Multifunctional nano-enzyme platform as well as preparation method and application thereof

Abstract

The invention belongs to the field of nano-drug preparations, and discloses a multifunctional nano-enzyme platform and a preparation method and application thereof, and the multifunctional nano-enzyme platform is mainly composed of a nano-enzyme, a carrier material and a targeting substance. The synthesis of the nano-enzyme, the loading of the nano-enzyme on a carrier material and the modification of a targeting substance on the surface of the nano-enzyme are carried out through methods such as stirring synthesis, coprecipitation, strong acid oxidation, chemical reduction, covalent binding, a solvothermal method and physical adsorption. The targeting substance on the surface of the nano-enzyme platform can be accurately and efficiently positioned to an inflammation part, the meta-acid microenvironment of the inflammation part enables the targeting substance and the carrier material to be subjected to structural depolymerization, so that various nano-enzymes are released, and the nano-enzymes not only realize self-cascade and synergistic catalysis enhancement effects of the enzymes at the inflammation part, but also can be used for preparing the nano-enzyme. And a substrate self-circulation catalysis system is also formed, so that the effect of efficiently relieving and treating various inflammation-related diseases is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug preparations, and in particular to a multifunctional nano enzyme platform and a preparation method and application thereof. Background Art

[0002] Inflammation is a complex biological response and is usually the body's natural defense mechanism against injury or harmful stimulation. It is a protective response that aims to remove pathogenic factors (such as pathogens, damaged cells) and initiate the repair process. When the body is damaged or infected, immune cells release inflammatory factors, which can regulate the inflammatory response, thereby causing local vasodilation and increased permeability, allowing more immune cells, nutrients and repair factors to enter the damaged area, leading to redness, swelling and fever. Inflammation is divided into acute inflammation and chronic inflammation, which mainly depends on the nature and duration of the injury. Acute inflammation can lead to the occurrence of diseases such as gout (manifested as acute arthritis), acute gastroenteritis, and acute wound infection, while diabetes, rheumatoid arthritis, ischemic stroke, atherosclerosis, colitis, myocardial infarction, etc. are chronic inflammations caused by prolonged inflammation. Among them, gout, as a more serious type of acute inflammation, has attracted much attention due to its short onset time and severe pain.

[0003] Gout is a long-term recurrent metabolic disease characterized by purine metabolism disorders and / or uric acid (UA) metabolism imbalances. A high-purine diet will lead to a continuous increase in UA, the final product of purine catabolism. When the UA concentration in the tissue continues to increase until saturation, it will cause monosodium urate crystals (MSU) to deposit around the joints. Since some mammals such as humans lack uricase, they cannot metabolize these excess UA, thus forming gout. Gout first affects the joints of the lower limbs (foot, ankle, knee, etc.), causing severe joint pain accompanied by varying degrees of redness, fever and swelling, and eventually developing into joint damage and deformity, and even disability in severe cases. Patients are restricted in their activities and have difficulty moving, which brings great trouble to their daily lives.

[0004] Currently, there are two main methods for treating gout attacks: (1) broad-spectrum anti-inflammatory drugs such as colchicine and nonsteroidal anti-inflammatory drugs, which mainly relieve pain associated with the inflammatory site and do not interfere with UA metabolism; (2) uric acid-lowering drugs such as allopurinol and febuxostat are used for long-term management of UA levels. However, these drugs can only inhibit the production of UA but cannot eliminate excess UA or MSU in gout patients.

[0005] Therefore, it is crucial to find alternative therapies that can directly decompose uric acid and its crystals while eliminating inflammation. In this regard, enzyme therapy has shown broad application prospects. Enzymes are protein molecules with catalytic activity synthesized by organisms. They can accelerate chemical reactions in the body and be reused without changing their own structure. Among them, the enzymes that play a major role in relieving and treating gout include urate oxidase and antioxidant enzymes (such as superoxide dismutase and catalase). Urate oxidase can degrade UA into more soluble allantoin, thereby reducing the accumulation of UA; antioxidant enzymes can reduce oxidative stress and various inflammatory reactions, thereby relieving gout symptoms. Compared with traditional drugs, the use of enzymes to treat gout has significant advantages in the efficiency, tolerance and anti-inflammatory effects of reducing UA.

