Responsive nano-enzyme for delivering KGN as well as preparation method and application of responsive nano-enzyme

Abstract

The invention discloses a responsive nano-enzyme for delivering KGN and a preparation method and application thereof, and the preparation method comprises the following steps: suspending zinc chloride and cerium nitrate in an N, N-dimethylformamide solution, and suspending meso-tetra (4-carboxyphenyl) porphin in the N, N-dimethylformamide solution; adding a proper amount of glacial acetic acid; the preparation method comprises the following steps: dissolving tannic acid in an N, N-dimethylformamide solution, and dissolving meso-tetra (4-carboxyl phenyl) porphin in the N, N-dimethylformamide solution to obtain meso-tetra (4-carboxyl phenyl) porphin-Zn < 2 + >-Ce < 3 + >; the preparation method comprises the following steps: adding modified mercaptoacetic acid-meso-tetra (4-carboxyl phenyl) porphin-Zn < 2 + >-Ce < 3 + > and cysteine modified CAP bone targeting peptide into triethylamine, and reacting to obtain meso-tetra (4-carboxyl phenyl) porphin-Zn < 2 + >-Ce < 3 + >-tannic acid-coated CAP peptide with targeting property; the preparation method comprises the following steps: dissolving KGN in dimethyl sulfoxide, diluting, and adding into an N, N-dimethylformamide solution of SH-TCPP-Zn < 2 + >-Ce < 3 + > (at) TA to obtain the sulfydryl-tetra (4-carboxylphenyl) porphin-Zn < 2 + >-Ce < 3 + > (at) tannic acid (at) KGN(at) CAP peptide. The invention relates to a biodegradable nano particle with a cartilage targeting function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a responsive nanozyme for delivering KGN, its preparation method, and its application. Background Technology

[0002] With the accelerating aging of the global population, age-related diseases are increasingly threatening human health. Osteoarthritis (OA), a common chronic joint disease, is seeing a year-on-year increase in both incidence and disability rates. In aging bodies, excessive free radicals, such as superoxide anions, accumulate, triggering oxidative stress, damaging the extracellular matrix of chondrocytes, and leading to cartilage degeneration and loss of joint function. Simultaneously, the abnormal expression of inflammatory factors further exacerbates joint damage, creating a vicious cycle. Traditional treatments, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and intra-articular injections of hyaluronic acid, can alleviate symptoms to some extent, but they cannot fundamentally reverse cartilage damage or halt disease progression. Therefore, the development of safe and effective new treatment strategies is urgently needed.

[0003] KGN, a synthetically produced small molecule drug, possesses a unique chemical structure and biological function. Its small molecule nature endows it with excellent cell membrane penetration capabilities, allowing it to smoothly enter chondrocytes. By activating signaling pathways such as Wnt / β-catenin and BMP / Smad, it effectively promotes chondrocyte proliferation and differentiation, inhibits chondrocyte apoptosis, and plays a crucial role in cartilage tissue repair. Despite the significant potential of KGN in cartilage therapy, its clinical application still faces numerous challenges. Firstly, KGN lacks specific recognition of diseased cartilage tissue, making it difficult to precisely accumulate at damaged joint sites after entering the body. Most of the drug diffuses into non-target tissues via blood circulation, reducing treatment efficiency and potentially causing systemic toxicity. Secondly, KGN has low bioavailability; it is rapidly degraded by metabolic enzymes in the liver and intestines, and is also affected by the first-pass effect, significantly reducing the amount of drug entering the bloodstream and reaching the lesion site, making it difficult to maintain effective therapeutic concentrations and severely limiting its therapeutic effect on osteoarthritis. Improving the targeting selectivity and bioavailability of KGN is key to overcoming the bottlenecks in its clinical application. Summary of the Invention

[0004] The purpose of this invention is to provide a responsive nanozyme for delivering KGN, its preparation method, and its application, in order to solve one or more technical problems existing in the prior art, or at least provide a beneficial option or create conditions.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a responsive nanozyme for delivering KGN, comprising:

[0007] S1, zinc chloride and cerium nitrate are suspended in N,N-dimethylformamide solution, and methyl-tetra(4-carboxyphenyl)porphyrin is suspended in N,N-dimethylformamide solution, and then stirred and sonicated respectively;

