobic drug by over 30% compared to traditional nanocarriers, and extending the sustained-release period to 2-3 weeks. The hydrogel preparation method provided by this invention adjusts the ratio of sodium alendronate to Ce... 3+The ratio allows for gelation time control within 5-30 minutes. The ROS-responsive oil-water two-phase hydrogel prepared using this invention exhibits an 80% increase in ROS scavenging efficiency (the hydrogel matrix itself can scavenge ROS, and the released Ce...). 3+ Further ROS clearance reduced the expression of inflammatory factors (IL-1β, TNF-α) by more than 50%, and increased the expression of cartilage-specific genes (SOX-9, Collagen II) by 2 times.

[0020] The ROS-responsive oil-water two-phase hydrogel provided by this invention has good biocompatibility. The oil phase uses natural anti-inflammatory essential oils, which have low cytotoxicity and can be metabolized through the lymphatic system, with no risk of long-term accumulation. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of the ROS-responsive oil-water two-phase hydrogel of this invention.

[0022] Figure 2 This is a comparison chart showing the solubility of the hydrophobic drug KGN used in this invention in cinnamon oil.

[0023] Figure 3 The images show the ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention, where Figure A is the hydrogel precursor solution and Figure B is a picture of the hydrogel after formation.

[0024] Figure 4 The ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention has an oil phase stained with Sudan III, mixed with the water phase, and after sonication, an observable distribution map of the oil and water phases can be obtained.

[0025] Figure 5 The figures show a comparison of the degradation behavior of the ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention in water and hydrogen peroxide solution. Figure A shows the original ROS-responsive oil-water two-phase hydrogel, Figure B shows the ROS-responsive oil-water two-phase hydrogel in hydrogen peroxide solution, and Figure C shows the ROS-responsive oil-water two-phase hydrogel in water.

[0026] Figure 6 The ROS-responsive degradation curve of the ROS-responsive oil-water hydrogel prepared in Example 1 of this invention in hydrogen peroxide solution.

[0027] Figure 7 In Comparative Example 1 of this invention, sodium alendronate and Ce... 3+ The proportions of the images show that the gel could not be formed. Image A shows the precursor solution before gelation, and image B shows the image after gelation.

[0028] Figure 8 In Comparative Example 2 of this invention, sodium alendronate and Ce... 3+The proportions of the images show that the gel could not be formed. Image A shows the precursor solution before gelation, and image B shows the image after gelation.

[0029] Figure 9 The images shown are of the preparation of the ROS-responsive blank hydrogel prepared in Comparative Example 3 of this invention. In Figure A, the precursor solution is shown before gelation, and in Figure B, the image is shown after gelation.

[0030] Figure 10 After modeling MIA, the mouse body weight changes were observed for 21 days using the ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention.

[0031] Figure 11 The images show a comparison of the degree of bone and joint swelling in MIA mice treated with the ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention. Figure A represents the Normal group, Figure B represents the Control group, and Figure C represents the CKCA group.

[0032] Figure 12 The images depict the in vivo bone repair and cartilage proliferation effects of the ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention. In the images, A represents H&E staining (A1 is the Normal group, A2 is the Control group, and A3 is the CKCA group); B represents Masson staining (B1 is the Normal group, B2 is the Control group, and B3 is the CKCA group); and C represents Safranin O staining (C1 is the Normal group, C2 is the Control group, and C3 is the CKCA group). Detailed Implementation

[0033] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention is further described below with reference to specific embodiments. However, the embodiments are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.

