Preparation method of cascade catalytic nano reaction system

Abstract

The application discloses a preparation method of a cascade catalytic nanoreaction system. The steps are as follows: 1) mixing Cu(NO3)2 anhydrous ethanol solution and potassium chloride, stirring overnight, then adding 2-methyl imidazole dissolved in ethanol, drying to form a precipitate, annealing under an inert atmosphere, etching with sulfuric acid to obtain CuNC single-atom nanoscale enzyme; 2) dispersing the nanoscale enzyme in ultrapure water by ultrasonic crushing, adding glucose oxidase, stirring and centrifuging to obtain nanoscale enzyme CuNC- GOx loaded with glucose oxidase; 3) mixing CuNC- GOx dissolved in ultrapure water with DSPE- PEG- NH2 to obtain the cascade catalytic nanoreaction system. The obtained cascade catalytic nanoreaction system is used for in-situ catalysis of endogenous biomolecules H2O2 in a tumor lesion area, ensures continuous generation of cytotoxic ROS, has a photothermal effect, has a high-efficiency synergistic anti-tumor effect of chemical kinetics and photothermal multi-strategy, and realizes high-efficiency anti-tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterials and tumor treatment, and in particular to a preparation method of a cascade nanoscale enzyme for synergistic action of cascade catalytic therapy and photothermal therapy. BACKGROUND

[0002] Tumor therapy induced by reactive oxygen species (ROS) has attracted great research interest due to its high efficiency and non-invasiveness. Excessive production of ROS (hydroxyl radical ·OH) can cause a series of cell injuries such as cytoskeleton contraction and deoxyribonucleic acid (DNA) chain breakage, and ultimately leads to tumor cell apoptosis. Enzymes can generate reactive oxygen species in vivo to achieve tumor therapy, and can also activate the body's immune capacity and enhance the function of the immune system. Enzyme therapy for generating ROS for tumor therapy has attracted the attention of researchers. However, natural enzymes have disadvantages such as high cost, poor stability, low yield, difficulty in preservation, and short half-life, which seriously limit the practical application of enzyme therapy.

[0003] Nanoscale enzymes, as a class of nanomaterials containing enzymatic properties, have formed a new field. Nanoscale enzymes have the advantages of economy, stability, and ease of mass production. More importantly, nanoscale enzymes combine the catalytic activity of enzymes and the unique physicochemical properties of nanomaterials, making them a substitute for natural enzymes and widely used in the biomedical field. Nanoscale enzymes can use the weak acidity in the tumor microenvironment as a reaction condition, and use intratumoral hydrogen peroxide (H2O2) as a reaction raw material to trigger Fenton / Fenton-like reactions, catalyze intratumoral H2O2 into toxic hydroxyl radicals (·OH), thereby inducing cell apoptosis and achieving chemical kinetic therapy of tumors. However, how to further improve the efficacy of chemical kinetic therapy still faces great challenges. Among them, the catalytic efficiency of the catalyst is an important factor that directly affects its therapeutic effect. Single-atom nanoscale enzymes refer to single-atom catalysts with enzymatic catalytic activity, in which metal atoms are monodispersed on the carrier as active sites, maximizing the utilization of atomic catalysis, so that they can trigger abnormally low or high levels of intracellular biomarkers to induce cancer-specific cytotoxicity, which can greatly improve the efficiency of chemical kinetic therapy. In addition, the carbon-loaded structure obtained based on high-temperature carbonization has strong near-infrared absorption, and this feature can be used for photothermal therapy of tumors to further enhance the therapeutic effect.

