Hyaluronic acid-based quercetin conjugate micelle as well as preparation method and application thereof

Abstract

The invention relates to a hyaluronic acid-based quercetin conjugate micelle and a preparation method and application thereof.The preparation method comprises the following steps that a coupling agent is added into a hyaluronic acid solution for an activation reaction, then a phenylboronic acid solution is added for a grafting reaction, and HA-PBA is obtained through purification treatment; the preparation method comprises the following steps: mixing HA-PBA and quercetin in an organic solvent, and 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 are subjected to an entrapment reaction to prepare a KGN-loaded hyaluronic acid-based quercetin conjugate micelle (HPQ at K); the HPQ and HPQ (at) K prepared by the invention have spherical structures, so that the bone can reach a super-lubrication state, the solubility of quercetin and KGN is improved, and the treatment and repair effects of osteoarthritis are improved.

Description

Technical Field

[0001] The invention belongs to the field of chemical pharmaceutical preparations, and specifically relates to a hyaluronic acid-quercetin conjugate micelle and a preparation method and application thereof. Background Art

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

[0003] To date, the treatment of early OA has mainly focused on single pain relief, including the use of oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of hyaluronic acid and steroids, etc., but the drugs are easily cleared by the body, resulting in poor treatment effects and easy recurrence of the disease. Based on the high ROS and low pH environment in osteoarthritis lesions, prodrug systems with ROS and pH responses have always been a hot topic in osteoarthritis research, but most drug delivery systems mainly target one or two aspects of lubrication, anti-inflammation and induction of chondrogenesis, which is still limited for the treatment of more severe OA. Therefore, it is urgent to develop a means to inhibit the development of OA from multiple aspects of lubrication, anti-inflammation and induction of chondrogenesis. Summary of the invention

[0004] The purpose of the present invention is to overcome the above technical deficiencies, provide a hyaluronic acid-based quercetin conjugate micelle and a preparation method and application thereof, and solve the technical problem that the prior art has a poor single-aspect therapeutic effect on osteoarthritis.

[0005] In order to achieve the above technical purpose, the technical solution provided by the present invention is: In a first aspect, the present invention provides a method for preparing hyaluronic acid-quercetin conjugate micelles, comprising the following steps: S1, adding a coupling agent to a hyaluronic acid solution for activation reaction, then adding a phenylboric acid solution for grafting reaction, and obtaining HA-PBA through purification; S2, mixing HA-PBA and quercetin in an organic solvent, and obtaining hyaluronic acid-quercetin conjugate micelles through grafting reaction and purification of the obtained mixed solution.

[0006] In a second aspect, the present invention provides a hyaluronic acid-quercetin conjugate micelle prepared by the above preparation method.

[0007] In a third aspect, the present invention provides a KGN-loaded hyaluronic acid-quercetin conjugate micelle, which is prepared by mixing the hyaluronic acid-quercetin conjugate micelle and KGN in a fourth solvent, and subjecting the mixture to encapsulation reaction and purification.

[0008] In a fourth aspect, the present invention provides a use of a hyaluronic acid-quercetin conjugate micelle or a KGN-loaded hyaluronic acid-quercetin conjugate micelle in the preparation of a drug for inhibiting / treating osteoarthritis.

