A tumor-targeting nano-enzyme and a preparation method and application thereof

Abstract

The application discloses a tumor-targeting nano-enzyme and a preparation method and application thereof, and belongs to the technical field of cell biology. Hollow MnO2 particles are prepared by using a template etching method, then Pt, Au and Ru metal particles are adsorbed, and finally hyaluronic acid is modified, so that the tumor-targeting nano-enzyme is obtained. The application establishes a tumor-targeting and tumor microenvironment-responsive nano-enzyme synthesis preparation method through nanoparticle self-assembly and targeting molecule modification, and explores the anti-colorectal cancer effect and targeting function of the nano-enzyme in vivo and in vitro, so that the problems of toxic side effects caused by the lack of targeting of traditional drugs and drug resistance of traditional chemotherapy drugs are greatly reduced. In addition, the tiny nano-enzyme can be discharged through the kidney, the damage to normal tissues is reduced, and a very promising treatment method is provided for tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field, in particular, to a tumor-targeting nano-enzyme and a preparation method and application thereof. BACKGROUND

[0002] In recent years, malignant tumors including colorectal cancer (CRC) have become the main diseases threatening human health, with poor prognosis and high mortality. At present, the main treatment methods for malignant tumors are surgery, radiotherapy and chemotherapy. However, these methods still have obvious shortcomings, such as limited anti-tumor effect and serious drug side effects.

[0003] Nano-enzyme is a kind of nanomaterial with natural enzyme activity. Compared with natural enzymes, nano-enzyme has the advantages of simple preparation, low price and high stability. Since Yan group reported the peroxidase (POD) mimetic activity based on Fe3O4 nanoparticles in 2007, various nano-enzymes have been reported for cancer treatment based on tumor microenvironment (TME) induction. Generally, the catalytic therapy of nano-enzyme mainly relies on the POD mimetic activity to convert H2O2 into ·OH, inducing tumor cell apoptosis, which is called chemodynamic therapy (CDT). In recent years, nano-enzymes with synergistic multiple enzyme activities have attracted extensive attention of researchers. By combining two or more nano-enzymes with POD (converting H2O2 into ·OH), catalase (CAT, decomposing H2O2 into O2), glucose oxidase (GOD, converting glucose into gluconic acid and H2O2), oxidase (OXD, converting O2 into O2· - ) mimetic activity, a cascade catalytic system is formed to induce cascade catalysis to improve tumor CDT.

[0004] Compared with single enzyme mimetic nano-enzyme, multifunctional nano-enzyme has synergistic effect and cascade catalysis and many other advantages, which can overcome the limitations of tumor microenvironment (TME). However, due to the mutual influence of multiple enzyme mimetic activities, it is difficult to rationally design multi-enzyme nano-enzyme with synergistic catalytic activity. SUMMARY

[0005] The purpose of the present application is to provide a tumor-targeting nano-enzyme and a preparation method and application thereof to solve the problems existing in the prior art.

[0006] To achieve the above purpose, the present application provides the following solutions.

[0007] One of the technical solutions of the present application is a preparation method of a tumor-targeting nano-enzyme, comprising the following steps: preparing hollow MnO2 particles by using a template etching method, then adsorbing Pt, Au and Ru metal particles, and finally modifying hyaluronic acid to obtain the tumor-targeting nano-enzyme.

[0008] The second technical solution of the present application is the tumor-targeting nano-enzyme MnO2@PtAuRu@HA prepared by the preparation method.

[0009] The third technical solution of the present application is the application of the tumor-targeting nano-enzyme MnO2@PtAuRu@HA in the preparation of an anti-tumor drug, wherein the tumor includes colorectal cancer.

[0010] The fourth technical solution of the present application is an anti-tumor drug, which comprises the tumor-targeting nano-enzyme MnO2@PtAuRu@HA.

