Preparation method of nano-enzyme preparation as well as product and application of nano-enzyme preparation

Abstract

The invention discloses a preparation method of a nano-enzyme preparation as well as a product and application of the nano-enzyme preparation. The synthesis method of the nano-enzyme preparation comprises the following steps: mixing a transmembrane protein solution and a solution containing one or more nano-enzymes to prepare the nano-enzyme preparation. The nano-enzyme can target a disease site, and on one hand, the nano-enzyme removes active oxygen and active nitrogen species on the disease site; on the other hand, the nano-enzyme can be combined with cytokines of disease parts, the promotion effect of the cytokines on diseases is inhibited, and the effect of preventing and treating colitis and colitis-related colon cancer diseases is achieved. The nano-enzyme developed by the invention not only can relieve oxidative stress of diseases, but also can reduce the pro-inflammatory and tumor-promoting effects of cytokines, and can effectively treat colitis-related colon cancer caused by deterioration of colitis.

Description

Technical Field

[0001] This invention relates to a method for preparing nanozyme preparations, as well as the products and applications thereof. Background Technology

[0002] Chronic inflammation, characterized by immune cells and inflammatory mediators, irreversibly damages human tissues and is an associated risk factor for many diseases, such as cardiovascular disease, diabetes, and cancer. Importantly, inflammation is closely related to all stages of development and malignant progression in most types of cancer, as well as the effectiveness of anticancer treatments. Colitis-associated colorectal cancer (CAC) is a deadly malignancy caused by long-term, recurrent inflammatory bowel disease (IBD). Unlike sporadic colorectal cancer, CAC develops through a unique pathogenic mechanism driven by chronic inflammation and has a worse prognosis. Ulcerative colitis increases the risk of colorectal cancer by up to 18% to 20% in patients with colitis lasting more than 30 years, while this risk increases by up to 8% in patients with Crohn's disease. The inflammatory microenvironment creates a favorable environment for carcinogenesis through multiple pathways. The excessive production of reactive oxygen species (ROS), reactive nitrogen species (RNS), and inflammatory mediators leads to DNA damage and gene mutations. In addition, excessive infiltration of neutrophils and macrophages can produce pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which activate cancer signaling pathways, thereby promoting epithelial cell proliferation, inhibiting apoptosis, and accelerating the clonal expansion of abnormal cells, thus promoting tumor development.

[0003] First-line drug therapy for inflammatory bowel disease typically includes nonsteroidal anti-inflammatory drugs (NSAIDs) and biologics (such as tumor necrosis factor inhibitors), whose main role is to reduce inflammation and provide temporary remission. However, for patients with long-term, severe disease, high inflammatory activity, or neutrophil and macrophage infiltration, simply suppressing inflammation may not be sufficient to stop the progression of chronic active colitis. Therefore, the ideal treatment for colitis should not only include immediate suppression of inflammation but also reduction of excessive immune cell infiltration. Our analysis of clinical samples and database data revealed elevated expression of CXCL2 and CXCR2 at sites of colitis and colorectal tumors. In intestinal epithelial cells and immune cells in chronic inflammation secrete CXCL2, which attracts neutrophils and macrophages by binding to CXCR2, thereby exacerbating chronic inflammation and increasing the risk of cancer. Furthermore, the chronic inflammatory environment continuously promotes CXCL2 secretion by neutrophils and macrophages, forming an inflammation-carcinogenesis feedback loop. In short, CXCL2 drives the transformation of colitis into cancer by attracting and activating neutrophils and macrophages, synergistically promoting the inflammatory microenvironment. Therefore, therapeutic strategies targeting CXCL2, neutrophils, and macrophages would be promising approaches to control the progression of precancerous lesions.

