Iron-modified zif8 nanoparticles encapsulating antitumor drugs, and preparation method and application thereof

Abstract

The application discloses an iron modified ZIF8 nanoparticle encapsulating an antitumor drug and a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method of the nanoparticle comprises the following steps: firstly, preparing a zinc hydroxide loaded antitumor drug, then adding 2-methyl imidazole to obtain a ZIF8 nanoparticle encapsulating the antitumor drug, and finally adding Fe 2+ A methanol solution of a salt is reacted to obtain the iron modified ZIF8 nanoparticle encapsulating the antitumor drug. Through process improvement, the application loads an antitumor chemotherapy drug in the ZIF8 material, so that the ZIF8 material has good acid responsive drug release capacity. Through iron element modification, the nanoparticle is endowed with peroxidase activity, so that the nanoparticle can realize acid and hypoxia responsive chemotherapy and chemical kinetics therapy. The nanoparticle and microorganisms capable of producing lactate oxidase are combined to show super strong non-external field responsive precise tumor treatment capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a kind of iron modified ZIF8 nanoparticles encapsulating antitumor drugs and its preparation method and application. BACKGROUND

[0002] It is a big difficulty in tumor treatment to develop precision therapeutic agents. Traditional chemotherapy drugs have poor selectivity for cancer cells, resulting in a large number of normal cells being killed when chemotherapy is performed, which produces significant side effects. This limits the application of cancer chemotherapy therapeutic agents. In recent years, with the in-depth study of the biochemical characteristics of tumors, our understanding of the tumor tissue microenvironment has become clearer, such as: tumor tissues exhibit a weak acid environment compared to normal tissues; due to the Warburg effect, tumor tissues have high lactic acid content and are severely hypoxic compared to normal tissues; tumor cells have high expression of hydrogen peroxide, etc. These characteristics of the tumor tissue microenvironment provide ideas for the development of a new generation of precision therapeutic agents based on the response of the tumor tissue microenvironment.

[0003] Chemodynamic therapy (CDT) has emerged as a new type of tumor treatment technology based on the conversion reaction of endogenous chemical products in tumors, i.e., using the tumor microenvironment to activate the Fenton reaction (Fenton-like reaction) to catalytically convert weakly oxidizing H2O2 into strongly oxidizing hydroxyl radicals for tumor-specific treatment. The essence of chemodynamic therapy is catalysis, and the catalytic metal ions (such as Fe 2+ ,Mn 2+ , etc.) released by the material in response to the acid environment of the tumor tissue or nanoscale enzymes with catalytic properties can catalyze the high expression of hydrogen peroxide in tumor cells into strongly cytotoxic hydroxyl radicals, achieving tumor killing. However, for cells in normal tissues, there is no serious impact due to inadequate pH and insufficient hydrogen peroxide concentration.

[0004] Acid-responsive drug carriers carrying responsive chemotherapy drugs are also an important development direction. Using materials that can respond to acid degradation to carry chemotherapy drugs to release locally in tumor tissues can reduce the toxicity of direct intravenous injection of chemotherapy drugs.

[0005] Zeolitic imidazolate framework-8 (ZIF8) is a metal-organic framework (MOF) material with imidazole groups, and its metal part is Zn 2+The ion, the organic connecting agent is 2-methylimidazole.The ZIF8 has acid response degradation characteristics, and the structure of the material collapses under acidic conditions, releasing the drug loaded in the MOF channel.Because its aqueous phase is relatively stable and has good biological safety, it has been widely concerned in recent years, and is applied to many fields as a drug carrier, for example, ZIF-8 nanomaterials have been reported to be used for various modes of tumor treatment, including chemotherapy, photodynamic therapy, photothermal therapy, chemical dynamic therapy, acoustic dynamic therapy, starvation therapy, etc.At present, the conventional way of loading drugs on ZIF-8 is to synthesize ZIF-8 first and then load drugs by using the porosity of the material, but this method has the problem of drug burst for some drugs.In addition, Zn 2+ The absence of intrinsic catalytic activity makes the ZIF8 material not have obvious hydrogen peroxide catalytic activity.

[0006] Based on the above background, if the ZIF8 material is modified and modified to make the nanoparticles have the ability to catalyze the overexpression of hydrogen peroxide in tumor tissue, the nanoparticles as a whole will have the ability to exhibit good chemotherapy and chemical dynamics therapy treatment ability for the tumor microenvironment response, effectively solving the problem of lack of precision of traditional drugs. SUMMARY

[0007] The purpose of the present application is to load antitumor drugs in ZIF8 materials to avoid the problem of drug burst by process design, and to give the nanoparticles peroxide enzyme activity by element modification, so that the nanoparticles can realize acid and hypoxic response chemotherapy and chemical dynamics therapy.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a preparation method of iron-modified ZIF8 nanoparticles encapsulating antitumor drugs, comprising the following steps:

[0010] (1) resuspend zinc hydroxide in a methanol solution containing antitumor drugs to obtain a resuspension;

[0011] (2) drop 2-methylimidazole methanol solution into the resuspension to react, and prepare ZIF8 nanoparticles encapsulating antitumor drugs;

[0012] (3) resuspend the ZIF8 nanoparticles encapsulating antitumor drugs in methanol, and add Fe 2+ salt methanol solution under the protection of a non-oxygen atmosphere to react, and prepare the iron-modified ZIF8 nanoparticles encapsulating antitumor drugs.

