Composite cell membrane vesicle as well as preparation method and application thereof

Abstract

The invention relates to a composite cell membrane vesicle as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The composite cell membrane vesicle provided by the invention contains BP-coated Cu and glucose oxidase; the surface of the vesicle contains a CD64 protein and an EGFR (epidermal growth factor receptor) monoclonal antibody. The invention proves that the composite cell membrane vesicles have a homologous-molecular dual-targeting effect on tumor cells, animal experiments prove that the composite cell membrane vesicles can target tumor cells and are taken by the tumor cells, and after the composite cell membrane vesicles are taken by the tumor cells, BP-coated Cu and GOx of the composite cell membrane vesicles synergistically induce the cells to generate copper death, so that the composite cell membrane vesicles have the effect of treating the tumor cells. The EGFR monoclonal antibody blocks the EGFR pathway, and the growth and activity of tumor cells are inhibited from two pathways, so that the anti-tumor effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a composite cell membrane vesicle and a preparation method and application thereof. Background Art

[0002] Cuproptosis is a newly discovered way of programmed cell death in 2022. Different from cell death pathways such as apoptosis, necrosis, pyroptosis, and ferroptosis, cuproptosis is a copper-dependent cell death pathway characterized by the aggregation of mitochondrial lipoylated proteins induced by excessive accumulation of intracellular copper ions and the downregulation of iron-sulfur cluster proteins. Due to its mechanism different from apoptosis and other cell death pathways, cuproptosis has broad application prospects in the treatment of tumors (especially drug-resistant tumors).

[0003] Black phosphorus nanosheets (BP) are a new type of two-dimensional nanomaterial with good photothermal performance, photodynamic performance, drug loading capacity, and biosecurity, and have shown great potential in the diagnosis and treatment of tumors, infections, neurodegenerative diseases, etc. in recent years.

[0004] Existing small molecule cuproptosis inducers have the following disadvantages: First, the copper ion loading and delivery ability is low. Due to their small molecular weight and limited copper ion binding groups, each small molecule copper ion carrier can only load one to several copper ions. In addition, monovalent Cu + has stronger cuproptosis induction activity than divalent Cu 2+ , but due to its poor stability in aqueous solution, it is difficult to deliver in vivo. Second, there is no tumor targeting. Small molecule cuproptosis inducers have no targeting in vivo, cannot be efficiently enriched at target sites such as tumors, affecting the treatment effect, and the distribution in normal tissues and organs brings a greater risk of side effects. Third, the pharmacokinetic performance is poor. Due to their small molecular weight, small molecule cuproptosis inducers are easily metabolized and excreted by cells and the body, have a short retention time at target sites such as tumors, are difficult to maintain an effective concentration, and are difficult to induce cuproptosis persistently and efficiently, and the curative effect is limited.

[0005] Therefore, there is an urgent need for a cuproptosis induction system with high transfer efficiency, good tumor targeting, and excellent in vivo pharmacokinetic characteristics. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a composite cell membrane vesicle with high copper element loading efficiency, tumor targeting, and good pharmacokinetic performance, and a preparation method and application thereof.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a composite cell membrane vesicle, which contains BP@Cu and glucose oxidase; the surface of the vesicle contains CD64 protein and EGFR monoclonal antibody.

[0009] The present invention loads BP@Cu and glucose oxidase into the cell membrane vesicle, and binds CD64 protein and EGFR monoclonal antibody to the membrane of the vesicle to construct a composite cell membrane vesicle, which has excellent anti-tumor efficacy, especially more prominent anti-nasopharyngeal carcinoma efficacy. The present invention confirms through experiments that BP@Cu can induce cuproptosis in tumor cells. BP@Cu and glucose oxidase are co-delivered to interfere with the energy metabolism of tumor cells, further enhancing the sensitivity of tumor cells to cuproptosis. At the same time, the present invention also confirms that the composite cell membrane vesicle has a homologous-molecular dual-targeting effect on tumor cells. Through animal experiments, it is confirmed that the composite cell membrane vesicle can target tumor cells and be taken up by tumor cells. After the composite cell membrane vesicle is taken up by tumor cells, the BP@Cu and glucose oxidase carried by it synergistically induce cuproptosis in cells, and the EGFR monoclonal antibody blocks the EGFR pathway, inhibiting the growth and development of tumor cells from two ways, thereby playing an anti-tumor effect.

[0010] As a preferred embodiment of the composite cell membrane vesicle of the present invention, the mass ratio of Cu element to BP in the BP@Cu is Cu:BP=(0.016 - 10):1. The present invention confirms through experiments that when Cu and BP are in the above ratio, Cu can be doped onto BP.

[0011] As a preferred embodiment of the composite cell membrane vesicle of the present invention, the mass ratio of Cu element to BP in the BP@Cu is Cu:BP=(0.08 - 2):1.

[0012] As a preferred embodiment of the composite cell membrane vesicle of the present invention, the mass ratio of Cu element to BP in the BP@Cu is Cu:BP = 0.4:1.

[0013] As a preferred embodiment of the composite cell membrane vesicle of the present invention, the mass ratio of Cu element to BP in the BP@Cu is Cu:BP = 2:1.

[0014] As a preferred embodiment of the composite cell membrane vesicle of the present invention, the BP in the BP@Cu is a nanosheet with a size of 100 - 200 nm.

[0015] As a preferred embodiment of the composite cell membrane vesicles of the present invention, the mass ratio of BP@Cu to glucose oxidase is BP@Cu:glucose oxidase = (400 - 500):(5 - 6). Within the preferred ratio range, the composite cell membrane vesicles of the present invention containing BP@Cu and glucose oxidase within the above mass ratio range can synergistically enhance the cuproptosis of tumor cells.

[0016] As a preferred embodiment of the composite cell membrane vesicles of the present invention, the mass ratio of BP@Cu to glucose oxidase is BP@Cu:glucose oxidase = 500:6.

[0017] In a second aspect, the present invention provides a method for preparing the above composite cell membrane vesicles, comprising the following steps:

[0018] (1) After constructing cells overexpressing CD64 protein through lentiviral infection technology, the cell membranes of the cells overexpressing CD64 protein are separated and purified by a hypotonic method to obtain cell membranes overexpressing CD64 protein;

[0019] (2) The cell membranes overexpressing CD64 protein obtained in step (1) are extruded to obtain cell membrane vesicles overexpressing CD64 protein;

[0020] (3) BP@Cu and glucose oxidase are loaded into the cell membrane vesicles overexpressing CD64 protein obtained in step (2) to obtain vesicle BP@Cu-GOx;

[0021] (4) The vesicle BP@Cu-GOx obtained in step (3) is mixed with EGFR monoclonal antibody and incubated to obtain composite cell membrane vesicles BP@Cu-GOx-CTX.

