Preparation method of cholesterol-consuming nano-reactor and application thereof

Abstract

The application provides a preparation method of a cholesterol-consuming nano reactor and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: adding a zinc acetate solution dropwise into a 2-methylimidazole solution to obtain a mixed solution; adding a cholesterol oxidase solution and a new indocyanine green solution dropwise into the mixed solution to perform a reaction, so as to obtain a reaction solution; centrifuging the reaction solution, washing the precipitate, and obtaining ZIF@COD / IR820 nanoparticles; adopting a membrane protein extraction method to obtain Hepa1-6 liver cancer cell membrane fragments and red blood cell membrane fragments; mixing the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZIF@COD / IR820 nanoparticles, and then placing the mixture in a liposome extruder to perform repeated extrusion for several times, so as to obtain HM / ZIF@COD / IR820. The nano reactor prepared by the method can intensify cholesterol consumption of tumor cells, promote tumor cell apoptosis, has excellent homologous targeting, and is beneficial to improving the treatment effect of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of a cholesterol-consuming nanoreactor. Background Art

[0002] In the existing patented technology for treating liver cancer with cholesterol oxidase, the synthesis of 2-methylimidazole zinc metal framework nanoreactor (named ZIF) and the loading of cholesterol oxidase in the drug synthesis process are mostly step-by-step operations, the synthesis steps are complex, and the cholesterol oxidase loading efficiency is low; the traditional 2-methylimidazole zinc metal framework nanoreactor lacks targeting and cannot reach tumor cells in a targeted manner, resulting in insignificant cholesterol consumption effect alone, which in turn affects the treatment effect of liver cancer. Summary of the Invention

[0003] In order to solve the problem of poor efficacy of existing liver cancer treatment drugs, the present invention provides a method for preparing a cholesterol-consuming nanoreactor. The nanoreactor prepared by this method can intensify the cholesterol consumption of tumor cells, promote tumor cell apoptosis, and has excellent targeting, which is beneficial to improving the treatment effect of liver cancer.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides a method for preparing a cholesterol-consuming nanoreactor, the preparation method comprising:

[0006] Add zinc acetate solution dropwise to the 2-methylimidazole solution to obtain a mixed solution;

[0007] adding cholesterol oxidase solution and new indocyanine green (IR820) solution dropwise to the mixed solution to react and obtain a reaction solution;

[0008] The reaction solution was centrifuged, and the precipitate was washed to obtain ZI F@COD / I R820 nanoparticles;

[0009] Membrane protein extraction was used to obtain Hepa1-6 liver cancer cell membrane fragments and red blood cell membrane fragments;

[0010] The Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZI F@COD / I R820 nanoparticles were dissolved together and placed in a liposome extruder. The extrusion was repeated several times to obtain a cholesterol-consuming nanoreactor, named HM / ZI F@COD / I R820.

[0011] Furthermore, the step of adding zinc acetate solution dropwise to the 2-methylimidazole solution to obtain a mixed solution specifically comprises:

[0012] Add zinc acetate solution dropwise at a uniform rate to a 2-methylimidazole solution having a concentration of 160±20 mmol / L to obtain a mixed solution;

[0013] The concentration of the zinc acetate solution is 10±3 mmol / L, and the volume ratio of the 2-methylimidazole solution to the zinc acetate solution is 1:1.

[0014] Furthermore, the cholesterol oxidase solution and the new indocyanine green solution are added dropwise to the mixed solution to react to obtain a reaction solution, which specifically includes:

[0015] Adding dropwise 1±0.3 mg / mL cholesterol oxidase solution and 1±0.4 mg / mL new indocyanine green solution to the mixed solution, stirring and reacting for a period of time to obtain a reaction solution;

[0016] The volume ratio of the mixed solution, the cholesterol oxidase solution and the new indocyanine green solution is 100:2:2.

