Preparation method and application of nano drug-loaded delivery system based on tumor microenvironment response

Abstract

The invention relates to the field of triple negative breast cancer, and discloses a preparation method and application of a nano drug-loaded delivery system based on tumor microenvironment response, and a ZIF-67 / MnO2 / DOX / BSA / SDS nano drug-loaded delivery system is prepared through oxidation reduction, metal-organic framework self-assembly and electrostatic adsorption reaction. The particle size, the potential, the infrared ray, the TEM, the XRD and the active oxygen generation capacity of the ZMDBS are investigated, and then the cell safety of the ZMDBS to human-derived normal liver cells LO2 and the in-vitro biological characteristics of the cellular uptake effect, the cell treatment effect, the active oxygen generation condition, the apoptosis condition, the endocytosis mechanism and the cell migration inhibition effect to triple-negative breast cancer cells 4T1 are investigated. Then, by constructing a Balb / c mouse subcutaneous triple negative breast cancer tumor model, the in-vivo safety, tissue distribution condition, treatment effect and multiple in-vivo biological characteristics of pathology of the ZMDBS in a 2 triple negative breast cancer tumor model mouse are evaluated.

Description

Technical Field

[0001] The present invention relates to the field of triple-negative breast cancer, and specifically to a preparation method and application of a nano drug delivery system based on tumor microenvironment response. Background Art

[0002] Triple-negative breast cancer refers to breast cancer in which the results of immunohistochemical examination of cancer tissue are negative for estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene Her-2. This type of breast cancer accounts for 10.0% to 20.8% of all breast cancer pathological types, has special biological behavior and clinical pathological characteristics, and has a worse prognosis than other types.

[0003] In the prior art, there is a nano drug system for treating triple-negative breast cancer, and its preparation method and application with announcement number CN108524470A. The invention provides a nano drug system for treating triple-negative breast cancer, and its preparation method and application, which belongs to the field of biotechnology. The system includes a ROS-responsive hyperbranched polymer, a photosensitizer and a chemotherapeutic drug. The structural formula of the hyperbranched polymer is shown in Formula I, the photosensitizer is dihydrochlorin e6, and the chemotherapeutic drug is sorafenib. The invention also provides a method for preparing a nano drug system for treating triple-negative breast cancer. The invention also provides the application of the above-mentioned nano drug delivery system in the preparation of drugs for treating triple-negative breast cancer. The nano drug system can not only be enriched in tumor tissue, but also increase the ROS level in tumor tissue under 660nm laser irradiation, so that the nano carrier in the tumor tissue disintegrates and the chemotherapeutic drug is quickly released, thereby synergistically enhancing the treatment of tumors, while reducing the dosage of chemotherapeutic drugs and alleviating toxic side effects.

[0004] Triple-negative breast cancer has a poor prognosis, high recurrence and metastasis rate, and high mortality rate. It has become the focus of breast cancer research and attention in recent years. Although a large number of related clinical trials have been conducted, no targeted treatment options have been found. Chemotherapy is one of its main treatment methods, but most traditional chemotherapy drugs have defects such as large toxic side effects and systemic circulation instability. In addition, most chemotherapy drugs cannot be enriched in tumor tissues in the body, or the drug concentration in tumor tissues is low. Therefore, the drug dosage will be increased, but the severe adverse reactions that follow also limit its therapeutic effect.

[0005] In view of the above problems, the present invention provides a preparation method and application of a nano drug delivery system based on tumor microenvironment response. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the shortcomings of the prior art, the present invention provides a preparation method and application of a nano drug delivery system based on tumor microenvironment response, which has the advantages of successfully synthesizing a ZIF-67 / MnO2 / DOX / BSA / SDS (abbreviated as ZMDBS) nano drug delivery system with suitable particle size potential, good stability and tumor microenvironment responsiveness, providing strong support and a solid foundation for subsequent biological evaluation, and solving the problems in the above-mentioned background technology.

[0008] (II) Technical solution

[0009] In order to achieve the above purpose, a cobalt / manganese-based nanodrug delivery system ZMDBS with suitable particle size potential, good stability and tumor microenvironment responsiveness was successfully synthesized, providing strong support and a solid foundation for subsequent biological evaluation.

[0010] In order to solve the above problems, the present invention provides the following technical solutions:

[0011] A method for preparing a nano drug delivery system based on tumor microenvironment response comprises the following steps:

[0012] Step S1, synthesis of nano drug delivery system;

[0013] Step S11, preparation of nano-MnO2;

[0014] Step S12, preparation of nano ZIF-67;

[0015] Step S13, preparation of ZIF-67 / MnO2 solution;

[0016] Step S14, preparation of ZMBS;

[0017] Step S15, preparation of ZMDBS;

[0018] Step S2, characterization of nano drug delivery system;

[0019] Step S21, particle size and potential measurement;

[0020] Step S22, determination of drug loading and encapsulation efficiency;

[0021] Step S23, measuring the UV-visible absorption spectrum;

[0022] Step S24, determination of Fourier infrared spectrum;

[0023] Step S25, observing the morphology of nanoparticles;

[0024] Step S26, X-ray diffraction measurement;

[0025] Step S27, element analysis XPS;

[0026] Step S28, GSH responsiveness;

[0027] Step S29, active oxygen generation capability;

[0028] Step S210, nanozyme catalytic activity;

[0029] Step S211, pH responsiveness;

[0030] Step S212, stability inspection;

[0031] Step S3: Evaluate the pharmacodynamic characteristics of the anticancer nano-drug delivery system through in vivo and in vitro experiments.

[0032] Preferably, the synthesis of the nano drug delivery system in step S1 specifically includes the following steps:

[0033] Step S11, preparation of nano-MnO2:

[0034] Take 0-5mL of KMnO4 solution (3.16mg / mL), add 5mL of polyallylamine hydrochloride solution (PAH, 6.32mg / mL) thereto, stir for 15-20min to make it fully react, the solution changes from purple to dark brown, then centrifuge with a 10kd ultrafiltration tube (5000rpm, 30min), and dilute the resulting concentrated solution to 5-10mL to obtain a prefabricated nano MnO2 solution;

[0035] Step S12, preparation of nano ZIF-67:

[0036] Take 0-1mL of Co(NO3)2·6H2O methanol solution (29.1mg / mL), add 0-5mL of 2-methylimidazole methanol solution (2-MeIM, 65.6mg / mL), stir for 10min to make it fully react, the solution turns blue-purple, stand for 6-12h, then centrifuge (10000rpm, 20min), wash with methanol 1-3 times, and vacuum dry at 55-65℃ for 6-12h to obtain blue-purple powder, which is nano ZIF-67;

[0037] Step S13, preparation of ZIF-67 / MnO2 solution:

[0038] Dissolve ZIF-67 in ethanol, sonicate for 25-30 minutes, then add an appropriate amount of ultrapure water to obtain a 2 mg / mL ZIF-67 solution; take 0-1 mL of the above solution, add 0-1 mL of ultrapure water and mix thoroughly, then add 0-1 mL of nano MnO2 solution and stir for 1-5 minutes to obtain a ZIF-67 / MnO2 solution;

[0039] Step S14, preparation of ZMBS:

[0040] Add 1-3 mL of BSA to ZM, stir for 1-2 min, then add 0-1 mL of SDS, continue stirring for 0.5-1.5 h, centrifuge to remove the precipitate, and obtain a stable nano ZMBS solution, which is then refrigerated at 0-4 °C.

[0041] Step S15, preparation of ZMDBS:

[0042] Dissolve ZIF-67 in ethanol, sonicate for 15-35 minutes, and then add an appropriate amount of ultrapure water to obtain a 2 mg / mL ZIF-67 solution; take 0-1 mL of the above solution, add 0-1 mL of ultrapure water and mix thoroughly, then add 0-1 mL of nano MnO2 solution, stir for 1-5 minutes, then add 1-3 mL of doxorubicin hydrochloride (DOX, 2 mg / mL), 1-3 mL of bovine serum albumin, stir for 1-2 minutes, then add 0-1 mL of SDS, stir for 0.5-1 h, centrifuge to remove the precipitate, and obtain a stable nano ZMDBS solution, which is refrigerated in a refrigerator at 0-4°C.

[0043] Preferably, the step S2, characterization of the nano drug delivery system, specifically includes the following steps:

[0044] Step S21, particle size and potential measurement;

[0045] Step S22, determination of drug loading and encapsulation efficiency;

[0046] Step S23, determination of UV-visible absorption spectrum:

[0047] Using a UV-visible spectrophotometer, the UV spectra of different groups of nanomaterials in the range of 200-800nm ​​were measured, and the synthesis of the nano drug delivery system ZMDBS was analyzed through the characteristic peaks presented on the spectra;

[0048] Step S24, determination of Fourier infrared spectrum:

[0049] Fourier transform infrared spectrometer was used to measure the different groups of nanomaterials at 500-4000cm -1 The infrared spectrum within the range was used to analyze the functional groups contained in ZMDBS through the characteristic peaks presented on the spectrum, and to confirm whether DOX was successfully encapsulated by the nanocarrier;

[0050] Step S25, observing the morphology of nanoparticles;

[0051] Step S26, X-ray diffraction measurement:

[0052] Use an X-ray diffractometer to perform X-ray diffraction on nanomaterials to obtain their diffraction patterns, and obtain the composition, crystal structure or morphology information of the materials through pattern analysis;

[0053] Step S27, elemental analysis XPS:

[0054] X-ray photoelectron spectroscopy (XPS) was used to qualitatively and quantitatively analyze the elemental composition of the nanomaterial ZMDBS.