[0034] The background of this invention mentions the following shortcomings of existing injectable hydrogels: First, traditional hydrogels utilize natural macromolecules or artificially synthesized polymers, resulting in complex structures, cumbersome synthesis, and a lack of clear pharmacological activity, requiring the introduction of multiple drugs to achieve the corresponding therapeutic function; second, hydrogels are difficult to degrade in vivo, and the degradation products lack clear pharmacological effects; third, hydrophobic drugs require encapsulation with complex nanocarriers (liposomes or nanoparticles), resulting in low drug loading and insufficient sustained-release efficiency; fourth, the gelation time of hydrogels is fixed, making it difficult to simultaneously meet the requirements of injection fluidity and in-situ molding; fifth, single-function systems (such as anti-inflammatory or lubricating only) cannot simultaneously intervene in the multiple pathological mechanisms of OA, including oxidative stress, cartilage degeneration, and synovial inflammation. To address the above technical problems, this invention provides a ROS-responsive oil-water two-phase hydrogel, its preparation method, and its applications.

[0035] The technical solution of the present invention will be described in detail below.

[0036] This invention first provides a method for preparing a ROS-responsive oil-water two-phase hydrogel, such as... Figure 1 As shown, it includes the following steps: S1, dissolve the hydrophobic drug in the anti-inflammatory essential oil to obtain an oil phase loaded with the hydrophobic drug.

[0037] S2, according to alendronate sodium and Ce 3+ A molar ratio of 1:0.5 to 3 was used to mix an aqueous solution of alendronate sodium with soluble Ce. 3+ The salt solution is mixed to obtain the aqueous phase.

[0038] S3, the oil phase is added to the aqueous phase, and emulsified by ultrasonication to obtain a precursor solution, which is then gelled. Sodium alendronate and Ce are then added. 3+ A gel matrix is ​​formed by chemical cross-linking, and the oil phase loaded with hydrophobic drugs is dispersed in the gel matrix in the form of droplets to obtain a ROS-responsive oil-water two-phase hydrogel.

[0039] In the above technical solution, alendronate sodium and Ce 3+ It forms a hydrogel matrix that degrades under ROS, reducing ROS at inflammatory sites, alendronate sodium, and soluble Ce. 3+ Ce in salt 3+ Chemically cross-linked hydrogels are formed between Ce through complexation. 3+ The interaction between alendronate sodium and ROS is disrupted. Since the oil-water two-phase hydrogel prepared in this invention is ROS-responsive, it degrades under high ROS conditions, such as at inflammatory sites, and can further release Ce, which has ROS-scavenging capabilities. 3 +It also contains pharmacologically active alendronate sodium, anti-inflammatory essential oils, and hydrophobic drugs, thereby alleviating oxidative stress, inhibiting osteoclasts, promoting subchondral bone repair, inhibiting synovial inflammation, and inducing mesenchymal stem cells to differentiate into cartilage, achieving a synergistic effect of multiple mechanisms of antioxidation, anti-inflammation, and repair.

[0040] To ensure the effectiveness of the hydrophobic drug, the content of the hydrophobic drug in the oil phase is 1% to 10%. When the content of the hydrophobic drug is less than 1%, the dosage cannot achieve the expected effect; when the content of the hydrophobic drug is more than 10%, precipitation will occur, which is not conducive to the effectiveness.

[0041] It should be noted that the hydrophobic drug used in this invention is a poorly soluble nonsteroidal anti-inflammatory drug or 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid. The anti-inflammatory essential oil used is cinnamon oil, clove oil, or turmeric oil. The soluble Ce used in this invention... 3+ The salt is cerium nitrate or cerium chloride.

[0042] To balance the morphology and efficacy of the hydrogel, the oil phase in the precursor solution is 1% to 10%. The oil phase ratio is selected based on certain criteria. The oil phase content is positively correlated with the drug loading. If the oil phase content is too low, the hydrophobic drug content loaded in the hydrogel will be insufficient, reducing the efficacy. On the other hand, if the oil phase content is too high, the mechanical properties of the hydrogel will be reduced, or even the hydrogel may not be able to form.

[0043] In this invention, the gelation time of the precursor solution is limited to 5 min to 30 min, based on the relationship between alendronate sodium and Ce. 3+ Different proportions, Ce 3+ The higher the mass percentage, the faster the gelation time.

