A hyaluronic acid-based quercetin conjugate micelle, a preparation method and application thereof

Abstract

The application relates to a hyaluronic acid-based quercetin conjugate micelle and a preparation method and application thereof, and comprises the following steps: adding a coupling agent into a hyaluronic acid solution to carry out an activation reaction, then adding a phenylboronic acid solution to carry out a grafting reaction, and carrying out purification treatment to obtain HA-PBA; mixing HA-PBA and quercetin in an organic solvent, carrying out grafting reaction and purification treatment on the obtained mixed solution to obtain the hyaluronic acid-based quercetin conjugate micelle (HPQ). The hyaluronic acid-based quercetin conjugate micelle and KGN can also be used to prepare the hyaluronic acid-based quercetin conjugate micelle loaded with KGN (HPQ@K) through a loading reaction; the prepared HPQ and HPQ@K have a spherical structure, can make the interosseous joints reach a super-lubrication state, and can improve the solubility of quercetin and KGN, and improve the treatment and repair effect of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical preparations, specifically relating to a hyaluronic acid-based quercetin conjugate micelle, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a prevalent, age-related joint disease characterized by degeneration of articular cartilage, osteophyte formation, and functional impairment. With the increasing aging of the population, OA is gradually becoming one of the most common chronic diseases.

[0003] To date, early-stage osteoarthritis (OA) treatment has primarily focused on single-faceted pain relief, including oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of hyaluronic acid, and steroids. However, these drugs are easily cleared by the body, leading to poor treatment efficacy and a high relapse rate. Given the high ROS and low pH environment at the site of osteoarthritis lesions, prodrug systems with ROS and pH responsiveness have been a hot topic in osteoarthritis research. However, most drug delivery systems primarily target one or two aspects of lubrication, anti-inflammation, and cartilage regeneration induction, which remains limited for treating more severe OA. Therefore, there is an urgent need to develop a method that simultaneously inhibits OA development through lubrication, anti-inflammation, and cartilage regeneration induction. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a hyaluronic acid-based quercetin conjugate micelle, its preparation method and application, thereby solving the technical problem that the single-sided treatment effect of osteoarthritis in the prior art is not good.

[0005] To achieve the above-mentioned technical objectives, the technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing hyaluronic acid-based quercetin conjugate micelles, comprising the following steps: S1, adding a coupling agent to a hyaluronic acid solution for activation reaction, then adding a phenylboronic acid solution for grafting reaction, and purifying the solution to obtain HA-PBA; S2, mixing HA-PBA and quercetin in an organic solvent, and obtaining the mixture by grafting reaction and purification to prepare hyaluronic acid-based quercetin conjugate micelles.

[0007] Secondly, the present invention provides a hyaluronic acid-based quercetin conjugate micelle prepared by the above-described preparation method.

[0008] Thirdly, the present invention provides a hyaluronic acid-based quercetin conjugate micelle loaded with KGN, which is prepared by mixing hyaluronic acid-based quercetin conjugate micelles and KGN in a fourth solvent, followed by an encapsulation reaction and purification.

[0009] Fourthly, the present invention provides the use of hyaluronic acid quercetin conjugate micelles or KGN-loaded hyaluronic acid quercetin conjugate micelles in the preparation of drugs for inhibiting / treating osteoarthritis.

[0010] Compared with the prior art, the beneficial effects of the present invention include:

[0011] This invention grafts quercetin onto hyaluronic acid via phenylboronic acid to form HPQ micelles, and encapsulates KGN to form drug-loaded micelles HPQ@K. The HPQ micelles and drug-loaded micelles HPQ@K prepared by this invention have a spherical structure, which can achieve a super-lubricated state between bones, improve the solubility of quercetin and KGN, enhance the bioavailability of quercetin and KGN, and exert the dual-drug synergistic effect of quercetin and KGN. This invention combines nanoparticle lubrication, QUT to reduce the expression of inflammatory factors and scavenge free radicals, and KGN to induce cartilage regeneration, thus treating osteoarthritis from multiple aspects and improving the treatment and repair effects of osteoarthritis. Attached Figure Description

[0012] Figure 1 The HP obtained in Example 1 of this invention 1 H NMR spectrum;

[0013] Figure 2 These are the infrared spectra (HA, PBA, HP) of the raw materials and products of HP prepared in Example 1 of this invention.

[0014] Figure 3 It is the HPQ obtained in Example 1 of this invention. 1 H NMR spectrum;

[0015] Figure 4 The infrared spectra of the raw materials and products (HA, QUT, KGN, HPQ, HPQ@K) in Example 2 of this invention are shown.

[0016] Figure 5 It is the critical micelle concentration of HPQ, the product of Example 1 of the present invention;

[0017] Figure 6 These are the particle size and potential diagrams of HPQ, the product of Example 1 of this invention, and HPQ@K after being loaded with HPQ and KGN at different mass ratios in Examples 2-4.

[0018] Figure 7 These are particle size distribution diagrams and electron micrographs of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of the present invention; wherein, (A) is HPQ obtained in Example 1, and (B) is HPQ@K obtained in Example 2;

[0019] Figure 8 This is the friction coefficient of HPQ@K obtained in Example 2 of the present invention at the simulated bone and joint environment under the drug administration concentration;

[0020] Figure 9 This is a graph showing the inhibition of hyaluronidase activity by HPQ@K obtained in Example 2 of the present invention; wherein, (A) the inhibition rate of hyaluronidase by HPQ@K; and (B) the change curve of hyaluronic acid viscosity after HPQ@K inhibits hyaluronidase.

[0021] Figure 10 This is a graph showing the test results of HPQ@K scavenging DPPH free radicals obtained in Example 2 of the present invention; wherein, (A) the scavenging rate of HPQ@K on DPPH free radicals; and (B) the ultraviolet spectrum of HPQ@K scavenging DPPH free radicals.