[0004] The research finds that the low content of endogenous H2O2 in the tumor microenvironment greatly limits the efficiency of Fenton / Fenton-like reaction, thereby reducing the effect of chemical kinetics therapy. An effective way to solve this problem is to increase the content of H2O2 in the tumor. Studies have confirmed that the tumor contains a large amount of glucose to support the rapid growth of the tumor. Based on this fact, on the one hand, glucose oxidase (GOx) can be used to catalyze the decomposition of glucose to produce a large amount of H2O2, providing enough reaction substrate for Fenton / Fenton-like reaction, thereby increasing the production of ROS and accelerating the catalytic therapy process; on the other hand, GOx can be used to deplete a large amount of glucose in the tumor microenvironment to achieve better tumor inhibition effect. Based on the above research results or facts, it is necessary to introduce nano-enzyme and GOx into the tumor tissue at the same time. Some scholars propose to use Fe3O4 nano-enzyme and GOx to construct a cascade reaction system. Some studies report that Fe3O4 nano-enzyme and GOx or gold nano-enzyme particles with GOx activity are assembled into a medium material to construct a double-enzyme triggered cascade reaction platform; after targeting the tumor microenvironment, GOx catalyzes the decomposition of excess glucose in the tumor tissue into excess H2O2, which is further degraded by Fe3O4 nano-enzyme into a large amount of ROS for tumor therapy. However, the encapsulation amount of GOx in this double-enzyme cascade reaction platform is limited, which makes it difficult to achieve the desired catalytic effect; and the efficiency of Fe3O4 nano-enzyme in triggering Fenton / Fenton-like reaction is low, which cannot fully release ROS to achieve more efficient tumor therapy. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a cascade catalytic nano-reaction system, which is used for in-situ catalysis of endogenous biomolecule H2O2 in tumor lesion area, ensuring continuous production of cytotoxic ROS, and having photothermal effect, chemical kinetics and photothermal multi-strategy synergistic anti-tumor effect, realizing efficient anti-tumor therapy.

[0006] In order to solve the above technical problems, the technical scheme adopted by the present application is:

[0007] A preparation method of a cascade catalytic nano-reaction system is provided, comprising the following steps:

[0008] 1) Synthesis of single-atom nano-enzyme: mix Cu(NO3)2 ethanol solution with potassium chloride and stir overnight, then add 2-methylimidazole dissolved in ethanol, dry to form a precipitate, anneal the precipitate under an inert atmosphere, and finally etch with sulfuric acid to obtain CuNC single-atom nano-enzyme;

[0009] 2) loading glucose oxidase (GOx): the CuNC monatomic nanoszyme obtained in step 1) is ultrasonically broken and dispersed in ultrapure water, glucose oxidase is added, stirred, and centrifuged to obtain the nanoszyme CuNC-GOx loaded with glucose oxidase;

[0010] 3) modifying the cascade catalytic nanoreaction system: the nanoszyme CuNC-GOx loaded with glucose oxidase obtained in step 2) is dissolved in ultrapure water and mixed with DSPE-PEG-NH2 to perform PEG modification, and a cascade catalytic nanoreaction system is obtained.

[0011] According to the above scheme, in step 1), the drying temperature is 60-80°C.

[0012] According to the above scheme, in step 1), the annealing temperature is 740-760°C, and the time is 1.5-2.5h. Preferably, the heating rate is 2-8°C / min.

[0013] According to the above scheme, in step 1), the mass-volume ratio of Cu(NO3)2 to anhydrous ethanol is 50-150mg:50mL.

[0014] According to the above scheme, in step 1), the mass ratio of Cu(NO3)2 to potassium chloride is 1:400-500.

[0015] According to the above scheme, in step 1), the mass ratio of Cu(NO3)2 to 2-methylimidazole is 1:1-1.3.

[0016] According to the above scheme, in step 1), the concentration of the sulfuric acid solution is 0.8-1.2mol / L.

[0017] According to the above scheme, in step 1), the etching time is 20-28h.

[0018] According to the above scheme, in step 1), the obtained monatomic nanoszyme CuNC has a nanosheet structure and contains abundant Cu lone pair active sites.

[0019] According to the above scheme, in step 1), the loading amount of Cu in the obtained monatomic nanoszyme CuNC is 4.8-5.2wt%.

[0020] According to the above scheme, in step 2), the activity of glucose oxidase is 100±5U / mg.

[0021] According to the above scheme, in step 2), the mass ratio of the CuNC monatomic nanoszyme to glucose oxidase is 1:2-3.

