ROS-responsive macrophage-chondrocyte dual-targeting nano-micelle as well as preparation method and application of ROS-responsive macrophage-chondrocyte dual-targeting nano-micelle

Abstract

The invention relates to an ROS-responsive macrophage-chondrocyte dual-targeting nano-micelle and a preparation method and application thereof, HA capable of targeting and highly expressing CD44 cells is taken as a carrier skeleton, chemical coupling with TK and CUR is carried out in sequence, self-assembly is carried out through intermolecular force to form a stable nano-micelle, and drugs JPH203 and KGN are entrapped at the same time, so that efficient loading and targeting delivery are realized. The HA can be specifically combined with RAW264.7 macrophages and articular cartilage cells for highly expressing a CD44 receptor in an inflammatory state, and is actively delivered in a targeting manner. The ROS response characteristic of TK is utilized, oxidative fracture is carried out in an OA high ROS microenvironment, and micelles are triggered to disintegrate and release drugs accurately. The released JPH203 can inhibit an inflammatory mTOR pathway and effectively resist inflammation, and KGN is beneficial to promoting cartilage cell proliferation and effectively collecting and promoting differentiation of mesenchymal stem cells into cartilage cells, so that in-situ cartilage regeneration is realized, and a more efficient solution with low side effects is provided for osteoarthritis treatment.

Description

Technical Field

[0001] This invention belongs to the technical field of precision-targeted arthritis treatment drug preparation, and specifically relates to a method for preparing and applying ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles. Background Technology

[0002] Osteoarthritis (OA), a chronic degenerative joint disease characterized by progressive degeneration of articular cartilage, synovial inflammation, and subchondral bone remodeling, is currently treated primarily with the goal of symptom relief and slowing disease progression. Common treatments include oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injection of hyaluronic acid (HA), physical therapy, and end-stage joint replacement surgery. However, existing treatment options have significant limitations: while NSAIDs can quickly relieve pain, long-term use can easily lead to gastrointestinal damage, cardiovascular risks, and nephrotoxicity, and they cannot improve the degenerative process of cartilage; intra-articular injection of exogenous HA can temporarily replenish the viscoelasticity of synovial fluid, but its short biological half-life, insufficient retention time in the joint cavity, and significant individual differences in efficacy make it difficult to achieve long-term cartilage protection; physical therapy can only help improve joint function and has no repair effect on damaged cartilage; and while joint replacement surgery is an effective option for end-stage OA, it is highly invasive, has a high revision rate, and is not suitable for young or highly active patients. Therefore, developing novel treatment strategies that combine long-acting targeting properties, anti-inflammatory enhancement, and cartilage repair is a key scientific issue in overcoming the bottleneck in OA treatment.

[0003] Nanomedicine delivery systems have demonstrated unique advantages in precision treatment of osteoarthritis (OA) due to their high drug loading efficiency, targeted enrichment capabilities, and sustained-release properties. For example, nanocarriers constructed using biocompatible carrier materials (such as biodegradable polylactic-co-glycolic acid copolymers, liposomes, and chitosan) can be loaded with functional drugs (such as small molecule chemical drugs, gene drugs, and protein / peptide drugs) possessing anti-inflammatory, antioxidant, or proliferative activities. This can significantly increase the local drug concentration in the affected joint and reduce systemic toxicity. However, current nanodelivery systems still face bottlenecks such as insufficient targeting (e.g., low targeted enrichment efficiency in affected joints and systemic toxicity caused by non-specific drug distribution), uncontrollable drug release (e.g., burst release or premature degradation), and carrier biosafety, which limit their clinical translation.

[0004] In summary, given the high incidence, pathological complexity, and limitations of existing treatments for osteoarthritis (OA), designing and optimizing a multifunctional nanomedicine delivery system that precisely targets articular cartilage, provides controllable drug release, and exhibits functional synergistic effects, based on the pathological microenvironmental characteristics of OA-affected joints (such as high expression of inflammatory factors, accumulation of reactive oxygen species, and metabolic imbalance of chondrocytes), has significant theoretical value and clinical translational prospects for improving the effectiveness and safety of OA treatment and promoting the transformation of OA treatment from "symptom relief" to "cartilage repair." Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a ROS-responsive macrophage-chondrocyte dual-targeting nanomicelle, its preparation method, and its application. This method provides mild and easy-to-operate preparation conditions, and the prepared ROS-responsive macrophage-chondrocyte dual-targeting nanomicelle exhibits good dispersibility and colloidal stability. The raw materials used are environmentally friendly, the preparation process is simple, and it is feasible for industrialization.