[0022] Figure 11 These are the cumulative release curves of HPQ@K against QUT and KGN under different conditions obtained in Example 2 of the present invention; (A) QUT, (B) KGN;

[0023] Figure 12 These are the particle sizes of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of the present invention in serum, and the particle size distribution curves of HPQ@K obtained in Example 2 under different conditions; wherein, (A) is the particle size of HPQ and HPQ@KGN in serum; (B) is the particle size distribution curve of HPQ@K within 8 hours under pH=7.4 and pH=5.0 conditions; (C) is the particle size distribution curve of HPQ@K within 8 hours under pH=7.4 and pH=7.4, c(H2O2)=0.1mM conditions; (D) is the particle size distribution curve of HPQ@K within 8 hours under pH=7.4 and pH=6.5 conditions.

[0024] Figure 13 The effects of different concentrations of HPQ (obtained in Example 1) and HPQ@K (obtained in Example 2) on chondrocyte viability were detected using CCK-8 assays; (A) shows the results of HPQ detection at 24h and 48h; (B) shows the results of HPQ@K detection at 24h and 48h.

[0025] Figure 14 The expression of inflammation-related genes, matrix degradation-related genes, and matrix synthesis-related genes of HPQ obtained in Example 1 of this invention after 24 hours of IL-1β stimulation;

[0026] Figure 15 The expression of inflammation-related genes, matrix degradation-related genes, and matrix synthesis-related genes of HPQ@K obtained in Example 2 of this invention after 24 hours of IL-1β stimulation;

[0027] Figure 16The results are Live / Dead staining of chondrocytes 24 hours after intervention with HPQ (0.1 mg / mL) obtained in Example 1 and HPQ@K (0.1 mg / mL) obtained in Example 2 of this invention;

[0028] Figure 17 The results are Live / Dead staining of chondrocytes 48 hours after intervention with HPQ (0.1 mg / mL) obtained in Example 1 and HPQ@K (0.1 mg / mL) obtained in Example 2 of this invention;

[0029] Figure 18 These are immunoblots of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of this invention after IL-1β stimulation of chondrocytes for 24 hours.

[0030] Figure 19 This is the quantitative analysis result of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of the present invention after IL-1β stimulation of chondrocytes for 24 hours;

[0031] Figure 20 These are immunoblots of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of this invention after 48 hours of IL-1β stimulation of chondrocytes;

[0032] Figure 21 This is the quantitative analysis result of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of the present invention after IL-1β stimulation of chondrocytes for 48 hours;

[0033] Figure 22 These are JC-1 staining images of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of this invention after IL-1β stimulation of chondrocytes;

[0034] Figure 23 These are transmission electron micrographs of mitochondria in chondrocytes after IL-1β stimulation of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of this invention.

[0035] Figure 24 These are immunoblots of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of this invention against key proteins in the antioxidant pathway;

[0036] Figure 25 Yes Figure 23 Quantitative analysis of immunoblotting;

[0037] Figure 26 It is mRFP-GFP-LC3 fluorescence detection;

[0038] Figure 27 These are the results of an autophagy level test;

[0039] Figure 28It is an expression of aging markers;

[0040] Figure 29 This is a diagram showing the results of SA-β-Gal staining;

[0041] Figure 30 This is an X-ray image showing how HPQ and HPQ@K improve cartilage degeneration in DMM model mice;

[0042] Figure 31 These are Micro-CT images showing how HPQ and HPQ@K improve cartilage degeneration in DMM model mice;

[0043] Figure 32 This is a staining diagram showing how HPQ and HPQ@K improve cartilage degeneration in DMM model mice;

[0044] Figure 33 These are the immunohistochemical results of Aggrecan, MMP13, p62, and p16 in HPQ and HPQ@K improving cartilage degeneration in DMM model mice;

[0045] Figure 34 This is a histological staining result and experimental diagram of OA cartilage; where (A) is the staining result and (B) is a schematic diagram of OA cartilage specimen grouping and processing.

[0046] Figure 35 The results show the immunohistochemical effects of HPQ@K on Aggrecan, MMP13, p62, and p16 in OA cartilage. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0048] Terminology Explanation

[0049] QUT: Quercetin;

[0050] HA: Hyaluronic acid;

[0051] KGN: kartogenin;

[0052] EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide;

[0053] NHS: N-hydroxysuccinimide;

[0054] PBA: 3-Aminophenylboronic acid;

[0055] DMSO: Dimethyl sulfoxide.

[0056] To address the shortcomings of most existing drug delivery systems for osteoarthritis, which primarily treat only one aspect and thus have poor efficacy, this invention provides a hyaluronic acid-based quercetin conjugate micelle, its preparation method, and its applications. This micelle is a ROS- and pH-responsive hyaluronic acid-based quercetin conjugate micelle, which can be selectively loaded with KGN. Hyaluronic acid (HA) lubricates the joints, quercetin (QUT) scavenges free radicals and has anti-inflammatory properties to treat cartilage damage, and KGN enhances CBFβ cell nuclear localization, induces hMSCs to differentiate into chondrocytes, and protects articular chondrocytes, thus achieving a certain therapeutic effect on OA.

[0057] However, since QUT and KGN are completely hydrophobic small molecule drugs, direct mixing hinders cellular uptake and results in low bioavailability. This invention grafts quercetin (QUT) onto hyaluronic acid (HA) via 3-aminophenylboronic acid to form HPQ micelles, and then encapsulates KGN using dialysis to form drug-loaded micelles HPQ@K. This invention utilizes the spherical structure of the micelles to achieve a super-lubricated state between bones, enhances the solubility of QUT and KGN, improves their bioavailability, and leverages the synergistic effect of QUT and KGN. This combines nanoparticle lubrication, QUT's reduction of inflammatory factor expression and free radical scavenging, and KGN's induction of cartilage regeneration for the treatment and repair of osteoarthritis, enhancing autophagy and anti-aging, and treating OA from multiple perspectives to achieve a synergistic effect.

[0058] In a first aspect, the present invention provides a method for preparing hyaluronic acid-based quercetin conjugate micelles, comprising the following steps:

[0059] S1, a coupling agent is added to a hyaluronic acid solution to activate the reaction, followed by the addition of a phenylboronic acid solution for grafting, and then HA-PBA is obtained after purification.

[0060] S2, HA-PBA and quercetin are mixed in an organic solvent, and the resulting mixture is subjected to grafting reaction and purification treatment to obtain hyaluronic acid-based quercetin conjugate micelles.

