Bionanosenzyme drug for treating gouty arthritis and preparation method and application thereof

Abstract

This invention relates to the field of biomedical nanomaterials and pharmaceutical formulations, and discloses a biomimetic nanoenzyme drug for treating gouty arthritis, its preparation method, and its application. The biomimetic nanoenzyme drug is a core-shell structured nanoparticle, comprising, from the innermost layer to the outermost layer: ① a catalytic and antioxidant core, formed by a composite of carbon dots and manganese dioxide; ② a molecularly imprinted polymer layer coating the core, the molecularly imprinted polymer being formed using uric acid as a template molecule; and ③ a platelet cell membrane layer coating the molecularly imprinted polymer layer. This invention utilizes the aforementioned biomimetic nanoenzyme drug for treating gouty arthritis, its preparation method, and its application. This drug, through a multi-level biomimetic assembly strategy, integrates carbon dots (CDs) to enhance antioxidant activity, molecularly imprinted polymers (MIPs) to impart uric acid selectivity, and platelet membrane (PLM) camouflage to improve targeting and compatibility, thereby achieving a safe and highly effective synergistic treatment for gouty arthritis.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials and pharmaceutical formulations, and in particular to biomimetic nanoenzyme drugs for the treatment of gouty arthritis, their preparation methods and applications. Background Technology

[0002] Gouty arthritis is a metabolic disease caused by excessively high levels of uric acid (UA) in the body, leading to the formation of monosodium urate (MSU) crystals that deposit in the joints and trigger a severe inflammatory response. Clinical treatment faces two major challenges: firstly, the need for highly efficient and specific reduction of UA and MSU levels at the lesion site; and secondly, the need to simultaneously control the excessive oxidative stress and inflammatory response induced by MSU crystal formation. Currently, recombinant uricases (such as polyethylene glycol-modified uricase) are effective uric acid-lowering biological agents, but they suffer from strong immunogenicity, high cost, and the potential to exacerbate oxidative stress by producing hydrogen peroxide (H2O2) during the catalytic process. In recent years, nanomaterials with enzyme-like activity (nanozymes) have provided new insights into gout treatment. Manganese dioxide (MnO2) nanomaterials have attracted attention due to their combined uricase-like and catalase (CAT) activities, enabling them to simultaneously degrade UA and scavenge reactive oxygen species (ROS). However, naked MnO2 nanozymes have the following inherent defects, severely limiting their clinical application: 1. Poor substrate selectivity: They non-specifically oxidize various essential reducing molecules in the body (such as glutathione GSH, vitamin C, etc.), disrupting intracellular redox homeostasis and producing off-target toxic side effects. 2. Poor biocompatibility: They are easily cleared by the immune system and have low accumulation efficiency at lesion sites; the Mn released during the catalytic process... 2+ Ions may produce pro-oxidation effects and pose potential toxicity. 3. Insufficient catalytic stability: They are easily over-reduced by the substrate during the reaction, leading to dissolution and inactivation. Therefore, developing a safe nanomedicine for gout treatment that combines high selectivity, good biocompatibility, efficient synergistic therapeutic function, and other key technical challenges is an urgent technical problem to be solved. Summary of the Invention

[0003] The purpose of this invention is to provide a biomimetic nanozyme drug for treating gouty arthritis, its preparation method, and its application, in order to solve the aforementioned technical problems. This drug, through a multi-level biomimetic assembly strategy, integrates carbon dots (CDs) to enhance antioxidant activity, molecular imprinting (MIP) to impart uric acid selectivity, and platelet membrane (PLM) camouflage to improve targeting and compatibility, thereby achieving a safe and efficient synergistic treatment for gouty arthritis.

[0004] To achieve the above objectives, this invention provides a biomimetic nanoenzyme drug for treating gouty arthritis. The biomimetic nanoenzyme drug is a core-shell structured nanoparticle, with the following layers from the innermost to the outermost: ① The core, which provides catalysis and antioxidant protection, is formed by the combination of carbon dots and manganese dioxide; ② A molecularly imprinted polymer layer covering the core, wherein the molecularly imprinted polymer is formed using uric acid as a template molecule; ③ The platelet cell membrane layer that covers the molecularly imprinted polymer layer.