[0044] In order to fully mix the oil phase and the aqueous phase, the present invention specifically defines ultrasonic emulsification as follows: emulsification at 25℃~35℃ with an ultrasonic power of 70W~80W for 1min~3min, thereby obtaining a precursor solution with better homogeneity.

[0045] The technical effects of the present invention will be described below through specific embodiments and comparative examples.

[0046] Example 1 A method for preparing a ROS-responsive oil-water two-phase hydrogel includes the following steps: S1, the hydrophobic drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid, i.e. KGN, is dissolved in cinnamon oil to obtain an oil phase with a KGN content of 10%.

[0047] S2, according to the molar ratio of sodium alendronate to cerium nitrate 1:1, an aqueous solution of sodium alendronate with a concentration of 33.7 mg / mL and an aqueous solution of cerium nitrate with a concentration of 43.4 mg / mL were mixed to obtain an aqueous phase.

[0048] S3, the oil phase is added to the aqueous phase, and emulsified by ultrasonication to obtain a precursor solution with an oil phase ratio of 5%. The solution is allowed to stand at room temperature for 15 minutes to form a gel. Sodium alendronate and Ce... 3+ A gel matrix is ​​formed by chemical cross-linking, and the oil phase loaded with hydrophobic drugs is dispersed in the gel matrix in the form of droplets to obtain a ROS-responsive oil-water two-phase hydrogel.

[0049] Example 2 A method for preparing a ROS-responsive oil-water two-phase hydrogel includes the following steps: S1, the hydrophobic drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid, i.e. KGN, is dissolved in cinnamon oil to obtain an oil phase with a KGN content of 10%.

[0050] S2, according to the molar ratio of sodium alendronate to cerium nitrate 1:2, an aqueous solution of sodium alendronate with a concentration of 33.7 mg / mL and an aqueous solution of cerium nitrate with a concentration of 43.4 mg / mL were mixed to obtain an aqueous phase.

[0051] S3, the oil phase is added to the aqueous phase, and emulsified by ultrasonication to obtain a precursor solution with an oil phase ratio of 5%. The solution is allowed to stand at room temperature for 10 minutes to form a gel. Sodium alendronate and Ce... 3+ A gel matrix is ​​formed by chemical cross-linking, and the oil phase loaded with hydrophobic drugs is dispersed in the gel matrix in the form of droplets to obtain a ROS-responsive oil-water two-phase hydrogel.

[0052] Example 3 A method for preparing a ROS-responsive oil-water two-phase hydrogel includes the following steps: S1, the hydrophobic drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid, i.e. KGN, is dissolved in cinnamon oil to obtain an oil phase with a KGN content of 10%.

[0053] S2, according to the molar ratio of sodium alendronate to cerium nitrate 1:3, an aqueous solution of sodium alendronate with a concentration of 33.7 mg / mL and an aqueous solution of cerium nitrate with a concentration of 43.4 mg / mL were mixed to obtain an aqueous phase.

[0054] S3, the oil phase is added to the aqueous phase, and emulsified by ultrasonication to obtain a precursor solution with an oil phase ratio of 5%. The solution is allowed to stand at room temperature for 5 minutes to form a gel. Sodium alendronate and Ce... 3+A gel matrix is ​​formed by chemical cross-linking, and the oil phase loaded with hydrophobic drugs is dispersed in the gel matrix in the form of droplets to obtain a ROS-responsive oil-water two-phase hydrogel.

[0055] Example 4 A method for preparing a ROS-responsive oil-water two-phase hydrogel includes the following steps: S1, the hydrophobic drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid, i.e. KGN, is dissolved in cinnamon oil to obtain an oil phase with a KGN content of 10%.

[0056] S2, according to the molar ratio of sodium alendronate to cerium nitrate 1:0.5, an aqueous solution of sodium alendronate with a concentration of 33.7 mg / mL and an aqueous solution of cerium nitrate with a concentration of 43.4 mg / mL were mixed to obtain an aqueous phase.