[0009] Compared with the prior art, the beneficial effects of the present invention include: The present invention grafts quercetin onto hyaluronic acid through 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 the present invention have a spherical structure, can make the bone space reach a super-lubricated state, and improve the solubility of quercetin and KGN, improve the bioavailability of quercetin and KGN, and bring into play the dual-drug synergistic effect of quercetin and KGN, and combine the lubrication of nanoparticles, the reduction of the expression of inflammatory factors and the scavenging of free radicals by QUT, and the induction of cartilage formation by KGN into one, so as to jointly treat osteoarthritis from multiple aspects and improve the treatment and repair effects of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the HP obtained in Example 1 of the present invention 1 H NMR spectrum; Figure 2 is the infrared spectra of the raw materials and products of HP prepared in Example 1 of the present invention (HA, PBA, HP); Figure 3 is the HPQ obtained in Example 1 of the present invention 1 H NMR spectrum; Figure 4 is the infrared spectra of the raw materials and products (HA, QUT, KGN, HPQ, HPQ@K) of Example 2 of the present invention; Figure 5 is the critical micelle concentration of the product HPQ of Example 1 of the present invention; Figure 6 The particle size and potential diagram of the product HPQ of Example 1 of the present invention and HPQ@K after being loaded with HPQ and KGN at different mass ratios in Examples 2-4; Figure 7 The figures are the particle size distribution diagram and electron microscope morphology diagram of HPQ obtained in Example 1 and HPQ@K obtained in Example 2 of the present invention; wherein, (A) HPQ obtained in Example 1, (B) HPQ@K obtained in Example 2; Figure 8 is the friction coefficient of HPQ@K obtained in Example 2 of the present invention at the dosage concentration simulating the bone joint environment; Fig. 9 1 is a graph showing the inhibition of hyaluronidase activity by HPQ@K obtained in Example 2 of the present invention; wherein (A) is the inhibition rate of hyaluronidase by HPQ@K; (B) is the viscosity change curve of hyaluronic acid after HPQ@K inhibits hyaluronidase; Fig.10 1 is a graph showing the test results of HPQ@K obtained in Example 2 of the present invention for scavenging DPPH free radicals; wherein (A) the scavenging rate of HPQ@K for DPPH free radicals; (B) the ultraviolet spectrum of HPQ@K for scavenging DPPH free radicals; Fig.11 This is the cumulative release curve of HPQ@K obtained in Example 2 of the present invention to QUT and KGN under different conditions; (A) QUT, (B) KGN; Fig.12 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 change 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 change curve of HPQ@K under pH=7.4 and pH=5.0 conditions within 8 hours; (C) is the particle size distribution change curve of HPQ@K under pH=7.4 and pH=7.4, c(H2O2)=0.1mM conditions within 8 hours; (D) is the particle size distribution change curve of HPQ@K under pH=7.4 and pH=6.5 conditions within 8 hours; Fig.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; wherein (A) is the detection result of HPQ at 24h and 48h; (B) is the detection result of HPQ@K at 24h and 48h; Fig.14 is the expression of inflammation-related genes, matrix decomposition-related genes and matrix synthesis-related genes after IL-1β stimulation for 24 hours in HPQ obtained in Example 1 of the present invention; Fig.15 The expression of inflammation-related genes, matrix decomposition-related genes and matrix synthesis-related genes in HPQ@K obtained in Example 2 of the present invention after IL-1β stimulation for 24 hours; Fig.16 Live / Dead staining results of chondrocytes after 24 hours of intervention with HPQ (0.1 mg / mL) obtained in Example 1 of the present invention and HPQ@K (0.1 mg / mL) obtained in Example 2; Fig.17 Live / Dead staining results of chondrocytes after 48 hours of intervention with HPQ (0.1 mg / mL) obtained in Example 1 of the present invention and HPQ@K (0.1 mg / mL) obtained in Example 2; Fig.18 It is the immunoblot of HPQ obtained in Example 1 of the present invention and HPQ@K obtained in Example 2 after IL-1β stimulated chondrocytes for 24 hours; Fig.19 It is the quantitative analysis result of HPQ obtained in Example 1 of the present invention and HPQ@K obtained in Example 2 after IL-1β stimulated chondrocytes for 24 hours; Fig. 20 It is the immunoblot of HPQ obtained in Example 1 of the present invention and HPQ@K obtained in Example 2 after IL-1β stimulated chondrocytes for 48 hours; Fig.21 It is the quantitative analysis result of HPQ obtained in Example 1 of the present invention and HPQ@K obtained in Example 2 after IL-1β stimulated chondrocytes for 48 hours; Fig. 22 This is a JC-1 staining image of HPQ obtained in Example 1 of the present invention and HPQ@K obtained in Example 2 after IL-1β stimulated chondrocytes; Fig.23 1 is a transmission electron microscopic image of mitochondria of HPQ obtained in Example 1 of the present invention and HPQ@K obtained in Example 2 after IL-1β stimulation of chondrocytes; Fig.24 It is the immunoblot of the key proteins of the antioxidant pathway of HPQ obtained in Example 1 of the present invention and HPQ@K obtained in Example 2; Fig.25 Yes Fig.23 Quantitative analysis of immunoblots; Fig.26 is mRFP-GFP-LC3 fluorescence detection; Fig. 27 It is the result of autophagy level test; Fig.28 is the expression of aging markers; Fig.29 is the SA-β-Gal staining result; Fig.30 This is an X-ray image showing that HPQ and HPQ@K improve cartilage degeneration in DMM model mice; Fig.31 This is the Micro-CT image of HPQ and HPQ@K improving cartilage degeneration in DMM model mice; Fig.32 This is the staining image showing that HPQ and HPQ@K improve cartilage degeneration in DMM model mice; Fig.33 The immunohistochemical results of Aggrecan, MMP13, p62 and p16 in improving cartilage degeneration in DMM model mice by HPQ and HPQ@K; Fig.34 The figure shows the histological staining results and experimental schematic diagram of OA cartilage; (A) is the staining result, and (B) is the schematic diagram of the grouping and processing flow of OA cartilage specimens; Fig.35The immunohistochemical results of Aggrecan, MMP13, p62 and p16 in improving OA cartilage by HPQ@K. DETAILED DESCRIPTION

[0011] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0012] Explanation of terms QUT: quercetin; HA: hyaluronic acid; KGN: kartogenin; EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide; NHS: N-hydroxysuccinimide; PBA: 3-aminophenylboronic acid; DMSO: dimethyl sulfoxide.

[0013] In view of the defect that most drug delivery systems in the prior art are mainly single-aspect treatments for osteoarthritis, resulting in poor effects, the present invention provides a hyaluronic acid-quercetin conjugate micelle and a preparation method and application thereof. The micelle is a hyaluronic acid-quercetin conjugate micelle with ROS and pH response, and KGN can be loaded according to the situation; wherein, hyaluronic acid (HA) plays a role in lubricating bones and joints, quercetin (QUT) has free radical scavenging and anti-inflammatory functions to treat cartilage damage, and KGN can enhance CBFβ cell nuclear localization, induce hMSCs to differentiate into chondrocytes, and protect articular chondrocytes, thereby achieving a certain effect of treating OA.

[0014] However, since QUT and KGN are completely hydrophobic small molecule drugs, if they are directly mixed, they are not conducive to cell uptake and both show low bioavailability. The present invention grafts quercetin (QUT) onto hyaluronic acid (HA) through 3-aminophenylboronic acid to form HPQ micelles, and encapsulates KGN through dialysis to form drug-loaded micelles HPQ@K. The present invention utilizes the spherical structure of the micelle to achieve a superlubricating state between bones, and improves the solubility of QUT and KGN, improves the bioavailability of QUT and KGN, and exerts the dual-drug synergistic effect of QUT and KGN, combining nanoparticle lubrication, QUT to reduce the expression of inflammatory factors and scavenge free radicals, and KGN to induce cartilage formation, which is used for the treatment and repair of osteoarthritis, enhances autophagy and anti-aging, and treats OA from multiple aspects to achieve synergy.