[0011] Based on the above technical solutions, the present application has the following technical effects:

[0012] The present application uses a template etching method to prepare hollow MnO2 particles, then polyethylene imine is used to mediate adsorption of Pt, Au and Ru metal particles, and finally hyaluronic acid is modified, so as to obtain the tumor-targeting nano-enzyme MnO2@PtAuRu@HA (MPARH). The present application establishes a synthesis and preparation method of a tumor-targeting and tumor microenvironment-responsive nano-enzyme through nanoparticle self-assembly and targeting molecule modification, and explores the anti-colorectal cancer effect and targeting function of the nano-enzyme in vivo and in vitro, which greatly reduces the toxic side effects caused by the lack of targeting of traditional drugs and the drug resistance problem of traditional chemotherapy drugs. In addition, the small nano-enzyme can be excreted through the kidney, reducing damage to normal tissues, and providing a very promising treatment method for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0014] Figure 1 TEM observation results of each stage in the synthesis process of the targeting nano-enzyme, wherein a is a hollow MnO2 nanoparticle, b is a MnO2@PtAuRu particle, c is a high-resolution TEM image of MnO2@PtAuRu, and d is a MnO2@PtAuRu@HA;

[0015] Figure 2 X-ray photoelectron spectroscopy, Fourier infrared spectroscopy and Zeta potential characterization results of the targeting nano-enzyme, wherein a is the characterization result of the nano-enzyme XPS, b is the characterization result of the nano-enzyme Fourier infrared spectroscopy, and c is a graph of the zeta potential value change of the products at each stage of the synthesis of the nano-enzyme;

[0016] Figure 3 Characterization of various enzyme activities of the targeted nanoscale enzyme, wherein a is a schematic diagram of a multi-enzyme cascade reaction, b is GOD enzyme activity verification, c is H2O2 production detection, d is CAT enzyme activity verification, e is POD enzyme activity characterization, f is GPx enzyme activity verification;

[0017] Figure 4 Characterization of the photothermal performance of the targeted nanoscale enzyme, wherein a is the temperature rise curve of the nanoscale enzyme solution at different concentrations, b is the calculation of the photothermal conversion efficiency, c is the characterization of the enhanced POD enzyme activity under laser irradiation;

[0018] Figure 5 Characterization of various enzyme activities of the targeted nanoscale enzyme in tumor cells, wherein a is the image of the change of O2 content over time, b is the image of the change of H2O2 content over time, c is the change of intracellular ROS, d is the change of intracellular GSH;

[0019] Figure 6 Verification of the inhibition effect of the targeted nanoscale enzyme on tumor cell growth, and detection of the apoptosis of tumor cells after treatment with the targeted nanoscale enzyme, wherein a is the cell viability detection of HCT116 and NCM460 cells after treatment with the nanoscale enzyme, b is the detection of the apoptosis of tumor cells after treatment with the nanoscale enzyme;

[0020] Figure 7 In vivo treatment effect and targeting verification of the targeted nanoscale enzyme, wherein a is the image of the tumor tissue ex vivo after treatment in different treatment groups, b is the change curve of the tumor size during treatment, c is the small animal live imaging result. DETAILED DESCRIPTION

[0021] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, features and embodiments of the present application.

[0022] It should be understood that the terms described in the present application are only for describing the particular embodiments, and are not intended to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0024] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0025] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0026] The technical solutions described in the present application are conventional solutions in the art, and the reagents or raw materials used are commercially available or have been disclosed, unless otherwise specified.

[0027] The embodiment of the present application provides a preparation method of a tumor-targeting nano-enzyme, comprising the following steps: hollow MnO2 particles are prepared by using a template etching method, then Pt, Au and Ru metal particles are adsorbed, and finally hyaluronic acid is modified, so that the tumor-targeting nano-enzyme is obtained.

[0028] In some specific embodiments, the following steps are included:

[0029] (1) ethanol, ammonia water and water are mixed, then tetraethyl orthosilicate is added, stirring reaction is carried out, and after the reaction is completed, SiO2 NPs are obtained by centrifugation; KMnO4 solution is added to the SiO2 NPs solution for ultrasonic reaction, and after the reaction is completed, stirring is continued; the reaction product is dissolved in Na2CO3 solution for stirring reaction, and hollow mesoporous MnO2 is obtained;

[0030] (2) polyethyleneimine solution is added to the hollow mesoporous MnO2, and ultrasonic reaction is carried out to obtain MnO2@PEI particles;

[0031] (3) the MnO2@PEI particles are added to PtAuRu solution, ultrasonic and centrifugal treatment are carried out, and MnO2@PtAuRu is obtained;

[0032] (4) the MnO2@PtAuRu solution is mixed with hyaluronic acid solution, stirring reaction is carried out, and centrifugal treatment is carried out, so that MnO2@PtAuRu@HA is obtained, that is, the tumor-targeting nano-enzyme.