[0006] Among the related research on gout treatment that has been disclosed so far, the authorization announcement number: CN115429774B discloses: a bionic membrane-wrapped uricase nanoparticle and a preparation method thereof, wherein the natural uricase wrapped in the prepared bionic membrane-wrapped nanoparticle is used to prolong the half-life of uricase in the body, thereby improving the bioavailability of uricase and reducing the immunogenicity; the authorization announcement number: CN115463109B discloses: a bionic fusion membrane-wrapped uricase and catalase nanoparticle and a preparation method thereof, wherein the natural uricase, natural catalase and polydopamine nanoparticles are wrapped with a bionic fusion membrane, and the photothermal effect cascade of the uricase and catalase activities in coordination with the polydopamine nanoparticles is used to achieve the effect of treating gout; the publication number: CN113768865A discloses a polymer microneedle for inhibiting the release of inflammatory factors to treat acute gout attacks and a preparation method thereof, and the invention uses nanomaterials to wrap serine protease inhibitors (anti-inflammatory) for sustained release to treat gout. Anqi Lin et al. explored the application of autocatalytic uricase / catalase mimics in relieving acute gout, and demonstrated that these autocatalytic catalysts can effectively degrade UA and eliminate H2O2 by simulating the dual functions of uricase and catalase, thereby reducing the inflammatory response during gout attacks. However, they can only remove ROS at a single site of inflammation and are not targeted, which greatly reduces their effectiveness in relieving and treating gout.

[0007] In summary, the current treatment options for gout and other diseases still have some defects: (1) The natural uricase and other natural enzymes are used in combination for targeted treatment of gout, but natural enzymes have disadvantages such as high cost and difficulty in preservation, making them difficult to use on a large scale. (2) Although the use of uricase mimetic enzymes in combination with hydrogen peroxide mimetic enzymes can remove H2O2 while reducing UA, they can only remove ROS at a single site of inflammation, and lack targeted treatment for gout sites, which greatly reduces their therapeutic effect. (3) When targeting gout sites, cell membranes are often used, which are difficult to prepare, difficult to preserve, and have high production costs. (4) Although there is a certain cascade effect between different enzymes, their action lines are single and cannot form a circulation pathway, resulting in limited effects. Therefore, new research is urgently needed to further improve the relief and treatment of inflammatory diseases such as gout. Summary of the invention

[0008] The purpose of the present invention is to solve the problems existing in the prior art, and propose a multifunctional nanozyme platform and its preparation method and application, which realizes the cascade and synergistic catalytic enhancement of nanozymes at the inflammatory site by preparing a multifunctional nanozyme platform, and at the same time forms a substrate self-circulation system, so as to achieve the effect of efficiently alleviating and treating various inflammatory-related diseases, and solves the problems of insufficient stability of natural enzymes, excessive specificity, i.e., single function, easy recognition and removal by the immune system, inability to cope with complex physiological environments, high synthesis and production costs, poor reusability, lack of targeting; low carrier material loading rate, poor environmental responsiveness; difficult preparation and storage of targeting substances, etc. At the same time, it also reduces the excess harmful byproducts produced by a single nanozyme, solves the problems of unsustainable treatment effects, low degradation and removal efficiency, etc.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A multifunctional nanozyme platform, comprising a nanozyme, a carrier material and a targeting substance, wherein the nanozyme comprises a catalase-like, a superoxide dismutase-like, a glucose oxidase-like, a glutathione peroxidase-like, a uricase-like, a esterase-like or other nanozymes with inflammation inhibition effect;

[0011] The carrier material includes MOF material, silicon nanomaterial, metal nanomaterial, polymer nanomaterial, lipid nanomaterial, and carbon nanomaterial according to the material composition;

[0012] The targeting substances include antibodies, cell membranes, peptides, and small biological molecules;

[0013] The nanozyme platform can target the site of inflammation. The microenvironment of the site of inflammation causes the targeting substance and the carrier material to depolymerize and release the nanozyme. The nanozyme can efficiently remove excess reactive oxygen and related harmful substances in the diseased site through self-cascading, synergistic enhancement and substrate self-circulation catalytic system, thereby achieving the effect of alleviating and treating inflammation-related diseases.