[0015] In a first aspect, the present invention provides a method for preparing hyaluronic acid-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 obtaining HA-PBA after purification; S2, HA-PBA and quercetin are mixed in an organic solvent, and the resulting mixture is subjected to grafting reaction and purification to obtain hyaluronic acid-quercetin conjugate micelles.

[0016] Preferably, in step S1, the hyaluronic acid solution is prepared by dissolving hyaluronic acid in ultrapure water, the molecular weight of the hyaluronic acid is 5000Da to 15000Da; the ratio between the carboxyl group of the hyaluronic acid and the ultrapure water is (0.5 to 1.5) mmol:30mL.

[0017] 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).

[0018] Preferably, in step S1, the activation reaction is carried out at 20-28° C. for 3.5-4.5 h.

[0019] Preferably, in step S1, the phenylboric acid solution is prepared by dissolving phenylboric acid in a first organic solvent, and the concentration of the phenylboric acid solution is 0.05 to 0.15 mmol / mL.

[0020] Further preferably, the first organic solvent includes dimethyl sulfoxide. DMSO used in the present invention is a highly polar solvent. Since the solubility of 3-aminophenylboronic acid in water is limited, part of DMSO can increase the solubility of 3-aminophenylboronic acid in the reaction process, and hyaluronic acid is a polysaccharide different from organic solvents, but it will not precipitate in the mixed system of pure water and DMSO, so the solvent system used in the present invention can improve the reaction yield.

[0021] Preferably, in step S1, the phenylboronic acid includes 3-aminophenylboronic acid, and the molar ratio of the carboxyl group of hyaluronic acid to the phenylboronic acid is (0.5-1.5): 1. More preferably, the molar ratio of the carboxyl group of hyaluronic acid to the phenylboronic acid is 1:1.

[0022] Preferably, in step S1, the grafting reaction is carried out at 20-28° C. for 20-30 hours.

[0023] Preferably, in step S1, the purification treatment includes ultrapure water dialysis for 48 to 72 hours and freeze-drying.

[0024] Preferably, in step S2, mixing HA-PBA and quercetin in an organic solvent specifically comprises: dissolving HA-PBA in a second organic solvent to obtain a 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.

[0025] Further preferably, the HA-PBA solution is obtained by adding HA-PBA to a second organic solvent under a protective atmosphere and stirring and dissolving at 40-45° C.; the second organic solvent includes formamide; and the third organic solvent includes dimethyl sulfoxide.

[0026] Preferably, in step S2, the molar ratio of -B(OH)2 group in HA-PBA to quercetin is (0.02-0.12):0.12.

[0027] Preferably, in step S2, the grafting reaction is carried out under a protective atmosphere at 35-45° C. for 40-55 hours.

[0028] Preferably, in step S2, the purification treatment is performed by dialyzing with ultrapure water for 48 to 72 hours, filtering with a 0.4 to 0.5 μm microporous filter membrane, and then freeze-drying.

[0029] The molecular weight cutoff of the dialysis bag in the dialysis process of the present invention is 800-1200Da. If the molecular weight is too low, small molecule drug monomers cannot be removed, and if the molecular weight is too large, the final product is lost.

[0030] In a second aspect, the present invention provides a hyaluronic acid-quercetin conjugate micelle prepared by the above preparation method.

[0031] In a third aspect, the present invention provides a KGN-loaded hyaluronic acid-quercetin conjugate micelle, which is prepared by mixing the hyaluronic acid-quercetin conjugate micelle and KGN in a fourth solvent, and subjecting the mixture to encapsulation reaction and purification.

[0032] Preferably, the mass ratio of hyaluronic acid-quercetin conjugate micelles to KGN is (5-15):1.

[0033] More preferably, the mass ratio of hyaluronan-quercetin conjugate micelles to KGN is (10-15):1.

[0034] Preferably, the fourth solvent comprises formamide.

[0035] Preferably, the encapsulation reaction is carried out at 20-28°C for 3-5 hours.

[0036] Preferably, the purification is performed by dialyzing for 40 to 60 hours using a dialysis bag with a molecular weight cutoff of not less than 800 Da, and then filtering using a 0.4 to 0.5 μm microporous filter membrane and freeze-drying.

[0037] In a fourth aspect, the present invention provides a use of a hyaluronic acid-quercetin conjugate micelle or a KGN-loaded hyaluronic acid-quercetin conjugate micelle in the preparation of a drug for inhibiting / treating osteoarthritis.

[0038] As a preferred embodiment, the main reaction route of the hyaluronic acid-quercetin conjugate micelle of the present invention includes: (1) Preparation of HP: hyaluronic acid (0.5mmol≤n (-COOH) ≤1.5mmol) in coupling agent (0.5mmol≤n EDC ≤1.5mmol, 0.5mmol≤n NHS ≤1.5mmol) to activate -COOH, and then graft with phenylboronic acid (PBA=1mmol). After the reaction, dialyze with ultrapure water, and then freeze-dry to obtain HA-PBA (HP) raw material, as shown in formula (I):

[0039] Formula (I) In formula (I), m=1, 0.1≤n≤0.3; (2) Preparation of HPQ: HP (where 0.02≤n (-B(OH)2 ≤0.12mmol) was dissolved in formamide (heated and stirred at 45°C under nitrogen atmosphere), then 36.24mg (0.12mmol) of quercetin was weighed and dissolved in DMSO, and added to the above solution after quercetin was dissolved. Under nitrogen atmosphere, react at 40°C for 48h, dialyze for 72h after 48h, and then filter with a 0.45μm microporous filter membrane to remove large aggregates to obtain HPQ, which was freeze-dried for storage; as shown in formula (II):

[0040] Formula (II) In formula (II), m=1, 0.1≤n≤0.3.