[0017] Furthermore, the reaction solution is centrifuged, and the precipitate is washed to obtain ZI F@COD / I R820 nanoparticles, which specifically includes:

[0018] The reaction solution was centrifuged at 12000 rpm for 20±5 min, and the precipitate was washed with water several times to obtain ZIF@COD / IR820 nanoparticles.

[0019] Furthermore, the membrane protein extraction method is used to obtain Hepa1-6 liver cancer cell membrane fragments and red blood cell membrane fragments, specifically comprising:

[0020] Hepa1-6 cells were dispersed in membrane protein extraction buffer and ground at 0 ± 4 °C to obtain a mixture;

[0021] The mixture was centrifuged, the supernatant was collected, and the centrifugation was continued. The obtained precipitate was freeze-dried to obtain Hepa1-6 liver cancer cell membrane fragments;

[0022] Red blood cells were added to 0.25× PBS solution and lysed at 0±4°C for 30±10 min. The red blood cells were then centrifuged and the blood ghosts were collected and washed twice. The red blood cell membrane fragments were obtained by freeze-drying.

[0023] Furthermore, the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZIF@COD / I R820 nanoparticles were dissolved together and placed in a liposome extruder, and extruded repeatedly several times to obtain a cholesterol-consuming nanoreactor, named HM / ZIF@COD / I R820, which specifically includes:

[0024] The Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments, and the ZI F@COD / I R820 nanoparticles were dissolved in water and placed in an Avant i microextruder. The extrusion was repeated 15 to 20 times to obtain a cholesterol-consuming nanoreactor, named HM / ZI F@COD / I R820.

[0025] The mass ratio of the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZI F@COD / I R820 nanoparticles is 1:1:5.

[0026] Based on the same inventive concept, the present invention provides a cholesterol-consuming nanoreactor, which is prepared by the above-mentioned preparation method of a cholesterol-consuming nanoreactor.

[0027] Based on the same inventive concept, the present invention provides an application of a cholesterol-consuming nanoreactor as or in the preparation of a drug for preventing and treating liver cancer.

[0028] Based on the same inventive concept, the present invention also provides an application of a cholesterol-consuming nanoreactor as or in the preparation of a tumor prevention and treatment drug.

[0029] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0030] 1. The present invention provides a method for preparing a cholesterol-depleting nanoreactor. The method uses a one-pot process to prepare ZIF@COD / IR820 nanoparticles loaded with cholesterol oxidase (COD) and IR820, uses a membrane protein extraction method to obtain Hepa1-6 liver cancer cell membrane fragments and erythrocyte membrane fragments, and finally, the ZIF@COD / IR820 nanoparticles, Hepa1-6 liver cancer cell membrane fragments, and erythrocyte membrane fragments are mixed and repeatedly extruded in a microextruder to obtain a nanoreactor HM / ZIF@COD / IR820. By adjusting the input ratios of zinc acetate, 2-methylimidazole, COD, and IR820, as well as the ratio of the two cell membrane fragments to ZIF@COD / IR820, the prepared HM / ZIF@COD / IR820 has excellent COD and IR820 encapsulation efficiency, and the nanoparticles are uniform and size-controllable. Compared with the traditional step-by-step loading method, the preparation process of the present method is simpler and easier to operate.

[0031] 2.The cholesterol-depleting nanoreactor of the present application, which is a 2-methylimidazole zinc metal framework (ZI F) loaded with COD and IR820, and is disguised with a hybrid cell membrane of red blood cell membrane-tumor cell membrane, can better target tumor cells, and can deplete cholesterol in tumor cells through COD, promote tumor cell apoptosis, and generate H2O2 through COD, which is catalyzed by catalase on the hybrid cell membrane to generate O2, thereby providing an O2 source for sonodynamic therapy, and the sonosensitizer IR820 stimulates O2 generation 1 O2 kills tumor cells and enhances the effect of sonodynamic therapy, thereby synergistically improving the therapeutic effect of tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0033] Figure 1 TEM image of ZI F loaded with COD and IR820 (ZI F@COD / IR820) nanoparticles.