[0055] Step S28, GSH responsiveness;

[0056] Step S29, active oxygen generation capability:

[0057] To verify that ZIF-67 and MnO2 in ZMDBS can produce reactive oxygen species and thus induce cell apoptosis, the reactive oxygen species production capacity of ZIF-67 / MnO2 was detected by TMB method;

[0058] Step S210, nanozyme catalytic activity:

[0059] Verify Co 2+ and Mn 2+ It can act as a Fenton-like catalyst to catalyze the generation of OH from hydrogen peroxide in the TNBC tumor microenvironment. The MB method was used to detect the OH generation ability of ZM;

[0060] Step S211, pH responsiveness;

[0061] Step S212, stability inspection:

[0062] Verify the stability of the nanodrug delivery system under physiological conditions.

[0063] Preferably, the step S21 further includes:

[0064] The particle size and potential of ZMDBS were measured by dynamic light scattering using an AntonPaar nanoparticle size and zeta potential analyzer;

[0065] Particle size determination: Take 1 mL of the solution containing ZMDBS in the particle size sample pool, and test the particle size and PDI of the nanomaterial on the machine. If the concentration of the nanomaterial to be tested is too high, it should be diluted;

[0066] Potential measurement: Place ZMDBS in a potential cell so that the solution can submerge the two metal sheets of the potential cell, and use a machine to detect the Zeta potential of the nanoparticles.

[0067] Preferably, the step S22 further comprises:

[0068] The content of DOX in ZMDBS was determined by UV-visible spectrophotometry. 2 mL of the ZMDBS solution after hydrolysis with hydrochloric acid was taken into a purple cuvette, and its absorbance at 480 nm was measured. The content of DOX was calculated according to the standard curve of DOX, and the drug loading and encapsulation efficiency of DOX were calculated as follows:

[0069]

[0070] Among them, M DOX It refers to the content of DOX successfully loaded on the nanocarrier ZMBS; M 总 Refers to the total mass of the nanomaterial ZMDBS; M add It refers to the mass of DOX added during the experiment.

[0071] Preferably, the step S25 further comprises:

[0072] Scanning electron microscopy and transmission electron microscopy are used to observe the microscopic morphology of nanomaterials;

[0073] SEM measurement: Place the nanomaterial to be tested on a microscope plate with conductive adhesive, spray gold with an ion sputtering instrument, and then load it into the sample chamber of a scanning electron microscope for observation;

[0074] TEM measurement: The nanomaterial to be tested was dispersed in 2 mL of ultrapure water and ultrasonicated for 30 min. Then 10 μL of the solution was dropped on a 300-mesh carbon support film and allowed to stand for 10 min. The excess liquid was then absorbed with filter paper. The solution to be tested was placed in the injection rod, which was then transferred to the TEM injection chamber for observation.

[0075] Preferably, the step S28 further comprises:

[0076] Step S281, verifying that ZMDBS has GSH responsiveness, using the characteristic that ZM has an obvious ultraviolet characteristic absorption peak at 300-400nm, using ZM to react with GSH of different concentrations, observing the ultraviolet absorption curve of ZM at 300-400nm, it can be preliminarily determined whether MnO2 reacts with GSH;

[0077] Step S282: In order to further verify the GSH responsiveness of ZM, the GSH consumption characteristics of ZM were evaluated by Ellman experiment;

[0078] Step S283: Using this principle, different concentrations of ZM were reacted with GSH (150 μM), and then DTNB (100 μM) was added to detect the amount of GSH remaining after the reaction, which can indirectly prove the GSH responsiveness of ZM.

[0079] Preferably, the step S211 further includes:

[0080] Take 2 portions of 3 mL of ZMDBS in 25KD renewable cellulose dialysis bags, seal them and place them in centrifuge tubes containing 30 mL of PBS buffer solutions of different pH values, and then incubate them in a 37°C shaking incubator. Take 200 μL of PBS buffer solution for testing at 0, 1, 2, 4, 6, 8, 12, 24, and 36 hours, respectively, and add an equal amount of PBS of the same pH. Measure its absorbance at 480 nm with a UV-visible spectrophotometer, convert the corresponding concentration according to the standard curve of DOX, and calculate the cumulative release rate of DOX. The formula is as follows:

[0081] CR (%) = (actually released DOX mass / total DOX mass loaded in the nanomaterial) * 100%.

[0082] A nanodrug delivery system based on tumor microenvironment response is applied to the treatment of triple-negative breast cancer.

[0083] (III) Beneficial effects

[0084] Compared with the prior art, the present invention provides a preparation method and application of a nano drug delivery system based on tumor microenvironment response, which has the following beneficial effects: the present invention has high stability, small toxic and side effects, good biocompatibility and tumor microenvironment TME responsiveness.

[0085] The present invention constructs a novel cobalt / manganese-based nanodrug delivery system ZMDBS that responds to the tumor microenvironment, and demonstrates the anti-TNBC efficacy of ZMDBS from multiple dimensions such as in vitro biological evaluation and in vivo biological evaluation. At the same time, the transcriptomics analysis method is used to successfully obtain the mechanism of action of ZMDBS inducing 4T1 cell apoptosis, providing a new reference idea for the clinical treatment of triple-negative breast cancer.

[0086] The present invention synthesizes MnO2 through redox reaction and synthesizes metal organic framework structure ZIF-67 through self-assembly; then ZIF-67 is used as a carrier to load the chemotherapy drug DOX, and MnO2 is added to relieve tumor hypoxia, BSA is added to increase the biocompatibility of the material, and the surfactant SDS is added to increase the stability of the material, and the cobalt / manganese-based nano drug delivery system ZMDBS is obtained through self-assembly.

[0087] The invention characterizes various properties of ZMDBS, such as particle size, potential, drug loading, encapsulation rate, functional group composition, morphology, crystal form, element composition, glutathione responsiveness, active oxygen generation capacity, pH responsiveness, drug sustained-release capacity and stability, by using various instruments, such as nanoparticle size and Zeta potential analyzer, ultraviolet spectrophotometer, Fourier transform infrared spectrometer, scanning electron microscope, transmission electron microscope, X-ray diffractometer, X-ray photoelectron spectrometer and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 It is a schematic diagram of the structure of the present invention.

[0089] Figure 2 Schematic diagram of the synthesis process of the nano drug delivery system of the present invention;

[0090] Figure 3 A schematic diagram of the characterization process of the nano drug delivery system of the present invention;

[0091] Figure 4 A schematic diagram of the specific process of synthesizing the nano drug delivery system of the present invention;

[0092] Figure 5 This is a diagram of the TMB experimental preparation of ZM of the present invention;

[0093] Figure 6 This is a diagram of the MB experimental preparation of the ZM of the present invention. DETAILED DESCRIPTION

[0094] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0095] Embodiment 1:

[0096] like Figure 1-6 As shown, a method for preparing a nano drug delivery system based on tumor microenvironment response comprises the following steps:

[0097] Step S1, synthesis of nano drug delivery system;

[0098] Step S11, preparation of nano-MnO2;

[0099] Take 0-5mL of KMnO4 solution, add 0-5mL of polyallylamine hydrochloride solution PAH, stir for 15-25min to make it fully react, the solution changes from purple to dark brown, then centrifuge with a 10kd ultrafiltration tube, and dilute the resulting concentrated solution to 5-10mL to obtain a prefabricated nano MnO2 solution;

[0100] Step S12, preparation of nano ZIF-67;

[0101] Take 0-1 mL of Co(NO3)2·6H2O methanol solution, add 0-5 mL of 2-methylimidazole methanol solution 2-MeIM, stir for 5-10 min to make it fully react, the solution turns blue-purple, stand for 6-12 h, then centrifuge, wash with methanol 1-3 times, and vacuum dry at 55-65 ° C for 6-12 h to obtain blue-purple powder, which is nano ZIF-67;

[0102] Step S13, preparation of ZIF-67 / MnO2 solution;

[0103] Dissolve ZIF-67 in ethanol, sonicate for 25-35 minutes, then add an appropriate amount of ultrapure water to obtain a 2 mg / mL ZIF-67 solution; take 0-1 mL of the above solution, add 0-1 mL of ultrapure water and mix thoroughly, then add 0-1 mL of nano MnO2 solution and stir for 1-5 minutes to obtain a ZIF-67 / MnO2 solution;

[0104] Step S14, preparation of ZMBS;

[0105] Add 1-3 mL of BSA to ZM, stir for 1-2 min, then add 0-1 mL of SDS, continue stirring for 0.5-1.5 h, centrifuge to remove the precipitate, and obtain a stable nano ZMBS solution, which is then refrigerated at 0-4 °C.