[0041] The present invention adopts a single reaction of phenylboronic acid and quercetin to avoid side reactions; and the reaction of o-diphenol hydroxyl group and phenylboronic acid to generate boric acid ester in formula II is a dehydration reaction. Compared with most studies that react in water, the present invention can significantly improve the reaction degree of phenylboronic acid and quercetin by using an organic solvent. 1 H NMR integration revealed that the degree of substitution of phenylboronic acid was consistent with that of quercetin, which indicated that the phenylboronic acid on the hyaluronic acid in the present invention reacted completely with quercetin.

[0042] As a preferred embodiment, the present invention also provides a KGN-loaded hyaluronic acid-based quercetin conjugate micelle (HPQ@K), which is prepared by dissolving the HPQ freeze-dried product, adding KGN (Kartogenin), stirring at room temperature for a period of time, transferring the solution to a dialysis bag, and dialyzing with ultrapure water to remove unencapsulated free small molecules. After the dialysis, the aggregates are removed by filtration through a microporous membrane, and the product is finally freeze-dried and stored. The unique structure of HPQ@K of the present invention gives the material ROS and pH responses, which is conducive to the accumulation of drugs in lesions, and integrates nano-lubrication, QUT anti-inflammatory, and KGN to promote cartilage formation, providing a new idea for OA treatment.

[0043] The present invention is further described in detail below through specific examples.

[0044] Example 1 (1) Preparation of HP: Weigh 379 mg of hyaluronic acid (HA) (n(-COOH) = 1 mmol) and place it in a beaker containing 30 mL of ultrapure water and stir. After HA is dissolved, add 155 mg (1 mmol) of EDC and 115 mg (1 mmol) of NHS, and activate -COOH at room temperature for 4 hours to obtain the first reaction solution. After 4 hours, weigh 137 mg (1 mol) of 3-aminophenylboronic acid (PBA) and dissolve it in 10 mL of DMSO, then add it to the above first reaction solution and react at room temperature for 24 hours to obtain the second reaction solution. The second reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, dialyzed with ultrapure water for 72 hours, and then freeze-dried to obtain the HA-PBA (abbreviated as HP) raw material.

[0045] (2) Preparation of HPQ: About 102 mg of HA-PBA (n(-B(OH)2) = 0.06 mmol) was weighed and dissolved in 10 mL of formamide (the solution was heated and stirred at 45°C under a nitrogen atmosphere) to obtain a HA-PBA solution; then 36.24 mg (0.12 mmol) of quercetin was weighed and dissolved in 5 mL of DMSO to obtain a quercetin solution; the quercetin solution was added to the HA-PBA solution, and the reaction was carried out at 40°C under a nitrogen atmosphere for 48 h. After 48 h, the solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, dialyzed with ultrapure water for 72 h, and then filtered with a 0.45 μm microporous filter membrane to remove large aggregates. The product HA-PBA-QUT (abbreviated as HPQ) was obtained by lyophilization.

[0046] Example 2 Preparation of HPQ@K: Weigh 45 mg of the lyophilized HPQ sample prepared in Example 1 and dissolve it in 10 mL of formamide, then add 4.5 mg of KGN (Kartogenin), that is, m(HPQ):m(KGN)=10:1, and stir at room temperature for 4 hours. After the reaction is completed, the solution is 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 the dialysis is completed, the aggregates are removed by filtration through a 0.45 μm microporous membrane, and the product HA-PBA-QUT@KGN (abbreviated as HPQ@K) is obtained by lyophilization.

[0047] Example 3 The only difference from Example 2 is that the amount of KGN is adjusted to 9 mg, that is, m(HPQ):m(KGN)=5:1, and the other steps and conditions are the same as Example 2.

[0048] Example 4 The only difference from Example 2 is that the amount of KGN is adjusted to 3 mg, that is, m(HPQ):m(KGN)=15:1, and the other steps and conditions are the same as Example 2.

[0049] Performance Testing 1. The HP prepared in Example 1 was subjected to NMR and infrared detection. 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 7.0-8.0ppm, which indicates the successful synthesis of HP. The absence of characteristic peaks of hydrogen atoms of hydroxyl (-OH) and amino (-NH2) on PBA in HP is because HP is measured in D2O, and the active atoms are easily replaced by deuterium atoms in the solution. Figure 2 It can be seen that in the infrared spectrum of HA, 2928cm -1 and 2882cm -1 Corresponding to the characteristic peaks of methyl (-CH3) and methylene (-CH2) stretching vibration in hyaluronic acid. 1622.8cm -1 The characteristic peak corresponding to the stretching vibration of C=O in the carboxyl group (-COOH) of hyaluronic acid is 1665.2 cm -1 The characteristic peak corresponding to the C=O stretching vibration of the imide (-CONH-) in hyaluronic acid is 1557.7 cm -1 The characteristic peaks corresponding to the in-plane bending vibration of NH in the imide (-CONH-) in hyaluronic acid. In HP, the characteristic peaks corresponding to HA can be found respectively. 1 HNMR and FT-IR can verify the successful preparation of HP.

[0050] 2. The HPQ obtained in Example 1 was subjected to NMR testing, and the raw materials and products in Example 1 and the raw materials and products in Example 2 were subjected to infrared testing. The results are as follows: Figure 3 to Figure 4 shown.

[0051] like Figure 3 As shown in the figure, the signals in the δ6.0-9.0 region are assigned to the signals of the aromatic hydrogen of QUT and PBA, indicating that HPQ was successfully synthesized. Peaks 1, 2, and 10 were used as integral positioning peaks, and the peak area of ​​the peak position region where PBA and QUT exist was integrated using MestRenova. The integral results are shown in the figure. Figure 3 The degree of substitution is calculated to be about 20% through the integration results, and the drug loading of QUT is about 12.9%. 1 H NMR confirmed the successful synthesis of HPQ.