[0022] As a preferred embodiment of the preparation method of the present invention, in step (1), the cells are tumor cells. In order to enable the composite cell membrane vesicles to have a homologous targeting effect on tumors, the cell membranes of tumor cells are used to prepare cell membrane vesicles, so that the cell membrane vesicles can homologously target the corresponding tumor cells in vivo, thereby achieving the effect of targeted anti-tumor. The types of tumor cells described in the present invention are not limited, and different types of tumor cells can be selected as needed to prepare cell membrane vesicles to achieve the function of homologous targeting.

[0023] As a preferred embodiment of the preparation method of the present invention, in step (1), the lentivirus infection technique is to construct a lentivirus containing the coding sequence of CD64 protein and the coding sequence of GFP protein, infect cells, and obtain cells stably overexpressing CD64 protein through puromycin screening. The Fc fragment on the IgG antibody can specifically bind to the CD64 protein. When the cells are incubated with the IgG antibody, due to the high affinity and specificity between the CD64 protein and Fc, the IgG antibody can be bound to the cell membrane. The present invention has experimentally confirmed that the expressed CD64 has a binding site for the Fc fragment and can specifically bind to the IgG antibody.

[0024] As a preferred embodiment of the preparation method of the present invention, in step (2), the extrusion is carried out in a micro syringe extruder.

[0025] As a preferred embodiment of the preparation method of the present invention, the micro syringe extruder contains a polycarbonate membrane with at least one of pore sizes of 1μm, 400nm, and 200nm.

[0026] As a preferred embodiment of the preparation method of the present invention, in step (3), the BP in BP@Cu can be prepared by liquid phase exfoliation method.

[0027] As a preferred embodiment of the preparation method of the present invention, the preparation method of BP@Cu is mainly to ultrasonically treat the aqueous dispersion of BP for 2 min, centrifuge at 4 °C and 18,000×g for 10 min, remove the supernatant, add an appropriate amount of copper ion solution, ultrasonically treat for 2 - 3 min, centrifuge at 4 °C and 18,000×g for 10 min, and wash twice with water to obtain BP@Cu. The present invention provides a preparation method of BP@Cu and characterizes BP@Cu, and finds that the Cu carried in BP@Cu + accounts for 85% of the total Cu element, indicating that BP can effectively deliver unstable Cu + , improving the induction efficiency of cuproptosis. At the same time, BP@Cu can passively target tumors through the enhanced permeability and retention effect. In addition, the surface modification of BP@Cu can further improve its targeting performance, realize enrichment at the tumor site, reduce non-specific enrichment in normal tissues and organs, improve the specificity of cuproptosis induction and disease treatment, and reduce the risk of side effects.

[0028] As a preferred embodiment of the preparation method of the present invention, the copper ion solution includes but is not limited to copper sulfate solution and copper chloride solution.

[0029] As a preferred embodiment of the preparation method of the present invention, in step (3), the BP@Cu is subjected to surface modification, and the surface modification includes but is not limited to at least one of polyethylene glycol modification, chitosan modification, mannose modification, and galactose modification. After surface modification, the stability, biocompatibility, and targeting of BP@Cu can be improved.

[0030] As a preferred embodiment of the preparation method of the present invention, in step (3), the mass ratio of BP@Cu to cell membrane vesicles is BP@Cu: cell membrane vesicles = 1: (1 - 10).

[0031] As a preferred embodiment of the preparation method of the present invention, in step (3), the mass ratio of BP@Cu to glucose oxidase in the vesicle BP@Cu is BP@Cu: glucose oxidase = (1 - 10): 1.

[0032] As a preferred embodiment of the preparation method of the present invention, in step (3), the mixing is carried out by ultrasonic treatment at a power of 110 - 120W for 2 - 5min.

[0033] As a preferred embodiment of the preparation method of the present invention, in step (4), the mass ratio of the vesicle BP@Cu-GOx to the anti-EGFR monoclonal antibody is vesicle BP@Cu-GOx: anti-EGFR monoclonal antibody = (10 - 20): 1.

[0034] As a preferred embodiment of the preparation method of the present invention, in step (4), the incubation conditions are incubation at 4°C for 25 - 30min.

[0035] In the third aspect, the present invention provides the use of the above-mentioned composite cell membrane vesicles in the preparation of anti-tumor drugs. The present invention has confirmed through cell experiments and animal experiments that the composite cell membrane vesicles can effectively induce tumor cell death and reduce the volume of solid tumors, and have excellent anti-tumor effects.

[0036] As a preferred embodiment of the use of the present invention, the anti-tumor drug is an anti-nasopharyngeal carcinoma drug.

[0037] In the fourth aspect, the present invention provides the use of the above-mentioned composite cell membrane vesicles combined with near-infrared light treatment in the preparation of anti-tumor drugs. The present invention has confirmed through animal experiments that the combination of composite cell membrane vesicles and near-infrared light treatment can effectively reduce the solid tumor of tumor-bearing mice to disappearance, and the anti-tumor effect is more excellent.

[0038] As a preferred embodiment of the use of the present invention, the wavelength of the near-infrared light is 805 - 810nm.

[0039] As a preferred embodiment of the use of the present invention, the wavelength of the near-infrared light is 808nm.

[0040] As a preferred embodiment of the application of the present invention, the conditions for near-infrared light treatment are 1 W / cm 2 Irradiate for 5 - 10 min.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] 1. In the present invention, BP@Cu and glucose oxidase are loaded into cell membrane vesicles, and CD64 protein and EGFR monoclonal antibody are conjugated to the membrane of the vesicles to construct a composite cell membrane vesicle, which has excellent anti-tumor efficacy, especially more prominent anti-nasopharyngeal carcinoma efficacy.

[0043] 2. The composite cell membrane vesicle provided by the present invention can achieve a homologous-molecular targeting effect, and can target and deliver BP@Cu and GOx to the tumor site, showing good energy metabolism intervention, copper death induction and tumor suppression effects. Compared with traditional small molecule copper death inducers, the copper death induction and tumor treatment effects of the composite cell membrane vesicle of the present invention are more excellent, effectively improving the defect of poor pharmacokinetic performance of BP@Cu, GOx and EGFR monoclonal antibody.