[0014] Preferably, the catalase-like enzymes include metal oxide nanozymes, metal nanozymes, MOF-like nanozymes, and carbon-based nanozymes.

[0015] Preferably, the superoxide dismutase-like enzymes include cerium oxide nanozymes, carbon material nanozymes, platinum nanozymes, gold nanozymes, manganese dioxide nanozymes, copper nanozymes, nickel nanozymes, copper zinc oxide nanozymes, iron nanozymes, and manganese nanozymes.

[0016] Preferably, the glucose oxidase-like enzymes include ferroferric oxide nanozymes, graphene oxide complex enzymes, copper oxide nanozymes, and cobalt-based nanozymes.

[0017] Preferably, the glutathione peroxidase-like enzymes include manganese dioxide nanozymes, cerium dioxide nanozymes, selenium nanozymes, metal organic frameworks, gold nanozymes, zinc oxide nanozymes, and vanadium pentoxide nanozymes.

[0018] Preferably, the uricase-like enzymes include platinum nanozymes, palladium nanozymes, rhodium nanozymes, iridium nanozymes, manganese oxide nanozymes, iron-based nanozymes, copper-based nanozymes, and cobalt-based nanozymes.

[0019] Preferably, the carrier material includes nanoparticles, nanorods, micelles, nanotubes, and hydrogels according to the material form.

[0020] A method for preparing a multifunctional nanozyme platform comprises the following steps:

[0021] S1. Synthesize the desired nanozymes a, b, and c, select gold nanozyme as nanozyme a, select carbon nanozyme as nanozyme b, and select platinum nanozyme as nanozyme c;

[0022] S2. The nanozymes a, b, and c obtained in step S1 are loaded with a carrier material to obtain a nanozyme platform P1;

[0023] S3. Connect the nanozyme platform P1 obtained in the above step S2 with the targeting substance T1 to obtain a multifunctional nanozyme platform.

[0024] Preferably, the synthesis, loading and connection methods described in S1-S3 include: chemical reduction method, stirring synthesis method, co-precipitation method, strong acid oxidation method, solvent thermal method, template method, electrochemical deposition method, microemulsion method, sol-gel method, physical adsorption method, hydrogen bond interaction, van der Waals force, and electrostatic interaction.

[0025] An application of a multifunctional nanozyme platform, the application including the relief and treatment of gout, hyperuricemia, acute gastroenteritis, acute wound infection, diabetes, rheumatoid arthritis, myocardial infarction, ischemic stroke, atherosclerosis, and colitis-related diseases.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This multifunctional nanozyme platform is a new multifunctional catalytic system that can play a significant role in anti-oxidation and degradation of uric acid at the site of inflammation. First, it removes excessive reactive oxygen species (ROS) in the local environment, thereby alleviating the inflammatory response caused by oxidative stress. At the same time, the nanozyme can target and degrade uric acid deposited in the joints of gout patients, effectively reducing its accumulation in tissues; during the degradation of MSU, the nanozyme will also generate hydrogen peroxide (H2O2). The nanozyme has a unique dual catalytic function, which can quickly decompose H2O2 into harmless oxygen (O2) and water. The release of O2 in turn provides an oxidative environment for the oxidative decomposition of uric acid, promoting the continuous decomposition of uric acid. The H2O2 produced by the decomposition of uric acid can continue to be catalyzed by the nanozyme to produce oxygen, thus forming a substrate self-circulating catalytic system. Through this coherent reaction chain, the nanozyme not only completes the continuous operation of removing ROS-uric acid degradation-decomposition of H2O2, but also forms a self-enhanced catalytic circulation system during the decomposition process. Ultimately, the entire process presents a cascade synergistic effect, achieving multi-level, efficient treatment of the inflammatory site, significantly improving the therapeutic effect, and effectively reducing local oxidative stress and uric acid accumulation. The construction of this self-circulating system makes nanozymes long-lasting and effective in the treatment of inflammation, providing a new targeted strategy for the treatment of inflammatory-related diseases such as gout, and has great clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a Fourier infrared (FT-IR) analysis diagram of the multifunctional nanozyme platform of the present invention;

[0029] Figure 2 This is a diagram of enzyme activity detection of the multifunctional nanozyme platform of the present invention;