[0106] Step S15, preparation of ZMDBS;

[0107] Dissolve ZIF-67 in ethanol, sonicate for 15-35 minutes, and then add an appropriate amount of ultrapure water to obtain a 2 mg / mL ZIF-67 solution; take 0-1 mL of the above solution, add 0-1 mL of ultrapure water and mix thoroughly, then add 0-1 mL of nano MnO2 solution, stir for 1-5 minutes, then add 1-3 mL of doxorubicin hydrochloride DOX, 1-3 mL of bovine serum albumin, stir for 1-2 minutes, then add 0-1 mL of SDS, stir for 0.5-1 h, centrifuge to remove the precipitate, and obtain a stable nano ZMDBS solution, which is refrigerated in a refrigerator at 0-4°C.

[0108] Embodiment 2:

[0109] A method for preparing a nano drug delivery system based on tumor microenvironment response, further comprising the following steps:

[0110] Step S2, characterization of nano drug delivery system;

[0111] Step S21, particle size and potential measurement;

[0112] The particle size and potential of ZMDBS were measured by dynamic light scattering using an AntonPaar nanoparticle size and zeta potential analyzer;

[0113] Particle size determination: Take 1 mL of the solution containing ZMDBS in the particle size sample pool, and test the particle size and PDI of the nanomaterial on the machine. If the concentration of the nanomaterial to be tested is too high, it should be diluted;

[0114] Potential measurement: Put ZMDBS in a potential cell so that the solution can submerge the two metal sheets of the potential cell, and then test the Zeta potential of the nanoparticles on a machine;

[0115] Step S22, determination of drug loading and encapsulation efficiency;

[0116] The content of DOX in ZMDBS was determined by UV-visible spectrophotometry. 2 mL of the ZMDBS solution after hydrolysis with hydrochloric acid was taken into a purple cuvette, and its absorbance at 480 nm was measured. The content of DOX was calculated according to the standard curve of DOX, and the drug loading and encapsulation efficiency of DOX were calculated as follows:

[0117]

[0118] Among them, M DOX It refers to the content of DOX successfully loaded on the nanocarrier ZMBS; M 总 Refers to the total mass of the nanomaterial ZMDBS; M add It refers to the mass of DOX added during the experiment;

[0119] Step S23, measuring the UV-visible absorption spectrum;

[0120] Using a UV-visible spectrophotometer, the UV spectra of different groups of nanomaterials in the range of 200-800 nm were measured, and the synthesis of the nano drug delivery system ZMDBS was analyzed through the characteristic peaks presented on the spectra;

[0121] Step S24, determination of Fourier infrared spectrum;

[0122] Fourier infrared spectrometer was used to measure the infrared spectra of different groups of nanomaterials in the range of 500-4000. The functional groups contained in ZMDBS were analyzed through the characteristic peaks presented on the spectra, and it was also confirmed whether DOX was successfully encapsulated by the nanocarriers.

[0123] Step S25, observing the morphology of nanoparticles;

[0124] Scanning electron microscopy and transmission electron microscopy are used to observe the microscopic morphology of nanomaterials;

[0125] SEM measurement: Place the nanomaterial to be tested on a microscope plate with conductive adhesive, spray gold with an ion sputtering instrument, and then load it into the sample chamber of a scanning electron microscope for observation;

[0126] TEM measurement: The nanomaterial to be tested was dispersed in 2 mL of ultrapure water and ultrasonicated for 30 min. Then 10 μL of the solution was dropped on a 300-mesh carbon support film and allowed to stand for 10 min. The excess liquid was then absorbed with filter paper. The solution to be tested was placed in the injection rod, which was then transferred to the TEM injection chamber for observation.

[0127] Step S26, X-ray diffraction measurement;

[0128] Use an X-ray diffractometer to perform X-ray diffraction on nanomaterials to obtain their diffraction patterns, and obtain the composition, crystal structure or morphology information of the materials through pattern analysis;

[0129] Experimental steps: Freeze ZMDBS in a -20℃ refrigerator and then put it into a freeze dryer for freeze drying. Spread the freeze-dried powder sample into the sample slot, press it lightly to make it flat, and then test it.

[0130] Parameter settings: voltage 40kV, current 40mA, step length 0.02°, test speed 0.1 sec / step, copper target, incident ray wavelength 0.15406nm; scanning range 5-90°.

[0131] Step S27, element analysis XPS;

[0132] X-ray photoelectron spectroscopy (XPS) was used to qualitatively and quantitatively analyze the elemental composition of the nanomaterial ZMDBS.

[0133] Experimental steps: Freeze ZMDBS in a -20°C refrigerator and then put it into a freeze dryer for freeze drying. Stick the freeze-dried powder sample on aluminum foil with double-sided tape, press it lightly into a sheet, and then test it.

[0134] Parameter settings: monochromatic AlKa (hv = 1486.6eV), power 150W, 500μm diameter beam spot; binding energy calibrated with C1s284.8.

[0135] Step S28, GSH responsiveness;

[0136] Step S281, verifying that ZMDBS has GSH responsiveness, using the characteristic that ZM has an obvious ultraviolet characteristic absorption peak at 300-400nm, using ZM and different concentrations of GSH (15.63, 31.25, 62.5, 125, 250, 500, 700, 1000μM) to react, observing the ultraviolet absorption curve of ZM at 300-400nm, it can be preliminarily determined whether MnO2 reacts with GSH;

[0137] Step S282: In order to further verify the GSH responsiveness of ZM, the GSH consumption characteristics of ZM were evaluated by Ellman experiment;

[0138] Step S283, using this principle, reacting ZM with different concentrations and GSH (150 μM), and then adding DTNB (100 μM) to detect the amount of GSH remaining after the reaction, which can indirectly prove the GSH responsiveness of ZM;

[0139] The principle of the Ellman experiment is: the characteristic ultraviolet absorption peak of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is around 323nm. When GSH is added and incubated for half an hour, DTNB reacts with GSH to generate a yellow product TNB. The ultraviolet absorption peak of DTNB at 323nm decreases or even disappears, and the characteristic ultraviolet absorption peak of TNB begins to appear at 412nm.

[0140] Step S29, active oxygen generation capability;

[0141] In order to verify that ZIF-67 in ZMDBS can produce reactive oxygen species and thus induce cell apoptosis, the TMB method was used to detect the reactive oxygen species production ability of ZIF-67 / (hereinafter referred to as ZM);

[0142] 3,3',5,5'-Tetramethylbenzidine is a white powder TMB, which is a colorless solution after being dissolved in ethanol. In the free radical reaction, TMB will undergo a redox reaction with active oxygen, and the oxidized TMB will form an unstable divalent cation radical intermediate (divalent cation radical). Two TMB divalent cation radicals can further react in two ways:

[0143] Two TMB divalent cation radicals combine with each other to form a blue binuclear tetravalent cation radical (ox-TMB). The UV-visible characteristic absorption peaks of ox-TMB are 370nm and 652nm, and it is blue. The TMB divalent cation radical undergoes a redox reaction with hydrogen peroxide to form a yellow TMB divalent cation. The absorption of light by this substance is strongest at 450nm and it is yellow.

[0144] according to Figure 3 The reagent preparation scheme for testing samples is as follows: to carry out this experiment, first take 1.6 mL of 30% hydrogen peroxide, add 0.2 mL of TMB solution (10 mM) thereto, mix well, then add 0.150 mL of PBS, then add 0.050 mL of ZM solution, mix well, let stand for 5 min for full reaction, and obtain sample No. 3. Take 2 mL of the sample in a constant volume of UV cuvette, put it into the instrument, and use a UV-visible spectrophotometer to detect its spectrum at 350-750 nm. Similarly, the remaining samples can be modified according to the corresponding volumes in the table and tested according to the above steps.

[0145] Step S210, nanozyme catalytic activity;

[0146] To further confirm the catalytic activity of ZM nanozymes, CO 2+ and Mn 2+ It can act as a Fenton-like catalyst to catalyze the excess H2O2H in the TNBC tumor microenvironment to generate OH. The MB method was used to detect the OH generation ability of ZM.

[0147] Methylene blue is a phenothiazine salt that forms a blue solution when dissolved in water. Its UV-visible characteristic absorption peak is at 665nm. MB can be oxidized by OH to generate a colorless and transparent product (MB-OH), thereby reducing or even eliminating the absorption peak at 665nm. The ability of hydrogen peroxide to generate OH after reacting with ZM can be detected by a UV-visible spectrophotometer.

[0148] The reaction with MB needs to be carried out in a buffer system, and a buffer solution needs to be prepared. The preparation of the buffer solution is as follows: dissolve it in ethanol to prepare a solution with a concentration of 100 mM.

[0149] according to Figure 4 To prepare the reagents for the test sample, for this experiment, first take 196 and 1.3 mL of 30% hydrogen peroxide solution, mix them thoroughly, let them stand for 1 hour to allow them to react fully, and obtain sample No. 3. Take 2 mL of the sample in a constant purple cuvette, put it into the instrument, and use a UV-visible spectrophotometer to detect its spectrum at 200-800 nm. Similarly, the remaining samples can be modified according to the table. The corresponding volume and tested according to the above steps.

[0150] Step S211, pH responsiveness;

[0151] The pH-responsive nano drug delivery system should be stable under normal physiological conditions (pH=7.4), but degrade and release the internal drug under the tumor microenvironment (pH=5.5). To this end, the present invention studies the drug release curve of the ZMDBS nano drug delivery system at different pH values ​​over time.

