Composite nano material as well as preparation method and application thereof

Abstract

The invention discloses a composite nano material as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The composite nano material comprises a metal organic framework, glucose oxidase (GOx) and catalase (CAT) modified by a polymer, wherein the glucose oxidase (GOx) and the catalase (CAT) are positioned in the metal organic framework. On one hand, the CAT covalently modified by the polymer can efficiently catalyze hydrogen peroxide (H2O2) to be decomposed into O2 and H2O, and on the other hand, the CAT covalently modified by the polymer can efficiently catalyze H2O2 to be converted into high-toxicity. OH. On one hand, the composite nanomaterial can catalyze glucose and O2 to generate gluconic acid and H2O2 through GOx, on the other hand, H2O2 is decomposed into water and O2 by CAT to further promote glucose consumption, on the other hand, H2O2 is converted into high-toxicity. OH by CAT, and the composite nanomaterial has a remarkable inhibition effect on tumor growth and has a wide application prospect in tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a composite nanomaterial and a preparation method and application thereof. BACKGROUND

[0002] Tumor is one of the global public health problems, which seriously affects the survival rate and quality of life of patients. Tumor cells produce adenosine-5'-triphosphate through extremely low-efficiency aerobic glycolysis, resulting in an abnormal increase in the demand for glucose. Chemical dynamic therapy (CDT) as a new tumor treatment method utilizes Fenton or Fenton-like reaction mediated by metal ions (such as Fe 2+ , Cu 2+ , Mn 2+ , etc.) to catalyze the generation of hydrogen peroxide (H2O2) in the tumor region into highly toxic hydroxyl radicals (·OH), thereby achieving efficient killing of tumor cells. CDT has pH and H2O2 dependence, and can achieve higher tumor specificity, while having less toxic side effects on normal tissues. However, the existing CDT therapy has limitations: on the one hand, the activity and substrate specificity of metal-based catalysts are low, which limits the treatment effect; on the other hand, the intake of exogenous metal substances may cause adverse body side reactions. To overcome these challenges, researchers introduced glucose oxidase (GOx), which accelerated the glycolysis of glucose at the tumor site, in situ generated a large amount of gluconic acid and H2O2, and provided a more ideal microenvironment for CDT. In addition, the glucose consumption caused by GOx also achieved starvation therapy (ST), further enhancing the treatment effect. This combined therapy of "ST+CDT" not only improves the specificity and effectiveness of treatment, but also reduces the damage to normal tissues, providing a new idea for tumor treatment. However, the CDT or ST+CDT therapy still has the problem of intake of exogenous metal substances which may cause adverse side reactions in the body, which needs to be solved urgently.

[0003] Enzyme is a biological catalyst with high catalytic selectivity, which is widely used due to its high catalytic efficiency and mild reaction conditions. Horseradish peroxidase (HRP) can efficiently convert H2O2 to ·OH. However, the endogenous O2 and H2O2 in tumors are limited, and how to maximize their therapeutic effect is a difficult problem. Studies have found that the introduction of catalase (CAT) in the GOx-HRP "ST+CDT" system can catalyze the decomposition of H2O2 into O2 to alleviate tumor hypoxia and enhance the synergistic therapeutic effect. After the nanocatalyst is taken up by the tumor, GOx catalyzes the generation of gluconic acid and H2O2 from glucose and O2, and H2O2 is decomposed into water and O2 by CAT to promote glucose consumption (related to ST), and is converted to ·OH by HRP (related to CDT). There are two major problems in the current "ST+CDT" synergistic therapy: first, the types of enzyme catalysts are numerous, the cost is high, and the separation and purification are further increased the application cost; second, there is competition between the ST and CDT reaction pathways, and how to rationally design the H2O2 catalytic conversion pathway to optimize the synergistic therapeutic effect needs further research.

