Preparation method and application of cholesterol consumption type nanoreactor

Abstract

The invention provides a preparation method and application of a cholesterol consumption type nanoreactor, and belongs to the technical field of biological medicines.The preparation method comprises the steps that a zinc acetate solution is dropwise added into a 2-methylimidazole solution, and a mixed solution is obtained; dropwise adding a cholesterol oxidase solution and a new indocyanine green solution into the mixed solution for reaction to obtain a reaction solution; and centrifuging the reaction liquid, and washing the precipitate to obtain the ZIF (at) COD / IR820 nano-particles. The method comprises the following steps: acquiring Hepa1-6 liver cancer cell membrane fragments and erythrocyte membrane fragments by adopting a membrane protein extraction method; and mixing the Hepa1-6 liver cancer cell membrane fragments, the erythrocyte membrane fragments and the ZIF (at) COD / IR820 nanoparticles, putting the mixture into a liposome extruder, and repeatedly extruding for a plurality of times to obtain the HM / ZIF (at) COD / IR820. The nano-reactor prepared by the method can aggravate cholesterol consumption of tumor cells and promote apoptosis of the tumor cells, has excellent homologous targeting, and is beneficial to improving the treatment effect of liver cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly 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 complicated, 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 the simple cholesterol consumption effect is not significant, 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] Adding 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 for reaction to 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 method 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] Further, the step of adding a 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] Wherein, 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 step of adding a cholesterol oxidase solution and a new indocyanine green solution to the mixed solution for reaction to obtain a reaction solution specifically comprises:

[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] Wherein, 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 for several times to obtain ZI F@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 is centrifuged, the supernatant is taken, and the centrifugation is continued. The obtained precipitate is freeze-dried to obtain Hepa1-6 liver cancer cell membrane fragments;

[0022] Add red blood cells into 0.25×PBS solution, lyse the red blood cells at 0±4℃ for 30±10min, centrifuge, collect blood ghosts, wash twice, and freeze-dry to obtain red blood cell membrane fragments.

[0023] Furthermore, the Hepa1-6 liver cancer cell membrane fragments, the red blood cell membrane fragments and the ZI F@COD / I R820 nanoparticles are dissolved together and placed in a liposome extruder, and extruded repeatedly several times to obtain a cholesterol-consuming nanoreactor, named HM / ZI F@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 micro-extruder, and extruded repeatedly for 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. A method for preparing a cholesterol-consuming nanoreactor of the present invention, wherein the method adopts a "one-pot method" to prepare ZIF@COD / IR820 nanoparticles loaded with cholesterol oxidase (COD) and IR820, adopts 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 micro-extruder to obtain a nanoreactor HM / ZIF@COD / IR820. The method adjusts the input ratio of zinc acetate, 2-methylimidazole, COD and IR820, and the ratio of the two cell membrane fragments to ZIF@COD / IR820, so that the prepared HM / ZIF@COD / IR820 has excellent COD and IR820 encapsulation rate, and the nanoparticles are uniform in size and controllable in size. 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 present invention discloses a cholesterol-consuming nanoreactor, which uses a 2-methylimidazole zinc metal framework (ZIF) disguised as a hybrid cell membrane of an erythrocyte membrane and a tumor cell membrane to load COD and IR820, and can better homologously target tumor cells, aggravate the cholesterol consumption of tumor cells through COD, and promote tumor cell apoptosis. At the same time, COD consumes H2O2 generated by cholesterol, and generates O2 under the catalysis of catalase on the hybrid cell membrane, providing an O2 source for sonodynamic therapy, and the sonosensitizer IR820 stimulates the generation of O2 1 O2 kills tumor cells, enhances the effect of sonodynamic therapy, and thus synergistically improves the therapeutic effect of tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, a brief introduction is given below to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

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

[0034] Figure 2 This is a fusion image of fluorescently labeled red blood cell membrane (red) and tumor cell membrane (green) observed under a microscope: the orange color indicates that the two cell membranes are completely fused to form a hybrid cell membrane.