[0030] Figure 3 This is a detection diagram of H2O2 accumulation after the multifunctional nanozyme platform of the present invention degrades UA;

[0031] Figure 4 This is a biocompatibility analysis diagram of the multifunctional nanozyme platform of the present invention (human epidermal immortalized cells HaCat);

[0032] Figure 5 This is a diagram showing the effect of intracellular ROS removal by the multifunctional nanozyme platform of the present invention;

[0033] Figure 6 This is a diagram showing the effect of the multifunctional nanozyme platform of the present invention on clearing inflammatory factors (IL-6, IL-1β, TNF-α) at ​​the cellular level;

[0034] Figure 7 The data and intuitive diagram of the degree of gout swelling treated by the multifunctional nanozyme platform of the present invention at the mouse level;

[0035] Figure 8 This is a blood routine and blood biochemical analysis chart of the multifunctional nanozyme platform of the present invention;

[0036] Fig. 9 This is a pathological analysis diagram of the multifunctional nanozyme platform of the present invention;

[0037] Fig.10 Schematic diagram of the multifunctional nanozyme platform of the present invention for treating gout in mice. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0039] Reference Figure 1-Figure 10 A multifunctional nanozyme platform and its preparation method and application include a multifunctional nanozyme platform, which includes a nanozyme, a carrier material and a targeting substance.

[0040] Further improved nanozymes include catalase-like, superoxide dismutase-like, glucose oxidase-like, glutathione peroxidase-like, uricase-like (UOD-like), esterase-like or other nanozymes that have the effects of degrading uric acid and inhibiting inflammation.

[0041] Further improved catalase-like enzymes include metal oxide nanozymes, metal nanozymes, MOF-like nanozymes, carbon-based nanozymes, and the like.

[0042] Further improved superoxide dismutase-like nanozymes include cerium oxide nanozymes, carbon material nanozymes, platinum nanozymes, gold nanozymes, manganese dioxide nanozymes, copper nanozymes, nickel nanozymes, copper zinc oxide nanozymes, iron nanozymes, manganese nanozymes, and the like.

[0043] Further improved glucose oxidase-like enzymes include ferroferric oxide nanozymes, graphene oxide complex enzymes, copper oxide nanozymes, cobalt-based nanozymes, and the like.

[0044] Further improved glutathione peroxidase-like enzymes include manganese dioxide nanozymes, cerium dioxide nanozymes, selenium nanozymes, hoof nanozymes, metal-organic frameworks, gold nanozymes, zinc oxide nanozymes, vanadium pentoxide nanozymes, and the like.

[0045] Further improved uricase-like enzymes include platinum nanozymes, palladium nanozymes, rhodium nanozymes, iridium nanozymes, manganese oxide nanozymes, iron-based nanozymes, copper-based nanozymes, cobalt-based nanozymes, and the like.

[0046] Further improved, the carrier material includes MOF materials, silicon nanomaterials, metal nanomaterials, polymer nanomaterials, lipid nanomaterials, magnetic nanomaterials, carbon nanomaterials, etc. according to the material composition.

[0047] For further improvement, the carrier material includes nanoparticles, nanorods, micelles, nanotubes, hydrogels, etc. according to the material form.

[0048] Further improved targeting substances include antibodies, cell membranes, peptides, small biological molecules, etc.

[0049] The present invention also discloses a method for preparing a multifunctional nanozyme platform, which comprises the following steps:

[0050] S1. Synthesize the desired nanozymes a, b, and c, select gold nanozyme as nanozyme a, select carbon nanozyme as nanozyme b, and select platinum nanozyme as nanozyme c;

[0051] S2. The nanozymes a, b, and c obtained in step S1 are loaded with a carrier material to obtain a nanozyme platform P1;

[0052] S3. The nanozyme platform P1 obtained in the above step S2 is connected with the targeting substance T1 to obtain a multifunctional nanozyme platform.

[0053] The synthesis, loading and connection methods described in S1-S3 include: chemical reduction method, stirring synthesis method, coprecipitation method, strong acid oxidation method, solvent thermal method, template method, electrochemical deposition method, microemulsion method, sol-gel method, physical adsorption method, hydrogen bond interaction, van der Waals force, electrostatic interaction, etc.