[0004] Applicant's previous studies have found that natural enzyme catalase (CAT) can only catalyze the decomposition of H2O2 into H2O and O2 in neutral or alkaline solution, i.e. it exhibits intrinsic catalytic properties, while in acidic conditions it can not only decompose H2O2 into O2 and H2O, but also convert it to ·OH, i.e. it exhibits HRP-like activity, and the stronger the acidity, the higher the HRP-like activity. Molecular dynamics simulation can analyze the interaction between protein and substrate at the atomic level, thereby analyzing at the micro level how to affect the conformation, catalytic activity and stability of the protein, and elucidating the exact interaction between the substrate and the protein, and the crucial influence of these interactions on the catalytic activity and stability of the enzyme. Based on this, the present application first analyzes the activity mechanism of CAT in different microenvironments through molecular dynamics simulation, and in view of this, a modified CAT with excellent intrinsic catalytic activity and HRP-like activity is rationally designed and prepared, and is used in combination with GOx for synergistic anti-tumor research. SUMMARY

[0005] The technical problem solved by the present application is to provide a composite nanomaterial, a preparation method and application thereof, which addresses the shortcomings of the prior art. The composite nanomaterial is a tumor synergistic therapy system based on molecular dynamics and enzyme microenvironment engineering simulation, which aims to solve the problem of the intake of exogenous metal substances in existing CDT or ST+CDT therapy, which may cause adverse side effects in the body.

[0006] To solve the above technical problems, the present application discloses a composite nanomaterial, a preparation method and application thereof. The specific technical solutions are as follows:

[0007] In a first aspect, the present application provides a preparation method of a composite nanomaterial, comprising the following steps:

[0008] (1) Preparation of a polymethacrylic acid modified catalase; that is, the catalase is obtained by moderate modification of a polymer containing a carboxyl group, which can efficiently catalyze the decomposition of hydrogen peroxide (H2O2) into O2 and H2O, and can also efficiently catalyze the conversion of the H2O2 into highly toxic ·OH.

[0009] (2) Mixing of zinc ions, 2-methylimidazole, glucose oxidase (GOx) and the polymethacrylic acid modified catalase prepared in step (1) to obtain the composite nanomaterial through a first reaction.

[0010] In step (1), the weight average molecular weight of the polymethacrylic acid is 4000-6000 Da, preferably 5000 Da.

[0011] In step (1), the polymethacrylic acid modified catalase is prepared by mixing polymethacrylic acid (PMAA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in a buffer solution, adding catalase (CAT), and obtaining the polymethacrylic acid modified catalase through a second reaction. The buffer solution is a PBS buffer solution with a concentration of 10-30 mM and a pH of 5-7; the molar ratio of the amino groups contained in the polymethacrylic acid and the catalase is 1-30:1, preferably 4:1; the mass ratio of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to the catalase is 5-10:1; the second reaction is carried out at a temperature of 18-30°C for a time of 3-10 h, preferably at 20°C for 5 h.

[0012] The polymethacrylic acid modified catalase is named CAT-n, where n is the molar ratio of the amino groups contained in the PMAA and the CAT. When the molar ratio is 4:1, it is CAT-4, which has both high overall enzyme activity and can efficiently produce ·OH and O2 at the same time. The CAT-4 is renamed as CATc.

[0013] Preferably, it is found by the present application that the catalase (CAT) has the best performance under simulated conditions at a pH of 5, at which the CAT has a variety of conformations, a flexible active center, multiple low free energy regions, key amino acid residues with a moderate distance, and a relatively stable structure, which ensures that the CAT can effectively perform functions, that is, the CAT has high enzyme activity in a moderate acidic microenvironment.

[0014] Further preferably, the CAT-n is prepared by the following method: a certain amount of PMAA is added into 18 mL of 20 mM pH 6 buffer, 40 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is added, the reaction is stirred for 15 min, 5 mg of CAT is added, the reaction is carried out for 2-24 h (20℃, 120 rpm), after the reaction is completed, ultrafiltration is carried out by using an ultrafiltration tube (with a molecular weight cut-off of 100 kDa), and the CAT-n is collected.