[0044] 3. The present invention confirms through animal experiments that the combination of the composite cell membrane vesicle and near-infrared light can effectively shrink the solid tumor of tumor-bearing mice to disappearance, and the anti-tumor effect is more excellent. Description of the Drawings

[0045] Figure 1 It is the preparation flow chart of the composite cell membrane vesicle of the present invention;

[0046] Figure 2 It is the STEM image of the elemental distribution of BP@Cu obtained in Example 1 of Effect Example 1 of the present invention;

[0047] Figure 3 It is the statistical result chart of the Cu element loading efficiency in BP@Cu with different ratios in Effect Example 1 of the present invention;

[0048] Figure 4 It is the XPS spectrum of BP@Cu obtained in Example 1 of Effect Example 1 of the present invention, where the upper figure is BP@Cu and the lower figure is BP nanosheets;

[0049] Figure 5 It is the fluorescence microscope image of the cells overexpressing CD64 obtained in Example 1 of Effect Example 1 of the present invention binding to fluorescently labeled IgG molecules;

[0050] Figure 6 It is the flow cytometer result chart of the cells overexpressing CD64 obtained in Example 1 of Effect Example 1 of the present invention;

[0051] Figure 7 Laser confocal microscopy images of the cells with stable overexpression of CD64 (a) and the cell membrane vesicles after binding with antibodies (b) obtained in Example 1 of Effect Example 1 of the present invention;

[0052] Figure 8 TEM images of the cell membrane vesicles (a) and the composite cell membrane vesicles BP@Cu-GOx-CTX (b) obtained in Example 1 of Effect Example 1 of the present invention;

[0053] Figure 9 Analysis graphs of the average size (a) and zeta potential (b) of the cell membrane vesicles (a) and the composite cell membrane vesicles BP@Cu-GOx-CTX obtained in Example 1 of Effect Example 1 of the present invention;

[0054] Figure 10 WB detection result graph of the loading of GOx, surface binding of CD64 and CTX in the composite cell membrane vesicles BP@Cu-GOx-CTX obtained in Example 1 of Effect Example 1 of the present invention;

[0055] Figure 11 Statistical result graphs of the Cu level (a) and cell viability (b) in NPC cells S18 with different treatments in Effect Example 2 of the present invention;

[0056] Figure 12 WB detection result graph of the copper death marker protein in NPC cells S18 with different treatments in Effect Example 2 of the present invention;

[0057] Figure 13 TEM images of the mitochondria of NPC cells S18 with different treatments in Effect Example 2 of the present invention;

[0058] Figure 14 Statistical result graphs of the glucose content (a) and pH (b) in different treatments in Effect Example 2 of the present invention;

[0059] Figure 15 Calcein AM / PI staining result graph of NPC cells S18 with different treatments in Effect Example 2 of the present invention;

[0060] Figure 16 TEM images of the mitochondria of NPC cells S18 with different treatments in Effect Example 2 of the present invention;

[0061] Figure 17 WB detection result graph of NPC cells S18 with different treatments in Effect Example 2 of the present invention;

[0062] Figure 18 Cell viability and calcein AM / PI staining result graph of NPC cells S18 with different treatments in Effect Example 2 of the present invention;

[0063] Figure 19 For the photothermal performance detection results of NPC cells S18 under different treatments in Effect Example 2 of the present invention (a), the photothermal performance detection results of NPC cells S18 treated with composite cell membrane vesicles BP@Cu-GOx-CTX at different doses (b), and the cell viability statistical results of NPC cells S18 under different treatments (c);

[0064] Figure 20 For the WB detection results of tumor tissues of mice under different treatments in Effect Example 2 of the present invention;

[0065] Figure 21 For the photothermal performance detection results of tumor tissues of mice under different treatments in Effect Example 2 of the present invention (a), the relative volume change results of tumor tissues of mice under different treatments (b), the representative diagrams of tumor tissues of mice under different treatments (c), and the tumor weight statistical results of mice under different treatments (d);

[0066] Figure 22 For the graph of body weight change results of mice under different treatments in Effect Example 2 of the present invention;

[0067] Figure 23 For the laser confocal microscope images and average fluorescence intensity statistical results of cells under different treatments in Effect Example 2 of the present invention;

[0068] Figure 24 For the in vivo imaging results of mice under different treatments in Effect Example 2 of the present invention;

[0069] Figure 25 For the WB detection results of NPC cells S18 under different treatments in Effect Example 2 of the present invention;

[0070] In the above figures, those with "*" all indicate significant differences between two groups, where "*" means p < 0.05, "**" means p < 0.01, and "***" means p < 0.001. Detailed implementation manners

[0071] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0072] Other materials, reagents, etc. used in the examples, comparative examples and effect examples can be obtained from commercial channels without special instructions.

[0073] The size of the black phosphorus nanosheets (BP) used in the present invention is 100 - 200 nm, which can be purchased through commercial channels. The present invention provides a preparation method by liquid-phase exfoliation, including the following steps:

[0074] Take 20 mg of bulk black phosphorus, grind it into powder, add it to 40 mL of isopropanol, sonicate for 45 s, with an interval of 15 s, centrifuge at 6000×g for 5 min, take the supernatant and centrifuge at 9000×g for 20 min, then take the supernatant and centrifuge at 12000×g for 20 min. Remove the supernatant, and disperse the obtained precipitate in isopropanol to obtain a dispersion of black phosphorus nanosheets (BP) in isopropanol.

[0075] The method for preparing the BP aqueous dispersion used in the present invention is as follows: After sonication of the BP isopropanol dispersion for 2 min, centrifuge at 4°C and 18000×g for 10 min. After removing the supernatant, add an appropriate amount of water and sonicate for 2 min to obtain the BP aqueous dispersion.

[0076] BP@Cu of the present invention can be surface-modified. In the following examples, PEG surface modification is carried out. The specific operation of PEG surface modification is as follows: Mix BP@Cu with an aqueous solution of polyethylene glycol (PEG) such that the mass ratio of BP@Cu to PEG is BP@Cu:BP = 1:10. After sonication at 4°C for 30 min, incubate at 4°C for 3 h. After the incubation, centrifuge at 4°C and 18000×g for 10 min, and remove the supernatant to obtain PEG-modified BP@Cu.