[0008] S2, mix the above solutions and add an appropriate amount of glacial acetic acid, stir and sonicate to form a homogeneous system, add the above mixture to a hydrothermal reactor for hydrothermal reaction, collect the collected samples and centrifuge, take the precipitate and wash with N,N-dimethylformamide solution and then wash with ethanol, and quantify by freeze drying;

[0009] S3, tannic acid is dissolved in N,N-dimethylformamide solution, and methyl-3-tetra(4-carboxyphenyl)porphyrin is dissolved in N,N-dimethylformamide solution. The mixture is added to a hydrothermal reactor for hydrothermal reaction. The collected sample is centrifuged, washed, and freeze-dried to obtain methyl-3-tetra(4-carboxyphenyl)porphyrin-Zn. 2+ -Ce 3+ ;

[0010] S4, neu-tetra(4-carboxyphenyl)porphyrin-Zn 2+ -Ce 3+ Activated overnight, then modified with mercaptoacetic acid;

[0011] S5, modified mercaptoacetic acid-tetra-tetra(4-carboxyphenyl)porphyrin-Zn 2+ -Ce 3+ The cysteine-modified CAP bone-targeting peptide was reacted thoroughly with triethylamine, and the free CAP bone-targeting peptide was removed by dialysis to obtain the targeted methyl-tetra(4-carboxyphenyl)porphyrin-Zn. 2+ -Ce 3+ @Tanolic acid@CAP peptide;

[0012] S6, dissolve KGN in dimethyl sulfoxide, dilute with N,N-dimethylformamide, and add the above solution to SH-TCPP-Zn. 2+ -Ce 3+ The solution of TA in N,N-dimethylformamide was stirred and dialyzed to remove free KGN, yielding mercapto-tetra(4-carboxyphenyl)porphyrin-Zn. 2+ -Ce 3+ @Tanolic acid@KGN@CAP peptide.

[0013] Furthermore, in S1, the molar ratio of cerium nitrate, zinc chloride, and medium-tetra(4-carboxyphenyl)porphyrin (TCPP) is set to 7.5:7.5:1.

[0014] Furthermore, in S1, the time for ultrasonic treatment ranges from 10 to 40 minutes.

[0015] Furthermore, in S2, the temperature range for the hydrothermal reaction is 50-120℃;

[0016] The centrifugation rate ranges from 8000 to 20000 rpm.

[0017] Further, in S4, the methyl-tetra(4-carboxyphenyl)porphyrin-Zn 2+ -Ce 3+ Activation overnight, followed by modification with mercaptoacetic acid, specifically including:

[0018] Mercaptoacetic acid was dissolved in N,N-dimethylformamide solution, and then methyl-tetra(4-carboxyphenyl)porphyrin-Zn was added. 2+ -Ce 3+ Ultrasonic treatment to fully disperse it;

[0019] The mixture was placed in a vacuum drying oven under a nitrogen atmosphere to obtain mercapto-medium-tetra(4-carboxyphenyl)porphyrin-Zn 2+ -Ce 3+ Wash several times with N,N-dimethylformamide and acetone solution, collect by centrifugation, and dry overnight in a vacuum oven.

[0020] Furthermore, in step S3, the activation temperature ranges from 120 to 180°C, and the activation time ranges from 8 to 16 hours.

[0021] The centrifugation rate ranges from 8000 to 20000 rpm;

[0022] The temperature range of the vacuum oven is 50-90℃.

[0023] Furthermore, in step S5, the modified 1-tetra(4-carboxyphenyl)porphyrin-Zn is added. 2+ -Ce 3+ The mass ratio of tannic acid to thiol-CAP bone-targeting peptide is 10:(0.5-2).

[0024] Furthermore, in step S5, KGN and methyl-tetra(4-carboxyphenyl)porphyrin-Zn are added. 2+ -Ce 3+ The mass ratio of tannic acid to mercapto-CAP peptide is 1:(6-8).

[0025] Secondly, the present invention also provides a responsive nanozyme for delivering KGN prepared by the above preparation method.