[0015] In step (2), the molar ratio of zinc ions to 2-methylimidazole is 1:3-5, and the amount of glucose oxidase is 1-5 g per mole of zinc ions; the mass ratio of the glucose oxidase to the polymethacrylic acid modified catalase is 1-16:1. In the first reaction, the reaction temperature is 15-30℃, and the reaction time is 20-50 min. Preferably, the molar ratio of zinc ions to 2-methylimidazole is 1:4, and the amount of glucose oxidase is 3 g per mole of zinc ions; the mass ratio of the glucose oxidase to the polymethacrylic acid modified catalase is 1:1. Further preferably, step (2) is carried out by the following method: after the zinc ions and 2-methylimidazole are mixed, the glucose oxidase and the polymethacrylic acid modified catalase are added to carry out the first reaction to obtain the composite nanomaterial. More preferably, in step (2), the composite nanomaterial is prepared by the following method: 1 mL of Zn(NO3)2·6H2O aqueous solution (20 mM) is added to 1 mL of 2-methylimidazole aqueous solution (80 mM), stirred uniformly, then 60 μg of GOx and 60 μg of CATc are added, and the reaction is stirred at room temperature for 30 min, followed by centrifugation (12000 rpm, 5 min) with water for 3 times, and the prepared composite nanomaterial (GOx+CATc)@aZIF-8 is collected.

[0016] In a second aspect, the present application provides a composite nanomaterial prepared by the preparation method of the first aspect. The composite nanomaterial comprises: a metal organic framework and glucose oxidase (GOx) and catalase (CAT) located inside the metal organic framework.

[0017] In a third aspect, the present application provides a use of the composite nanomaterial of the second aspect in the preparation of a tumor treatment drug. The composite nanomaterial is a tumor synergistic treatment system, and after enrichment in the tumor site, the GOx and the CATc are released, the GOx catalyzes the generation of gluconic acid and H2O2 from glucose and O2, the H2O2 is decomposed into water and O2 by the CATc to further promote the consumption of glucose, and on the other hand, the H2O2 is converted into highly toxic ·OH by the CATc, and the composite nanomaterial has a significant inhibitory effect on tumor growth.

[0018] The tumor includes esophageal squamous cell carcinoma.

[0019] Beneficial effects:

[0020] 1) The preparation process of the present application is mild and simple;

[0021] 2) The use of natural enzymes CAT and GOx instead of exogenous metal nano-enzymes reduces the adverse side effects of the body;

[0022] 3) The presence of the intrinsic catalytic activity of CAT and the HRP-like catalytic activity regulated by the microenvironment in the present application enables CAT to simultaneously play two roles, reduces the types of enzyme catalysts in the tumor treatment process, and reduces the cost;

[0023] 4) The mechanism analysis and rational design of the activity change of CAT under different microenvironments through molecular dynamics simulation not only help to deeply understand the activity change mechanism of the enzyme, but also guide the optimization design of the enzyme, promote the research progress in related fields, improve the research efficiency and accuracy, and provide support for industrial and medical applications;

[0024] 5) The tumor synergistic treatment system based on enzyme microenvironment engineering prepared by the method of the present application has good biocompatibility and can significantly inhibit tumor growth, and has a broad application prospect in tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0025] The above and / or other aspects of the present application will become apparent from the following detailed description of the application taken in conjunction with the accompanying drawings.

[0026] Figure 1 The ability of CAT to decompose H2O2 under different acidic conditions. From left to right, the three test tubes are pH 5, 7 and 8 buffer solutions.

[0027] Figure 2Figure 1 is a series of molecular dynamics simulation graphs, wherein a is the change of RMSD with simulation time, b is a three-dimensional structure simulation graph of the influence of different pH on H74, N147 and Y357 at simulation time 100 ns, red is pH 7, blue is pH 5, and green is pH 3, c is a free energy landscape graph at pH 7, d is a free energy landscape graph at pH 5, e is a free energy landscape graph at pH 3, f is the change of the center-of-mass distance between H74 and N147 residues with simulation time, g is the change of the center-of-mass distance between H74 and Y357 residues with simulation time, and h is the change of the center-of-mass distance between N147 and Y357 residues with simulation time.

[0028] Figure 3 Figure 2 is a series of protein structure simulation analysis graphs, wherein a is the change of the number of hydrogen bonds with simulation time, b is the change of the radius of gyration Rg with simulation time, and c is the change of the solvent accessible surface area SASA with simulation time.