[0152] Take 2 portions of 3 mL of ZMDBS in 25KD renewable cellulose dialysis bags, seal them and place them in centrifuge tubes containing 30 mL of PBS buffer solutions of different pH values, and then incubate them in a 37°C shaking incubator. Take 200 μL of PBS buffer solution for testing at 0, 1, 2, 4, 6, 8, 12, 24, and 36 hours, respectively, and add an equal amount of PBS of the same pH. Measure its absorbance at 480 nm with a UV-visible spectrophotometer, convert the corresponding concentration according to the standard curve of DOX, and calculate the cumulative release rate of DOX. The formula is as follows:

[0153] CR (%) = (actually released DOX mass / total DOX mass loaded in nanomaterials) * 100%;

[0154] Step S212, stability inspection;

[0155] Verify the stability of the nanodrug delivery system under physiological conditions.

[0156] Combined with Examples 1 and 2, the results are as follows:

[0157] Firstly, nano MnO2 was synthesized through redox reaction, and characterized by UV-visible spectrophotometer, nanoparticle size and Zeta potential analyzer, transmission electron microscope, X-ray diffractometer and other analytical instruments. KMnO4:PAH (m / m) = 1:2 was determined as the optimal feed ratio for synthesizing nano MnO2. Subsequently, the average particle size of nano MnO2 was determined to be 76.39±2.43nm, the average potential was +20.7±2.3mV, and the XRD phenotype of MnO2 was λ-MnO2. At the same time, the metal organic framework structure ZIF-67 is synthesized by self-assembly, and is characterized by transmission electron microscope, nanoparticle size and Zeta potential analyzer, scanning electron microscope, Fourier infrared spectrometer, X-ray diffractometer and other analytical instruments, and 2-MIM: Co(N03)2·6H20(n / n)=40:1 is determined as the optimal feed ratio for synthesizing nano ZIF-67, and then the structure of ZIF-67 is determined to be a polyhedral structure with good crystal form, and its average particle size is 228.9±15.45nm, and the potential is +6.29mV. Then, the present invention uses ZIF-67 as a carrier, loads the chemotherapy drug DOX, and adds MnO2 to relieve tumor hypoxia, adds BSA to increase the biocompatibility of the material, and adds the surfactant SDS to increase the stability of the material, and obtains the cobalt / manganese-based nano drug delivery system ZMDBS through self-assembly. The present invention measures the standard curve of DOX by UV-spectrophotometer, and further determines that the encapsulation rate of DOX in ZMDBS is 95.03±2.27%; the drug loading is 11.40±0.28%. Subsequently, TEM, potential, infrared and UV characterization of ZMDBS are carried out, and it is determined that DOX is successfully encapsulated by ZMBS, and the morphology of the obtained ZMDBS is spherical, the particle size is between 100-200nm, and the potential is -12.45mV. Then, the present invention performs elemental analysis of ZMDBS by XPS technology. The results show that ZMDBS is mainly composed of carbon (C) element, oxygen (O) element, nitrogen (N) element, manganese (Mn) element and cobalt (Co) element, which confirms that ZMDBS contains Mn2+ and Co2+, laying a theoretical foundation for subsequent detection of active oxygen generation capacity, identification of nanozyme activity, etc.

[0158] Next, the present invention evaluates the GSH responsiveness, reactive oxygen generation ability, nanozyme catalytic activity, and pH responsiveness of ZIF-67 / MnO2, and the results show that ZIF-67 / MnO2 has good GSH responsiveness, reactive oxygen generation ability, nanozyme catalytic activity, pH responsiveness and other properties. Finally, the present invention evaluates the stability of ZMDBS, and the results show that the ZMDBS nano solution is stable in nature within 7 days and no precipitation occurs. In summary, the present invention successfully synthesizes a cobalt / manganese-based nano drug delivery system ZMDBS with suitable particle size potential, good stability, and tumor microenvironment responsiveness, providing strong support and a solid foundation for subsequent biological evaluation.

[0159] Embodiment 3:

[0160] Step S3, evaluating the pharmacodynamic characteristics of the anticancer nano-drug delivery system through in vivo and in vitro experiments;

[0161] Step S31, analyzing the pharmacodynamic characteristics of the organism in vitro;

[0162] Using laser confocal microscopy, flow cytometer, inverted fluorescence microscope, microplate reader and other instruments, we investigated the cell uptake behavior, anti-4T1 efficacy, cell safety, reactive oxygen species production, cell apoptosis, endocytosis mechanism, cell migration inhibition effect and other in vitro properties of ZMDBS from the perspective of in vitro biology.

[0163] Step S311, in vitro experiment based on nano drug delivery system ZMDBS;

[0164] Step S3111, cell culture: mouse triple-negative breast cancer cells 4T1 and human normal liver cells LO2 were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% double antibody. The cells were cultured in an incubator at 37° C. and 5% CO 2 .

[0165] Step S3112, cell recovery: Take out the frozen cell line from the liquid nitrogen tank, immediately place it in a constant temperature water bath at 37°C, and shake it gently. During the process, be careful to avoid the tip of the freezing tube from touching the liquid surface. After the cells are thawed, transfer the cells to a centrifuge tube on a sterile operating table, centrifuge at 1000r / min for 5min, and then transfer to a sterile cell culture dish. Gently shake to evenly disperse the cells and place them in a 37°C, 5% CO2 incubator for culture.

[0166] Step S3113, cell passage: When the cells are spread to 70% to 80% density in the culture dish, they can be passaged. Take out the cells in the incubator and put them in the clean bench, discard the original culture solution, add 3mL of PBS, shake gently, wash twice, discard PBS, add pancreatin after warming to 37°C, mix evenly, so that it covers the entire culture dish, and then put it in a 37°C incubator for incubation for 2min. After digestion, the cells become round and can be detached by gently blowing. Then immediately add 3mL of RMPI-1640 complete medium, mix evenly, transfer the cells to a centrifuge tube immediately, centrifuge at 1000r / min for 5min, discard the supernatant, add 1mL of complete medium to resuspend, and perform cell passage at a ratio of 1:5. The cell suspension is divided into 5 culture dishes containing 10mL of RPMI-1640 complete culture solution, evenly dispersed, and placed in a 37°C, 5% CO2 incubator for culture, which is the passage process.

[0167] Step S3114, cell freezing: When the cells are spread to 70% to 80% density in the culture dish, they can be frozen. Take out the cells in the incubator and put them in the clean bench, discard the original culture medium, add 3mL of PBS, shake gently, wash twice, discard the PBS, add trypsin that has been warmed to 37°C, mix well so that it covers the entire culture dish, and then put it in a 37°C incubator for incubation for 2 minutes. After digestion, the cells become round and can be detached by gently blowing. Then immediately add 3mL of RMPI-1640 complete culture medium, mix well, and immediately transfer the cells to a centrifuge tube. Centrifuge at 1000r / min for 5 minutes, discard the supernatant, add freezing solution containing 10% DMSO and 90% FBS, resuspend the cells, and then transfer the cells to cryopreservation tubes, mark them, and perform gradient freezing. First, store the cryopreservation tubes at 4°C;

[0168] Step S3115, evaluation of cell uptake behavior: effective cell uptake is a prerequisite for the efficacy of nanomaterials. To study the uptake of nano ZMDBS in 4T1 cells, 4T1 cells in the logarithmic growth phase were plated in a confocal dish at a density of 5.0×104 / dish, and cultured overnight in a 37°C, 5% CO2 incubator. After observing that they were completely attached to the wall under a microscope, the culture medium was discarded, and a complete culture medium containing the same concentration of nano ZMDBS (2.5 μg / mL, based on DOX concentration) was added to each well. After incubation in the incubator for 3h, 6h, and 9h, the culture medium was discarded, and P was added to each well. The cells were washed twice with BS, and then fixed with 4% paraformaldehyde solution for 15 minutes. The paraformaldehyde was then discarded and the cells were washed twice with PBS. Subsequently, according to the instructions of the DAPI kit, the prepared DAPI solution (a nuclear dye) was added for staining for 10 minutes. The cells were then washed twice with PBS. Finally, 500 μL of PBS solution was added to each well to keep the cells moist. The fluorescence intensity of 4T1 cells under different incubation times with nano-ZMDBS was observed under a laser confocal microscope (CLSM). Quantitative analysis of uptake at different times: 4T1 cells in the logarithmic growth phase were plated in a 12-well plate at a density of 1.0×105 / well, and cultured overnight in a 37°C, 5% CO2 incubator. After observing their complete attachment under a microscope, complete culture medium containing the nanodrug delivery system ZMDBS (2.5 μg / mL, based on DOX concentration) with the same DOX concentration was added at different time periods. After incubation in the incubator for 3h, 6h, and 9h, the culture medium was discarded, the cells were digested with trypsin, washed twice, transferred to a flow tube, and the cells were quantitatively analyzed using a flow cytometer. Quantitative analysis of drug uptake at different drug concentrations: 4T1 cells in the logarithmic growth phase were plated in a 12-well plate at a density of 1.0×105 / well, and cultured overnight in a 37°C, 5% CO2 incubator. After observing their complete adhesion under a microscope, complete culture medium containing the nano-drug delivery system ZMDBS with different DOX concentrations (1, 2, 3, 4, 5 μg / mL, based on DOX concentration) was added, and the cells were incubated in an incubator for 6 hours, then the culture medium was discarded, the cells were digested with trypsin, washed twice, transferred to a flow tube, and the cells were quantitatively analyzed using a flow cytometer.