[0035] Figure 3 Transmission electron microscopy image of hybrid cell membrane-camouflaged ZI F-loaded COD and IR820 (HM / ZI F@COD / IR820) nanoparticles.

[0036] Figure 4 Statistical graph of Hepa1-6 cell survival rate after treatment with HM / ZI F@COD / I R820.

[0037] Figure 5 This is a statistical chart of the hemolysis rate of red blood cells after treatment with HM / ZI F@COD / I R820.

[0038] Figure 6 Filipin dye was used to label the cholesterol content in Hepa1-6 liver cancer cells.

[0039] Figure 7 DCFH-DA probe is used to label reactive oxygen species in Hepa1-6 cells.

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

[0041] The present invention will be described in detail below in conjunction with specific implementations 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 implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0042] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes 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, and cholesterol oxidase (COD) and a new indocyanine green (IR820) with sonodynamic therapy effect are simultaneously loaded into the 2-methylimidazole zinc metal frame nanoreactor during the synthesis process, thereby simplifying the synthesis process, greatly improving the loading efficiency of cholesterol oxidase, and treating liver cancer by cholesterol consumption and sonodynamic therapy. The present invention uses a hybrid cell membrane (named HM) of liver cancer tumor cell membrane and erythrocyte membrane to disguise a 2-methylimidazole zinc metal frame nanoreactor (named HM / ZIF@COD / IR820) loaded with COD and IR820, the liver cancer tumor cell membrane has homologous tumor cell targeting, and the erythrocyte membrane has a rich peroxidase catalytic system to promote the generation of oxygen from hydrogen peroxide in the tumor site for sonodynamic therapy, giving the new HM / ZIF@COD / IR820 nanoreactor a homologous targeting function, and 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. adding cholesterol oxidase solution and new indocyanine green solution dropwise to the mixed solution to react and obtain a reaction solution;

[0048] S3. The reaction solution is centrifuged, and the precipitate is 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 are dissolved together and placed in a liposome extruder. The extrusion is repeated several times to obtain a cholesterol-consuming nanoreactor, which is 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] Wherein, 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 concentration of the 2-methylimidazole solution is 160±20 mmol / L and the concentration of the zinc acetate solution is 10±3 mmol / L, which has the advantage that the size of the generated 2-methylimidazole zinc metal framework is about 100 nm, which is conducive to the subsequent entry into tumor cells. The volume ratio of the 2-methylimidazole solution to the zinc acetate solution is 1:1, which has the advantage that the reaction raw materials are fully utilized and the yield of the 2-methylimidazole zinc metal framework is improved.

[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] Wherein, 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 for several times to obtain ZI F@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 finally 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 is centrifuged, the supernatant is taken, and the centrifugation is continued. The obtained precipitate is freeze-dried to obtain Hepa1-6 liver cancer cell membrane fragments;

[0065] Add red blood cells into 0.25×PBS solution, lyse the red blood cells at 0±4℃ for 30±10min, centrifuge, collect blood ghosts, wash twice, and freeze-dry to obtain red blood cell membrane fragments.

[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 micro-extruder, and extruded repeatedly for 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 repeatedly extruding 15 to 20 times is that through multiple extrusions, the hybrid cell membrane is evenly wrapped on the ZI F@COD / I R820 nanoparticles, thereby obtaining HM / ZI F@COD / I R820 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, which has the advantage of making full use of the three raw materials to obtain HM / ZI F@COD / I R820 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 in combination with examples and experimental data.

[0072] Example 1

[0073] The present 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 to the reaction system. After reacting for 60 s, 2 mL of 1 mg / mL COD solution and 2 ml of 1 mg / mL IR820 solution were added to the system and stirred at 1200 rpm for 8 min.