[0026] Thirdly, the present invention also provides the application of the above-described responsive nanozyme for delivering KGN in a medicament for treating osteoarthritis.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention designs a biodegradable nanoparticle with cartilage targeting, utilizing the chelating properties of a carrier to achieve metal element chelation. The CAP peptide with cartilage targeting is further modified by carrying the drug 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (KGN). Intra-articular injection of the nanomedicine, in the osteoarthritis (OA) microenvironment with high levels of reactive oxygen species (ROS), effectively promotes ion release, macrophage polarization, and chondrocyte repair, providing a foundation for further development of osteoarthritis treatment drugs. Attached Figure Description

[0029] Figure 1 (a) is TCPP-Zn in Embodiment 3 of the present invention. 2+ -Ce 3+ TEM image;

[0030] Figure 1 (b~c) represent TCPP-Zn in Embodiment 3 of the present invention. 2+ -Ce 3+ TEM image of @TA@KGN@CAP;

[0031] Figure 2 TCPP-Zn in Embodiment 3 of the present invention 2+ -Ce 3+ Mapping spectra of @TA@KGN@CAP;

[0032] Figure 3 (a) is TCPP-Zn in Embodiment 1 of the present invention. 2+ DLS spectrum;

[0033] Figure 3 (b) is TCPP-Ce in Embodiment 2 of the present invention. 3+ DLS spectrum;

[0034] Figure 3 (c) TCPP-Zn in Embodiment 3 of the present invention 2+ -Ce 3+ DLS spectrum;

[0035] Figure 3 (d) is TCPP-Zn in Embodiment 3 of the present invention. 2+ -Ce 3+ DLS spectrum of @KGN@CAP;

[0036] Figure 4 From left to right: Example 1 TCPP-Zn 2+ Example 2 TCPP-Ce 3+ In Example 3, TCPP-Zn 2+ -Ce 3+ and TCPP-Zn 2+ -Ce 3+ Appearance image of @KGN@CAP nanozyme;

[0037] Figure 5 This is Example 1 of the present invention, TCPP-Zn. 2+ Example 2 TCPP-Ce 3+ And TCPP-Zn in Example 3 2+ -Ce 3+ @TA nanozyme UV-Vis absorption spectrum;

[0038] Figure 6 TCPP-Zn in Embodiment 4 of the present invention 2+ -Ce 3+ @TA@KGN@CAP nanozyme release curves of KGN over 72 hours under different conditions;

[0039] Figure 7 a represents TCPP-Zn in Embodiment 5 of this invention. 2+ TCPP-Ce 3+ and TCPP-Zn 2+ -Ce 3+ CAT-like enzyme activity diagram of nanozymes;

[0040] Figure 7 b represents TCPP-Zn under different concentration conditions in Example 5 of this invention. 2+ -Ce 3+ CAT-like enzyme activity diagram of @KGN@CAP nanozymes;

[0041] Figure 8 TCPP-Zn at different concentrations in Example 6 of this invention 2+ TCPP-Ce 3+ and TCPP-Zn 2+ -Ce 3+ and TCPP-Zn 2+ -Ce 3+ SOD-like enzyme activity diagram of @KGN@CAP nanozymes;

[0042] Figure 9 This is a diagram showing the effect of different nanozymes on the activity of C28 / I2 cells in Example 7 of the present invention. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Unless otherwise specified, all materials and instruments used in this invention are readily available to those skilled in the art, and all methods employed are well-known to those skilled in the art. Unless otherwise specified, "room temperature" in this invention refers to 25±2℃.

[0047] Metal-organic frameworks (MOFs) have shown great promise in the biomedical field due to their unique structure and tunable properties. In this application, metal ions Zn... 2+ With Ce 3+ TCPP-Zn was constructed by coordinating porphyrin-based MOFs material TCPP (tetracarboxyphenylporphyrin). 2+ -Ce 3+ Nanozymes have extremely high SOD enzyme activity and good safety.