[0029] Figure 4 Figure 3 is a series of CAT-n H2O2 consumption reaction graphs, wherein a is the total reaction rate of CAT-n H2O2 consumption b is the ratio of the reaction rates of CAT-n H2O2 consumption to generate ·OH and O2, respectively (V 652 / V 240 ), n is the molar ratio of the amino groups contained in PMAA and CAT, including CAT-0, CAT-1, CAT-4, CAT-8, CAT-16 and CAT-30.

[0030] Figure 5 Figure 4 is a series of material characterization graphs, wherein a is a scanning electron microscope graph, b is an XRD graph, and c is the potential of each substance.

[0031] Figure 6 Figure 5 is a series of cell KYSE450 survival rate graphs, wherein a is the survival rate of cell KYSE450 incubated with different concentrations of aZIF-8, b is the survival rate of cell KYSE450 incubated with GOx+CAT, (GOx+CAT)@aZIF-8 and (GOx+CATc)@aZIF-8.

[0032] Figure 7 Figure 6 is a series of in vivo dynamic fluorescence imaging after tail vein injection of saline, DiD and DiD@aZIF-8 and ex vivo fluorescence imaging of main organs and tumors 24 h after injection, He is heart, Li is liver, Sp is spleen, Lu is lung, Ki is kidney, and Tu is tumor.

[0033] Figure 8The relevant parameters of tumor-bearing mice after receiving various treatments are shown in a, b, c, d and e. a is a schematic diagram of mouse experiments, b is a tumor growth curve of tumor-bearing mice after receiving various treatments, c is a tumor image of each treatment group at the end of the experiment, d is the corresponding tumor weight, and e is a body weight change curve of each treatment group. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application and are not intended to limit the scope of the application. Any modification or replacement of the method, step or condition of the application without departing from the spirit and essence of the application shall fall within the scope of the application.

[0035] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0036] The catalase CAT used in the following examples was purchased from Beijing Solabio Technology Co., Ltd. with a specification of 1 g / bottle and a purity of 68%. The purchased CAT had a titer of 5000 U / mg. The CAT was a tetramer with a relative molecular mass of about 250 kDa, and the CAT contained 4 subunits, each of which contained an amount of amino groups of 60 (the PDB number of CAT was 4BLC, and the simulation was obtained using PyMOL software).

[0037] The glucose oxidase GOx used in the following examples was purchased from Aldrich with a specification of 0.1 MU / bottle.

[0038] Example 1 Investigation of the ability of catalase to decompose H2O2 under different acidic conditions

[0039] The ability of catalase (CAT) to decompose H2O2 under different acidic conditions was investigated. 1.5 mL of prepared buffer solutions of pH 5 (20 mM acetic acid-sodium acetate), pH 7 (20 mM PBS) and pH 8 (20 mM PBS) were added to three centrifuge tubes, respectively, and 6 μL of H2O2 aqueous solution with a mass concentration of 30%, 4 μL of TMB (25 mg / mL) and 10 μL of CAT (1 mg / mL) were sequentially added to each centrifuge tube, and mixed. After about 2 min, the color change of the solution in the test tube was observed. As shown in FIG. 1, the blue color of the solution gradually became lighter with the increase of pH, and the ability of the solution to produce bubbles gradually became larger with the increase of pH, wherein the blue color represents the ability of CAT to decompose H2O2 to produce ·OH, i.e. the HRP-like activity of CAT, and the bubbles represent the ability of CAT to decompose H2O2 to produce O2, i.e. the intrinsic catalytic activity of CAT. This result shows that the pH of the environment of CAT enzyme has a significant influence on the reaction pathway of the enzyme catalyzing H2O2. Figure 1 ​

[0040] Example 2 Mechanism analysis and rational design by molecular dynamics simulation

[0041] In view of the different intrinsic catalytic activity and HRP-like catalytic activity of CAT under different microenvironment conditions found in Example 1, the mechanism hidden behind it was analyzed by molecular dynamics simulation. Specifically, the activity of CAT in three microenvironment systems of pH 7, pH 5 and pH 3 was simulated.