[0005] Furthermore, in the core, the carbon dots are synthesized using polyethylene polyamine as the carbon source; the manganese dioxide is an amorphous or low-crystallinity nanostructure rich in oxygen vacancies.

[0006] Furthermore, the molecularly imprinted polymer layer is polymerized using a urea-containing silane coupling agent as a functional monomer and tetraethyl orthosilicate as a crosslinking agent; the urea-containing silane coupling agent is ureapropyltriethoxysilane.

[0007] Furthermore, the volume ratio of the functional monomer to the crosslinking agent is 1:1 to 1:4.

[0008] Furthermore, the present invention also provides a method for preparing biomimetic nanozyme drugs, comprising the following steps: Step S1: Prepare carbon dot-manganese dioxide composite CM; Step S2: On the surface of the carbon dot-manganese dioxide complex, using uric acid as a template molecule, a molecularly imprinted polymer layer is formed by polymerization to obtain the intermediate complex CMM. Step S3: Co-incubate the intermediate complex with the platelet membrane or sonicate it to coat the platelet membrane onto its surface, thereby obtaining the biomimetic nanoenzyme drug CMMP.

[0009] Furthermore, the specific operation of step S1 is as follows: First, carbon dot-loaded silica nanospheres (SC) were synthesized. In the presence of SC, potassium permanganate was used for in-situ reduction to deposit a MnO2 layer on the surface of SC, thus obtaining the precursor. The silica template was removed by etching with sodium carbonate solution to obtain a carbon dot-manganese dioxide composite (CM) with a hollow structure.

[0010] Furthermore, the specific operation of step S2 is as follows: The carbon dot-manganese dioxide complex CM was dispersed in an alcohol solvent; the template molecule uric acid UA, the functional monomer UPTEOS, and the crosslinking agent TEOS were added, and a sol-gel reaction was carried out under the action of an alkaline catalyst to polymerize on the surface of CM to form a SiO2-based molecularly imprinted polymer layer; the template molecule UA was eluted to remove it, and a CMM with UA-specific recognition cavity was obtained.

[0011] Furthermore, the specific operation of step S3 is as follows: First, platelets are extracted and platelet membrane vesicles (PLM) are prepared. The intermediate complex (CMM) is mixed with PLM, and PLM is spontaneously coated onto the surface of CMM by ultrasonic extrusion to form the biomimetic nanoenzyme drug CMMP.

[0012] Furthermore, the present invention also provides the application of the above-mentioned biomimetic nanozyme drug in the preparation of a drug for treating gouty arthritis.

[0013] Furthermore, the present invention also provides a pharmaceutical composition for treating gouty arthritis, the pharmaceutical composition comprising the above-described biomimetic nanoenzyme drug, and a pharmaceutically acceptable carrier or excipient.

[0014] The functions of the drug include: specifically degrading uric acid and monosodium urate crystals, scavenging reactive oxygen species, inhibiting the release of pro-inflammatory factors, and / or reducing joint inflammation. The drug is in the form of an injection.

[0015] The advantages and positive effects of the biomimetic nanoenzyme drug for treating gouty arthritis, its preparation method, and its application described in this invention are as follows: 1. The introduction of CDs in this invention not only enhances the antioxidant properties, but also inhibits Mn 2+ The pro-oxidative side effects were reduced, improving the biosafety of the material. PLM modification significantly enhanced the enrichment and cellular uptake efficiency of nanomedicines at inflamed joints, thereby amplifying the therapeutic effect.

[0016] 2. The introduction of the MIP layer in this invention is a pioneering application of molecular imprinting technology to regulate the substrate selectivity of therapeutic nanozymes. It transforms the nanozyme from a "broad-spectrum oxidant" into a "precise UA degrader," significantly reducing the loss of important antioxidant molecules such as GSH in vivo, thus minimizing off-target toxicity at its source. The PLM outer layer further enhances biocompatibility and blood compatibility.

