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CN113995732A - Preparation method and application of drug-loaded zinc-iron oxide nanocomposite coated with cancer cell membrane - Google Patents

Preparation method and application of drug-loaded zinc-iron oxide nanocomposite coated with cancer cell membrane Download PDF

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CN113995732A
CN113995732A CN202111346320.9A CN202111346320A CN113995732A CN 113995732 A CN113995732 A CN 113995732A CN 202111346320 A CN202111346320 A CN 202111346320A CN 113995732 A CN113995732 A CN 113995732A
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iron oxide
zinc
cancer cell
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侯琳
张振中
杨静
张萌
王芮婷
张红岭
何玉萍
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Zhengzhou University
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Abstract

A preparation method and application of a zinc-iron oxide nanocomposite coated with cancer cell membranes and loaded with drugs are disclosed, wherein mesoporous zinc-iron oxide nanoparticles are used for physically loading DNA damage drugs, and then the cancer cell membranes are coated on the surfaces of the mesoporous zinc-iron oxide nanoparticles to form the zinc-iron oxide nanocomposite coated with the cancer cell membranes, and the preparation method specifically comprises the following steps: (1) preparing zinc-iron oxide nanoparticles; (2) preparing a zinc-iron oxide nano composite loaded with the DNA injury medicine; (3) preparing a cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite; the method is convenient to operate, the method is stable and reliable, the prepared cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite has the advantages of good biocompatibility, small toxic and side effects and the like, can play a role in targeting and activating a cGAS-STING pathway in the aspect of tumor treatment to achieve the effect of immunotherapy, is an innovation in tumor immunotherapy drugs, and has great economic and social benefits.

Description

Preparation method and application of cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite
Technical Field
The invention relates to medicine, in particular to a preparation method and application of a cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite.
Background
Since the twentieth century, the environment has been worsened, people have come into closer contact with carcinogenic factors, the incidence of malignant tumors has increased year by year, and the health of human beings is seriously affected. In recent years, tumor immunotherapy has achieved great success, and a large number of immune checkpoint inhibitor drugs including anti-PD-1, anti-PD-L1 and anti-CTLA-4 are applied clinically. While these T cell-based immunotherapies have proven to be an effective strategy in a variety of tumors, they are only partially effective in patients with low immune response activation, and non-responsive patients often suffer from non-T cell-inflamed tumors, lacking markers associated with activation of adaptive anti-tumor immune responses, making such therapies less than ideal for the treatment of metastatic tumors. By effectively activating the innate immune response in tumor tissue, the transition of the tumor from immune-unresponsive to immune-responsive is expected to be achieved. For this reason, in recent years, the interest in tumor immunotherapy has gradually shifted to the innate immunity.
When external antigens enter a body, cytoplasmic DNA is combined with the cGAS and then is transmitted to second messenger cGAMP, the dimerized STING is immediately combined with the cGAMP, conformation change occurs, TBK1 protein is collected after the dimerization of the dimeric STING from autophagosome through endoplasmic reticulum and Golgi apparatus, and the dimeric STING is ubiquitinated, so that Interferon Regulatory Factors (IRFs) and NF-kB are phosphorylated and activated, and the latter can induce the expression of type I interferon and other immune response genes. The cGAS-STING pathway plays an important role in the development of anti-viral innate immune responses, anti-tumor immunity, and autoimmune diseases. Zinc ions enhance the activity of cGAS enzyme in vitro and in cells by promoting the separation of cGAS-DNA, promote STING activation, and generate a large amount of type I interferon, so that the zinc ions play a vital role in the body.
Genotoxic stress caused by DNA damaging processes (e.g., reactive oxygen species ROS, DNA damaging drugs platinum, paclitaxel, etc.) produces chromosomal fragments that are recognized by the nucleic acid sensor cyclic GMP-amp (cgamp) synthase (cGAS). The zinc ions promote cGAS-DNA phase separation to promote STING activation, generate a large amount of type I interferon, and further improve the tumor immune response through a synergistic effect.
In order to realize the effect of positioning and releasing zinc ions and DNA injury medicaments in tumor cells, the mesoporous zinc-iron oxide nanoparticles loaded with the DNA injury medicaments are wrapped by cancer cell membranes, the cancer cell membranes can be degraded in the acidic environment of tumors, and the zinc-iron oxide responds to glutathione highly expressed in the tumor cells to release zinc and iron ions (ROS is generated through Fenton reaction) and the medicaments to enhance the tumor immunotherapy. Therefore, the research on activating the cGAS-STING pathway to generate the type I IFN to achieve the tumor immunotherapy has great significance and value.
Disclosure of Invention
Aiming at the situation and overcoming the defects of the prior art, the invention aims to provide a preparation method and application of a cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite, which can effectively solve the medication problem of cancer immunotherapy.