[0048] To further improve the therapeutic effect on osteoarthritis, it is necessary to achieve efficient loading of KGN. Tannic acid, as a natural polyphenol compound, has unique chemical properties and bioactivity. This application utilizes tannic acid to load TCPP-Zn 2+ -Ce 3+ The etching process provides more space for KGN loading; simultaneously, by modifying the carrier system with CAP peptides that have cartilage targeting properties, a ROS-responsive nanozyme targeted delivery system was constructed. Tannic acid itself has multiple effects such as anti-oxidation and anti-inflammation, and it is compatible with TCPP-Zn.2+ -Ce 3+ The synergistic effect of SOD enzyme activity can more comprehensively regulate the oxidative stress and inflammatory microenvironment within the joint, creating favorable conditions for cartilage repair. The carrier can achieve controlled release of KGN in the high-concentration reactive oxygen species microenvironment of osteoarthritis, enabling it to exert a cartilage-promoting effect locally in the joint, thereby successfully reversing age-related osteoarthritis.

[0049] This application presents a responsive nanozyme system for delivering KGN, integrating multiple functions such as efficient free radical scavenging, promoting cartilage regeneration, anti-oxidation and anti-inflammation, and precise targeting. This provides an innovative multi-mechanism synergistic therapeutic strategy for age-related osteoarthritis. This application will detail the preparation method of this nanozyme and explore its application potential in the treatment of osteoarthritis.

[0050] According to one aspect of the present invention, the present invention provides a method for preparing a responsive nanozyme for delivering KGN, specifically comprising:

[0051] Example 1

[0052] Reference Figure 3 (a) Figure 4 and Figure 5 ,include:

[0053] (1) TCPP-Zn 2+ preparation

[0054] 6.44 mg of zinc chloride (0.04725 mmol) was suspended in 5 mL of N,N-dimethylformamide (DMF) solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Similarly, 5 mg of mes-tetra(4-carboxyphenyl)porphyrin TCPP (790.77 g / mol, 0.0063 mmol) was suspended in 5 mL of DMF solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Then, the above solutions were mixed and 200 μL of glacial acetic acid was added, stirred for 1 h (1000 rpm), and sonicated for 30 min to form a homogeneous system. Finally, the mixture was added to a hydrothermal reactor and heated at 65 °C for 16 h. The collected samples were collected, centrifuged at 10000 rpm for 20 min, and the precipitate was washed twice with N,N-dimethylformamide (DMF) solution, then washed once with ethanol, and finally quantified by freeze-drying. The molar ratio of zinc chloride to TCPP is set at 15:1.

[0055] (2) TCPP-Zn 2+ @TA

[0056] 5.36 mg of tannic acid (0.00315 mmol) was suspended in 5 mL of N,N-dimethylformamide (DMF) solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Similarly, 5 mg of TCPP-Zn... 2+ (790.77 g / mol, 0.0063 mmol) was suspended in 5 mL of DMF solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Then, the mixture was stirred for 1 h (1000 rpm) and sonicated for 30 min to form a homogeneous system. Finally, the mixture was added to a hydrothermal reactor and heated at 65 °C for 16 h. The collected samples were collected, centrifuged at 10000 rpm for 20 min, and the precipitate was washed three times with N,N-dimethylformamide (DMF) solution. Finally, quantification was performed by freeze-drying. Tannic acid and TCPP-Zn 2+ The molar ratio is approximately 1:2.

[0057] Example 2

[0058] Reference Figure 3 (b) Figure 4 and Figure 5 ,include:

[0059] (1) TCPP-Ce 3+ preparation

[0060] 41.04 mg of cerium nitrate (0.0945 mmol) was suspended in 5 mL of N,N-dimethylformamide (DMF) solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Similarly, 5 mg of methyl-tetra(4-carboxyphenyl)porphyrin TCPP (790.77 g / mol, 0.0063 mmol) was suspended in 5 mL of DMF solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Then, the above solutions were mixed and 200 μL of glacial acetic acid was added, stirred for 1 h (1000 rpm), and sonicated for 30 min to form a homogeneous system. Finally, the mixture was added to a hydrothermal reactor and heated at 65 °C for 16 h. The collected samples were collected, centrifuged at 10000 rpm for 20 min, and the precipitate was washed twice with N,N-dimethylformamide (DMF) solution, then washed once with ethanol, and finally quantified by freeze-drying. The molar ratio of cerium nitrate to TCPP was set at 15:1.