[0169] Step S3116, live / dead cell staining experiment: The live / dead cell staining experiment can intuitively feel the toxicity or therapeutic effect of the drug on the cell in the form of pictures, and is one of the important indicators of the in vitro biological evaluation of nanomedicines. The live / dead cell staining experiment of nanomaterials was performed using a cell activity and toxicity detection kit. The 4T1 cells (mouse breast cancer cells) that had grown to the logarithmic growth phase and were in good condition were digested with trypsin and inoculated into a 24-well plate at a density of 4×104 cells / well, and then the well plate was placed in a 37°C, 5% CO2 incubator and allowed to stand overnight to allow the cells to adhere to the wall. Then, RPMI-1640 complete medium was used to dilute DOX, ZMBS, and ZMDBS to 5μg / mL, 38.85μg / mL, and 43.85μg / mL (the corresponding DOX concentrations were all 5μg / mL). The old culture solution in the well plate was discarded, and the unattached cells were washed with PBS, and then a new culture solution containing the above-mentioned liquid was added, and placed in a 37°C, 5% CO2 incubator and allowed to stand for 24h. After the drug solution acted on 4T1 cells for 24 hours, the culture medium was discarded and the diluted Calcein-AM&PI mixed solution was added to the 4T1. After 30 minutes of action, the excess fluorescent dye was washed away with PBS, and then the well plate was placed under an inverted fluorescence microscope for observation and photographing of the cell staining.

[0170] Step S3117, in vitro efficacy experiment: The MTT method was used to examine the effects of different groups (Control, DOX, ZMBS, ZMDBS) on the viability of mouse breast cancer cells 4T1, and to evaluate the in vitro efficacy of ZMDBS. The principle of the MTT method is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorption value is measured at a wavelength of 490nm or 570nm using an enzyme marker, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells. Take 4T1 cells in logarithmic growth phase and in good condition, and inoculate them in a 96-well plate at a density of 4×103 cells / well, 100 μL per well, set the zeroing well A0 at the same time, and fill the edge wells of the 96-well plate with sterile PBS. After the cells were placed in an incubator and incubated overnight, the supernatant in the wells was discarded, and free DOX and nano ZMDBS solutions containing DOX concentrations of 20.00, 10.00, 5.00, 2.50, 1.25, 0.63, 0.31, 0.16, and 0.08 μg / mL were added to each well, and the corresponding concentration of ZMBS solution was added at the same time. A control group (Control group) was set up, and the same volume of RPMI-1640 complete medium was added. A parallel group (n=3) was set up, and the 96-well plate was placed in an incubator and incubated for 24 hours, and the supernatant was discarded, and 100 μL of MTT solution (concentration of 0.5 mg / mL) was added to each well, and then the supernatant was discarded, and 150 μL of DMSO was added to each well, and the cells were shaken at a low speed on a shaker for 10 minutes to fully dissolve the crystals. The absorbance (A) at 570 nm was detected using an enzyme marker, and the cell survival rate was calculated according to the formula, which is as follows:

[0171]

[0172] Among them, A sample refers to the absorbance of the sample group, A0 refers to the absorbance of the zero-adjusting well, and A control Refers to the absorbance of the control group.

[0173] Step S3118, cell safety experiment: The MTT method was used to examine the effects of different groups (Control, DOX, ZMBS, ZMDBS) on the viability of human normal liver cells LO2, so as to compare the cytotoxicity of ZMDBS and DOX. Take LO2 cells in the logarithmic growth phase and inoculate them in a 96-well plate at a density of 8×103 cells / well, 100 μL per well, set the zero well A0 at the same time, and fill the edge wells of the 96-well plate with sterile PBS. After the cells were placed in an incubator and incubated overnight, the supernatant in the wells was discarded, and free DOX and nano ZMDBS solutions containing DOX concentrations of 10.00, 5.00, 2.50, 1.25, 0.63, 0.31, 0.16, and 0.08 μg / mL were added to each well. At the same time, the corresponding concentration of ZMBS solution was added to set up a control group (Control group), and the same volume of RPMI-1640 complete medium was added to set up a parallel group (n=3). After the 96-well plate was placed in an incubator for 24 hours, the supernatant was discarded, and 100 μL of MTT solution (concentration of 0.5 mg / mL) was added to each well, and then the supernatant was discarded, and 150 μL of DMSO was added to each well, and the cells were shaken at a low speed on a shaker for 10 minutes to fully dissolve the crystals. The absorbance (A) at 570 nm was detected using an enzyme marker, and the cell survival rate was calculated according to the formula.

[0174] Step S3119, investigation of reactive oxygen generation ability: The DCFH-DA fluorescent probe is used to detect the level of reactive oxygen generated by the material. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to generate DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. The reactive oxygen in the cell can oxidize the non-fluorescent DCFH to generate fluorescent DCF. By detecting the fluorescence of DCF, the level of reactive oxygen in the cell can be known. Breast cancer cells 4T1 were inoculated in a 24-well plate at a density of 5.0×104 cells / well and left to stand in an incubator for 12 hours. DOX, ZMBS, and ZMDBS were diluted to 3μg / mL, 23.32μg / mL, and 26.32μg / mL (the corresponding DOX concentrations were all 3μg / mL) using RPMI-1640 complete medium. The old culture medium in the well plate was discarded, and the non-adherent cells were washed with PBS, and then new culture medium containing the above-mentioned drug solution was added, and placed in a 37°C 5% CO2 incubator for 24 hours. After the drug solution acted on 4T1 cells for 24 hours, the culture medium was discarded, and the diluted DCFH-DA fluorescent probe was added to 4T1. After 30 minutes, it was washed 3 times with PBS, and then the cells were infiltrated with PBS. The well plate was placed under an inverted fluorescence microscope for observation and the cell staining was photographed. Using the same experimental method, the fluorescence intensity of ROS was quantitatively analyzed using a flow cytometer.

[0175] Step S3120, apoptosis experiment: Annexin V-FITC / PI apoptosis detection kit was used to investigate the effects of different groups (Control, DOX, ZMBS, ZMDBS) on the apoptosis of mouse breast cancer cell 4T1. Annexin V selectively binds to phosphatidylserine (PS). PS is mainly distributed on the inner side of the cell membrane. In the early stage of cell apoptosis, different types of cells will turn PS outward to the cell surface, that is, the outer side of the cell membrane. When PS is exposed to the cell surface, it promotes coagulation and inflammatory response. Annexin V can block the procoagulant and proinflammatory activity of PS after binding to PS everted to the cell surface. Annexin V-FITC, which is labeled with the green fluorescent probe FITC, can be used to detect the eversion of phosphatidylserine, an important feature of apoptosis, very simply and directly using a flow cytometer or a fluorescence microscope. Propidium iodide (PI) can stain necrotic cells or cells that have lost cell membrane integrity in the late stage of apoptosis, showing red fluorescence. For necrotic cells, since the integrity of the cell membrane has been lost, Annexin V-FITC can enter the cytoplasm and bind to the phosphatidylserine located on the inner side of the cell membrane, thereby also making the necrotic cells present green fluorescence. Based on the above principle, 4T1 cells in the logarithmic growth phase were taken and plated in a 12-well plate at a density of 1.0×105 / well, and cultured overnight in a 37°C, 5% CO2 incubator. After observing that they were completely attached to the wall under a microscope, DOX, ZMBS, and ZMDBS were diluted to 2.50μg / mL, 19.43μg / mL, and 21.93μg / mL (the corresponding DOX concentration was 2.50μg / mL) using RPMI-1640 complete medium. The old culture medium in the well plate was discarded, and the unattached cells were washed with PBS, and then the new culture medium containing the above-mentioned drug solution was added, and a blank control group was set up, and RPMI-1640 complete medium without drug solution was added. Place the cells that have been replaced with culture medium in a 37°C, 5% CO2 incubator for 12 hours. After the drug solution has acted on the 4T1 cells for 12 hours, aspirate the cell culture medium in the well plate into a suitable centrifuge tube, mark it, wash the adherent cells once with PBS, and add an appropriate amount of trypsin cell digestion solution (which may contain EDTA) to digest the cells. Incubate at room temperature until the adherent cells can be blown off by gently blowing, add the culture medium in the corresponding centrifuge tube to each well, blow gently to completely digest the cells, then transfer to the corresponding centrifuge tube, centrifuge (1000r / min, 5min), pour off the supernatant, add 195μL of the binding solution in the kit to resuspend the cells, and add 5μL of Annexin V-FITC and 10μL of PI, and mix gently.Incubate at room temperature in the dark for 10-20 min, then perform detection using a flow cytometer.