In order to achieve the purpose, the technical scheme of the invention is that the preparation method of the zinc-iron oxide nano composite coated with the cancer cell membrane comprises the following steps of physically loading a DNA injury medicament by using mesoporous zinc-iron oxide nano particles, and then coating the surface of the cancer cell membrane to form the zinc-iron oxide nano composite coated with the cancer cell membrane:
(1) preparing zinc-iron oxide nano particles: 1-3 mmol of FeCl3·6H2O and 0.5-1.5 mmol ZnCl2Dissolving in 40mL of ethylene glycol to obtain a solution, adding 1.8-5.4 g of NaAc and 1.0g of polyethylene glycol, violently stirring the mixture for 30min, sealing in a Teflon-lined high-pressure kettle (the volume is 100 mL), sealing in a muffle furnace, reacting at the temperature of 200 ℃ for 12-20 h, cooling the high-pressure kettle to room temperature, collecting a solid product by using a magnet, washing the solid product for 3 times by using distilled water and ethanol respectively to obtain a sample, and drying the sample in a vacuum oven at the temperature of 75-85 ℃ for 7.5-8.5h to obtain zinc-iron oxide nanoparticles;
(2) preparing a DNA injury drug-loaded zinc-iron oxide nano composite: dispersing 2-5 mg of zinc-iron oxide nanoparticles prepared in the step (1) in 3-6 mL of PBS (phosphate buffer solution) with the pH value of 7.4 to form a first mixed solution; dispersing 4-10 mg of DNA damage drug in 1-2 mL of solvent to form a second mixed solution; dropwise adding the second mixed solution into the first mixed solution under stirring, stirring at room temperature for 24 hours, centrifuging at 12000-15000 rpm for 10-20 min, and drying at 70-80 ℃ in vacuum for 5-6 hours to obtain a zinc-iron oxide nano compound loaded with the DNA damage drug;
the DNA damage drug is one of paclitaxel, adriamycin or platinum drugs;
the solvent is ultrapure water, PBS buffer solution with pH = 7.4, absolute ethyl alcohol, DMSO or formamide;
(3) preparing a cancer cell membrane-wrapped drug-loaded zinc-iron oxide nano compound: extracting cancer cell membranes by differential centrifugation, centrifuging the cells for 10min at 660g, washing the cells for 3 times with PBS (pH 7.4), suspending the cell pellets in hypotonic lysis buffer for mechanical disruption, centrifuging for 5min at 3200g, collecting supernatant, repeating the process, centrifuging the cells for 6min at 3200g again, combining the supernatants, centrifuging for 25min at 21000g at 4 ℃, collecting supernatant, centrifuging the supernatant for 5min at 45000g in a super-speed centrifuge at 4 ℃, discarding supernatant, quantifying the extracted cell membrane fragments with BCA protein quantification kit, dispersing the DNA damaging drug-loaded zinc-iron oxide nanocomposite prepared in step (2) in PBS (pH 7.4), mixing the cell membrane fragments with the prepared cell membranes at 1: 0.125-4, ultrasonically treating the mixture in a water bath ultrasonic instrument for 28-32min, extruding the mixture for 5-15 times through a polycarbonate membrane to obtain a product, and centrifuging the product at 12000-15000 rpm for 10-20 min to obtain the cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite;
the hypotonic lysis buffer solution is prepared from 20mM Tris-HCl, 10mM KCl and 2mM MgCl2And 1mM PMSF.
Furthermore, the particle size of the zinc-iron oxide nanoparticles in the step (1) is 80-160 nm, and the particle size of the cancer cell membrane-coated drug-loaded zinc-iron oxide nanocomposite in the step (3) is 100-180 nm.
The polycarbonate film in the step (3) is a polycarbonate porous film with the thickness of 200-400 nm.
The cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite prepared by the method is applied to preparation of an antitumor drug injection.
The cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite prepared by the method is applied to preparation of a drug for enhancing a cGAS-STING immune signal pathway.
The cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite prepared by the method is applied to preparation of a mesoporous zinc-iron oxide tumor microenvironment drug based on pH sensitivity and glutathione response.
The method is used for preparing the cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite and is applied to preparing chemotherapeutic and immunotherapy combined antitumor drugs.
The method is convenient to operate, the method is stable and reliable, the prepared cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite has the advantages of good biocompatibility, small toxic and side effects and the like, can play a role in targeting and activating a cGAS-STING pathway in the aspect of tumor treatment to achieve the effect of immunotherapy, is an innovation in tumor immunotherapy drugs, and has great economic and social benefits.
Detailed Description
The following examples are provided to explain the present invention in detail.
In particular, the invention may be embodied as set forth in the following examples.
Example 1
A preparation method of a cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite comprises the following steps:
(1) preparing zinc-iron oxide nanoparticles: 3mmol of FeCl3·6H2O and 1.5mmol ZnCl2Dissolving in 40mL of ethylene glycol to obtain a solution, adding 1.8g of NaAc and 1.0g of polyethylene glycol, violently stirring the mixture for 30min, sealing in a Teflon-lined high-pressure kettle, sealing in a muffle furnace, reacting at 200 ℃ for 12h, cooling the high-pressure kettle to room temperature, collecting a solid product by using a magnet, washing the solid product by using distilled water and ethanol for 3 times respectively to obtain a sample, and drying the sample in a vacuum oven at 75 ℃ for 8.5h to obtain zinc-iron oxide nanoparticles;
(2) preparing a zinc-iron oxide nano composite loaded with paclitaxel: dispersing 2mg of zinc-iron oxide nanoparticles prepared in the step (1) into 3mL of PBS (phosphate buffer solution) with the pH value of 7.4 to form a first mixed solution; dispersing 4mg of paclitaxel in 1mL of absolute ethanol to form a second mixed solution; dropwise adding the second mixed solution into the first mixed solution under stirring, stirring at room temperature for 24h, centrifuging at 12000rpm for 20min, and drying at 70 ℃ under vacuum for 6h to obtain a paclitaxel-loaded zinc-iron oxide nano compound;
(3) preparing a cancer cell membrane-wrapped drug-loaded zinc-iron oxide nano compound: extracting cancer cell membranes by differential centrifugation, centrifuging the cells for 10min at 660g, washing the cells for 3 times with PBS (pH 7.4), resuspending the cell pellet in hypotonic lysis buffer for mechanical disruption, centrifuging for 5min at 3200g, collecting the supernatant, repeating the process, centrifuging the cells for 6min at 3200g again, combining the supernatants, centrifuging for 25min at 21000g at 4 ℃, collecting the supernatant, centrifuging the supernatant for 5min at 45000g in a super-speed centrifuge at 4 ℃, discarding the supernatant, quantifying the extracted cell membrane fragments with a BCA protein quantification kit, dispersing the paclitaxel-loaded zinc-iron oxide nanocomposite prepared in step (2) in PBS (pH 7.4) at 1: 1, carrying out ultrasonic treatment on the mixture for 28min in a water bath ultrasonic instrument, extruding the mixture for 7 times through a polycarbonate membrane to obtain a product, and centrifuging the product at 12000rpm for 20min to obtain the cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite.