[0013] In the preparation of nanoparticles, the Zn precursor is first prepared into zinc hydroxide to load the antitumor drug, then 2-methyl imidazole is added to the matrix, and the reaction is stirred to obtain ZIF8 nanoparticles encapsulating the antitumor drug. The metal organic framework nanoparticles prepared by the method have good acid-responsive drug release capacity, and the problem of drug burst existing in the synthesis of ZIF8 by the usual method and then stirring to load the antitumor drug is avoided. Further, Fe 2+ Replace part of the outer layer Zn in ZIF8 2 + , to obtain Fe-modified ZIF8 nanoparticles encapsulating the antitumor drug, the introduction of Fe element endows the nanoparticles with peroxidase activity, so that the prepared nanoparticles have good application potential in chemical kinetic therapy.

[0014] In the present application, methanol is used as the solvent. Studies have shown that, compared with other organic solvents such as DMSO and ethanol, methanol solvent system is more conducive to the formation of the nanoparticles.

[0015] In step (1), the Zn precursor is prepared into zinc hydroxide, the zinc hydroxide is resuspended in a methanol solution containing an antitumor drug, and a resuspension is prepared by stirring.

[0016] Further, the Zn precursor is dissolved in water, excess sodium hydroxide is added, and the Zn 2+ is completely converted into Zn(OH)2, and Zn(OH)2 is obtained by centrifugation; the Zn precursor is Zn 2+ salt, which can be but is not limited to any one of ZnCl2, ZnSO4 and Zn(NO3)2.

[0017] The antitumor drug can be but is not limited to an oxygen-responsive chemotherapeutic drug, and specifically, tirapazamine (TPZ) can be used, which is converted into a strong toxic benzotriazine group (BTZ) under hypoxic conditions to achieve enhanced oxygen-responsive cytotoxicity. The present application uses ZIF8 nanoparticles to carry TPZ to endow it with acid-responsive and hypoxia-enhanced chemotherapeutic capacity.

[0018] Further, the zinc hydroxide is resuspended in a methanol solution containing an antitumor drug, and stirred at 200-600 rpm for at least 5 minutes. By stirring, the zinc hydroxide dispersion solution is dispersed and the antitumor drug is allowed to fully contact with it.

[0019] In step (2), the 2-methyl imidazole methanol solution is added dropwise to the resuspension prepared in step (1) for reaction, and zinc ions and organic ligand 2-methyl imidazole are assembled to form a zeolite imidazole framework structure through coordination. It can be observed that the reaction solution is changed into orange red, the precipitate is collected by centrifugation, and the drug-loaded ZIF8 nanoparticles are prepared after methanol washing.

[0020] Furthermore, the molar ratio of Zn(OH)₂ to 2-methylimidazole is 0.02-0.05:1; the reaction time is 5-10 minutes. Stirring is performed at 200-600 rpm for 5-10 minutes.

[0021] In step (3), under a non-oxygen atmosphere, Fe, which has a stronger affinity for 2-methylimidazole, is used. 2+ To replace part of the Zn in ZIF8 2+ This achieves Fe modification.

[0022] The non-oxygen atmosphere can be, but is not limited to, nitrogen.

[0023] Furthermore, Fe 2+ Salt with Fe 2+ The mass ratio of ZIF8 nanoparticles to encapsulated antitumor drugs was 1:10-20; Fe was added to the methanol solution of nanoparticles at 200-600 rpm. 2+ Salt methanol solution; reaction conditions: room temperature, reaction time: 2-5 minutes.

[0024] The Fe 2+ Salts can be, but are not limited to, FeCl2, Fe(NO3)2, and FeSO4.

[0025] This invention provides ZIF8 nanoparticles with iron-modified encapsulation of antitumor drugs, prepared by the aforementioned method.

[0026] The present invention also provides the application of the iron-modified ZIF8 nanoparticles encapsulating antitumor drugs in the preparation of tumor therapeutic drugs.

[0027] Studies have shown that the iron-modified ZIF8 nanoparticles encapsulating antitumor drugs provided by this invention have significant acid-responsive drug release characteristics and exhibit acid-excited peroxidase activity. They can localize the release of chemotherapeutic drugs in the acidic tumor microenvironment to exert their chemotherapeutic effect, while catalyzing the hydrogen peroxide highly expressed in tumor cells to become a highly cytotoxic hydroxyl radical to exert its chemokinetic therapeutic effect, thus jointly achieving tumor killing.

[0028] Furthermore, the tumor is a solid tumor. The solid tumor can be, but is not limited to, breast cancer, pancreatic cancer, liver cancer, colon cancer, etc.

[0029] Another object of the present invention is to provide a biohybrid material composed of iron-modified ZIF8 nanoparticles encapsulating antitumor drugs and microorganisms that secrete lactate oxidase.

[0030] Lactate oxidase is an enzyme of biological origin, which can catalyze the oxidation of lactic acid or lactate into hydrogen peroxide, while realizing oxygen consumption and hydrogen peroxide production. Based on the characteristics that some microorganisms can produce lactate oxidase, the iron-modified ZIF8 nanoparticles encapsulating antitumor drugs are combined with such microorganisms, and the lactate oxidase produced by the microorganisms is used to catalyze the production of hydrogen peroxide from excess lactic acid in tumor tissues, thereby increasing the levels of hypoxia and hydrogen peroxide in the tumor microenvironment, and further enhancing the tumor treatment effect of the nanomaterials.