[0006] A ROS-responsive macrophage-chondrocyte dual-targeting nanomicelle uses a ROS-responsive dual-targeting shell as a carrier. The carrier is constructed by coupling hyaluronic acid (HA) with curcumin (CUR) via ROS-responsive unit TK. It is used to encapsulate JPH203 and KGN drugs and forms a nanomicelle delivery system that combines targeted delivery to chondrocytes and macrophages, ROS-responsive drug release, and osteoarthritis treatment through self-assembly.

[0007] Preferably, the dual targeting of the present invention is a hyaluronic acid-modified targeting molecule.

[0008] A method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles includes the following steps: Step S1: Synthesize HA-TK-CUR. First, dissolve the ROS-responsive unit TK, EDC·HCl, and NHS in water and stir at room temperature for 2 h. Then, slowly add a dimethyl sulfoxide solution of curcumin CUR to the above solution and continue stirring at room temperature for 1 d. Put the solution into a dialysis bag and dialyze for 3 d. Take out the liquid from the dialysis bag and freeze-dry it to obtain a yellow solid powder intermediate TK-CUR. Step S2: Dissolve the TK-CUR intermediate solid with dimethyl sulfoxide, add EDC·HCl and NHS to the solution, stir at room temperature for 2 h, then slowly add an aqueous solution of HA to the above solution, continue stirring at room temperature for 1 d, dialyze the solution for 3 d, take out the liquid from the dialysis bag and freeze dry to obtain yellow solid powder HA-TK-CUR; Step S3: Dissolve the obtained HA-TK-CUR in a mixed solution of tetrahydrofuran and water at a ratio of 1:9; dissolve KGN and JPH203 in tetrahydrofuran; Step S4: Add the KGN and JPH203 mixed solution from the above steps to the solution containing HA-TK-CUR under ultrasonic conditions, and sonicate for 30 min; Step S5: Use a rotary evaporator to fully evaporate the tetrahydrofuran; Step S6: Filter using a PES membrane filtration device; load the filtered solution into an ultrafiltration tube for dialyzing to obtain ROS-responsive nanomicelles.

[0009] Preferably, in step S1 of the present invention, CUR is 36.8 mg, 0.1 mmol; EDC·HCl is 19.2 mg, 0.1 mmol; NHS is 11.6 mg, 0.1 mmol; TK is 22.5 mg, 0.1 mmol; and the molar ratio of HA:EDC·HCl:NHS:TK dissolved in water is 1:5:5:5.

[0010] Preferably, in step S2 of the present invention, HA is 30 mg, 0.02 mmol; EDC·HCl is 19.2 mg, 0.1 mmol; NHS is 11.6 mg, 0.1 mmol; the molar ratio of HA-TK:EDC·HCl:NHS:CUR dissolved in a mixed solution of water and dimethyl sulfoxide is 1:1:1:1; the molecular weight cutoff of the dialysis bag is 3500D; and the molecular weight cutoff of the ultrafiltration tube is 50000D.

[0011] Preferably, in step S3 of the present invention, HA-TK-CUR is dissolved in 9 mL of ultrapure water, KGN and JPH203 are dissolved in tetrahydrofuran, and the mass ratio of HA-TK:JPH203:KGN is 10:0.5:0.5; the aqueous solution of tetrahydrofuran has a tetrahydrofuran to ultrapure water ratio of 1:9 and a total volume of 10 mL.

[0012] Preferably, the rotary evaporation time in step S5 of the present invention is 2-3 h; the filter membrane pore size of the PES membrane filtration device used is 0.22 μm; Preferably, the conditions for membrane dialysis in step S6 of the present invention are as follows: the molecular weight cutoff of the dialysis bag used is 3500 D; the dialysis time is 72 h; and the stirring conditions are: stirring at room temperature for 24 h.

[0013] Preferably, the ultrafiltration tube used in step S6 of the present invention has a rejection capacity of 50,000 D; and the centrifuge parameters are: 2000 rpm, 4 ℃, 1 h.

[0014] The application of a ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles in the combined treatment of arthritis using metabolic regulation therapy and immunomodulation therapy.

[0015] The application of the ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles of the present invention in the treatment of arthritis includes the precise delivery of nanoimmunotherapy drugs to the inflamed area by targeting macrophages-chondrocytes and the treatment of osteoarthritis.

[0016] Compared with existing technologies, the ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles and their preparation method of the present invention are as follows: 1. The preparation method of this invention is simple, the main materials are easy to obtain, and the biosafety is good; 2. The nano-immunotherapy prepared by this invention can precisely target the inflamed area and effectively treat osteoarthritis; 3. This invention uses novel materials and enhances the combined therapeutic effect through cascade reactions, thereby maximizing the efficacy of the drug. 4. The drug encapsulated in this invention has a controlled-release effect, which can improve the safety, efficacy and utilization rate of the drug. Attached Figure Description

[0017] Figure 1 This is a flowchart of a ROS-responsive macrophage-chondrocyte dual-targeting nanomicelle prepared according to the present invention.