[0054] The following uses the treatment of gout in mice as an example to further illustrate the specific implementation steps of the preparation method and application of the multifunctional nanozyme platform:

[0055] Step 1

[0056] Preparation of Pt NPs:

[0057] Under vigorous stirring at room temperature, 1 mL of 5 mM chloroplatinic acid hexahydrate (H2PtCl6·6H2O) was mixed with 1 mL of 50 mM polyvinylpyrrolidone (PVP), and then 1 mL of 50 mM sodium borohydride (NaBH4) was added to the mixture, and the reaction system was reconstituted to a final volume of 10 mL with double distilled water. The reaction was allowed to react for a period of time to form PtNPs.

[0058] Preparation of CNDs:

[0059] Mix 50 mL of concentrated nitric acid (HNO3) and concentrated sulfuric acid (H2SO4) (V HNO3 :V H2SO4 =1:1), add activated carbon, start heating and reflux, collect the corresponding solution containing CNDs, neutralize with NaHCO3 to neutrality, purify the obtained solution by filtration, and then dialyze with a dialysis bag with a molecular cutoff of 3500Da, and then concentrate the CNDs solution with a centrifugal filter device, and filter with a molecular weight cutoff membrane (MWCO) of 100kDa. CNDs are obtained.

[0060] Preparation of Au@CNDs:

[0061] 150ul of tetrachloroauric acid (HAuCl4) aqueous solution (0.83mg / mL) was added to 3mL (0.03mg / mL) of CNDs suspension and reacted at 100°C for a period of time to obtain a stable Au@CNDs suspension.

[0062] Preparation of Au@CNDs-Pt-ZIF-8(ACPZ):

[0063] The prepared Au@CNDs and PVP-Pt NPs were mixed with 12 mL of 2-methylimidazole (2-MIM) methanol solution (7.9 mmol / L). After stirring for a period of time, the above mixture was added with 20 mL of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) methanol solution (0.98 mmol), stirred, centrifuged to obtain Au@CNDs-Pt-ZIF-8 (ACPZ), and washed to obtain ACPZ.

[0064] Preparation of PEG-FA-Au@CNDs-Pt-ZIF-8(FACPZ):

[0065] Dissolve 10 mg of PEG-FA in 5 mL of deionized water, then add ACPZ to the PEG-FA solution, treat with ultrasound, and react at room temperature in the dark. Wash the resulting solution with deionized water several times to remove the clean FACPZ.

[0066] The results are as follows Figure 1 As shown in the FACPZ spectrum, at 3860 cm -1(-CH2), 1650cm -1 (C=O), 1100cm -1 The characteristic peak of PEG-FA appeared on (COC), indicating that PEG-FA loading was successful.

[0067] Step 2

[0068] Detection of catalase activity (CAT-like) of nanozymes

[0069] Test method:

[0070] In 20 mL PBS (pH = 7.4), in the presence of 1 M H2O2, the O2 content generated by 1 mL 100 ug / mL of different samples (ACZ, PZ, ACPZ, FACPZ) was calculated by a dissolved oxygen meter.

[0071] The results are as follows Figure 2 As shown in a, the amount of oxygen (O2) produced by the decomposition of H2O2 by different nanozymes at the same concentration is significantly different. The CAT-like of ZIF-8 wrapped with Au@CNDs and Pt NPs alone is average, but when both are present, that is, ACPZ, the amount of O2 produced by decomposing H2O2 in 20 minutes can reach 48mg / L, indicating that Au@CNDs and Pt NPs have a synergistic enhancement effect on CAT-like enzyme activity. However, its activity has decreased to a certain extent after being wrapped with PEG-FA, which may be due to the fact that the encapsulation of organic matter reduces the mutual contact between it and the substrate, which reduces its enzyme activity to a certain extent.

[0072] Detection of superoxide dismutase activity (SOD-like) of nanozymes

[0073] Test method: DOJINDO # S311-100T SOD enzyme detection kit was used to detect the superoxide dismutase activity of different samples (ACZ, PZ, ACPZ, FACPZ) at the same concentration (200ug / mL).