[0176] Step S3121, study of endocytosis mechanism: In order to study the endocytosis form of ZMDBS entering cells, several common endocytosis inhibitors were used for investigation: ① Chlorpromazine hydrochloride (CPZ for short): clathrin-mediated endocytosis pathway inhibitor. ② Amiloride hydrochloride (AMI for short): macropinocytosis endocytosis pathway inhibitor. ③ Genistein (GEN for short): caveolin-mediated endocytosis pathway inhibitor. ④ 4°C: Inhibition of energy-dependent endocytosis pathway. Based on the above principle, 4T1 cells in the logarithmic growth phase were taken and plated in a 12-well plate at a density of 1.0×105 / well, cultured overnight in a 37°C, 5% CO2 incubator, and observed under a microscope that they were completely attached to the wall, and then divided into 5 groups: blank control group (Control group), chlorpromazine hydrochloride group, genistein group, amiloride hydrochloride group, 4°C group, and 3 duplicate wells were set. Each group was added with 1 mL of RPM I-1640 unmixed culture medium without serum, RPM I-1640 unmixed culture medium containing 50 μM chlorpromazine hydrochloride, RPMI-1640 unmixed culture medium containing 200 μM genistein, and RPM I-1640 unmixed culture medium containing 25 μM amiloride hydrochloride, respectively, and incubated in the incubator for 2 h. The 4°C group was added with RPM I-1640 unmixed culture medium without serum and incubated in a 4°C refrigerator for 2 h. After the incubation, the culture medium was aspirated, and 1 mL of ZMDBS solution diluted to 21.93 μg / mL with RPM I-1640 complete culture medium was added to each well and incubated for 3 h. After the incubation, the culture medium was discarded, washed twice with PBS, digested with trypsin, and the cells were collected in a flow tube, and the DOX fluorescence intensity was immediately detected using a flow cytometer.

[0177] Step S3122, evaluation of the inhibitory effect on cell migration: Cell migration is one of the basic functions of normal cells, and the cell migration of tumor cells is more significant. The cell scratch test is a simple and economical in vitro experimental method to study the effect of cell migration. The principle is: when the cells grow to fuse into a monolayer state, an artificial blank area is created on the fused monolayer of cells, called a "scratch". The cells in the central part are artificially removed, and then the culture is continued. After a certain period of time, the cells at the edge of the scratch will leave the crowded area and gradually enter the blank area, thereby healing the "scratch". The experimenter observes whether the surrounding cells migrate to the central scratch area to judge the growth and migration ability of the cells. The scratch experiment simulates the process of cell migration in vivo to a certain extent.

[0178] Based on the above principle, first use a marker to draw a horizontal line every 0.5-1cm behind each well of a 6-well plate, so that there are 3 horizontal lines behind each well. Then take 4T1 cells in the logarithmic growth phase, spread them in a 6-well plate at a density of 2.0×105 / well, and culture them overnight in a 37°C, 5% CO2 incubator. When the fusion rate reaches 100% under a microscope, use a 200μL gun tip perpendicular to the cell plane to scratch the cell layer along the line drawn with a marker on the back of the plate the day before. After the scratching is completed, discard the old culture medium and wash the cells 3 times with sterile PBS to remove non-adherent cells so that the scratches left by the scratching are clearly visible. Then use serum-free RPM I-1640 culture medium to dilute DOX, ZMBS, and ZMDBS to 2.50μg / mL, 19.43μg / mL, and 21.93μg / mL, respectively (the corresponding DOX concentrations are all 2.50μg / mL). Four groups were set up, including Control group, DOX group, ZMBS group and ZMDBS group, and 2 mL of serum-free culture medium (Control group), serum-free culture medium containing DOX (DOX group), serum-free culture medium containing ZMBS (ZMBS group), and serum-free culture medium containing ZMDBS (ZMDBS group) were added respectively. At 0 h and 24 h, the cells at the scratch site were observed under an inverted fluorescence microscope and photographed.

[0179] Step S32: Analyze the biological efficacy characteristics in vivo:

[0180] The in vivo biological evaluation of ZMDBS was carried out by constructing a subcutaneous triple-negative breast cancer tumor model in Balb / c mice. The in vivo biological evaluations of ZMDBS in triple-negative breast cancer tumor model mice were investigated, including in vivo safety evaluation, tissue distribution, therapeutic effect, pathological evaluation, ability to promote tumor cell apoptosis and ability to inhibit tumor cell proliferation.

[0181] Step S321, in vivo experiment based on nano drug delivery system ZMDBS;

[0182] Step S3211, hemolysis experiment: blood was collected from the eyeball of the mouse, placed in a 5 mL anticoagulant tube, mixed thoroughly to prevent coagulation, and then centrifuged (1500 rpm, 4°C, 15 min), the supernatant was discarded, and the red blood cell sediment at the bottom was washed 3 times with saline (0.9% NaCl solution). The washed red blood cells were resuspended in saline to prepare a 2% red blood cell suspension (v / v). ZMDBS was prepared into 5 concentration gradients (based on the concentration of DOX) with saline: 20, 40, 60, 80, 100 μg / mL, and a positive control group and a negative control group were set up at the same time. Three parallel groups (n=3) were set up for all groups. The sample addition details of the sample group, positive control group, and negative control group are as follows. Each group of samples was placed in a 37°C incubator for 2 hours, and then centrifuged (10,000 rpm, 4°C, 15 minutes) to precipitate the nanomaterials. The supernatant was collected and diluted 1-fold with physiological saline. The absorbance of each group at 540 nm was detected using an enzyme marker, and the hemolysis rate was calculated according to the following formula:

[0183]

[0184] Among them, A sample is the absorbance of the ZMDBS sample set, A negative is the absorbance of the negative control group, A positive is the absorbance of the positive control group.

[0185] Step S3212, construction of triple-negative breast cancer subcutaneous tumor model: The triple-negative breast cancer cells 4T1 in the logarithmic growth phase and in good condition were digested with trypsin and counted with a cell counter. The cell concentration was adjusted to 6×106 / mL with PBS. The PBS solution containing cells was divided into sterile EP tubes according to this concentration and placed in an ice box for standby use. Female BALB / c with a growth cycle of about 6 weeks were raised in a sterile barrier environment for 7 days to adapt to the environment. Then, the cells were inoculated into the subcutaneous part of the right hind buttocks of the mouse at a dose of 100 μL of PBS solution containing cells per mouse (i.e., 600,000 cells per mouse). After the syringe was drawn out, the injection site was pressed with a sterile cotton swab for about ten seconds to prevent leakage. The growth of mouse tumors was observed daily, and when the tumor volume grew to about 100 mm3, it could be used for experimental research on subcutaneous tumor models.

[0186] Step S3213, drug tissue distribution study: A small animal fluorescence luminescence three-dimensional in vivo imaging system was used to detect the distribution and metabolism of drugs in mice. Since the fluorescence of DOX in mice is weak, indocyanine green (ICG) with stronger fluorescence was used instead of DOX to study the drug tissue distribution of ZMDBS in vivo. The synthesis method of ICG-ZMBS refers to the synthesis method of ZMDBS, only DOX was changed to ICG, and the concentration was still 2 mg / mL.

[0187] (1) Investigating the tissue distribution of ZMDBS at different time points in living animals

[0188] Ten female BALB / c mice with tumor volumes of about 100 mm3 were selected, fasted for 24 hours in advance, and then divided into two groups, 5 in each group, respectively, the ICG group and the ICG-ZMBS group. The ICG group mice were injected with 100 μL of ICG solution at a dose of 7.5 mg / kg through the tail vein. Similarly, the ICG-ZMBS group mice were injected with 100 μL of ICG-ZMBS with the same dose of ICG through the tail vein. Then, the drug distribution in the 10 mice was recorded at 3h, 6h, 12h and 24h using the small animal fluorescence luminescence three-dimensional living imaging system, and the fluorescence intensity of the tumor site in the mice was quantified using the Living Image software.

[0189] (2) Investigate the tissue distribution of ZMDBS in ex vivo organs and tumor tissues at different time points

[0190] Twenty-four female BALB / c mice with tumor volumes of about 100 mm3 were selected, fasted for 24 hours in advance, and then divided into 8 groups, 3 mice in each group, namely ICG (3h) group, ICG (6h) group, ICG (12h) group, ICG (24h) group, ICG-ZMBS (3h) group, ICG-ZMBS (6h) group, ICG-ZMBS (12h) group, and ICG-ZMBS (24h) group. The mice in the ICG group were injected with 100 μL of ICG solution at a dose of 7.5 mg / kg through the tail vein. Similarly, the mice in the ICGZMBS group were injected with 100 μL of ICG-ZMBS with the same dose of ICG through the tail vein. The ICG (3h) group and the ICG-ZMBS (3h) group were killed immediately 3h after administration and the mouse heart, liver, spleen, lung, kidney and tumor tissues were obtained by dissection. The organs were washed with physiological saline, the floating blood on the surface was removed, and they were placed on black cardboard in order and placed in the observation room of the small animal fluorescence luminescence three-dimensional living imaging system to record the fluorescence distribution of the isolated organs and tissues. The other groups were killed and dissected at 6h, 12h and 24h, respectively, and the fluorescence distribution of the isolated organs and tissues was recorded using the small animal fluorescence luminescence three-dimensional living imaging system. The fluorescence intensity of the isolated organs and tissues was quantified using the Living Image software.

[0191] Step S3214, in vivo pharmacodynamic evaluation: 20 female BALB / c mice with tumor size of about 100 mm3 were selected and randomly divided into 4 groups, namely, saline group (Control group), ZMBS group, DOX group and ZMDBS group, with 5 mice in each group. Day 0 was counted from the first tail vein administration, the dosage was 1.0 mg / kg, and the drug was given once every 2 days, for a total of 3 times. Starting from day 0, the weight of each mouse was weighed and recorded every 2 days, and the changes in the tumor volume of mice in each experimental group were observed. The length and width of the tumor were measured with a vernier caliper, and the mouse tumor volume was calculated according to the following formula:

[0192] V=1 / 2*L*W 2

[0193] Among them, V represents the volume of the mouse tumor, L represents the length of the tumor, and W represents the width of the tumor.