[0077] In the following examples, comparative examples, the cell membrane vesicles are obtained by mechanical extrusion. The mechanical extrusion is completed using a micro syringe extrusion instrument. The assembly method of the micro syringe extrusion instrument is as follows:

[0078] After washing two internal membrane cylindrical supports with PBS, place them flat on the experimental table with the black ring surface facing up, and then place one of them into the outer shell of the A-type extruder;

[0079] After pre-wetting two filter supports with PBS, place them respectively above the holes of the inner membrane support; After pre-wetting polycarbonate membranes of different sizes (1 μm, 400 nm, 200 nm) with PBS, place them above the filter supports;

[0080] Then align and tighten the outer shells of the two extruders A and B by hand, and at this time, the assembly of the micro syringe extrusion instrument is completed.

[0081] In the following examples, comparative examples, and effectiveness examples, each 1 mL of the mixed membrane protein extraction reagent contains 10 μL of protease inhibitor, 10 μL of phosphatase inhibitor, and 980 μL of membrane protein extractant.

[0082] In the following effectiveness examples, the Balb / c nude mice are purchased from Guangdong Pharmaron Biotechnology Co., Ltd., and the breeding conditions are SPF level.

[0083] In the following effectiveness examples, the nasopharyngeal carcinoma (NPC) cell line S18 is purchased from Geneo Biotech, and the product number is JNO-H0098.

[0084] In the following examples, comparative examples, and effect examples, the experimental methods not specifically noted are conventional methods in the art, and reference can be made to "Experimental Zoology" (edited by Ding Yuqiang, published by Fudan University Press), "Medical Experimental Zoology (2nd Edition)" (published by Science Press), "Principles and Techniques of Medical Immunology Experiments" (edited by Chu Yiwei and Lu Qing, published by Fudan University Press), "Molecular Cloning Experiment Guide (4th Edition)", etc.

[0085] In the immunoblotting experiments in the following effect examples, the antibodies selected are all conventional selections by those skilled in the art according to the experimental purpose. The present invention does not specifically limit the types and commercial sources of the reagents used.

[0086] Example 1

[0087] Example 1 provides a composite cell membrane vesicle and a preparation method thereof. The preparation process of the cell membrane vesicle is as Figure 1 shown. The preparation method includes the following steps:

[0088] S1. Centrifuge the BP aqueous dispersion at 4°C and 18,000×g for 10 min. After removing the supernatant, add an appropriate amount of copper chloride solution so that the mass ratio of Cu element to BP is Cu:BP = 2:1. Ultrasonic for 2 min, and stir the solution during ultrasonic treatment to make Cu and BP fully combine. After the ultrasonic treatment, centrifuge at 4°C and 18,000×g for 10 min, wash twice with water, and perform PEG surface modification on the BP loaded with Cu element to obtain BP@Cu;

[0089] S2. Clone the gene sequence encoding the CD64 protein from human cDNA, use the BamHI and Xbal restriction enzyme cleavage sites for enzyme cleavage and ligation to insert the C-terminal green fluorescent protein GFP, and assemble it into the pLV vector to construct the lentiviral vector pLV-CD64-GFP;

[0090] S3. Transfer the lentiviral vector in step S2 into human embryonic kidney 293 cells to induce lentivirus containing the CD64-GFP fusion gene. Use the lentivirus containing the CD64-GFP fusion gene to infect nasopharyngeal carcinoma cells S18 to obtain nasopharyngeal carcinoma cells overexpressing the CD64-GFP protein. At this time, CD64-GFP is localized to the cell membrane, and a cell line stably overexpressing the CD64 protein is obtained by screening with puromycin;

[0091] S4. After culturing the cells overexpressing CD64-GFP protein obtained in step S3 in a 15-cm culture dish until the density reaches 95%, wash them 3 times with pre-cooled PBS. Add 4 mL of PBS to collect the cells into a centrifuge tube, centrifuge at 4°C and 1000×g for 5 min, remove the supernatant, repeat the centrifugation 2 times. Resuspend with 5 mL of PBS and centrifuge at 4°C and 600×g for 5 min, remove the supernatant, then add 5 mL of PBS to resuspend and centrifuge at 4°C and 600×g for 1 min, remove the supernatant to obtain the treated cells;

[0092] S5. Resuspend the treated cells obtained in step S4 with an appropriate amount of mixed membrane protein extraction reagent and incubate on ice for 40 min until the cells absorb water and swell. Pipette 1.5 mL of the cell suspension into a pre-cooled glass homogenizer and homogenize about 70 times to obtain cell membrane fragments;

[0093] S6. Centrifuge the cell membrane fragments obtained in step S5 at 4°C and 700×g for 10 min, collect the supernatant, and then centrifuge at 4°C and 18000×g for 30 min, remove the supernatant to obtain the cell membrane overexpressing CD64-GFP protein;

[0094] S7. Use a microsyringe to extrude the syringe in the instrument (which has been washed with PBS), suck up the cell membrane suspension of the cell membrane overexpressing CD64-GFP protein obtained in step S6, insert it into both ends of the extruder, transfer the a syringe filled with the cell membrane suspension to the b syringe through the extruder, and then push the b syringe to push the liquid back into the a syringe. Repeat the extrusion step to make the cell membrane suspension pass through polycarbonate membranes with pore sizes of 1 μm, 400 nm, and 200 nm respectively, and extrude back and forth 11 times to ensure that the final cell membrane suspension is in the b syringe. The cell membrane suspension in the b syringe is the cell membrane vesicle suspension;

[0095] S8. Mix the BP@Cu obtained in step S1 with the cell membrane vesicles obtained in step S7 so that the mass ratio of BP@Cu to cell membrane vesicles is BP@Cu: cell membrane vesicles = 1:1, and ultrasonicate for 5 min with the ultrasonic power set at 120 W to obtain vesicle BP@Cu;

[0096] S9. Add glucose oxidase (hereinafter referred to as GOx) to the vesicle BP@Cu obtained in step S8 so that the mass ratio of BP@Cu to GOx is BP@Cu: GOx = 1:1, ultrasonicate for 2 min with the ultrasonic power set at 120 W, centrifuge at 4°C and 18000×g for 10 min. The obtained precipitate is vesicle BP@Cu-GOx, and add PBS to resuspend it to obtain a vesicle BP@Cu-GOx suspension;

[0097] S10. Mix the EGFR monoclonal antibody Cetuximab (hereinafter referred to as CTX) with the vesicle BP@Cu-GOx suspension obtained in step S9, so that the mass ratio of vesicle BP@Cu-GOx to CTX is vesicle BP@Cu-GOx:CTX = 10:1. Incubate at 4 °C for 30 min. After the incubation, centrifuge at 4 °C and 18,000×g for 10 min, remove the supernatant. The obtained precipitate is the composite cell membrane vesicle BP@Cu-GOx-CTX. Wash it three times with PBS to remove free GOx and CTX, and resuspend it with PBS for standby.