[0018] Figure 2 The HC and HC prepared by this invention J HC K and HC JK The spectral spectrum.

[0019] Figure 3 The HC and HC prepared by this invention J HC K and HC JK Hydrated particle size and morphology diagram.

[0020] Figure 4 The HC and HC prepared by this invention J HC K and HC JK Surface potential diagram.

[0021] Figure 5 This refers to the percentage of CUR / JPH203 / KGN released by nanomicelles at different times within 24 hours under simulated inflammatory conditions with high ROS content, as determined by high performance liquid chromatography (HPLC).

[0022] Figure 6 RAW264.7 cells tested by the CCK-8 assay were subjected to PBS buffer (control) and HC and HC prepared according to this invention. J HC K and HC JKA schematic diagram showing the comparison of cell viability after 24 hours of treatment.

[0023] Figure 7 The fluorescence intensity in HC and RAW264.7 cells prepared in this invention was measured by flow cytometry after being co-incubated with different concentrations of free HA for 12 h.

[0024] Figure 8 These are fluorescence images of chondrocytes in an inflammatory state, captured by an inverted fluorescence microscope. After co-incubation with different concentrations of free HA, and then with PBS and HC prepared in this invention for 12 h, the chondrocytes were incubated with CUR.

[0025] Figure 9 The results were obtained by flow cytometry analysis of RAW264.7 cells induced by LPS, PBS, and HC and HC prepared in this invention. J HC K and HC JK The percentage content of M1 and M2 states in RAW264.7 cells after co-incubation for 12 h.

[0026] Figure 10 RAW264.7 cells captured by an inverted fluorescence microscope were induced with LPS and then reacted with PBS and HC and HC prepared in this invention. J HC K and HC JK Fluorescence images of ROS levels after 12 h of co-incubation.

[0027] Figure 11 These are images of chondrocytes in an inflammatory state, captured by an inverted fluorescence microscope, with PBS and HC and HC prepared according to this invention. J HC K and HC JK Fluorescent images of cell proliferation levels labeled with Calcein / PI dye after 12 h of co-incubation.

[0028] Figure 12 This is a graph showing the weight changes of an arthritis model mouse within 30 days after treatment. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] like Figure 1 As shown, a method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles includes the following steps: To synthesize HA-TK-CUR, TK, EDC·HCl, and NHS were first dissolved in water and stirred at room temperature for 2 h. Then, a dimethyl sulfoxide solution of CUR was slowly added to the solution, and stirring continued at room temperature for 1 day. The solution was then placed in a dialysis bag and dialyzed for 3 days. The liquid in the dialysis bag was then removed and freeze-dried to obtain a yellow solid powder intermediate, TK-CUR. The HA-TK intermediate solid was dissolved in dimethyl sulfoxide, and EDC·HCl and NHS were added to the solution. The mixture was stirred at room temperature for 2 h. Subsequently, an aqueous solution of HA was slowly added to the solution, and stirring continued at room temperature for 1 day. The solution was dialyzed for 3 days, and the liquid in the dialysis bag was then removed and freeze-dried to obtain a yellow solid powder, HA-TK-CUR. The molar ratio of HA:EDC·HCl:NHS:TK was 1:5:5:5; while the molar ratio of HA-TK:EDC·HCl:NHS:CUR was 1:1:1:1; the mass ratio of HA-TK-CUR:JPH203:KGN was 10:0.5:0.5; the volume of tetrahydrofuran was 1 mL; the ratio of tetrahydrofuran to ultrapure water in the tetrahydrofuran aqueous solution was 1:9, and the total volume was 10 mL; the sonication time was 30 min; the rotary evaporator settings were: 40 ℃, 70 rpm, 1 h; the molecular cutoff of the PES membrane filtration device was 0.22 μm; the molecular cutoff of the dialysis bag was 3500 D; and the dialysis process parameters were: room temperature, 36 h.

[0031] The JPH203 and KGN obtained in the above steps were dissolved in tetrahydrofuran; HA-TK-CUR was dissolved in a mixed solution of ultrapure water and tetrahydrofuran at a ratio of 9:1. The above solution was added to the aqueous solution of tetrahydrofuran under ultrasonic conditions and sonicated; the above solution was placed in a rotary evaporator, and after the tetrahydrofuran had fully evaporated, it was filtered using a PES membrane filtration device; the filtered solution was placed in a dialysis bag for dialyzing to obtain the final material JPH203 / KGN@HA-TK-CUR.

[0032] The process parameters for the stirring reaction are: room temperature, 24 h; the molecular weight cutoff of the ultrafiltration tube is 50,000 D; the process parameters in the ultrafiltration instruction manual are: 4℃, 2000 rpm, 1 h; repeated 4-6 times.