Example 2
A preparation method of a cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite comprises the following steps:
(1) preparing zinc-iron oxide nanoparticles: 2mmol of FeCl3·6H2O and 1mmol ZnCl2Dissolving in 40mL of ethylene glycol to obtain a solution, adding 3.6g of NaAc and 1.0g of polyethylene glycol, vigorously stirring the mixture for 30min, sealing in a Teflon-lined autoclave, sealing in a muffle furnace, reacting at 180 ℃ for 16h, cooling the autoclave to room temperature, collecting a solid product by using a magnet, washing the solid product for 3 times by using distilled water and ethanol respectively to obtain a sample, and drying the sample in a vacuum oven at 80 ℃ for 7h to obtain zinc-iron oxide nanoparticles;
(2) preparing the zinc-iron oxide nanocomposite loaded with the adriamycin: dispersing 4mg of zinc-iron oxide nanoparticles prepared in the step (1) in 5mL of PBS (phosphate buffer solution) with the pH value of 7.4 to form a first mixed solution; dispersing 7mg of adriamycin in 1mL of ultrapure water to form a second mixed solution; dropwise adding the second mixed solution into the first mixed solution under stirring, stirring at room temperature for 24h, centrifuging at 13500rpm for 15 min, and drying at 75 ℃ under vacuum for 5.5h to obtain a zinc-iron oxide nano compound loaded with adriamycin;
(3) preparing a cancer cell membrane-wrapped drug-loaded zinc-iron oxide nano compound: extracting cancer cell membranes by differential centrifugation, centrifuging the cells for 10min at 660g, washing the cells for 3 times with PBS (pH 7.4), resuspending the cell pellet in hypotonic lysis buffer for mechanical disruption, centrifuging for 5min at 3200g, collecting the supernatant, repeating the process, centrifuging the cells for 6min at 3200g again, combining the supernatants, centrifuging for 25min at 21000g at 4 ℃, collecting the supernatant, centrifuging the supernatant for 5min at 45000g in a super-speed centrifuge at 4 ℃, discarding the supernatant, quantifying the extracted cell membrane fragments with a BCA protein quantification kit, dispersing the adriamycin-loaded zinc-iron oxide nanocomposite prepared in step (2) in PBS (pH 7.4) at 1: 2, performing ultrasonic treatment on the mixture in a water bath ultrasonic instrument for 30min, extruding the mixture for 11 times through a polycarbonate membrane to obtain a product, and centrifuging the product at 13500rpm for 15 min to obtain the cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite.
Example 3
A preparation method of a cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite comprises the following steps:
(1) preparing zinc-iron oxide nanoparticles: 1mmol of FeCl3·6H2O and 0.5mmol ZnCl2Dissolving in 40mL of ethylene glycol to obtain a solution, adding 5.4g of NaAc and 1.0g of polyethylene glycol, vigorously stirring the mixture for 30min, sealing in a Teflon-lined autoclave, sealing in a muffle furnace, reacting at 200 ℃ for 12h, cooling the autoclave to room temperature, collecting a solid product by using a magnet, washing the solid product for 3 times by using distilled water and ethanol respectively to obtain a sample, and drying the sample in a vacuum oven at 85 ℃ for 7.5h to obtain zinc-iron oxide nanoparticles;
(2) preparing a zinc-iron oxide nano composite loaded with paclitaxel: dispersing 5mg of zinc-iron oxide nanoparticles prepared in the step (1) into 6mL of PBS buffer solution with pH7.4 to form a first mixed solution; dispersing 10mg of paclitaxel in 2 mL of formamide to form a second mixed solution; dropwise adding the second mixed solution into the first mixed solution under stirring, stirring at room temperature for 24h, centrifuging at 15000rpm for 10min, and drying at 80 ℃ in vacuum for 5h to obtain a paclitaxel-loaded zinc-iron oxide nano composite;
(3) preparing a cancer cell membrane-wrapped drug-loaded zinc-iron oxide nano compound: extracting cancer cell membranes by differential centrifugation, centrifuging the cells for 10min at 660g, washing the cells for 3 times with PBS (pH 7.4), resuspending the cell pellet in hypotonic lysis buffer for mechanical disruption, centrifuging for 5min at 3200g, collecting the supernatant, repeating the process, centrifuging the cells for 6min at 3200g again, combining the supernatants, centrifuging for 25min at 21000g at 4 ℃, collecting the supernatant, centrifuging the supernatant for 5min at 45000g in a super-speed centrifuge at 4 ℃, discarding the supernatant, quantifying the extracted cell membrane fragments with a BCA protein quantification kit, dispersing the paclitaxel-loaded zinc-iron oxide nanocomposite prepared in step (2) in PBS (pH 7.4) at 1: 4, performing ultrasonic treatment on the mixture in a water bath ultrasonic instrument for 32min, extruding the mixture for 15 times through a polycarbonate membrane to obtain a product, and centrifuging the product at 15000rpm for 10min to obtain the cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite.