[0033] In some specific embodiments, in step (1), the ethanol is anhydrous ethanol; the mass concentration of the ammonia water is 25%; the volume ratio of the ethanol, ammonia water, water and tetraethyl orthosilicate is (25-30):(1-2):(3-5):(1-2); the ultrasonic reaction is carried out at a frequency of 100 kHz for 30-40 min; and the stirring time is 5-6 h.

[0034] In some specific embodiments, in step (1), the concentration of the Na2CO3 solution is 2 M; and the stirring reaction of the reaction product in the Na2CO3 solution is carried out at a reaction temperature of 60-70 °C for 12-16 h at a stirring frequency of 550-650 rpm.

[0035] In some specific embodiments, in step (2), the concentration of the polyethyleneimine solution is 200 μg / mL; and the ultrasonic reaction is carried out at a frequency of 100 kHz for 30-40 min.

[0036] In some specific embodiments, in step (3), the mass ratio of the MnO2@PEI particles to PtAuRu is (1-1.1):(2-2.2); and the PtAuRu solution is prepared by mixing HAuCl4·4H2O, H2PtCl6·6H2O, RuCl3 solution and sodium citrate solution in water, and then adding NaBH4 solution and stirring for 3-4 h.

[0037] In some specific embodiments, the PtAuRu solution is prepared by adding 560 μL of 0.01 g / mL HAuCl4·4H2O, 700 μL of 0.01 g / mL H2PtCl6·6H2O, 300 μL of 0.01 g / mL RuCl3 solution and 1.5 mL of 0.01 g / mL sodium citrate solution into 100 mL of deionized water. After the solution is mixed well, 5 mL of 6 mg / mL NaBH4 (30 mg) solution is added and stirred for 4 h.

[0038] In some specific embodiments, in step (4), the concentration of the MnO2@PtAuRu solution is 1 mg / mL; the concentration of the hyaluronic acid solution is 10 mg / mL; and the volume ratio of the MnO2@PtAuRu solution to the hyaluronic acid solution is (1-1.1):(1-1.1).

[0039] In some specific embodiments, in step (4), the stirring reaction is carried out at a frequency of 600 rpm for 6 h; and the centrifugation is carried out at a frequency of 4800 rpm for 5 min.

[0040] The application further provides the tumor-targeting nano-enzyme MnO2@PtAuRu@HA prepared by the preparation method.

[0041] The application further provides application of the tumor-targeting nano-enzyme MnO2@PtAuRu@HA in preparation of an anti-tumor drug, wherein the tumor includes colorectal cancer.

[0042] The application further provides an anti-tumor drug, which comprises the tumor-targeting nano-enzyme MnO2@PtAuRu@HA.

[0043] The main materials used in the embodiments and sources thereof are as follows:

[0044] SPF level Balb / c nude mice (for establishing a mouse subcutaneous tumor model): Beijing Vital River Laboratory Animal Technology Co., Ltd. The method for constructing a mouse subcutaneous tumor model is as follows: 100 μL of HCT116 cell suspension (1*10 8 cell / mL) is subcutaneously injected into the rib of a mouse.

[0045] Anhydrous ethanol: National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10009218.

[0046] Ammonia (NH3·H2O): National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10002108.

[0047] Tetraethyl orthosilicate (TEOS): Merck (Germany), item number 131903.

[0048] Potassium permanganate (KMnO4): National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10017318.

[0049] Sodium carbonate (Na2CO3): Shanghai Macklin Biochemical Technology Co., Ltd., item number S818014.

[0050] Polyethyleneimine (PEI): Merck (Germany), item number 408719.

[0051] Chloroauric acid, tetrahydrate (HAuCl4·4H2O): National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10010711.

[0052] Chloroplatinic acid, hexahydrate (H2PtCl6·6H2O): National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 51006311.

[0053] Ruthenium (III) chloride hydrate (RuCl3·xH2O): Merck (Germany), item number 206229.

[0054] Trisodium citrate: National Pharmaceutical Group Chemical Reagent Co., Ltd., Catalog No. 10019408.

[0055] Sodium borohydride: National Pharmaceutical Group Chemical Reagent Co., Ltd., Catalog No. 80115816.

[0056] Hyaluronic acid: Shanghai Macklin Biochemical Technology Co., Ltd., Catalog No. H823435.