[0052] like Figure 4 As shown in the infrared spectrum of HA, 1622.8 cm -1 The characteristic peak corresponding to the C=O stretching vibration of the carboxyl group (-COOH) in hyaluronic acid is 1665.2 cm -1 Corresponding to the stretching vibration peak of (-CONH-)C=O in hyaluronic acid, 1557.7cm -1 The peak corresponding to the in-plane bending vibration of (-CONH-)NH can be clearly observed in the spectrum of HPQ, which is 1622.8cm -1 The absorption peak at the carboxyl position was weakened, and 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 the amino group were covalently bonded and the basic structure of HA was preserved. -1 ~900cm -1 and 1100cm -1 ~1500cm -1 Within this range, a large number of characteristic peaks of QUT appeared in the infrared spectrum of HPQ, proving the successful synthesis of HPQ.

[0053] At the same time, by Figure 4 The infrared spectrum of HPQ@K shows that compared with HPQ, HPQ@K obviously has the aromatic carboxyl group (Ar-COOH) on KGN at 1719.7 cm -1 The characteristic peaks of amide (-CONH-) and amide (-CONH-) are at 3318.8 cm -1 The characteristic peak of NH stretching vibration indicates that KGN was successfully encapsulated.

[0054] 3. Figure 5The 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 be presented in nanoform in water at a lower concentration. The low CMC value means that the micelles can self-assemble at very dilute concentrations, which will maintain the stability of the micelles in the blood circulation.

[0055] 4. The particle size (d), potential, drug loading (DL), and encapsulation efficiency (EE) of the product HPQ of Example 1 and HPQ@K encapsulated with different mass ratios of HPQ and KGN in Examples 2-4 were tested, wherein the particle size and potential were measured by a particle size analyzer (DLS), and the drug loading and encapsulation efficiency were quantitatively measured by HPLC. The results are shown in Tables 1 and Figure 6 shown.

[0056] Table 1 Particle size (d), potential, drug loading, and encapsulation efficiency of different encapsulation ratios

[0057] like Figure 6 As shown in Table 1, it is the result of optimizing the ratio in the preparation process of HPQ@K. When the dosage of hyaluronic acid is 379 mg, the dosage of EDC is 155 mg, and the dosage of NHS is 115 mg, the micelle particle size is about 150.53±3.14 nm, and the Zeta potential is about -48.26±0.95 mv. The prepared HPQ is most suitable. 1 HNMR determined that the ratio of repeating units containing QUT in HPQ was 0.2.

[0058] With the increase of KGN ratio, the particle size and Zeta potential of drug-loaded micelles gradually increased. The particle size of HPQ was 150.53nm and the Zeta potential was -48.26mV; when m(HPQ):m(KGN)=15:1, the particle size was 164.33nm and the Zeta potential was -38.63mV; when m(HPQ):m(KGN)=10:1, the particle size was 178.63nm and the Zeta potential was -32.40mV; when m(HPQ):m(KGN)=5:1, the particle size reached 344.16nm and the Zeta potential was -25.63mV. The above results show that KGN was successfully encapsulated. However, when m(HPQ):m(KGN)=5:1, the particle size increased sharply, exceeding the encapsulation upper limit of HPQ. When the particle size of nanoparticles is too large, it is not conducive to cell uptake and may cause cell necrosis, which is not conducive to subsequent experiments.

[0059] Both the groups with m(HPQ):m(KGN)=15:1 and 10:1 showed excellent particle size results. In order 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, the encapsulation efficiency and drug loading were reduced. The encapsulation efficiency was lower than that of m(HPQ):m(KGN)=10:1. This may be due to the low concentration of KGN in the solution, which could not fully contact the hydrophobic segment, resulting in a low drug encapsulation efficiency during self-assembly. In summary, the particle size, potential, encapsulation efficiency and drug loading of HPQ@K obtained when m(HPQ):m(KGN)=10:1 were the most suitable, so HPQ@K with m(HPQ):m(KGN)=10:1 was selected for subsequent testing.

[0060] 5. Morphology and particle size distribution of HPQ and HPQ@K Figure 7 (A) and (B) show the particle size distribution and electron microscopy morphology of empty shell micelles HPQ and drug-loaded micelles HPQ@K. It can be found that both HPQ and HPQ@K have a uniform distribution. From the transmission electron microscopy image, it can be observed that both have a relatively regular circular morphology. Whether from the particle size distribution or the transmission electron microscopy image, it can be observed that the particle size of HPQ@K is slightly larger than that of HPQ, indicating that KGN is successfully encapsulated. There is a certain difference between the nanosize measured by the transmission electron microscope and the results measured by the particle size analyzer (DLS). The particle size measured by TEM is smaller than that measured by DLS. The difference in particle size may be due to the shrinkage of the micelle shell when the TEM sample is prepared during the drying process, while DLS measures the size of the hydrated particles.

[0061] 6. Determination of friction coefficient of HPQ@K in simulated bone and joint environment In order to determine the friction coefficient of HPQ@K at the drug concentration (1 mg / mL), after the micelles were formed, due to the advantages of the spherical structure, a reciprocating mode was adopted with a reciprocating frequency of 5 Hz, an amplitude of 4 mm, a loading force of 5 N, and an upper sample of a polyethylene (PE) ball with a diameter of 8 mm to simulate the upper part of the joint. The lower sample was a polished Ti6Al4V disk with a surface roughness of 1.7 nm to simulate the lower part of the joint. The HPQ@K micelles were located between the upper and lower samples. The reciprocating time was 40 min to simulate the bone joint environment.