[0004] Artificial enzyme mimics aim to replicate the unique catalytic activity of natural enzymes using engineered materials. With advancements in nanotechnology, nanomaterials exhibiting enzyme-mimicking catalytic activity have been discovered—these are nanozymes. Researchers have identified nanozymes that mimic the activities of various natural enzymes, including superoxide dismutase (SOD)-like, catalase (CAT)-like, peroxidase (POD)-like, oxidase-like, and hydrolase-like nanozymes. Compared to natural enzymes, nanozymes offer advantages such as stability, low cost, recyclability, tunable activity, and long-lasting catalysis. Therefore, an increasing number of nanozymes are being developed and applied in the biomedical field. Antioxidant nanozymes have shown significant therapeutic potential in various inflammation-related diseases, including inflammatory bowel disease, cardiovascular disease, rheumatoid arthritis, and gouty arthritis. Gu et al. discovered that Prussian blue nanozymes (PB) can mimic the catalytic activities of SOD, CAT, and POD. PB, possessing multiple enzyme-like activities, can protect cells by scavenging intracellular ROS. In vivo experiments have also demonstrated that PB has a significant therapeutic effect on a lipopolysaccharide-induced mouse hepatitis model. This result indicates that nanozymes can alleviate ROS-related inflammation or damage in vivo. Furthermore, PB, as an active molecule, has been approved by the FDA as an antidote for radioactive cesium and thallium, demonstrating its good biocompatibility and supporting its application in vivo for the treatment of inflammation-related diseases. However, PB itself lacks targeting specificity; therefore, it is necessary to utilize other scientific methods to endow it with targeting capabilities.

[0005] In 1994, Gaudreault first prepared erythrocyte vesicles and used them as drug carriers to study their anti-tumor effects. In 2011, Zhang's research group first adopted a top-down strategy, combining erythrocyte membranes with nanoparticles to prepare erythrocyte membrane-encapsulated nanoparticles as a biomimetic platform for nanoparticle delivery. Currently, cell membranes, as a delivery system, can deliver nanoparticles to lesion sites. Compared with traditional delivery systems, these cell membrane-encapsulated mimics exhibit excellent biocompatibility and retain active targeting cellular properties through receptor-ligand interactions. Macrophages are part of the innate immune system, clearing unwanted or foreign substances / bacteria / viruses from the body. In inflammation-related diseases, inflammatory tissues secrete various cytokines and chemokines. Due to receptors such as CCR2, CXCR1, and CCR7 on their surface, macrophage membranes can bind to chemokines, promoting their recruitment at inflammatory sites, thus exhibiting inflammation targeting. Meanwhile, receptors on macrophage membranes, such as TNFR and IL-1R, can bind to pro-inflammatory factors TNF-α and IL-1β, clearing pro-inflammatory factors from inflamed sites and alleviating inflammation. Therefore, macrophage membrane modification can endow nanoparticles with the ability to target inflammation and clear pro-inflammatory factors, showing great potential in the treatment of inflammatory diseases. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a method for preparing a nanoenzyme preparation with both disease prevention and treatment functions, as well as its product and application, which inhibits the transformation of enteritis into colon cancer through the dual effects of reducing oxidative stress and reducing the infiltration of immune cells.

[0007] Technical solution: This invention provides a method for synthesizing nanoenzyme preparations, comprising the following steps: (1) The cells are modified by cell engineering technology to express the target membrane protein required, the cell membrane is extracted and dispersed in solution A; (2) Disperse one or more nanozymes in a biocompatible solution A; (3) Mix the solutions obtained in steps (1) and (2) and prepare nanoenzyme preparations using ultrasonic equipment, microfluidic equipment and co-extrusion equipment.

[0008] The synthesis method further includes collecting the product from step (3) and performing one or more treatments such as centrifugation and cryopreservation.

[0009] Among them, the transmembrane protein mentioned in step (1) includes one or more combinations of CCR2, CCR4, CCR8, PD-L1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CD47, CD55, CD59, GPIbα, GPIV, GPV, GPVI, GPPIX, CLEC-2, TNF-αR, IL-1R, LFA-1, etc.

[0010] Preferably, the transmembrane protein is CXCR2.

[0011] The cells used to express the transmembrane protein in step (1) include one or more combinations of cell lines such as RAW264.7, HL-60, Caco-2, HT-29, HCT116, SW480, J774, 4T1, MC-38, CT-26, HUVEC, 3T3-L1, THP-1, and 293T.

[0012] Preferably, the cells with good targeting of inflammatory sites are RAW264.7 macrophage cell lines.

[0013] In step (1), solution A includes one or more combinations of phosphate buffer, physiological saline, or water.

[0014] The nanozymes mentioned in step (2) include oxaloacetic nanozymes, nitrogenous nanozymes, carbonaceous nanozymes, metal nanozymes, metal compound-based nanozymes, organometallic framework-based nanozymes, metal coordination nanozymes, single-atom nanozymes, or composite material nanozymes (Note: Nanozymes are prepared by mixing nanozymes with cell membranes and then co-extruding them using equipment. The size of the nanozymes needs to be less than half the size of the pores of the extruded membrane. According to the nanozyme synthesis method, the size of the above nanozymes can be controlled within the corresponding size).