[0057] S3, the oil phase is added to the aqueous phase, and emulsified by ultrasonication to obtain a precursor solution with an oil phase ratio of 5%. The solution is allowed to stand at room temperature for 30 minutes to form a gel. Sodium alendronate and Ce... 3+ A gel matrix is ​​formed by chemical cross-linking, and the oil phase loaded with hydrophobic drugs is dispersed in the gel matrix in the form of droplets to obtain a ROS-responsive oil-water two-phase hydrogel.

[0058] To further demonstrate the technical effects of the present invention, a comparative example is also provided, as follows: Comparative Example 1 A method for preparing a hydrogel includes the following steps: S1, the hydrophobic drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid, i.e. KGN, is dissolved in cinnamon oil to obtain an oil phase with a KGN content of 10%.

[0059] S2, according to the molar ratio of sodium alendronate to cerium nitrate 1:0.33, an aqueous solution of sodium alendronate with a concentration of 33.7 mg / mL and an aqueous solution of cerium nitrate with a concentration of 43.4 mg / mL were mixed to obtain an aqueous phase.

[0060] S3, the oil phase is added to the aqueous phase and emulsified by ultrasonication to obtain a precursor solution with an oil phase ratio of 5%. It is difficult to gel at room temperature for 3 hours, indicating that sodium alendronate and Ce... 3+ The formation of a gel matrix through chemical cross-linking requires a suitable ratio.

[0061] Comparative Example 2 A method for preparing a hydrogel includes the following steps: S1, the hydrophobic drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid, i.e. KGN, is dissolved in cinnamon oil to obtain an oil phase with a KGN content of 10%.

[0062] S2, according to the molar ratio of sodium alendronate to cerium nitrate 1:0.25, an aqueous solution of sodium alendronate with a concentration of 33.7 mg / mL and an aqueous solution of cerium nitrate with a concentration of 43.4 mg / mL were mixed to obtain an aqueous phase.

[0063] S3, the oil phase is added to the aqueous phase and emulsified by ultrasonication to obtain a precursor solution with an oil phase ratio of 5%. Gelation is difficult after standing at room temperature for 3 hours or more, indicating that sodium alendronate and Ce... 3+ The formation of a gel matrix through chemical cross-linking requires a suitable ratio.

[0064] Comparative Example 3 A method for preparing a hydrogel includes the following steps: A sodium alendronate aqueous solution with a concentration of 33.7 mg / mL and a cerium nitrate aqueous solution with a concentration of 43.4 mg / mL were mixed according to a 1:1 molar ratio of sodium alendronate to cerium nitrate. After standing, a blank hydrogel without an oil phase was obtained.

[0065] The performance of the ROS-responsive oil-water two-phase hydrogel prepared in the embodiments of the present invention and the hydrogel prepared in the comparative example were tested, and the specific results are as follows.

[0066] Figure 2 This is a comparison chart showing the solubility of the hydrophobic drug KGN used in this invention in cinnamon oil. Figure 2 It can be seen that when the hydrophobic drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid was dissolved in cinnamon oil at mass percentages of 1%, 3%, 5%, 10%, 15%, 20%, and 30%, respectively, it was found that KGN exceeding 10% was poorly soluble in the oil phase. Therefore, 10% KGN was selected as the content of the hydrophobic drug in the oil phase.

[0067] Figure 3 This is a diagram of the ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention. Figure 3 It is known that dispersing cinnamon oil containing KGN in sodium alendronate and soluble Ce... 3+ Ce in salt 3+ In the chemically cross-linked hydrogel, after ultrasonic emulsification, microscopic observation revealed that cinnamon oil containing KGN was relatively uniformly dispersed within the hydrogel. Figure 3 As can be seen from B, the hydrogel forms a complete solid, adheres to the bottom of the bottle, and will not fall off.

[0068] Figure 4 The ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention has an oil phase stained with Sudan III, mixed with the water phase, and after sonication, an observable distribution map of the oil and water phases can be obtained.