[0031] The research of the present application shows that the combination of ZIF8 nanoparticles and microorganisms can reduce the proliferation activity of microorganisms by forming oxidative stress, and significantly improve the biological safety, but the combination process does not affect the metabolic function of the microorganisms themselves. The biological hybrid material exhibits significant antitumor performance.

[0032] The microorganism secreting lactate oxidase can be a natural microorganism or an artificially constructed strain reported in the prior art, which has the function of secreting lactate oxidase and can be applied to the body.

[0033] Further, the microorganism secreting lactate oxidase can be an engineered E. coli strain secreting lactate oxidase under hypoxia induction, which is constructed by the method described in the patent document with reference to application number 2022116167019.

[0034] Further, the preparation method of the biological hybrid material comprises: adding a gamma-polyglutamic acid aqueous solution to the methanol solution of the iron-modified ZIF8 nanoparticles encapsulating antitumor drugs, stirring and then centrifuging to collect the product; then adding an aqueous solution containing 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) and N-hydroxysuccinimide (NHS) to activate the product, and collecting the activated product after centrifugation; then adding an aqueous solution containing microorganisms secreting lactate oxidase to react to prepare the biological hybrid material.

[0035] In the above preparation method, gamma-polyglutamic acid (gamma-PGA) is first coated on the surface of the iron-modified ZIF8 nanoparticles encapsulating antitumor drugs, then the gamma-PGA is activated by NHS / EDC, and then the nanoparticles are combined with the microorganisms secreting lactate oxidase based on the amidation reaction.

[0036] Further, 50-100 mg of the iron-modified ZIF8 nanoparticles encapsulating the antitumor drug are dissolved in 10-20 mL of methanol, 1 mL of 10 mg / mL of a gamma-PGA aqueous solution is added under stirring at 500 rpm, and the product is collected by centrifugation after 3-5 min of continuous stirring. Subsequently, 2 mL of an aqueous solution containing 20-30 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 5-15 mg of N-hydroxysuccinimide is added to 5-10 mg of the product, oscillation is performed at a rate of 180 rpm for 3-5 min, and then centrifugation is performed, after which the product is washed with deionized water and centrifuged once, 1-2 mL of 5*10 7 CFU / mL of a microorganism solution secreting lactate oxidase is added, oscillation is continued for 5-10 min, and the biohybrid material is obtained.

[0037] Further, the antitumor drug encapsulated in the ZIF8 nanoparticles is the hypoxia-responsive chemotherapeutic drug tirapazamine.

[0038] The application also provides use of the biohybrid material in the preparation of a tumor treatment drug.

[0039] The research of the application shows that the biohybrid material has a strong killing effect on tumor cells at a very low effective concentration in a hypoxic environment. The microorganism producing lactate oxidase and the iron-modified ZIF8 nanoparticles synergistically achieve the tumor killing function.

[0040] The application has the following beneficial effects:

[0041] (1) The application improves the process to load an antitumor chemotherapeutic drug in ZIF8 material. Specifically, zinc hydroxide is first prepared to load the chemotherapeutic drug, and then 2-methylimidazole is added to react to prepare ZIF8 nanoparticles encapsulating the chemotherapeutic drug. The nanoparticles have good acid-responsive drug release capacity, avoiding the burst release problem existing in the synthesis of ZIF8 and then the stirring loading of the drug; further, the nanoparticles are modified by iron elements to have peroxidase activity, so that the nanoparticles can realize acid and hypoxia-responsive chemotherapy and chemical kinetics therapy.

[0042] (2) Compared with ordinary cancer treatment drugs, the nano material provided by the application has the following advantages: (i) the nano material is targeted to the microenvironment characteristics of tumors to achieve precise treatment of tumors; (ii) the nano material simultaneously combines chemical kinetics therapy and chemotherapy on one treatment platform, so that the nano material exhibits good tumor treatment effect; (iii) the nano material is linked to microorganisms capable of producing lactate oxidase based on functional design, and the combination of the two exhibits super-strong non-external-field-responsive precise tumor treatment capacity. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1SEM image of Fe-ZIF8-TPZ prepared in Example 1.

[0044] Figure 2 Elemental analysis results of Fe-ZIF8-TPZ prepared in Example 1.

[0045] Figure 3 UV-Vis spectrum of TPZ standard solution.

[0046] Figure 4 TPZ standard curve.

[0047] Figure 5 TPZ release of Fe-ZIF8-TPZ under different pH conditions.

[0048] Figure 6 Detection results of peroxidase-like activity of Fe-ZIF8-TPZ under different conditions.

[0049] Figure 7 Cytotoxicity of Fe-ZIF8-TPZ on 4T1 cells under different conditions.

[0050] Figure 8 Pictures of Fe-ZIF8-TPZ before and after complexing with engineered E. coli, where the left image is before complexing and the right image is after complexing.

[0051] Figure 9 Cytotoxicity of PP3244@Fe-ZT on 4T1 cells under hypoxic and normoxic conditions.