[0033] This invention uses ultraviolet-visible absorption spectroscopy (UV-Vis), fluorescence spectroscopy, transmission electron microscopy (TEM), zeta potential, and dynamic light scattering analysis (DLS) to characterize the prepared nano-immunotherapy drugs (HC, HC). J HC K and HC JKThe chemokinetic properties of a ROS-responsive macrophage-chondrocyte dual-targeting nanomicelle were determined by high-performance liquid chromatography (HPLC). Cell viability analysis (CCK-8 assay) was then used to evaluate the cytotoxicity and cell-killing effects of the ROS-responsive macrophage-chondrocyte dual-targeting nanomicelle. Flow cytometry was used to monitor the targeting effect of the nanomedicine on RAW264.7 cells in an inflammatory state. Inverted fluorescence microscopy was used to observe and verify the antioxidant effect of the nanoimmunotherapy in cells and its targeting and proliferative effects on chondrocytes in an inflammatory state. The viability and inflammatory status of mice were assessed using body weight change curves.

[0034] Example 1: A method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles, comprising the following steps: TK (22.5 mg, 0.1 mmol), EDC·HCl (19.2 mg, 0.1 mmol), and NHS (11.6 mg, 0.1 mmol) were dissolved in water and stirred at room temperature for 2 h. Then, a dimethyl sulfoxide solution of CUR (30 mg, 0.02 mmol) was slowly added to the above solution, and stirring was continued at room temperature for 3 days. The remaining solution was dialyzed (molecular weight cutoff of 3500 D) for 3 days, then lyophilized to remove water, yielding a yellow solid powder TK-CUR. The HA-TK intermediate solid was dissolved in dimethyl sulfoxide, and EDC·HCl (19.2 mg, 0.1 mmol) and NHS (11.6 mg, 0.1 mmol) were added to the solution. The mixture was stirred at room temperature for 2 h, and then a dimethyl sulfoxide solution of CUR (36.8 mg, 0.1 mmol) was slowly added to the above solution. The mixture was stirred at room temperature for 1 day, and the solution was dialyzed for 3 days. The liquid in the dialysis bag was then removed and lyophilized to obtain a yellow solid powder HA-TK-CUR. The JPH203 and KGN obtained in the above steps were dissolved in tetrahydrofuran, and HA-TK-CUR was dissolved in a mixed solution of ultrapure water and tetrahydrofuran at a ratio of 9:1. This solution was then added to the tetrahydrofuran aqueous solution under ultrasonic conditions and sonicated. The solution was placed in a rotary evaporator, and after the tetrahydrofuran had fully evaporated, it was filtered using a PE membrane filter. The filtered solution was then dialyzed in a dialysis bag to obtain the final material JPH203 / KGN@HA-TK-CUR (HC, HC J and HC K The synthesis steps are similar to those described above. Test Result 1: See Appendix Figure 2 The test results of ultraviolet-visible absorption spectroscopy (UV-Vis) and fluorescence absorption spectroscopy were compared with those of HC and HC prepared in this invention. JHC K and HC JK The ultraviolet-visible absorption spectrum, Figure 2 The left side of the middle section shows HC and HC prepared according to this invention. J HC K and HC JK The ultraviolet-visible spectrum, Figure 2 The right side of the middle section shows HC and HC prepared according to this invention. J HC K and HC JK The fluorescence emission spectrum of HC and HC prepared in this invention was observed. J HC K and HC JK A similar absorption peak was observed at 280 nm, which is a characteristic peak of CUR, indicating that the delivery carrier successfully externalized CUR and had no significant impact on the UV-Vis absorption of CUR. This was compared with the HC and HC prepared in this invention. J HC K and HC JK The fluorescence absorption spectra of HC and HC prepared in this invention were observed. J HC K and HC JK A similar absorption peak was observed at 500 nm, which is a characteristic peak of CUR, indicating that the delivery carrier has no significant effect on the fluorescence absorption of CUR.

[0035] Test Result 2: See Appendix Figure 3 Based on the hydrodynamic particle size and transmission electron microscopy (TEM) test results, the HC and HC prepared in this invention... J HC K and HC JK The hydrated particle size and morphology were observed. Figure 3 In this invention, 'a' refers to HC and HC prepared by this invention. J HC K and HC JK Transmission electron microscope images, Figure 3 b is the HC and HC prepared in this invention. J HC K and HC JK Hydrated particle size distribution. Hydrodynamic particle size analysis results show that the HC and HC prepared in this invention... J HC K and HC JK The hydrated particle sizes were 201.5 nm, 202.6 nm, 201.8 nm, and 201.7 nm, respectively, with no significant difference. The HC and HC prepared in this invention... J HC K and HC JK TEM images show the HC and HC prepared in this invention.J HC K and HC JK It appears as uniformly dispersed spherical nanoparticles.

