A multifunctional nanozyme platform and its preparation method and application

Abstract

The present invention belongs to the field of nano drug preparations, and discloses a multifunctional nanozyme platform and its preparation method and application, which is mainly composed of nanozymes, carrier materials and targeting substances. The synthesis of nanozymes, the loading of nanozymes on carrier materials and the modification of their surface targeting substances are carried out by methods such as stirring synthesis, coprecipitation, strong acid oxidation, chemical reduction, covalent bonding, solvent thermal method, physical adsorption, etc. The targeting substance on the surface of the nanozyme platform can be accurately and efficiently positioned to the inflammation site, and the acidic microenvironment of the inflammation site causes the targeting substance and the carrier material to undergo structural depolymerization, thereby releasing a variety of nanozymes, which not only realize the self-cascade and synergistic catalytic enhancement of enzymes at the inflammation site, but also form a substrate self-circulation catalytic system, thereby achieving the effect of efficiently alleviating and treating various inflammation-related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug preparations, and in particular to a multifunctional nanoenzyme 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 stimuli. It is a protective response aimed at removing pathogenic factors (such as pathogens, damaged cells) and initiating the repair process. When the body is injured or infected, immune cells release inflammatory factors, which can regulate the inflammatory response, 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 leads to a continuous increase in UA, the end product of purine catabolism. When the UA concentration in tissues continues to increase until saturation, it causes monosodium urate crystals (MSU) to deposit around the joints. Since some mammals, such as humans, lack uricase, they are unable to metabolize this excess UA, leading to gout. Gout first affects the joints of the lower limbs (feet, ankles, knees, etc.), causing severe joint pain accompanied by varying degrees of redness, heat, and swelling, eventually developing into joint damage and deformity, and even disability in severe cases. Patients are restricted in their activities and have difficulty moving, causing great distress in their daily lives.

[0004] Currently, there are two main approaches to treat 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 without interfering with UA metabolism; and (2) uric acid-lowering drugs such as allopurinol and febuxostat, which 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 also eliminating inflammation. Enzyme therapy shows broad application prospects in this regard. Enzymes are catalytically active protein molecules synthesized by organisms. They accelerate chemical reactions in the body and can be reused without changing their own structure. Urate oxidase and antioxidant enzymes (such as superoxide dismutase and catalase) are the enzymes that play a major role in alleviating and treating gout. Urate oxidase degrades UA into the more soluble allantoin, thereby reducing UA accumulation; antioxidant enzymes reduce oxidative stress and various inflammatory responses, thereby alleviating gout symptoms. Compared with traditional medications, enzymatic treatment of gout has significant advantages in UA-lowering efficiency, tolerability, and anti-inflammatory effects.

[0006] Among the currently published research on gout treatment, Authorization Announcement No. CN115429774B discloses: a biomimetic membrane-coated uricase nanoparticle and a preparation method thereof, wherein the prepared biomimetic membrane-coated nanoparticles are used to encapsulate natural uricase in the body's circulation half-life, thereby improving the bioavailability of uricase and reducing immunogenicity; Authorization Announcement No. CN115463109B discloses: a biomimetic fusion membrane-coated uricase and catalase nanoparticle and a preparation method thereof, wherein the biomimetic fusion membrane is used to encapsulate natural uricase, natural catalase, and polydopamine nanoparticles, and the cascade effect of the photothermal effect of the uricase and catalase activities in conjunction with the polydopamine nanoparticles is utilized to achieve the therapeutic effect of gout; Publication No. CN113768865A discloses a polymer microneedle for inhibiting the release of inflammatory factors to treat acute gout attacks and a preparation method thereof, wherein the invention uses nanomaterials to encapsulate serine protease inhibitors (anti-inflammatory) for sustained-release treatment of gout. Anqi Lin et al. explored the use of autocatalytic uricase / catalase mimics in alleviating acute gout. They demonstrated that these autocatalytic catalysts, by mimicking the dual functions of uricase and catalase, can effectively degrade UA and eliminate H₂O₂, thereby alleviating the inflammatory response during gout attacks. However, they can only scavenge ROS at a single site of inflammation and are non-targeted, significantly reducing their effectiveness in alleviating and treating gout.

