Quercetin nano-enzyme for diagnosis and treatment of osteoarthritis and preparation method of quercetin nano-enzyme

Abstract

The invention discloses quercetin nano-enzyme for osteoarthritis diagnosis and treatment and a preparation method and application thereof, and belongs to the technical field of biological medicine. Based on the principle of weak negative electricity of cartilage in joint inflammation, quercetin, meso-tetra (4-carboxyphenyl) porphin, cerium nitrate hexahydrate, gadolinium nitrate hexahydrate, benzoic acid and polyethylene glycol are creatively and jointly heated for 5-10 h at 70-150 DEG C, and the ultra-small water-soluble quercetin nano-enzyme with negative electricity is prepared. The quercetin nano-enzyme prepared by the invention can accurately target an OA cartilage part under the electrostatic action, and an inflammation position is accurately judged through a magnetic resonance / fluorescence multi-mode imaging technology. Besides, the quercetin nano-enzyme can also simulate the activity of three important antioxidant enzymes in vivo, reduce inflammation by removing ROS (reactive oxygen species) and inflammatory factors, promote anti-inflammatory immune response and achieve the effect of remodeling the OA microenvironment. Therefore, the quercetin nano-enzyme composed of natural traditional Chinese medicines and metal ions is expected to provide a new thought for clinical early diagnosis and precise treatment of OA.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of advanced functional materials, traditional Chinese medicine and biological medicine, and particularly relates to a quercetin nanoenzyme for diagnosis and treatment of osteoarthritis and a preparation method thereof. BACKGROUND

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease with complex etiology and dynamic pathological evolution, characterized by progressive degradation and loss of articular cartilage, subchondral bone sclerosis, cyst formation and osteophyte proliferation. The treatment of OA aims to relieve pain and inflammation, improve joint function, and develop reasonable intervention strategies at different stages of the disease, covering various means such as non-drug (e.g. exercise, dietary adjustment), drug and surgery. Compared with systemic administration, intra-articular injection has unique advantages. However, traditional intra-articular therapeutic drugs (such as analgesics, glucocorticoids, hyaluronic acid, etc.) have significant limitations: their efficacy often needs to be maintained by repeated injections, and is accompanied by risks of kidney and cardiovascular toxicity; long-term systemic administration can easily cause serious adverse reactions such as gastrointestinal damage and osteoporosis. In addition, the non-vascular nature of synovial joints leads to rapid drug clearance, and combined with the difficulty in targeting lesions when administered systemically, it is often necessary to increase the dose, further exacerbating the inherent cytotoxicity of these exogenous synthetic compounds. It is worth noting that the significant effect of some treatment methods may be influenced by other factors, including the placebo effect. Therefore, how to effectively control inflammation while maximizing patient safety has always been a core challenge in the clinical and scientific fields.

[0003] With the rise of the concept of 'precision medicine', nanomedicine emerges as a new diagnostic and therapeutic carrier. The core of nanomedicine is to use nanotechnology to target the delivery of diagnostic agents or therapeutic drugs to inflammatory tissues, thereby improving efficacy and reducing side effects. This drug delivery system is usually constructed by natural or synthetic polymer materials, inorganic materials, etc. as carriers through chemical bonding or non-covalent interaction. The application of biomaterial matrix significantly enhances the lesion targeting of drugs, prolongs the action time and promotes the realization of precision diagnosis and treatment. In osteoarthritis (OA), pro-inflammatory factors released by macrophages are key pathogenic factors, and abnormal neovascularization at the inflammatory site (manifested as excessive proliferation of endothelial cells, lack of pericytes, and incomplete basement membrane) leads to abnormal increase in vascular permeability. This pathological feature enables nanoparticles (NPs) to be passively enriched in inflammatory lesions by 'leakage and retention effect' (ELVIS effect). Compared with traditional drugs, nanomedicine exhibits multiple advantages in inflammation diagnosis and treatment: 1) improving the solubility and bioavailability of hydrophobic drugs; 2) enhancing the targeted accumulation of drugs in specific cells or tissues; 3) promoting the endocytosis of cells and the efficiency of transmembrane transport; 4) supporting the synergistic co-delivery of multiple drugs or therapeutic strategies; 5) enabling the design of intelligent responsive formulations in response to changes in the lesion microenvironment (such as pH, temperature, and specific enzyme activity). In summary, osteoarthritis diagnosis and treatment strategies based on nanotechnology are driving the field towards microscale, non-invasive operation, functional integration, intelligent response, and dynamic monitoring, and have become an important way to achieve precise intervention of inflammation.