The preparation method is simple, the tumor part is targeted through the homologous targeting effect of the cancer cell membrane, the cancer cell membrane wrapped drug-loaded zinc-iron oxide nano-composite has good biocompatibility, zinc ions are released by the acidic environment and the high-expression glutathione response in the tumor cell, active oxygen is generated, the cytoplasmic DNA content is increased, the cGAS-STING passage in the cell is strongly activated, the STING activation is further promoted, a large amount of I-type interferon is generated, and the tumor immune response is further improved through the synergistic effect. After repeated experiments, consistent results were obtained, taking example 2 as an example, the experimental data are as follows:
firstly, characterization test of cancer cell membrane coated drug-loaded zinc-iron oxide nanocomposite:
1. the determination of the content of paclitaxel in the cancer cell membrane-coated drug-loaded zinc-iron oxide nanocomposite comprises the following steps:
and measuring the content of the DNA damage medicament at the wavelength of 227nm by adopting an ultraviolet spectrophotometry. Calculating the drug loading rate of the sample by the formula (1), wherein the drug loading rate reaches about 38.4%;
Figure 408027DEST_PATH_IMAGE001
formula (1);
2. the particle size and the potential of the cancer cell membrane-coated drug-loaded zinc-iron oxide nanocomposite are measured:
dispersing a proper amount of the cancer cell membrane-coated drug-loaded zinc-iron oxide Nano-composite into water, and measuring the hydration particle size and the potential of the Nano-composite by using a Nano-ZS90 type laser Nano-particle size analyzer to be 157.8nm and 23.8mV respectively;
3. characterization of cancer cell membrane-wrapped drug-loaded zinc-iron oxide nanocomposite transmission electron microscope:
dissolving the drug compound in ultrapure water to prepare a solution with the concentration of 50 mug/mL, dripping 1 drop of the solution on a common carbon supporting film, repeating the operation for 5 times after the liquid is evaporated, and shooting by a transmission electron microscopy electron microscope (TalosF200S) type to obtain the drug compound with the particle size of 100-180 nm.
Secondly, in-vitro degradation experiment of the cancer cell membrane-coated drug-loaded zinc-iron oxide nano composite:
wrapping cancer cell membrane with zinc-iron oxide nano-composite (CM @ ZnFe) carrying medicine2O4PTX) was added to PBS (pH 7.4), PBS (pH 5.4) and PBS (pH 5.4, containing 10mM GSH) at a concentration of 50. mu.g/mL, and the particle size was measured at 30min, 1h, 2h and 4h, respectively. The results show CM @ ZnFe after 4h2O4The PTX particle size reaches about 1000nm, which better indicates that the preparation achieves the positioning release in tumor cells.
Thirdly, detecting related protein expression experiments of tumor tissues by Western Blotting technology:
culturing melanoma B16F10 cells, inoculating to the right hind limb of C57BL/6 mouse subcutaneously until the tumor volume reaches 100mm3In the meantime, the administration treatment was performed, and the mice were randomly divided into 6 groups, specifically: PBS, PTX, ZnCl2+FeCl3，ZnCl2+FeCl3+PTX，CM@ZnFe2O4，CM@ZnFe2O4Six groups of PTX, tail vein injections, once every other day, 5 times total. PTX was administered at a dose of 4 mg/kg.
Extracting tumor tissues of tumor-bearing mice, cracking by using a lysate to obtain protein, quantifying the protein content by a BCA quantitative method, detecting the protein expression amounts of I-type interferon, pIFR3 and IRF3, and comparing a physiological saline group with other control groups, wherein the preparation can activate a cGAS-STING pathway to generate a large amount of I-type interferon, and improve the tumor immune response to achieve the anti-tumor effect.
The same experiment as in the above experiment method was carried out for examples 1 and 3, and the same or similar results were obtained, which are not listed here.
From the above experiments, the invention has the following outstanding beneficial technical effects compared with the prior art:
(1) the cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite provided by the invention has good biocompatibility and stability, can physically load DNA damage drugs, improves the drug-loading rate, and solves the problem of drug application of many fat-soluble drugs; meanwhile, the homologous targeting effect of the cancer cell membrane can enable the preparation to be positioned at a tumor part, and the positioning release is realized under the conditions of an acid environment and high-expression GSH;
(2) the DNA damage drug and the iron ions provided by the invention can damage DNA at low dose to release the DNA into cytoplasm, and the cGAS-STING signal pathway senses dsDNA and Zn in cytoplasm2+Can strongly enhance the cGAS-STING pathway in cells to promote STING activation, generate a large amount of type I interferon, and further improve the tumor immune response through synergistic action;
(3) the DNA damage medicament provided by the invention can play a better anti-tumor effect under the action of low dose, and can avoid adverse side effects in chemotherapy.
The method has the advantages of convenient operation and stable and reliable method, the prepared cancer cell membrane-coated drug-loaded zinc-iron oxide nano-composite has the advantages of good biocompatibility, small toxic and side effects and the like, can play the roles of GSH and pH response, positioning and releasing the drug to reach tumor cells in the aspect of tumor treatment, simultaneously activates a cGAS-STING pathway in the cells to improve immune response, realizes the synergistic effect of chemotherapy and immune combination of tumors, improves the treatment effect of the tumors, is an innovation on tumor treatment drugs, and has great economic and social benefits.