[0015] The oxoanthropoietin nanozymes include one or more combinations of oxide-based nanozymes, sulfide-based nanozymes, selenide-based nanozymes, or telluride-based nanozymes.

[0016] Furthermore, oxide-based nanozymes include one or more combinations of cerium oxide-based nanozymes, manganese oxide-based nanozymes, copper oxide-based nanozymes, iron oxide-based nanozymes, nickel oxide-based nanozymes, cobalt oxide-based nanozymes, zirconium oxide-based nanozymes, hafnium oxide-based nanozymes, etc.

[0017] The nitrogen-based nanozymes include one or more combinations of nitrogen-based nanozymes or phosphorus-based nanozymes.

[0018] The carbon-based nanoenzymes include one or more combinations of fullerene-based nanoenzymes, carbon fiber-based nanoenzymes, carbon nanotube-based nanoenzymes, graphene-based nanoenzymes, or other carbon-based nanoenzymes.

[0019] The metal nanozymes include one or more combinations of gold nanozymes, copper nanozymes, silver nanozymes, platinum nanozymes, palladium nanozymes, rhodium nanozymes, ruthenium nanozymes, or alloy nanozymes.

[0020] The metal compound-based nanozymes include one or more of the following: iron oxide nanozymes, titanium dioxide-based nanozymes, manganese oxide nanozymes, vanadium oxide nanozymes, molybdenum sulfide-based nanozymes, iron sulfide-based nanozymes, copper sulfide-based nanozymes, metal selenide-based nanozymes, and metal phosphide nanozymes.

[0021] Among them, organometallic framework-based nanozymes include one or more combinations of iron-based organometallic framework nanozymes, manganese-based organometallic framework nanozymes, zinc-based organometallic framework nanozymes, copper-based organometallic framework nanozymes, gallium-based organometallic framework nanozymes, zirconium-based organometallic framework nanozymes, hafnium-based organometallic framework nanozymes, vanadium-based organometallic framework nanozymes, and metal-doped organometallic framework nanozymes.

[0022] Preferably, the nanozyme is an organometallic framework-based nanozyme; the organometallic framework-based nanozyme is an iron-based organometallic framework nanozyme.

[0023] The precursor of the iron-based organometallic framework nanozyme includes one or more combinations of organometallic salts, halide metal salts, nitrate metal salts, acetate metal salts, sulfate metal salts, or metal cyanide complexes.

[0024] The halide metal salts include bromide metal salts.

[0025] The metal cyanide complex includes one or more combinations of mononuclear metal cyanide complexes, bimetallic cyanide complexes, transition metal cyanide complexes, noble metal cyanide complexes, or polynuclear metal cyanides.

[0026] Preferably, the precursor of the iron-based organometallic framework nanozyme is a metal cyanide complex or a metal chloride salt or a combination thereof.

[0027] The metal cyanide complex includes one or more combinations of mononuclear metal cyanide complexes, mixed ligand metal cyanide complexes, or multimetal cyanide complexes.

[0028] Preferably, the metal cyanide complex is a mononuclear metal cyanide complex.

[0029] The mononuclear metal cyanide complex includes one or more combinations of ferrocyanide and ferricyanide.

[0030] Preferably, the mononuclear metal cyanide complex is potassium ferricyanide.

[0031] Among them, the precursors of non-ferrous metal elements contained in iron-based organometallic framework nanozymes include one or more combinations of organometallic salts, halide metal salts, nitrate metal salts, acetate metal salts, sulfate metal salts, or metal cyanide complexes.

[0032] The halide metal salts include metal chloride salts and metal bromide salts.

[0033] The iron-based organometallic framework nanoenzyme contains one or more of the following non-ferrous metal elements: lithium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, gallium, germanium, rubidium, strontium, iridium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, tin, antimony, cerium, hafnium, iridium, platinum, gold, or bismuth.

[0034] The precursors of the non-ferrous metal elements include one or more combinations of organometallic salts, halide metal salts, nitrate metal salts, acetate metal salts, sulfate metal salts, or metal cyanide complexes.

[0035] In step (2), solution A is one or more combinations of phosphate buffer, physiological saline and ultrapure water.