[0042] From a in FIG. 11, Figure 2 From a in FIG. 11, the root mean square deviation (RMSD) of the system reaches a steady state within 40 ns under different pH conditions, proving that the simulation is complete and the simulation is effective. In addition, the smaller the RMSD value, the more stable the protein structure, from b in FIG. 11. Figure 2 From a in FIG. 11, the RMSD value of CAT is larger and basically similar at pH 7 and pH 3, and the RMSD value of CAT is smaller at pH 5, indicating that a suitable acidic environment can improve the structural stability of the CAT enzyme protein. At pH 7, Figure 2 From c in FIG. 11, the RMSD and radius of gyration (Rg) range of the low free energy region is relatively narrow, at pH 5, Figure 2 From d in FIG. 11, the low free energy region is relatively dispersed, and compared with pH 7, the RMSD and Rg range of values are wider, at pH 3, Figure 2 From e in FIG. 11, the low free energy region shows a relatively continuous distribution trend, and forms an obvious low energy channel in a specific RMSD and Rg range. It can be seen that at pH 5, CAT has suitable conformational diversity, active center flexibility and multiple low free energy regions, which are conducive to promoting substrate binding and providing moderate activation energy.

[0043] According to the literature, H74, N147 and Y357 are key amino acid residues for the catalytic activity of CAT, and the dynamic changes of the center-of-mass distance between the three key amino acid residues with the simulation time under different pH conditions are shown in f-h in FIG. 12. Figure 2 From f-h in FIG. 12, the various curves fluctuate frequently, indicating that the relative positions between the amino acid residues in the system are constantly changing, and are in dynamic conformation adjustment. At a simulation time of 100 ns, the center-of-mass distance between H74, N147 and Y357 residues under different pH conditions is basically consistent, indicating that the local structure of the enzyme is relatively stable at this time. In addition, from b in FIG. 11, Figure 2 From b in FIG. 11, compared with the overall position change of the three key amino acids at 100 ns, the overall position change of the three key amino acids at 100 ns under pH 3 conditions is larger, indicating that the structure of the protein has relatively large changes at pH 3.

[0044] Hydrogen bonds are one of the important factors for stabilizing the secondary structure and tertiary structure of proteins, from g in FIG. 11, Figure 3As can be seen from a in FIG. 6, the number of hydrogen bonds in CAT enzyme protein gradually decreases as the pH value decreases, indicating that the hydrogen bonds inside CAT decrease more at a lower pH (pH 3), which weakens the interaction within the protein molecule and is not conducive to the stability of the enzyme protein. The gyration radius Rg value reflects the volume and shape of the protein molecule, and the larger the value, the more puffy the protein structure is, and the smaller the value, the more compact the protein structure is. As can be seen from b in FIG. 6, the Rg value of CAT is smaller at pH 7, and the Rg values of CAT at pH 5 and pH 3 are larger and similar, but overall, the Rg value at pH 3 is slightly larger than the Rg value at pH 5, indicating that the structure of CAT is more compact at pH 7, and the structure of CAT is looser at pH 3 and pH 5, which is consistent with the change in the number of hydrogen bonds. Figure 3 As can be seen from c in FIG. 6, overall, the SASA value of CAT slightly increases as the pH decreases, which is consistent with the change in Rg. As can be seen from the above, compared with pH 7 and pH 3, CAT has a relatively stable structure with moderate tightness, reasonable hydrophilic and hydrophobic properties at pH 5. Figure 3

[0045] In summary, the centroid distance between the three key amino acid residues of CAT is in a specific range and fluctuates relatively reasonably at pH 5, at which time the moderate distance and conformational flexibility of the protein ensure that the protein molecule is neither too rigid to adapt to substrate binding and structural fine-tuning during the reaction process, nor too flexible to cause structural instability, providing a structural basis for protein activity (both maintaining the stability of the structure and allowing necessary flexibility), so that the protein can effectively perform functions. As can be seen from the above, CAT has higher enzyme activity in a moderate acidic microenvironment.