[0036] Test Result 3: See Appendix Figure 4 The HC and HC prepared by this invention are shown. J HC K and HC JK The surface potential map was used to determine HC and HC using dynamic light scattering (DLS). J HC K and HC JK The surface potentials were -20.77 mV, -18.01 mV, -20.95 mV, and -20.81 mV, respectively, with no significant difference.

[0037] Test Result 4: See Appendix Figure 5 The chemical kinetic performance test results were obtained, and the HC and HC prepared in this invention were detected by high performance liquid chromatography (HPLC). J HC K and HC JK Release efficiency of CUR / JPH2O3 / KGN in a simulated inflammatory environment with high ROS content within 24 h. 50 μg / mL HC and HC were prepared in ultrapure water. J HC K and HC JK H₂O₂ was added to the solution to achieve a concentration of 10 μmol / L, creating a high-ROS inflammatory environment. The HC and HC₂ prepared according to this invention were detected by high-performance liquid chromatography (HPLC). J HC K and HC JK Chemical kinetic properties. For example... Figure 8 As shown, the percentage of each drug released continuously increased at 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h and 24 h in the H2O2 environment. The results indicate that the longer the ROS stimulation time, the more fully CUR / JPH2O3 / KGN is released, indicating that the nanomaterial has excellent controllable release effect. Figure 5 This study demonstrates the percentage release of CUR / JPH203 / KGN from nanomicelles at different times within 24 h under simulated inflammatory conditions with high ROS content, as detected by high performance liquid chromatography (HPLC).

[0038] Test Result 5: See Appendix Figure 6 The results of the CCK-8 cell viability test were used to evaluate the HC and HC prepared in this invention, using RAW264.7 cells as a model cell. J HC K and HCJK The effects of four types of nanoparticles on cell proliferation. Figure 6 The presentation showcases RAW264.7 cells tested using the CCK-8 assay after passing through PBS buffer (control) and HC and HC prepared according to this invention. J HC K and HC JK Schematic diagram comparing cell viability after 24 h of treatment. Prepared with sterile PBS. Different concentrations of HC and HC from different manufacturer's instructions (7%). J HC K and HC JK Nanoparticle solution, sterilized by overnight UV irradiation. Prepare HC, HC... J HC K and HC JK Nanoparticle solutions (CUR concentrations of 0, 1, 2, 5, 10, 20, 50, and 100 μg / mL) were prepared in 5 replicates per group, with a control group included. PBS buffer was added to the outermost wells to minimize the impact of evaporation. Cells were then cultured at 37 °C in a 5% CO2 incubator for 24 h. After culture, the nanoparticles were aspirated, and the cells were gently rinsed twice with PBS. A culture medium containing 10% CCK-8 solution was added to the wells, avoiding air bubbles during addition. The plates were then incubated at 37 °C in a 5% CO2 incubator for another 2–4 h. The 96-well plates were then removed, and the OD value at 450 nm was measured using a microplate reader. The effect of different concentrations of the material on cell proliferation was compared with PBS buffer as a control, and cell viability was calculated based on these values. Figure 6 (As shown). Compared with the PBS control group, HC, HC J HC K and HC JK There was no significant difference in the effect of PFODBT concentration from 0 to 100 μg / mL on the survival rate of RAW264.7 cells; cell survival rates remained above 80%. This fully demonstrates that the synthetic HC and HC... J HC K and HC JK It has good biocompatibility and can be used for in vivo therapy.

[0039] Test Result 6: See Appendix Figure 7 The fluorescence of nanomedicine contained in RAW264.7 cells was detected by flow cytometry, thereby evaluating the targeting effect of nanoimmunotherapy on RAW264.7 cells in the M1 state. Figure 7The fluorescence intensity in RAW264.7 cells prepared according to this invention was measured by flow cytometry after co-incubation for 12 h with HC prepared according to this invention and RAW264.7 cells with different concentrations of free HA. First, RAW264.7 cells were seeded in 6-well plates and cultured to 70% confluence. LPS was added to induce M1 state, and the cells were incubated for 12 h. Then, free HA (concentration gradient: 0, 10, 20, 50, 100, 200 ppm) was added, and the cells were incubated at 37°C for 1 h (allowing free HA to occupy the CD44 receptor first). The supernatant was then discarded. Next, the LPS-induced RAW264.7 cells were co-incubated with PBS and HC prepared according to this invention for 12 h, and then cultured in a 37°C, 5% CO2 cell culture incubator for 24 h. After culturing, the nanomedicine was aspirated, gently washed once with PBS, centrifuged, the supernatant was discarded, PBS was added and mixed, centrifuged again, the supernatant was discarded, and PBS was added and mixed again. The resulting cell suspension was loaded into flow cytometry tubes, and the fluorescence in RAW264.7 cells was detected by flow cytometry to evaluate the targeting effect of the nanoimmunotherapy on M1-state RAW264.7 cells. As the concentration of free HA decreased, the fluorescence intensity in RAW264.7 cells increased accordingly, indicating that the synthesized HA... JK By competing with free HA for binding to the CD44 receptor on RAW264.7 cells, the targeting effect of HC on M1 state RAW264.7 cells was fully verified.