[0036] Preferably, solution A is a phosphate buffer solution.

[0037] The present invention also provides a nanoenzyme preparation prepared by the method, wherein the nanoenzyme preparation has a core-shell structure, with an outer layer of cell membrane and a core of nanoenzyme.

[0038] The present invention also provides the application of the nanoenzyme preparations in the treatment, relief and / or prevention of colonic diseases, etc.

[0039] The colonic diseases mentioned above are diseases that broadly involve the colon, including chronic enteritis, acute enteritis, colitis, and other related diseases.

[0040] The chronic enteritis diseases mentioned include ulcerative colitis, Crohn's disease, etc.

[0041] The acute enteritis mentioned above includes pseudomembranous enteritis, bacterial or viral enteritis, etc.

[0042] The colitis-related diseases mentioned include colitis-associated colon cancer, intestinal stricture, intestinal polyps, or toxic megacolon.

[0043] In one implementation, the colitis-related diseases include ulcerative colitis, Crohn's disease, colitis-related colon cancer, etc.

[0044] Preferably, the disease is colitis-associated colon cancer.

[0045] The nanozyme preparation comprises a targeted cell membrane and a nanozyme. The targeted cell membrane originates from macrophages with inflammation-targeting capabilities. The nanozyme is a nanozyme capable of scavenging ROS. The nanozyme preparation refers to a material co-constructed from a cell membrane and a nanozyme.

[0046] This invention utilizes the targeting and ROS elimination capabilities of nanozyme preparations to target the disease site of colitis-related colon cancer. On the one hand, nanozymes remove ROS to achieve an antioxidant effect, thereby alleviating the excessive inflammatory state at the pathological site of the disease. On the other hand, the modified cell membrane inhibits the recruitment of neutrophils and macrophages by removing CXCL2 at the disease site, reducing damage to the disease site and alleviating the malignant transformation of colitis into colon cancer.

[0047] This invention modifies macrophages to highly express CXCR2 on their membrane surface, enabling them to clear CXCL2 from disease sites and inhibit the infiltration of neutrophils and macrophages. Subsequently, a nanozyme formulation is prepared by encapsulating PB nanozymes in the cell membrane. This cell-like artificial enzyme nanoformulation inherits the antioxidant catalytic activity of its internal core and the biointerface characteristics of its source cell. The enzyme-like activity of the nanoformulation can significantly inhibit inflammation by scavenging ROS, while its biointerface characteristics can be used to neutralize immunomodulatory molecules such as cytokines and chemokines through specific receptor-ligand binding. Artificial enzymes with cell-like characteristics have great potential in addressing the progression from colitis to colon cancer, representing a method that can simultaneously prevent and treat colitis-related colon cancer.

[0048] This invention is the first to propose using a modified cell membrane to encapsulate nanozymes. By utilizing the multiple antioxidant enzyme activities (SOD-like, CAT-like, etc.) of the nanozyme preparation and its scavenging of CXCL2, the ecological niche of pathological sites in colitis-associated colon cancer can be regulated. The constructed macrophage membrane is used to achieve the targeting of the nanozymes. On the one hand, it can effectively remove ROS from the disease site and alleviate oxidative stress. On the other hand, by using the CXCR2 receptor on the cell membrane to clear CXCL2, it can inhibit the infiltration of neutrophils and macrophages, thereby repairing the pathological microenvironment of the colon. This can achieve the treatment, relief, and / or prevention of colitis-associated colon cancer.

[0049] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Combining Disease Prevention and Treatment Functions: This invention utilizes the constructed pathological environment targeting module and the anti-inflammatory properties of the nanozyme itself to prevent and treat colitis-related colon cancer. On one hand, the nanozyme preparation constructed in this invention targets the lesion site in the colon, clearing ROS at the disease site and alleviating oxidative stress; on the other hand, the cell membrane targeting module can clear CXCL2 at the disease site, inhibit the infiltration of neutrophils and macrophages, restore the colonic niche, and prevent and treat colitis-related colon cancer.

[0050] 2. Safe to use and meets clinical needs: The nanozymes used in this invention have core components that have received FDA approval and possess high safety. The antioxidant-active nanozymes alleviate the worsening of colitis and mitigate the disease symptoms of colitis-related colon cancer, thus meeting clinical treatment needs.