[0034] Figure 2 Microscopic observation of the fusion of fluorescently labeled red blood cell membranes (red) and tumor cell membranes (green): orange represents complete fusion of the two cell membranes to form a hybrid cell membrane.

[0035] Figure 3 TEM image of ZI F loaded with COD and IR820 (ZI F@COD / IR820) nanoparticles.

[0036] Figure 4 Survival rate of Hepa1-6 cells after treatment with HM / ZI F@COD / IR820.

[0037] Figure 5 Statistical graph of the hemolysis rate of red blood cells after treatment with HM / ZI F@COD / IR820.

[0038] Figure 6 Cholesterol content in Hepa1-6 hepatoma cells labeled with Filipin staining solution.

[0039] Figure 7 Active oxygen in Hepa1-6 cells labeled with DCFH-DA probe.

[0040] Figure 8AM / PI staining marks the death of Hepa1-6 cells after HM / ZI F@COD / I R820 treatment combined with ultrasonic treatment. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0042] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0043] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0044] The technical principles of the present invention are as follows:

[0045] The present invention adopts a "one-pot" method to simultaneously load cholesterol oxidase (COD) and a new indocyanine green (IR820) with sonodynamic therapy effect into the 2-methylimidazole zinc metal framework nanoreactor during its synthesis, simplifying the synthesis process and greatly improving the loading efficiency of cholesterol oxidase. The method combines cholesterol consumption and sonodynamic therapy to treat liver cancer. The present invention utilizes a hybrid cell membrane of liver cancer tumor cell membrane and red blood cell membrane (named HM) to disguise the 2-methylimidazole zinc metal framework nanoreactor (named HM / ZIF@COD / IR820) loaded with COD and IR820. The liver cancer tumor cell membrane has homologous tumor cell targeting, and the red blood cell membrane has a rich peroxidase catalytic system to promote the generation of oxygen from hydrogen peroxide at the tumor site for sonodynamic therapy. This gives the new HM / ZIF@COD / IR820 nanoreactor a homologous targeting function, while simultaneously combining cholesterol consumption and sonodynamic therapy to treat liver cancer, thereby improving the therapeutic effect of liver cancer.

[0046] The present invention provides a method for preparing a cholesterol-consuming nanoreactor, the method comprising: S1. adding a zinc acetate solution dropwise to a 2-methylimidazole solution to obtain a mixed solution;

[0047] S2. Cholesterol oxidase solution and new indocyanine green solution were added dropwise to the mixture to react to obtain a reaction solution;

[0048] S3. The reaction solution was centrifuged, and the precipitate was washed to obtain ZI F@COD / I R820 nanoparticles;

[0049] S4. Use membrane protein extraction method to obtain Hepa1-6 liver cancer cell membrane fragments and red blood cell membrane fragments;

[0050] S5. The Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZI F@COD / I R820 nanoparticles were dissolved together and placed in a liposome extruder. The extrusion was repeated several times to obtain a cholesterol-consuming nanoreactor, named HM / ZI F@COD / I R820.

[0051] Step S1 specifically includes:

[0052] Add zinc acetate solution dropwise at a uniform rate to a 2-methylimidazole solution having a concentration of 160±20 mmol / L to obtain a mixed solution;

[0053] The concentration of the zinc acetate solution is 10±3 mmol / L, and the volume ratio of the 2-methylimidazole solution to the zinc acetate solution is 1:1.

[0054] In the present invention, the advantage of using a 2-methylimidazole solution concentration of 160±20 mmol / L and a zinc acetate solution concentration of 10±3 mmol / L is that the size of the generated 2-methylimidazole zinc metal framework is about 100 nm, which is conducive to subsequent entry into tumor cells. The advantage of using a volume ratio of the 2-methylimidazole solution to the zinc acetate solution of 1:1 is that the reaction raw materials are fully utilized and the yield of the 2-methylimidazole zinc metal framework is increased.