[0004] The activity of endogenous antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) decreases in patients with osteoarthritis (OA). The use of artificial nanoscale enzymes with biological catalytic function for treatment has become a new research direction. Nanoscale enzymes have excellent thermal stability and biological stability, multifunctionality, easy preparation and activity adjustability. Among the many nanomaterials with ROS scavenging ability (such as manganese dioxide, platinum and other inorganic nanoparticles), cerium-mediated nanometallic materials (CeO2 NPs) have attracted much attention in the biomedical field due to their good biocompatibility and multi-enzyme activity (mimic SOD, CAT and hydroxyl radical scavenging). Studies have shown that these characteristics make them effective preparations for the treatment of ROS-related diseases such as inflammatory diseases. For example, Professor Wei's research group used a dextran sulfate sodium-induced mouse intestinal inflammation model to confirm that cerium-mediated nanometallic materials exhibited higher ROS scavenging ability than non-catalytic antioxidants, and their stability was better than that of free enzymes. Pharmacokinetic studies further revealed that cerium-based SOD mimics had more advantages than natural Cu / Zn SOD and Fe-SOD in the treatment of chronic inflammation. However, the positive charge and non-specificity of this material may lead to rapid absorption through the gastrointestinal wall after oral administration, increasing the risk of systemic exposure and potentially causing side effects. Therefore, developing new cerium-based nanoscale enzymes to achieve precise individualized drug delivery at the molecular level to avoid the toxicity of ineffective treatment and reduce the burden on patients is the most promising strategy for the future treatment of osteoarthritis.

[0005] Quercetin is a natural flavonoid compound derived from traditional Chinese medicine (widely exists in various fruits and vegetables), named after Quercus, with significant antioxidant and anti-inflammatory activities. In the pathogenesis of osteoarthritis (OA), the key pro-inflammatory factor IL-1β is closely related to tissue injury. It can inhibit the synthesis of cartilage extracellular matrix (ECM), accelerate its degradation, and stimulate the expression of matrix metalloproteinases (MMPs), thereby promoting cartilage destruction and hindering repair. Studies have shown that quercetin, as an active ingredient of traditional Chinese medicine, can effectively inhibit the production of inflammatory mediators nitric oxide (NO) and MMPs by IL-1β-induced chondrocytes, while increasing the level of superoxide dismutase (SOD), thereby reducing oxidative stress and reducing ECM degradation. In addition, it can also up-regulate the expression of transforming growth factor-β (TGF-β) and insulin-like growth factor, promoting the synthesis of glycosaminoglycan (GAG), thereby playing a dual role of anti-inflammatory and promoting cartilage repair. However, quercetin as a traditional Chinese medicine molecule has the limitation of poor water solubility, which restricts its bioavailability. For example, Wang Xianwen's research group coupled quercetin with low-toxicity iron ions, partially simulating the function of intracellular antioxidant enzymes and improving its protective ability against ROS-mediated damage. It is worth noting that the characteristic change in the early stage of OA is the loss of GAG with strong negative charge, resulting in a decrease in the negative charge within the joint. This change in the electric microenvironment has important implications: on the one hand, it explains the increased distribution of negatively charged contrast agents (such as Gd-DTPA) in diseased cartilage (due to the weakening of repulsion with normal cartilage negative charge); on the other hand, studies have confirmed that negatively charged nanosystems can specifically target and anchor inflammatory tissues (such as inflamed colonic mucosa), while positively charged carriers are more likely to adhere to healthy tissues. This suggests that we can use the poorly water-soluble traditional Chinese medicine quercetin as the active core to construct a nanoscale enzyme delivery system with a negative surface charge, which is expected to achieve precise treatment of osteoarthritis based on charge targeting. SUMMARY