[0069] Figure 5This is a comparison of the degradation behavior of the ROS-responsive oil-water two-phase hydrogel prepared in Example 1 of this invention in water and hydrogen peroxide solution. Figure 5 It can be seen that when 500 mg of the hydrogel from Example 1 was added to a 400 µM H₂O₂ aqueous solution, the hydrogel gradually changed from milky white to yellow over time, and was eventually completely degraded. Simultaneously, the H₂O₂ was oxidized, releasing O₂. This is because ROS disrupts the chemical cross-linking of the hydrogel, and Ce... 3+ Released and reduced to Ce 4+ This demonstrates that the hydrogel prepared by the present invention is a ROS-responsive hydrogel.

[0070] Figure 6 The ROS-responsive degradation curve of the ROS-responsive oil-water hydrogel prepared in Example 1 of this invention is shown in hydrogen peroxide solution. 900 mg of the hydrogel from Example 1 was placed in a centrifuge tube and continuously immersed in a 400 µM H2O2 aqueous solution. The gel was placed in a constant temperature shaker (37°C, 100 rpm) and removed at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. The surface of the hydrogel was wiped with filter paper to remove the H2O2 aqueous solution, and the wet weight of the hydrogel was measured. The residual mass ratio of the hydrogel was calculated. Figure 6 It can be seen that the hydrogel gradually degrades over time, proving its ROS responsiveness.

[0071] Figure 7 In Comparative Example 1 of this invention, sodium alendronate and Ce... 3+ The proportions of the image prevent it from forming a gel. Figure 8 In Comparative Example 2 of this invention, sodium alendronate and Ce... 3+ The proportions of the image prevent it from forming a gel. Figure 9 This is a diagram showing the preparation of the ROS-responsive blank hydrogel prepared in Comparative Example 3 of this invention. Figures 7-9 It can be seen that Comparative Examples 1 and 2 of the present invention could not form hydrogels. Comparative Example 3, due to the absence of an oil phase component, formed a colorless and transparent hydrogel. Figure 9 As can be seen from B, it formed a blank hydrogel without drug loading, and the solid morphology remained unchanged when the glass bottle was tilted.

[0072] Figure 10 To conduct animal experiments on the ROS-responsive oil-water hydrogel prepared in Example 1 of this invention, we constructed an osteoarthritis MIA mouse model to evaluate osteoarthritis treatment in vivo and monitored changes in mouse body weight over 21 days. Figure 11 Animal experiments were conducted on the ROS-responsive oil-water hydrogel prepared in Example 1 of this invention, and the degree of bone and joint swelling in mice with different components was compared.

[0073] Figure 12Animal experiments were conducted using the ROS-responsive oil-water hydrogel prepared in Example 1 of this invention. Mice were photographed and euthanized on day 21, and the affected areas were sectioned. Further analysis of the histological changes in cartilage was performed using H&E, Masson, and Safranin O staining. In the Normal group, the articular cartilage structure was intact and smooth, with uniform chondrocyte distribution and uniform and clear staining of the cartilage matrix, exhibiting normal cartilage tissue characteristics. In the Control group, the articular cartilage showed severe damage, reduced chondrocytes, and significantly thinner cartilage layer, displaying typical OA pathological features. Compared to the Control group, the CKCA group showed a more uniform cell distribution, no significant cartilage matrix degradation, and significantly better staining intensity of the extracellular matrix of chondrocytes than the Control group. Therefore, the prepared ROS-responsive oil-water hydrogel can effectively protect cartilage structure, reduce cartilage matrix degradation, promote cartilage tissue proliferation, and maintain chondrocyte homeostasis, thus providing better treatment in osteoarthritis.

[0074] In vivo osteoarthritis repair assessment This invention uses MIA mice to conduct in vivo osteoarthritis repair research, and the specific testing methods are as follows: Mice were randomly divided into three groups of four each: Normal, Control, and CKCA.