[0057] DMEM medium: Thermo Fisher Scientific, Catalog No. 11965092.

[0058] PBS buffer: Thermo Fisher Scientific, Catalog No. 10010002.

[0059] Fetal bovine serum: Nanjing Nvigan Biotech Co., Ltd., Catalog No. F101-01.

[0060] Penicillin-streptomycin double antibody: Thermo Fisher Scientific, Catalog No. 15140122

[0061] 100mm cell culture dish: China Select Co., Ltd., Catalog No. 12310.

[0062] Trypsin-EDTA (0.25%): Thermo Fisher Scientific, Catalog No. 25200072.

[0063] Hydrogen peroxide (H2O2): National Pharmaceutical Group Chemical Reagent Co., Ltd., Catalog No. 10011208.

[0064] Glutathione (GSH): Merck, Germany, Catalog No. G6013.

[0065] 3,3',5,5'-tetramethylbenzidine (TMB): Merck, Germany, Catalog No. 860336.

[0066] 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB): Merck, Germany, Catalog No. D218200.

[0067] Tris(2,2-dipyridyl)dichlororuthenium ([Ru(dpp)3 2+ Cl2): Shanghai Bide Pharmaceutical Co., Ltd., Catalog No. BD406203.

[0068] ThiolTrace TM Violet 500: AAT Bioquest, USA, Catalog No. AAT-B22280.

[0069] 2',7'-dichlorofluorescin diacetate (DCFH-DA): Shanghai Biyun Tian Co., Ltd., Cat. No. S0033S.

[0070] Annexin V-FITC Apoptosis Detection Kit: Shanghai Biyun Tian Co., Ltd., Cat. No. C1062M.

[0071] CCK-8 Kit: Shanghai Biyun Tian Co., Ltd., Cat. No. C0037.

[0072] Horseradish Peroxidase (HRP): Merck, Cat. No. P8375.

[0073] Amplex Red (Fluorescent Red Stain): Shanghai Biyun Tian Co., Ltd., Cat. No. ST010.

[0074] Hoechst 33342: Shanghai Biyun Tian Co., Ltd., Cat. No. C1022.

[0075] Glucose Assay Kit: Shanghai Biyun Tian Co., Ltd., Cat. No. S0201S.

[0076] HCT116 and NCM460 cell lines: purchased from ATCC cell bank.

[0077] TEM images were taken using JEM-2100 and Philips Tecnai G2 F20 microscopes equipped with STEM units, UV-Vis absorption spectra were measured using a Shimadzu 2600 spectrometer, zeta potential of nanomaterials was detected using a Malvern Nano-ZS90 Zetasizer. X-ray photoelectron spectroscopy (XPS) was collected by ESCA lab 250 (Thermal Scientific, US). O2 concentration changes were detected using a JPB-607A dissolved oxygen meter. Fluorescence images were taken using a Zeiss LSM880 laser confocal microscope equipped with an Airyscan unit. Thermal images were obtained by Sinuodar Fotric 225S infrared thermal imager.

[0078] Example 1

[0079] A preparation method of a tumor-targeting nanozyme, comprising the following steps:

[0080] (1) Preparation of hollow MnO2 nanoparticles: First, 140 mL of absolute ethanol, 5 mL of 25% ammonia water (NH3-H2O) and 20 mL of deionized water were mixed and stirred at 600 rpm for 5 min. Then 5 mL of TEOS was added, and after stirring at 600 rpm for 2 h, centrifugation was performed at 6500 rpm for 5 min, and ethanol washing was performed twice to obtain SiO2 NPs. Next, an aqueous solution containing 300 mg of KMnO4 (5 mg / mL) was gradually added dropwise to the prepared SiO2 NPs (80 mg, 2 mg / mL) solution under ultrasonic conditions at 100 kHz and 25°C for 30 min, and stirring was continued for 6 h. Unreacted KMnO4 was removed by centrifugation at 5000 rpm for 5 min, and was redissolved in a 2 M aqueous Na2CO3 solution, and stirring was performed at 600 rpm at 60°C for 12 h. After centrifugation at 5500 rpm for 5 min, the product was washed twice with water to obtain hollow mesoporous MnO2 nanoparticles.