Claims (10)

1.一种癌细胞膜包裹载药锌铁氧化物纳米复合物的制备方法，其特征在于，利用介孔锌铁氧化物纳米粒物理负载DNA损伤药物，然后将癌细胞膜包裹其表面，构成癌细胞膜包裹锌铁氧化物纳米复合物，具体包括以下步骤：1. a preparation method of cancer cell membrane wrapping drug-loaded zinc-iron oxide nanocomposite, is characterized in that, utilizes mesoporous zinc-iron oxide nanoparticles to physically load DNA damage medicine, then cancer cell membrane is wrapped its surface, constitutes cancer cell membrane The encapsulation of the zinc-iron oxide nanocomposite specifically includes the following steps: （1）、制备锌铁氧化物纳米粒：将1～3mmol FeCl3·6H2O和0.5～1.5mmol ZnCl2溶解在40mL乙二醇中，得溶液，然后加入1.8～5.4g的NaAc和1.0g聚乙二醇，将混合物剧烈搅拌30min，然后密封在特氟隆衬里高压釜中，再放入马弗炉中密封，160-200°C反应12～20h，随后将高压釜冷却至室温，用磁体收集固体产物，固体产物分别用蒸馏水和乙醇洗涤3次，得样品，将样品在真空烘箱中75-85℃干燥7.5-8.5h，得锌铁氧化物纳米粒；(1) Preparation of zinc-iron oxide nanoparticles: Dissolve 1-3 mmol FeCl 3 ·6H 2 O and 0.5-1.5 mmol ZnCl 2 in 40 mL of ethylene glycol to obtain a solution, and then add 1.8-5.4 g of NaAc and 1.0 g polyethylene glycol, the mixture was vigorously stirred for 30min, then sealed in a Teflon-lined autoclave, then put into a muffle furnace and sealed, reacted at 160-200°C for 12-20h, and then the autoclave was cooled to room temperature, The solid product was collected by a magnet, and the solid product was washed three times with distilled water and ethanol, respectively, to obtain a sample, and the sample was dried in a vacuum oven at 75-85 ° C for 7.5-8.5 h to obtain zinc-iron oxide nanoparticles; （2）、制备负载DNA损伤药物的锌铁氧化物纳米复合物：将步骤（1）制备的锌铁氧化物纳米粒2～5mg分散于pH7.4的PBS缓冲液3～6mL中，形成第一混合液；将4～10mg DNA损伤药物分散于1～2 mL溶剂中，形成第二混合液；将第二混合液在搅拌下滴加于第一混合液中，室温搅拌24h，然后12000～15000rpm离心10～20 min，真空70-80℃干燥5-6h，得负载DNA损伤药物的锌铁氧化物纳米复合物；(2) Preparation of zinc-iron oxide nanocomposites loaded with DNA damaging drugs: Disperse 2-5 mg of zinc-iron oxide nanoparticles prepared in step (1) in 3-6 mL of pH 7.4 PBS buffer to form the first step. A mixed solution; 4-10 mg of DNA-damaging drugs are dispersed in 1-2 mL of solvent to form a second mixed solution; the second mixed solution is added dropwise to the first mixed solution under stirring, stirred at room temperature for 24 hours, and then 12000- Centrifuge at 15,000 rpm for 10-20 min, and dry in a vacuum at 70-80 °C for 5-6 h to obtain a DNA-damaging drug-loaded zinc-iron oxide nanocomposite; 所述的DNA损伤药物为紫杉醇、阿霉素或铂类药物中的一种；The DNA damage drug is one of paclitaxel, doxorubicin or platinum drugs; 所述溶剂为超纯水、pH=7 .4的PBS缓冲液、无水乙醇、DMSO或甲酰胺的一种；Described solvent is a kind of ultrapure water, the PBS buffer solution of pH=7.4, dehydrated alcohol, DMSO or formamide; （3）、制备癌细胞膜包裹载药锌铁氧化物纳米复合物：通过差速离心法提取癌细胞膜，将细胞以660g离心10min，并用pH7.4的PBS 洗涤3次，将细胞沉淀重悬在低渗裂解缓冲液中进行机械破坏，以3200g离心5min，收集上清液，并重复该过程，将细胞再次以3200g离心6min，合并上清液，在4 ℃条件下以 21000g离心25min，收集上清液，将上清液在超高速离心机中4℃条件下以45000g离心5min，弃去上清液，用BCA蛋白质定量试剂盒对提取的细胞膜碎片进行量化，将步骤（2）制备的负载DNA损伤药物的锌铁氧化物纳米复合物分散于pH7.4的PBS中，与制备的细胞膜以1：0.125～4的质量比混合，在水浴超声仪中将混合物超声处理28-32min，通过聚碳酸酯膜挤压5～15次，得产物，将产物12000～15000rpm离心10～20min，即得癌细胞膜包裹载药锌铁氧化物纳米复合物；(3) Preparation of drug-loaded zinc-iron oxide nanocomposites encapsulated in cancer cell membranes: The cancer cell membranes were extracted by differential centrifugation, the cells were centrifuged at 660 g for 10 min, washed three times with PBS pH 7.4, and the cell pellets were resuspended in Perform mechanical disruption in hypotonic lysis buffer, centrifuge at 3200g for 5min, collect the supernatant, and repeat the process, centrifuge the cells again at 3200g for 6min, combine the supernatant, centrifuge at 21000g for 25min at 4 °C, and collect the supernatant. The supernatant was centrifuged at 45,000g for 5 min at 4°C in an ultracentrifuge, the supernatant was discarded, and the extracted cell membrane debris was quantified with the BCA protein quantification kit. The zinc-iron oxide nanocomplexes of DNA-damaging drugs were dispersed in PBS with pH 7.4, mixed with the prepared cell membrane at a mass ratio of 1:0.125-4, and the mixture was sonicated in a water bath sonicator for 28-32 min. The carbonate membrane is extruded for 5 to 15 times to obtain a product, and the product is centrifuged at 12000 to 15000 rpm for 10 to 20 minutes to obtain a drug-loaded zinc-iron oxide nanocomposite wrapped in the cancer cell membrane; 所述的低渗裂解缓冲液是由20mM Tris-HCl、10mM KCl、2mM MgCl2和1mM PMSF混合制成。