[0006] The purpose of the present application is to provide a quercetin nanoscale enzyme for the diagnosis and treatment of osteoarthritis and a preparation method thereof. The preparation method is stable, reliable, and has good reproducibility, with the advantages of simple steps, abundant yield, safe operation, and low economic cost. The prepared quercetin nanoscale enzyme not only can realize targeted magnetic resonance / fluorescence imaging, but also can inhibit inflammation by simulating antioxidant enzymes, thus constructing a nanoscale probe with simple preparation method, high imaging efficiency, and good diagnosis and treatment effect, to realize precise diagnosis and treatment of osteoarthritis.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows: a quercetin nanoscale enzyme for the diagnosis and treatment of osteoarthritis and a preparation method thereof, comprising the following steps:

[0008] (1) sequentially adding meso-tetra(4-carboxyphenyl) porphyrin, cerium nitrate hexahydrate, gadolinium nitrate hexahydrate, benzoic acid, quercetin and polyethylene glycol into N,N-dimethylformamide to obtain a reaction solution after fully dissolving;

[0009] (2) after heating the reaction solution to 70-150℃, stirring and reacting in the dark for 5-10h;

[0010] (3) after the reaction is completed, cooling the solution to room temperature and dialyzing for 2-5 days to obtain water-soluble quercetin nanoparticles, which are quercetin nanoscale enzymes for diagnosis and treatment of osteoarthritis.

[0011] Preferably, in step (1), the concentration of meso-tetra(4-carboxyphenyl) porphyrin is 0.5-1.5mg / mL.

[0012] Preferably, in step (1), the mass ratio between meso-tetra(4-carboxyphenyl) porphyrin and cerium nitrate hexahydrate is 1:1-1:7.

[0013] Preferably, in step (1), the mass ratio between meso-tetra(4-carboxyphenyl) porphyrin and gadolinium nitrate hexahydrate is 1:1-1:10.

[0014] Preferably, in step (1), the mass ratio between meso-tetra(4-carboxyphenyl) porphyrin and benzoic acid is 1:1-1:177.

[0015] Preferably, in step (1), the mass ratio between meso-tetra(4-carboxyphenyl) porphyrin and polyethylene glycol is 1:40-1:60.

[0016] Preferably, in step (1), the mass ratio between meso-tetra(4-carboxyphenyl) porphyrin and quercetin is 1:1-1:5.

[0017] Preferably, in step (1), the polyethylene glycol is one of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000 and polyethylene glycol 5000.

[0018] Preferably, in step (2), the reaction temperature is 70-150℃ and the reaction time is 5-10h.

[0019] Preferably, in step (3), the dialysis bag has a molecular weight of 5000-14000 and the dialysis time is 2-5 days.

[0020] The application takes quercetin and porphyrin photosensitizer (TCPP) as an organic linker, takes cerium ions and gadolinium ions as metal nodes, and takes polyethylene glycol as a surfactant, and a water-soluble quercetin nano metal organic probe is prepared by a simple one-step method. Once entering the inside of osteoarthritis, the negative charge of the quercetin nano enzyme penetrates into the cartilage degeneration area, and the T1 MRI / fluorescence multimodal imaging technology is used to accurately determine the OA lesion site. In the treatment of OA, the quercetin nano enzyme simulates the activities of three important antioxidant enzymes, namely superoxide dismutase, catalase and peroxidase, and exhibits excellent ROS scavenging capacity. At the same time, the release of cerium ions can effectively convert pro-inflammatory macrophages (M1) into anti-inflammatory macrophages (M2), thereby repairing the damaged OA epithelial barrier.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] (1) The nano diagnosis and treatment agent prepared by the application is synthesized by a one-step method, and the preparation method is stable and reliable, has good repeatability, and has the advantages of simple steps, safe operation, rich yield and low economic cost;