[0081] (2) Polyethyleneimine (PEI) modification: 100 μL of a 200 μg / mL PEI aqueous solution was added to the MnO2 nanoparticles prepared above, and ultrasonic treatment was performed at 100 kHz and 25°C for 30 min. Centrifugation was performed at 5200 rpm for 5 min, and the residual PEI was removed by washing twice with deionized water to obtain MnO2@PEI particles.

[0082] (3) Preparation and adsorption of PtAuRu particles: 560 μL of a 0.01 g / mL HAuCl4-4H2O solution, 700 μL of a 0.01 g / mL H2PtCl6-6H2O solution, 300 μL of a 0.01 g / mL RuCl3 solution and 1.5 mL of a 0.01 g / mL sodium citrate solution were added to 100 mL of deionized water. After the solution was mixed well, 5 mL of a 6 mg / mL NaBH4 (30 mg) solution was added and stirred for 4 h. Subsequently, 5 mg of MnO2@PEI was added to the prepared PtAuRu solution, and ultrasonic treatment was performed for 30 min. The solution was centrifuged at 4800 rpm for 5 min, and was washed twice with deionized water to obtain MnO2@PtAuRu.

[0083] (4) Modification of hyaluronic acid (HA): Hyaluronic acid powder (50 mg) was dissolved in 5 mL of deionized water under ultrasonic conditions. 5 mL of a 1 mg / mL MnO2@PtAuRu solution was mixed with the HA aqueous solution. After stirring at 600 rpm for 6 h, the precipitate was obtained by centrifugation at 4800 rpm for 5 min, and was washed twice. Finally, MnO2@PtAuRu@HA (MPARH) was resuspended in a PBS solution and stored at 4°C.

[0084] As Figure 1The results of the characterization of the nanoscale enzyme are shown in Figure 1. a is the hollow MnO2nanoparticle prepared, the central color is lower than the edge; b is the MnO2@PtAuRu particle, after adsorption of the nanoscale enzyme particle, small particles can be seen attached to the surface; c is the high-definition transmission electron microscopy image of the MnO2@PtAuRu, the lattice spacing characteristic of Pt, Au and Ru can be seen; d is the MnO2@PtAuRu@HA, a transparent shell layer of about 3 nm is formed on the surface.

[0085] Figure 2 The results of the characterization of the nanoscale enzyme are shown in Figure 1. a is the hollow MnO2nanoparticle prepared, the central color is lower than the edge; b is the MnO2@PtAuRu particle, after adsorption of the nanoscale enzyme particle, small particles can be seen attached to the surface; c is the high-definition transmission electron microscopy image of the MnO2@PtAuRu, the lattice spacing characteristic of Pt, Au and Ru can be seen; d is the MnO2@PtAuRu@HA, a transparent shell layer of about 3 nm is formed on the surface. -1 The results of the characterization of the nanoscale enzyme are shown in Figure 1. a is the hollow MnO2nanoparticle prepared, the central color is lower than the edge; b is the MnO2@PtAuRu particle, after adsorption of the nanoscale enzyme particle, small particles can be seen attached to the surface; c is the high-definition transmission electron microscopy image of the MnO2@PtAuRu, the lattice spacing characteristic of Pt, Au and Ru can be seen; d is the MnO2@PtAuRu@HA, a transparent shell layer of about 3 nm is formed on the surface.

[0086] Example 2

[0087] 1. Verification of multi-enzyme activity of tumor-targeting nanoscale enzyme

[0088] The experimental steps are as follows:

[0089] (1) GOD enzyme activity verification: different concentrations of MPARH nanoscale enzyme (0, 25, 50, 100, 200 μg / mL) were added to 1 mg / mL glucose solution, shaken at 37°C, and the glucose consumption was observed. After 12 h of culture, the glucose concentration was detected by glucose detection kit (o-toluidine method) after centrifugation. The absorbance value at 630 nm was substituted into the standard curve to calculate the glucose concentration; in order to detect the generation of H2O2, different concentrations of MPARH nanoscale enzyme (0, 0.5, 1, 2.5, 5 mg / mL) were incubated with MPARH nanoscale enzyme for 2 h. After centrifugation at 8000 rpm, Amplex Red and HRP were added to the mixture. The absorbance value at 571 nm was substituted into the standard curve to calculate the H2O2concentration.