[0007] In summary, the current treatment options for gout and other diseases still have some defects: (1) The natural uricase is used in combination with other natural enzymes 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) The use of uricase mimics in combination with hydrogen peroxide mimics, although they can remove H2O2 while reducing UA, can only remove ROS in a single inflammatory site, and lack targeted treatment for the gout site, which greatly reduces its therapeutic effect. (3) When targeting the gout site, 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 routes are single and cannot form a circulation pathway, resulting in limited effect. 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. By preparing a multifunctional nanozyme platform, it realizes the cascade and synergistic catalytic enhancement of nanozymes at the site of inflammation, and at the same time forms a substrate self-circulation system, achieving the effect of effectively alleviating and treating various inflammatory-related diseases. It solves the shortcomings of natural enzymes such as insufficient stability, excessive specificity (i.e., single function), easy recognition and clearance 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; and difficult preparation and storage of targeting substances. At the same time, it also reduces the excess harmful byproducts produced by single nanozymes and solves the problems of unsustainable therapeutic effects and low degradation and clearance efficiency.

[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, superoxide dismutase-like, glucose oxidase-like, glutathione peroxidase-like, uricase-like, esterase-like, or other nanozyme with inflammation-inhibiting effects;

[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 inflammatory site causes the targeting substance and the carrier material to depolymerize and release the nanozyme. The nanozyme efficiently removes 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 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 enzyme includes ferroferric oxide nanozyme, graphene oxide complex enzyme, copper oxide nanozyme, and cobalt-based nanozyme.

[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, 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.

[0025] A multifunctional nanozyme platform is used for the relief and treatment of diseases related to gout, hyperuricemia, acute gastroenteritis, acute wound infection, diabetes, rheumatoid arthritis, myocardial infarction, ischemic stroke, atherosclerosis, and colitis.

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

[0027] This multifunctional nanozyme platform is a novel, multifunctional catalytic system that can exert significant antioxidant and uric acid degradation effects at sites of inflammation. First, it removes excess reactive oxygen species (ROS) in the local environment, thereby alleviating the inflammatory response triggered by oxidative stress. Simultaneously, the nanozyme can target and degrade uric acid deposited in the joints of gout patients, effectively reducing its accumulation in tissues. During the MSU degradation process, the nanozyme also generates hydrogen peroxide (H2O2). This nanozyme possesses a unique dual catalytic function, rapidly decomposing H2O2 into harmless oxygen (O2) and water. This O2 release, in turn, provides an oxidative environment for the oxidative decomposition of uric acid, promoting its continued decomposition. The H2O2 produced by uric acid decomposition can then 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 ROS removal, uric acid degradation, and H2O2 decomposition, but also forms a self-reinforcing catalytic cycle during the decomposition process. Ultimately, the entire process exhibits a cascading synergistic effect, achieving multi-level, highly effective treatment of the inflammatory site, significantly improving treatment efficacy and effectively reducing local oxidative stress and uric acid accumulation. This self-circulating system enables nanozymes to be long-lasting and effective in treating inflammation, providing a new targeted strategy for the treatment of inflammatory diseases such as gout, and possessing enormous clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 2 This is a diagram showing 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 ROS clearance effect of 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 in mice;

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

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

[0037] Figure 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 clearly and completely described 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 Figures 1-10 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 with the effects of degrading uric acid and inhibiting inflammation.

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

[0042] Further improved 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, 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] Further improved, 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 to 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 (HPtCl6·6H2O) was mixed with 1 mL of 50 mM polyvinylpyrrolidone (PVP). 1 mL of 50 mM sodium borohydride (NaBH4) was then 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] 50mL concentrated nitric acid (HNO3) and concentrated sulfuric acid (H2SO4) (V HNO3 :V H2SO4 =1:1) was mixed, added to activated carbon, and the temperature was raised to reflux. The corresponding solution containing CNDs was collected and neutralized with NaHCO3 until neutral. The resulting solution was purified by filtration and then dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da. The CNDs solution was then concentrated using a centrifugal filter device and filtered using a 100 kDa molecular weight cutoff (MWCO) membrane to obtain CNDs.

[0060] Preparation of Au@CNDs:

[0061] 150 μl of HAuCl4 aqueous solution (0.83 mg / mL) was added to 3 mL (0.03 mg / 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 mixture was added with 20 mL of zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O) methanol solution (0.98 mmol). The mixture was stirred and centrifuged to obtain Au@CNDs-Pt-ZIF-8 (ACPZ), which was then 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, ultrasonicate, and react at room temperature in the dark. Wash the resulting solution with deionized water several times to remove the 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 using a dissolved oxygen meter.