[0046] Example 3: Experimental verification that CAT has higher enzyme activity in a moderate acidic microenvironment

[0047] ​The enzyme was modified to different degrees using polymethacrylic acid (PMAA, weight average molecular weight 5000 Da) containing carboxyl groups, labeled CAT-n, where n is the molar ratio of PMAA added to the amino group of CAT in the reaction system. A certain amount of PMAA was added to 18 mL of 20 mM pH 6 PBS buffer according to different ratios, followed by 40 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred for 15 min, then 5 mg of CAT was added, and the reaction was continued for 5 h (20℃, 120 rpm). After the reaction, CAT-n was collected by ultrafiltration using an ultrafiltration tube (molecular weight cutoff of 100 kDa). In this example, CAT-0, CAT-1, CAT-4, CAT-8, CAT-16, and CAT-30 were prepared with PMAA additions of 0, 17.5, 70, 140, 280, and 525 mg, respectively. The total H2O2 consumption rate of CAT-n was detected using a microplate reader. and the ratio of the reaction rates of H2O2 consuming to produce ·OH and O2 respectively (V 652 / V 240 The overall reaction rate was determined by the Ti(SO4)2 method.

[0048] Depend on Figure 4 It can be seen that, in CAT-4, although CAT's It has decreased somewhat, but at this point its V 652 / V 240 At its highest level, CAT-4 exhibited both high overall enzyme activity and efficient production of ·OH and O2, consistent with previous simulation results. Therefore, CAT-4 was selected for subsequent experiments and renamed CATc.

[0049] Example 4: Material Preparation and Characterization

[0050] Because zeolite imidazole ester framework-8 (ZIF-8) is simple and rapid to synthesize and easy to functionalize, and because the coordination bonds of ZIF-8 are sensitive to the acidic tumor microenvironment in tumor therapy, facilitating the release of substances loaded within it, ZIF-8 was chosen as the carrier in this invention. Specific method: 1 mL of Zn(NO3)2·6H2O (20 mM) aqueous solution was added to 1 mL of 2-methylimidazole (80 mM) aqueous solution and stirred until homogeneous. Then, 60 μg each of glucose oxidase GOx and CATC were added, and the reaction was stirred at room temperature for 30 min. The mixture was then washed three times by centrifugation (12000 rpm, 5 min) and the prepared material (GOx+CATc)@aZIF-8 was collected.

[0051] As a control, replacing CATc with unmodified CAT of the same quality yielded (GOx+CAT)@aZIF-8;

[0052] aZIF-8 is obtained without the addition of GOx and CATC during the reaction process;

[0053] Adding only CATC without adding GOX during the reaction yields CATC@aZIF-8.

[0054] The prepared materials were characterized by... Figure 5 As can be seen from 'a', aZIF-8, (GOx+CAT)@aZIF-8, and (GOx+CATc)@aZIF-8 are spherical, with average particle sizes of 92.7±13.5 nm, 79.5±6.7 nm, and 103±10.6 nm, respectively. X-ray diffraction (XRD) of each material shows that... Figure 5 In step b), the prepared ZIF-8 has an amorphous structure. The Zeta potential of each substance was measured in aqueous solution, and the results are as follows... Figure 5 As shown in c, the changes in potential between the substances indicate that the protein was successfully encapsulated within aZIF-8. Furthermore, the encapsulation efficiency of GOx, CAT, and CATC was measured to be approximately 85%.

[0055] Example 5: MTT assay for cytotoxicity

[0056] The effects of different drug treatments on the survival of esophageal cancer cells KYSE450 were evaluated using the standard MTT assay. Human esophageal cancer cells KYSE450 were diluted with 1640 medium and stored at 3 × 10⁶ cells per well. 4 The cells were seeded at high density into 96-well plates and incubated for 12 hours. Then, the old culture medium was aspirated, and 1640 medium containing different concentrations of aZIF-8, GOx+CAT, (GOx+CAT)@aZIF-8, and (GOx+CATc)@aZIF-8 were added to each well, and the cells were incubated for another 24 hours. Next, the old culture medium was aspirated, and 100 μL of 1640 medium solution containing 5 mg / mL MTT was added to each well, and the cells were incubated for another 4 hours. Finally, the absorbance of each well at 490 nm was measured using a microplate reader. Using 1640 medium as a blank, the cell viability was calculated using the following formula: Cell viability (%) = (sample OD0) / (sample OD0)0. 490 / Blank OD 490 )×100%.