[0090] (2) CAT enzyme activity verification: in order to evaluate the activity of the CAT mimic, different concentrations of MPARH nanoscale enzyme (25, 50, 100 μg / mL) were mixed with H2O2in PBS solution at pH 7.4. At room temperature, the oxygen content of the mixture was measured every 30 s for 10 min using a dissolved oxygen meter, and the oxygen concentration curve was drawn to compare the oxygen production of different groups.

[0091] (3) POD enzyme activity verification: different concentrations of H2O2(0.5, 1, 2, 4 mM) solution were added to PBS solution (pH 6.5) containing MPARH nanoscale enzyme. Then active oxygen indicator TMB was added, and the absorbance curve was recorded.

[0092] (4) GPx enzyme activity verification: MPARH nanoscale enzyme (50 μg / mL) was added to the PBS solution containing glutathione (100 mM). The mixed solution was shaken and incubated for different times (0, 1, 2, 3, 4 min). Then 10 μL of DTNB solution (50 mM) was added. Finally, the absorbance change curve was determined, and the glutathione depletion level was observed.

[0093] Figure 3 is the enzyme activity verification of nanoscale enzyme. a is the schematic diagram of multi-enzyme cascade reaction, b and c are GOD enzyme activity, which can be seen that the glucose concentration decreases and the H2O2 concentration increases; d is the characterization of CAT enzyme activity, the results show that as the concentration of nanoscale enzyme increases, the generation rate of oxygen also increases, e is the characterization of POD enzyme activity, after the nanoscale enzyme is co-incubated with H2O2, the active oxygen content in the solution increases significantly, f is the GPx enzyme activity verification, and the results show that the nanoscale enzyme consumes glutathione.

[0094] 2. Characterization of photothermal performance of tumor-targeting nanoscale enzyme

[0095] The experimental steps are as follows:

[0096] (1) MPARH nanoscale enzyme solution (50 μg / mL, 100 μg / mL, 200 μg / mL) was irradiated with 808 nm laser (1 W / cm 2 ) for 5 min, and the temperature change per second was recorded with Uni-trend thermometer (UT325) and the change curve was drawn.

[0097] (2) The photothermal conversion efficiency of nanoscale enzyme was calculated from the temperature change value of the cooling section.

[0098] (3) MPARH nanoscale enzyme solution (50 μg / mL) containing H2O2 was irradiated with 808 nm laser (1 W / cm 2 ), and after adding TMB, the absorbance value at 650 nm was detected at 0, 1, 2.5, 5 min, respectively, and a column chart was drawn.

[0099] Figure 4 is the characterization of photothermal performance of nanoscale enzyme. a is the temperature rise curve of nanoscale enzyme solution with different concentrations, and the temperature rise rate increases with the increase of concentration, b is the calculation of photothermal conversion efficiency; the photothermal conversion efficiency of nanoscale enzyme is 43.3%, c is the characterization of laser irradiation enhancing POD enzyme activity, after the nanoscale enzyme is co-incubated with H2O2, the active oxygen content in the solution increases significantly, and the active oxygen content after laser irradiation is higher than that of the unirradiated and control groups.

[0100] 3. Intracellular multi-enzyme activity verification of tumor-targeting nanoscale enzyme

[0101] The experimental steps are as follows:

[0102] (1)HCT116 cell line was cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37℃ with 5% CO2 in a humidified incubator. The medium was updated every two days. When the cells grew to 80-90% confluence, they were subcultured at 1:3 with 0.25% trypsin.

[0103] (2)HCT116 cells (1*10 5 cells per well) were seeded in 28mm confocal dishes and incubated for 12h to adhere. HCT116 cells were treated with different solutions: ① PBS, ② MnO2, ③ PtAuRu, ④ MnO2@PtAuRu, ⑤ MPARH, ⑥ MPARH + 808nm laser.

[0104] After 3h incubation, cells were irradiated with 808nm laser (1W / cm 2 ) for 5min. Intracellular GSH consumption was detected by ThiolTrace TM Violet 500 (1:500 dilution) according to the instructions for 30min, then stained with Hoechst 33342 (5μg / mL) for 15min, and washed with PBS. Intracellular ROS detection was performed by DCFH-DA (10mM) diluted with serum-free medium at 1:1000 to label ROS. After incubation at 37℃ for 20min, cells were washed with PBS twice and stained with Hoechst 33342 for 15min. Fluorescence images were collected by confocal laser scanning microscope (Nikon).