[0062] The results are as follows Figure 8 As shown, the measured friction coefficient is only 0.008, reaching a super-lubricated state, which can effectively slow down the wear of bone and joint activities and the destruction of surface articular cartilage, and is beneficial to the recovery of osteoarthritis.

[0063] 7. Inhibition of hyaluronidase by HPQ@K The presence of QUT in the HPQ@K structure gives HPQ@K a certain ability to inhibit hyaluronidase activity, which is very beneficial for promoting the clearance of inflammatory factors in interosseous joints and maintaining the micelle morphology. Five groups of HPQ@K with concentrations of 5 mg / mL, 2.5 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.1 mg / mL were set up for enzyme inhibition experiments (the hyaluronidase concentration was 8u / mL; and in all experimental groups, the concentration of hyaluronic acid was 10 mg / mL), and a control group was set up (the only difference was that no hyaluronidase was added); the hyaluronidase inhibition effect was calculated by measuring the ultraviolet absorption intensity at 540 nm, and the viscosity of the hyaluronic acid in each group was determined by a rotational rheometer.

[0064] Fig. 9 (A) shows the inhibition results of HPQ@K on hyaluronidase. When the concentration of HPQ@K reached 2.5 mg / mL, the inhibition rate reached 53.86%.

[0065] Fig. 9 (B) shows the change in hyaluronic acid viscosity in the experimental system after the hyaluronidase inhibition experiment. Compared with the control group without enzyme addition, as the concentration of HPQ@K decreases, the inhibitory effect on hyaluronidase decreases, and the viscosity of the sample gradually decreases, indicating that hyaluronic acid has been degraded, proving that HPQ@K has an inhibitory effect on hyaluronidase.

[0066] 8. HPQ@K scavenges free radicals Five groups were set up for free radical scavenging experiments according to the concentrations of HPQ@K, namely 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL. The concentration of DPPH in each group was 150 μM, and the free radical scavenging effect was calculated by measuring the ultraviolet absorption intensity at 517 nm.

[0067] Fig.10 (B) shows the free radical scavenging ability of HPQ@K. Fig.10 (A) Yes Fig.10 From the quantitative results at 517 nm in (B), we can see that when the concentration of HPQ@K is only 0.1 mg / mL, the DPPH scavenging rate can reach 68.86%, and it can be as high as nearly 90% at 0.25 mg / mL. The high DPPH scavenging rate at lower concentrations demonstrates the powerful free radical scavenging ability of HPQ@K, which is due to the powerful free radical scavenging ability of QUT. It can have a great positive impact on the recovery of bone and joint diseases, indicating the excellent potential of HPQ@K in the treatment of osteoarthritis.

[0068] 9. pH and ROS responsiveness of HPQ@K The following experimental groups were set up: (1) HPQ@K was added to PBS buffer at pH 5.0; (2) HPQ@K was added to PBS buffer with a pH of 7.4 and 0.1 mM H2O2; (3) HPQ@K was added to PBS buffer at pH 6.5; (4) HPQ@K was added to PBS buffer at pH 7.4; The concentration of HPQ@K in each experimental group was 1 mg / mL, and the cumulative release curves of QUT and KGN in each experimental group within 72 h were determined by HPLC.

[0069] The results are as follows Fig.11 As shown in the figure, under the condition of pH 5.0, the cumulative release of QUT and KGN in 72h was 76.06% and 82.61%, respectively. Under the condition of pH 7.4 + 0.1mM H2O2, the cumulative release of QUT and KGN in 72h was 65.60% and 67.56%, respectively; HPQ@K showed excellent pH and ROS response ability, which was beneficial to the accumulation of drugs in lesions. In comparison, under the condition of pH 7.4, the cumulative release of QUT and KGN in 72h was only 10.15% and 10.61%, respectively. Therefore, HPQ@K of the present invention has excellent dual response potential.

[0070] 10. Serum stability of HPQ@K and particle size changes under different conditions The particle size and potential of the micelles under physiological conditions (simulation conditions are the same as those of experimental group 4 in the previous article, i.e., PBS solution with pH = 7.4) as well as the particle size change curve of the micelles under simulated lesion microenvironment (simulation conditions are the same as those of experimental groups 1-3 in the previous article) were measured by a laser particle size analyzer.

[0071] Fig.12 (A) Particle sizes of HPQ and HPQ@K in serum within 7 days, due to the electrostatic repulsion of negatively charged micelles, which reduces the nonspecific adsorption between micelles and negatively charged proteins, indicating the excellent stability of HPQ and HPQ@K under normal physiological conditions. Fig.12 (B-D) are the results of the particle size changes of HPQ@K exposed to various simulated environments for 8 hours. For HPQ@K micelles, no obvious changes in size over time were observed at pH 7.4. On the contrary, when HPQ@K micelles were incubated in PBS at pH 5.0, the size of HPQ@K micelles was observed to increase to 591.4nm, and when incubated in a pH 7.4+0.1mM H2O2 environment, the size of HPQ@K micelles was observed to increase to 538.0nm. The results show that HPQ@K micelles can maintain structural integrity in blood circulation and have pH and ROS responsiveness, which is conducive to the controlled release of drugs.

[0072] 11. To verify the effects of HPQ and HPQ@K in reducing toxicity and regulating inflammatory response, the present invention detected their effects on chondrocyte viability through CCK-8 experiment and compared them with free drugs KGN and QUT.