[0057] Depend on Figure 6 As can be seen from 'a', cell viability is relatively high when the concentration of aZIF-8 is 3.125-25 μg / mL, but decreases sharply when the concentration reaches 50 μg / mL. This phenomenon is likely related to the release of Zn from aZIF-8.2+ Related. By Figure 6 As shown in b, cell viability decreased with increasing concentrations of GOx+CAT, (GOx+CAT)@aZIF-8, and (GOx+CATc)@aZIF-8. All concentrations mentioned are equivalent CATC concentrations. GOx+CAT significantly inhibited cell proliferation at 15 ng / mL (equivalent CATC concentration), with cell viability <50%. Its high cytotoxicity may be related to the spatial separation of GOx and CAT. The H2O2 produced by GOx cannot be consumed by CAT in time, leading to H2O2 accumulation and oxidative stress damage to cells. When GOx and CAT are confined together, i.e., (GOx+CAT)@aZIF-8, they are spatially close. On the one hand, CAT can effectively eliminate the H2O2 produced by GOx, thus weakening its damage to cells; on the other hand, the O2 produced by CAT catalyzing the decomposition of H2O2 can further promote GOx's consumption of glucose. Figure 6 As shown in b, within the range of 1.667-15 ng / mL (equivalent CATC concentration), the interaction between the two processes compared to GOx+CAT resulted in a slightly higher cell survival rate for (GOx+CAT)@aZIF-8. Further increasing the material concentration did not significantly affect cell survival between GOx+CAT and (GOx+CAT)@aZIF-8. At 5 ng / mL (equivalent CATC concentration), the cell survival rate of (GOx+CATc)@aZIF-8 was <40%. Compared to GOx+CAT and (GOx+CAT)@aZIF-8, (GOx+CATc)@aZIF-8 exhibited higher cytotoxicity, which can be attributed to two aspects: firstly, similar to CAT, CATC can consume H2O2 to generate O2, further promoting GOx's consumption of glucose; secondly, CATC can also consume H2O2 to generate highly toxic ·OH.

[0058] Example 6: In vivo experiment

[0059] Four-week-old male BALB / c nude mice were inoculated with KYSE 450 cells (10T) under their right axilla. 6 (Each tumor per individual). When the tumor volume reaches 100mm... 3The mice were randomly divided into 3 groups (n=5) at the time of 80-100mm3. To evaluate the performance of the carrier economically and effectively, the fluorescent dye octadecyl indocyanine green perchlorate DiD was selected instead of the enzyme to prepare free DiD and DiD-loaded aZIF-8 nanoparticles (DiD@aZIF-8), and 100 μL of normal saline, free DiD and DiD@aZIF-8 (DiD dose 100 μg / kg) were injected into the tail vein, respectively. The fluorescence was detected by a small animal living imaging system (Ex=625 nm, Em=680 nm), and the distribution of DiD in the mouse body was determined by isoflurane anesthesia at 0, 3, 6, 12, 24 h after the tail vein injection. To study their distribution in the main organs and tumors, some mice were sacrificed and dissected at 24 h, and the tumors and main organs were obtained to observe the fluorescence intensity of DiD in the ex vivo tissue organs.

[0060] The premise of in vivo anti-tumor study is that the carrier can carry the drug to the tumor site and can accumulate in the tumor, so it is necessary to study the in vivo distribution of the carrier. As shown in Figure 7 Fig. 6, no obvious fluorescence signal was observed in the tumor site throughout the process after the injection of free DiD into the tail vein; after the injection of DiD@aZIF-8 into the tail vein, the fluorescence signal was quickly distributed in the tumor, and the fluorescence signal in the tumor reached a maximum at 12 h after the injection. Therefore, aZIF-8 is beneficial to increase the accumulation of drugs in the tumor site.