[0075] Mice were anesthetized with gas. After routine disinfection, the right knee joint was bent to 90° to fully open the joint cavity. A 1mL syringe was inserted from the side; a feeling of air drop indicated the needle had entered the joint cavity. After aspiration without blood, the Normal group was injected with 15µL of sterile saline. The other two groups were injected with sodium iodoacetate (2mg / mL). After needle withdrawal, hemostasis was achieved by squeezing with a sterile cotton ball to establish the OA model. Mice were housed in cages for one week after disinfection, ensuring free access to food and movement, and the degree of joint swelling was observed. One week after MIA injection, a three-week treatment period was initiated. Mice were anesthetized with gas, and intra-articular administration was performed as described previously. The Normal and Control groups were injected with 15µL of sterile saline, while the CKCA group received weekly injections of 15µL of ROS-responsive oil-water two-phase hydrogel for OA treatment. After injection, the needle was withdrawn and disinfected with an alcohol swab. The degree of knee joint swelling and the mice's behavior were observed during treatment.

[0076] After treatment, the mice were euthanized, and decalcified sections of the affected areas were stained. Differences between groups were analyzed, and the results were examined. It was observed that the swelling in the affected areas of the mice gradually decreased after treatment until it returned to normal. Analysis of the stained sections revealed severe damage to the articular cartilage of the mice after modeling, with a reduction in chondrocytes and a significantly thinner cartilage layer, exhibiting typical pathological features of osteoarthritis (OA). After treatment, the CKCA group showed a more uniform cell distribution, no significant degradation of the cartilage matrix, and significantly higher staining intensity of the extracellular matrix of the chondrocytes compared to the Control group.

[0077] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a ROS-responsive oil-water two-phase hydrogel, characterized in that, Includes the following steps: Hydrophobic drugs were dissolved in anti-inflammatory essential oils to obtain an oil phase loaded with hydrophobic drugs; According to alendronate sodium and Ce 3+ A molar ratio of 1:0.5 to 3 was used to mix an aqueous solution of alendronate sodium with soluble Ce. 3+ The salt solution was mixed to obtain the aqueous phase; The oil phase was added to the aqueous phase and ultrasonically emulsified to obtain a precursor solution, which was then gelled. Sodium alendronate and Ce were then added. 3+ A gel matrix is ​​formed by chemical cross-linking, and the oil phase loaded with hydrophobic drugs is dispersed in the gel matrix in the form of droplets to obtain a ROS-responsive oil-water two-phase hydrogel.

2. The method for preparing ROS-responsive oil-water two-phase hydrogel according to claim 1, characterized in that, The hydrophobic drug is present in the oil phase loaded with the hydrophobic drug at a mass ratio of 1% to 10%.

3. The method for preparing ROS-responsive oil-water two-phase hydrogel according to claim 1, characterized in that, The hydrophobic drug is a poorly soluble nonsteroidal anti-inflammatory drug or 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid.

4. The method for preparing ROS-responsive oil-water two-phase hydrogel according to claim 1, characterized in that, The anti-inflammatory essential oil is cinnamon oil, clove oil, or turmeric oil.

5. The method for preparing ROS-responsive oil-water two-phase hydrogel according to claim 1, characterized in that, The oil phase in the precursor solution accounts for 1% to 10% of the total mass.

6. The method for preparing ROS-responsive oil-water two-phase hydrogel according to claim 1, characterized in that, The gelation time is 5 min to 30 min.

7. The method for preparing ROS-responsive oil-water two-phase hydrogel according to claim 1, characterized in that, The soluble Ce 3+ The salt is cerium nitrate or cerium chloride.

8. The method for preparing ROS-responsive oil-water two-phase hydrogel according to claim 1, characterized in that, The ultrasonic emulsification is performed at 25℃~35℃ with an ultrasonic power of 70W~80W for 1min~3min.

9. A ROS-responsive oil-water two-phase hydrogel, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the ROS-responsive oil-water two-phase hydrogel of claim 9 in the preparation of a drug for treating osteoarthritis.

Images