[0061] Preferably, in step S1, the hyaluronic acid solution is prepared by dissolving hyaluronic acid in ultrapure water, and the molecular weight of the hyaluronic acid is 5000 Da to 15000 Da; the ratio between the carboxyl group of the hyaluronic acid and the ultrapure water is (0.5 to 1.5) mmol: 30 mL.

[0062] Preferably, in step S1, the coupling agent includes EDC and NHS, and the molar ratio of the carboxyl group of hyaluronic acid to EDC and NHS is (0.5-1.5):(0.5-1.5):(0.5-1.5).

[0063] Preferably, in step S1, the activation reaction is carried out at 20–28°C for 3.5–4.5 h.

[0064] Preferably, in step S1, the phenylboronic acid solution is prepared by dissolving phenylboronic acid in a first organic solvent, and the concentration of the phenylboronic acid solution is 0.05 to 0.15 mmol / mL.

[0065] More preferably, the first organic solvent includes dimethyl sulfoxide (DMSO). The DMSO used in this invention is a highly polar solvent. Since the solubility of 3-aminophenylboronic acid in water is limited, a portion of the DMSO can increase the solubility of 3-aminophenylboronic acid during the reaction process. Furthermore, hyaluronic acid is a polysaccharide different from that in organic solvents, but it will not precipitate in a mixture of pure water and DMSO. Therefore, the solvent system used in this invention can improve the reaction yield.

[0066] Preferably, in step S1, the phenylboronic acid includes 3-aminophenylboronic acid; the molar ratio of the carboxyl group of hyaluronic acid to phenylboronic acid is (0.5-1.5):1. More preferably, the molar ratio of the carboxyl group of hyaluronic acid to phenylboronic acid is 1:1.

[0067] Preferably, in step S1, the grafting reaction is carried out at 20–28°C for 20–30 h.

[0068] Preferably, in step S1, the purification process includes ultrapure water dialysis for 48–72 h and lyophilization.

[0069] Preferably, in step S2, mixing HA-PBA and quercetin in an organic solvent specifically includes: dissolving HA-PBA in a second organic solvent to obtain an HA-PBA solution; dissolving quercetin in a third organic solvent to obtain a quercetin solution; and then mixing the HA-PBA solution and the quercetin solution.

[0070] More preferably, the HA-PBA solution is obtained by adding HA-PBA to a second organic solvent under a protective atmosphere and stirring to dissolve it at 40-45°C; the second organic solvent includes formamide; and the third organic solvent includes dimethyl sulfoxide.

[0071] Preferably, in step S2, the molar ratio of -B(OH)2 group in HA-PBA to quercetin is (0.02~0.12):0.12.

[0072] Preferably, in step S2, the grafting reaction is carried out under a protective atmosphere at 35–45°C for 40–55 h.

[0073] Preferably, in step S2, the purification process involves dialyzing with ultrapure water for 48–72 hours, followed by filtration through a 0.4–0.5 μm microporous membrane and then freeze-drying.

[0074] In the dialysis process of this invention, the molecular weight cutoff of the dialysis bag is 800-1200 Da. If the molecular weight is too low, small molecule drug monomers cannot be removed; if the molecular weight is too high, the final product will be lost.

[0075] Secondly, the present invention provides a hyaluronic acid-based quercetin conjugate micelle prepared by the above-described preparation method.

[0076] Thirdly, the present invention provides a hyaluronic acid-based quercetin conjugate micelle loaded with KGN, which is prepared by mixing hyaluronic acid-based quercetin conjugate micelles and KGN in a fourth solvent, followed by an encapsulation reaction and purification.

[0077] Preferably, the mass ratio of hyaluronic acid-based quercetin conjugate micelles to KGN is (5-15):1.

[0078] A further preferred embodiment is that the mass ratio of hyaluronic acid-based quercetin conjugate micelles to KGN is (10-15):1.

[0079] Preferably, the fourth solvent includes formamide.

[0080] Preferably, the encapsulation reaction is carried out at 20–28°C for 3–5 hours.

[0081] Preferably, purification is performed by dialyzing with a dialysis bag containing a molecular weight cutoff of not less than 800 Da for 40–60 h, followed by filtration through a 0.4–0.5 μm microporous membrane and then lyophilization.

[0082] Fourthly, the present invention provides the use of hyaluronic acid quercetin conjugate micelles or KGN-loaded hyaluronic acid quercetin conjugate micelles in the preparation of drugs for inhibiting / treating osteoarthritis.

[0083] As a preferred embodiment, the main reaction route of the hyaluronic acid-based quercetin conjugate micelles of the present invention includes:

[0084] (1) Preparation of HP: Hyaluronic acid (where 0.5 mmol ≤ n (-COOH) ≤1.5mmol) in coupling agent (0.5mmol≤n EDC ≤1.5mmol, 0.5mmol≤n NHS The -COOH group was activated under the action of ≤1.5mmol, and then grafted with phenylboronic acid (PBA=1mmol). After the reaction was completed, the mixture was dialyzed with ultrapure water and then lyophilized to obtain HA-PBA(HP) raw material, as shown in formula (I):

[0085]

[0086] Formula (I)

[0087] In equation (I), m=1, 0.1≤n≤0.3;

[0088] (2) Preparation of HPQ: HP (where 0.02≤n) (-B(OH)2 ≤0.12 mmol) was dissolved in formamide (the solution was heated and stirred at 45°C under a nitrogen atmosphere). Then, 36.24 mg (0.12 mmol) of quercetin was weighed and dissolved in DMSO. After the quercetin dissolved, it was added to the above solution. The reaction was carried out at 40°C for 48 h under a nitrogen atmosphere. After 48 h, the mixture was dialyzed for 72 h. Then, the large aggregates were removed by filtration through a 0.45 μm microporous membrane to obtain HPQ, which was then lyophilized and stored as shown in formula (II).

[0089]

[0090] Equation (II)

[0091] In equation (II), m=1, 0.1≤n≤0.3.