[0017] 3. In this invention, CMMP integrates three major functions: UA-specific degradation (through MnO2 and MIP), potent antioxidant / anti-inflammatory (through the synergy of MnO2 and CDs), and inflammation-targeted delivery (through PLM). It provides synergistic intervention for gout from multiple pathological stages, with significantly better efficacy than single-function materials. At the same time, the preparation process of the biomimetic nanoenzyme drug is clear, the functions of each component are well-defined, the structure is characterizable, and it has good reproducibility and transformation potential.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the preparation of biomimetic nanozyme drug (CMMP) in an embodiment of the present invention; Figure 2 The images shown are transmission electron microscope (TEM) images and elemental distribution diagrams of CMMP in this embodiment of the invention. Figure 3The figures show the in vitro kinetic curves of CMMP's uricase-like activity in embodiments of the present invention, where A represents the relationship between the initial rate (v) of the uric acid reaction catalyzed by CM and CMM and the uric acid concentration ([UA]). B is a double reciprocal plot of the oxidative decomposition of uric acid catalyzed by CM and CMM. Figure 4 These are optical microscope images of the in vitro degradation of MSU crystals by CMMP in an embodiment of the present invention, wherein A is an actual image, B is an optical microscope image of MSU crystals, C is an optical microscope image of MSU crystals after co-incubation with CM, and D is an optical microscope image of MSU crystals after co-incubation with CMM. Figure 5 These are fluorescence micrographs of macrophages treated with CMMP to alleviate MSU-induced oxidative stress in an embodiment of the present invention. In the images, A is intracellular ROS fluorescence imaging after treatment with only basal culture medium, B is intracellular ROS fluorescence imaging after treatment with monosodium urate crystals, C is intracellular ROS fluorescence imaging after treatment with monosodium urate crystals and CM, D is intracellular ROS imaging after treatment with monosodium urate crystals and CMM, E is intracellular ROS imaging after treatment with monosodium urate crystals and CMMP, and F is a quantitative comparison of the fluorescence intensity values ​​in A, B, C, D, and E. Figure 6 This is a curve showing the effect of CMMP treatment on paw swelling rate in a mouse model of gouty arthritis, as described in this embodiment of the invention. Figure 7 The effect of CMMP treatment on the level of inflammatory factors in mouse serum is shown in the embodiments of the present invention, wherein A is TNF-α, B is IL-1β, C is IL-10, and D is IL-6; Figure 8 The images show tissue sections (H&E staining) of major organs in mice after CMMP treatment in this embodiment of the invention, demonstrating its biocompatibility. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0023] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0024] This invention discloses a biomimetic nanoenzyme drug for treating gouty arthritis. The biomimetic nanoenzyme drug has core-shell structured nanoparticles, comprising, from the inside out: Catalytic and Antioxidant Core: Hollow or solid nanospheres (denoted as CM) formed by the composite of carbon dots (CDs) and manganese dioxide (MnO2). CDs not only possess antioxidant activity themselves, but also effectively complex and shield the pro-oxidant Mn released during MnO2 catalysis. 2+ This synergistically enhances the overall antioxidant capacity of the complex (e.g., scavenging •OH, O2•). − , H2O2) ability.

[0025] Selective Recognition Intermediate Layer: A molecularly imprinted polymer (MIP) layer (collectively denoted as CMM) encapsulating the CM core. This MIP layer is polymerized using uric acid (UA) as a template molecule and a urea-containing silane coupling agent (preferably ureapropyltriethoxysilane, UPTEOS) as a functional monomer. This layer endows the nanoparticles with the ability to specifically recognize and bind to UA molecules, while also acting as a physical barrier to significantly reduce the non-specific oxidation of non-target biomolecules such as GSH by the nanozyme, thereby improving the targeting and safety of the therapy.

[0026] Biological Targeting and Camouflage Outer Layer: The outermost layer is the platelet cell membrane (PLM) (collectively denoted as CMMP). PLM is extracted from natural platelets, and its surface retains characteristic proteins such as CD61. This membrane layer provides nanoparticles with immune evasion capabilities, prolonging their circulation time in vivo; simultaneously, utilizing the natural targeting of platelets to inflamed sites, it actively targets and accumulates at the lesions of gouty arthritis, and promotes drug uptake by inflammatory cells such as macrophages, enhancing the local therapeutic effect.

[0027] The carbon dots (CDs) are preferably synthesized via a solvothermal method using polyethylene polyamine as the carbon source, exhibiting blue fluorescence and antioxidant properties. The manganese dioxide (MnO2) is an amorphous or low-crystallinity nanostructure rich in oxygen vacancies, possessing excellent uricase-like and catalase-like activities. In the molecularly imprinted polymer (MIP) layer, the volume ratio of the functional monomer UPTEOS to the crosslinking agent tetraethyl orthosilicate (TEOS) is preferably 1:1 to 1:4, more preferably 1:2. The platelet membrane (PLM) is extracted from platelets of mammals (such as mice, rats, or humans) using a repeated freeze-thaw method.