[0061] (2) TCPP-Ce 3+ @TA preparation

[0062] 5.36 mg of tannic acid (0.00315 mmol) was suspended in 5 mL of N,N-dimethylformamide (DMF) solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Similarly, 5 mg of TCPP-Ce... 3+ The sample was suspended in 5 mL of DMF solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Then, it was mixed and stirred for 1 h (1000 rpm) and sonicated for 30 min to form a homogeneous system. Finally, the mixture was added to a hydrothermal reactor and heated at 65 °C for 16 h. The collected sample was centrifuged at 10000 rpm for 20 min, and the precipitate was washed three times with N,N-dimethylformamide (DMF) solution. Finally, quantification was performed by freeze-drying. Tannic acid and TCPP-Ce were also analyzed. 3+ The molar ratio is set to approximately 1:2.

[0063] Example 3

[0064] Reference Figure 1 (a~c) Figure 2 , Figure 3 (c) Figure 3 (d) Figure 4 and Figure 5 ,include:

[0065] (1) TCPP-Zn 2+ -Ce 3+ preparation

[0066] 6.44 mg of zinc chloride (0.04725 mmol) and 20.52 mg of cerium nitrate (0.04725 mmol) were suspended in 5 mL of N,N-dimethylformamide (DMF) solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Similarly, 5 mg of mes-tetra(4-carboxyphenyl)porphyrin TCPP (790.77 g / mol, 0.0063 mmol) was suspended in 5 mL of DMF solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Then, the above solutions were mixed and 200 μL of glacial acetic acid was added, stirred for 1 h (1000 rpm), and sonicated for 30 min to form a homogeneous system. Finally, the mixture was added to a hydrothermal reactor and heated at 65 °C for 16 h. The collected samples were centrifuged at 10,000 rpm for 20 min. The precipitate was washed twice with N,N-dimethylformamide (DMF) solution, then once with ethanol, and finally quantified by freeze-drying. The molar ratio of cerium nitrate, zinc chloride, and TCPP was set at 7.5:7.5:1.

[0067] (2) TCPP-Zn 2+ -Ce 3+@TA preparation

[0068] 5.36 mg of tannic acid (0.00315 mmol) was suspended in 5 mL of N,N-dimethylformamide (DMF) solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. Similarly, 5 mg of methyl-tetra(4-carboxyphenyl)porphyrin TCPP (790.77 g / mol, 0.0063 mmol) was suspended in 5 mL of DMF solution, stirred for 1 h (1000 rpm), and sonicated for 10 min. The mixture was then stirred for 1 h (1000 rpm) and sonicated for 30 min to form a homogeneous system. Finally, the mixture was added to a hydrothermal reactor and heated at 65 °C for 16 h. The collected samples were collected, centrifuged at 10000 rpm for 20 min, and the precipitate was washed three times with N,N-dimethylformamide (DMF) solution. The final quantification was achieved by freeze-drying. (Tannic acid and TCPP-Zn) 2+ -Ce 3+ The molar ratio is set to 1:2.

[0069] (3) TCPP-Zn 2+ -Ce 3+ @TA@CAP

[0070] Synthetic TCPP-Zn 2+ -Ce 3+ @TA was activated overnight at 150°C, and then further modified with mercaptoacetic acid. In short, 2 mL of mercaptoacetic acid was dissolved in 10 mL of N,N-dimethylformamide (DMF) solution, and then 100 mg of TCPP-Zn was added. 2+ -Ce 3+ The mixture was ultrasonically treated for 30 min to ensure thorough dispersion. Under a nitrogen (N2) atmosphere, the mixture was placed in a vacuum drying oven (70°C, 24 h) to obtain SH-TCPP-Zn. 2+ -Ce 3+ Subsequently, the modified SH-TCPP-Zn 2+ -Ce 3+ The sample was washed three times with DMF and acetone, centrifuged (10,000 rpm, 20 min), and collected. It was then reacted with triethylamine (TEA) for 4 h (640 rpm), followed by dialysis (MWCO: 3500 Da) to remove free CAP-Cys, yielding targeted TCPP-Zn. 2+ -Ce 3+ @CAP.