[0023] (2) The water-soluble quercetin nano probe modified by PEG has the advantages of good biocompatibility, biological safety, water solubility and modifiability, etc., and improves the problem of poor water solubility of traditional Chinese medicine quercetin, so as to be better applied to the human body;

[0024] (3) The quercetin nano probe prepared by the application assembles quercetin, gadolinium ions with imaging function and cerium ions with antioxidant function to form a metal organic nano enzyme, and removes active oxygen and inflammatory factors by simulating the activities of superoxide dismutase, catalase and peroxidase, so as to achieve the purpose of protecting chondrocytes from ROS damage;

[0025] (4) The quercetin nano probe prepared by the application first attempts to construct a negatively charged traditional Chinese medicine nano probe to realize efficient penetration and retention of the quercetin nano probe in the degenerated cartilage;

[0026] (5) The quercetin nano probe prepared by the application has diversified functions: the quercetin therein has excellent antioxidant and anti-inflammatory properties, can effectively scavenge excess active oxygen and inflammatory factors in the body, and reduce oxidative stress damage; at the same time, the cerium ions contained therein can specifically regulate the phenotype of macrophages, efficiently convert pro-inflammatory M1 type macrophages into anti-inflammatory M2 type macrophages, and thereby relieve inflammatory reaction. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a transmission electron microscope graph of the quercetin nano particle of the application (scale 200 nm);

[0028] Figure 2 Hydrodynamic size plot of different nanoparticles of the present invention, Ce-QGd and Ce-PQGd;

[0029] Figure 3 Surface charge plot of different nanoparticles of the present invention, PEG, Ce-QGd and Ce-PQGd;

[0030] Figure 4 UV spectrum plot of different nanoparticles of the present invention, TCPP, Gd(NO3)3.6H2O and Ce-PQGd;

[0031] Figure 5 SOD-like activity of quercetin nanoparticles of the present invention;

[0032] Figure 6 CAT-like activity of quercetin nanoparticles of the present invention;

[0033] Figure 7 POD-like activity of quercetin nanoparticles of the present invention;

[0034] Figure 8 DPPH-based radical scavenging rate plot of quercetin nanoparticles of the present invention;

[0035] Figure 9 ABTS-based radical scavenging rate plot of quercetin nanoparticles of the present invention;

[0036] Figure 10 MB-based hydroxyl radical scavenging rate plot of quercetin nanoparticles of the present invention;

[0037] Figure 11 Fluorescence imaging and fluorescence signal correlation plot of quercetin nanoparticles of the present invention; (A) Fluorescence imaging capability plot, (B) Fluorescence signal intensity change with increasing concentration plot;

[0038] Figure 12 Magnetic resonance imaging correlation plot of quercetin nanoparticles of the present invention; (A) T1 magnetic resonance imaging signal plot, (B) T1 magnetic resonance imaging relaxation rate curve plot;

[0039] Figure 13 Cell toxicity correlation plot of quercetin nanoparticles of the present invention;

[0040] Figure 14 Blood compatibility of quercetin nanoparticles of the present invention;

[0041] Figure 15To evaluate the quercetin nanoparticles of this invention at the cellular level using magnetic resonance imaging: (A) T1-weighted images and pseudo-color images of ATDC5 chondrocytes after incubation with aqueous solutions of quercetin nanoenzymes at different concentrations; (B) Measurement of T1-weighted image signal values ​​after incubation with aqueous solutions of quercetin nanoenzymes at different concentrations and ATDC5 chondrocytes.

[0042] Figure 16 To evaluate the fluorescence imaging level of quercetin nanoparticles at the cellular level in this invention: (A) Fluorescence images of quercetin nanozyme aqueous solution after co-incubation with IL-1β-induced ATDC5 cells for different times; (B) Fluorescence images of quercetin nanozyme aqueous solution after co-incubation with IL-1β-inducible ATDC5 cells for different times.