[0028] This invention also provides a method for preparing the above-mentioned biomimetic nanozyme drug (CMMP), comprising the following steps ( Figure 1 (as shown) S1. Synthesis of carbon dot-manganese dioxide complex (CM): a. Synthesis of silica nanospheres loaded with carbon dots (SC); b. In the presence of SC, potassium permanganate (KMnO4) is used for in-situ reduction to deposit a MnO2 layer on the surface of SC, thus obtaining the precursor; c. The silica template is removed by etching with sodium carbonate solution to obtain a carbon dot-manganese dioxide composite (CM) with a hollow structure.

[0029] S2. Constructing the molecularly imprinted layer (obtaining the CMM): a. Disperse CM in an alcohol solvent; b. Add template molecule uric acid (UA), functional monomer UPTEOS and crosslinking agent TEOS, and carry out sol-gel reaction under the action of alkaline catalyst (such as ammonia water) to polymerize on the CM surface to form a SiO2-based molecularly imprinted polymer layer. c. Elution removes the template molecule UA to obtain a CMM with UA-specific recognition of the cavity.

[0030] S3, Platelet Membrane Disguise (obtaining CMMP): a. Extracting platelets and preparing platelet membrane vesicles (PLM). b. Mix CMM and PLM, and use ultrasonic extrusion to allow PLM to spontaneously coat the surface of CMM, forming the final biomimetic nanoenzyme drug CMMP.

[0031] This invention also provides the application of the above-mentioned biomimetic nanoenzyme drug in the preparation of a drug for treating gouty arthritis. The drug can be used for: ① It specifically degrades uric acid (UA) and monosodium urate (MSU) crystals in the joint cavity and blood.

[0032] ② Clear excess reactive oxygen species (ROS) from inflamed areas and alleviate oxidative stress.

[0033] ③ It inhibits the release of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6) from inflammatory cells such as macrophages and promotes the release of anti-inflammatory factors (such as IL-10).

[0034] ④ Reduce joint swelling, inflammatory cell infiltration, and tissue damage in animal models of gouty arthritis.

[0035] In this invention, CMMP integrates three major functions: UA-specific degradation (through MnO2 and MIP), potent antioxidant / anti-inflammatory (through the synergy of MnO2 and CDs), and inflammation-targeted delivery (through PLM). It provides synergistic intervention for gout from multiple pathological stages, and its therapeutic effect is significantly better than that of single-function materials.

[0036] The introduction of the MIP layer in this invention is a pioneering application of molecular imprinting technology to regulate the substrate selectivity of therapeutic nanozymes. It transforms the nanozyme from a "broad-spectrum oxidant" into a "precise UA degrader," significantly reducing the loss of important antioxidant molecules such as GSH in vivo, thus minimizing off-target toxicity at its source. The PLM outer layer further enhances biocompatibility and blood compatibility.

[0037] The introduction of CDs in this invention not only enhances antioxidant properties but also inhibits Mn. 2+ The pro-oxidative side effects were reduced, improving the biosafety of the material. PLM modification significantly enhanced the enrichment and cellular uptake efficiency of nanomedicines at inflamed joints, thereby amplifying the therapeutic effect.

[0038] The preparation process of the biomimetic nanoenzyme drug in this invention is clear, the function of each component is well-defined, the structure is characterizable, and it has good reproducibility and transformation potential.

[0039] The following examples provide a detailed explanation.

[0040] Example 1: Preparation and Characterization of CMMP Preparation of S1, carbon dot-manganese dioxide complex (CM): a) Synthesis of carbon dots (CDs): 10 mL of polyethylene polyamine was placed in a polytetrafluoroethylene reactor liner and reacted at 200 °C for 12 hours. After the reaction was completed, the product was dissolved in ultrapure water and dialyzed in a dialysis bag with a molecular weight cutoff of 500 Da for 48 hours. After freeze-drying, solid CDs were obtained.