[0022] According to the above scheme, in the step 2), the CuNC single-atom nanoscale enzyme is dispersed in ultrapure water by ultrasonic crushing to obtain a CuNC single-atom nanoscale enzyme solution of 0.9-1.1 mg / mL.

[0023] According to the above scheme, in the step 2), the centrifugal speed is 13000-15000 rpm.

[0024] According to the above scheme, in the step 2), the glucose oxidase loading capacity of the obtained nanoscale enzyme loaded with glucose oxidase is 56-60%.

[0025] According to the above scheme, in the step 3), the mass ratio of CuNC single-atom nanoscale enzyme to DSPE-PEG-NH2 is 4:10-15.

[0026] According to the above scheme, in the step 3), the centrifugal speed is 13000-15000 rpm.

[0027] The application provides an application of the obtained cascade catalytic nanoreaction system prepared by the preparation method in preparation of an antitumor drug.

[0028] The application has the following beneficial effects:

[0029] 1. The application provides a preparation method of a cascade catalytic nanoreaction system, which is prepared by embedding Cu lone electron through a salt template method to obtain CuNC single-atom nanoscale enzyme, which has an ultrathin sheet structure, high Cu loading capacity, rich Cu lone electron active sites, provides high-density catalytic active sites and loading sites, and has a photothermal effect, promotes chemical kinetic therapy, ensures solid tumor site ablation, and greatly improves the catalytic environment; then a large amount of natural glucose oxidase is loaded on the CuNC single-atom nanoscale enzyme sheet structure, which can effectively deplete glucose in the tumor microenvironment, reduce the pH, and provide sufficient H2O2 for the next step of catalysis, greatly promoting the catalytic therapy efficiency; finally, DSPE-PEG-NH2 is further encapsulated, so that the CuNC single-atom nanoscale enzyme and the glucose oxidase are fixed in the structure and are not easy to leak, and the water solubility is improved, so that the system can smoothly reach the tumor site to play a role; the obtained cascade catalytic nanoreaction system of the application has composite catalytic activity by loading GOx on the CuNC single-atom nanoscale enzyme, has a photothermal effect, is suitable for triggering a cascade catalytic reaction in situ in a tumor lesion area, realizes efficient synergistic antitumor effect of chemical kinetics and photothermal multi-strategy, effectively avoids the problem of poor treatment effect due to resistance of malignant tumors to cell apoptosis, realizes efficient antitumor therapy, and has important application prospect.

[0030] 2. The preparation method has simple process, short production cycle, cheap and readily available raw materials, saves production cost, does not use toxic raw materials or volatile chemical reagents in the production process, is safe and environmentally friendly, and is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Transmission electron microscopy image of CuNC single-atom nanoszyme prepared in Example 1.

[0032] Figure 2 Spherical aberration-corrected transmission electron microscopy image of CuNC single-atom nanoszyme prepared in Example 1.

[0033] Figure 3 Fourier-transformed X-ray fine structure spectrum of CuNC and copper foil prepared in Example 1.

[0034] Figure 4 X-ray absorption near-edge spectrum of CuNC single-atom nanoszyme and copper foil prepared in Example 1.

[0035] Figure 5 Infrared spectrum of CuNC single-atom nanoszyme, CGP nanoreaction system, and glucose oxidase prepared in Example 1.

[0036] Figure 6 GOx standard curve measured by BCA kit, used for quantifying glucose oxidase loaded on the CGP nanosystem prepared in Example 1.

[0037] Figure 7 UV-visible absorption spectrum of POD activity test of CuNC single-atom nanoszyme prepared in Example 1.

[0038] Figure 8 UV-visible absorption spectrum of POD activity test of CuNC single-atom nanoszyme prepared in Example 1 under different material concentrations.

[0039] Figure 9 UV-visible absorption spectrum of CGP nanosystem cascade catalytic reaction prepared in Example 1 under different concentrations of glucose.