[0071] (4) TCPP-Zn 2+ -Ce 3+ @TA@KGN@CAP Preparation

[0072] Dissolve 1.5 mg Kartogenin (KGN) (BIDE, BD247584) in 0.2 mL of dimethyl sulfoxide TCPP-Zn 2+ -Ce 3 + @TA@KGN@CAP was added to a DMF solution (10 mg) and stirred for 24 h (640 rpm), followed by dialysis (MWCO: 2500 Da) to remove free KGN, yielding TCPP-Zn. 2+ -Ce 3+ @TA@KGN@CAP.

[0073] Example 4

[0074] Reference Figure 6 Drug release experiment:

[0075] TCPP-Zn was evaluated 2+ -Ce 3+ The ROS-responsive release capacity of @TA@KGN@CAP nanozymes was assessed by dialyzing 2 mg of nanozyme in 20 mg PBS solution (H2O2, 0, 10 mM, and 50 mM) for 72 h. The dialysis bag MWCO: 3500 Da. 1 mL of dialysate was collected sequentially at 0, 1, 2, 4, 8, 24, 48, and 72 h, and the same volume of PBS was added back to the solution. The release of KGN in the samples was determined by HPLC.

[0076] Example 5

[0077] Reference Figure 7 (a) and Figure 7 (b) CAT enzyme mimicry activity of nanozymes:

[0078] With TCPP-Zn 2+ and TCPP-Ce 3+ As a control, TCPP-Zn was measured. 2+ -Ce 3+ The concentration change of H2O2 after enzyme decomposition. In short, after adding a certain amount of H2O2 (10mM) solution at room temperature, the concentration of O2 generated after the reaction with the same concentration (20μg / mL) of nanozyme was measured using a portable dissolved oxygen meter (JPB-607, Nanjing Kehuan Analytical Instrument Co., Ltd.). Simultaneously, the concentrations of TCPP-Zn at different concentrations were also measured. 2+ -Ce 3+ The concentration of O2 generated after the reaction of @TA@KGN@CAP nanozymes (5, 10, 20, 50 μg / mL).

[0079] Example 6

[0080] Reference Figure 8CAT enzyme mimicry activity of nanozymes:

[0081] SOD enzyme mimicry activity of nanozymes: TCPP-Zn was measured using a commercial colorimetric SOD assay kit (S311-10, Dojindo Molecular Technologies). 2+ TCPP-Ce 3+ and TCPP-Zn 2+ -Ce 3+ SOD-like activity assay. The experiment was performed according to the manufacturer's instructions, and SOD-like activity at a series of concentrations (0-50 μg / mL, concentration gradient 5 μg / mL) was expressed as the percentage of inhibition of the competitive WST reaction. Absorbance was measured at 450 nm using a microplate reader (Synergy™ Neo2, USA). The SOD inhibition rate (%) was calculated according to a formula.

[0082]

[0083] Control 1 is a blank control (without inhibitor), Control 2 is a sample blank control, and Control 3 is a blank reagent control.

[0084] Example 7

[0085] Reference Figure 9 CCK-8 assay for cell viability:

[0086] The effect of different concentrations of nanozymes on the activity of C28 / I2 human chondrocytes was determined using the CCK-8 assay. Cells were cultured at a concentration of 1×10⁻⁶. 5 Cells were seeded per well in 12-well plates and incubated overnight. Subsequently, the cells were treated with different concentrations (0, 5, 10, 20, 50 μg / mL) of nanoparticles for 24 h, followed by treatment with CCK-8 solution for 1 h. Finally, the absorbance at OD 450 nm in each well was measured using a microplate reader, and the percentage of viable cells was plotted using GraphPad Prism software.

[0087] According to another aspect of the present invention, the present invention also provides a responsive nanozyme for delivering KGN prepared by the above preparation method.