[0105] (3)HCT116 cells (1*10 5 cells per well) were cultured in 28mm confocal dishes for 12h, and MPARH nanoszyme was added to the culture medium for 0, 1, 2, and 3h, respectively, and then stained with [Ru(dpp)3] 2+ Cl2 (20μM) at 37℃ for 30min. Nuclei were also stained with Hoechst 33342. Intracellular H2O2 was detected by Amplex Red (10μg / mL) at 37℃ for 30min. Other steps were the same as oxygen detection; fluorescence images were collected by confocal laser scanning microscope (Nikon).

[0106] Figure 5 Intracellular multi-enzyme activity verification of nanoszyme, a is the image of O2 content change over time, b is the image of H2O2 content change over time, c is the change of intracellular ROS, and d is the change of intracellular GSH, which proves that nanoszyme can still maintain good enzyme catalytic activity in cells.

[0107] 4. In vitro anti-tumor performance verification of tumor-targeting nanoszyme

[0108] The experimental steps are as follows:

[0109] (1) HCT116 and NCM460 cell lines were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO2. The medium was replaced every two days. When the cells grew to 80-90%, they were subcultured at 1:3 with 0.25% trypsin.

[0110] (2) HCT116 and NCM460 cells were placed in a 96-well plate. After 24h of incubation, the medium was replaced according to the grouping: ① PBS, ② MnO2, ③ PtAuRu, ④ MnO2@PtAuRu, ⑤ MPARH, ⑥ MPARH+808nm, and incubated for another 24h. The 808nm group was irradiated with an 808nm laser (1W / cm 2 ) for 3h, and then incubated with 100μL of medium containing CCK-8 solution for 2h. The absorbance at 450nm was measured with a microplate reader, and the cell viability was calculated.

[0111] (3) The Annexin V-FITC / PI cell apoptosis detection kit (Beyotime) was used to quantify the apoptosis level of HCT116 cells treated with nanozymes. HCT116 and NCM460 cells were first seeded in a 6-well plate and incubated for 12h according to the grouping: ① PBS, ② MnO2, ③ PtAuRu, ④ MnO2@PtAuRu, ⑤ MPARH, ⑥ MPARH+808nm. Then the cells were washed, collected, counted, and stained according to the instructions. Finally, flow cytometry was used to detect the apoptosis and necrosis levels of the cells.

[0112] Figure 6 a is the result of the change in cell viability after different treatments of HCT116 and NCM460 cells. It can be seen that the activity of tumor cells treated with nanozymes decreased significantly, while the normal cells were not significantly affected. b is the detection of tumor cell apoptosis after treatment with nanozymes. The results show that the nanozyme treatment group has the highest apoptosis rate, proving that the nanozyme anti-tumor effect is significant.

[0113] 5. In vivo anti-tumor performance verification of tumor-targeting nanozymes

[0114] Female 6-8 week old BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised in a specific pathogen-free (SPF) environment. All animal experiments were approved by the Capital Medical University Animal Ethics Committee. Subsequently, HCT116 tumor cells (5*10 6 / 100μL, 5*10

[0115] When the tumor volume reaches approximately 100 mm 3 Mice bearing tumors were randomly divided into 6 groups and intravenously injected with different solutions: ①PBS, ②MnO2, ③PtAuRu, ④MnO2@PtAuRu, ⑤MPARH, ⑥MPARH+808nm; the drugs were administered every two days, and changes in tumor size were recorded. Six hours after each injection, group 6 was irradiated with an 808nm laser (1W / cm²). 2 After 5 minutes and 9 days of treatment, the mice were sacrificed, and the major organs (heart, liver, spleen, lung, kidney) and tumors were isolated and fixed with 4% paraformaldehyde solution.

[0116] Two subcutaneously tumor-bearing mice were constructed using the same steps described above. They were injected intravenously with Cy5.5-labeled MnO2@PtAuRu and MPARH, respectively. Six hours after injection, the fluorescence distribution in vivo and in vitro organs was observed using a small animal in vivo imaging system.

[0117] Figure 7 Image a shows images of ex vivo tumor tissue after treatment in different treatment groups. Image b shows the curve of tumor size change during treatment. It can be seen that the tumor-targeting nanozyme injection group has the best anti-tumor effect, and the tumor almost disappears. Image c shows the results of in vivo imaging in small animals. The fluorescence of the unmodified hyaluronic acid nanozyme at the tumor site is weak. After modification with hyaluronic acid, the tumor-targeting effect is significantly enhanced.