[0040] Figure 10 Temperature change of photothermal effect of CuNC single-atom nanoszyme prepared in Example 1 under different material concentrations.

[0041] Figure 11 Temperature change of photothermal effect of CuNC single-atom nanoszyme prepared in Example 1 under different irradiation powers.

[0042] Figure 12 Live and dead cell fluorescence images of CGP nanosystem prepared in Example 1 after different treatments under acidic conditions.

[0043] Figure 13 Figure 4 is a plot of the change in tumor volume for mice.

[0044] Figure 14 Figure 5 is a plot of the change in body weight for mice.

[0045] Figure 15 Figure 6 is a histological analysis of hematoxylin-eosin (H&E) stained tissue sections, Ki-67 antigen and TUNEL immunostaining. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0047] Example 1

[0048] A preparation method of a cascade catalytic nanoreaction system is provided, comprising the following steps:

[0049] First step, synthesis of CuNC single-atom nanoszyme

[0050] 0.1 g of Cu(NO3)2 was dissolved in 50 mL of anhydrous ethanol at room temperature, and 50 g of KCl was added as a template and stirred overnight. Then, the obtained solution was added to 50 mL of anhydrous ethanol containing 0.1 g of 2-methylimidazole, and the mixture was dried at 60°C to obtain a blue powder. The powder was annealed at 750°C for 2 h under a nitrogen atmosphere, with a heating rate of 5°C / min, and finally the sample was etched with a 1M sulfuric acid solution for 24 h to obtain the single-atom nanoszyme CuNC.

[0051] Second step, loading of glucose oxidase (GOx)

[0052] The CuNC single-atom nanoszyme was ultrasonically broken for 20 min at 80% power to obtain a 4 mL solution of 1 mg / mL dispersed in ultrapure water (resistivity of 18.2 MΩ·cm), and 10 mg of glucose oxidase (activity of 100 U / mg) was added to the solution and stirred overnight, and then centrifuged at a speed of 15000 rpm to obtain the nanoszyme CuNC-GOx loaded with glucose oxidase.

[0053] Third step, modification of the cascade catalytic nanoreaction system

[0054] The glucose oxidase-loaded nanoscale enzyme precipitate CuNC-GOx was resuspended in ultrapure water to obtain 4 mL of a 1 mg / mL CuNC-GOx solution, then 15 mg of DSPE-PEG-NH2 was added and stirred overnight. The next day, centrifugation was performed at a speed of 15000 rpm, and the black precipitate was washed with water. The precipitate was resuspended to 4 mL to obtain a 1 mg / mL solution in terms of CuNC, that is, the CGP nanoreaction system capable of cascade catalytic reaction was obtained by further modification with PEG.

[0055] As shown in the transmission electron microscopy image, the CuNC monatomic nanoscale enzyme can be observed to have an ultrathin nanosheet structure. Figure 1

[0056] As shown in the spherical aberration-corrected transmission electron microscopy image, a large number of bright spots can be observed, indicating the presence of a large number of isolated monatomic Cu sites. The present application uses methyl imidazole as a precursor, and by optimizing the process, the copper loading is greatly improved. The copper content measured by ICP can reach 5.0 wt%. Figure 2

[0057] As shown in the spherical aberration-corrected transmission electron microscopy image, a large number of bright spots can be observed, indicating the presence of a large number of isolated monatomic Cu sites. The present application uses methyl imidazole as a precursor, and by optimizing the process, the copper loading is greatly improved. The copper content measured by ICP can reach 5.0 wt%. Figure 3

[0058] As shown in the spherical aberration-corrected transmission electron microscopy image, a large number of bright spots can be observed, indicating the presence of a large number of isolated monatomic Cu sites. The present application uses methyl imidazole as a precursor, and by optimizing the process, the copper loading is greatly improved. The copper content measured by ICP can reach 5.0 wt%. Figure 4 0 2+

[0059] As shown in the spherical aberration-corrected transmission electron microscopy image, a large number of bright spots can be observed, indicating the presence of a large number of isolated monatomic Cu sites. The present application uses methyl imidazole as a precursor, and by optimizing the process, the copper loading is greatly improved. The copper content measured by ICP can reach 5.0 wt%. Figure 5 -1 -1 As shown in the spherical aberration-corrected transmission electron microscopy image, a large number of bright spots can be observed, indicating the presence of a large number of isolated monatomic Cu sites. The present application uses methyl imidazole as a precursor, and by optimizing the process, the copper loading is greatly improved. The copper content measured by ICP can reach 5.0 wt%.