[0088] According to another aspect of the invention, the invention also provides the use of the above-described responsive nanozyme for delivering KGN in a medicament for treating osteoarthritis.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a responsive nanoscale enzyme for delivering KGN, characterized in that, The preparation method comprises the following steps: S1, suspending zinc chloride and cerium nitrate in an N,N-dimethylformamide solution, suspending meso-tetra(4-carboxyphenyl) porphyrin in an N,N-dimethylformamide solution, stirring and ultrasonic treatment respectively; S2, mixing the above solutions, adding an appropriate amount of glacial acetic acid, stirring and ultrasonic treatment, forming a uniform system, adding the mixture to a hydrothermal reactor for hydrothermal reaction, collecting the sample, centrifuging, taking the precipitate, washing with N,N-dimethylformamide solution and then with ethanol, and quantifying by freeze-drying; S3, dissolving tannic acid in N,N-dimethylformamide solution, dissolving meso-tetra(4-carboxyphenyl) porphine in N,N-dimethylformamide solution, adding the mixture to a hydrothermal reactor for hydrothermal reaction, collecting the collected sample for centrifugal washing, and obtaining meso-tetra(4-carboxyphenyl) porphine-Zn by freeze-drying 2+ -Ce 3+ ; S4, meso-tetra(4-carboxyphenyl)porphine-Zn 2+ -Ce 3+ Activated overnight, modified with mercaptoacetic acid; S5, the modified mercaptoacetic acid- meso-tetrakis (4-carboxyphenyl) porphyrin-Zn 2+ -Ce 3+ and cysteine modified CAP bone targeting peptide under the condition of adding triethylamine, and reacting sufficiently, removing free CAP bone targeting peptide by dialysis to obtain meso-tetrakis (4-carboxyphenyl) porphyrin-Zn with targeting property 2+ -Ce 3+ @tannic acid @CAP peptide; S6, KGN was dissolved in dimethyl sulfoxide, diluted in N,N-dimethylformamide, and the above solution was added to SH-TCPP-Zn 2+ -Ce 3+ @TA was stirred in N,N-dimethylformamide and dialyzed to remove free KGN, to obtain mercapto meso-tetrakis(4-carboxyphenyl) porphyrin-Zn 2+ -Ce 3+ @Tannic acid KGN CAP peptide.

2. The method of claim 1, wherein the responsive nanoscale enzyme delivering KGN is prepared by, In the S1, the molar ratio of cerium nitrate, zinc chloride and meso-tetra(4-carboxyphenyl) porphyrin is set to 7.5:7.5:

1.

3. The method of claim 1, wherein the responsive nanoscale enzyme delivering KGN is prepared by, In the S1, the ultrasonic treatment time is 10-40 min.

4. The method of claim 1, wherein the responsive nanoscale enzyme delivering KGN is prepared by, In the S2, the hydrothermal reaction temperature is 50-120℃; The centrifugation rate is 8000-20000 rpm.

5. The method of claim 1, wherein the responsive nanoscale enzyme delivering KGN is prepared by, In S4, meso-tetrakis(4-carboxyphenyl)porphyrin-Zn 2+ -Ce 3+ Activated overnight, modified with mercaptoacetic acid, specifically including: Mercaptoacetic acid was dissolved in N,N-dimethylformamide solution, and meso-tetra(4-carboxyphenyl)porphine-Zn was added 2+ -Ce 3+ ultrasonicated to disperse it sufficiently; The mixture was left in a vacuum oven under nitrogen atmosphere to give the mercapto-meso-tetra(4-carboxyphenyl)porphyrin-Zn 2+ -Ce 3+ Washed several times with N,N-dimethylformamide and acetone solution, centrifuged and dried in a vacuum oven overnight.

6. The method of claim 1, wherein the responsive nanoscale enzyme delivering KGN is prepared by, In the S3, the activation temperature is 120-180℃, and the activation time is 8-16 h; The centrifugation rate is 8000-20000 rpm; The temperature of the vacuum oven is 50-90℃.

7. The method of claim 1, wherein the responsive nanoscale enzyme delivering KGN is prepared by, In S5, the modified meso-tetra(4-carboxyphenyl)porphine-Zn is added 2+ -Ce 3+ @Tannic acid@Thiol-CAP bone-targeting peptide mass ratio is 10:(0.5-2).

8. The method of claim 1, wherein the responsive nanoscale enzyme delivering KGN is prepared by, In S5, the KGN and meso-tetra(4-carboxyphenyl) porphyrin-Zn are added 2+ -Ce 3+ @The mass ratio of tannic acid and thiol-CAP peptide is 1 :(6-8).

9. The responsive nano-enzyme for delivering KGN prepared by the preparation method of any one of claims 1-8.

10. The responsive nano-enzyme for delivering KGN of claim 9 in the treatment of osteoarthritis.

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