[0098] Example 2

[0099] Example 2 provides a composite cell membrane vesicle and its preparation method. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0100] In step S8, the mass ratio of BP@Cu to the cell membrane vesicle is adjusted to BP@Cu:cell membrane vesicle = 1:10;

[0101] In step S9, the mass ratio of BP@Cu to GOx is adjusted to BP@Cu:GOx = 10:1;

[0102] In step S10, the mass ratio of vesicle BP@Cu-GOx to CTX is adjusted to vesicle BP@Cu-GOx:CTX = 20:1;

[0103] The remaining steps and their parameter conditions remain unchanged.

[0104] Example 3

[0105] Example 3 provides a composite cell membrane vesicle and its preparation method. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0106] In step S1, the mass ratio of Cu element to BP is adjusted to Cu:BP = 0.016:1;

[0107] The remaining steps and parameter conditions remain unchanged.

[0108] Example 4

[0109] Example 4 provides a composite cell membrane vesicle and its preparation method. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0110] In step S1, the mass ratio of Cu element to BP is adjusted to Cu:BP = 0.08:1;

[0111] The remaining steps and parameter conditions remain unchanged.

[0112] Example 5

[0113] Example 5 provides a composite cell membrane vesicle and a preparation method thereof. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0114] In step S1, the mass ratio of Cu element to BP is adjusted to Cu:BP = 0.4:1;

[0115] The remaining steps and parameter conditions remain unchanged.

[0116] Example 6

[0117] Example 6 provides a composite cell membrane vesicle and a preparation method thereof. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0118] In step S1, the mass ratio of Cu element to BP is adjusted to Cu:BP = 10:1;

[0119] The remaining steps and parameter conditions remain unchanged.

[0120] Comparative Example 1

[0121] Comparative Example 1 provides a composite cell membrane vesicle and a preparation method thereof. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0122] In step S10, CTX is replaced with IgG to prepare a composite cell membrane vesicle BP@Cu-GOx-IgG;

[0123] The remaining steps and parameter conditions remain unchanged.

[0124] Comparative Example 2

[0125] Comparative Example 2 provides a composite cell membrane vesicle and a preparation method thereof. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0126] In step S9, GOx is not added, and finally a composite cell membrane vesicle BP@Cu-CTX is prepared;

[0127] The remaining steps and parameter conditions remain unchanged.

[0128] Comparative Example 3

[0129] Comparative Example 3 provides a composite cell membrane vesicle and a preparation method thereof. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0130] In step S8, BP@Cu is not added, and finally a composite cell membrane vesicle GOx-CTX is prepared;

[0131] The remaining steps and parameter conditions remain unchanged.

[0132] Comparative Example 4

[0133] Comparative Example 4 provides a composite cell membrane vesicle and a preparation method thereof. The preparation method of the cell membrane vesicle is similar to that of Example 1, except that:

[0134] In step S8, BP@Cu is not added;

[0135] In step S9, GOx is not added, and finally the composite cell membrane vesicle CTX is prepared;

[0136] The remaining steps and parameter conditions remain unchanged.

[0137] Effect Example 1

[0138] Characterize the composite cell membrane vesicles obtained in Examples 1, 3 - 6. The specific scheme is as follows:

[0139] 1 Characterize BP@Cu obtained in step S1 of Examples 1, 3 - 6

[0140] 1.1 Use a transmission scanning electron microscope (STEM) to observe the distribution of P and Cu elements (element mapping) in BP@Cu obtained in Example 1. The results are shown in Figure 2 .

[0141] As Figure 2 shown, the method of the present invention can successfully load Cu elements onto BP nanosheets to prepare Cu - doped BP nanosheets BP@Cu.

[0142] 1.2 Use inductively coupled plasma mass spectrometry (ICP - MS) to determine the content of Cu in BP@Cu obtained in step S1 of Examples 1, 3 - 6. The results are shown in Figure 3 .

[0143] As Figure 3 shown, as the content of Cu elements increases, the Cu doping amount in BP@Cu also increases. When the mass ratio of Cu elements to BP is 2:1, the highest value of Cu doping amount is reached. When the mass ratio of Cu elements to BP is 10:1, the Cu doping amount decreases. The above results indicate that further increasing the content of Cu elements during the preparation process will not affect its doping content on BP.

[0144] 1.3 Perform X - ray photoelectron spectroscopy (XPS) detection on BP@Cu obtained in Example 1. The results are shown in Figure 4 .

[0145] As Figure 4 shown, the energy spectrum of BP@Cu shows characteristic bands at 130.2 eV (P 2p3 / 2) and 131.1 eV (P 2p1 / 2), and at the same time, Cu also appears2+ (935.6 eV) and Cu + (932.9 eV) characteristic peaks, which are 15% and 85% respectively, indicating that most Cu 2+ is reduced to Cu + , and BP nanosheets are ideal carriers for loading and delivering highly unstable Cu + .

[0146] 2 Verify the composite cell membrane vesicles obtained in Example 1

[0147] 2.1 CD64 is a receptor for the IgG Fc fragment and can bind to various IgG antibodies through the Fc fragment. This binding has high affinity and specificity and can be achieved in a physiological environment, so that IgG antibodies targeting tumors such as CTX can be bound to the cell membrane vesicles. Therefore, the cells stably overexpressing the CD64 protein obtained in Example 1 were incubated with IgG carrying Alexa Flour568 (AF568) for 5 min and then observed under a fluorescence microscope to confirm whether the CD64 protein can specifically bind to IgG. During the preparation process, it was designed that the C-terminus of CD64 carried GFP. Whether the lentivirus of CD64-GFP was successfully infected could be judged by observing whether the nasopharyngeal carcinoma cell S18 carried green fluorescence under a fluorescence microscope; AF568 carried red fluorescence, and whether the IgG antibody was bound to the cell membrane could be judged by observing whether the cells incubated with the IgG antibody carried red fluorescence under a fluorescence microscope. The results are shown in Figure 5 ; Whether CD64-GFP was successfully overexpressed in the cell line stably overexpressing CD64 was analyzed by flow cytometry. The results are shown in Figure 6 ; Whether the CD64-GFP protein was successfully localized to the cell membrane after binding to IgG in the cell line stably overexpressing CD64 was observed by laser confocal microscopy. The results are shown in Figure 7 .