[0092] This invention employs a single reaction between phenylboronic acid and quercetin, avoiding side reactions. Furthermore, the reaction of the ortho-dihydroxyl group in Formula II with phenylboronic acid to form a borate ester is a dehydration reaction. Compared to most studies that involve reactions in water, this invention, by using an organic solvent, significantly enhances the reaction degree between phenylboronic acid and quercetin. 1 ¹H NMR integration revealed that the degree of substitution of phenylboronic acid was consistent with that of quercetin, indicating that the phenylboronic acid on the hyaluronic acid in this invention reacted completely with quercetin.

[0093] As a preferred embodiment, the present invention also provides hyaluronic acid-based quercetin conjugate micelles loaded with KGN (HPQ@K). The HPQ lyophilized product is dissolved, KGN (Kartogenin) is added, and the mixture is stirred at room temperature for a period of time. The solution is then transferred to a dialysis bag and dialyzed with ultrapure water to remove unloaded free small molecules. After dialysis, aggregates are removed by filtration through a microporous membrane, and the product is finally lyophilized for storage. The unique structure of HPQ@K in this invention endows the material with ROS and pH responsiveness, which is beneficial for drug accumulation in lesions. It integrates nano-lubrication, QUT anti-inflammatory properties, and KGN-promoted cartilage regeneration, providing a new approach for OA treatment.

[0094] The present invention will be further described in detail below through specific embodiments.

[0095] Example 1

[0096] (1) Preparation of HP: 379 mg of hyaluronic acid (HA) (n(-COOH) = 1 mmol) was weighed and placed in a beaker containing 30 mL of ultrapure water and stirred. After the HA dissolved, 155 mg (1 mmol) of EDC and 115 mg (1 mmol) of NHS were added. The -COOH was activated at room temperature for 4 h to obtain the first reaction solution. After 4 h, 137 mg (1 mol) of 3-aminophenylboronic acid (PBA) was weighed and dissolved in 10 mL of DMSO. Then it was added to the first reaction solution and reacted at room temperature for 24 h to obtain the second reaction solution. The second reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with ultrapure water for 72 h. Then it was freeze-dried to obtain HA-PBA (HP) raw material.

[0097] (2) Preparation of HPQ: Weigh about 102 mg of HA-PBA (n(-B(OH)2) = 0.06 mmol) and dissolve it in 10 mL of formamide (heat and stir the solution at 45 °C under nitrogen atmosphere) to obtain HA-PBA solution; then weigh 36.24 mg (0.12 mmol) of quercetin and dissolve it in 5 mL of DMSO to obtain quercetin solution; add quercetin solution to HA-PBA solution and react at 40 °C for 48 h under nitrogen atmosphere. After 48 h, transfer it to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze it with ultrapure water for 72 h. Then filter it with a 0.45 μm microporous membrane to remove large aggregates and freeze dry to obtain product HA-PBA-QUT (abbreviated as HPQ).

[0098] Example 2

[0099] Preparation of HPQ@K: 45 mg of the lyophilized HPQ sample prepared in Example 1 was weighed and dissolved in 10 mL of formamide. Then, 4.5 mg of KGN (Kartogenin) was added, resulting in a ratio of m(HPQ):m(KGN) = 10:1. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with ultrapure water for 48 hours to remove unencapsulated free small molecules. After dialysis, the aggregates were removed by filtration through a 0.45 μm microporous membrane, and the product HA-PBA-QUT@KGN (abbreviated as HPQ@K) was obtained by lyophilization.

[0100] Example 3

[0101] The only difference from Example 2 is that the amount of KGN is adjusted to 9 mg, i.e., m(HPQ):m(KGN) = 5:1. The other steps and conditions are the same as in Example 2.

[0102] Example 4

[0103] The only difference from Example 2 is that the amount of KGN is adjusted to 3 mg, i.e., m(HPQ):m(KGN) = 15:1. The other steps and conditions are the same as in Example 2.

[0104] Performance testing

[0105] 1. The HP prepared in Example 1 was subjected to NMR and infrared detection, and the results are as follows: Figure 1 and Figure 2 As shown, by Figure 1 It can be seen that the characteristic peaks of hydrogen atoms on the aromatic ring of PBA can be found in the range of 7.0–8.0 ppm, indicating the successful synthesis of HP. The absence of characteristic hydrogen atom peaks for the hydroxyl (-OH) and amino (-NH2) groups on PBA in HP is because the measurement in HP is performed in D2O, where the active atoms readily replace deuterium atoms in the solution. Figure 2 It can be seen that in the infrared spectrum of HA, 2928 cm⁻¹ -1 and 2882cm -1 This corresponds to the characteristic peaks of the stretching vibrations of the methyl (-CH3) and methylene (-CH2) groups in hyaluronic acid. 1622.8 cm⁻¹ -1 The peak at 1665.2 cm⁻¹ corresponds to the characteristic peak of the C=O stretching vibration in the carboxyl group (-COOH) of hyaluronic acid. -1 The characteristic peak corresponding to the C=O stretching vibration of the imide (-CONH-) in hyaluronic acid is 1557.7 cm⁻¹. -1 The characteristic peaks correspond to the in-plane bending vibrations of NH in the imide (-CONH-) group of hyaluronic acid. In HP, the characteristic peaks corresponding to HA can be found separately. Therefore, from... 1 ¹H NMR and FT-IR can verify the successful preparation of HP.

[0106] 2. The HPQ obtained in Example 1 was subjected to NMR testing, and the raw materials and products in Example 1 and Example 2 were subjected to infrared detection. The results are as follows: Figures 3 to 4 As shown.

[0107] like Figure 3 As shown, the signal in the δ 6.0–9.0 region was assigned as the signals of the aromatic hydrogens of QUT and PBA, indicating successful synthesis of HPQ. Using peaks 1, 2, and 10 as integration localization peaks, peak area integration was performed using MestRenova on the peak regions where PBA and QUT were present. The integration results are shown below. Figure 3 The degree of substitution was calculated to be approximately 20% based on the integral results, thus the drug loading of the QUT was approximately 12.9%. 1 1H NMR confirmed the successful synthesis of HPQ.