[0060] As shown in the spherical aberration-corrected transmission electron microscopy image, a large number of bright spots can be observed, indicating the presence of a large number of isolated monatomic Cu sites. The present application uses methyl imidazole as a precursor, and by optimizing the process, the copper loading is greatly improved. The copper content measured by ICP can reach 5.0 wt%. Figure 6

[0061] ​​​​​​​​​A cascade catalytic nanoreaction system prepared by the present embodiment is used to test enzyme activity and in vitro and in vivo anti-tumor efficacy (other embodiments have the same test effect as the present embodiment), and the method is as follows:

[0062] (1) POD-like activity test

[0063] POD-like activity of the embodiment 1 is determined by colorimetry in 0.1M acetic acid-sodium acetate (HAc-NaAc) buffer solution (pH = 4.0) with 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate. In 0.20 mL of HAc-NaAc buffer solution, nanocatalyst solution (10 μL, 100 μg / mL), hydrogen peroxide (H2O2 aqueous solution, 100 μL, 1 mM) and TMB solution (50 μL, 10 mM) are sequentially added and mixed thoroughly. The mixed solution is incubated at 37°C in the dark for 1 minute. The final concentrations of nanocatalyst, hydrogen peroxide and TMB are fixed at 100 μg / mL, 100 mM and 1 mM, respectively. The ultraviolet-visible absorption spectrum of the test solution is recorded by a multifunctional enzyme marker.

[0064] The results are shown in Figure 7 , and TMB molecules are oxidized by ·OH, with a maximum absorbance of 652 nm. To further explore the relationship between concentration and catalytic efficiency, the results are shown in Figure 8 , and the absorbance value increases with the increase of H2O2 concentration in the presence of CuNC single-atom nanocatalyst. The above tests prove that the embodiment 1 exhibits significant POD-like activity.

[0065] (2) Cascade catalytic activity test

[0066] Using the characteristics of glucose oxidase that can catalyze glucose to glucose acid and H2O2, a cascade catalytic nanoreaction system is added to a glucose solution of different concentrations, and then TMB is used as a color developing agent. It is observed that the rate of TMB conversion to blue oxTMB changes with the change of glucose concentration. The POD activity is also enhanced, further indicating that GOx still maintains excellent catalytic activity in the system.

[0067] The results are shown in Figure 9 , and the absorbance value of the solution at 652 nm gradually increases with the increase of glucose concentration, showing significant POD-GOx-like activity.

[0068] (3) Photothermal performance test

[0069] The temperature change of CuNC single-atom nanocatalyst under near-infrared irradiation is determined by a thermometer.

[0070] CuNC monatomic nanoszyme was dispersed into different concentrations after sufficient ultrasonic disruption. 1 mL of the dispersion was placed in an ep tube and irradiated by a near-infrared irradiation instrument at 1.5 W / cm 2 The temperature change of the solution within ten minutes was obtained under the irradiation of the near-infrared irradiation instrument at different powers Figure 10 As the concentration of the dispersion increased, the rate of temperature rise of the solution also increased, indicating that the photothermal performance of the material was dependent on the concentration of the material. In order to further explore the effect of the power of the near-infrared irradiation instrument on the photothermal efficiency of the CuNC monatomic nanoszyme, the temperature change curve of the dispersion at 100 ug / mL under different powers was obtained, as shown in Figure 11 As the power increased, the rate of temperature rise of the solution also increased, indicating that the photothermal performance of the material was dependent on the power of the near-infrared irradiation instrument.