[0061] Example 7 In vivo anti-tumor experiment

[0062] Four-week-old male BALB / c nude mice were used, and KYSE 450 cells (10 6 mm3, the mice were randomly divided into 5 groups (n=5): 3

[0063] (I) PBS group; (II) aZIF-8 group; (III) GOx+CAT group; (IV) (GOx+CAT)@aZIF-8 group; (V) (GOx+CATc)@aZIF-8 group.

[0064] From the 0th day, 100 μL was administered through the tail vein, and subsequent administration was performed on the 2nd, 4th, 6th, 8th, 10th and 12th days, and the mice were sacrificed on the 14th day (a) in Figure 8 The amount of administration was 10 mg of material / kg. In addition, the tumor volume was measured with a vernier caliper from the 0th day, and the body weight of the mouse was detected, and the tumor volume was calculated according to the formula V=(a×b 2 ​) / 2, where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). The tumor volume was measured at the same time as the mouse body weight, and the final measurement was performed on day 14.

[0065] The aZIF-8 group, GOx+CAT group, (GOx+CAT)@aZIF-8 group, and (GOx+CATc)@aZIF-8 group exert their effects by enhancing penetration and retention at the tumor site. Figure 8 As shown in b, the PBS and aZIF-8 groups exhibited the fastest tumor growth rates. Mild tumor growth inhibition was observed in the GOx+CAT group, while moderate tumor growth inhibition was observed in the (GOx+CAT)@aZIF-8 group. Mice treated with (GOx+CAT)@aZIF-8 showed the most significant tumor growth inhibition. At the end of the tumor growth monitoring period, the tumors were removed and weighed, and the trend in tumor weight was similar to the above results. Figure 8 c and Figure 8 (d) To investigate the systemic toxicity of the nanoreactor, changes in mouse body weight were recorded during treatment. Figure 8 In the results (e), no significant differences in body weight were observed among the groups of mice. The good anti-tumor effect of (GOx+CATc)@aZIF-8 observed in these experiments indicates that the nanoreactor effectively maintains enzyme activity, prolongs its residence time within the tumor, and achieves a combination of starvation therapy and chemokinetic therapy.

[0066] This invention provides a composite nanomaterial, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing composite nanomaterials, characterized in that, Includes the following steps: (1) Preparation of catalase modified with polymethacrylic acid; (2) Zinc ions, 2-methylimidazole, glucose oxidase and the catalase modified with polymethacrylic acid prepared in step (1) are mixed and subjected to a first reaction to obtain the composite nanomaterial.

2. The preparation method according to claim 1, characterized in that, In step (1), the polymethacrylic acid has a weight-average molecular weight of 4000-6000 Da.

3. The preparation method according to claim 1, characterized in that, In step (1), the polymethacrylic acid modified catalase is prepared by the following method: polymethacrylic acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are mixed in a buffer solution, catalase is added, and the polymethacrylic acid modified catalase is obtained by a second reaction.

4. The preparation method according to claim 3, characterized in that, The buffer solution is a 10-30 mM PBS buffer with a pH of 5-7; The molar ratio of amino groups in the polymethacrylic acid and catalase is 1-30:1; The mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to catalase is 5-10:1; The second reaction is carried out at a temperature of 18-30℃ for 3-10 hours.

5. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of zinc ions to 2-methylimidazole is 1:3-5, and the amount of glucose oxidase is calculated as 1-5g of glucose oxidase per mole of zinc ions.

6. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of glucose oxidase and polymethacrylic acid modified catalase is 1-16:

1.

7. The preparation method according to claim 1, characterized in that, In step (2), the first reaction is carried out at a temperature of 15-30℃ and for a time of 20-50 min.

8. The composite nanomaterial prepared by the preparation method according to any one of claims 1-7.

9. The application of the composite nanomaterial according to claim 8 in the preparation of drugs for treating tumors.

10. The application according to claim 9, characterized in that, The tumors mentioned include esophageal squamous cell carcinoma.

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