[0108] like Figure 4As shown, in the infrared spectrum of HA, 1622.8 cm⁻¹ -1 The value corresponds to the characteristic peak of the C=O stretching vibration of the carboxyl group (-COOH) in hyaluronic acid, at 1665.2 cm⁻¹. -1 The corresponding stretching vibration peak of (-CONH-)C=O in hyaluronic acid is 1557.7 cm⁻¹. -1 This corresponds to the in-plane bending vibration peak of (-CONH-)NH. In the HPQ spectrum, a peak at 1622.8 cm⁻¹ is clearly observed compared to HA. -1 The absorption peak at the carboxyl group position was weakened, while the stretching vibration peak of (-CONH-)C=O and the in-plane bending vibration peak of (-CONH-)NH were enhanced, indicating that the carboxyl group and amino group were covalently bonded, and the basic structure of HA was preserved. Comparing the infrared spectra of HA, QUT, and HPQ, it was found that the absorption peak at 500 cm⁻¹ was significantly enhanced. -1 ~900cm -1 and 1100cm -1 ~1500cm -1 Within the specified range, numerous characteristic peaks of QUT appeared in the infrared spectrum of HPQ, proving the successful synthesis of HPQ.

[0109] At the same time, by Figure 4 Infrared spectra of HPQ@K show that, compared to HPQ, HPQ@K clearly exhibits an aromatic carboxyl group (Ar-COOH) on KGN at 1719.7 cm⁻¹. -1 The characteristic peaks and imine (-CONH-) are at 3318.8 cm⁻¹. -1 The characteristic peaks of NH stretching vibration indicate that KGN was successfully encapsulated.

[0110] 3. Figure 5 The critical micelle concentration (CMC) of HPQ prepared in Example 1 was measured by a fluorescent probe. The CMC of HPQ micelles was 20.4 μg / ml, indicating that HPQ can exist in nanoscale form in water at a low concentration. The low CMC value means that micelles can self-assemble at very dilute concentrations, which will maintain the stability of micelles in blood circulation.

[0111] 4. The particle size (d), potential, drug loading (DL), and encapsulation efficiency (EE) of HPQ@K after different mass ratios of HPQ and KGN in Examples 2-4 were tested. Particle size and potential were measured using a particle size analyzer (DLS), and drug loading and encapsulation efficiency were quantitatively determined by HPLC. The results are shown in Table 1 below. Figure 6 As shown.

[0112] Table 1. Particle size (d), potential, drug loading, and encapsulation efficiency at different loading ratios.

[0113]

[0114] like Figure 6 Table 1 shows the results of the optimized proportions in the preparation process of HPQ@K. When the amount of hyaluronic acid is 379 mg, the amount of EDC is 155 mg, and the amount of NHS is 115 mg, the micelle size is approximately 150.53 ± 3.14 nm, and the Zeta potential is approximately -48.26 ± 0.95 mV, the prepared HPQ is most suitable. This was confirmed by NMR. 1 HNMR determined that the proportion of repeating units containing QUT in HPQ was 0.2.

[0115] With increasing KGN ratio, the particle size and zeta potential of the drug-loaded micelles gradually increased. The HPQ particle size was 150.53 nm and the zeta potential was -48.26 mV; when m(HPQ):m(KGN) = 15:1, the particle size was 164.33 nm and the zeta potential was -38.63 mV; when m(HPQ):m(KGN) = 10:1, the particle size was 178.63 nm and the zeta potential was -32.40 mV; when m(HPQ):m(KGN) = 5:1, the particle size reached 344.16 nm and the zeta potential was -25.63 mV. These results show that KGN was successfully loaded. However, when m(HPQ):m(KGN) = 5:1, the particle size increased sharply, exceeding the upper limit of HPQ loading. When the nanoparticle size is too large, it is not conducive to cell uptake and may lead to cell necrosis, thus hindering subsequent experiments.

[0116] Both groups with m(HPQ):m(KGN) ratios of 15:1 and 10:1 exhibited excellent particle size results. To select the optimal experimental group, the drug loading and encapsulation efficiency at different ratios were determined by HPLC, as shown in Table 1. When m(HPQ):m(KGN) = 10:1, it had better encapsulation efficiency and drug loading. When m(HPQ):m(KGN) = 15:1, both encapsulation efficiency and drug loading decreased, with the encapsulation efficiency being lower than that of m(HPQ):m(KGN) = 10:1. This may be due to the lower concentration of KGN in the solution, which could not fully contact the hydrophobic segment, resulting in a lower drug loading during self-assembly. In summary, the HPQ@K obtained with m(HPQ):m(KGN) = 10:1 had the most suitable particle size, potential, encapsulation efficiency, and drug loading. Therefore, HPQ@K with m(HPQ):m(KGN) = 10:1 was selected for subsequent testing.

[0117] 5. Morphology and particle size distribution of HPQ and HPQ@K

[0118] Figure 7Images (A) and (B) show the particle size distribution and electron microscopy morphology of empty-shell micelles HPQ and drug-loaded micelles HPQ@K. Both HPQ and HPQ@K exhibit uniform distribution and relatively regular circular morphologies as observed in the transmission electron microscopy (TEM) images. Both particle size distribution and TEM images show that HPQ@K particles are slightly larger than HPQ particles, indicating successful KGN loading. The nanoscale dimensions obtained by TEM differ somewhat from those measured by a particle size analyzer (DLS). The particle size measured by TEM is smaller than that measured by DLS. This difference in particle size may be due to the shrinkage of the micelle shells during the drying process of the TEM sample, while DLS measures the size of hydrated particles.

[0119] 6. Determination of the friction coefficient of HPQ@K in a simulated bone and joint environment

[0120] To determine the friction coefficient of HPQ@K at a drug concentration (1 mg / mL), a reciprocating mode was used after micelle formation due to the advantage of the spherical structure. The reciprocating frequency was 5 Hz, the amplitude was 4 mm, and the loading force was 5 N. The upper sample was a polyethylene (PE) sphere with a diameter of 8 mm, simulating the upper part of the joint. The lower sample was a polished Ti6Al4V disk with a surface roughness of 1.7 nm, simulating the lower part of the joint. The HPQ@K micelles were located between the upper and lower samples. The reciprocating time was 40 min, simulating the bone joint environment.