[0041] b) Synthesis of CDs-loaded silica nanospheres (SC): 10 mg of the above-mentioned CDs were dispersed in 20 mL of anhydrous ethanol, 2 mL of 25% (w / w) ammonia solution was added, followed by 0.3 mL of tetraethyl orthosilicate (TEOS). The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 10 minutes and washed three times with ultrapure water to obtain SC.

[0042] c) Synthesis of CM: SC was dispersed in ultrapure water to prepare a dispersion of 0.5 mg / mL. Potassium permanganate (KMnO4) was added to this dispersion to a final concentration of 5 mM, and the mixture was stirred at room temperature for 24 hours. Subsequently, the brownish-black precipitate was collected by centrifugation at 10,000 rpm and washed with ultrapure water. The precipitate was redispersed in a 2 M sodium carbonate (Na2CO3) solution and stirred at 60 °C for 3 hours to etch the silica template. After etching, the mixture was centrifuged and washed with water to obtain the carbon dot-manganese dioxide complex (CM).

[0043] S2. Preparation of molecularly imprinted layer (CMM): 50 mg of the above-mentioned CM was dispersed in 50 mL of anhydrous ethanol. In a separate container, 0.1 mL of ureapropyltriethoxysilane (UPTEOS, 50% by mass) and 2 mg of uric acid (UA) were dissolved in 10 mL of methanol and sonicated for 30 minutes to form a pre-assembled solution. This pre-assembled solution was added to the ethanol dispersion of CM, followed by 0.1 mL of TEOS and 0.25 mL of concentrated ammonia (25% by mass). The mixture was stirred and polymerized at room temperature for 24 hours. After the reaction was completed, the solid was collected by centrifugation and repeatedly washed with a solution containing 0.1% ammonia until the washing solution showed no UV absorption at 290 nm (indicating that the template molecule UA was completely eluted), yielding the carbon dot-manganese dioxide-molecularly imprinted polymer complex (CMM).

[0044] S3. Preparation of platelet membrane camouflage (CMMP): Whole blood was collected from mice using heparin-anticoagulated tubes. The plasma was centrifuged at 300 rpm for 10 minutes to remove red blood cells, and the supernatant platelet-rich plasma was collected. This plasma was then centrifuged at 2000 rpm for 10 minutes, and the precipitate was platelets. The platelets were resuspended in sterile water and frozen at -80 °C for 20 minutes, followed by thawing at room temperature. This process was repeated three times to disrupt the cells. Finally, the precipitate was collected by centrifugation at 15000 rpm, yielding platelet membrane (PLM) vesicles. 10 mg of CMM and the equivalent of 1 mL of whole blood-derived PLM vesicles were co-dispersed in 1 mL of PBS buffer and sonicated (100 W for 10 minutes) to coat the CMM surface with PLM, thus obtaining the final biomimetic nanozyme drug CMMP.

[0045] Characterization: An aqueous solution of CMMP was dropped onto a carbon-supported copper grid, and the structure of CMMP was observed using transmission electron microscopy. Under the electron microscope, CMMP exhibited a hollow spherical structure. The elemental distribution diagram shows ( Figure 2 CMMP has characteristic elements such as Mn, O, Si, P, and S, which correspond to manganese dioxide, silicon dioxide-based molecular imprints, and platelet membrane components, respectively, and form a core-shell structure from the inside out.

[0046] Example 2: Enzyme-like activity and selectivity test of CMM The CMM prepared in Example 1 was used as a substrate, and its uricase oxidase activity was measured in phosphate-buffered saline (PBS) at pH 7.4 at 37°C. The reaction rate was calculated by monitoring the decrease in absorbance at 290 nm. Using a Michaelis-Menten kinetic model, the apparent Michaelis constant (Km) was calculated to be 86.01 μM, and the maximum reaction rate (Vmax) was 35.08 μM / min.

[0047] To examine its selectivity, glutathione (GSH), ascorbic acid (VC), and cysteine ​​(Cys) were tested under the same conditions. The results are as follows: Figure 3 As shown, CMM retained 90% of the catalytic activity of UA as CM (the unmodified MIP control), while its catalytic activity for GSH, VC, and Cys decreased to 11%, 8%, and 15% of that of CM, respectively. This indicates that the molecularly imprinted layer effectively endows CMM with a highly selective oxidation capacity for uric acid.