[0073] The results are as follows Fig.13 As shown in Figure 2, HPQ and HPQ@K showed no significant toxicity at concentrations ≤ 0.1 mg / ml, and only showed a decrease in cell viability at 0.2 mg / mL ( Fig.13 (A and B), indicating that they significantly reduced the toxicity of KGN and QUT.

[0074] The regulatory effects of HPQ and HPQ@K on chondrocyte gene expression under IL-1β stimulation after 24-hour intervention were further analyzed by PCR. Fig.14 and Fig.15 PCR results showed that after 24 hours of IL-1β stimulation, HPQ and HPQ@K nanoparticles significantly downregulated the expression of inflammation-related genes (iNOS, COX2) and matrix decomposition-related genes (MMP3, MMP13), and significantly upregulated the expression of matrix synthesis-related genes (Aggrecan, Collagen Ⅱ, SOX9), showing concentration dependence, reaching the best at 0.1 mg / ml.

[0075] In addition, Live / Dead staining results further verified the safety of HPQ and HPQ@K at a concentration of 0.1 mg / mL. Fig.16 and Fig.17 As shown, green represents surviving cells and red represents dead cells. After 24 and 48 hours of treatment, the survival rate of chondrocytes remained high, and no obvious cell death was observed. In summary, HPQ and HPQ@K reduced the toxicity of free KGN and QUT, enhanced their anti-inflammatory and matrix protection effects with increasing concentrations, and achieved the best effect at 0.1 mg / ml.

[0076] 12. In order to verify the sustained-release effect of HPQ and HPQ@K and their regulatory effect on IL-1β-stimulated chondrocytes at the protein level, the present invention used a concentration of 0.1 mg / mL to intervene in 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.

[0077] Western blot results showed that ( Fig.18 and Fig.19After 24 hours of intervention, both HPQ and HPQ@K significantly restored the expression of matrix-related 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 ( Fig.21 and Fig.21 ), the regulatory effects of HPQ and HPQ@K were further enhanced, as shown by more significant upregulation of matrix protein Aggrecan, Collagen II, and SOX9 (promoting matrix protein recovery), and stronger inhibition of pro-inflammatory and decomposition protein expression. 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 continue to exert regulatory effects with extended action time. Among them, the regulatory effect of HPQ@K at 48 hours was significantly better than that of HPQ, especially in enhancing matrix protein recovery and inhibiting pro-inflammatory and decomposition protein expression.

[0078] 13. In order to evaluate the regulatory effect of HPQ and HPQ@K on IL-1β-induced chondrocyte damage, the present invention systematically analyzed the indicators related to oxidative stress, autophagy and aging with mitochondrial function as the core.

[0079] Fig. 22 JC-1 staining and mitochondrial ATP detection showed that both HPQ and HPQ@K could significantly reverse IL-1β-induced mitochondrial membrane potential depolarization and functional damage, and the effect of HPQ@K was more significant. Fig.23 Transmission electron microscopy showed that HPQ and HPQ@K significantly improved the mitochondrial structural damage caused by IL-1β, and HPQ@K had a better protective effect, which was beneficial to improving the mitochondrial structural integrity.

[0080] In addition, if Fig.24 and Fig.25 As shown in the results, HPQ and HPQ@K significantly inhibited ROS generation and enhanced antioxidant capacity by upregulating the expressions of key antioxidant pathway proteins NRF2, GCLC, HO-1, and GCLM, with HPQ@K having a stronger effect.

[0081] like Fig.26 and Fig. 27 As shown in the figure, in terms of autophagy, dual fluorescence labeling and Western blot analysis showed that HPQ and HPQ@K could restore the decreased autophagy level induced by IL-1β, and significantly enhance the autophagy activity by downregulating p62 and upregulating Beclin-1, Atg3 and LC3 II / I ratio, and the enhancing effect of HPQ@K was particularly prominent.

[0082] In addition, if Fig.28As shown in Figure 2, HPQ and HPQ@K significantly inhibited the expression of senescence markers p21 and p16. Fig.29 The SA-β-Gal staining results showed that HPQ and HPQ@K significantly reduced the IL-1β-induced chondrocyte senescence rate, and the anti-aging effect of HPQ@K was better than that of HPQ.

[0083] Therefore, HPQ and HPQ@K alleviated IL-1β-induced mitochondrial dysfunction, enhanced antioxidant pathways, promoted autophagy, and exhibited anti-aging effects.

[0084] 14. Prolonging the duration of drug release in the joint cavity is crucial for intra-articular administration. Based on the above experimental results, the present invention further verified the in vivo protective effects of HPQ and HPQ@K in DMM model mice, and evaluated their ROS and pH response characteristics in vivo retention and drug release performance, as well as their biosafety.

[0085] Cy5 active dye was encapsulated into HPQ to become HPQ@Cy5. Cy5 and HPQ@Cy5 were subjected to in vivo fluorescence imaging analysis. The Cy5 concentration in the control group and the experimental group was 10μM in the injected solution, and 200μL was injected. The results showed that free Cy5 was quickly cleared from the joint cavity after injection (the D1 signal decreased significantly). This performance may be due to the ROS and pH responsiveness of HPQ, which achieves precise responsive drug release in the DMM model arthritis environment, thereby prolonging the drug action time and enhancing the therapeutic effect.

[0086] Based on the previous studies that HPQ and HPQ@K significantly reversed IL-1β-induced chondrocyte inflammation and matrix metabolism imbalance in vitro, this section further explored their protective effects in DMM model mice.

[0087] Fig.30 The X-ray imaging results showed that the cartilage gap in the DMM model group was reduced and osteophyte formation was significant, while after treatment with HPQ and HPQ@K, the cartilage morphology was significantly improved, and the effect of HPQ@K was better.