[0077] 3) After the reaction, centrifuge at 12000 rpm for 20 min to obtain a precipitate, wash twice with ultrapure water to obtain purified ZIF@COD / IR820 nanoparticles. Detect COD and IR820 in the supernatant, and calculate the encapsulation efficiency of the two based on the 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 ZI F / I R820 nanoparticles: The preparation method of ZI F / I R820 nanoparticles is the same as the above steps 1)-3), except that in step 2), "add 2 mL of 1 mg / mL COD solution and 2 mL of 1 mg / mL IR820 solution after reacting for 60 s" is replaced by "add 2 mL of 1 mg / mL IR820 solution after reacting for 60 s", thereby obtaining ZI F / I R820 nanoparticles.

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

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

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

[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 to 20 times through an Avant i micro extruder (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 the 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 / well density was inoculated in a 96-well plate and cultured overnight. 100 μg / mL of HM / ZI F@COD / I R820 was added, and the cell viability was measured by CCK8 assay to evaluate the cytotoxicity of HM / ZI F@COD / I R820 probe.

[0088] 2. Take 2mL of fresh mouse blood and centrifuge at 3500rpm for 5min to obtain red blood cells. Wash with PBS 5 times and dilute to 20mL. Use PBS and deionized water as negative and positive controls. Add different concentrations (5, 10, 20, 50, 100μg / mL) of Zn 2+The nanoreactor HM / ZI F@COD / I R820 was fixed 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 at 550 nm was measured 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 effect 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 buffer, ZIF@COD / IR820, and HM / ZIF@COD / IR820 for 24 h (n=3). Ultrasound stimulation was used to accelerate the degradation of nanoparticles while allowing IR820 to exert sonodynamic therapeutic effects, and then washed with PBS. Filipin dye was used to label intracellular cholesterol, and the depletion of intracellular cholesterol in Hepa1-6 cells by intracellular COD after different treatments was compared.

[0094] Results: Filipin staining solution was used to mark intracellular cholesterol under a microscope, which showed that the cholesterol content of Hepa1-6 cells was significantly reduced after treatment with HM / ZI F@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 inoculated into a six-well plate at 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 24h. The old culture medium was discarded, rinsed with phosphate buffer, and 0.5w / cm 2 Ultrasonic stimulation was performed at a power of 1000 nm for 30 s, and the DCFH-DA reactive oxygen species detection probe was added. After 15-20 min, 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, inoculate the Hepa1-6 cells in good condition into a six-well plate at 200,000 / well, shake well and put into a cell culture incubator for overnight culture. When the cell density reaches about 80%, discard the old culture medium, 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 and alive) to compare 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 ultrasound 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 series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are 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 other 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 can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a cholesterol-consuming nanoreactor, characterized in that: The preparation method comprises: Adding 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 is centrifuged, and the precipitate is washed to obtain ZIF@COD / IR820 nanoparticles; Membrane protein extraction method 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, and extruded repeatedly for 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 a 2-methylimidazole solution to obtain a mixed solution specifically comprises: 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; Wherein, 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 for reaction 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; Wherein, 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 step of centrifuging the reaction solution and washing the precipitate to obtain ZIF@COD / IR820 nanoparticles specifically comprises: The reaction solution was centrifuged at 12000 rpm for 20±5 min, and the precipitate was washed with water for 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 is centrifuged, the supernatant is taken, and the centrifugation is continued. The obtained precipitate is freeze-dried to obtain Hepa1-6 liver cancer cell membrane fragments; Add red blood cells into 0.25×PBS solution, lyse the red blood cells at 0±4℃ for 30±10min, centrifuge, collect blood ghosts, wash twice, and freeze-dry to obtain red blood cell membrane fragments.

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 for 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 micro-extruder, and extruded repeatedly for 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 a cholesterol-consuming nanoreactor as claimed in claim 7 as or in the preparation of a drug for preventing and treating liver cancer.

9. Use of a cholesterol-consuming nanoreactor as claimed in claim 7 as or in the preparation of a tumor prevention and treatment drug.

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