[0043] Figure 17 The imaging evaluation of the quercetin nanoparticles of the present invention in tumor tissue at the in vivo level includes: (A) in vivo fluorescence imaging of small animals in the quercetin nanozyme model group and control group at different time points; (B) semi-quantitative analysis of fluorescence intensity in small animals using in vivo fluorescence imaging; and (C) T1WI images of small animals in the quercetin nanozyme model group and control group at different time points. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] All raw materials and reagents in the embodiments of this application were purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0046] (1) Dissolve 0.025g of racemic tetra(4-carboxyphenyl)porphyrin, 0.096g of cerium nitrate hexahydrate, 0.09982g of gadolinium nitrate hexahydrate, 0.683g of benzoic acid, 0.0478g of quercetin and 1.246g of polyethylene glycol 2000 (Mw=2000) in 25mL of N,N-dimethylformamide (DMF) to obtain a reaction solution;

[0047] (2) Heat the reaction solution to 95°C and react for 7 hours with stirring at 800 rpm in the dark.

[0048] (3) After the reaction is complete, the solution is cooled to room temperature and dialyzed for 2 days using a dialysis bag with a molecular weight of 8000 to obtain quercetin nanoparticles.

[0049] like Figure 1 As shown, the prepared quercetin nanoparticles are spherical with a diameter of approximately 14 nm, exhibiting relatively uniform size and good dispersibility. The hydrated particle size of the quercetin nanoenzyme nanoparticles, as measured by a particle size analyzer, is approximately 106 nm. Figure 2). In addition, the surface charge of PEG, Ce-QGd and Ce-PQGd (PEG-coated Ce-QGd, i.e. quercetin nanosensor) was tested by the instrument, and the results are shown in Figure 3 . PEG and Ce-QGd are negatively charged, and the coating of PEG increases the negative charge on the surface of quercetin nanosensor. In order to further verify the successful preparation of quercetin nanosensor, ultraviolet spectrum test was carried out on different probes, and the results are shown in Figure 4 . The spectrum of quercetin nanosensor not only shows the characteristic absorption peak of TCPP, but also shows the characteristic absorption peak of Gd 3+ , which further proves the successful preparation of quercetin nanosensor.

[0050] In order to verify the enzyme activity of quercetin nanosensor, the present application tests the superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity of quercetin nanosensor. By using WST-1 method to determine the SOD activity, it is found that with the increase of the concentration of quercetin nanosensor, the SOD value increases Figure 5 , which indicates that the higher the concentration of quercetin nanosensor, the stronger the inhibition effect on O2 ·- . In addition, with the increase of the concentration of quercetin nanosensor, the clearance rate of hydrogen peroxide (H2O2) gradually increases in a concentration-dependent manner Figure 6 . This shows that quercetin nanosensor has CAT activity and can decompose H2O2 into H2O and O2. In order to verify the POD activity of quercetin nanosensor, the concentration change of the natural substrate TMB of POD is used to detect the POD activity of quercetin nanosensor. As shown in Figure 7 , with the increase of the concentration of quercetin nanosensor, more and more TMB oxidation products are produced, and the solution color gradually deepens. The results show that quercetin nanosensor has POD-like activity and is concentration-dependent.