The hypotonic lysis buffer was prepared by mixing 20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl 2 and 1 mM PMSF. 2.根据权利要求1所述的癌细胞膜包裹载药锌铁氧化物纳米复合物的制备方法，其特征在于，包括以下步骤：2. the preparation method of cancer cell membrane package drug-loaded zinc-iron oxide nanocomposite according to claim 1, is characterized in that, comprises the following steps: （1）、制备锌铁氧化物纳米粒：将3mmol FeCl3·6H2O和1.5mmol ZnCl2溶解在40mL乙二醇中，得溶液，然后加入1.8g的NaAc和1.0 g聚乙二醇，将混合物剧烈搅拌30min，然后密封在特氟隆衬里高压釜中，再放入马弗炉中密封，200°C反应12h，随后将高压釜冷却至室温，用磁体收集固体产物，固体产物分别用蒸馏水和乙醇洗涤3次，得样品，将样品在真空烘箱中75℃干燥8.5h，即得锌铁氧化物纳米粒；(1) Preparation of zinc-iron oxide nanoparticles: Dissolve 3mmol FeCl 3 6H 2 O and 1.5mmol ZnCl 2 in 40mL of ethylene glycol to obtain a solution, then add 1.8g of NaAc and 1.0g of polyethylene glycol, The mixture was vigorously stirred for 30min, then sealed in a Teflon-lined autoclave, then put into a muffle furnace and sealed, reacted at 200° C. for 12h, then the autoclave was cooled to room temperature, and the solid products were collected with a magnet. Distilled water and ethanol were washed 3 times to obtain a sample, and the sample was dried in a vacuum oven at 75°C for 8.5h to obtain zinc-iron oxide nanoparticles; （2）、制备负载紫杉醇的锌铁氧化物纳米复合物：将步骤（1）制备的锌铁氧化物纳米粒2mg分散于pH7.4的PBS缓冲液3mL中，形成第一混合液；将4 mg紫杉醇分散于1mL无水乙醇中，形成第二混合液；将第二混合液在搅拌下滴加于第一混合液中，室温搅拌24h，然后12000rpm离心20min，真空70℃干燥6h，得负载紫杉醇的锌铁氧化物纳米复合物；(2) Preparation of paclitaxel-loaded zinc-iron oxide nanocomposite: Disperse 2 mg of zinc-iron oxide nanoparticles prepared in step (1) in 3 mL of pH 7.4 PBS buffer to form a first mixed solution; mg paclitaxel was dispersed in 1 mL of absolute ethanol to form a second mixed solution; the second mixed solution was added dropwise to the first mixed solution with stirring, stirred at room temperature for 24 h, then centrifuged at 12,000 rpm for 20 min, and dried in vacuum at 70 °C for 6 h to obtain a load A zinc-iron oxide nanocomplex of paclitaxel; （3）、制备癌细胞膜包裹载药锌铁氧化物纳米复合物：通过差速离心法提取癌细胞膜，将细胞以660g离心10min，并用pH 7.4的PBS洗涤3次，将细胞沉淀重悬在低渗裂解缓冲液中进行机械破坏，以3200g离心5min，收集上清液，并重复该过程，将细胞再次以3200g离心6min，合并上清液，在4 ℃条件下以 21000g离心25min，收集上清液，将上清液在超高速离心机中4℃条件下以45000g离心5min，弃去上清液，用BCA蛋白质定量试剂盒对提取的细胞膜碎片进行量化，将步骤（2）制备的负载紫杉醇的锌铁氧化物纳米复合物分散于pH 7.4的PBS中，与制备的细胞膜以1：1的质量比混合，在水浴超声仪中将混合物超声处理28min，通过聚碳酸酯膜挤压7次，得产物，将产物12000rpm离心20 min，即得癌细胞膜包裹载药锌铁氧化物纳米复合物。(3) Preparation of drug-loaded zinc-iron oxide nanocomposites encapsulated in cancer cell membranes: the cancer cell membranes were extracted by differential centrifugation, the cells were centrifuged at 660 g for 10 min, washed three times with PBS pH 7.4, and the cell pellets were resuspended in a low Perform mechanical disruption in lysis buffer, centrifuge at 3200g for 5min, collect the supernatant, and repeat the process, centrifuge the cells again at 3200g for 6min, combine the supernatant, centrifuge at 21000g for 25min at 4 °C, and collect the supernatant The supernatant was centrifuged at 45000g for 5 min at 4°C in an ultracentrifuge, the supernatant was discarded, and the extracted cell membrane fragments were quantified with BCA protein quantification kit. The zinc-iron oxide nanocomposite was dispersed in PBS with pH 7.4, mixed with the prepared cell membrane at a mass ratio of 1:1, the mixture was sonicated in a water bath sonicator for 28 min, and extruded through a polycarbonate membrane for 7 times, A product was obtained, and the product was centrifuged at 12,000 rpm for 20 min to obtain a drug-loaded zinc-iron oxide nanocomposite encapsulated by the cancer cell membrane. 3.