[0052] Figure 10 Hydroxyl radical generation of PP3244@FT and 4T1 cells under hypoxic culture in different concentrations of lactic acid.

[0053] Figure 11 Comparison of tumor growth inhibition performance of Fe-ZIF8-TPZ and PP3244@Fe-ZT.

[0054] Figure 12 Physical picture of tumor growth inhibition of PP3244@Fe-ZT.

[0055] Figure 13 Biological safety evaluation of PP3244@Fe-ZT.

[0056] Figure 14 Metabolomics characterization of PP3244@Fe-ZT.

[0057] Figure 15 Comparison of proliferation activity of PP3244 and PP3244@Fe-ZT.

[0058] Figure 16SEM image of Fe-ZIF8-TPZ prepared for Example 3.

[0059] Figure 17 TEM image of Fe-ZIF8-TPZ prepared for Comparative Example 1.

[0060] Figure 18 TPZ release of TPZ@Fe-ZIF8 prepared for Comparative Example 2 under different pH conditions.

[0061] Figure 19 SEM image of the product prepared for Comparative Example 3. DETAILED DESCRIPTION

[0062] The application will be further described in conjunction with specific examples. The following examples are intended to illustrate the application and not to limit the scope of the application. Modifications or substitutions of the methods, steps or conditions of the application, which do not depart from the spirit and essence of the application, are intended to fall within the scope of the application.

[0063] 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.

[0064] Example 1: Preparation and characterization of Fe-ZIF8-TPZ

[0065] 1. Preparation of iron-modified ZIF8 nanoparticles encapsulating taplazamine

[0066] Dissolve 200 mg of Zn(NO3)2·6H2O in 0.4 mL of water, and under the condition of stirring at 500 rpm, add 0.25 mL of 8M aqueous sodium hydroxide solution to it, so that the Zn ions are completely changed into white hydroxide precipitate. Then centrifuge and resuspend the zinc hydroxide precipitate with 0.5 mL of methanol. Add 1 mL of taplazamine (TPZ, CAS number 27314-97-2) methanol solution with a concentration of 1 mg / mL to it, and stir at 500 rpm for 5 min. Then slowly add 5 mL of 2-methylimidazole methanol solution with a concentration of 0.4 g / mL to it, and stir at 500 rpm for 5 min. Observe that the solution turns orange-red, centrifuge and wash twice with methanol to obtain ZIF8-TPZ.

[0067] Then, take 50 mg of ZIF8-TPZ nanoparticles and resuspend them in 5 mL of methanol, and under the condition of nitrogen protection and stirring at 500 rpm, add 1 mL of 5 mg / mL FeCl2 to it, and react for 3 min. Centrifuge to obtain Fe-ZIF8-TPZ.

[0068] 2. Morphology and elemental analysis of Fe-ZIF8-TPZ

[0069] SEM images of the product prepared in step 1 are attached. Figure 1 As shown, the Fe-ZIF8-TPZ nanoparticles have a size of about 100 nm, and the size of each particle is similar, indicating good dispersibility.

[0070] The elemental distribution of the products is characterized using the EDS mapping pattern of STEM, such as... Figure 2 As shown in the figure, Fe was shown to be uniformly distributed around Zn and coincident with N, demonstrating that Fe successfully replaced Zn near the surface of ZIF8 particles, thus achieving Fe modification.

[0071] 3. Acid-responsive TPZ release performance of Fe-ZIF8-TPZ

[0072] ZIF materials are unstable in acidic aqueous environments, which provides a basis for drug loading with 2-methylimidazolium-based MOF materials for acidic controlled-release drugs.

[0073] The acid-responsive TPZ release performance of Fe-ZIF8-TPZ was tested as follows: TPZ was dissolved in PBS to obtain TPZ solutions of 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256, and 1 / 512 mg / mL. The UV-Vis spectra were measured using a microplate reader, as shown in the attached image. Figure 3 As shown, a standard curve was constructed using its characteristic peak at 460nm. Figure 4 The relationship between the concentration x of TPZ and the absorption peak intensity at 460 nm in PBS solution was found to be y = 0.1834x + 0.048.

[0074] Resuspend 10 mg of Fe-ZIF8-TPZ in PBS solutions of pH=5 and pH=7, respectively, and place them in a shaker at 37℃ and 180 rpm. At different time points, take 0.5 mL of the sample, centrifuge, collect the supernatant, and measure the absorption intensity at 460 nm using a microplate reader to obtain the TPZ release concentration at the corresponding time points. Figure 5 .like Figure 5 As shown, Fe-ZIP8-TPZ exhibited significant acid-responsive TPZ release.

[0075] 4. Peroxidase activity of Fe-ZIF8-TPZ

[0076] Fe 2+ The introduction of [Zn] replaces part of the original Zn. 2+As the metal part of the MOF material, Fe-ZIF8-TPZ nanoparticles provide active sites for the catalytic reaction of hydrogen peroxide, making Fe-ZIF8-TPZ a kind of peroxidase-like nanoscale enzyme that can catalyze hydrogen peroxide to produce hydroxyl radicals.