[0051] Considering the free radical scavenging activity of quercetin as a natural antioxidant, in order to verify whether quercetin nanosensor has free radical scavenging ability, the present application uses 1-1 diphenyl-2-trinitrobenzene hydrazine (DPPH), 1-1 diphenyl-2-trinitrobenzene hydrazine (ABTS) and methylene blue to detect the free radical scavenging ability of quercetin nanosensor. As a stable nitrogen-centered free radical, the single electron of DPPH will be color lightened due to being captured, which leads to the decrease of its absorbance value at the maximum light absorption wavelength, and the decrease is linear. Studies have shown that the decrease of DPPH absorbance level indicates the increase of antioxidant activity, which is used to evaluate the antioxidant capacity of the sample. As shown in Figure 8As shown, with increasing quercetin nanozyme concentration, the reaction solution gradually changed from purple to pale yellow, exhibiting a concentration-dependent effect. This indicates that quercetin nanozyme possesses DPPH free radical scavenging ability, and the free radical scavenging rate gradually increases with increasing nanozyme concentration. It has been reported that the absorbance value of the free radical absorption peak at 734 nm in ABTS decreases with a decrease in the number of free radicals, and the blue-green color of the solution also becomes lighter. Therefore, ABTS can be used to assess the free radical scavenging ability of quercetin nanozyme. Figure 9 As shown, the blue-green color of the reaction solution gradually lightens with increasing quercetin nanozyme concentration, indicating an increase in its free radical scavenging ability. Furthermore, the ·OH free radicals generated by the Fenton reaction cause MB to change color. Therefore, the ·OH scavenging ability of quercetin nanozymes can be tested using methylene blue (MB). Figure 10 As shown, the color change of the MB solution decreased with increasing quercetin nanozyme concentration, indicating that quercetin nanozyme has a strong scavenging ability for ·OH, and this ability is concentration-dependent.

[0052] To verify the T1 magnetic resonance and fluorescence imaging capabilities of quercetin nanozymes, we prepared a series of quercetin nanozyme solutions with varying concentrations and performed magnetic resonance and fluorescence imaging on them. Simultaneously, we measured the fluorescence intensity at an emission wavelength of 650 nm using a fluorescence spectrophotometer. Figure 11 As shown, the fluorescence intensity gradually increases with increasing quercetin nanozyme concentration. With increasing Gd... 3+ As the concentration increases, the aqueous solution containing quercetin nanozyme becomes increasingly bright. Figure 12 The higher the T1 WI signal, the stronger the T1 imaging capability.

[0053] To select safe and effective drug concentrations for subsequent experiments, we used the CCK-8 assay to evaluate the cytotoxicity of different concentrations of quercetin nanozymes. Figure 13 As shown, cell viability decreased slightly after 24 hours of co-incubation with quercetin nanozyme with increasing concentration. At concentrations as high as 400 μg / mL, ATDC5 chondrocyte viability remained above 80%, and macrophage viability remained above 70%, indicating low cytotoxicity of the quercetin nanozyme. Furthermore, compared to erythrocytes treated with deionized water, the hemolysis rate of rat erythrocytes treated with different concentrations of quercetin nanozyme (0–500 μg / mL) remained below 5%. Figure 14 This further confirms the good biocompatibility of quercetin nanozyme in biological environments.

[0054] To verify the magnetic resonance and fluorescence intracellular imaging capabilities of quercetin nanozymes, we performed fluorescence and magnetic resonance imaging on cells co-incubated with different concentrations of quercetin nanozymes. Figure 15As shown in Figure A, with increasing quercetin nanozyme concentration, ATDC5 chondrocytes phagocytosed by quercetin nanozymes became increasingly bright, and the statistically significant T1-weighted image signal also increased. Figure 15 B) indicates that the quercetin nanozyme can effectively perform magnetic resonance imaging within ATDC5 chondrocytes. For example... Figure 16 As shown in Figure A, the fluorescence signal intensity of the quercetin nanozyme in ATDC5 chondrocytes gradually increased with concentration, indicating that this nanozyme has excellent intracellular fluorescence imaging capabilities. Notably, compared to the red fluorescence signal induced by IL-1β in ATDC5 chondrocytes, the red fluorescence signal in ATDC5 chondrocytes without IL-1β induction was weaker. Figure 16 B) This indicates that the quercetin nanozyme has targeting properties for degenerated chondrocytes.