[0074] The results are as follows Figure 2 As shown in b, similar to the trend of CAT-like activity, the ACPZ formed when Au@CNDs combined with PtNPs had a strong activity against O2 at 200ug / mL. ·- The clearance rate of Au@CNDs can reach about 98%, while that of ACZ and PZ can only reach about 50%, which directly indicates that Au@CNDs and PtNPs also have a synergistic enhancement effect on SOD-like enzyme activity.

[0075] Detection of uricase activity of nanozymes (UOD-like)

[0076] Test method: Since UA has an absorption peak at 292nm, the UA clearance can be judged by observing the absorbance at 292nm. Take 100ul FACPZ (50ug / mL) and add 100ul UA (200uM) and mix evenly. Continuously monitor its absorbance at 292nm at 37℃.

[0077] The results are as follows Figure 2 As shown in Figure c, 50ug / mL FACPZ can degrade 175.8uM UA to 12.5uM within 68h, indicating that the UA degradation rate of FACPZ can reach 92.88%, indicating that the nanozyme has a good ability to remove UA.

[0078] Detection of H2O2 accumulation after nanozyme degradation of UA

[0079] Test method: Take 500ul FACPZ (500ug / mL) and 500ul UA (200uM) and mix them evenly. Degrade them at 37℃ for 120min. Centrifuge at 8000rpm for 5min, take the supernatant and add it to FOX reagent (25mM H2SO4) containing 250μM (NH4)2Fe(SO4)2, 100μM dimethylol orange and 100mM D-sorbitol, and record the absorbance at 560nm. If there is no H2O2, FOX reagent will maintain its bright yellow color. When H2O2 accumulates, as the concentration of H2O2 changes from low to high, the color of FOX reagent changes from yellow to red, and then from purple to purple.

[0080] The results are as follows Figure 3 As shown in the figure, the H2O2 produced by simple uricase in the process of reducing UA will increase the absorbance of FOX at 560nm, while FACPZ can remove the produced H2O2 while removing UA, making it have a lower absorbance at 560nm. This shows that a self-circulating system is formed between nanozymes.

[0081] Step 3

[0082] Detection of cell viability of nanozymes

[0083] Test method: The FACPZ prepared in step 1 was prepared into solutions of 5, 10, 20, 30, and 40 ug / mL using culture medium, and the nanozymes of different concentrations (5, 10, 20, 30, and 40 ug / mL) were incubated with human immortalized epidermal cells (HaCat) for 24 hours, and the cell viability was detected using the CCK-8 method.

[0084] The results are as follows Figure 4As shown, nanozymes at different concentrations had no obvious toxicity to HaCat cells, and the cell viability of each group was maintained above 90%, indicating that the nanozymes had no obvious damage to the cells within this concentration range.

[0085] Step 4

[0086] Detection of the ability of nanozymes to remove intracellular ROS

[0087] Test method: Macrophages (RAW264.7) were placed in 24-well culture medium and treated with MSU at a concentration of 50ug / mL. After 24h of treatment, different samples (PBS, native uricase, ACZ, PZ, ACPZ, FACPZ) (20ug / mL) were used for another 24h. Then, they were treated with DCFH-DA (ratio of 1:1000) for 30min, rinsed with serum-free culture medium, and then imaged under a fluorescence microscope.

[0088] The results are as follows Figure 5 As shown, the MSU group produced a large amount of ROS, that is, a large amount of fluorescence. After a period of treatment with the FACPZ group, compared with the other groups, its ROS was almost completely eliminated and almost no fluorescence appeared, indicating that FACPZ can eliminate a large amount of ROS generated in the cell.

[0089] Step 5

[0090] Detection of nanozyme cell level to clear inflammatory factor levels

[0091] Test method: IL-6, IL-1β, and TNF-α ELISA detection kits were used to detect inflammatory factors at the cellular level.

[0092] The results are as follows Figure 5 As shown, FACPZ has an excellent effect in clearing intracellular inflammatory factors (IL-6, IL-1β, TNF-α).

[0093] Step 6

[0094] Detection of the effect of nanozyme on gout treatment in mice:

[0095] Test method: The experiment was divided into seven groups, with 8 mice in each group, including 6-8-week-old KM mice (SPF, -30g). (1) Blank control group; (2) Negative group; (3) Colchicine group; (4) ACZ group; (5) PZ group; (6) ACPZ group; (7) FACPZ group. During the experiment, the thickness of the mouse foot pad was measured with a vernier caliper and photographed.