[0051] 3. Highly efficient ROS elimination capacity: Unlike traditional antioxidant drugs and natural enzymes, nanozymes eliminate ROS by consuming large amounts of drug molecules. Natural antioxidant enzymes suffer from volatilization and high cost. The ROS-eliminating nanozyme developed in this invention is an enzyme mimic with SOD and CAT-like activities, capable of continuous catalysis in vivo. This cascade catalysis efficiently removes ROS, thus treating colitis-related diseases with high efficacy and low side effects.

[0052] 4. It has advantages such as simple preparation process and low raw material cost, which facilitates industrial production and clinical translation. Attached Figure Description

[0053] Figure 1 : Fluorescence image of CXCR2-RAW cell line after lentiviral transfection (scale bar, 50 μm); Figure 2 XRD patterns of Prussian blue PB and nanozyme preparation PB@ECM; Figure 3 XPS spectra of Prussian blue PB and nanozyme preparation PB@ECM; Figure 4 TEM images of Prussian blue PB and nanozyme preparation PB@ECM; Figure 5 Prussian blue PB-like SOD activity (A) and CAT-like activity (B); Figure 6 : SOD-like activity (A) and CAT-like activity (B) of the nanozyme preparation PB@ECM; Figure 7 PB and PB@ECM nanozymes· Detection of OH scavenging activity; Figure 8 Fluorescent images of PB and PB@ECM nanozymes that slow down ROS production in macrophages (scale bar, 100 μm). Figure 9 Flow cytometry results of PB and PB@ECM nanozymes in reducing ROS production in macrophages; Figure 10 Schematic diagram of the construction of an animal model of colitis-related colorectal cancer; Figure 11 Images of PB@ECM nanozyme formulations targeting lesion sites; Figure 12 Colon photographs of different groups in a colitis-associated colon cancer model after treatment; Figure 13 Statistical graph of colon length after treatment in different groups of a colitis-associated colon cancer model; Figure 14 : Colon pathological sections (top) (scale bar, 200 μm) and fluorescence images of neutrophils (LY-6G) (middle) and macrophages (bottom) after treatment in different groups of a colitis-associated colon cancer model (F4 / 80), scale bar, 100 μm; Figure 15 Pathological sections of mouse heart, liver, spleen, lungs and kidneys. Detailed Implementation

[0054] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0055] Example 1: Preparation of nanozymes (PB nanozymes) 1. Prepare K3[Fe(CN)6] (3.3 mg / mL) -1 Prepare a solution of 0.01M HCl for later use; 2. Take 6g of polyvinylpyrrolidone (PVP-K30, Meilun Biotechnology, catalog number: MB1924) and add it to 80mL of K3[Fe(CN)6] (3.3mg / mL). -1 Stir in the mixture for 10 minutes; 3. Add 80 mL of 0.01 M HCl to the system from step 2 and stir for 5 minutes; 4. Transfer the solution from step 3 to an 80°C oil bath and let it stand for 20 hours.

[0056] 5. Centrifuge the liquid obtained in step 4 at 10000 rpm for 20 minutes, repeat this step 3 times; and wash with deionized water three times.

[0057] 6. After vacuum drying the sample from step 5 for 24 hours, it is ready for use.

[0058] Example 2: Construction of Nanozyme Preparation 1. Using lentiviral transfection technology, CXCR2 protein was transfected into the RAW264.7 cell line to construct an engineered CXCR2-RAW cell line stably expressing the membrane protein CXCR2. Figure 1 ).

[0059] 2. Collect CXCR2-RAW cells, resuspend the cells in 1×PBS (pH=7.4), centrifuge at 1500 rpm for 5 minutes to wash the cells, and mix the centrifuged cells with 1 mL of lysis buffer (containing 20 mmol / L Tris-HCl, 10 mmol / L KCl, 2 mmol / L MgCl2, and a protease inhibitor without EDTA) for 5 minutes. Then centrifuge at 6000 rpm for 5 minutes and collect the supernatant. Add 250 μL of the above lysis buffer to resuspend the cells, and centrifuge at 6000 rpm for 5 minutes and collect the supernatant. Centrifuge the supernatant at 20000 rpm for 25 minutes and collect the supernatant. Then centrifuge the supernatant at 100000 rpm for 35 minutes and collect the pellet, which is the cell membrane. Store the cell membrane at -80℃.