[0048] Furthermore, CMM exhibits significant catalase-like activity, capable of catalyzing the decomposition of H₂O₂ to produce oxygen. It also exhibits activity against hydroxyl radicals (•OH) and superoxide anions (O₂•OH). − The clearance rates of ) reached 85% and 78% at a concentration of 100 μg / mL, respectively.

[0049] To assess the degradation ability of CMM on monosodium urate crystals, the morphology of the crystals was observed after co-incubation of CMM and monosodium urate (MSU) crystals. Sodium urate crystals were prepared as follows: 50 mg of uric acid was dissolved in 40 mL of 15 mM sodium hydroxide solution, and the pH was adjusted to 7.0; 526 mg of sodium chloride was dissolved in 40 mL of ultrapure water. The uric acid and sodium chloride solutions were mixed at a ratio of 1:5 and incubated at 37 °C overnight. The resulting white precipitate was centrifuged, resuspended in ethanol, sonicated for 30 min, and centrifuged again. The precipitate was then dried to obtain MSU. 6 mg of MSU was dispersed in 10 mL of PBS buffer (pH=6), and either CM or CMM was added. After incubation at 37 °C for 30 min, the MSU in each group was observed under an optical microscope. The results are as follows: Figure 4 As shown, MSU exhibits a needle-like morphology. After incubation with CM and CMM, the needle-like crystals disappear, indicating that CM and CMM can effectively degrade MSU.

[0050] Example 3: Study on the cell compatibility and anti-inflammatory effects of CMMP Mouse mononuclear macrophage leukemia cells (RAW264.7) were used for testing. The MTT assay results showed that when the CMMP concentration (as Mn) was as high as 200 μM, the cell viability was still greater than 85%, indicating that it has low cytotoxicity.

[0051] To simulate a gout environment, RAW264.7 cells were stimulated with 0.25 mg / mL monosodium urate (MSU) crystals for 4 hours, followed by co-culturing with 100 μg / mL CMMP for 12 hours. The reactive oxygen species (ROS) fluorescent probe DCFH-DA was used to detect... Figure 5 As shown in the figure, compared with the MSU stimulation group alone, the intracellular ROS fluorescence intensity in the CMMP treatment group was reduced by approximately 70%. Enzyme-linked immunosorbent assay (ELISA) results showed that CMMP treatment significantly inhibited the release of MSU-induced pro-inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), reducing them by 65% ​​and 58%, respectively, while increasing the level of the anti-inflammatory factor interleukin-10 (IL-10) by 2.1-fold.

[0052] Example 4: Evaluation of the therapeutic effect of CMMP on gouty arthritis in mice.

[0053] C57BL / 6 mice were randomly divided into 5 groups (n=5): blank control group, model group (MSU), MSU+CM group, MSU+CMM group, and MSU+CMMP group. Except for the blank group, which was injected with saline, the other groups were injected with 25 μL of 20 mg / mL MSU suspension in the left hind paw to induce acute gouty arthritis. Two hours after modeling, the treatment groups were injected with 25 μL of CM, CMM, or CMMP dispersion containing an equal amount of manganese (0.5 mg Mn / kg) at the same site, while the model group and blank group were injected with an equal volume of saline.

[0054] The thickness of the mouse paws was measured periodically using vernier calipers. Results are as follows: Figure 6 As shown, after MSU injection, the paws of the model group swelled rapidly, reaching peak swelling at 12 hours. The CMMP treatment group showed the least swelling, with a significantly lower swelling rate than the model group at 24 hours (P<0.01), and the swelling essentially returned to normal at 72 hours. The CM and CMM groups also showed some relief, but both were inferior to the CMMP group.

[0055] Serum was collected from mice 72 hours after treatment. ELISA analysis showed ( Figure 7 As shown in the figure, compared with the model group, the serum levels of TNF-α, IL-1β and IL-6 in the CMMP treatment group decreased by 72%, 68% and 61%, respectively, while the IL-10 level increased by 1.8 times.

[0056] The heart, liver, spleen, lungs, kidneys, and other major organs of mice were collected for hematoxylin-eosin (H&E) staining and pathological examination. Figure 8 (As shown in the figure). The results showed that the organ tissues of mice in each treatment group were intact and no obvious pathological changes were observed, indicating that CMMP has good in vivo biocompatibility at the experimental dose.