[0096] The results are as follows Figure 6As shown, after 10 hours of intravenous injection of nanozyme treatment, the swelling level dropped from the original 30% to 10%, which was better than the colchicine group within the same treatment time. It can also be observed from the intuitive graph that the FACPZ group had a better therapeutic effect.

[0097] Step 7

[0098] Detection of Nanozymes Biosafety Evaluation

[0099] Test method: Blood biochemistry: Take blood from 3 mice in each group, put it in a clean EP tube, let it stand at room temperature, wait for the blood to fully coagulate, centrifuge it at 4°C, absorb the supernatant into a clean EP tube, centrifuge it again, absorb the supernatant into a new EP tube, and measure the indicators of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood uric acid nitrogen (BUN), and creatinine (Crea). Routine blood test: Take blood from mice and put it into an EP tube containing sodium heparin for white blood cell count analysis. H&E staining: Take mice for dissection, take heart, liver, spleen, lung, and kidney tissues, wash them thoroughly in physiological saline, put them into a marked 10mL EP tube (fixed with 4% paraformaldehyde) after washing, and send them for section analysis.

[0100] The results are as follows Figure 8 and 9 As shown in the results of blood biochemistry, it can be seen that the indexes of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the colchicine group were significantly higher than those in the blank group, indicating that colchicine has a greater hepatotoxicity to mice; according to the indexes of urea nitrogen (BUN) and creatinine (Crea), it can be seen that the FACPZ group has no significant effect on the renal function of mice. According to blood biochemical analysis, FACPZ has no significant effect on the liver and renal functions of mice.

[0101] At the same time, according to the blood routine analysis, there was no significant change in the number of white blood cells, indicating that the FACPZ group had a good anti-inflammatory effect. The H&E staining results also showed that the nanozyme had no obvious toxicity to the heart, liver, spleen, lung, and kidney tissues of mice, indicating that FACPZ has good biological safety.

[0102] In summary, through this coherent reaction chain, the multifunctional nanozyme platform not only completes the continuous operation of removing ROS-degrading MSU-decomposing hydrogen peroxide, but also forms a self-enhancing catalytic cycle system during the decomposition process. In the end, the whole process presents a cascade synergistic effect, achieving multi-level and efficient treatment of inflammatory sites, significantly improving the treatment effect, and effectively reducing local oxidative stress and the accumulation of uric acid crystals. This invention combines four therapeutic methods of targeting, catalysis, cascade, and synergy for the efficient treatment of gout. The nanozyme successfully overcomes the limitations of natural enzymes themselves, such as low stability, high cost, difficulty in storage and difficulty in real application, and the lack of intercommunication and non-targeting between single nanozymes.

[0103] Compared with natural enzymes, nanozymes are widely used in biomedicine due to their high stability, low cost, and controllable enzyme activity. After the multifunctional nanozyme platform targets the gout site, the carrier material first depolymerizes, thereby releasing a variety of nanozymes inside. These enzymes cooperate with each other to play unique functions in different steps, forming a highly coordinated cascade reaction system.

[0104] First, the platinum nanoparticles (Pt NPs) in the nanozyme system are activated, and they can effectively remove urate crystals (MSU) deposited in the gout site. However, during this degradation process, the platinum nanoparticles will release hydrogen peroxide (H2O2), and this byproduct may cause further damage to the tissue if it is not treated. At the same time, the carbon quantum dots (CNDs) in the system begin to play a role. CNDs can catalyze the excess superoxide anions (O2 ·- ) into H2O2, which not only effectively reduces O2 ·- The harmful effects of chlorination also produce more H2O2 through conversion.

[0105] Finally, the gold nanoparticles (Au NPs) in the system can further decompose the H2O2 produced by CNDs and Pt NPs into oxygen and water. This oxygen release process not only eliminates the side effects of hydrogen peroxide, but also enhances the degradation effect of Pt NPs on MSU by improving the local oxidation environment.