[0060] 3. Resuspend the cell membranes collected in step 2 using PBS, and add the same volume of 500 μL PB nanozyme solution (1 mg / mL). -1 Mix and sonicate for 5 minutes.

[0061] 4. Co-extrude the mixture from step 3 using equipment (Mini-Extruder) to obtain PB@ECM nanozyme preparation.

[0062] 5. Store the PB@ECM nanozyme preparation obtained in step 4 at -80℃.

[0063] Example 3: Evaluation of the synthesis and antioxidant activity of nanozymes in Examples 1 and 2 In Examples 1 and 2, PB was synthesized using K3[Fe(CN)6] as a precursor, and subsequently, PB@ECM nanozyme formulations were successfully prepared. Figure 2 and Figure 3 The PB@ECM nanozyme maintained a particle size of 200 nm and exhibited good dispersibility. Figure 4 ).

[0064] Subsequently, the enzyme-like activity of the nanozymes prepared in Examples 1 and 2 was investigated. •− O2 is a free radical with asymmetric electrons. Due to the asymmetry of its electronic configuration, O2... •− It causes extremely high damage to biological systems. Nanozymes target O2. •− The elimination rate can be achieved through WST-1 and O2.•− The interaction formed by the formazan dye was used for detection (WST-1 kit) when the nanozyme reacted with O2. •− When elimination occurs, its absorbance decreases. The scavenging rate of the nanozyme at concentrations of 10, 20, 50, 100, and 150 μg / mL was investigated, and the results are as follows: Figure 5 As shown in Figures A and 6A, the nanozymes constructed in this invention exhibit good SOD enzyme activity, especially at a concentration of 150 μg / mL, where the scavenging rate of PB nanozymes is 60% and that of PB@ECM nanozymes is 50%. Subsequently, CAT-like activity was assessed. In a hypoxic environment, the self-polymerization of dopamine induced by H2O2 is inhibited. However, in the presence of CAT or CAT-like nanozymes, the O2 generated from the decomposition of H2O2 accelerates the self-polymerization of dopamine, resulting in a colorimetric reaction. Therefore, a dopamine-based CAT-like activity assay was used to evaluate the second step of the antioxidant cascade reaction, namely the decomposition of H2O2. The results are as follows: Figure 5 As shown in B and 6B, both nanozymes exhibited a significant concentration-dependent decomposition of H2O2. EPR was used to detect the scavenging activity of the nanozymes. · The ability of OH to scavenge OH has been demonstrated experimentally, showing that the nanozyme constructed in this invention has excellent scavenging properties. · The ability of OH ( Figure 7 ).

[0065] Example 4: Verification of Antioxidant Function at the Cell Level 1. RAW 264.7 cells were spaced at a density of 1 × 10⁻⁶ cells per well. 5 One seed was inoculated into a 6-well plate and incubated overnight.

[0066] 2. Add 50 μg / mL nanozyme (PB, PB@ECM) solution to the cells from step 1 and incubate for 2 hours. Wash the cells three times with PBS, then add 100 μM H2O2 and incubate for 0.5 hours to stimulate intracellular ROS production.

[0067] 3. After washing three times with PBS, DMEM medium containing 10 μM DCFH-DA (a reactive oxygen species fluorescent probe, Beyotime Biotechnology Co., Ltd.) was added to the cells and incubated for 30 minutes. After washing with PBS, fluorescence images were acquired using a laser scanning confocal microscope, and flow cytometry was used for semi-quantitative analysis of intracellular ROS levels. Fluorescence images are shown below. Figure 8 As shown, nanozymes with pathological microenvironment response exhibit good antioxidant activity.

[0068] Meanwhile, the quantitative detection results of flow cytometry are as follows: Figure 9 As shown, the nanoenzyme preparation material constructed in this invention also demonstrates that it has excellent reactive oxygen species scavenging ability.