[0121] The results are as follows Figure 8 As shown, the measured coefficient of friction is only 0.008, reaching a super-lubricated state. This can effectively reduce the wear and tear of joints and the damage to the surface articular cartilage, which is beneficial to the recovery of osteoarthritis.

[0122] 7. Inhibition of hyaluronidase by HPQ@K

[0123] The presence of QUT in the HPQ@K structure endows HPQ@K with a certain inhibitory effect on hyaluronidase activity, which is very beneficial for promoting the clearance of osteoarthritis inflammatory factors and maintaining micelle morphology. Five groups were set up with HPQ@K concentrations of 5 mg / mL, 2.5 mg / mL, 1 mg / mL, 0.5 mg / mL and 0.1 mg / mL for enzyme inhibition experiments (hyaluronidase concentration was 8u / mL in all experimental groups; and the concentration of hyaluronic acid was 10 mg / mL in all experimental groups). A control group was also set up (the only difference was that no hyaluronidase was added). The inhibitory effect of hyaluronidase was calculated by measuring the ultraviolet absorption intensity at 540 nm, and the viscosity of hyaluronic acid in each group was measured by rotational rheometer.

[0124] Figure 9 (A) shows the inhibitory effect of HPQ@K on hyaluronidase, with an inhibition rate of 53.86% when the concentration of HPQ@K reaches 2.5 mg / mL.

[0125] Figure 9 (B) shows the change in hyaluronic acid viscosity in the experimental system after the hyaluronidase inhibition experiment. Compared with the control group without added enzyme, the inhibitory effect on hyaluronidase decreased as the concentration of HPQ@K decreased, and the viscosity of the sample gradually decreased, indicating that hyaluronic acid was degraded, proving that HPQ@K has an inhibitory effect on hyaluronidase.

[0126] 8. Scavenging of free radicals by HPQ@K

[0127] Five groups were set up for free radical scavenging experiments with HPQ@K concentrations of 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, respectively. The concentration of DPPH in each group was 150 μM. The free radical scavenging effect was calculated by measuring the ultraviolet absorption intensity at 517 nm.

[0128] Figure 10 (B) demonstrates HPQ@K's ability to scavenge free radicals. Figure 10 (A) is Figure 10 The quantification results at 517 nm in (B) show that when the concentration of HPQ@K is only 0.1 mg / mL, the DPPH scavenging rate can reach 68.86%, and at 0.25 mg / mL it can reach nearly 90%. The high DPPH scavenging rate at a low concentration demonstrates the strong free radical scavenging ability of HPQ@K. This is due to the strong free radical scavenging ability of QUT, which can have a great positive impact on the recovery of osteoarthritis and shows the excellent potential of HPQ@K in the treatment of osteoarthritis.

[0129] 9. pH and ROS responsiveness of HPQ@K

[0130] The following experimental groups were set up respectively:

[0131] (1) HPQ@K was added to PBS buffer at pH 5.0;

[0132] (2) HPQ@K was added to PBS buffer at pH 7.4 containing 0.1 mM H2O2;

[0133] (3) HPQ@K was added to PBS buffer at pH 6.5;

[0134] (4) HPQ@K was added to PBS buffer at pH 7.4;

[0135] The concentration of HPQ@K in each experimental group was 1 mg / mL. The cumulative release curves of QUT and KGN in each experimental group within 72 h were determined by HPLC.

[0136] The results are as follows Figure 11 As shown, at pH 5.0, the cumulative release rates of QUT and KGN after 72 hours were 76.06% and 82.61%, respectively; at pH 7.4 + 0.1 mM H₂O₂, the cumulative release rates were 65.60% and 67.56%, respectively. This demonstrates the excellent pH and ROS responsiveness of HPQ@K, which is beneficial for drug accumulation in lesions. In contrast, at pH 7.4, the cumulative release rates of QUT and KGN after 72 hours were only 10.15% and 10.61%, respectively. Therefore, HPQ@K of this invention possesses excellent dual-response potential.

[0137] 10. Serum stability and particle size changes of HPQ@K under different conditions

[0138] The particle size and potential of micelles were measured using a laser particle size analyzer under physiological conditions (simulated conditions are the same as in Experiment 4 above, i.e., PBS solution with pH=7.4), as well as the particle size change curves of micelles under simulated lesion microenvironment (simulated conditions are the same as in Experiment 1-3 above).

[0139] Figure 12 (A) shows the particle size of HPQ and HPQ@K in serum over 7 days. Due to the electrostatic repulsion of negatively charged micelles, this reduces the non-specific adsorption between micelles and negatively charged proteins, indicating the excellent stability of HPQ and HPQ@K under normal physiological conditions. Figure 12 (B-D) show the particle size changes of HPQ@K micelles exposed to various simulated environments for 8 hours. No significant size change over time was observed for HPQ@K micelles at pH 7.4. Conversely, when HPQ@K micelles were incubated in PBS at pH 5.0, the size increased to 591.4 nm, and when incubated at pH 7.4 with 0.1 mM H₂O₂, the size increased to 538.0 nm. These results indicate that HPQ@K micelles maintain structural integrity in blood circulation and exhibit pH and ROS responsiveness, which is beneficial for controlled drug release.

[0140] 11. To verify the effects of HPQ and HPQ@K in reducing toxicity and regulating inflammatory response, this invention tested their effects on chondrocyte viability using a CCK-8 assay and compared them with free drugs KGN and QUT.

[0141] The results are as follows Figure 13 As shown, HPQ and HPQ@K did not exhibit significant toxicity at concentrations ≤0.1 mg / mL, with a decrease in cell viability only observed at 0.2 mg / mL. Figure 13 (A and B) indicate that they significantly reduced the toxicity of KGN and QUT.

[0142] Further analysis using PCR investigated the regulatory effects of HPQ and HPQ@K intervention on chondrocyte gene expression after 24 hours of IL-1β stimulation. Results are shown below. Figure 14 and Figure 15 PCR results showed that HPQ and HPQ@K nanoparticles significantly downregulated the expression of inflammation-related genes (iNOS, COX2) and matrix degradation-related genes (MMP3, MMP13) after IL-1β stimulation for 24 hours, while significantly upregulating the expression of matrix synthesis-related genes (Aggrecan, Collagen II, SOX9) in a concentration-dependent manner, reaching the optimal concentration at 0.1 mg / ml.