[0055] To evaluate the targeting and imaging properties of quercetin nanozymes to articular cartilage in an in vivo rat model of early osteoarthritis using magnetic resonance / fluorescence imaging, we performed small animal in vivo fluorescence and magnetic resonance imaging on rats with early osteoarthritis on day 14 post-modeling. Figure 17 A. After injection of quercetin nanozyme, the fluorescence intensity of both normal articular cartilage and early degenerative cartilage in the knee joint showed a trend of first increasing and then decreasing. After testing the fluorescence intensity, it was found that ( Figure 17 B) The quercetin nanozyme showed stronger fluorescence signal intensity and a slower decay rate in the OA group compared to the normal group. This indicates that the quercetin nanozyme can target and increase its effective penetration into cartilage through electrostatic interactions, thereby increasing its retention time. Furthermore, magnetic resonance imaging images were acquired at five time points: 0h, 2h, 4h, 6h, and 24h. Figure 17 As shown in Figure C, the T1WI signal intensity of both normal and degenerated articular cartilage gradually increased over time. The highest signal enhancement was observed in the degenerated cartilage two hours after quercetin nanozyme injection, indicating that the nanozyme effectively penetrated the cartilage. The signal intensity in the degenerated cartilage gradually decreased over time. Compared to the T1 MRI signal intensity in early-stage degenerated cartilage, normal cartilage showed lower signal enhancement upon nanoparticle injection. This demonstrates that the quercetin nanozyme not only penetrates rapidly into degenerated cartilage but also remains for an extended period, achieving highly efficient imaging.

[0056] For those skilled in the art, the present invention is not limited to the details of the exemplary embodiments described above. The embodiments are illustrative and not restrictive, and the scope of protection of the present invention is defined by the appended claims rather than the foregoing description. Therefore, no reference numerals in the claims should be construed as limiting the scope of the claims.

[0057] The above merely provides an example of one or more embodiments of the present specification and is not intended to limit one or more embodiments of the present specification. One or more embodiments of the present specification can have various changes and modifications for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present specification shall be included in the scope of claims.

Claims

1. A quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis and a preparation method thereof, characterized in that, Comprising the following steps: (1) meso-tetra(4-carboxyphenyl) porphyrin, cerium nitrate hexahydrate, gadolinium nitrate hexahydrate, benzoic acid, quercetin and polyethylene glycol are sequentially added to N,N-dimethylformamide to obtain a reaction solution after being fully dissolved; (2) the reaction solution is heated to 70-150℃, and then stirred in the dark for 5-10h; (3) after the reaction is completed, the solution is cooled to room temperature and dialyzed for 2-5 days to obtain water-soluble quercetin nanoparticles, which are quercetin nanoszymes for the diagnosis and treatment of osteoarthritis.

2. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis and its preparation method according to claim 1, characterized in that, The concentration of meso-tetra(4-carboxyphenyl) porphyrin in step (1) is 0.5-1.5mg / mL.

3. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1, wherein, The mass ratio of meso-tetra(4-carboxyphenyl) porphyrin to cerium nitrate hexahydrate in step (1) is 1:1-1:

7.

4. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1, and a preparation method thereof, characterized in that, The mass ratio of meso-tetra(4-carboxyphenyl) porphyrin to gadolinium nitrate hexahydrate in step (1) is 1:1-1:

10.

5. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1, wherein, The mass ratio of meso-tetra(4-carboxyphenyl) porphyrin to benzoic acid in step (1) is 1:1-1:

177.

6. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1, wherein, The mass ratio of meso-tetra(4-carboxyphenyl) porphyrin to quercetin in step (1) is 1:1-1:

5.

7. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1, wherein, The polyethylene glycol in step (1) is one of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 3000, polyethylene glycol 4000 and polyethylene glycol 5000.

8. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1 and a preparation method thereof, characterized in that, The dialysis bag in step (3) has a molecular weight of 5000-14000.

9. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1 and a preparation method thereof, characterized in that, The particle size of the quercetin nanoszyme prepared in step (3) is 14nm, and the surface charge is-20--25mV.

10. The quercetin nanoenzyme for the diagnosis and treatment of osteoarthritis according to claim 1 and a preparation method thereof, characterized in that, The quercetin nanoszyme prepared in step (3) targets the osteoarthritis inflammation site under magnetic resonance / fluorescence guidance, and simulates antioxidant enzymes under the action of quercetin, reduces inflammation by removing ROS and inflammatory factors, while promoting anti-inflammatory immune response, and achieves the effect of remodeling the microenvironment of osteoarthritis.

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