[0055] Step S2 specifically includes:

[0056] Adding dropwise 1±0.3 mg / mL cholesterol oxidase solution and 1±0.4 mg / mL new indocyanine green solution to the mixed solution, stirring and reacting for a period of time to obtain a reaction solution;

[0057] The volume ratio of the mixed solution, the cholesterol oxidase solution and the new indocyanine green solution is 100:2:2.

[0058] In the present invention, the cholesterol oxidase solution and the new indocyanine green solution adopt the above concentration and volume ratio, which has the advantage of being able to load cholesterol oxidase and IR820 to the maximum extent, thereby improving the loading efficiency of both.

[0059] Step S3 specifically includes:

[0060] The reaction solution was centrifuged at 12000 rpm for 20±5 min, and the precipitate was washed with water several times to obtain ZIF@COD / IR820 nanoparticles.

[0061] In the present invention, the roles of 2-methylimidazole and zinc acetate in the ZIF@COD / IR820 nanoparticles are to provide an imidazole framework and zinc ions, respectively, and ultimately form a 2-methylimidazole zinc metal framework.

[0062] Step S4 specifically includes:

[0063] Hepa1-6 cells were dispersed in membrane protein extraction buffer and ground at 0 ± 4 °C to obtain a mixture;

[0064] The mixture was centrifuged, the supernatant was collected, and the centrifugation was continued. The obtained precipitate was freeze-dried to obtain Hepa1-6 liver cancer cell membrane fragments;

[0065] Red blood cells were added to 0.25× PBS solution and lysed at 0±4°C for 30±10 min. The red blood cells were then centrifuged and the blood ghosts were collected and washed twice. The red blood cell membrane fragments were obtained by freeze-drying.

[0066] Step S5 specifically includes:

[0067] The Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments, and the ZI F@COD / I R820 nanoparticles were dissolved in water and placed in an Avant i microextruder. The extrusion was repeated 15 to 20 times to obtain a cholesterol-consuming nanoreactor, named HM / ZI F@COD / I R820.

[0068] The mass ratio of the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZI F@COD / I R820 nanoparticles is 1:1:5.

[0069] In the present invention, the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZI F@COD / IR820 nanoparticles are dissolved in water and placed in an Avant i micro-extruder. The advantage of repeated extrusion for 15 to 20 times is that the hybrid cell membrane is evenly wrapped on the ZI F@COD / IR820 nanoparticles through multiple extrusions, thereby obtaining HM / ZI F@COD / IR820 with uniform size and hybrid cell membrane camouflage.

[0070] In the present invention, the mass ratio of the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZI F@COD / IR820 nanoparticles is 1:1:5. The advantage is that the three raw materials are fully utilized to obtain HM / ZI F@COD / IR820 nanoparticles with uniform hybrid cell membranes, while ensuring that the size of the nanomaterials is appropriate, inheriting the function of homologous targeting of the hybrid cell membrane.

[0071] The preparation method and application of a cholesterol-consuming nanoreactor of the present application will be described in detail below with reference to examples and experimental data.

[0072] Example 1

[0073] This embodiment provides a method for preparing a cholesterol-consuming nanoreactor, comprising:

[0074] 1. One-pot synthesis of ZI F@COD / I R820:

[0075] 1) Weigh 656.8 mg of 2-methylimidazole and dissolve it in 50 mL of ultrapure water to a concentration of 160 mM. Weigh 148.7 mg of zinc acetate dihydrate powder and dissolve it in 50 mL of ultrapure water to a concentration of 10 mM.

[0076] 2) 50 mL of 2-methylimidazole solution was added to a round-bottom flask and magnetically stirred at room temperature for 5 min. 50 mL of zinc acetate dihydrate solution was uniformly added dropwise to the reaction system. After 60 s of reaction, 2 mL of 1 mg / mL COD solution and 2 mL of 1 mg / mL IR820 solution were added dropwise to the system, and the mixture was stirred at 1200 rpm for 8 min.