[0077] 3,3',5,5'-tetramethylbenzidine (TMB) was used as a chromogenic agent to indicate the peroxidase-like activity of Fe-ZIF8-TPZ under different conditions. Specifically, 0.1M acetic acid buffer with different pH (pH = 5.0, 6.0, 7.4) was used as the test buffer to simulate the pH of the late endosome, early endosome and normal environment after the particles were endocytosed by cancer cells. 50μL of 3M hydrogen peroxide, 300μL of 8M TMB solution and 50μL of 3mg / mL Fe-ZIF8-TPZ or ZIF8-TPZ were added to 2.6mL of buffer with different pH. After ten minutes, the spectra of each group were recorded using UV-visible spectroscopy. The peak near 652nm is the peak of TMB after oxidation by hydroxyl radicals, indicating the peroxidase-like activity of the material.

[0078] The results, as shown in Figure 6 Fe-ZIF8-TPZ showed significant acid-enhanced peroxidase-like activity, which is consistent with the weakly acidic pH of tumor tissue, indicating that Fe-ZIF8-TPZ nanoscale enzyme has the ability to kill tumors based on chemical kinetic therapy. In contrast, ZIF8-TPZ did not show significant peroxidase activity even at pH = 6 because it did not have a metal element with the potential to catalyze POD enzymes.

[0079] 5. Responsive cytotoxicity of Fe-ZIF8-TPZ

[0080] The cytotoxicity of Fe-ZIF8-TPZ was analyzed using the mouse breast cancer cell 4T1 cell line. 4T1 cells were tested in vitro at 37°C in a 5% CO2 environment. The culture medium used was RPMI 1640 medium containing 10% fetal bovine serum. The normoxic experiment was set at an oxygen concentration of 21%, the hypoxic experiment was set at an oxygen concentration of 4%, and the acidic condition was adjusted to pH 6.5 using 1M hydrochloric acid.

[0081] Specifically, 4T1 cells were digested and inoculated into a 96-well plate at a density of 4000 cells per well. After 12 hours of culture in a normoxic environment, the original culture medium was removed, and 200 μL of fresh culture medium containing PBS or different concentrations of Fe-ZIF8-TPZ was added to each well. The cell plate was placed in an anoxic or normoxic environment as required. After 24 hours, the culture medium was removed, and the absorbance of each well was measured by the CCK-8 method. The normoxic PBS group was used as a control to obtain the survival rate of 4T1 cells co-cultured with Fe-ZIF8-TPZ under different conditions.

[0082] The results of the cytotoxicity of Fe-ZIF8-TPZ on 4T1 cells under different conditions are shown in Figure 7 As shown, both anoxic and acidic environments significantly enhance the cytotoxicity of Fe-ZIF8-TPZ on 4T1 cells, which is due to the enhanced anoxic-responsive toxicity of TPZ and the acid-responsive peroxidase-like activity of nanoparticles.

[0083] Example 2: Complexing of Fe-ZIF8-TPZ with lactic acid oxidase

[0084] 1. Complexing of Fe-ZIF8-TPZ with an engineered E. coli capable of producing lactic acid oxidase

[0085] Fe-ZIF8-TPZ was coated with γ-PGA, and then based on the amidation reaction, the nanoparticles were complexed with an engineered E. coli PP3244 capable of producing lactic acid oxidase under anoxic response, as disclosed in application No. 2022116167019.

[0086] Specifically, 100 mg of Fe-ZIF8-TPZ nanoparticles were dissolved in 20 mL of methanol, and 1 mL of 10 mg / mL γ-PGA aqueous solution was added at 500 rpm. After stirring for 10 min, the product was collected at a speed of 10,000 rpm. Then, 2 mL of an aqueous solution containing 20 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) and 10 mg of N-hydroxysuccinimide (NHS) was added to 5 mg of the product, and oscillated at a speed of 180 rpm for 5 min. After washing with deionized water and centrifugation once, 2 mL of 5 x 10 7 CFU / mL of PP3244 aqueous solution was added, and oscillation was continued for 5 min to obtain the complex, which was denoted as PP3244@Fe-ZT.

[0087] The electron micrographs before and after complexing are shown in Figure 8 As shown, the surface of the original engineered E. coli PP3244 is smooth, while the surface of the nanoparticles loaded with Fe-ZIF8-TPZ has obvious protruding particles, i.e., Fe-ZIF8-TPZ particles.

[0088] 2. Cytotoxicity of PP3244@Fe-ZT

[0089] The cytotoxicity of PP3244@Fe-ZT was analyzed using mouse breast cancer cell 4T1 cell line. 4T1 cells were tested in vitro at 37℃, 5% CO2 environment. The culture medium used was RPMI 1640 medium containing 10% fetal bovine serum. The oxygen concentration was set to 21% for normoxic experiments and 4% for hypoxic experiments.

[0090] Specifically, 4T1 cells were digested and inoculated into a 24-well plate at a density of 12000 cells per well. After 12 hours of normoxic culture, the original culture medium was removed, 600 μL of fresh culture medium was added to each well, and a Transwell @ chamber was placed in the well. 400 μL of culture medium containing PBS or different concentrations of PP3244@Fe-ZT was added to the chamber. The cell plate was cultured under hypoxic and normoxic conditions, respectively. After 24 hours, the chamber was removed, and the absorbance of each well was measured by CCK-8 method. The normoxic PBS group was used as a control, and the survival rate of 4T1 cells co-cultured with PP3244@Fe-ZT under each condition was obtained.