[0069] Example 5: Physiological performance of nanoenzyme preparations in a colitis-associated colon cancer model 1. C57 mice (Violentia Laboratory Animal Technology Co., Ltd.) were divided into four groups: a healthy group, a disease group, a cell membrane group, a PB nanozyme group, and a PB@ECM nanozyme preparation group, with 5 mice in each group. The healthy group (Normal) received no treatment, while the disease group underwent azomethane / dextran sulfate sodium modeling (model construction method as described below). Figure 10 As shown: On day 1, mice were intraperitoneally injected with azomethane (10 mg / kg); starting on day 7, mice were given 2.5% sodium dextran sulfate solution for one week, followed by two weeks of normal water, repeated three times. This process lasted 10 weeks to establish the azomethane / sodium dextran sulfate model. The cell membrane group (ECM), PB nanozyme group (PB), and PB@ECM nanozyme group (PB@ECM) were administered intravenously with the same dose of the corresponding different drugs (15 mg / kg, note: ECM converted to the corresponding concentration) in addition to the treatments given to the model group. Treatment was administered twice a week (with a three-day interval between treatments) for 10 weeks. During this period, the disease targeting of PB@ECM was observed, and the experiment demonstrated that PB@ECM had superior inflammation targeting (…). Figure 11 The experiment ended on day 71. The mice were euthanized by carbon dioxide asphyxiation, and their colons and major organs were removed via laparotomy.

[0070] 2. Observation and detection of colon length and lesion tissue were performed to observe changes in colon length and the number of colon tumors. Detection included colonic CXCL2 levels, H&E staining, F4 / 80 staining, and LY-6G staining. The results were statistically analyzed, including colon length and tumor growth. Statistical analysis of the detection results was also performed, including F4 / 80 positive expression and LY-6G positive expression. Compared with the model group and other drug treatment groups, mice treated with nanozyme preparations showed a significantly lower number of colon tumors than other groups, with almost none (…). Figure 12 The shortening of the colon caused by the disease was relieved. Figure 13 The colon length (cm) for each group is shown in Table 1 (* indicates mice that died before sample collection): Table 1

[0071] Compared with the model group and other drug treatment groups, mice treated with nanozyme preparations showed fewer tumors and inflammation in their colon pathology. Figure 14 The lowest F4 / 80 and LY-6G expression ( Figure 14The inflammation level in mice was lower than that in the control group and other drug groups, indicating that targeted delivery of nanozymes can effectively alleviate inflammation at the pathological sites of colitis-associated colon cancer and inhibit the progression of colitis to colon cancer. Furthermore, no biotoxicity was observed in other organs after administration to mice. Figure 15 This indicates that it has good biosafety.

[0072] In summary, the targeted nanozymes prepared using this invention can alleviate and treat colitis-related colorectal cancer, and have a certain degree of universality, making them potentially applicable in clinical practice.

Claims

1. A method for synthesizing a nanoenzyme preparation, characterized in that, Includes the following steps: (1) Disperse the transmembrane protein in solution A, which has good biocompatibility; (2) Disperse one or more nanozymes in a biocompatible solution A; (3) Mix the solutions obtained in step (1) and step (2) to prepare nanozyme preparations.

2. The synthesis method according to claim 1, characterized in that, The transmembrane protein mentioned in step (1) is obtained by modifying cells through cell engineering technology.

3. The synthesis method according to claim 2, characterized in that, Engineering technologies include transfection technology; transfection technology includes one or more combinations of lentivirus transfection, adenovirus transfection, and CRISPR-Cas9 technology.

4. The synthesis method according to claim 1, characterized in that, The transmembrane proteins mentioned in step (1) include one or more combinations of CCR2, CCR4, CCR8, PD-L1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CD47, CD55, CD59, GPIbα, GPIV, GPV, GPVI, GPPIX, CLEC-2, TNF-αR, IL-1R, and LFA-1.

5. The synthesis method according to claim 1, characterized in that, Cells used to express the transmembrane protein described in step (1) comprise one or more combinations of the following cell lines: RAW264.7, HL-60, Caco-2, HT-29, HCT116, SW480, J774, 4T1, MC-38, CT-26, HUVEC, 3T3-L1, THP-1, and 293T.

6. The synthesis method according to claim 1, characterized in that, Solution A includes one or more combinations of phosphate buffer, physiological saline, or water.

7. The synthesis method according to claim 1, characterized in that, The nanozymes mentioned in step (2) include one or more of the following: oxo-compound nanozymes, metal nanozymes, organometallic framework nanozymes, and single-atom nanozymes.

8. A nanozyme preparation prepared by the synthesis method according to any one of claims 1 to 7.

9. The use of the nanozyme preparation according to claim 8 in the preparation of treatments, relief and / or prevention of colonic diseases.

10. The application according to claim 9, characterized in that, The diseases mentioned include chronic colitis, colitis-associated colon cancer, acute colitis, and localized or specific colonic lesions.

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