[0071] (4) In vitro efficacy test

[0072] The state of the cells was observed and determined by using a Calcein-AM / PI live cell / dead cell double staining kit.

[0073] After the 4T1 cells were digested with 0.25% trypsin-EDTA, the cells were collected and washed with 1x Assay Buffer, centrifuged at 450g for 5 min, twice, to remove residual esterase. The cell pellet obtained by centrifugation was resuspended with 1x Assay Buffer, and the cell suspension after resuspension was counted to have a cell amount of 1x10 5～6 6 / ml. 1-2 ul of Calcein-AM (stock solution) was added to each 1 ml of cell amount, and the mixture was mixed by blowing, incubated at 37°C in the dark for 20-25 min; 3-5 ul of PI stock solution provided in the kit was added to the above stained cells, and the cells were stained at room temperature in the dark for 5 min; the cells after incubation with fluorescence were centrifuged at 450g for 5 min to remove the staining solution; the cells were washed with 1x PBS at 450g for 5 min, and then resuspended with 1x PBS after centrifugation; 3-5 ul was dropped on a clean glass slide, and a clean cover glass was used to press the slide, and then fluorescence microscopy was performed in time; under the fluorescence microscope, 490±10 nm excitation filter was used to detect live cells (yellow-green fluorescence) and dead cells (red fluorescence) at the same time. In addition, only dead cells can be observed using a 545 nm emission filter. Direct detection can also be performed using appropriate filters under a fluorescence microplate reader. It is recommended to avoid light throughout the process of fluorescence staining of cells. The results are shown in Figure 12As shown, by observing that the cells incubated with CuNC monatomic nanoszyme were slightly affected compared to untreated cells; but when the cells were incubated with the cascade catalytic nanoreaction system CGP, nearly half of the cells were apoptotic; while further adding infrared irradiation at the same time of incubating with the cascade catalytic nanoreaction system CGP, the tumor cells were almost completely apoptotic. It shows that this cascade catalytic nanoreaction system prepared in this way has good anti-tumor effect, and the anti-tumor efficiency is obviously improved after combined with near-infrared irradiation.

[0074] (4) In vivo efficacy test

[0075] Female albino laboratory mice (BALB / c) nude mice (6 weeks old, body weight about 20 g) were selected as animal models. The animal experiments in this study were strictly in accordance with the approved protocol of the Ethics Committee of Wuhan University of Science and Technology Medical College (Approval No: 2024136). The right back was subcutaneously injected with mouse breast cancer cells (4T1 cells (8 x 10 5 ))· The 4T1 tumor-bearing mice were randomly divided into 3 groups (4 in each group): control group and Example 1 group and Example 1 combined with near-infrared group. Each group was given 10 mg / Kg, once, and light was added after 24 h interval. During the treatment, the body weight and tumor size were measured every other day and recorded.

[0076] The results are shown in Figure 13 , which are in sharp contrast to the control group. After injecting the synthetic nanoszyme, the tumor growth was effectively inhibited. Among them, the inhibition rate of Example 1 was higher. Combined with the activity test of the nanoszyme, we can conclude that the excellent therapeutic effect of Example 1 is due to the synergistic effect between glucose depletion and cascade catalysis. It is worth mentioning that the tumor inhibition rate in the Example 1 combined with near-infrared group was further improved, which can be attributed to the fact that near-infrared improves a certain catalytic efficiency and plays a certain role in ablation of the tumor. As Figure 14 shown, the body weight of mice in each group remained basically unchanged within 18 days, which further indicates that the toxicity and side effects of these synthetic nanoszymes on mice can be ignored.

[0077] Next, the tumor tissue was subjected to histopathological analysis, and the degree of damage of the nanoszyme to the tumor tissue was evaluated by hematoxylin-eosin (H&E) staining; the tumor cell proliferation was detected by Ki-67 antigen; and the tumor cell apoptosis was detected by TUNEL antigen.