[0077] 3) After the reaction, the mixture was centrifuged at 12,000 rpm for 20 minutes to obtain a precipitate, which was then washed twice with ultrapure water to obtain purified ZIF@COD / IR820 nanoparticles. The COD and IR820 contents in the supernatant were assayed, and the encapsulation efficiencies of the two were calculated using a standard curve.

[0078] Test results: ZI F@COD / I R820 nanoparticles were successfully prepared ( Figure 1 ), among which, the encapsulation efficiency of COD was 89.2% and that of IR820 was 93.5%.

[0079] Preparation of ZIF / IR820 nanoparticles: The preparation method of ZIF / IR820 nanoparticles is the same as steps 1) to 3) above, except that in step 2), "after reacting for 60 seconds, 2 mL of 1 mg / mL COD solution and 2 mL of 1 mg / mL IR820 solution are added" is replaced by "after reacting for 60 seconds, 2 mL of 1 mg / mL IR820 solution are added" to obtain ZIF / IR820 nanoparticles.

[0080] 2. Cell membrane extraction and preparation of hybrid cell membrane camouflaged nanoreactors:

[0081] 1) Hepa1-6 cells were cultured and harvested, dispersed in membrane protein extraction buffer (purchased from Beijing Solebold Technology Co., Ltd.), and ground in an ice bath. The mixture was centrifuged at 3000 rpm for 10 minutes, the supernatant removed, and centrifuged at 8000 rpm for 10 minutes to obtain a precipitate, which was lyophilized to obtain tumor cell membrane fragments.

[0082] 2) Blood was collected from the mouse orbits and centrifuged at 3500 rpm for 5 min at 4°C to remove plasma. Red blood cells were washed with pre-chilled 1× PBS and lysed with 5 volumes of 0.25× PBS on ice for 30 min. The blood was centrifuged at 8000 rpm for 10 min, and blood ghosts were collected, washed twice, and freeze-dried to obtain red blood cell membrane fragments.

[0083] 3) 1 mg of erythrocyte membrane fragments, 1 mg of tumor cell membrane fragments, and 5 mg of ZIF@COD / IR820 or ZIF / IR820 were dissolved in 1 ml of water and repeatedly extruded 15-20 times through an Avant i microextruder (400 nm polycarbonate porous membrane) to obtain hybrid cell membrane-camouflaged HM / ZIF@COD / IR820 or HM / ZIF / IR820.

[0084] like Figure 2 As shown, the red blood cell membrane and tumor cell membrane in HM / ZI F@COD / I R820 successfully fused, and electron microscopy results showed that the cell membrane was coated on the ZI F@COD / I R820 nanoparticles ( Figure 3 ), the arrow at the outer edge indicates the cell membrane.

[0085] Example 2

[0086] This example tests the safety of HM / ZI F@COD / I R820 prepared in Example 1.

[0087] 1. Hepa1-6 cells were cultured at 1×10 4 Cells were seeded at a density of 100 μg / mL in 96-well plates and cultured overnight. Cells were then added to a 100 μg / mL HM / ZI F@COD / I R820 solution. Cell viability was determined using a CCK8 assay to assess the cytotoxicity of the HM / ZI F@COD / I R820 probe.

[0088] 2. Take 2 mL of fresh mouse blood and centrifuge at 3500 rpm for 5 minutes to obtain red blood cells. Wash with PBS 5 times and dilute to 20 mL. Use PBS and deionized water as negative and positive controls. Add different concentrations (5, 10, 20, 50, 100 μg / mL) of Zn 2+The nanoreactor HM / ZI F@COD / I R820 was diluted to 0.5 mL of PBS and 0.2 mL of red blood cells was added. After incubation at 37°C for 1, 2, 4, and 8 hours, the mixture was centrifuged at 3000 rpm and the absorbance of the supernatant was measured at 550 nm using a UV-visible spectrophotometer. The hemolysis rate was calculated as follows: Hemolysis rate (%) = (OD test -OD negative cotol ) / (OD positive control -OD negative cotol )100%.