[0091] The cytotoxicity of PP3244@Fe-ZT on 4T1 cells under hypoxic and normoxic conditions is shown in Figure 9 As shown in the table, very low concentrations of PP3244@Fe-ZT can produce strong killing effect on cancer cells under hypoxic environment, but not under normoxic conditions.

[0092] When the same concentration of PP3244@Fe-ZT was co-cultured with 4T1 cells under hypoxic conditions, different concentrations of lactic acid were added to the culture medium, and the content of hydroxyl radicals in the cells was also observed to increase significantly Figure 10 ).

[0093] Combining the data of hypoxic enhanced toxicity from the cell experiment and the data of increased free radical content caused by the addition of lactic acid indicated by the DCFH-DA probe, it is proved that the killing of PP3244@Fe-ZT on cells is partly due to the synergy of their functions, that is, PP3244 produces hydrogen peroxide by catalyzing lactic acid with lactic acid oxidase, and then Fe-ZIF8-TPZ catalyzes it to produce highly toxic hydroxyl radicals.

[0094] 3. Anti-tumor performance test of Fe-ZIF8-TPZ and PP3244@Fe-ZT

[0095] To demonstrate the application prospect of Fe-ZIF8-TPZ and PP3244@Fe-ZT hybrid materials in the field of tumor treatment, the in vivo anti-tumor model is used to evaluate the tumor treatment ability of the hybrid. The tumor growth inhibition of animals (mice) is used to illustrate the application of materials in the field of tumor treatment.

[0096] The 3-week-old balb / c female mice are injected with 10 6 4T1 cancer cells on the back to construct a 4T1 tumor model after being fed in a 12h light / 12h dark environment for 1 week to adapt to the environment. After the tumor grows to about 100mm 3 , the mice with 4T1 tumor are randomly divided into four groups, and the day is recorded as day 0. On day 0, 2, and 4, the mice in each group are treated. The first group of mice is injected with PBS solution, the second group of mice is injected with 0.25mg Fe-ZIF8-TPZ particles, the third group of mice is injected with 5×10 6 engineered E. coli PP3244, and the fourth group of mice is injected with 5×10 6 PP3244@Fe-ZT hybrid materials. The tumor size is recorded every day since the first injection, and the results are shown in Figure 11 .

[0097] As can be seen from Figure 11 , compared with the control group, the tumor volume of mice injected with Fe-ZIF8-TPZ and PP3244 is inhibited to a certain extent, and the tumor inhibition performance of PP3244@Fe-ZT is extremely strong, and the tumor of the mice injected with PP3244@Fe-ZT hybrid materials almost disappeared in a few days before treatment Figure 12 .

[0098] In summary, Fe-ZIF8-TPZ and PP3244@Fe-ZT have been proved to have certain tumor growth inhibition ability, and the tumor growth inhibition ability of PP3244@Fe-ZT is extremely amazing, and almost realizes tumor ablation without external field stimulation.

[0099] 4、Fe-ZIF8-TPZ for the biological safety of engineered microorganisms

[0100] When the materials related to the present application are used to treat mice, it is found that after 14 days of treatment with pure engineered microorganisms, the liver of the mouse has obvious lesions, the color is white, and the tissue section has obvious vacuoles Figure 13). This indicates that intravenous injection of PP3244 can seriously damage the liver of mice. Interestingly, the mice treated with PP3244@Fe-ZT do not show this phenomenon. This means that the loading of Fe-ZIF8-TPZ may change the microbial activity in the bio-hybrid material PP3244@Fe-ZT.

[0101] We performed metabolomics characterization on PP3244 and PP3244@Fe-ZT, respectively, and the heat map of the proportion of the content of the partial metabolites of the microorganisms in PP3244@Fe-ZT compared with the corresponding metabolites in PP3244 is shown in FIG. 2. Figure 14 As can be seen from the figure, the concentration of signal molecules such as indoline inside the microorganisms is still normal, indicating that the microorganisms can still carry out normal physiological activities, but the significant increase in the concentration of bacterial antioxidant-related substances CoA, the significant decrease in the content of guanine and cytosine, etc. indicate that the loading of Fe-ZIF8-TPZ may increase the antioxidant stress of the cells, reduce the ability of the bacteria to synthesize genetic material, and thus cause a certain decrease in the proliferation activity of the bacteria. This phenomenon is also reflected in the proliferation activity analysis of PP3244 and PP3244@Fe-ZT Figure 15 ), and the proliferation rate of PP3244@Fe-ZT is significantly lower than that of PP3244. This partial inhibition of the activity of the microorganisms by the hybrid material can effectively reduce the safety risk to the patient caused by the excessive proliferation of the microorganisms, while not affecting the treatment function of the microorganisms injected into the patient's body.