[0040] Test Result 7: See Appendix Figure 8 To evaluate the targeting effect of HC nanomicelles on chondrocytes under inflammatory conditions. Figure 8These are fluorescence images of chondrocytes in an inflammatory state, captured by an inverted fluorescence microscope. After co-incubation with different concentrations of free HA, and then with PBS and HC prepared according to this invention for 12 h, the fluorescence of the nanomedicine within the chondrocytes was observed using an inverted fluorescence microscope. By detecting changes in the fluorescence intensity of the nanomedicine within the cells after treatment with different concentrations of free HA, the competitive effect of free HA and nanomedicine on the CD44 receptor of inflammatory cells was analyzed, thereby evaluating the targeting effect of the aforementioned nanomicelles. First, chondrocytes were seeded in 6-well plates and cultured to 70% confluence. LPS was added to induce an inflammatory state, and the cells were incubated for 12 h. Then, free HA (concentration gradient: 0, 10, 20, 50, 100, 200 ppm) was added, and the cells were incubated at 37°C for 1 h (allowing free HA to first occupy the CD44 receptor). Next, the TNF-α-induced chondrocytes were co-incubated with PBS and HC prepared according to this invention for 12 h and then cultured in a 37°C, 5% CO2 cell culture incubator for 24 h. Cells were then collected, and the fluorescence intensity of the nanomedicine in chondrocytes was captured using an inverted fluorescence microscope. As the concentration of free HA decreased, the fluorescence intensity in chondrocytes increased accordingly. This indicates that the synthesized HC competes with free HA for binding to the CD44 receptor on chondrocytes, fully verifying the targeting effect of HC on chondrocytes under inflammatory conditions and demonstrating the dual-targeting effect of the nanomedicine.

[0041] Test Result 8: Reference Appendix Figure 9 The percentage of M1 and M2 states in RAW264.7 cells was detected by flow cytometry to evaluate the polarization-promoting effect of nanoimmunotherapy on RAW264.7 cells. Figure 9 The results were obtained by flow cytometry analysis of RAW264.7 cells induced by LPS, PBS, and HC and HC prepared in this invention. J HC K and HC JK The percentage of M1 and M2 states in RAW264.7 cells after 12 h of co-incubation. HC at a concentration of 50 μg / mL. J HC K and HC JK The nanoparticle solution was sterilized by overnight UV irradiation. Then, LPS-induced RAW264.7 cells were mixed with PBS and the HC and HC solutions prepared in this invention. J HC K and HC JKCells were co-incubated for 12 h and then cultured for 24 h in a 37 ℃, 5% CO2 cell culture incubator. Twelve h after the end of treatment, cells were collected and washed with PBS. A culture medium containing 0.1% CD86 and 0.1% CD206 was added to wells of a plate to label M1 and M2 cells. Cells were then collected, washed with PBS, and placed in flow cytometry tubes. The percentage of M1 and M2 states in RAW264.7 cells was detected using flow cytometry. Figure 8 It can be seen from this that HC J and HC JK Group compared to HC, HC K The proportion of M1 cells in the treatment group was significantly lower than that in the PBS group, while the proportion of M2 cells was significantly higher in the treatment group; this indicates that the treatment group can promote the polarization of RAW264.7 cells from M1 to M2, while HC J and HC JK The presence of JPH203 in the sample makes the effect of promoting the polarization of M1 to M2 more prominent.