[0089] Test results: CCK8 test results showed that after HM / ZI F@COD / I R820 treatment, the cell survival rate reached more than 85%, indicating that the nanoparticles are safe ( Figure 4 Hemolysis experiments showed that the hemolysis rate of red blood cells after HM / ZI F@COD / I R820 treatment met the international standard of 5%, indicating that the nanoparticles can be used for in vivo research ( Figure 5 ).

[0090] Example 3

[0091] In this example, the cholesterol consumption and sonodynamic therapy effects of HM / ZI F@COD / I R820 were evaluated.

[0092] 1. Cholesterol consumption in Hepa1-6 cells:

[0093] Hepa1-6 cells were incubated with phosphate buffered saline, ZIF@COD / IR820, and HM / ZIF@COD / IR820 for 24 hours (n=3). Ultrasound stimulation was used to accelerate the degradation of nanoparticles while simultaneously enabling the sonodynamic therapeutic effect of IR820. After treatment, cells were washed with PBS. Filipin stain was used to label intracellular cholesterol, and the effect of COD on cholesterol depletion in Hepa1-6 cells after the different treatments was compared.

[0094] Results: Microscopic observation of intracellular cholesterol marked by Filipin staining showed that the cholesterol content in Hepa1-6 cells was significantly reduced after treatment with HM / ZIF@COD / IR820 ( Figure 6 blue markers).

[0095] 2. Evaluation of Hepa1-6 cell killing effect in vitro:

[0096] Hepa1-6 cells in good condition were seeded into a six-well plate at a rate of 200,000 / well, shaken and placed in a cell culture incubator for overnight culture. When the cell density reached about 80%, the old culture medium was discarded, and fresh cell culture medium containing 100μg / mL nanoreactor HM / ZI F@COD / I R820 and fresh cell culture medium without nanoreactor were added to the six-well plate and cultured for another 24h. The old culture medium was discarded, washed with phosphate buffer, and 0.5w / cm 2 Ultrasonic stimulation was performed at a power of 100 nm for 30 seconds, and DCFH-DA reactive oxygen species detection probe was added. After 15-20 minutes, the cells were photographed and observed under a fluorescence microscope, and the fluorescence intensity was analyzed to detect the intracellular 1 Production of O2.

[0097] Test results: After HM / ZI F@COD / I R820 treatment combined with ultrasonic treatment, a large amount of 1 O2 generation ( Figure 7 .green mark).

[0098] 3. Take another Hepa1-6 cell and seed the Hepa1-6 cells in good condition into a six-well plate at 200,000 / well. After shaking, place the cells in a cell culture incubator and culture overnight. When the cell density reaches about 80%, discard the old culture medium and add fresh culture medium containing 100 μg / mL phosphate buffer, fresh cell culture medium of HM / ZI F / I R820 nanoreactor (the synthesis method of the nanoparticles is shown in Example 1), fresh cell culture medium of HM / ZI F@COD / I R820 nanoreactor and fresh cell culture medium without nanoreactor HM / ZI F@COD / I R820 combined with ultrasonic stimulation and continue to culture for 24 hours. The cells were stained with calcein / propidium iodide (dead or alive) and compared with the group without depletion of cholesterol and only sonodynamic therapy (0.5w / cm 2 Ultrasound stimulation for 30 seconds) and cholesterol depletion by nanoreactor plus sonodynamic therapy (0.5w / cm 2 The ultrasonic power of the group (ultrasound stimulation for 30 seconds) killed the Hepa1-6 cells.

[0099] The experimental results showed that after HM / ZI F@COD / I R820 treatment combined with ultrasonic treatment, compared with the control group, almost all cells in the experimental group died, and the tumor cell killing efficiency was significantly improved ( Figure 8 , red represents dead cells, green represents live cells).