根据权利要求1所述的癌细胞膜包裹载药锌铁氧化物纳米复合物的制备方法，其特征在于，包括以下步骤：3. the preparation method of cancer cell membrane package drug-loaded zinc-iron oxide nanocomposite according to claim 1, is characterized in that, comprises the following steps: （1）、制备锌铁氧化物纳米粒：将2mmol FeCl3·6H2O和1mmol ZnCl2溶解在40mL乙二醇中，得溶液，然后加入3.6g的NaAc和1.0g聚乙二醇，将混合物剧烈搅拌30min，然后密封在特氟隆衬里高压釜中，再放入马弗炉中密封，180°C反应16h，随后将高压釜冷却至室温，用磁体收集固体产物，固体产物分别用蒸馏水和乙醇洗涤3次，得样品，将样品在真空烘箱中80℃干燥7h，得锌铁氧化物纳米粒；(1) Preparation of zinc-iron oxide nanoparticles: Dissolve 2mmol FeCl 3 6H 2 O and 1mmol ZnCl 2 in 40mL of ethylene glycol to obtain a solution, then add 3.6g of NaAc and 1.0g of polyethylene glycol, The mixture was vigorously stirred for 30min, then sealed in a Teflon-lined autoclave, then put into a muffle furnace and sealed, reacted at 180° C. for 16h, then the autoclave was cooled to room temperature, and the solid product was collected with a magnet, and the solid product was treated with distilled water respectively. Washed with ethanol for 3 times to obtain a sample, which was dried in a vacuum oven at 80 °C for 7 h to obtain zinc-iron oxide nanoparticles; （2）、制备负载阿霉素的锌铁氧化物纳米复合物：将步骤（1）制备的锌铁氧化物纳米粒4mg分散于pH7.4的PBS缓冲液5mL中，形成第一混合液；将7mg阿霉素分散于1mL超纯水中，形成第二混合液；将第二混合液在搅拌下滴加于第一混合液中，室温搅拌24h，然后13500rpm离心15 min，真空75℃干燥5.5h，得负载阿霉素的锌铁氧化物纳米复合物；(2) Preparation of doxorubicin-loaded zinc-iron oxide nanocomposite: Disperse 4 mg of zinc-iron oxide nanoparticles prepared in step (1) in 5 mL of pH 7.4 PBS buffer to form a first mixed solution; Disperse 7 mg of doxorubicin in 1 mL of ultrapure water to form a second mixed solution; add the second mixed solution dropwise to the first mixed solution with stirring, stir at room temperature for 24 h, then centrifuge at 13,500 rpm for 15 min, and dry at 75°C under vacuum 5.5h to obtain doxorubicin-loaded zinc-iron oxide nanocomposite; （3）、制备癌细胞膜包裹载药锌铁氧化物纳米复合物：通过差速离心法提取癌细胞膜，将细胞以660g离心10min，并用pH 7.4的PBS洗涤3次，将细胞沉淀重悬在低渗裂解缓冲液中进行机械破坏，以3200g离心5min，收集上清液，并重复该过程，将细胞再次以3200g离心6min，合并上清液，在4 ℃条件下以 21000g离心25min，收集上清液，将上清液在超高速离心机中4℃条件下以45000g离心5min，弃去上清液，用BCA蛋白质定量试剂盒对提取的细胞膜碎片进行量化，将步骤（2）制备的负载阿霉素的锌铁氧化物纳米复合物分散于pH 7.4的PBS中，与制备的细胞膜以1：2的质量比混合，在水浴超声仪中将混合物超声处理30min，通过聚碳酸酯膜挤压11次，得产物，将产物13500rpm离心15 min，即得癌细胞膜包裹载药锌铁氧化物纳米复合物。(3) Preparation of drug-loaded zinc-iron oxide nanocomposites encapsulated in cancer cell membranes: the cancer cell membranes were extracted by differential centrifugation, the cells were centrifuged at 660 g for 10 min, washed three times with PBS pH 7.4, and the cell pellets were resuspended in a low Perform mechanical disruption in lysis buffer, centrifuge at 3200g for 5min, collect the supernatant, and repeat the process, centrifuge the cells again at 3200g for 6min, combine the supernatant, centrifuge at 21000g for 25min at 4 °C, and collect the supernatant The supernatant was centrifuged at 45000g for 5 min at 4°C in an ultracentrifuge, the supernatant was discarded, and the extracted cell membrane fragments were quantified with BCA protein quantification kit. The zinc-iron oxide nanocomposite of mycin was dispersed in PBS at pH 7.4, mixed with the prepared cell membrane at a mass ratio of 1:2, the mixture was sonicated in a water bath sonicator for 30 min, and extruded through a polycarbonate membrane for 11 Next, the product was obtained, and the product was centrifuged at 13,500 rpm for 15 min to obtain the drug-loaded zinc-iron oxide nanocomposite encapsulated by the cancer cell membrane. 4.根据权利要求1所述的癌细胞膜包裹载药锌铁氧化物纳米复合物的制备方法，其特征在于，包括以下步骤：4. the preparation method of cancer cell membrane package drug-loaded zinc-iron oxide nanocomposite according to claim 1, is characterized in that, comprises the following steps: （1）、制备锌铁氧化物纳米粒：将1mmol FeCl3·6H2O和0.5mmol ZnCl2溶解在40mL乙二醇中，得溶液，然后加入5.4g的NaAc和1.0g聚乙二醇，将混合物剧烈搅拌30min，然后密封在特氟隆衬里高压釜中，再放入马弗炉中密封，200°C反应12h，随后将高压釜冷却至室温，用磁体收集固体产物，固体产物分别用蒸馏水和乙醇洗涤3次，得样品，将样品在真空烘箱中85℃干燥7.5h，得锌铁氧化物纳米粒；(1) Preparation of zinc-iron oxide nanoparticles: Dissolve 1 mmol FeCl 3 6H 2 O and 0.5 mmol ZnCl 2 in 40 mL of ethylene glycol to obtain a solution, then add 5.4 g of NaAc and 1.0 g of polyethylene glycol, The mixture was vigorously stirred for 30min, then sealed in a Teflon-lined autoclave, then put into a muffle furnace and sealed, reacted at 200° C. for 12h, then the autoclave was cooled to room temperature, and the solid products were collected with a magnet. Washed with distilled water and ethanol for 3 times to obtain a sample, which was dried in a vacuum oven at 85 °C for 7.5 h to obtain zinc-iron oxide nanoparticles; （2）、制备负载紫杉醇的锌铁氧化物纳米复合物：将步骤（1）制备的锌铁氧化物纳米粒5mg分散于pH7.4的PBS缓冲液6mL中，形成第一混合液；将10mg紫杉醇分散于2 mL甲酰胺中，形成第二混合液；将第二混合液在搅拌下滴加于第一混合液中，室温搅拌24h，然后15000rpm离心10min，真空80℃干燥5h，得负载紫杉醇的锌铁氧化物纳米复合物；(2) Preparation