[0148] As Figure 5 shown, the cells overexpressing the CD64-GFP protein emitted green fluorescence and red fluorescence under a fluorescence microscope, proving that CD64 was stably expressed in the cells and IgG could specifically bind to CD64 on the cell membrane; as Figure 6 - 7 shown, the flow cytometry test results showed that a relatively high GFP fluorescence intensity was presented in the cells, indicating that the CD64-GFP cells were successfully overexpressed in the cells, while the laser confocal microscope confirmed that the CD64-GFP protein was successfully localized to the cell membrane.

[0149] 2.2 The cell membrane vesicles obtained in step S7 of Example 1 and the composite cell membrane vesicles BP@Cu-GOx-CTX obtained in step S10 were observed using a transmission electron microscope (TEM), and the size and zeta potential of the cell membrane vesicles were analyzed by dynamic light scattering (DLS). Figure 8 - 9 .

[0150] like Figure 8 As shown in the figure, the product prepared by extruding the cell membrane back and forth through the micro syringe extrusion instrument appears as a vesicle under TEM, indicating that the cell membrane vesicles were successfully prepared ( Figure 8 -a); BP@Cu-GOx-CTX after treatment still maintains vesicle shape, indicating that the composite cell membrane vesicles were successfully prepared ( Figure 8 -b). and cell membrane vesicles ( Figure 8 -a), the obtained composite cell membrane vesicles ( Figure 8 -b) There is an obvious high electron density area inside, and the cell membrane structure is covered on the outside, indicating that BP@Cu has successfully entered the cell membrane vesicles. Figure 9 As shown, the average size of cell membrane vesicles is 133.0 nm and the zeta potential is -8.4 mV; the average size of BP@Cu-GOx-CTX is 168.8 nm, slightly larger than the cell membrane vesicles, and the zeta potential is reduced to -16.6 mV, which is consistent with the surface binding of CTX.

[0151] 2.3 The contents of GOx and CTX in the vesicles BP@Cu-GOx obtained in step S9 and the vesicles BP@Cu-GOx-CTX obtained in step S10 of Example 1 were quantified by immunoblotting (WB). Figure 10 .

[0152] like Figure 10 As shown, GOx and CTX were successfully loaded or bound to the cell membrane vesicles, and quantitative spot calculation showed that 50 μg of BP@Cu could load about 0.60 μg of GOx, and about 0.57 μg of CTX was successfully bound to the surface of the composite cell membrane vesicles, giving the composite cell membrane vesicles the function of homologous / molecular dual targeting.

[0153] Effect Example 2

[0154] In order to explore the anti-tumor efficacy and mechanism of BP@Cu, BP@Cu-GOx, and BP@Cu-GOx-CTX, cell experiments and animal experiments were conducted to verify the efficacy. The specific scheme is as follows:

[0155] In the following experiments, unless otherwise specified, NPC cells S18 (hereinafter referred to as S18) were used as experimental subjects for cell experiments.

[0156] 1BP@Cu anti-tumor experiment and exploration of its mechanism of action

[0157] 1.1 S18 cells were co-cultured with 12.5 μg / mL BP@Cu for 2 h and analyzed by ICP-MS. The copper death inducer Elesclomol (ES) (dosage: 10 nM ES + 1 μm Cu) 2+ ) is a positive control, and the results are shown in Figure 11 -a; BP or BP@Cu obtained in Example 1 with different concentrations were co-cultured with S18 for 2 h, washed off, and replaced with new culture medium and cultured for 24 h. The activity of S18 was detected by CCK-8 detection kit. The results are shown in Figure 11 -b.

[0158] like Figure 11 As shown, the copper level in S18 cells increased significantly to about 154.8 ng / 10 5 cells, much higher than the copper level of the small molecule copper death inducer ES ( Figure 11 -a); BP and BP@Cu inhibited the activity of S18 in a dose-dependent manner, and the S18 activity of BP@Cu was significantly lower than that of BP( Figure 11 -b), indicating that BP doped with Cu can further increase the Cu + The delivery effect induced cell death and significantly inhibited cell activity.

[0159] 1.2 The oligomerization level of DLAT, a key marker of copper death, and the expression levels of Heat shock protein 70 (HSP70), Fe-S cluster protein Ferredoxin 1 (FDX1), and LIAS in S18 cells treated with BP@Cu obtained in Example 1 at different concentrations were analyzed by WB technology, and the loss of mitochondria in cells was observed by TEM. The results are shown in Figure 12 - 13 , the same concentrations of BP were used as the control group, with concentrations of 3.125, 6.25, and 12.5 μg / mL, respectively.

[0160] like Figure 12 As shown in Figure 3, after S18 cells were treated with BP@Cu, the oligomerization of DLAT increased, FDX1 and LIAS were downregulated, and HSP70 was upregulated, indicating that BP@Cu has the efficacy of inducing cell death by copper. Figure 13 As shown in the figure, the mitochondria of cells treated with BP showed partial damage, which is consistent with the mitochondrial damage in the process of copper death. At the same time, the treatment of BP@Cu caused more significant changes in the expression of copper death marker proteins and mitochondrial morphology, which is consistent with the enhanced copper death induction activity. In summary, BP nanosheets can efficiently load and deliver Cu into cells. + To induce copper death of NPC cells.

[0161] Antitumor Experiment of 2BP@Cu-GOx-CTX and Exploration of Its Mechanism of Action

[0162] 2.1 Incubate 2 mg / mL glucose solution with 50 μg / mL BP@Cu-GOx-CTX obtained in Example 1, and detect the glucose concentration and pH value at different time points. Co-culture S18 with 12.5 μg / mL BP@Cu-GOx-CTX obtained in Example 1 and BP@Cu-CTX obtained in Comparative Example 2 for 2 h respectively. Wash away the materials, add fresh medium and continue to culture for 24 h. Stain the cells with calcein AM / PI, detect the mitochondrial damage of the cells by TEM, and detect the oligomerization of DLAT, the expression levels of HSP70, aconitase 2 (Aco2), mitofusin 1 (MFN-1), citrate synthase (CS), and the key subunit UQCRC2 of mitochondrial complex III in the cells by WB. The results are shown in Figure 14 - 17 .