[0071] The results are as follows Figure 2 As shown in Figure a, the amount of oxygen (O2) produced by the decomposition of H2O2 by different nanozymes at the same concentration showed significant differences. The CAT-like activity of ZIF-8 encapsulated with Au@CNDs and Pt NPs alone was average, but when both were present, that is, ACPZ, the amount of O2 produced by the decomposition of H2O2 in 20 minutes could reach 48 mg / L, indicating that Au@CNDs and Pt NPs have a synergistic effect on CAT-like enzyme activity. However, its activity decreased after being encapsulated with PEG-FA, which may be because the encapsulation of organic matter reduced the mutual contact between it and the substrate, resulting in a certain decrease in its enzyme activity.

[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 high activity against O2 at 200 μg / mL. ·- The clearance rate of Au@CNDs can reach about 98%, while 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 (UOD-like) of nanozymes

[0076] Test method: Since UA has an absorption peak at 292nm, the UA clearance status can be judged by observing the absorbance value at 292nm. Take 100ul FACPZ (50ug / mL) and add 100ul UA (200uM) and mix evenly. The absorbance value at 292nm is continuously monitored 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 clear UA.

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

[0079] Test Method: Mix 500 μl of FACPZ (500 μg / mL) and 500 μl of UA (200 μM) and degrade at 37°C for 120 minutes. Centrifuge at 8000 rpm for 5 minutes. Add the supernatant to FOX reagent (25 mM H2SO4) containing 250 μM (NH4)2Fe(SO4)2, 100 μM xylenol orange, and 100 mM D-sorbitol, and record the absorbance at 560 nm. In the absence of H2O2, FOX reagent maintains its bright yellow color. When H2O2 accumulates, the color of FOX reagent changes from yellow to red, and then from purple to violet, as the H2O2 concentration increases from low to high.

[0080] The results are as follows Figure 3 As shown in the figure, the H2O2 produced by uricase during the process of reducing UA increases the absorbance of FOX at 560nm, while FACPZ can also remove the generated H2O2 while removing UA, resulting in a lower absorbance at 560nm. This indicates that a self-circulating system is formed between the 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. Nanozymes of different concentrations (5, 10, 20, 30, and 40 ug / mL) were incubated with human immortalized epidermal cells (HaCat) for 24 hours, and 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 remained above 90%, indicating that the nanozyme had no obvious damage to the cells within this concentration range.

[0085] Step 4

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

[0087] Assay Method: Macrophages (RAW264.7) were plated in 24-well plates and treated with MSU at a concentration of 50 μg / mL. After 24 hours of treatment, cells were treated with various samples (PBS, native uricase, ACZ, PZ, ACPZ, FACPZ) (20 μg / mL) for an additional 24 hours. Cells were then treated with DCFH-DA (1:1000) for 30 minutes, rinsed with serum-free medium, and imaged under a fluorescence microscope.

[0088] The results are as follows Figure 5 As shown in the figure, the MSU group produced a large amount of ROS, that is, a large amount of fluorescence. After being treated with FACPZ for a period of time, the ROS in the group were almost completely eliminated compared with the other groups, and almost no fluorescence appeared, indicating that FACPZ can eliminate a large amount of ROS produced in the cells.

[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 in the results, 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, each with 8 mice, 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 footpads 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 seen from the intuitive graph that the FACPZ group has a better therapeutic effect.

[0097] Step 7

[0098] Detection of nanozymes and biosafety evaluation

[0099] Test method: Blood biochemistry: Blood was collected from 3 mice in each group and placed in clean EP tubes at room temperature. After the blood was fully coagulated, it was centrifuged at 4°C and the supernatant was aspirated into a clean EP tube. After a second centrifugation, the supernatant was aspirated into a new EP tube and the indicators of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood uric acid nitrogen (BUN), and creatinine (Crea) were measured. Routine blood test: Blood was collected from mice and placed in EP tubes containing sodium heparin for white blood cell count analysis. H&E staining: The mice were dissected and the heart, liver, spleen, lung, and kidney tissues were thoroughly washed in physiological saline. After washing, they were placed in labeled 10mL EP tubes (fixed with 4% paraformaldehyde) and sent for section analysis.