[0106] Through this series of mutually coordinated catalytic reactions, the nanozyme system forms a cascade-coordinated enhanced self-circulating system. This system ensures that the nanozymes can not only gradually eliminate harmful substances at the site of inflammation, but also continuously improve their therapeutic effects through a self-enhanced reaction chain. This design provides a more durable and efficient targeted treatment strategy for diseases related to uric acid deposition such as gout, and has significant clinical application potential. This sophisticated nanozyme combination not only achieves multi-level disease intervention, but also greatly reduces possible side effects, reflecting the cutting-edge development of nanomedicine in the field of targeted therapy.

[0107] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A multifunctional nanozyme platform, comprising a nanozyme, a carrier material and a targeting substance, characterized in that: The nanozymes include catalase-like, superoxide dismutase-like, glucose oxidase-like, glutathione peroxidase-like, uricase-like, and esterase-like; The carrier material includes MOF material, silicon nanomaterial, metal nanomaterial, polymer nanomaterial, lipid nanomaterial, and carbon nanomaterial according to the material composition; The targeting substances include antibodies, cell membranes, peptides, and small biological molecules; The nanozyme platform can target the site of inflammation. The microenvironment of the site of inflammation causes the targeting substance and the carrier material to depolymerize and release the nanozyme. The nanozyme can efficiently remove excess reactive oxygen and related harmful substances in the diseased site through self-cascading, synergistic enhancement and substrate self-circulation catalytic system, thereby achieving the effect of alleviating and treating inflammation-related diseases.

2. A multifunctional nanozyme platform according to claim 1, characterized in that: The catalase-like enzymes include metal oxide nanozymes, metal nanozymes, MOF-like nanozymes, and carbon-based nanozymes.

3. A multifunctional nanozyme platform according to claim 1, characterized in that: The superoxide dismutase-like enzymes include cerium oxide nanozymes, carbon material nanozymes, platinum nanozymes, gold nanozymes, manganese dioxide nanozymes, copper nanozymes, nickel nanozymes, copper zinc oxide nanozymes, iron nanozymes, and manganese nanozymes.

4. A multifunctional nanozyme platform according to claim 1, characterized in that: The glucose oxidase-like enzymes include ferroferric oxide nanozymes, graphene oxide complex enzymes, copper oxide nanozymes, and cobalt-based nanozymes.

5. A multifunctional nanozyme platform according to claim 1, characterized in that: The glutathione peroxidase-like enzymes include manganese dioxide nanozymes, cerium dioxide nanozymes, selenium nanozymes, metal organic frameworks, gold nanozymes, zinc oxide nanozymes, and vanadium pentoxide nanozymes.

6. A multifunctional nanozyme platform according to claim 1, characterized in that: The uricase-like enzymes include platinum nanozymes, palladium nanozymes, rhodium nanozymes, iridium nanozymes, manganese oxide nanozymes, iron-based nanozymes, copper-based nanozymes, and cobalt-based nanozymes.

7. A multifunctional nanozyme platform according to claim 1, characterized in that: The carrier material includes nanoparticles, nanorods, micelles, nanotubes, and hydrogels according to the material form.

8. A method for preparing a multifunctional nanozyme platform according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Synthesize the desired nanozymes a, b, and c, select gold nanozyme as nanozyme a, select carbon nanozyme as nanozyme b, and select platinum nanozyme as nanozyme c; S2. The nanozymes a, b, and c obtained in step S1 are loaded with a carrier material to obtain a nanozyme platform P1; S3. Connect the nanozyme platform P1 obtained in step S2 with the targeting substance T1 to obtain a multifunctional nanozyme platform.

9. The method for preparing a multifunctional nanozyme platform according to claim 8, characterized in that: The synthesis, loading and connection methods described in S1-S3 include: chemical reduction method, stirring synthesis method, coprecipitation method, strong acid oxidation method, solvent thermal method, template method, electrochemical deposition method, microemulsion method, sol-gel method, physical adsorption method, hydrogen bond interaction, van der Waals force, and electrostatic interaction.

10. The use of a multifunctional nanozyme platform according to any one of claims 1 to 9, characterized in that: The applications include the relief and treatment of gout, hyperuricemia, acute gastroenteritis, acute wound infection, diabetes, rheumatoid arthritis, myocardial infarction, ischemic stroke, atherosclerosis, and colitis-related diseases.

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