[0143] Furthermore, Live / Dead staining results further validated the safety of HPQ and HPQ@K at a concentration of 0.1 mg / mL. Figure 16 and Figure 17 As shown, green represents surviving cells and red represents dead cells. After 24 and 48 hours of treatment, chondrocyte survival remained high, and no significant cell death was observed. In summary, HPQ and HPQ@K enhance their anti-inflammatory and matrix-protective effects by reducing the toxicity of free KGN and QUT, with the best effect achieved at 0.1 mg / ml.

[0144] 12. To verify the sustained-release effect of HPQ and HPQ@K and their regulatory role on IL-1β-stimulated chondrocytes at the protein level, this invention used a concentration of 0.1 mg / mL to intervene chondrocytes for 24 hours and 48 hours, respectively, and analyzed the expression of matrix-related proteins and inflammation-related proteins by protein immunoblotting and immunofluorescence.

[0145] Western blot results showed that ( Figure 18 and Figure 19 After 24 hours of intervention, both HPQ and HPQ@K significantly restored the expression of matrix-associated proteins Aggrecan, Collagen II, and SOX9, while inhibiting the expression of pro-inflammatory proteins iNOS and COX2, and degradative proteins MMP13 and MMP3. After 48 hours of intervention ( Figure 21 and Figure 21The regulatory effects of HPQ and HPQ@K were further enhanced, manifested in more significant upregulation of the expression of matrix proteins Aggrecan, Collagen II, and SOX9 (promoting matrix protein recovery), and a stronger inhibitory effect on the expression of pro-inflammatory and degradative proteins. Compared with the 24-hour results, the regulatory effect at 48 hours was significantly enhanced, indicating that HPQ and HPQ@K have good sustained-release properties and can exert their regulatory effects continuously with prolonged action time. Among them, HPQ@K showed a significantly better regulatory effect than HPQ at 48 hours, especially in enhancing matrix protein recovery and inhibiting the expression of pro-inflammatory and degradative proteins.

[0146] 13. To evaluate the regulatory effects of HPQ and HPQ@K on IL-1β-induced chondrocyte damage, this invention focuses on mitochondrial function and systematically analyzes oxidative stress, autophagy, and aging-related indicators.

[0147] Figure 22 JC-1 staining and mitochondrial ATP assays showed that both HPQ and HPQ@K could significantly reverse IL-1β-induced mitochondrial membrane potential depolarization and functional damage, with HPQ@K showing a more significant effect. Figure 23 Transmission electron microscopy showed that HPQ and HPQ@K significantly improved IL-1β-induced mitochondrial structural damage, and HPQ@K had a better protective effect, which is beneficial to improving mitochondrial structural integrity.

[0148] In addition, such as Figure 24 and Figure 25 As shown, HPQ and HPQ@K significantly inhibited ROS generation and enhanced antioxidant capacity by upregulating the expression of key antioxidant pathway proteins NRF2, GCLC, HO-1 and GCLM, with HPQ@K showing stronger effects.

[0149] like Figure 26 and Figure 27 As shown, in terms of autophagy, both dual fluorescent labeling and Western blot analysis showed that HPQ and HPQ@K could restore the IL-1β-induced decrease in autophagy levels and significantly enhance autophagy activity by downregulating p62 and upregulating Beclin-1, Atg3 and LC3 II / I ratio, with HPQ@K showing a particularly prominent enhancing effect.

[0150] In addition, such as Figure 28 As shown, HPQ and HPQ@K significantly inhibited the expression of aging biomarkers p21 and p16, and as... Figure 29 SA-β-Gal staining results showed that HPQ and HPQ@K significantly reduced the proportion of IL-1β-induced senescent chondrocytes, with HPQ@K showing better anti-aging effects than HPQ.

[0151] Therefore, HPQ and HPQ@K alleviate IL-1β-induced mitochondrial dysfunction, enhance antioxidant pathways, promote autophagy, and exhibit anti-aging effects.

[0152] 14. Utilizing sustained-release properties to prolong the duration of drug reactivity within the joint cavity is crucial for intra-articular drug delivery. Based on the above experimental results, this invention further validates the in vivo protective effects of HPQ and HPQ@K in DMM model mice, while also evaluating their ROS and pH response characteristics in vivo, drug release performance, and biosafety.

[0153] Cy5 reactive dye was encapsulated in HPQ to form HPQ@Cy5. In vivo fluorescence imaging analysis was performed on Cy5 and HPQ@Cy5. The Cy5 concentration in both the control and experimental groups was 10 μM, and 200 μL was injected. Results showed that free Cy5 was rapidly cleared from the joint cavity after injection (a significant decrease in D1 signal). This performance may be attributed to the ROS and pH-responsive properties of HPQ, enabling precise responsive drug release in the DMM model of arthritis, thereby prolonging the duration of drug action and enhancing therapeutic efficacy.

[0154] Based on the significant role of HPQ and HPQ@K in reversing IL-1β-induced chondrocyte inflammation and matrix metabolic imbalance in vitro in previous studies, this section further explores their protective effect in DMM model mice.

[0155] Figure 30 The X-ray imaging results showed that the cartilage gaps in the DMM model group were reduced and osteophyte formation was significant, while the cartilage morphology was significantly improved after HPQ and HPQ@K treatment, with HPQ@K showing better results.

[0156] Figure 31 Micro-CT results further validated this finding. The DMM group showed pathological features such as rough cartilage surface and osteoporosis, while HPQ and HPQ@K significantly alleviated the above-mentioned lesions, with HPQ@K showing a stronger protective effect.

[0157] Figure 32 HE staining, Safranin-O staining, and toluidine blue staining showed that the DMM group had significantly reduced cartilage matrix staining and aggravated degeneration, while HPQ and HPQ@K significantly restored the matrix staining characteristics and alleviated cartilage degeneration. Quantitative analysis showed that HPQ and HPQ@K of the present invention significantly reduced the Mankin score, OARSI score, and Safranin-O score, with HPQ@K showing better results.