[0100] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0101] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0102] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing a cholesterol-consuming nanoreactor, characterized in that: The preparation method comprises: Add zinc acetate solution dropwise to the 2-methylimidazole solution to obtain a mixed solution; adding cholesterol oxidase solution and new indocyanine green solution dropwise to the mixed solution for reaction to obtain a reaction solution; The reaction solution was centrifuged, and the precipitate was washed to obtain ZIF@COD / IR820 nanoparticles; Membrane protein extraction was used to obtain Hepa1-6 liver cancer cell membrane fragments and red blood cell membrane fragments; The Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZIF@COD / IR820 nanoparticles were dissolved together and placed in a liposome extruder. The extrusion was repeated several times to obtain a cholesterol-consuming nanoreactor, named HM / ZIF@COD / IR820.

2. The method for preparing a cholesterol-consuming nanoreactor according to claim 1, characterized in that: The step of adding a zinc acetate solution dropwise to the 2-methylimidazole solution to obtain a mixed solution specifically comprises: Add zinc acetate solution dropwise at a constant rate to a 2-methylimidazole solution with a concentration of 160±20 mmol / L to obtain a mixed solution; The concentration of the zinc acetate solution is 10±3 mmol / L, and the volume ratio of the 2-methylimidazole solution to the zinc acetate solution is 1:

1.

3. The method for preparing a cholesterol-consuming nanoreactor according to claim 1, characterized in that: The step of adding a cholesterol oxidase solution and a new indocyanine green solution to the mixed solution to react to obtain a reaction solution specifically comprises: Adding dropwise 1±0.3 mg / mL cholesterol oxidase solution and 1±0.4 mg / mL new indocyanine green solution to the mixed solution, stirring and reacting for a period of time to obtain a reaction solution; The volume ratio of the mixed solution, the cholesterol oxidase solution and the new indocyanine green solution is 100:2:

2.

4. The method for preparing a cholesterol-consuming nanoreactor according to claim 1, characterized in that: The reaction solution is centrifuged, and the precipitate is washed to obtain ZIF@COD / IR820 nanoparticles, specifically comprising: The reaction solution was centrifuged at 12000 rpm for 20±5 min, and the precipitate was washed with water several times to obtain ZIF@COD / IR820 nanoparticles.

5. The method for preparing a cholesterol-consuming nanoreactor according to claim 1, characterized in that: The membrane protein extraction method is used to obtain Hepa1-6 liver cancer cell membrane fragments and red blood cell membrane fragments, specifically comprising: Hepa1-6 cells were dispersed in membrane protein extraction buffer and ground at 0 ± 4 °C to obtain a mixture; The mixture was centrifuged, the supernatant was collected, and the centrifugation was continued. The obtained precipitate was freeze-dried to obtain Hepa1-6 liver cancer cell membrane fragments; Red blood cells were added to 0.25× PBS solution and lysed at 0±4°C for 30±10 min. The red blood cells were then centrifuged and the blood ghosts were collected and washed twice. The red blood cell membrane fragments were obtained by freeze-drying.

6. The method for preparing a cholesterol-consuming nanoreactor according to claim 1, characterized in that: The Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZIF@COD / IR820 nanoparticles are dissolved together and placed in a liposome extruder, and extruded repeatedly several times to obtain a cholesterol-consuming nanoreactor named HM / ZIF@COD / IR820, which specifically includes: The Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments, and the ZIF@COD / IR820 nanoparticles were dissolved in water and placed in an Avanti microextruder. The extrusion was repeated 15 to 20 times to obtain a cholesterol-consuming nanoreactor, named HM / ZIF@COD / IR820. The mass ratio of the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZIF@COD / IR820 nanoparticles is 1:1:

5.

7. A cholesterol-consuming nanoreactor, characterized in that: The nanoreactor is prepared by the preparation method of a cholesterol-consuming nanoreactor according to any one of claims 1 to 6.

8. Use of the cholesterol-consuming nanoreactor according to claim 7 in the preparation of drugs for preventing and treating liver cancer.

9. Use of the cholesterol-consuming nanoreactor according to claim 7 in the preparation of tumor prevention and treatment drugs.

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