[0042] Test Result 9: Reference Appendix Figure 10 Evaluation of nanomicelles (HC, HC J HC K and HC JK The results of the detection of the antioxidant effect of ) Figure 10 RAW264.7 cells captured by an inverted fluorescence microscope were induced with LPS and then reacted with PBS and HC and HC prepared in this invention. J HC K and HC JK Fluorescent images of ROS levels after 12 h of co-incubation. This experiment used an inverted fluorescence microscope to observe the levels of reactive oxygen species (ROS) labeled with the DCFH-DA fluorescent probe in RAW264.7 macrophages. Figure 9 The antioxidant activity of these nanomicelles was evaluated by analyzing the changes in intracellular ROS fluorescence intensity after treatment with different nanomicelles, and further analyzing their ability to scavenge intracellular ROS. (HC, HC, 50 μg / mL concentration) J HC K and HC JK The nanoparticle solution was sterilized overnight by UV irradiation, and then the cells were cultured in a 37 °C, 5% CO2 cell culture incubator for 24 h. RAW264.7 cells induced by LPS were then cultured with PBS and HC and HC prepared according to this invention. J HC K and HC JKAfter 12 hours of co-incubation, cells were collected and washed with PBS 12 hours after the treatment ended. RAW264.7 cells were then incubated with 0.2% DCFH-DA solution in a 37°C, 5% CO2 cell culture incubator for 30 minutes to label ROS in the cells. Subsequently, cells were collected and washed with PBS to remove the dye, and ROS in the RAW264.7 cells were imaged using an inverted fluorescence microscope. Figure 9 It can be seen from this that HC and HC... J HC K and HC JK The ROS content in the cells of the treatment group was significantly lower than that in the PBS group, indicating that the treatment group could effectively clear ROS in RAW264.7 cells, while HC J and HC JK The JPH203 in it further enhances its antioxidant effect.

[0043] Test Result 10: Reference Appendix Figure 11 Evaluation of nanomicelles (HC, HC J HC K and HC JK The results of the detection of the effect on promoting chondrocyte proliferation, Figure 11 These are images of chondrocytes in an inflammatory state, captured by an inverted fluorescence microscope, with PBS and HC and HC prepared according to this invention. J HC K and HC JK Fluorescence images of cell proliferation levels after co-incubation with Calcein / PI dye. In this experiment, cells were labeled using the Calcein-AM / PI double staining method, and the state of chondrocytes after different nanomicelle treatments was observed using an inverted fluorescence microscope. The effects of different nanomicelles on chondrocyte survival and proliferation were analyzed by counting the number of live cells (Calcein-AM labeled, green fluorescence) and the ratio of live to dead cells, thereby evaluating their proliferative biological activity. 50 μg / mL HC, HC... J HC K and HC JK The nanoparticle solution was sterilized overnight by UV irradiation, and then the cells were cultured in a 37 °C, 5% CO2 cell culture incubator for 24 h. RAW264.7 cells induced by LPS were then cultured with PBS and HC and HC prepared according to this invention. J HC K and HC JKAfter a 12-hour incubation period, cells were collected and washed with PBS 12 hours after treatment. RAW264.7 cells were then incubated with Calcein-AM (a live cell dye) and Propidium Iodide (a dead cell dye) in a 37°C, 5% CO2 cell culture incubator for 30 minutes to label live and dead cells. Cells were then collected and washed with PBS to remove the cell dyes. The number of live cells in each group was observed using an inverted fluorescence microscope. Figure 10 It can be seen from this that HC K and HC JK The number of live cells in the treatment group was significantly higher than that in other treatment groups, indicating that KGN can promote significant growth of chondrocytes.

[0044] Test Result 11: See Appendix Figure 12 The mouse weight monitoring results shown are as follows: Figure 12 This is a graph showing the weight changes of arthritis model mice within 30 days after treatment. The weights all showed a steady increase with no abnormalities.

[0045] This invention relates to the fields of drug delivery systems and osteoarthritis treatment, specifically to a multifunctional micelle system based on hyaluronic acid (HA)-TK (thionone bond)-curcumin (CUR) for targeted delivery of JPH203 (a LAT1 inhibitor) and KGN (a chondrocyte differentiation inducer) to treat osteoarthritis (OA). This micelle system uses HA, which can target CD44-expressing cells, as a carrier backbone. It is sequentially chemically coupled with TK and CUR, and self-assembles into stable nanomicelles through intermolecular forces, simultaneously encapsulating the drugs JPH203 and KGN, thus achieving efficient loading and targeted delivery. The core innovation lies in the dual-targeting characteristics of HA: HA can actively target and deliver drugs by specifically binding to RAW264.7 macrophages and articular chondrocytes that highly express CD44 receptors under inflammatory conditions. Simultaneously, utilizing the ROS-responsive properties of TK, it undergoes oxidative cleavage in the high-ROS microenvironment of OA, triggering micelle disintegration and precise drug release. By reversing the inflammatory microenvironment through macrophage phenotype remodeling, and simultaneously releasing KGN to promote chondrocyte proliferation and effectively recruit and promote mesenchymal stem cells to differentiate into chondrocytes, this invention achieves in situ cartilage regeneration, providing a more efficient and less side-effect-prone solution for osteoarthritis treatment. CUR stabilizes the nanostructure and enhances therapeutic efficacy through anti-inflammatory and antioxidant effects. This invention constructs an integrated "targeting-response-repair" delivery system by utilizing HA dual-targeting of RAW264.7 cells and chondrocytes under inflammatory conditions, TK drug release via ROS-responsive fragmentation, and the synergistic effect of CUR. This provides a novel strategy for precise and synergistic treatment of OA and has broad application prospects.