of paclitaxel-loaded zinc-iron oxide nanocomposite: 5 mg of zinc-iron oxide nanoparticles prepared in step (1) were dispersed in 6 mL of PBS buffer at pH 7.4 to form a first mixed solution; 10 mg of Paclitaxel was dispersed in 2 mL of formamide to form a second mixed solution; the second mixed solution was added dropwise to the first mixed solution under stirring, stirred at room temperature for 24 hours, then centrifuged at 15,000 rpm for 10 min, and dried in vacuum at 80 °C for 5 hours to obtain the loaded paclitaxel ZnFeO nanocomposites; （3）、制备癌细胞膜包裹载药锌铁氧化物纳米复合物：通过差速离心法提取癌细胞膜，将细胞以660g离心10min，并用pH 7.4的PBS洗涤3次，将细胞沉淀重悬在低渗裂解缓冲液中进行机械破坏，以3200g离心5min，收集上清液，并重复该过程，将细胞再次以3200g离心6min，合并上清液，在4 ℃条件下以 21000g离心25min，收集上清液，将上清液在超高速离心机中4℃条件下以45000g离心5min，弃去上清液，用BCA蛋白质定量试剂盒对提取的细胞膜碎片进行量化，将步骤（2）制备的负载紫杉醇的锌铁氧化物纳米复合物分散于pH 7.4的PBS中，与制备的细胞膜以1：4的质量比混合，在水浴超声仪中将混合物超声处理32min，通过聚碳酸酯膜挤压15次，得产物，将产物15000rpm离心10min，即得癌细胞膜包裹载药锌铁氧化物纳米复合物。(3) Preparation of drug-loaded zinc-iron oxide nanocomposites encapsulated in cancer cell membranes: the cancer cell membranes were extracted by differential centrifugation, the cells were centrifuged at 660 g for 10 min, washed three times with PBS pH 7.4, and the cell pellets were resuspended in a low Perform mechanical disruption in lysis buffer, centrifuge at 3200g for 5min, collect the supernatant, and repeat the process, centrifuge the cells again at 3200g for 6min, combine the supernatant, centrifuge at 21000g for 25min at 4 °C, and collect the supernatant The supernatant was centrifuged at 45000g for 5 min at 4°C in an ultracentrifuge, the supernatant was discarded, and the extracted cell membrane fragments were quantified with BCA protein quantification kit. The zinc-iron oxide nanocomposite was dispersed in PBS with pH 7.4, mixed with the prepared cell membrane at a mass ratio of 1:4, the mixture was sonicated in a water bath sonicator for 32 min, and extruded through a polycarbonate membrane for 15 times, A product was obtained, and the product was centrifuged at 15,000 rpm for 10 min to obtain a drug-loaded zinc-iron oxide nanocomposite encapsulated by the cancer cell membrane. 5.根据权利要求1-4任一所述的癌细胞膜包裹载药锌铁氧化物纳米复合物的制备方法，其特征在于，所述的步骤（1）中锌铁氧化物纳米颗粒的粒径为80～160 nm，步骤（3）中癌细胞膜包裹载药锌铁氧化物纳米复合物的粒径为100～180 nm。5 . The method for preparing a drug-loaded zinc-iron oxide nanocomposite coated with a cancer cell membrane according to any one of claims 1-4, wherein the particle size of the zinc-iron oxide nanoparticles in the step (1) is is 80-160 nm, and the particle size of the drug-loaded zinc-iron oxide nanocomposite wrapped in the cancer cell membrane in step (3) is 100-180 nm. 6.根据权利要求1-4任一所述的癌细胞膜包裹载药锌铁氧化物纳米复合物的制备方法，其特征在于，所述的步骤（3）中聚碳酸酯膜为200～400 nm的聚碳酸酯多孔膜。6. The method for preparing a drug-loaded zinc-iron oxide nanocomposite coated by a cancer cell membrane according to any one of claims 1-4, wherein the polycarbonate membrane in the step (3) is 200-400 nm of polycarbonate porous membrane. 7.权利要求1-4任一所述方法制备的癌细胞膜包裹载药锌铁氧化物纳米复合物在制备抗肿瘤药物注射剂中的应用。7. Application of the cancer cell membrane-coated drug-loaded zinc-iron oxide nanocomposite prepared by any one of the methods of claims 1-4 in the preparation of antitumor drug injections. 8.权利要求1-4任一所述方法制备的癌细胞膜包裹载药锌铁氧化物纳米复合物在制备增强cGAS-STING免疫信号通路药物中的应用。8. Application of the cancer cell membrane-coated drug-loaded zinc-iron oxide nanocomposite prepared by any one of the methods of claims 1-4 in the preparation of a drug for enhancing cGAS-STING immune signaling pathway. 9.权利要求1-4任一所述方法制备的癌细胞膜包裹载药锌铁氧化物纳米复合物在制备基于pH敏感，谷胱甘肽响应的介孔锌铁氧化物肿瘤微环境药物中的应用。9. The cancer cell membrane-coated drug-loaded zinc-iron oxide nanocomposite prepared by any one of claims 1-4 is used in the preparation of a pH-sensitive, glutathione-responsive mesoporous zinc-iron oxide tumor microenvironment drug. application. 10.权利要求1-4任一所述方法制备癌细胞膜包裹载药锌铁氧化物纳米复合物在制备化疗联合免疫治疗抗肿瘤药物中的应用。10. Application of the method of any one of claims 1 to 4 to prepare cancer cell membrane-encapsulated drug-loaded zinc-iron oxide nanocomposites in the preparation of chemotherapy combined with immunotherapy antitumor drugs.
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