[0194] After the body weight and tumor volume were recorded until the 12th day, blood was collected from the mouse eyeballs and the mice were killed by cervical dislocation. The mice were dissected, the tumor masses were removed and weighed, and the heart, liver, spleen, lung, kidney and tumor were placed in EP tubes containing 4% paraformaldehyde and stored in a 4°C refrigerator for subsequent experiments.

[0195] Step S3215, blood routine index examination: on the last day, blood was collected from the eyeballs of the mice in each group in 4.2.4 and placed in an anticoagulant tube with its own anticoagulant, and gently mixed to allow the anticoagulant to fully contact the anticoagulant tube. Subsequently, various blood routine indexes were tested using a fully automatic blood analyzer.

[0196] Step S3216, liver and kidney function index assessment: on the last day, blood was collected from the eyeballs of the mice in each group in 4.2.4 and placed in a sterile EP tube. After being placed at room temperature for 2 hours, the blood was centrifuged at 3000 rpm / min at 2-8°C for 15 minutes. The precipitate was discarded and the supernatant was placed in an automatic biochemical analyzer for detection.

[0197] Step S3217, hematoxylin-eosin (H&E) staining: After killing the mice in each group in 4.2.4, the heart, liver, spleen, lung, kidney and tumor tissues were taken and sent to Wuhan Saiweier Biotechnology Co., Ltd. for hematoxylin-eosin (H&E) staining. The specific experimental steps are as follows:

[0198] 1. Sampling: Fix fresh tissue with fixative for more than 24 hours. Take the tissue out of the fixative and trim the target area with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in a dehydration box.

[0199] 2. Dehydration and wax dipping: Place the dehydration box into the dehydrator and dehydrate with graded alcohols in sequence. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65℃ for 1 hour, paraffin II melted at 65℃ for 1 hour, paraffin III melted at 65℃ for 1 hour.

[0200] 3. Embedding: Embed the wax-soaked tissue in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool it in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0201] 4. Slice: Place the trimmed wax block in a -20℃ freezer to cool, and then place the cooled wax block in a paraffin slicer to slice it, with a thickness of 4μm. Float the slices on the 40℃ warm water of the slicer to flatten the tissue, pick up the tissue with a slide, and bake the slices in a 60℃ oven. After the wax is baked dry, take it out and store it at room temperature for later use.

[0202] 5. Dewax the paraffin sections to water: put the sections into xylene I for 20 minutes; xylene II for 20 minutes; anhydrous ethanol I for 5 minutes; anhydrous ethanol II for 5 minutes; 75% alcohol for 5 minutes, and wash with tap water.

[0203] 6. Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0204] 7. Eosin staining: Dehydrate the sections in 85% and 95% graded alcohol for 5 minutes each, and then stain them in eosin solution for 5 minutes.

[0205] 8. Dehydration and sealing: Put the slices into anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene I for 5 minutes, xylene II for 5 minutes until transparent, and seal with neutral gum.

[0206] 9. Microscope examination, image acquisition and analysis.

[0207] Step S3218, TUNEL immunofluorescence staining:

[0208] After the mice in each group in 4.2.4 were killed, the tumor tissues were sent to Wuhan Saiweier Biotechnology Co., Ltd. for TUNEL fluorescence staining. The specific experimental steps are as follows:

[0209] 1. Sampling: Fix fresh tissue with fixative for more than 24 hours. Take the tissue out of the fixative and trim the target area with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in a dehydration box.

[0210] 2. Dehydration and wax dipping: Place the dehydration box into the dehydrator and dehydrate with graded alcohols in sequence. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65℃ for 1 hour, paraffin II melted at 65℃ for 1 hour, paraffin III melted at 65℃ for 1 hour.

[0211] 3. Embedding: Embed the wax-soaked tissue in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool it in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0212] 4. Slice: Place the trimmed wax block in a -20℃ freezer to cool, and then place the cooled wax block in a paraffin slicer to slice it, with a thickness of 4μm. Float the slices on the 40℃ warm water of the slicer to flatten the tissue, pick up the tissue with a slide, and bake the slices in a 60℃ oven. After the wax is baked dry, take it out and store it at room temperature for later use.

[0213] 5. Dewax the paraffin sections to water: put the sections into xylene I for 10 minutes, xylene II for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, and wash with distilled water. (The dewaxing time should be appropriately extended in winter)

[0214] 6. Proteinase K repair: After the slices are slightly dried, use a tissue pen to draw a circle around the tissue (to prevent the liquid from flowing away), add proteinase K working solution to the circle to cover the tissue, and incubate in a 37°C incubator for 22 minutes. Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, each time for 5 minutes. (Protease K working solution preparation method, stock solution: PBS = 1:9)

[0215] 7. Membrane permeabilization: After the slices are slightly dried, add the membrane permeabilization working solution to cover the tissue in the circle, incubate at room temperature for 20 minutes, place the slides in PBS (pH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. (The membrane permeabilization solution is 0.1% triton, the preparation method is triton stock solution: PBS = 1:1000)

[0216] 8. Room temperature balance: After the slices are slightly dried, add buffer to cover the tissue and incubate the buffer at room temperature for 10 minutes.

[0217] 9. Add reaction solution: according to the number of slices and tissue size, take an appropriate amount of TDT enzyme, dUTP, and buffer in the TUNEL kit and mix them in a ratio of 1:5:50. Add them to the circle to cover the tissue. Lay the slices flat in a humidified box and incubate them in a 37°C constant temperature box for 2 hours. Add a small amount of water to the humidified box to maintain humidity.

[0218] 10. DAPI counterstaining of cell nuclei: Wash the sections with PBS (pH 7.4) for 3 times, 5 min each time. After removing PBS, add DAPI staining solution in the circle and incubate at room temperature for 10 min away from light.

[0219] 11. Sealing: Place the slides in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time. Slightly dry the sections and seal them with anti-fluorescence quenching sealing medium.

[0220] 12. Microscopic examination and photography: Observe the sections under a fluorescence microscope and collect images. (DAPI ultraviolet excitation wavelength is 330-380nm, emission wavelength is 420nm, emitting blue light; FITC excitation wavelength is 465-495nm, emission wavelength is 515-555nm, emitting green light.

[0221] Step S3219, Ki67 immunohistochemical staining: After killing the mice in each group in 4.2.4, the tumor tissues were sent to Wuhan Saiweier Biotechnology Co., Ltd. for Ki67 staining. The specific experimental steps are as follows:

[0222] 1. Sampling: Fix fresh tissue with fixative for more than 24 hours. Take the tissue out of the fixative and trim the target area with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in a dehydration box.

[0223] 2. Dehydration and wax dipping: Place the dehydration box into the dehydrator and dehydrate with graded alcohols in sequence. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65℃ for 1 hour, paraffin II melted at 65℃ for 1 hour, paraffin III melted at 65℃ for 1 hour.

[0224] 3. Embedding: Embed the wax-soaked tissue in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool it in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0225] 4. Slice: Place the trimmed wax block in a -20℃ freezer to cool, and then place the cooled wax block in a paraffin slicer to slice it, with a thickness of 4μm. Float the slices on the 40℃ warm water of the slicer to flatten the tissue, pick up the tissue with a slide, and bake the slices in a 60℃ oven. After the wax is baked dry, take it out and store it at room temperature for later use.

[0226] 5. Dewax the paraffin sections to water: put the sections into environmentally friendly dewaxing liquid I for 10 minutes, environmentally friendly dewaxing liquid II for 10 minutes, environmentally friendly dewaxing liquid III for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, and wash with distilled water.

[0227] 6. Antigen repair: During this process, the buffer should be prevented from evaporating excessively and the slides should not be dried. After cooling naturally, place the slides in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time. (The repair solution and repair conditions are determined according to the tissue).

[0228] 7. Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, each time for 5 minutes.

[0229] 8. Serum blocking: Add 3% BSA in the tissue circle to evenly cover the tissue, and block at room temperature for 30 minutes. (If the primary antibody is from goat, use rabbit serum to block, and other sources use BSA to block)

[0230] 9. Add primary antibody: Gently shake off the blocking solution, add primary antibody prepared in a certain proportion with PBS on the slices, and incubate the slices flat in a humidified box at 4°C overnight.

[0231] 10. Add secondary antibody: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP labeled) of the same species as the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0232] 11. DAB color development: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle, and control the color development time under a microscope. The positive color is brown-yellow, and the slices are rinsed with tap water to stop the color development.

[0233] 12. Re-staining of cell nuclei: Re-stain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin bluing solution, and rinse with running water.

[0234] 13. Dehydration and sealing: Place the slices in 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-n-butanol for 5 min-xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with sealing glue.

[0235] 14. Microscopic examination: The results are interpreted under a white light microscope. Hematoxylin stains the cell nucleus in blue, and DAB shows positive expression in brown-yellow.