[0158] Figure 33Further immunohistochemical results showed that Aggrecan expression was significantly decreased in the DMM group, while HPQ and HPQ@K treatments significantly restored Aggrecan levels, with HPQ@K showing a stronger effect. Conversely, MMP13 expression was significantly increased in the DMM group, while HPQ and HPQ@K treatments significantly inhibited MMP13 expression, with HPQ@K showing a more significant inhibitory effect. Furthermore, p62 immunohistochemical results showed that p62, an autophagy inhibition marker, was significantly increased in the DMM group, while HPQ and HPQ@K significantly decreased p62 expression, with HPQ@K showing a more significant enhancement of autophagy. Finally, p16 immunohistochemical results showed that p16, a cellular senescence marker, was significantly increased in the DMM group, while HPQ and HPQ@K significantly inhibited p16 expression, with HPQ@K exhibiting a stronger anti-aging effect.

[0159] This indicates that HPQ and HPQ@K improve cartilage degeneration in DMM model mice by enhancing autophagy and anti-aging effects, and demonstrate good in vivo persistence and biosafety.

[0160] 15. To evaluate the potential of HPQ@K in treating cartilage in human osteoarthritis (OA), this invention uses femoral medial condyle cartilage specimens derived from patients who have undergone total knee arthroplasty (TKA) as a model (both bones and joints were sourced from Tongji Medical College, Huazhong University of Science and Technology).

[0161] Figure 34 (A) H&E and Safranin-O staining revealed differences in the matrix structure of cartilage in mild and severe osteoarthritis (OA). Histological staining results showed that the cartilage matrix structure in mild OA cartilage was relatively intact, while severe OA cartilage exhibited significant matrix loss and tissue structure destruction. In subsequent experiments, OA cartilage specimens from the same total karate endocarditis (TKA) patient were divided into two groups. One group was maintained in a medium containing 50 ng / mL IL-1β to maintain gene expression of the OA phenotype for 72 hours, while the other group was simultaneously supplemented with 50 ng / mL IL-1β and 1 mg / mL HPQ@K for 72 hours. Figure 34 As shown in (B).

[0162] Figure 35Immunohistochemical results showed that HPQ@K significantly improved cartilage matrix metabolism and inhibited catabolism. In the IL-1β treatment group, aggrecan expression was significantly decreased, while HPQ@K effectively restored its expression. Conversely, MMP-13 expression was significantly increased in the IL-1β treatment group, while HPQ@K significantly inhibited MMP-13 expression. Furthermore, HPQ@K significantly enhanced chondrocyte autophagy activity by reducing the expression of the autophagy inhibition marker p62. Simultaneously, HPQ@K significantly reduced the expression of the cellular senescence marker p16, indicating a significant anti-aging effect.

[0163] This demonstrates that the HPQ@K of this invention improves the metabolism, autophagy, and aging properties of human osteoarthritis cartilage matrix, thereby achieving an effective therapeutic effect on osteoarthritis.

[0164] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin, characterized in that, The hyaluronic acid quercetin conjugate micelles loaded with kartogenin were prepared by mixing hyaluronic acid quercetin conjugate micelles and kartogenin in a fourth solvent, followed by an encapsulation reaction and purification. The mass ratio of the hyaluronic acid-based quercetin conjugate micelles to kartogenin is (5-15):1; The fourth solvent includes formamide; The encapsulation reaction was carried out at 20–28°C for 3–5 hours. The purification process includes dialysis, microporous membrane filtration, and lyophilization. The preparation method of the hyaluronic acid-based quercetin conjugate micelles includes the following steps: S1, a coupling agent is added to a hyaluronic acid solution to activate the reaction, followed by the addition of a phenylboronic acid solution for grafting, and then HA-PBA is obtained after purification. S2, HA-PBA and quercetin are mixed in an organic solvent, and the resulting mixture is subjected to grafting reaction and purification treatment to obtain hyaluronic acid-based quercetin conjugate micelles. The coupling agent includes EDC and NHS, and the phenylboronic acid includes 3-aminophenylboronic acid.

2. The method for preparing hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin according to claim 1, characterized in that, In step S1, the hyaluronic acid solution is prepared by dissolving hyaluronic acid in ultrapure water; the ratio between the carboxyl group of the hyaluronic acid and the ultrapure water is (0.5~1.5) mmol: 30 mL; The molar ratio of the carboxyl group of the hyaluronic acid to EDC and NHS is (0.5-1.5):(0.5-1.5):(0.5-1.5). The phenylboronic acid solution is prepared by dissolving phenylboronic acid in a first organic solvent, and the concentration of the phenylboronic acid solution is 0.05–0.15 mmol / mL. The molar ratio of the carboxyl group of the hyaluronic acid to the phenylboronic acid is (0.5-1.5):

1.

3. The method for preparing hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin according to claim 1, characterized in that, In step S1, the activation reaction is carried out at 20–28°C for 3.5–4.5 h; The grafting reaction described in step S1 is carried out at 20–28°C for 20–30 h. The purification process described in step S1 includes ultrapure water dialysis and lyophilization.

4. The method for preparing hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin according to claim 1, characterized in that, In step S2, the mixing of HA-PBA and quercetin in an organic solvent specifically includes: dissolving HA-PBA in a second organic solvent to obtain an HA-PBA solution; dissolving quercetin in a third organic solvent to obtain a quercetin solution; and then mixing the HA-PBA solution and the quercetin solution. The second organic solvent includes formamide; the third organic solvent includes dimethyl sulfoxide.

5. The method for preparing hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin according to claim 1, characterized in that, In step S2, the molar ratio of -B(OH)2 group and quercetin in the HA-PBA is (0.02~0.12):0.

12.

6. The method for preparing hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin according to claim 1, characterized in that, The grafting reaction described in step S2 is carried out under a protective atmosphere at 35–45°C for 40–55 h. The purification process described in step S2 includes dialysis, microporous membrane filtration, and lyophilization.

7. Hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin prepared by any one of claims 1-6.

8. The use of the hyaluronic acid-based quercetin conjugate micelles loaded with kartogenin as described in claim 7 in the preparation of a medicament for treating osteoarthritis.

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