[0163] GOx can catalyze the degradation of glucose to produce gluconic acid and H2O2. As Figure 14 shown, the BP@Cu-GOx-CTX nanocomposite can effectively degrade glucose in a time-dependent manner, and a decrease in pH was also observed during this process, which is consistent with the generation of gluconic acid; as Figure 15 shown, compared with BP@Cu-CTX (Comparative Example 2), the tumor cell death induced by BP@Cu-GOx-CTX containing GOx was more significant (green for live cells, red for dead cells); as Figure 16 shown, the oligomerization of DLAT and mitochondrial damage occurred in cells treated with BP@Cu-GOx-CTX, indicating that more significant cuproptosis was induced during the co-delivery of GOx.

[0164] Aco2 is located in the mitochondrial matrix and is a key enzyme in the TCA cycle; MFN-1 is a key regulator of mitochondrial fusion and maintenance of mitochondrial morphology, and it is also involved in the regulation of the TCA cycle and energy supply; CS synthesizes citrate using acetyl-CoA and oxaloacetic acid as substrates, and this process is crucial for energy production in the TCA cycle; UQCRC2 is a key subunit of mitochondrial complex III. As Figure 17 shown, treatment with the nanocomposite without GOx (BP@Cu-CTX, Comparative Example 2) decreased the expression of proteins involved in the TCA cycle and oxidative phosphorylation. However, the co-delivery of GOx and the BP@Cu-GOx-CTX nanocomposite partially restored the expression of these proteins. Given the key role of the TCA cycle in the occurrence of cuproptosis, it can be speculated that the co-delivered GOx enhanced the sensitivity of tumor cells to BP@Cu-induced cuproptosis by manipulating energy metabolism.

[0165] 2.2 Co-cultured S18 with BP@Cu-GOx-CTX obtained in Example 1 at different concentrations and BP@Cu-GOx-IgG obtained in Comparative Example 1 for 24 h respectively. The cell viability was detected by calcein AM / PI staining and CCK-8 kit. The results are shown in Figure 18 .

[0166] As Figure 18 shown, compared with BP@Cu-GOx-IgG, BP@Cu-GOx-CTX significantly improved the efficiency of inhibiting tumor cell activity in a dose-dependent manner. Similarly, the tumor cell death induced by BP@Cu-GOx-CTX nanocomposite was more significant, indicating that BP@Cu-GOx-CTX of the present invention has excellent anti-tumor efficacy.

[0167] 2.3 Exposed BP, BP@Cu obtained in Example 1, BP@Cu-GOx, BP@Cu-GOx-CTX to 808 nm near-infrared laser (NIR) irradiation for 0 - 300 s, and measured their temperatures. The results are shown in Figure 19 -a; Exposed BP@Cu-GOx-CTX obtained in Example 1 at different concentrations to 808 nm NIR for 0 - 300 s, and measured their temperatures. The results are shown in Figure 19 -b; Co-cultured BP@Cu-GOx-CTX obtained in Example 1 at different concentrations with S18, and irradiated one group with 808 nm NIR for 300 s. The cell viability of S18 was measured by CCK-8 detection kit. The results are shown in Figure 19 -c.

[0168] As Figure 19 shown, copper doping further enhanced the photothermal performance of BP nanosheets under 808 nm NIR irradiation, and this photothermal performance was not significantly affected by the presence of GOx, cell membrane vesicles or CTX. At the same time, BP@Cu-GOx-CTX showed a dose-dependent significant increase in temperature, indicating its potential in photothermal therapy (PTT) of NPC. In addition, irradiating NPC cells with 808 nm NIR after treatment with BP@Cu-GOx-CTX nanocomposite could significantly increase the tumor cell mortality and effectively reduce the activity of tumor cells.

[0169] 2.4 Divided Balb / c nude mice into 6 treatment groups, namely control group, control + irradiation group, IgG group, IgG + irradiation group, CTX group, CTX + irradiation group. Subcutaneously injected S18 into the mice of the above groups. When the tumor volume reached about 100 mm 3At that time, normal saline was intravenously injected (control group / control + irradiation group), BP@Cu-GOx-IgG obtained in Comparative Example 1 (IgG group / IgG + irradiation group), or BP@Cu-GOx-CTX obtained in Example 1 (CTX group / CTX + irradiation group). After injection, the mice in the control + irradiation group, IgG + irradiation group, and CTX + irradiation group were irradiated with 808 nm NIR (1 W / cm 2 ) for 10 min. After normal breeding for 24 h, the tumors were collected for WB detection of the oligomerization of DLAT and the expression level of HSP70, and the body temperature of the irradiated mice was measured. The results are shown in Figure 20 . They were continuously bred until the 10th day. The tumor volume and body weight of the mice were measured every day. On the 10th day, the tumor tissues were removed and weighed. The results are shown in Figure 21 - 22 .

[0170] As Figure 20 shown, compared with the mice injected with normal saline control group, significant increases in DLAT oligomerization and HSP70 expression were shown in the mice injected with BP@Cu-GOx-IgG, which was consistent with the delivery of BP@Cu and GOx into the tumor by homologous targeting. At the same time, more significant increases in DLAT oligomerization and HSP70 expression were observed in the tumors treated with BP@Cu-GOx-CTX. This result indicates that the composite cell membrane vesicle BP@Cu-GOx-CTX can efficiently induce cuproptosis of NPC in vivo.

[0171] As Figure 21 shown, after 808 nm laser irradiation, compared with the mice injected with BP@Cu-GOx-IgG, a more significant increase in temperature was observed in the tumors of the mice injected with BP@Cu-GOx-CTX ( Figure 21 -a), which was consistent with the enhanced NPC targeting ability mediated by CTX. Subsequently, during the measurement of the tumor size of the mice within 10 days, a slight inhibition of tumor growth was observed in the mice injected with BP@Cu-GOx-IgG, while a more significant inhibition of tumor growth was observed in the mice injected with BP@Cu-GOx-CTX. This may be due to the BP@Cu-GOx-CTX nanocomposite through efficient tumor targeting, EGFR signaling pathway inhibition, and cuproptosis induction. When combined with 808 nm laser irradiation, all tumors of the mice injected with BP@Cu-GOx-CTX were effectively inhibited ( Figure 21 -b, 21-c, 21-d). At the same time, there was no significant change in the body weight of the mice ( Figure 22 ), indicating that the composite cell membrane vesicle of the present invention has certain anti-tumor properties and certain safety.