[0236] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0237] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a nano drug delivery system based on tumor microenvironment response, characterized in that: The following steps are involved: Step S1, synthesis of nano drug delivery system; Step S11, preparation of nano-MnO2; Step S12, preparation of nano ZIF-67; Step S13, preparation of ZIF-67 / MnO2 solution; Step S14, preparation of ZMBS; Step S15, preparation of ZMDBS; Step S2, characterization of nano drug delivery system; Step S21, particle size and potential measurement; Step S22, determination of drug loading and encapsulation efficiency; Step S23, measuring the UV-visible absorption spectrum; Step S24, determination of Fourier infrared spectrum; Step S25, observing the morphology of nanoparticles; Step S26, X-ray diffraction measurement; Step S27, element analysis XPS; Step S28, GSH responsiveness; Step S29, active oxygen generation capability; Step S210, nanozyme catalytic activity; Step S211, pH responsiveness; Step S212, stability inspection; Step S3: Evaluate the pharmacodynamic characteristics of the anticancer nano-drug delivery system through in vivo and in vitro experiments.

2. The method for preparing a nano drug delivery system based on tumor microenvironment response according to claim 1, characterized in that The step S1, the synthesis of the nano drug delivery system, specifically includes the following steps: Step S11, preparation of nano-MnO2: Take 0-5mL of KMnO4 solution, add 0-5mL of polyallylamine hydrochloride solution PAH, stir for 15-25min to make it fully react, the solution changes from purple to dark brown, then centrifuge with a 10kd ultrafiltration tube, and dilute the resulting concentrated solution to 5-10mL to obtain a prefabricated nano MnO2 solution; Step S12, preparation of nano ZIF-67: Take 0-1 mL of Co(NO3)2·6H2O methanol solution, add 0-5 mL of 2-methylimidazole methanol solution 2-MeIM, stir for 5-10 min to make it fully react, the solution turns blue-purple, stand for 6-12 h, then centrifuge, wash with methanol 1-3 times, and vacuum dry at 55-65 ° C for 6-12 h to obtain blue-purple powder, which is nano ZIF-67; Step S13, preparation of ZIF-67 / MnO2 solution: Dissolve ZIF-67 in ethanol, sonicate for 25-35 minutes, then add an appropriate amount of ultrapure water to obtain a 2 mg / mL ZIF-67 solution; take 0-1 mL of the above solution, add 0-1 mL of ultrapure water and mix thoroughly, then add 0-1 mL of nano MnO2 solution and stir for 1-5 minutes to obtain a ZIF-67 / MnO2 solution; Step S14, preparation of ZMBS: Add 1-3 mL of BSA to ZM, stir for 1-2 min, then add 0-1 mL of SDS, continue stirring for 0.5-1.5 h, centrifuge to remove the precipitate, and obtain a stable nano ZMBS solution, which is then refrigerated at 0-4 °C. Step S15, preparation of ZMDBS: Dissolve ZIF-67 in ethanol, sonicate for 15-35 minutes, and then add an appropriate amount of ultrapure water to obtain a 2 mg / mL ZIF-67 solution; take 0-1 mL of the above solution, add 0-1 mL of ultrapure water and mix thoroughly, then add 0-1 mL of nano MnO2 solution, stir for 1-5 minutes, then add 1-3 mL of doxorubicin hydrochloride DOX, 1-3 mL of bovine serum albumin, stir for 1-2 minutes, then add 0-1 mL of SDS, stir for 0.5-1 h, centrifuge to remove the precipitate, and obtain a stable nano ZMDBS solution, which is refrigerated in a refrigerator at 0-4°C.

3. The method for preparing a nano drug delivery system based on tumor microenvironment response according to claim 1, characterized in that The step S2, characterization of the nano drug delivery system, specifically includes the following steps: Step S21, particle size and potential measurement; Step S22, determination of drug loading and encapsulation efficiency; Step S23, determination of UV-visible absorption spectrum: Using a UV-visible spectrophotometer, the UV spectra of different groups of nanomaterials in the range of 200-800 nm were measured, and the synthesis of the nano drug delivery system ZMDBS was analyzed through the characteristic peaks presented on the spectra; Step S24, determination of Fourier infrared spectrum: Fourier transform infrared spectrometer was used to measure the different groups of nanomaterials at 500-4000cm -1 The infrared spectrum within the range was used to analyze the functional groups contained in ZMDBS through the characteristic peaks presented on the spectrum, and to confirm whether DOX was successfully encapsulated by the nanocarrier; Step S25, observing the morphology of nanoparticles; Step S26, X-ray diffraction measurement: Use an X-ray diffractometer to perform X-ray diffraction on nanomaterials to obtain their diffraction patterns, and obtain the composition, crystal structure or morphology information of the materials through pattern analysis; Step S27, elemental analysis XPS: X-ray photoelectron spectroscopy (XPS) was used to qualitatively and quantitatively analyze the elemental composition of the nanomaterial ZMDBS. Step S28, GSH responsiveness; Step S29, active oxygen generation capability: To verify that ZIF-67 and MnO2 in ZMDBS can produce reactive oxygen species and thus induce cell apoptosis, the reactive oxygen species production capacity of ZIF-67 / MnO2 was detected by TMB method; Step S210, nanozyme catalytic activity: Verify Co 2+ and Mn 2+ It can act as a Fenton-like catalyst to catalyze hydrogen peroxide in the TNBC tumor microenvironment to generate OH. The MB method was used to detect the OH generation ability of ZM; Step S211, pH responsiveness; Step S212, stability inspection: Verify the stability of the nanodrug delivery system under physiological conditions.

4. The method for preparing a nano drug delivery system based on tumor microenvironment response according to claim 3, characterized in that: The step S21 further comprises: The particle size and potential of ZMDBS were measured by dynamic light scattering using an AntonPaar nanoparticle size and zeta potential analyzer; Particle size determination: Take 1 mL of the solution containing ZMDBS in the particle size sample pool, and test the particle size and PDI of the nanomaterial on the machine. If the concentration of the nanomaterial to be tested is too high, it should be diluted; Potential measurement: Place ZMDBS in a potential cell so that the solution can submerge the two metal sheets of the potential cell, and use a machine to detect the Zeta potential of the nanoparticles.

5. The method for preparing a nano drug delivery system based on tumor microenvironment response according to claim 3, characterized in that: The step S22 further comprises: The content of DOX in ZMDBS was determined by UV-visible spectrophotometry. 2 mL of the ZMDBS solution after hydrolysis with hydrochloric acid was taken into a purple cuvette, and its absorbance at 480 nm was measured. The content of DOX was calculated according to the standard curve of DOX, and the drug loading and encapsulation efficiency of DOX were calculated as follows: Among them, M DOX It refers to the content of DOX successfully loaded on the nanocarrier ZMBS; M 总 Refers to the total mass of the nanomaterial ZMDBS; M add It refers to the mass of DOX added during the experiment.

6. The method for preparing a nano drug delivery system based on tumor microenvironment response according to claim 3, characterized in that: The step S25 further comprises: Scanning electron microscopy and transmission electron microscopy are used to observe the microscopic morphology of nanomaterials; SEM measurement: Place the nanomaterial to be tested on a microscope plate with conductive adhesive, spray gold with an ion sputtering instrument, and then load it into the sample chamber of a scanning electron microscope for observation; TEM measurement: The nanomaterial to be tested was dispersed in 2 mL of ultrapure water and ultrasonicated for 30 min. Then 10 μL of the solution was dropped on a 300-mesh carbon support film and allowed to stand for 10 min. The excess liquid was then absorbed with filter paper. The solution to be tested was placed in the injection rod, which was then transferred to the TEM injection chamber for observation.

7. The method for preparing a nano drug delivery system based on tumor microenvironment response according to claim 3, characterized in that: The step S28 further comprises: Step S281, verifying that ZMDBS has GSH responsiveness, using the characteristic that ZM has an obvious ultraviolet characteristic absorption peak at 300-400nm, using ZM to react with GSH of different concentrations, observing the ultraviolet absorption curve of ZM at 300-400nm, it can be preliminarily determined whether MnO2 reacts with GSH; Step S282: In order to further verify the GSH responsiveness of ZM, the GSH consumption characteristics of ZM were evaluated by using the Elman experiment; Step S283: Using this principle, different concentrations of ZM are reacted with GSH, and then DTNB (100 μM) is added to detect the amount of GSH remaining after the reaction, which can indirectly prove the GSH responsiveness of ZM.

8. The method for preparing a nano drug delivery system based on tumor microenvironment response according to claim 3, characterized in that: The step S211 further comprises: Take 2 portions of 3 mL of ZMDBS in 25KD renewable cellulose dialysis bags, seal them and place them in centrifuge tubes containing 30 mL of PBS buffer solutions of different pH values, and then incubate them in a 37°C shaking incubator. Take 200 μL of PBS buffer solution for testing at 0, 1, 2, 4, 6, 8, 12, 24, and 36 hours, respectively, and add an equal amount of PBS of the same pH. Measure its absorbance at 480 nm with a UV-visible spectrophotometer, convert the corresponding concentration according to the standard curve of DOX, and calculate the cumulative release rate of DOX. The formula is as follows: CR (%) = (actually released DOX mass / total DOX mass loaded in the nanomaterial) * 100%.

9. A nano drug delivery system based on tumor microenvironment response as described in any one of claims 2 to 8 is used in the treatment of triple-negative breast cancer.

Images