[0100] The results are as follows Figure 8 and 9 As shown in the blood biochemistry results, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the colchicine group were significantly higher than those in the blank group, indicating that colchicine is highly hepatotoxic to mice. Blood urea nitrogen (BUN) and creatinine (Crea) levels indicate that the FACPZ group did not significantly affect the renal function of mice. Blood biochemistry analysis shows that FACPZ has no significant effect on the liver and kidney functions of mice.

[0101] Routine blood analysis also showed no significant changes in white blood cell counts, indicating that the FACPZ group had a good anti-inflammatory effect. H&E staining also showed that the nanozyme had no significant toxicity to the heart, liver, spleen, lung, and kidney tissues of mice, demonstrating the good biosafety of FACPZ.

[0102] In summary, through this coherent reaction chain, the multifunctional nanozyme platform not only completes the continuous operation of removing ROS, degrading MSU, and decomposing hydrogen peroxide, but also forms a self-enhancing catalytic cycle system during the decomposition process. Ultimately, the entire process presents a cascade synergistic effect, achieving multi-level and efficient treatment of inflammatory sites, significantly improving the therapeutic effect, and effectively reducing local oxidative stress and the accumulation of uric acid crystals. This invention combines four therapeutic methods, namely 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 actual application, and the lack of intercommunication and targeting between single nanozymes.

[0103] Compared with natural enzymes, nanozymes are widely used in biomedicine and other fields 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 and play unique functions in different steps to form 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 area. However, during this degradation process, the platinum nanoparticles will release hydrogen peroxide (H2O2). If this byproduct is not treated, it may cause further damage to the tissue. 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 HO are eliminated and more H2O2 is produced 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 oxidative environment.

[0106] Through this series of coordinated catalytic reactions, the nanozyme system forms a cascade-like, enhanced, self-circulating system. This system ensures that the nanozymes not only gradually eliminate harmful substances at the site of inflammation but also continuously enhance their therapeutic efficacy through a self-reinforcing 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 potential side effects, demonstrating the cutting-edge development of nanomedicine in the field of targeted therapy.

[0107] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional nanozyme platform, characterized in that: The preparation method comprises the following steps: Preparation of Pt NPs: 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. The reaction system was reconstituted to a final volume of 10 mL with double distilled water and reacted for a period of time to form Pt NPs. Preparation of CNDs: 50 mL of concentrated nitric acid (HNO3) and concentrated sulfuric acid (H2SO4) were mixed in a volume ratio of 1:1 and added to activated carbon. The temperature was raised and refluxed, and the corresponding solution containing CNDs was collected and neutralized with NaHCO3 until neutral. The resulting solution was purified by filtration and then dialyzed using a dialysis bag with a molecular cutoff of 3500 Da. The CNDs solution was then concentrated using a centrifugal filter device and filtered using a 100 kDa molecular weight cutoff membrane (MWCO) to obtain CNDs. Preparation of Au@CNDs: 150 μl of 0.83 mg / mL HAuCl4 aqueous solution was added to 3 mL of 0.03 mg / mL CNDs suspension and reacted at 100 °C for a period of time to obtain a stable Au@CNDs suspension; Preparation of Au@CNDs-Pt-ZIF-8 (ACPZ): The prepared Au@CNDs and PVP-Pt NPs were mixed with 12 mL of 7.9 mmol / L 2-methylimidazole (2-MIM) methanol solution. After stirring for a period of time, the mixture was added with 20 mL of 0.98 mmol of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) methanol solution. The mixture was stirred and centrifuged to obtain Au@CNDs-Pt-ZIF-8 (ACPZ), which was then washed to obtain ACPZ. Preparation of PEG-FA-Au@CNDs-Pt-ZIF-8 (FACPZ): Dissolve 10 mg of PEG-FA in 5 mL of deionized water, then add ACPZ to the PEG-FA solution, ultrasonicate, and react at room temperature in the dark. Wash the resulting solution with deionized water several times to remove the FACPZ. The preparation method of the multifunctional nanozyme platform prepares a multifunctional nanozyme platform.

2. An application of a multifunctional nanozyme platform, characterized in that: A multifunctional nanozyme platform prepared using the preparation method of the multifunctional nanozyme platform in claim 1, and the use of the multifunctional nanozyme platform in the preparation of drugs for alleviating and treating gout and hyperuricemia.

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