Claims

1. A ROS-responsive macrophage-chondrocyte dual-targeting nanomicelle, characterized in that, Using a ROS-responsive dual-targeting shell as a carrier, the carrier is constructed by coupling hyaluronic acid (HA) with curcumin (CUR) via ROS-responsive unit (TK) to encapsulate JPH203 and KGN drugs. Through self-assembly, a nanomicelle delivery system is formed that combines targeted delivery to chondrocytes and macrophages, ROS-responsive drug release, and osteoarthritis treatment.

2. The ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles according to claim 1, characterized in that... The dual targeting property refers to a hyaluronic acid-modified targeting molecule.

3. A method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles as described in claim 1 or 2, characterized in that... Includes the following steps: Step S1: Synthesize HA-TK-CUR. First, dissolve the ROS-responsive unit TK, EDC·HCl, and NHS in water and stir at room temperature for 2 hours. Then, slowly add a dimethyl sulfoxide solution of curcumin CUR to the above solution and continue stirring at room temperature for 1 day. Put the solution into a dialysis bag and dialyze for 3 days. Take out the liquid from the dialysis bag and freeze-dry it to obtain a yellow solid powder intermediate TK-CUR. Step S2: Dissolve the TK-CUR intermediate solid with dimethyl sulfoxide, add EDC·HCl and NHS to the solution, stir at room temperature for 2 h, then slowly add an aqueous solution of HA to the above solution, continue stirring at room temperature for 1 d, dialyze the solution for 3 d, take out the liquid from the dialysis bag and freeze dry to obtain yellow solid powder HA-TK-CUR; Step S3: Dissolve the obtained HA-TK-CUR in a mixed solution of tetrahydrofuran and water at a ratio of 1:9; dissolve KGN and JPH203 in tetrahydrofuran; Step S4: Add the KGN and JPH203 mixed solution from the above steps to the solution containing HA-TK-CUR under ultrasonic conditions, and sonicate for 30 min; Step S5: Use a rotary evaporator to fully evaporate the tetrahydrofuran; Step S6: Filter using a PES membrane filtration device; load the filtered solution into an ultrafiltration tube for dialyzing to obtain ROS-responsive nanomicelles.

4. The method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles according to claim 3, characterized in that... In step S1, CUR is 36.8 mg, 0.1 mmol; EDC·HCl is 19.2 mg, 0.1 mmol; NHS is 11.6 mg, 0.1 mmol; TK is 22.5 mg, 0.1 mmol; and the molar ratio of HA:EDC·HCl:NHS:TK dissolved in water is 1:5:5:

5.

5. The method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles according to claim 3, characterized in that... In step S2, HA was 30 mg, 0.02 mmol; EDC·HCl was 19.2 mg, 0.1 mmol; NHS was 11.6 mg, 0.1 mmol; the molar ratio of HA-TK:EDC·HCl:NHS:CUR dissolved in a mixed solution of water and dimethyl sulfoxide was 1:1:1:1; the molecular weight cutoff of the dialysis bag was 3500D, and the molecular weight cutoff of the ultrafiltration tube was 50000D.

6. The method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles according to claim 4, characterized in that, In step S3, HA-TK-CUR is dissolved in 9 mL of ultrapure water, and KGN and JPH203 are dissolved in tetrahydrofuran. The mass ratio of HA-TK:JPH203:KGN is 10:0.5:0.

5. The aqueous solution of tetrahydrofuran has a tetrahydrofuran to ultrapure water ratio of 1:9 and a total volume of 10 mL.

7. The method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles according to claim 4, characterized in that, The rotary evaporation time in step S5 is 2-3 hours; the pore size of the PES membrane filtration device used is 0.22 μm.

8. The method for preparing ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles according to claim 4, characterized in that... The conditions for membrane dialysis in step S6 are as follows: the molecular weight cutoff of the dialysis bag used is 3500D; the dialysis time is 72 h; and the stirring conditions are: stirring at room temperature for 24 h.

9. The preparation method according to claim 4, characterized in that, In step S6, the ultrafiltration tube used for ultrafiltration has a rejection capacity of 50,000; the centrifuge parameters are: 2000 rpm, 4 ℃, 1 h.

10. An application of ROS-responsive macrophage-chondrocyte dual-targeting nanomicelles, characterized in that, By enhancing the affinity of nanomicelles for chondrocytes and macrophages with hyaluronic acid, local drug concentrations are increased and JPH203 and KGN are delivered synergistically, achieving a synergistic treatment of inflammation suppression and cartilage regeneration.

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