[0102] Example 3: Preparation of Fe-ZIF8-TPZ

[0103] 1. Preparation of iron-modified ZIF8 nanoparticles encapsulating tirapazamine

[0104] Dissolve 200 mg of ZnSO4·H2O in 0.4 mL of water, and under the condition of stirring at 400 rpm, add 0.25 mL of 10M sodium hydroxide aqueous solution to it, so that the Zn ions are completely changed into white hydroxide precipitate. Then centrifuge and resuspend the zinc hydroxide precipitate with 0.5 mL of methanol. Add 1 mL of TPZ methanol solution with a concentration of 0.5 mg / mL to it, and stir at 400 rpm for 5 min. Then slowly add 4 mL of 2-methylimidazole methanol solution with a concentration of 0.4 g / mL to it, and stir at 400 rpm for 5 min. Observe that the solution turns orange-red, centrifuge and wash twice with methanol to obtain ZIF8-TPZ. Then, take 50 mg of ZIF8-TPZ nanoparticles and resuspend them in 5 mL of methanol, and under the condition of nitrogen protection and stirring at 500 rpm, add 1 mL of 10 mg / mL FeCl2 to it, and react for 3 min. Centrifuge to obtain Fe-ZIF8-TPZ.

[0105] 2. Morphology analysis of Fe-ZIF8-TPZ

[0106] SEM image of Fe-ZIF8-TPZ prepared with zinc sulfate heptahydrate is shown in Figure 2, which has no obvious difference in structure from Example 1, but the size has grown slightly. Figure 16

[0107] Example 4: Preparation of Fe-ZIF8-TPZ

[0108] 1. Preparation of Fe-ZIF8-TPZ

[0109] Dissolve 200 mg of ZnCl2in 0.4 mL of water, and add 0.25 mL of 10M sodium hydroxide aqueous solution to it under the condition of stirring at 400 rpm, so that the Zn ion is completely changed into white hydroxide precipitate. Then centrifuge, and resuspend the zinc hydroxide precipitate with 0.5 mL of methanol. Add 1 mL of TPZ methanol solution with a concentration of 2 mg / mL to it, and stir at 500 rpm for 5 min. Then slowly add 4 mL of 2-methylimidazole methanol solution with a concentration of 0.5 g / mL to it, and stir at 500 rpm for 3 min, and observe that the solution turns into orange red. Centrifuge, and wash with methanol twice to obtain ZIF8-TPZ. Then, take 50 mg of ZIF8-TPZ nanoparticles, resuspend them in 5 mL of methanol, and add 1 mL of 10 mg / mL FeCl2to it under the condition of nitrogen protection and stirring at 500 rpm, and react for 3 min. Centrifuge to obtain Fe-ZIF8-TPZ.

[0110] 2. Morphology analysis of Fe-ZIF8-TPZ

[0111] The product is observed by scanning electron microscope, and the structure of Fe-ZIF8-TPZ prepared by the above method has no obvious difference from Example 1.

[0112] Comparative Example 1

[0113] ​1. Dissolve 200 mg Zn(N03)2-6H20 in 0.4 mL water, under the condition of stirring at 500 rpm, add 0.25 mL of 8M sodium hydroxide aqueous solution to it, so that the Zn ion is completely changed into white hydroxide precipitate. Then centrifuge, resuspend the zinc hydroxide precipitate with 0.5 mL of methanol. Add 1 mL of TPZ methanol solution with a concentration of 1 mg / mL to it, stir at 500 rpm for 5 min. Then slowly add 5 mL of 2-methylimidazole methanol solution with a concentration of 0.4 g / mL to it, stir at 500 rpm for 5 min, and observe that the solution turns into orange red. Centrifuge, wash twice with methanol to obtain ZIF8-TPZ. Then, take 50 mg of ZIF8-TPZ nanoparticles, resuspend them in 5 mL of water, under the condition of nitrogen protection, add 1 mL of 5 mg / mL FeCl2 aqueous solution to it at 500 rpm, react for 3 min, and centrifuge to obtain the product.

[0114] 2. The TEM photo of the product is shown in the following figure. Figure 17 It can be seen that obvious etching of ZIF8-TPZ particles occurs and many irregular particles are generated. This indicates that when the reaction system is aqueous phase, the generated particles cannot meet our requirements.

[0115] Comparative Example 2

[0116] 1. Preparation of TPZ@Fe-ZIF8

[0117] Dissolve 200 mg Zn(N03)2-6H20 in 0.4 mL water, under the condition of stirring at 500 rpm, add 0.25 mL of 8M sodium hydroxide aqueous solution to it, so that the Zn ion is completely changed into white hydroxide precipitate. Then centrifuge, resuspend the zinc hydroxide precipitate with 0.5 mL of methanol. Slowly add 5 mL of 2-methylimidazole methanol solution with a concentration of 0.4 g / mL to it, stir at 500 rpm for 5 min, centrifuge, and wash twice with methanol to obtain ZIF8. Then, take 50 mg of ZIF8-TPZ nanoparticles, resuspend them in 5 mL of methanol, under the condition of nitrogen protection, add 1 mL of 5 mg / mL FeCl2 methanol solution to it at 500 rpm, react for 3 min, and centrifuge to obtain the product. Resuspend the centrifuged product in methanol, at this time, add 1 mL of TPZ methanol solution with a concentration of 1 mg / mL to it, stir at 500 rpm for 5 min, centrifuge, and obtain TPZ@Fe-ZIF8 loaded by adsorption.

[0118] 2. Drug release of TPZ@Fe-ZIF8

[0119] The total loading amount of TPZ in TPZ@Fe-ZIF8 can be obtained by subtracting the unloaded amount of TPZ in the supernatant after centrifugation from the amount of TPZ added.