[0172] 2.5 Co-culture S18 with BP@Cu-GOx-CTX obtained in Example 1 or BP@Cu-GOx-IgG obtained in Comparative Example 1 for 1 - 4 h, and observe and analyze the targeting effect between different cells by confocal laser microscopy. The results are shown in Figure 23 .

[0173] As Figure 23 shown, the uptake of BP@Cu-GOx-IgG by cells in the negative control increased with time, which might be mediated by homologous targeting. Meanwhile, BP@Cu-GOx-CTX was more effectively taken up by NPC cells, which might be due to CTX enhancing the targeting effect on EGFR. By quantifying the mean fluorescence intensity (MFI) of cells, it was estimated that after 2 h of incubation, the amount of CTX-conjugated vesicles taken up by cells was approximately 1.42 times more than that of IgG-conjugated vesicles.

[0174] 2.6 Female Balb / c nude mice at 5 - 7 weeks of age were subcutaneously injected with S18 (1×10 7 cells per mouse) to establish NPC model mice. When the tumor volume of the mice increased to approximately 100 mm 3 , they were randomly grouped and intravenously injected with 2.5 mg / kg (200 μL) of BP@Cu-GOx-CTX obtained in Example 1 or BP@Cu-GOx-IgG obtained in Comparative Example 1. After normal feeding for 4 h, in vivo imaging was performed. They were continuously fed until 24 h, then the mice were sacrificed, and tumors and major organs were taken for in vivo imaging. The results are shown in Figure 24 .

[0175] As Figure 24 shown, the mice treated with BP@Cu-GOx-CTX showed higher NPC targeting efficiency ( Figure 24 -a), and imaging of the major organs and tumor tissues of the mice further confirmed the NPC targeting ability of BP@Cu-GOx-CTX ( Figure 24 -b). The NPC targeting ability of BP@Cu-GOx-CTX was also confirmed in vivo in mice ( Figure 24 -c). Therefore, CD64 vesicles can serve as an effective homologous / molecular dual-targeted nanoplatform to achieve targeted drug delivery for NPC through CTX conjugation.

[0176] 2.7 Co-culture S18 with 12.5 μg / mL of BP@Cu-GOx-CTX obtained in Example 1 and BP@Cu-GOx-IgG obtained in Comparative Example 1 for 24 h respectively, and detect the phosphorylation levels of EGFR, AKT (a key downstream effector of EGFR), and ERK (a key downstream effector of EGFR) of S18 by WB technology. The results are shown in Figure 25 .

[0177] As Figure 25As shown, compared with the negative control BP@Cu-GOx-IgG, BP@Cu-GOx-CTX significantly inhibited the phosphorylation of EGFR at the tyrosine 1068 residue, which is crucial for EGFR activation and signal transduction. Correspondingly, the phosphorylation of two key downstream effectors of the EGFR signaling pathway, AKT and ERK, was also inhibited. In addition, efficient inhibition of EGFR phosphorylation was also observed in NPC cells treated with BP@Cu-GOx-CTX( Figure 25 -c). The above results indicate that the composite cell membrane vesicle BP@Cu-GOx-CTX of the present invention can block ligand binding, autophosphorylation and activation of EGFR, thereby inhibiting tumors.

[0178] In summary, the composite cell membrane BP@Cu-GOx-CTX of the present invention is effective in anti-tumor, especially anti-nasopharyngeal carcinoma, by targeting NPC for EGFR blockade and cuproptosis induction. At the same time, the composite cell membrane BP@Cu-GOx-CTX of the present invention can also synergistically perform photothermal therapy to further inhibit tumor growth.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composite cell membrane vesicle, characterized in that, The composite cell membrane vesicles contain BP@Cu and glucose oxidase; the surface of the vesicles contains CD64 protein and EGFR monoclonal antibody.

2. The composite cell membrane vesicle according to claim 1, wherein The mass ratio of Cu element to BP in the BP@Cu is Cu:BP = (0.016 - 10):

1.

3. The composite cell membrane vesicle according to claim 1, characterized in that The mass ratio of BP@Cu to glucose oxidase is BP@Cu:glucose oxidase = (400 - 500):(5 - 6).

4. The method for preparing the composite cell membrane vesicle according to any one of claims 1-4, characterized in that, It includes the following steps: (1) Cells overexpressing CD64 protein are constructed by lentivirus infection technology, and the cell membrane overexpressing CD64 protein is isolated and purified by a hypotonic method. (2) The cell membrane overexpressing CD64 protein obtained in step (1) is extruded to obtain cell membrane vesicles overexpressing CD64 protein. (3) BP@Cu and glucose oxidase are loaded into the cell membrane vesicles overexpressing CD64 protein obtained in step (2) to obtain vesicles BP@Cu-GOx. (4) The vesicles BP@Cu-GOx obtained in step (3) are mixed with EGFR monoclonal antibody and incubated to obtain composite cell membrane vesicles BP@Cu-GOx-CTX.

5. The preparation method according to claim 4, characterized in that, It includes at least one of the following (Ⅰ) - (Ⅱ): (Ⅰ) In step (1), the cells are tumor cells. (Ⅱ) In step (1), the lentivirus infection technology is to construct a lentivirus containing the CD64 protein coding sequence and the GFP protein coding sequence, infect the cells, and screen through puromycin to obtain cells stably overexpressing CD64 protein.

6. The preparation method according to claim 4, characterized in that, It includes at least one of the following (Ⅲ) - (Ⅳ): (Ⅲ) In step (3), the mass ratio of BP@Cu to glucose oxidase in the vesicles BP@Cu is BP@Cu:glucose oxidase = (1 - 10):

1. (Ⅳ) In step (3), the mixing is by ultrasonic treatment at a power of 110 - 120W for 2 - 5 min.

7. The preparation method according to claim 4, characterized in that, It includes at least one of the following (Ⅴ) - (Ⅵ): (Ⅴ) In step (4), the mass ratio of vesicles BP@Cu-GOx to EGFR monoclonal antibody is vesicles BP@Cu-GOx:EGFR monoclonal antibody = (10 - 20):

1. (Ⅵ) In step (4), the incubation conditions are incubation at 4°C for 25 - 30 min.

8. Use of the composite cell membrane vesicles according to any one of claims 1 - 3 in the preparation of anti-tumor drugs.

9. Use of the composite cell membrane vesicles according to any one of claims 1 - 3 in combination with near-infrared light treatment in the preparation of anti-tumor drugs.

10. The application according to claim 9, characterized in that, The wavelength of the near-infrared light is 805 - 810 nm.

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