[0057] Therefore, the present invention employs the above-mentioned biomimetic nanoenzyme drug for treating gouty arthritis, its preparation method and application. This drug integrates carbon dots (CDs) to enhance antioxidant activity, molecular imprinting (MIP) to impart uric acid selectivity, and platelet membrane (PLM) camouflage to improve targeting and compatibility through a multi-level biomimetic assembly strategy, thereby achieving safe and efficient synergistic treatment for gouty arthritis.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A biomimetic nanoscale enzyme drug for treating gouty arthritis, characterized in that, Biomimetic nanoenzyme drugs are core-shell structured nanoparticles, with the following layers from the innermost to the outermost: ① The core, which provides catalysis and antioxidant protection, is formed by the combination of carbon dots and manganese dioxide; In the core, the carbon dots are synthesized using polyethylene polyamine as the carbon source; the manganese dioxide is an amorphous or low-crystallinity nanostructure rich in oxygen vacancies. ② A molecularly imprinted polymer layer covering the core, wherein the molecularly imprinted polymer is formed using uric acid as a template molecule; ③ The platelet cell membrane layer that covers the molecularly imprinted polymer layer.

2. The biomimetic nanoscale enzyme drug of claim 1, wherein: The molecularly imprinted polymer layer is formed by polymerization using a urea-containing silane coupling agent as a functional monomer and tetraethyl orthosilicate as a crosslinking agent; the urea-containing silane coupling agent is ureapropyltriethoxysilane.

3. The biomimetic nanoscale enzyme drug of claim 2, wherein, The volume ratio of the functional monomer to the crosslinking agent is 1:1 to 1:

4.

4. A method for the preparation of a biomimetic nanoscale enzyme drug according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step S1: Prepare carbon dot-manganese dioxide composite CM; First, carbon-dot-loaded silica nanospheres (SC) were synthesized; then, in the presence of SC, potassium permanganate was used for in-situ reduction to deposit a MnO2 layer on the SC surface, yielding the precursor. The silica template was removed by etching with sodium carbonate solution to obtain a carbon dot-manganese dioxide complex CM with a hollow structure. Step S2: On the surface of the carbon dot-manganese dioxide complex, using uric acid as a template molecule, a molecularly imprinted polymer layer is formed by polymerization to obtain the intermediate complex CMM. Step S3: Co-incubate the intermediate complex with the platelet membrane or sonicate it to coat the platelet membrane onto its surface, thereby obtaining the biomimetic nanoenzyme drug CMMP.

5. The preparation method according to claim 4, characterized in that, The specific operation of step S2 is as follows: The carbon dot-manganese dioxide complex CM was dispersed in an alcohol solvent; the template molecule uric acid UA, the functional monomer UPTEOS, and the crosslinking agent TEOS were added, and a sol-gel reaction was carried out under the action of an alkaline catalyst to polymerize on the surface of CM to form a SiO2-based molecularly imprinted polymer layer; the template molecule UA was eluted to remove it, and a CMM with UA-specific recognition cavity was obtained.

6. The preparation method according to claim 4, characterized in that, The specific operation of step S3 is as follows: First, platelets are extracted and platelet membrane vesicles (PLM) are prepared. The intermediate complex (CMM) is mixed with PLM, and PLM is spontaneously coated onto the surface of CMM by ultrasonic extrusion to form the biomimetic nanoenzyme drug CMMP.

7. Use of the biomimetic nanoscale enzyme drug according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of gouty arthritis, characterized in that, Biomimetic nanoenzyme drugs are core-shell structured nanoparticles, with the following layers from the innermost to the outermost: ① The core, which provides catalysis and antioxidant protection, is formed by the combination of carbon dots and manganese dioxide; In the core, the carbon dots are synthesized using polyethylene polyamine as the carbon source; the manganese dioxide is an amorphous or low-crystallinity nanostructure rich in oxygen vacancies. ② A molecularly imprinted polymer layer covering the core, wherein the molecularly imprinted polymer is formed using uric acid as a template molecule; ③ The platelet cell membrane layer that covers the molecularly imprinted polymer layer.

8. A pharmaceutical composition for treating gouty arthritis, characterized by, The pharmaceutical composition comprises the biomimetic nanozyme drug according to any one of claims 1-3, and a pharmaceutically acceptable carrier.

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