[0120] Take 10 mg of TPZ@Fe-ZIF8 and resuspend in PBS solution with pH = 5, pH = 6 and pH = 7 respectively, and place in a 37°C 180 rpm shaker. At different time points, take 0.5 mL of sample, centrifuge to obtain the supernatant, and use an enzyme marker to test the absorbance intensity at 460 nm to obtain the solution TPZ release concentration at the corresponding time point.

[0121] Combined with the total loading amount data, the results are as follows: Figure 18 The results show that the TPZ loaded by this scheme occurs burst release under both acidic and neutral conditions, which cannot meet our acid-responsive release requirements.

[0122] Comparative Example 3

[0123] Dissolve 100 mg of ZnCl2 and 10 mg of FeCl2 in 0.4 mL of water, slowly add 5 mL of 2-methylimidazole methanol solution with a concentration of 0.4 g / mL under nitrogen protection, stir at 500 rpm for 5 min, centrifuge, and wash twice with methanol to obtain a yellow centrifugation product.

[0124] The SEM photo of the above product is shown in Figure 19 The generated particles contain a complex of iron and 2-methylimidazole, and the morphology is not uniform, which cannot meet our requirements. The EDS results prove that there is almost no zinc in it, which may be because the affinity of Fe 2+ with 2-methylimidazole is stronger, making it difficult for the complex to form a uniform structure of MOF.

[0125] This comparative example proves that it is not feasible to directly add Fe 2+ during the preparation of MOF, and it is necessary to replace part of Zn 2+ in ZIF8 with Fe 2+ with stronger affinity to 2-methylimidazole after the preparation of ZIF8 is completed to achieve Fe modification.

[0126] Conclusion: Fe-ZIF8-TPZ is a simple synthetic dual-mode chemotherapeutic and chemo-dynamic therapeutic agent. It releases TPZ in acidic environment and exhibits acid-triggered peroxygenase activity. It exhibits significant hypoxia- and acid-responsive cytotoxicity at cellular level and shows certain anti-tumor performance in vivo. When combined with engineered E. coli PP3244 that can produce lactate oxidase in response to hypoxia, Fe-ZIF8-TPZ reduces the proliferation activity of PP3244 by forming oxidative stress to PP3244, which significantly improves the safety of PP3244@Fe-ZT compared with pure bacteria. However, the combination process does not affect the metabolic function of the microorganism itself, which makes PP3244@Fe-ZT exhibit significant anti-tumor performance in vitro and in vivo, and even achieve tumor ablation almost without external field stimulation in vivo. This provides a way for the development of a new generation of hybrid materials.

[0127] The above merely describes preferred embodiments of the present application but is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a biological hybrid material in the preparation of a tumor treatment drug, characterized in that the biological hybrid material is a complex of iron-modified ZIF8 nanoparticles encapsulating an antitumor drug and a microorganism secreting lactic acid oxidase; the iron-modified ZIF8 nanoparticles encapsulating an antitumor drug are prepared by the following steps: (1) resuspending zinc hydroxide in a methanol solution containing an antitumor drug to obtain a resuspension; the antitumor drug is tirapazamine; (2) adding a 2-methylimidazole methanol solution dropwise to the resuspension to obtain ZIF8 nanoparticles encapsulating an antitumor drug; the biological hybrid material is prepared by the following steps: adding a γ-polyglutamic acid aqueous solution to an iron-modified ZIF8 nanoparticle methanol solution encapsulating an antitumor drug, stirring, and then centrifuging to collect the product; adding an aqueous solution containing 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to the product to activate it, centrifuging, and then collecting the activated product; and adding an aqueous solution containing a microorganism secreting lactic acid oxidase to the activated product to obtain the biological hybrid material; the microorganism is an engineered Escherichia coli; and the tumor is a solid tumor. In step (1), the zinc hydroxide is resuspended in a methanol solution containing an antitumor drug and stirred at 200-600 rpm for at least 5 minutes. In step (2), the molar ratio of zinc hydroxide to 2-methylimidazole is 0.02-0.05:1; and the reaction time is 5-10 minutes. ​ ​ (3) The ZIF8 nanoparticles encapsulating anti-tumor drugs were resuspended in methanol, and Fe 2+ was added under the protection of a non-oxygen atmosphere to react with the methanol solution of the salt to obtain the iron-modified ZIF8 nanoparticles encapsulating anti-tumor drugs. ​ ​ 2. Use according to claim 1, wherein In step (1), the Zn precursor is dissolved in water, an excess of sodium hydroxide is added to make the Zn completely transform into Zn(OH)2, and centrifugation is performed to obtain Zn(OH)2; the Zn precursor is any one of ZnCl2, ZnSO4, and Zn(NO3)2. 2+ In step (1), the Zn precursor is dissolved in water, an excess of sodium hydroxide is added to make the Zn completely transform into Zn(OH)2, and centrifugation is performed to obtain Zn(OH)2; the Zn precursor is any one of ZnCl2, ZnSO4, and Zn(NO3)2.

3. The use according to claim 1, wherein ​ 4. The use according to claim 1, wherein ​ 5. The use according to claim 1, wherein In step (3), Fe 2+ The salt is Fe 2+ The mass ratio of the salt to the ZIF8 nanoparticles encapsulating the antitumor drug is 1:10-20; the reaction condition is room temperature, and the reaction time is 2-5 minutes.

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