[0088] Fig.31 The Micro-CT results further verified 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, and HPQ@K showed a stronger protective effect.

[0089] Fig.32HE staining, Safranin-O staining and toluidine blue staining showed that the cartilage matrix staining in the DMM group was significantly reduced and the degeneration was aggravated, 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, and HPQ@K had a better effect.

[0090] Fig.33 The immunohistochemical results further showed that the Aggrecan immunohistochemical results showed that the expression of Aggrecan in the DMM group decreased significantly, while HPQ and HPQ@K treatment significantly restored the Aggrecan level, and HPQ@K had a stronger effect. On the contrary, MMP13 immunohistochemistry showed that the expression of MMP13 in the DMM group increased significantly, while HPQ and HPQ@K treatment significantly inhibited the expression of MMP13, and the inhibitory effect of HPQ@K was more significant. In addition, the p62 immunohistochemical results showed that the autophagy inhibition marker p62 in the DMM group increased significantly, while HPQ and HPQ@K significantly reduced the expression of p62, and HPQ@K enhanced autophagy more significantly. Finally, the p16 immunohistochemical results showed that the cell senescence marker p16 in the DMM group increased significantly, while HPQ and HPQ@K significantly inhibited the expression of p16, and HPQ@K showed a stronger anti-aging effect.

[0091] These results suggest that HPQ and HPQ@K improve cartilage degeneration in DMM model mice by enhancing autophagy and anti-aging effects, and exhibit good in vivo persistence and biosafety.

[0092] 15. To evaluate the potential of HPQ@K in treating human osteoarthritis (OA) cartilage, the present invention uses the medial femoral condyle cartilage specimens from total knee arthroplasty (TKA) patients as a model (the bone joints are from Tongji Medical College of Huazhong University of Science and Technology).

[0093] Fig.34 (A) H&E and Safranin-O staining showed the difference in matrix structure between mild and severe OA cartilage. Histological staining results showed that the cartilage matrix structure in mild OA cartilage was relatively intact, while severe OA cartilage showed significant matrix loss and tissue structure destruction. In subsequent experiments, OA cartilage specimens from the same TKA patient were divided into two groups. One group was maintained in a culture medium containing 50 ng / mL IL-1β to maintain gene expression of the OA phenotype for 72 hours, and the other group was simultaneously added with 50 ng / mL IL-1β and 1 mg / mL HPQ@K within 72 hours. Fig.34 (B) shown.

[0094] Fig.35The immunohistochemical results showed that HPQ@K significantly improved cartilage matrix metabolism and inhibited catabolic activity. In the IL-1β-treated group, the expression of Aggrecan was significantly decreased, while HPQ@K effectively restored its expression. In contrast, the expression of MMP-13 was significantly increased in the IL-1β-treated group, while HPQ@K significantly inhibited the expression of MMP-13. In addition, HPQ@K significantly enhanced the autophagic activity of chondrocytes by reducing the expression of the autophagy inhibition marker p62. At the same time, HPQ@K significantly reduced the expression of the cell senescence marker p16, indicating that it has a significant anti-aging effect.

[0095] This indicates that the HPQ@K of the present invention has the ability to improve the metabolism, autophagy and aging characteristics of human OA cartilage matrix, thereby achieving the effect of effectively treating osteoarthritis.

[0096] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing hyaluronic acid-quercetin conjugate micelles, characterized in that: The following steps are involved: S1, adding a coupling agent to a hyaluronic acid solution for activation reaction, then adding a phenylboronic acid solution for grafting reaction, and obtaining HA-PBA after purification; S2, HA-PBA and quercetin are mixed in an organic solvent, and the resulting mixture is subjected to grafting reaction and purification to obtain hyaluronic acid-quercetin conjugate micelles.

2. The method for preparing hyaluronic acid-quercetin conjugate micelles 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 coupling agent includes EDC and NHS, and 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 phenylboric acid solution is prepared by dissolving phenylboric acid in a first organic solvent, and the concentration of the phenylboric acid solution is 0.05-0.15 mmol / mL; The phenylboronic acid includes 3-aminophenylboronic acid; 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-quercetin conjugate micelles 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 in step S1 is carried out at 20-28° C. for 20-30 hours; The purification process in step S1 includes ultrapure water dialysis and freeze-drying.

4. The method for preparing hyaluronic acid-quercetin conjugate micelles 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 a 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; and the third organic solvent includes dimethyl sulfoxide.

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

12.

6. The method for preparing hyaluronic acid-quercetin conjugate micelles according to claim 1, characterized in that: The grafting reaction in step S2 is carried out under a protective atmosphere at 35-45° C. for 40-55 hours; The purification treatment in step S2 includes dialysis, microporous membrane filtration and freeze-drying.

7. Hyaluronic acid-quercetin conjugate micelles prepared by the preparation method according to any one of claims 1 to 6.

8. A KGN-loaded hyaluronic acid-quercetin conjugate micelle, characterized in that: The hyaluronic acid-based quercetin conjugate micelles described in claim 7 and KGN are mixed in a fourth solvent, and the mixture is prepared by encapsulation reaction and purification.

9. The KGN-loaded hyaluronic acid-quercetin conjugate micelle according to claim 8, characterized in that: The mass ratio of the hyaluronan-based quercetin conjugate micelle to KGN is (5-15):1; The fourth solvent comprises formamide; The encapsulation reaction is carried out at 20-28°C for 3-5h; The purification includes dialysis, microporous membrane filtration and lyophilization.

10. Use of the hyaluronan-quercetin conjugate micelle according to claim 7 or the KGN-loaded hyaluronan-quercetin conjugate micelle according to any one of claims 8 to 9 in the preparation of a drug for inhibiting / treating osteoarthritis.

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