[0078] The results are shown in Figure 15 , which are in sharp contrast to the control group. After injecting the synthetic nanoszyme, the tumor growth was effectively inhibited. Among them, the inhibition rate of Example 1 was higher. Combined with the activity test of the nanoszyme, we can conclude that the excellent therapeutic effect of Example 1 is due to the synergistic effect between glucose depletion and cascade catalysis. It is worth mentioning that the tumor inhibition rate in the Example 1 combined with near-infrared group was further improved, which can be attributed to the fact that near-infrared improves a certain catalytic efficiency and plays a certain role in ablation of the tumor. As Figure 15The leftmost column) is more pronounced after Example 1 combined with near-infrared treatment. As shown by immunohistochemical staining of the antigen Ki-67, after Example 1 combined with near-infrared treatment, the expression of Ki-67 was significantly reduced, and the tumor proliferation trend was weakened Figure 15 The middle column), only after Example 1 treatment still maintained a certain expression level. Immunohistochemical analysis of TUNEL revealed the apoptosis of cells, and the expression level of TUNEL in the tumor of the mouse treated with Example 1 combined with near-infrared was significantly increased, indicating that apoptosis occurred Figure 15 The rightmost column) is more pronounced after Example 1 combined with near-infrared treatment. In summary, the ROS accumulation level in the tumor of the mouse treated with Example 1 combined with near-infrared is high, indicating that the apoptosis is strong. The material of the application can be successfully enriched in the tumor site, and is specifically adapted to the tumor microenvironment to trigger efficient in situ treatment, and further enhances the anti-tumor performance under the assistance of near-infrared.

[0079] The above examples and comparative examples illustrate that the preparation method of the cascade catalytic nanoreaction system is to continuously try the amount of raw materials, so as to make the obtained nanoscale enzyme show high catalytic efficiency in the smallest range of raw material amount; strict control of each process link, after many practices, a cascade catalytic nanoreaction system which can be used as a cascade catalytic nanoreaction system for synergistic photothermal therapy in the field of tumor treatment is finally developed.

Claims

1. A method for preparing a cascade catalytic nanoreaction system, characterized in that, The method comprises the following steps: 1) mixing Cu(NO3)2 anhydrous ethanol solution with potassium chloride and stirring overnight, then adding 2-methylimidazole dissolved in ethanol, drying to form a precipitate, annealing the precipitate under an inert atmosphere, and finally etching with sulfuric acid to obtain CuNC monatomic nanoscale enzyme, wherein the loading amount of Cu is 4.8-5.2 wt%; 2) ultrasonic crushing of the CuNC monatomic nanoscale enzyme obtained in step 1) and dispersing in ultrapure water, adding glucose oxidase, stirring, and centrifuging to obtain nanoscale enzyme CuNC-GOx loaded with glucose oxidase; wherein the mass ratio of CuNC monatomic nanoscale enzyme to glucose oxidase is 1:2-3; and the glucose oxidase loading amount is 56-60%; 3) mixing the CuNC-GOx loaded with glucose oxidase obtained in step 2) with DSPE-PEG-NH2 after dissolving in ultrapure water to perform PEG modification, to obtain a cascade catalytic nanoreaction system; wherein the mass ratio of CuNC monatomic nanoscale enzyme to DSPE-PEG-NH2 is 4:10-15.

2. The production method according to claim 1, characterized by, In the step 1), the annealing temperature is 740-760℃, and the time is 1.5-2.5 h.

3. The production method according to claim 1, characterized by, In the step 1), the mass ratio of Cu(NO3)2 to potassium chloride is 1:400-500; and the mass ratio of Cu(NO3)2 to 2-methylimidazole is 1:1-1.

3.

4. The method of claim 1, wherein, In the step 1), the concentration of the sulfuric acid solution is 0.8-1.2 mol / L.

5. The preparation method according to claim 1, characterized in that, In the step 1), the etching time is 20-28 h.

6. The method of claim 1, wherein, In the steps 2) and 3), the centrifugal speed is 13000-15000 rpm.

7. Use of the cascade catalytic nanoreaction system prepared by the preparation method of claim 1 in the preparation of an anti-4T1 cell drug.

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