[0118] In summary, this invention discloses a MnO2@PtAuRu@HA targeted nanozyme that achieves oxygen self-supply and sustainable cascade catalysis, as well as synergistic tumor therapy. This nanozyme has three advantages. First, a small-sized PtAuRu nanozyme (~2 nm) with cascade catalytic activity was synthesized through rational design; the alloyed PtAuRu simultaneously possesses GOD, CAT, and POD enzyme activities. Glucose in cancer cells can be converted to H2O2 through GOD activity, and H2O2 can be converted to ·OH through POD activity, thereby inducing apoptosis. Irradiation with an 808 nm laser can also significantly enhance the cascade catalytic ability of the nanozyme. Second, PtAuRu nanoclusters are assembled using hollow MnO2. Due to its TME-responsive capability, the nanozyme releases the PtAuRu nanoclusters after reaching the tumor site. Furthermore, the high levels of GSH in the tumor can be degraded by MnO2. 2+ Third, the MnO2@PtAuRu@HA nanozyme exhibits excellent targeting properties, enabling precise delivery to tumor sites.

[0119] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a tumor-targeting nanozyme, characterized in that, Includes the following steps: Hollow MnO2 particles were prepared using a template etching method, followed by the adsorption of three metal particles, Pt, Au, and Ru, and finally modified with hyaluronic acid to obtain the tumor-targeting nanozyme. Includes the following steps: (1) Mix ethanol, ammonia and water, then add tetraethyl orthosilicate, stir and react. After the reaction is complete, centrifuge to obtain SiO2 NPs; add KMnO4 solution to SiO2 NPs solution for ultrasonic reaction, continue stirring after the reaction is complete, dissolve the reaction product in Na2CO3 solution and stir to obtain hollow mesoporous MnO2. (2) Add the polyethyleneimine solution to the hollow mesoporous MnO2 and react with ultrasound to obtain MnO2@PEI particles; (3) Add the MnO2@PEI particles to the PtAuRu solution, sonicate and centrifuge to obtain MnO2@PtAuRu; (4) Mix the MnO2@PtAuRu solution with the hyaluronic acid solution, stir and react, centrifuge to obtain MnO2@PtAuRu@HA, which is the tumor-targeting nanozyme.

2. The preparation method according to claim 1, characterized in that, In step (1), the ethanol is anhydrous ethanol; the mass concentration of the ammonia is 25%; the volume ratio of ethanol, ammonia, water and tetraethyl orthosilicate is (25-30):(1-2):(3-5):(1-2); the conditions for the ultrasonic reaction are: frequency of 100kHz and time of 30-40min; the stirring time is 5-6h.

3. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the Na2CO3 solution is 2M; the reaction conditions for dissolving the reaction product in the Na2CO3 solution and stirring are: reaction temperature of 60-70℃, time of 12-16h, and stirring frequency of 550-650rpm.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the polyethyleneimine solution is 200 μg / mL; the conditions for the ultrasonic reaction are: frequency of 100 kHz and time of 30 to 40 min.

5. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of MnO2@PEI particles to PtAuRu is (1-1.1):(2-2.2); the PtAuRu solution is prepared by adding HAuCl4·4H2O, H2PtCl6·6H2O, RuCl3 solution and sodium citrate solution to water and mixing them, and then adding NaBH4 solution and stirring for 3-4 hours.

6. The preparation method according to claim 1, characterized in that, In step (4), the concentration of the MnO2@PtAuRu solution is 1 mg / mL; the concentration of the hyaluronic acid solution is 10 mg / mL; and the volume ratio of the MnO2@PtAuRu solution to the hyaluronic acid solution is (1-1.1):(1-1.1).

7. The tumor-targeting nanozyme MnO2@PtAuRu@HA prepared by the preparation method according to any one of claims 1-6.

8. The application of the tumor-targeting nanozyme MnO2@PtAuRu@HA as described in claim 7 in the preparation of antitumor drugs, characterized in that, The tumors include colorectal cancer.

9. An antitumor drug, characterized in that, Includes the tumor-targeting nanozyme MnO2@PtAuRu@HA as described in claim 7.

Images